Bifunctional Cytotoxic Agents

专利摘要:

公开号:ES2722103T9
申请号:ES15708273T
申请日:2015-01-14
公开日:2020-02-11
发明作者:Andreas Maderna；Matthew David Doroski；Zecheng Chen；Hud Lawrence Risley；Jeffrey Michael Casavant；Christopher John O'donnell；Alexander M Porte；Chakrapani Subramanyam
申请人:Pfizer Inc；
IPC主号:

专利说明:

[0001]
[0002] Bifunctional Cytotoxic Agents
[0003]
[0004] Field of the Invention
[0005]
[0006] The present invention relates to novel CBI and CPI dimers useful for the treatment of proliferative diseases. The dimers can function as individual drugs, fillers in the antibody and drug conjugates (ADC), and linker-loading compounds useful with respect to the production or administration of said ADCs. The present invention also relates to compositions that include the dimers, linker-type compounds and ADCs mentioned above, for use in pathological conditions, including cancer.
[0007]
[0008] Background
[0009]
[0010] CPI-based monomers have been the subject of recent publications. For example, compounds (+) - CC-1065 and duocarmycins are natural products isolated from the culture broth of Streptomyces species , which have been shown to exert an ultra-potent activity against cultured cancer cells and in experimental animals. The (+) - yatakemycin has been isolated from Streptomyces sp. and represents the most powerful member of this class of natural products. It is believed that the biological activity of these natural products is related to a specific DNA alkylation of the N3 adenine sequence at sites rich in AT by the less substituted carbon atom of the activated cyclopropane. It is believed that this minor cleft junction initiates a cascade of cellular events that produces apoptosis as seen for duocarmicins ("Chemical and Biological Explorations of the Family of CC-1065 and the Duocarmycin Natural Products", Current Topics in Medicinal Chemistry , 2009, 9, 1494-1524). The key structural reason in these and other related analogs is the CPI structure, which is the active reactive group that the ADM rents:
[0011]
[0012]
[0013]
[0014]
[0015] The prodrug form of the CPI is converted into the active drug species in the biological medium by an intramolecular cyclization reaction. (The term "CPI" is derived from the chemical name: 1,2,8,8-tetrahydrocyclopropa [c] pyrrolo [3,2-e] indole-4 (5H) -one). In this way, the prodrug of the CPI is converted into the active drug species by an intramolecular cyclization reaction. Synthetic phenol precursors (prodrug form) have non-distinguishable biological properties (efficiency and selectivity of DNA alkylation, in vitro cytotoxic activity , in vivo antitumor activity ) compared to cyclopropane derivatives (active form) ("Design, Synthesis, and Evaluation of Duocarmycin O-Amino Phenol Prodrugs Subject to Tunable Reductive Activation ", J. Med. Chem. 2010, 53, 7731-7738). In other words, it does not matter if the applied CPI is in its active form of cyclopropane or in its prodrug form. It is important to highlight that, in these compounds, only one CPI motif is present, therefore, these compounds act as DNA monoalkylants. Subsequently, other synthetic analogs of CPI structures have been developed, that is, those shown in ("Chemical and Biological Explorations of the Family of CC-1065 and the Duocarmycin Natural Products", Current Topics in Medicinal Chemistry, 2009, 9, 1494-1524 ). Of interest in this reference are the synthetic analogs CBI, Cpzl, CFI, CI and CBQ. Alkylating duocarmycin analogs have been extensively studied in preclinical and clinical studies ("Chemical and Biological Explorations of the Family of CC-1065 and the Duocarmycin Natural Products, Current Topics in Medicinal Chemistry, 2009, 9, 1494-1524).
[0016]
[0017] An independent, but related, class of compounds are bifunctional analogs that contain two reasons for DNA alkylation, (i.e., a CPI). These compounds are different from conventional duocarmycins in that they lack the rest comprised between duocarmycins, which acts as a reason for DNA recognition. In contrast, these bifunctional compounds contain only two alkylation points (ie, two CPI motifs) condensed together. Due to the presence of two reactive alkylation motifs, these compounds are active crosslinkers of the Asset, while compounds that only have one alkylation motif (all duocarmycins) are only DNA monoalkylating agents.
[0018]
[0019]
[0020] C P I-d im e ro (B ize le s in a) C C P I-d im e ro D
[0021]
[0022] The compounds shown above are representative examples of the literature, and cytotoxins have been reported to be potent: A ("Glycosidic Prodrugs of Highly Potent Bifunctional Duocarmycin Derivatives for Selective Treatment of Cancer", Angew. Chem. Int. Ed. 2010, 49 , 7336-7339; "Duocarmycin Analogues Target Aldehyde Dehydrogenase 1 in LungCancer Cells", Angew. Chem. Int. Ed. 2012, 51,2874-2877; "Bifunctional prodrugs and drugs", document WO 2011/054837, document DE 10 2009 051 799; "The Two Faces of Potent Antitumor Duocarmycin-Based Drugs: A Structural Dissection Reveals Disparate Motifs for DNA versus Aldehyde Dehydrogenase 1 Affinity", Angew. Chem. Int. Ed. 2013, 52, 1-6. B ("Interstrand DNA Cross-linking with Dimers of the Spirocyclopropyl Alkylating Moiety of CC-1065 ", J. Am. Chem. SOC. 1989, 11 1,6428-6429;" CC-1065 analogs having two CPI subunits useful as antitumor agents and ultraviolet light absorbers ", European patent application (1990), EP 359454, also also for compounds C and D; C ("Synthesis and DNA Cross-Linking by a Rigid CPI Dimer", J. Am. Chem. SOC. 1991,113, 8994-8995; "Nucleotide Preferences for DNA Interstrand Cross-Linking Induced by the Cyclopropylpyrroloindole Analogue U-77,779" , Biochemistry 1993, 32, 2592-2600; "Determination of the Structural Role of the Internal Guanine-Cytosine Base Pair in Recognition of a Seven-Base-Pair Sequence Cross-Linked by Bizelesin", Biochemistry 1995, 34, 11005-11016; "Analysis of the Monoalkylation and Cross-Linking Sequence Specificity of Bizelesin, a Bifunctional Alkylation AgentRelated to (+) - CC-1065", J. Am. Chem. SOC. 1993,115, 5925 5933; "Mapping of DNA Alkylation Sites Induced by Adozelesin and Bizelesin in Human Cells by Ligation-Mediated Polymerase Chain Reaction ", Biochemistry 1994, 33, 6024-6030;" DNA Interstrand Cross-Links Induced by the Cyclopropylpyrroloindole Antitumor Agent Bizelesin Are Reversible upon Exposure to Alkali ", Biochemistry 1993, 32 , 9108-9114; "Replacement of the Bizelesin Ureadiyl Li nkage by a Guanidinium Moiety Retards Translocation from Monoalkylation to Cross-Linking Sites on DNA ", J. Am. Chem. Soc. 1997, 119, 3434-3442; "DNA interstrand crosslinking, DNA sequence specificity, and induced conformational changes produced by a dimeric analog of (+) - CC-1065", Anti-Cancer Drug Design (1991), 6, 427-452; "A phase I study of bizelesin, a highly potent and selective DNAinteractive agent, in patients with advanced solid malignancies", Ann Oncol. May 2003; 14 (5): 775-782; "A Phase I study of bizelesin (NSC 615291) in patients with advanced solid tumors", Clin Cancer Res. 2002, 3, 712-717; "Solution conformation of a bizelesin A-tract duplex adduct: DNA-DNA cross-linking of an A-tract straightens out bent DNA", J Mol Biol. 1995, 252, 86-101; "Preclinical pharmacology of bizelesin, a potent bifunctional analog of the DNA-binding antibiotic CC-1065", Cancer Chemother Pharmacol. 1994, 34, 317-322. D ("CC-1065 analogs having two CPI subunits useful as antitumor agents and ultraviolet light absorbers", European patent application (1990), EP 359454. The reason for active alkylation of DNA may exist, in principle, as well as prodrug that converts into the active drug in the biological medium, or in its active state that requires additional conversion.The conversion of the prodrug into an active drug for the bifunctional crosslinkers is illustrated for the CBI dimer shown below:
[0023]
[0024]
[0025]
[0026]
[0027] The same conversion occurs for all bifunctional crosslinkers that exist in their prodrug states. Other bifunctional cross-linking relationships have been reported. ("Chemical and Biological Explorations of the Family of CC-1065 and the Duocarmycin Natural Products", Current Topics in Medicinal Chemistry, 2009, 9, 1494-1524; "DNA interstrand crosslinking agents and their chemotherapeutic potential", Curr Med Chem. 2012 , 19, 364-385; "Design and Synthesis of a Novel DNA-DNA Interstrand Adenine-Guanine Cross-Linking Agent", J. Am. Chem. Soc. 2001, 123, 4865-4866; "Effect of base sequence on the DNA cross-linking properties of pyrrolobenzodiazepine (PBD) dimers ", Nucleic Acids Res. 2011,39, 5800-5812; "Sequence-selective recognition of DNA duplex through covalent interstrand cross-linking: kinetic and molecular modeling studies with pyrrolobenzodiazepine dimers", Biochemistry.
[0028] 2003, 42, 8232-8239; "Bifunctional alkylating agents derived from duocarmycin SA: potent antitumor activity with altered sequence selectivity", Bioorg Med Chem Lett. 2000, 10, 495-498; "Design, Synthesis and Cytotoxicity Evaluation of 1-Chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] indole (seco-CBI) Dimers", Bioorganic & Medicinal Chemistry 2000, 8, 1607-1617.
[0029]
[0030] A strategy for phosphate prodrugs for cytotoxins containing dry-monomeric CBI has been described in Zhao et al. ("Synthesis and biological evaluation of antibody conjugates of phosphate prodrugs of cytotoxic DNA alkylators for the targeted treatment of cancer", J. Med. Chem. 2012, 55, 766-782) and Zhang et al. ("Immunoconjugates containing phosphate-prodrugged DNA minor groove binding agents, compositions containing them, and methods of making them and their use for treating cancer", WO 2012/162482).
[0031]
[0032] None of the above-mentioned compounds, which have two CBI and / or CPI nuclei linked together to form a dimeric species (so-called cBl dimers, CPI dimers, or CBI / CPI dimers), have been considered for use in antibody conjugates and drug (ADC) as cargo.
[0033]
[0034] The conjugation of drugs with antibodies, both directly and through linkers, involves the consideration of a variety of factors, including the identity and location of the chemical group for the conjugation of the drug, the mechanism of drug release, the structural elements that provide the drug release, and structural modification towards the free drug released. In addition, if the drug is to be released after the internalization of the antibody, the drug release mechanism must correspond to the intracellular traffic of the conjugate.
[0035]
[0036] Although numerous different classes of drugs have been tested for administration by antibodies, only a few classes of drugs have proven effective as antibody and drug conjugates, while maintaining an adequate toxicity profile. One of the aforementioned classes is that of auristatins, derived from the natural product dolaestatin 10. Representative auristatins include (N-methylvaline-valinadolaisoleuinadolaprolina-norefedrina) and (N-methylvalina-valine-dolaisoleuina-dolaprolina-phenylalanine). Other tubulin-binding agents include maitansins (for example, see "Cell-binding agent-maytansinoid conjugates linked via a noncleavable linker, preparation methods, and methods using them for targeting specific cell populations" published as WO 2005/037992) . Other cytotoxic drugs that have been used in antibody binding include DNA binding drugs such as calicamycin that produces the cleavage of the sequence-specific double stranded DNA. Another class of cytotoxic DNA-binding drugs used in ADCs includes dimeric pyrrolobenzodiazepines (for example, see "Preparation of unsymmetrical pyrrolobenzodiazepines dimers for inclusion in targeted conjugates" published as document WO2013 / 041606). Another class of drugs where antibody administration has been attempted is that of DNA-binding alkylating agents, such as the duocarmycin analogs CC-1065 (see "Preparation of CC-1065 analogs and their conjugates for treatment of cancer" published as WO2010 / 062171) and related compounds (see "Antibody-drug peptide conjugates for use as cytotoxins in cancer treatment" published as WO 2007/038658, and "Immunoconjugates containing phosphate-prodrugged DNA minor groove binding agents, compositions containing them, and methods of making them and their use for treating cancer "published as document WO2012 / 162482). However, all these drugs have limitations regarding the indications of the disease and the treatment profile and therefore, there is still a need for additional drugs with improved properties that can be administered by antibody conjugation. Therefore, the present invention provides novel ADCs that are dimers as fillers.
[0037]
[0038] Summary of the invention
[0039]
[0040] The invention describes new analogs of structural dimers containing novel linker elements. These new separating motifs transmit different biological properties to the compounds, for example improved activities in cell proliferation assays and plasma stabilities. The present invention also describes new separator elements for the corresponding CPI dimers and CBI-CPI mixed structures.
[0041] Furthermore, the present invention is the first to disclose such compounds linked to an ADC in one embodiment, and incorporating these compounds into a targeted ADC is a significant advance.
[0042]
[0043] The present invention relates to cytotoxic dimers comprising CBI and / or CPI subunits (including dry forms of CBI and / or CPI, as detailed herein, antibody and drug conjugates comprising said dimers, and the same for use in the treatment of cancer Both CBI and CPI structures can be represented by their dry form and can be substituted and derivatized as detailed herein.
[0044]
[0045] Therefore, the present invention relates to compounds and pharmaceutical compositions containing them, and to the compounds for use, principally but not exclusively, as anti-cancer agents. According to one aspect, the present invention relates to "filler" compounds of Formula I:
[0046]
[0047] F1-L1-T-L2-F2 (Formula I)
[0048] a pharmaceutically acceptable salt thereof, in which:
[0049] each of F1 and F2 is independently selected from ring systems A, B, C and D:
[0050]
[0051] (Ring system A)
[0052]
[0053]
[0054]
[0055] (Ring C system)
[0056]
[0057]
[0058] each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ -alkenyl , C ² -C ⁶ -alkynyl , C ² -C ^6, halo, hydroxy, alkoxy, ^-NH2, -NH ( C ¹ C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ² , -NO ² , -C6-C14 aryl and C ⁶ -C ¹⁴ heteroaryl, in which two or more R are optionally joined to form a ring or rings, and wherein said Ca-C ¹⁴ -aryl and -heteroaryl -C ⁶ -C ¹⁴ optionally substituted with 1 to 5 substituents independently selected from alkyl ^C1 - ^{C10, C1} - ^C10 alkoxy , -halo, -C ¹ -C ¹⁰ alkylthio, -trifluoromethyl, -NH ² , -NH (C ¹ -C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ² , -C ¹ -C ¹⁰ alkyl N (C ¹ -C ⁸ alkyl) ² , -C ¹ -C ³ alkylthio, -NO ² or -C ¹ -C ¹⁰ heterocyclyl, for each ring system in which R appears;
[0059] each V1 is independently a link, O, N (R) or S, for each ring system in which V1 appears; each V2 is independently O, N (R) or S, for each ring system in which V2 appears;
[0060] each of W 1 and W2 is independently H, -C1-C5 alkyl for each ring system in which W 1 and W2 appear;
[0061] each X is independently -OH, -O-acyl, azido, halo, cyanate, thiocyanate, isocyanate, thioisocyanate, or
[0062]
[0063]
[0064]
[0065] for each ring system in which X appears;
[0066] each Y is independently selected from the group consisting of H, -C ¹ -C ⁶ -RA alkyl, -C (O) RA, -C (S) RA, -C (O) ORA, -S (O) ² ORa, -C (O) N (Ra) ² , -C (S) N (Ra) ² , glycosyl, -NO ² and -PO (ORA) ² , for each ring system in which Y appears, in the that each RA is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ -aryl, aralkyl, -C ¹ -C ¹⁰ -heterocyclyl, -carbocyclyl C ³ -C ⁸ and -C ¹ -C ²⁰ alkyl (R) ² , wherein said -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ aryl, aralkyl, - C ¹ -C ¹⁰ heterocyclyl, C ³ -C ⁸ -carbocyclyl and -C ¹ -C ²⁰ N (R ^{) 2} alkyl are optionally substituted with 1 to 3 substituents independently selected from R;
[0067] each Z is independently selected from the group consisting of H, and -C ¹ -C ⁸ alkyl and wherein each of said C ¹ -C ⁸ is optionally substituted with 1 to 3 substituents independently selected from R, for each ring system in which Z appears;
[0068] each of L1 and L2 is independent a direct link;
[0069] T is selected from:
[0070] -C (A1) X1-T2-X1C (B1) -, where T2 is:
[0071]
[0072]
[0073]
[0074] in which each X1 is independently a bond, in which each of A1 and B1 are independently = O in which each of gyj are independently 0 and m is 1, and in which D is bicyclo (1.1.1) pentane or Cuban, where said bicyclo (1.1.1) pentane or Cuban is optionally substituted with -RE, -C (O) RE, -C (O) ORE, -N (RE) ² , -N (R) C (O ) RE or -N (R) C (O) Or e, and D is additionally optionally substituted with 1 to 2 R, and
[0075] wherein each RE is independently selected from the group consisting of H, C ¹ -C ⁸ -alkyl, C ¹ -C ⁸ -heteroalquilo, -aryl C ⁶ -C ¹⁴ -aralkyl, ^C1 - ^C10 alkylene- , -C ³ -C ⁸ -carbocyclyl, -C (O) -C ¹ -C ⁸ alkyl, -C (O) N (C ¹ -C ⁸ alkyl) ² , and -C (O) -halo, and in the that each RE is optionally substituted with 1 to 3 substituents independently selected from R.
[0076] In other embodiments of the invention the variable -Y- is C (O) N (R ^{A) 2} or C (S) N (R ^{A) 2} where one RA is hydrogen or -C ¹ -C ²⁰ alkyl and the other RA it is -C ¹ -C ²⁰ -N (R) ² alkyl, so that the structure:
[0077]
[0078]
[0079]
[0080] it forms, where A is oxygen or sulfur.
[0081] As mentioned above, the embodiments of the present invention include those where R1, R2, R3 and R4 are each bonds to different carbon atoms in D. When D is a 6-membered carbocyclic ring (in bold, below) , this embodiment can take the form of a Cuban:
[0082]
[0083]
[0084]
[0085] Other forms of Cubans (for example substituted forms as detailed herein) and non-Cubans are also possible and are included within the invention.
[0086] According to another aspect of the invention, a "payload linker" compound of Formula IIA is provided:
[0087] L-P (Formula IIA)
[0088] or a pharmaceutically acceptable salt or solvate thereof, in which:
[0089] P is:
[0090] F1-L1-T-L2-F2
[0091] in which:
[0092] each of F1 and F2 is independently selected from ring systems A, B, C and D:
[0093]
[0094]
[0095]
[0096] (Ring system A)
[0097]
[0098]
[0099]
[0100] (Ring C system)
[0101]
[0102]
[0103]
[0104] each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ -alkenyl , C ² -C ⁶ -alkynyl , C ² -C ^6, halo, hydroxy, alkoxy, ^-NH2, -NH ( C ¹ -C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ² , -NO ² , -C ⁶ -C ¹⁴ -aryl and C ⁶ -C ¹⁴ -heteroaryl, in which two or more R are optionally linked to form a ring or rings, and wherein said -C ⁶ -C ¹⁴ aryl and -C ⁶ -C ¹⁴ heteroaryl are optionally substituted with 1 to 5 substituents independently selected from -C ¹ -C ¹⁰ alkyl, -alkoxy C ¹ -C ¹⁰ , -halo, -C ¹ -C ¹⁰ -alkylthio, -trifluoromethyl, -NH ² , -NH (C ¹ -C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ² , -C alkyl ¹ -C ¹⁰ -N (C ¹ -C ⁸ alkyl) ² , -C ¹ -C ³ alkylthio, -NO ² or -C ¹ -C ¹⁰ heterocyclyl, for each ring system in which R appears; each V ¹ is independently a link, O, N (R) or S, for each ring system in which V ¹ appears; each V ² is independently O, N (R) or S, for each ring system in which V ² appears;
[0105] each of W ¹ and W ² is independently H, -C ¹ -C ⁵ alkyl for each ring system in which W ¹ and W ² appear; each X is independently selected from -OH, -O-acyl, azido, halo, cyanate, thiocyanate, isocyanate, thioisocyanate, or
[0106]
[0107]
[0108]
[0109] for each ring system in which X appears;
[0110] each Y is independently selected from a link, H, -C (O) R ^A , -C (S) R ^A , -C (O) OR ^A , -S (O) ² OR ^A , -C (O) N (R ^A ) ² , -C (S) N (R ^a ) ² , glycosyl, -NO ² and -P (O) (OR ^A ) ² for each ring system in which Y appears, in which each R ^A is independently selected from H, -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ -aryl, aralkyl, -C ¹ -C ¹⁰ -heterocyclyl, -C ³ -C ⁸ -carbocyclyl, - C ¹ -C ²⁰ N (R) ² alkyl, C ¹ -C ²⁰ alkylene, C ¹ -C ⁸ heteroalkylene, C ⁶ -C ¹⁴ -arylene, aralkylene, heterocycle C ¹ -C ¹⁰ , -carbocycle C ³ -C ⁸ and alkyl-alkyl C ¹ -C ²⁰ N (R) -, and RF where said RA is optionally substituted with 1 to 3 substituents independently selected from R and in the that a Y is divalent and is linked to L, RF is -N (R6) QN (R5) C (O) - and is attached to L in the carbonyl adjacent to N (R5), in which R5 and R6 each is independently selected from the group consisting of H, C ¹ -C ⁸ -alkyl, C ¹ -C ⁸ -heteroalquilo, -aryl C ⁶ -C ¹⁴ -aralkyl, ^C1 - ^C10 heterocyclyl and carbocyclyl -C ³ -C ⁸ , or R5 or R6 bind to a carbon atom substituted on Q to form a C ¹ -C ¹⁰ heterocycle ring or C ⁶ -C ¹⁴ -heteroaryl ring, or R5 and R6 bind to each other to form a C ¹ -C ¹⁰ -heterocyclic or C ⁶ -C ^14- heteroaryl ring system, and where Q is -C ¹ -C ⁸ alkylene -, -C ¹ -C ⁸ -alkylene, -C ⁶ -C -arylene ¹⁴ -, - alkylene -, - C ¹ -C ¹⁰ heterocycle - or - C ³ -C ⁸ carbocycle -, in which each of Q, R5 and R6 are substituted tuidos independently and optionally with 1 to 3 substituents independently selected from R;
[0111] each Z is independently selected from the group consisting of H, -C ¹ -C ⁸ alkyl and wherein said C ¹ -C ⁸ alkyl, each is optionally substituted with 1 to 3 substituents independently selected from R, for each ring system in which Z appears;
[0112] each of L1 and L2 is independently selected from a direct link
[0113] T is selected from:
[0114] -C (A1) X1-T2-X1C (B1) -, where T2 is:
[0115]
[0116]
[0117]
[0118]
[0119] in which each X1 is independently a link in which each of A1 and B1 is independently = O in which each of gyj are independently 0 and m is 1, and in which D is bicyclo (1.1.1) pentane or Cuban where said bicyclo (1.1.1) pentane or Cuban is optionally substituted with -RE, -C (O) RE, -C (O) ORE, -N (RE) ² , -N (R) C (O) RE or -N (R) C (O) ORE, and D is additionally optionally substituted with 1 to 2 R, and
[0120] wherein each RE is independently selected from the group consisting of H, C ¹ -C ⁸ -alkyl, C ¹ -C ⁸ -heteroalquilo, aryl, -aralkyl, ^C1 - ^C10 heterocyclyl, -carbociclilo C ³ - C ⁸ , -c (o) -C ¹ -C ⁸ alkyl, -C (O) N (C- ^1- C ⁸ alkyl) ² , and -C (O) -halo, and in which each RE is optionally substituted with 1 to 3 substituents independently selected from R,
[0121] L is La-Lb- (Lc) - ^1-3 , in which LA is selected from the group consisting of -halo, -N (R) ² , -CON (R) ² , -S-aryl optionally substituted with -NO ² or -CON (R) ² , -S-heteroaryl optionally substituted with -NO ² , alkyl-SO ² -heteroaryl, arylSO ² -heteroaryl-,
[0122]
[0123]
[0124]
[0125]
[0126] Lb is LB1-LB2-LB3 in which LB1 is absent or is one or more components selected from the group consisting of -C (O) -, -C (S) -, -C (O) NR-, -C ( O) ^C1 - ^C6 alkyl -, -C (O) ^C1 - ^C6 NRalquil -, -C ¹ -C ⁶ -alkyl - (OCH ² CH ^{2) 1-6} -, -C (O) C ¹ alkyl -C6NRC (O) -, -C (O) C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ¹ - ⁶ -, -C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ¹ - ⁶ -C ( O) -, C ¹ C ⁶ -alkyl -SS-alkyl ^C1 - ^C6 NRC (O) CH ² -, -C ¹ -C ⁶ -alkyl - (OCH ² CH ^{2) 1-6} NRC (O) CH ² -, -C (O) alkyl ^C1 - ^C6 -NRC (O) -C ^{1. 6} -, -N = CR-phenyl-O-C ¹ -C ⁶ alkyl, - -N = CR-phenyl-O-C ¹ -Car alkyl (O) -, -C (O) -C ¹ -C alkyl ⁶ - (OCH ² CH ² ) ¹ - ⁶ NRC (O) -, -C (O) C ¹ -C ⁶ alkyl -phenyl (NR-C (O) C ¹ -C ⁶ alkyl) ¹ - ⁴ -, - C (O) ^C1 - ^C6 alkyl - (OCH ² CH ^{2) 1-6} -NR C (O) -C ^ -a yl lq or ^C1 - ^C6 -, -S-, -C (O) -CH (NR-C (O) ^C1 - ^C6 alkyl) -C ¹ -C ⁶ -alkyl and - (- CH ² -CH ² -O-) ^{1 to 20,}
[0127] wherein LB2 is AA ^0-12, wherein AA is a natural amino acid, an unnatural amino acid or - (CR15) HMO (CR15) p where o and p are each independently an integer of 1 to 20,
[0128] LB3 is -PABA-, -PABC- or is absent;
[0129] LC is absent or independently selected from the group consisting of -C ¹ -C ⁶ alkylene -, - NR heterocyclyl C ³ C ⁸ -NR-, -NRcarbocyclyl C ³ -C ⁸ -NR-, -NRalkyl C ⁱ -C ^to NR-, -NRalkylene C ⁱ -C ^a -, -S-, -NR-, -NRNR-, -O (CR ² ) ^{i -4} SS (CR ² ) ^i-4 N (R) -, -NRalkylene C ⁱ -C ^to -phenyleneNR-, -NRfenilen C ⁱ -C ^to SO ² NR-, -Oalkyl C ⁱ -C S-C ⁱ -C Salquil C (COOR) NR-, -NRC (COOR) alkyl C ⁱ -C S-C ⁱ -C Salquil ^to O-,
[0130]
[0131]
[0132]
[0133] in which
[0134] Xa is CR or N,
[0135] XB is CH, CR (C (R) 2) i-3NR, CR (C (R) 2) i-3O, CR (C (R) 2) i-3C (O) NR, CR- (C (R ) 2) i-3C (O) NRNR, CR (C (R) 2) i-3SO2NR, CR (C (R) ² ) i- ³ NRNR, CR (C (R) ² ) i- ³ NRC (O ) or N,
[0136] each XC is R,
[0137] each XD is - (CH ² ) i- ⁵ -, or is absent;
[0138] XE is O, S, C (R) ² , C (R) (C (R) 2) i-3-NR2 or NR and
[0139] Each XF is (C (R) ² ) i- ³ -NR or C (R) ² - (C (R) ² ) i- ³ -O.
[0140] In other embodiments of the invention the variable -Y- is C (O) N (R ^{A) 2} or C (S) N (R ^{A) 2} where one RA is hydrogen or -Ci-C20 alkyl and the other RA is -alkyl Ci -C20-N (R) -, so that the structure:
[0141]
[0142]
[0143]
[0144] it is formed, where each A is independently oxygen or sulfur.
[0145] In accordance with yet another aspect of the invention there is provided an antibody and drug conjugate compound of Formula MIA:
[0146] AB- (LP) i ^-20 (Formula IIIA)
[0147] or a pharmaceutically acceptable salt or solvate thereof, in which:
[0148] AB is an antibody;
[0149] P is:
[0150]
[0151] f 1-l 1-t -l 2-f2
[0152] in which:
[0153] each of F1 and F2 is independently selected from ring systems A, B, C and D:
[0154]
[0155] (Ring system A)
[0156]
[0157] (Ring B system)
[0158]
[0159]
[0160]
[0161] (Ring D system);
[0162] each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ -alkenyl , C ² -C ⁶ -alkynyl , C ² -C ^6, halo, hydroxy, alkoxy, ^-NH2, -NH ( C ¹ -C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ² , -NO ² , -C ⁶ -C ¹⁴ -aryl and C ⁶ -C ¹⁴ -heteroaryl, in which two or more R are optionally linked to form a ring or rings, and wherein said -C-C ¹⁴ -aryl and -C ⁶ -C ¹⁴ heteroaryl are optionally substituted with 1 to 5 substituents independently selected from -C ¹ -C ¹⁰ alkyl, -C-alkoxy ¹ -C ¹⁰ -halo, ^C1 - ^C10 alkylthio, -trifluoromethyl, -NH ^2, -NH (C ¹ -C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ^2, -C ¹ -C ¹⁰ -N (C ¹ -C ⁸ alkyl) ² , -C ¹ -C ³ alkylthio, -NO ² or -C ¹ -C ¹⁰ heterocyclyl, for each ring system in which R appears;
[0163] each V1 is independently a link, O, N (R) or S, for each ring system in which V1 appears; each V2 is independently O, N (R) or S, for each ring system in which V2 appears;
[0164] each of W 1 and W2 is independently H, -C ¹ -C ⁵ alkyl for each ring system in which W 1 and W2 appear;
[0165] each X is independently selected from -OH, -O-acyl, azido, halo, cyanate, thiocyanate, isocyanate, thioisocyanate, or
[0166]
[0167]
[0168]
[0169]
[0170] for each ring system in which X appears;
[0171] each Y is independently selected from a link, H, -C (O) RA, -C (S) RA, -C (O) ORA, -S (O) ² ORA, -C (O) N (Ra) ² , -C (S) N (Ra) ² , glycosyl, -NO ² and -P (O) (ORA) ² for each ring system in which Y appears, in which each RA is independently selected from H, - C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ -aryl, aralkyl, -C ¹ -C ¹⁰ heterocyclyl, -C ³ -C ⁸ -carbocyclyl, -C ¹ -C ²⁰ N -alkyl ( R) ² , -C ¹ -C ²⁰ alkylene, -C ¹ -C ⁸ -alkylene, -C- ¹⁴ -arylene, aralkylene, -C ¹ -C ¹⁰ -cyclocycle, -C ³ -C ⁸ -carbocycle and C-alkyl-C ¹ -alkyl -C ²⁰ N (R) -, and RF where said RA is optionally substituted with 1 to 3 substituents independently selected from R and in which a Y is divalent and is linked to L,
[0172] RF is -N (R6) QN (R5) C (O) - and is attached to L in the carbonyl adjacent to N (R5), in which R5 and R6 each independently selected from the group consisting of H, C ¹ -C ⁸ -alkyl, C ¹ -C ⁸ -heteroalquilo, C6-C ¹⁴ -aryl, -aralkyl, ^C1 - ^C10 heterocyclyl and carbocyclyl -C ³ -C ⁸ , or R ⁵ or R ⁶ bind to a carbon atom substituted on Q to form a C ¹ -C ¹⁰ heterocycle ring or C ⁶ -C ¹⁴ -heteroaryl ring, or R5 and R6 join together to form a C ¹ -C ¹⁰ hetero-cyclic or C ⁶ -C ¹⁴ heteroaryl ring system, and where Q is -C ¹ -C ⁸ alkylene -, - C ¹ -C ⁸ -alkylene alkylene, - C ⁶ -C ¹⁴ -arylene -, -aralkylene-, -C ¹ -C ¹⁰ heterocycle - or -C ³ -C ⁸ carbocycle -, in which each of Q, R5 and R6 are independently and optionally substituted with 1 to 3 independently selected substituents between R;
[0173]
[0174] each Z is independently selected from the group consisting of H, -C ¹ -C ⁸ alkyl and wherein said C ¹ -C ⁸ alkyl, each is optionally substituted with 1 to 3 substituents independently selected from R, for each ring system in which Z appears;
[0175]
[0176] each of L1 and L2 is independent a direct link
[0177]
[0178] T is selected from:
[0179]
[0180] -C (A1) X1-T2-X1C (B1) -, where T2 is:
[0181]
[0182]
[0183]
[0184]
[0185] in which each X1 is independently a bond, in which each of A1 and B1 are independently = O in which each of gyj are independently 0 and m is 1, and in which D is bicyclo (1.1.1) pentane or Cuban, where said bicyclo (1.1.1) pentane or Cuban is optionally substituted with -RE, -C (O) RE, -C (O) ORE, -N (RE) ² , -N (R) C (O ) RE or -N (R) C (O) ORE, and D is additionally optionally substituted with 1 to 2 R, and
[0186]
[0187] wherein each RE is independently selected from the group consisting of H, C ¹ -C ⁸ -alkyl, C ¹ -C ⁸ -heteroalquilo, -aryl, -aralkyl, ^C1 - ^C10 heterocyclyl, C ³ -carbociclilo -C ⁸ , -C (O) -C ¹ -C ⁸ alkyl, -C (O) N (C ¹ -C ⁸ alkyl) ² , and -C (O) -halo, and in which each RE is optionally substituted with 1 to 3 substituents independently selected from R,
[0188]
[0189] L is La-Lb- (Lc) ^1-3 ;
[0190]
[0191] LA is selected from: a bond to AB, -NR- (bond to AB), alkyl-SO ² -heteroaryl, arylSO ² -heteroaryl-,
[0192]
[0193]
[0194]
[0195]
[0196] LB is LB1-LB2-LB3
[0197]
[0198] in which LB1 is absent or is one or more components selected from the group consisting of -C (O) -, -C (S) -, -C (O) NR-, -C (O) C ¹ -C alkyl ⁶ -, -C (O) ^C1 - ^C6 NRalquil -, -C ¹ -C ⁶ -alkyl - (OCH ² CH ^{2) 1} -er, -C (O) alkyl ^C1 - ^C6 NRC (O) - , -C (O) C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ¹ - ⁶ -, -C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ¹ - ⁶ -C (O) -, -alkyl C ¹ C ⁶ -SS-alkyl C, -CaNRC (O) CH ² -, -alkyl C ¹ -C ⁶ - (OCH ² CH ² ) ^1-6 NRC (O) CH ² -, -C (O) alkyl ^C1 - ^C6 -NRC (O) -C ^1-6 -, -N = CR-phenyl-O-C ¹ .C ⁶ alkyl -, -N = CR-phenyl-O-alkyl C ¹ -Ca- C (O) -, -C (O) -C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ¹ - ⁶ NRC (O) -, -C (O) C ¹ C ⁶ alkyl -phenyl (NR-C (O) -C ¹ C ⁶ alkyl) ¹ - ⁴ -, -C (O) C ¹ alkyl Ca- (OCH ² CH ² ) ¹ -a-NRC (O) -C ¹ alkyl -Ca-C ¹ alkyl - C ⁶ -, -S-, -C (O) -CH (NR-C (O) -C _1- C alkyl) -C ¹ -C ⁶ alkyl - and (-CH ² -CH ² -O-) ^{1 -20} ;
[0199]
[0200] LB2 is AA ^0-12 , in which AA is a natural amino acid, an unnatural amino acid or - (CR15) or SS- (CR15) p where o and p are each independently an integer from 1 to 20,
[0201]
[0202] LB3 is -PABA-, -PABC- or is absent,
[0203]
[0204] LC is absent or independently selected from the group consisting of -C ¹ -C ⁶ alkylene -, -NRheterocyclyl C ³ -C ⁸ -NR-, -NRcarbocyclyl C ³ -C ⁸ -NR-, -NRalkyl C ¹ -C ⁶ NR-, -NRalkylene C ¹ -C ⁶ -, -S-, -NR-, -NRNR-, O (CR ² ) ^i-4 SS (CR ² ) ^i-4 N (R) -, -NRalkylene C ⁱ -C ⁶ -phenyleneNR-, -NRfenilen C ¹ -C ⁶ SO ² NR-, -Oalkyl C ⁱ - C ⁶ S-Salquil C ¹ -C ^to C (COOR) NR-, -NRC (COOR) alkyl C ⁱ -C ^to S-Salquil C ⁱ -C ^to O-,
[0205]
[0206]
[0207]
[0208] in which
[0209] Xa is CR or N,
[0210] XB is CH, CR (C (R) 2) 1-3NR, CR (C (R) 2) 1-3O, CR (C (R) 2) 1-3C (O) NR, CR- (C (R ) 2) 1-3C (O) NRNR, CR (C (R) 2) 1-3SO2NR, CR (C (R) ² ) i- ³ NRNR, CR (C (R) ² ) i- ³ NRC (O ) or N,
[0211] each X ^C is R;
[0212] each X ^D is - (CH ² ) ^1-5 -, or is absent;
[0213] X ^E is O, S, C (R) ² , C (R) (C (R) ² ) ^1-3 -NR ² or NR, and
[0214] Each X ^F is (C (R) ² ) ^1-3 -NR or C (R) ² - (C (R) ² ) ^1-3 -O.
[0215] In accordance with another aspect of the invention a "payload linker" compound of Formula IIB is provided:
[0216]
[0217]
[0218] or a pharmaceutically acceptable salt or solvate thereof, in which:
[0219] each of F1 and F2 is independently selected from ring systems A, B, C and D:
[0220]
[0221]
[0222]
[0223] (Ring B system)
[0224]
[0225] (Ring D system);
[0226] each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ -alkenyl , C ² -C ⁶ -alkynyl , C ² -C ^6, halo, hydroxy, alkoxy, ^-NH2, -NH ( C ¹ -C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ² , -NO ² , -C6-C-My -heteroaryl C6-C14, in which two or more R are optionally joined to form a ring or rings, and wherein said aryl and C6-C14-C14 heteroaryl C6 are optionally substituted with 1 to 5 substituents independently selected from alkyl ^C1 - ^{C10, C1} - ^C10 alkoxy, - halo, -C ¹ -C ¹⁰ alkylthio, -trifluoromethyl, -NH ² , -NH (C ¹ -C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ² , -CiC ¹⁰ -N alkyl (C ¹ alkyl -C ⁸ ) ² , -C ¹ -C ³ alkylthio, -NO ² or -C ¹ -C ¹⁰ heterocyclyl, for each ring system in which R appears;
[0227] each V1 is independently a link, O, N (R) or S, for each ring system in which V1 appears; each V2 is independently O, N (R) or S, for each ring system in which V2 appears;
[0228] each of W1 and W2 is independently H, -C ¹ -C ⁵ alkyl for each ring system in which W1 and W2 appear;
[0229] each X is independently -OH, -O-acyl, azido, halo, cyanate, thiocyanate, isocyanate, thioisocyanate, or
[0230]
[0231]
[0232]
[0233] for each ring system in which X appears;
[0234] each Y is independently selected from the group consisting of H, -C ¹ -C ⁶ alkyl -RA -C (O) RA, -C (S) RA, -C (O) ORA, -S (O) ² ORa , -C (O) N (Ra) ² , -C (S) N (Ra) ² , glycosyl, -NO ² and -PO (ORA) ² , for each ring system in which Y appears, in which each RA is independently selected from the group consisting of H, -C1-C20 alkyl, -C1-C8 heteroalkyl, -C6-C14 alkyl, aralkyl, -C1-C10 heterocyclyl, -C3-C8 carboxycyl and -C1-C20 alkyl 1N (R) 2, wherein said -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ -aryl, aralkyl, -C ¹ -C ¹⁰ -heterocyclyl, -C ³ -C carboxycyl ⁸ and -C ¹ -C ²⁰ N (R) ² alkyl are optionally substituted with 1 to 3 substituents independently selected from R;
[0235] each Z is independently selected from the group consisting of H, -C ¹ -C ⁸ alkyl and wherein said C ¹ -C ⁸ alkyl, each is optionally substituted with 1 to 3 substituents independently selected from R, for each ring system in which Z appears;
[0236] each of L1 and L2 is independent a direct link
[0237] T is selected from:
[0238] -C (A1) X1-T2-X1C (B1) -, where T2 is:
[0239]
[0240]
[0241] in which each X ¹ is independently a bond, in which each of A ¹ and B ¹ are independently = O, in which each of gyj are independently 0 and m is 1, and in which D is a bicycles (1.1 .1) pentane or Cuban where said bicyclo (1.1.1) pentane or Cuban is substituted with a member of the group selected from N (R ^E ) C (O) - where the carbonyl is attached to L, and -C (O) - where the carbonyl is attached to L, and additionally is optionally substituted with 1 to 2 R;
[0242] wherein each R ^E is independently selected from the group consisting of H, C ¹ -C ⁸ -alkyl, C ¹ -C ⁸ -heteroalquilo, -aryl, -aralkyl, ^C1 - ^C10 heterocyclyl, -carbociclilo C ³ - C ⁸ , -C (O) -C ¹ -C ⁸ alkyl, -C (O) N (C ¹ -C ⁸ alkyl) ² , and -C (O) -halo, and in which each R ^E is optionally substituted with 1 to 3 substituents independently selected from R;
[0243] L is La-Lb (Lc) ^1-3;
[0244] L ^A is selected from -halo, -N (R) ^2, -CON (R) ^2, -S-aryl , optionally substituted with -NO ² or -CONR ^2, -S-heteroaryl optionally substituted with ^-NO2, alkyl- SO ² -heteroaryl, arylSO ² -heteroaryl-,
[0245]
[0246]
[0247]
[0248]
[0249] L ^B is L ^B1 -L ^B2 -L ^B3
[0250] in which L ^B1 is absent or is one or more components selected from the group consisting of -C (O) -, -C (S) -, -C (O) NR-, -C (O) C ¹ alkyl - C ⁶ -, -C (O) ^C1 - ^C6 NRalquil -, C ¹ -C ^s -alkyl - (OCH ² CH ^{2) 1-6} -, -C (O) C ¹ -C ^s alkyl NRC (O ) -, -C (O) C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ¹ - ⁶ -, -C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ¹ - ⁶ -C (O) -, -C ¹ -C ⁶ alkyl -SS-C ¹ -C ⁶ alkyl NRC (O) CH ² -, - C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ^1-6 NRC (O) CH ² -, - C (O) alkyl ^C1 - ^C6 -NRC (O) -C ^1-6 -, -N = CR-phenyl-O-alkyl C ^1- C ⁶ -, -N = CR-phenyl-O-alkyl C ^1- C ⁶ -C (O) -, -C (O) -C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ¹ - ⁶ NRC (O) -, -C (O) C ¹ C ⁶ alkyl -phenyl (NR-C (O) -C ¹ -C ⁶ -alkyl) ^1-4 -, -C (O) C ¹ C ⁶ alkyl - (OCH ² CH ^{2) 1-6} -NR C (O) -C -C ¹ -C ⁶ alkyl ^C1 - ^C6 -, -S-, -C (O) -CH (NR-C (O) -alkyl Cr C ⁶⁾ -alkyl ^C1 - ^C6 - and (-CH ² -CH ² -O-) ^1-20 ;
[0251] L ^B2 is AA ^{or -12} , in which AA is a natural amino acid, an unnatural amino acid or - (CR ¹⁵ ) ^or -SS- (CR ¹⁵ ) ^p where oyp are each independently an integer from 1 to 20,
[0252] L ^B3 is -PABA-, -PABC- or is absent;
[0253] L ^C is absent or independently selected from the group consisting of -C ^1- C ⁶ alkylene -, -NRheterocyclyl C ³ -C ⁸ -NR-, -NR Carbocyclyl C ³ -C ⁸ -NR-, -NRalkyl C ¹ - C ⁶ NR-, -NRalkylene C ¹ -C ⁶ -, -S-, -NR-, -NRNR-, -O (CR ² ) ^1-4 SS (CR ² ) ^1-4 N (R) -, - NRalkylene C ¹ -C ⁶ -phenylene NR-, -NRphenylene C ¹ -C ⁶ SO ² NR-, -O-C ¹ -C ⁶ S-Salkyl C ¹ -C ⁶ C (COOR) NR-, -NRC (COOR) alkyl C ^ S -S a lq u il C ¹ -C ⁶ O-,
[0254]
[0255]
[0256]
[0257] in which
[0258] Xa is CR or N,
[0259] XB is CH, CR (C (R) ² ) ¹ - ³ NR, CR (C (R) ² ) ¹ -sO, CR (C (R) ² ) ¹ -sC (O) NR, CR- (C ( R) ² ) ¹ - ³ C (O) NRNR, CR (C (R) ² ) ¹ - ³ SO ² NR, CR (C (R) ² ) i- ³ NRNR, CR (C (R) ² ) i- ³ NRC (O) or N;
[0260] each XC is R;
[0261] each XD is - (CH ² ) i - ⁵ -, or is absent;
[0262] XE is O, S, C (R) ² , C (R) (C (R2) i -3-NR2 or NR, and
[0263] Each XF is (C (R) ² ) ¹ - ³ -NR or C (R) ² - (C (R) ² ) ¹ - ³ -O.
[0264] In accordance with a further aspect of the invention there is provided an antibody and drug conjugate compound of Formula IIIB:
[0265]
[0266]
[0267]
[0268] (Formula IIIB)
[0269] or a pharmaceutically acceptable salt or solvate thereof, in which:
[0270] AB is an antibody;
[0271] each of F1 and F2 is independently selected from ring systems A, B, C and D:
[0272]
[0273]
[0274]
[0275] stema e an o;
[0276] each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ -alkenyl , C ² -C ⁶ -alkynyl , C ² -C ^6, halo, hydroxy, alkoxy, ^-NH2, -NH ( C ¹ -C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ² , -NO ² , -C ⁶ -C ¹⁴ -aryl and C ⁶ -C ¹⁴ -heteroaryl, in which two or more R are optionally linked to form a ring or rings, and wherein said -C ⁶ -C ¹⁴ aryl and -C ⁶ -C ¹⁴ heteroaryl are optionally substituted with 1 to 5 substituents independently selected from -C ¹ -C ¹⁰ alkyl, -alkoxy C ¹ -C ¹⁰ , -halo, -C ¹ -C ¹⁰ -alkylthio, -trifluoromethyl, -NH ² , -NH (C ¹ -C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ² , -C alkyl ¹ -C ¹⁰ -N (C ¹ -C ⁸ alkyl) ² , -C ¹ -C ³ alkylthio, -NO ² or -C ¹ -C ¹⁰ heterocyclyl, for each ring system in which R appears;
[0277] each V ¹ is independently a link, O, N (R) or S, for each ring system in which V ¹ appears; each V ² is independently O, N (R) or S, for each ring system in which V ² appears;
[0278] each of W ¹ and W ² is independently H, -C ¹ -C ⁵ alkyl for each ring system in which W ¹ and W ² appear;
[0279]
[0280] each X is independently -OH, -O-acyl, azido, halo, cyanate, thiocyanate, isocyanate, thioisocyanate, or
[0281]
[0282]
[0283] s ii
[0284] f ' —O - S ii - R
[0285]
[0286]
[0287] for each ring system in which X appears;
[0288]
[0289] Each Y is independently selected from the group consisting of H, -C ¹ -C ⁶ -RA-C (O) RA, -C (S) RA, -C (O) ORA, -S (O) ² ORa , -C (O) N (Ra) ² , -C (S) N (Ra) ² , glycosyl, -NO ² and -PO (ORA) ² , for each ring system in which Y appears, in which each RA is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ -aryl, aralkyl, -C ¹ -C ¹⁰ -heterocyclyl, -carbocyclyl C ³ -C ⁸ and -C ¹ -C ²⁰ alkyl (R) ² , wherein said -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ -aryl, aralkyl, -heterocyclyl C ¹ -C ¹⁰ , -C ³ -C ⁸ -carbocyclyl and -C ¹ -C ²⁰ N (R ^{) 2} alkyl are optionally substituted with 1 to 3 substituents independently selected from R;
[0290]
[0291] each Z is independently selected from the group consisting of H, -C ¹ -C ⁸ alkyl and wherein said C1-C8 alkyl, each is optionally substituted with 1 to 3 substituents independently selected from R, for each system of ring in which Z appears;
[0292]
[0293] Each of L1 and L2 is independent a direct link.
[0294]
[0295] T is selected from:
[0296]
[0297] -C (A1) X1-T2-X1 C (B1) -,
[0298]
[0299] where T2 is:
[0300]
[0301]
[0302]
[0303]
[0304] in which each X1 is independently a bond in which each of A1 and B1 are independently = O in which each of gyj are independently 0 and m is 1, and in which D is bicyclo (1.1.1) pentane or Cuban where said bicyclo (1.1.1) pentane or Cuban is substituted with a member of the group selected from N (RE) C (O) - where the carbonyl is attached to L, and -C (O) - where the carbonyl is attached to L, and additionally it is optionally substituted with 1 to 2 R;
[0305]
[0306] wherein each RE is independently selected from the group consisting of H, C ¹ -C ⁸ -alkyl, C ¹ -C ⁸ -heteroalquilo, -aryl, -aralkyl, ^C1 - ^C10 heterocyclyl, C ³ -C -carbociclilo ⁸ , -C (O) -C ¹ -C ⁸ alkyl, -C (O) N (C ¹ -C ⁸ alkyl) ² , and -C (O) -halo, and in which each RE is optionally substituted with 1 to 3 substituents independently selected from R;
[0307]
[0308] L is La-Lb (Lc) ^1-3;
[0309]
[0310] LA is selected from: an AB bond, -NR- (AB bond), alkyl-SO2-heteroaryl, arylSO2-heteroaryl-,
[0311]
[0312]
[0313]
[0314]
[0315] LB is LB1-LB2-LB3
[0316]
[0317] in which LB1 is absent or is one or more components selected from the group consisting of -C (O) -, -C (S) -, -C (O) NR-, -C (O) C ¹ -C alkyl ⁶ -, -C (O) ^C1 - ^C6 NRalquil -, -C ¹ -C ⁶ -alkyl - (OCH ² CH ^{2) 1-6} -, -C (O) alkyl ^C1 - ^C6 NRC (O) - C (O) alkyl Ci-C ⁶ - (OCH ² CH ² ) i ^-6 - -alkyl Ci-Ca- (OCH ² CH ² ) iaC (O) -, -alkyl Ci-Ca-SS-alkyl Ci-CaNRC (O) CH ² -, - alkyl Ci-Ca- (OCH ² CH ² ) i-aNRC (O) CH ² -, -C (O) alkyl Ci-Ca-NRC (O) -alkyl Ci-a-, -N = CR-phenyl-O-Ci-Ca- alkyl, -N = CR-phenyl-O-Ci-Ca-C (O) alkyl -, -C (O) -Ci-Ca- alkyl (OCH ² CH ² ) i-aNRC (O) -, -C (O) alkyl Ci-Ca-phenyl (NR-C (O) alkyl Ci-Ca) i ^-4 -, -C (O) alkyl CiCa- (OCH ² CH ² ) ia-NRC (O) -Ci-Ca- alkyl, -Ci-Ca- alkyl, -S-, -C (O) -CH (NR-C (O) -Ci-Ca alkyl) -Ci-alkyl Ca- y (-CH ² -CH ² -O-) i - ² or;
[0318] LB2 is AAo-i ² , in which AA is a natural amino acid, an unnatural amino acid or - (CRi5) or SS- (CRi5) p where oyp are each independently an integer from 1 to 20,
[0319] LB3 is -PABA-, -PABC- or is absent;
[0320] L ^c is absent or independently selected from the group consisting of -alkylene C ⁱ -C ^a -, -NRheterocyclyl C ³ -C ⁸ -NR-, -NRcarbocyclyl C ³ -C ⁸ -NR-, -NRalkyl C ⁱ - C ^to NR-, -NRalkylene C ⁱ -C ^to -, -S-, -NR-, -NRNR-, -O (CR ² ) i - ⁴ -S (CR ² ) i - ⁴ N (R) -, -NRalkylene C ⁱ -C ^to -phenylene NR-, -NRphenylene C ⁱ -C ^to SO ² NR-, -O-alkyl C ^i. C ^a -S-Salquil C ^{i -} C ^a -C (CO-OR) NR-, -NRC (COOR) alkyl C ^{i -} C ^to S-Salquil C ^{i -} C ^to O-,
[0321]
[0322]
[0323]
[0324] in which
[0325] Xa is CR or N,
[0326] XB is CH, CR (C (R) 2) i -3NR, CR (C (R) 2) i-3O, CR (C (R) 2) i-3C (O) NR, CR- (C (R ) 2) i -3C (O) NRNR, CR (C (R) 2) i -3S02NR, CR (C (R) ² ) and ^-3 NRNR, CR (C (R) ² ) and ^-3 NRC (O ) or N;
[0327] each XC is R;
[0328] each XD is - (CH ² ) and ^-5 -, or is absent;
[0329] XE is O, S, C (R) ² , C (R) (C (R) 2) and -3-NR2 or NR, and
[0330] Each XF is (C (R) ² ) and ^-3 -NR or C (R) 2- (C (R) 2) and -3-0.
[0331] Additional aspects of the invention include compounds such as those mentioned herein where
[0332] each R is independently selected from the group consisting of H, -Ci-C ²⁰ alkyl and -NH ² ; each V1 is independently O or N (R) for each ring system in which V1 appears;
[0333] each V2 is independently O or N (R) for each ring system in which V2 appears;
[0334] each of W 1 and W2 is independently H, -C ¹ -C ⁵ alkyl, for each ring system in which W 1 and W2 appear;
[0335] each X is independently halo, for each ring system in which X appears;
[0336] each Y is independently selected from the group consisting of H, -C (O) Ra, -C (O) N (Ra) ² , glycosyl, -NO ² and -PO (ORa) ² , for each ring system in which Y appears, in which each RA is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ³ -C ⁸ -carbocyclyl and -C ¹ -alkyl C ²⁰ 1 (R) ² , wherein said -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ³ -C ⁸ -carbocyclyl and -C ¹ -C ^20- N (R) ² alkyl are optionally substituted with ia 3 substituents independently selected from R;
[0337] each of L1 and L2 is independently selected from a direct link; Y
[0338] T is selected from:
[0339] -C (A1) X1-T2-X1C (B1) -,
[0340] where T2 is:
[0341]
[0342] in which each X1 is a link, in which each of A1 and B1 are independently = O, and in which D is bicyclo (1.1.1) pentane or Cuban where said bicyclo (1.1.1) pentane or Cuban are optionally substituted with -NH ² , -N (R) C (O) H or -N (R) C (O) OH.
[0343] Additional aspects of the invention include compounds such as those mentioned herein where two or more Rs optionally join to form one or more rings.
[0344] Additional aspects of the invention include compounds such as those mentioned herein where
[0345] each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl and -NH ² ;
[0346] each V1 is independently O or N (R) for each ring system in which V1 appears;
[0347] each V2 is independently O or N (R) for each ring system in which V2 appears;
[0348] each of W 1 and W2 is independently H, -C ¹ -C ⁵ alkyl, for each ring system in which W 1 and W2 appear;
[0349] each X is independently halo, for each ring system in which X appears;
[0350] Each Y is independently selected from a link, H, -C (O) RA, -C (S) RA, -C (O) ORA, -S (O) ² ORA, -C (O) N (RA) ² , -C (S) N (Ra) ² , glycosyl, -NO ² and -P (O) (ORA) ² for each ring system in which Y appears, in which each RA is independently selected from H, - C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ -aryl, aralkyl, -C ¹ -C ¹⁰ heterocyclyl, -C ³ -C ⁸ -carbocyclyl, -C ¹ -C ²⁰ N -alkyl ( R) ² , -C ¹ -C ²⁰ alkylene, -C ¹ -C ⁸ -alkylene, -C ⁶ -C ¹⁴ -arylene, aralkylene, -C ¹ -C ¹⁰ heterocycle, -C ³ -C ⁸ -carbocycle and C-alkyl-C ¹ -C ²⁰ N (R) -, and RF where said RA is optionally substituted with 1 to 3 substituents independently selected from R and in which a Y is divalent and is linked to L, RF is -N (R6) QN (R5) C (O) - and is attached to L in the carbonyl adjacent to N (R5), in which each of R5 and R6 is independently selected from the group consisting of H, -C ¹ -C ⁸ alkyl , and heteroalkyl -C ¹ -C ⁸ , or R5 or R6 binds with a carbon atom substituted Q - heterocyclic ring to form a ^C1 - ^C10 heteroaryl or C6-ring ^C14, or R5 and R6 are joined together to form a ring system - heterocyclic ^C1 - ^C10 or of heteroaryl -C ⁶ -C ¹⁴ , and where Q is -alkylene -C ¹ -C ⁸ -, -arylene C ⁶ -C ¹⁴ - or carbocycle -C ³ -C ⁸ -, in which each of Q, R5 and R6 are independently and optionally substituted with 1 to 3 substituents independently selected from R;
[0351] each of L1 and L2 is independently selected from a direct link; Y
[0352] T is selected from:
[0353] -C (A1) X1-T2-X1C (B1) -,
[0354] where T2 is:
[0355]
[0356]
[0357]
[0358] in which each X1 is a link, in which each of A1 and B1 are independently = O, and in which D is bicyclo (1.1.1) pentane or Cuban where said bicyclo (1.1.1) pentane or Cuban are optionally substituted with -NH ² , -N (R) C (O) H or -N (R) C (O) OH.
[0359] Additional aspects of the invention include compounds such as those mentioned herein where
[0360] each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl and -NH ² ;
[0361] each V1 is independently O or N (R) for each ring system in which V1 appears;
[0362] each V2 is independently O or N (R) for each ring system in which V2 appears;
[0363] each of W 1 and W2 is independently H, -C ¹ -C ⁵ alkyl, for each ring system in which W 1 and W2 appear;
[0364] each X is independently halo, for each ring system in which X appears;
[0365] each Y is independently selected from the group consisting of H, -C (O) RA, -C (O) N (RA) ² , glycosyl, -NO ² and -PO (ORa) ² , for each ring system in which Y appears, in which each RA is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ³ -C ⁸ -carbocyclyl and -C ¹ -alkyl C ²⁰ 1 (R) ² , wherein said -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ³ -C ⁸ -carbocyclyl and -C ¹ -C ^20- N (R) ² alkyl are optionally substituted with 1 to 3 substituents independently selected from R;
[0366] each of L1 and L2 is independently selected from a direct link; Y
[0367] T is -C (A1) X1-T2-X1C (B1) -, where T2 is:
[0368]
[0369]
[0370]
[0371] in which each X1 is a link, in which each of A1 and B1 are independently = O, and in which D is bicyclo (1.1.1) pentane or Cuban where said bicyclo (1.1.1) pentane or Cuban are optionally substituted with -NH ² , -N (R) C (O) H or -N (R) C (O) OH.
[0372] Additional aspects of the invention include compounds such as those mentioned herein where
[0373] L ^A is selected from the group consisting of -halo, -N (R) ² , -CON (R) ² , -S-aryl optionally substituted with -NO ² or -CON (R) ² , -S-heteroaryl optionally substituted with -NO ² , alkyl-SO ² -heteroaryl, arylSO ² -heteroaryl-, and
[0374]
[0375]
[0376]
[0377] Lb is lB1-LB2-LB3 in which L ^B1 is absent or is one or more components selected from the group consisting of -C (O) -, -C (S) -, -C (O) NR-, -C (O) C ¹ -C ⁶ alkyl -, -C (O) NR C ¹ -C ⁶ -, - C _1- C alkyl ⁶ - (OCH ² CH ² ) ^1-6 -, -C (O) C alkyl ¹ -C ^to NRC (O) -, -C (O) C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ^1-6 -, -alkyl C ⁶ - (OCH ² CH ² ) ^1-6 -C (O) -, -C ¹ -C ⁶ alkyl -SS-C ¹ -C ⁶ alkyl NRC (O) CH ² -, -C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ^1-6 NRC (O) CH ² -, -C (O) C ¹ -C ⁶ alkyl -NRC (O) -C ^1.6 alkyl -, -C (O) -C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ^1-6 NRC ( O) -, -C (O) C ¹ -C ⁶ alkyl-phenyl (NR-C (O) C ¹ -C ⁶ K ⁴ alkyl -, -C (O) C ¹ alkyl ^. C ⁶ (OCH ² CH ² ) ^01.06 -NRC (O) -alkyl ^C1 - ^C6 -, -C ¹ -C ⁶ -alkyl -, -S-, -C (O) -CH (NR-C (O) C ¹ -C alkyl ⁶ ) -alkyl C ¹ -C ⁶ - and (-CH ² -CH ² -O-) ^1-20 , in which L ^B2 is AA ^0-12 , in which AA is a natural amino acid, and an amino acid is not natural or - (CR ¹⁵ ) ^or -SS- (CR ¹⁵ ) ^p where each of oyp are independently an integer from 1 to 20, and L ^B3 is -PABA-, -PABC- or is absent Y
[0378] L ^C is absent.
[0379] Additional aspects of the invention include antibody and drug conjugates such as those mentioned herein where L ^A is selected from: an AB bond, -NR- (AB bond), alkyl-SO ² -heteroaryl , arilSO ² -heteroaryl-,
[0380] Lb is L ^B1 -L ^B2 -L ^B3 in which L ^B1 is absent or is one or more components selected from the group consisting of -C (O) -, -C (S) -, -C (O) NR- , -C (O) -C ⁱ alkyl C ^a -, -C (O) C ⁱ -C NRalquil ^a -, -C ⁱ -C ^a - (OCH ² CH ^{2) i-6} -, -C (O ) C ¹ -C alkyl ^to NRC (O) -, -C (O) C ¹ -C alkyl ^to - (OCH ² CH ² ) ^ia -, -C ¹ -C alkyl ^to - (OCH ² CH ² ) ^ia - C (O) -, -alkyl C ⁱ -C ^to -SS-alkyl C ⁱ -C ^to NRC (O) CH ² -, -alkyl C ⁱ -C ^to - (OCH ² CH ² ) ^{i - to} NRC (O ) CH ² -, -C (O) C ¹ -C alkyl ^to -NRC (O) -C ^ia alkyl -, -C (O) -C ¹ -C alkyl ^to - (OCH ² CH ² ) ^{i - to} NRC (O) -, -C (O) alkyl C ^{i -} C -phenyl (NR-C (O) C ⁱ -C alkyl ^{a) i-4} -, -C (O) alkyl C ^{i -} C (OCH ² CH ^{2) y} -NRC (O) -C ⁱ -C ^a -, -C ⁱ -C ^a -, -S-, -C (O) -CH (NR-C (O) alkyl C ⁱ - C ^a ) -alkyl C ⁱ -C ^a - and (-CH ² -CH ² -O-) ^i-20 , in which L ^B2 is AA ^{or -i2} , in which AA is a natural amino acid, and an amino acid unnatural or - (CR ⁱ⁵ ) ^or -SS- (CR ⁱ⁵ ) ^p where each of oyp are independently an integer of ia 20, and L ^B3 is -PABA-, -PA BC- or is absent; Y
[0381]
[0382] L ^C is absent.
[0383]
[0384] Additional aspects of the invention include compounds such as those mentioned herein where R ^F is selected from:
[0385]
[0386]
[0387]
[0388]
[0389] Additional aspects of the invention include compounds such as those mentioned herein where one or more W is C ¹ -C ³ alkyl.
[0390]
[0391] Additional aspects of the invention include compounds such as those mentioned herein where X is chlorine.
[0392]
[0393] Additional aspects of the invention include compounds such as those mentioned herein where a Y is H or -C (O) C ¹ -C ^alquilo alkyl.
[0394]
[0395] Additional aspects of the invention include compounds such as those mentioned herein where one or more Z is H.
[0396]
[0397] Additional aspects of the invention include compounds such as those mentioned herein where the amide is -C (O) NH- or -n Hc (O) -.
[0398]
[0399] Additional aspects of the invention include compounds such as those mentioned herein where het is a heteroaryl selected from pyrrole-2-, 5-diyl-, fur-2,5-diyl-, indole-2,5 -diyl, benzofuran-2,5-diyl, and 3, a-dihydrobenzo [i, 2-b: 4, 3-b] dipyrrol-2,7-diyl.
[0400]
[0401] Additional aspects of the invention include compounds such as those mentioned herein where L ⁱ and L ² are selected from carbonyl, 2-carbonylindole-5-yl, 2-carbonyl-a-hydroxy-7-methoxyindole- 5-yl, 2-carbonyl-i, 2,3, a-tetrahydrobenzo [i, 2-b: 4,3-b] dipyrrol-7-yl, 2-carbonyl-4-hydroxy-5-methoxy-i, 2,3, atetrahydrobenzo [i, 2-b: 4,3-b '] dipyrrol-7-yl, and 2-carbonyl-4-hydroxy-5-methoxy-i, 2,3, a-tetrahydrobenzo [i, 2-b: 4,3-b '] dipyrrol-7-yl.
[0402]
[0403] Additional aspects of the invention are those compounds mentioned herein where one or more of the following are applied: W is methyl; X is a halogen; Y is hydrogen or -COR where R is C ¹ -C 10 ^alkyl ; and Z is hydrogen.
[0404]
[0405] Also included in the embodiments of the invention are those compounds described herein where L ⁱ and L ² are selected from 2-carbonylindole-5-yl; 2-carbonyl-a-hydroxy-7-methoxyindole-5-yl; 2-carbonyl-i, 2, 3, a-tetrahydrobenzo [i, 2-b: 4, 3-b] dipyrrol-7-yl; 2-carbonyl-4-hydroxy-5-methoxy-i, 2, 3, a-tetrahydrobenzo [i, 2-b: 4, 3-b '] dipyrrol-7-yl; and 2-carbonyl-4-hydroxy-5-methoxy-i, 2, 3, a-tetrahydrobenzo [i, 2-b: 4, 3-b '] dipyrrol-7-yl.
[0406]
[0407] Another aspect of the invention includes compounds as described herein where L ^A is The invention also includes linker-payload or antibody-drug conjugates comprising a radical of the payload compounds described herein.
[0408]
[0409] It should be noted that, the invention includes pharmaceutical compositions of the compounds of the invention, and any pharmaceutically acceptable salts thereof, described herein, wherein the pharmaceutical composition includes a pharmaceutically acceptable excipient.
[0410]
[0411] The invention also relates to one or more compounds of the invention or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer described herein, or a pharmaceutical composition or compositions comprising one or more of these compounds. of the invention or a pharmaceutically acceptable salt thereof.
[0412]
[0413] Some compounds, including payloads, linker-payloads and ADCs represented herein, are shown in a specific stereoisomeric form. The invention, however, is understood to include all stereoisomeric forms of said compounds. For example, a compound with two stereoisomeric centers can be represented as the R, S form of the compound, but the invention provides all stereoisomeric forms, for example, R, R; R, S; S, R and S, S.
[0414]
[0415] Detailed description
[0416]
[0417] The present invention relates to cytotoxic bifunctional compounds, drug-antibody (ADC) conjugates comprising said cytotoxic bifunctional compounds, and the same for use in the treatment of cancer and other pathological conditions. Methods of using said compounds and / or conjugates in vitro, in situ, and in vivo for the detection, diagnosis or treatment of mammalian cells, or the associated pathological conditions are also disclosed.
[0418]
[0419] Definitions and Abbreviations
[0420]
[0421] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. When trade names are used herein, the trade name includes the formulation product, the generic drug, and the active pharmaceutical principle (s) of the product of the trade name, unless the context indicate otherwise.
[0422]
[0423] The term "antibody" (or "Ab") herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments. that present the desired biological activity. An intact antibody has mainly two regions: a variable region and a constant region. The variable region binds to and interacts with a target antigen. The variable region includes a complementarity determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region can be recognized and interact with the immune system (see, for example, Janeway et al., 2001, Immuno. Biology, 5th ed., Garland Publishing, New York). An antibody can be of any type or class (for example, IgG, IgE, IgM, IgD, and IgA) or subclass (for example, IgG 1, IgG2, IgG3, IgG4, IgA1 and IgA2). The antibody can be derived from any suitable species. In some embodiments, the antibody is of human origin or of murine origin. An antibody can be, for example, human, humanized or chimeric.
[0424]
[0425] The terms "specifically binds" and "specific binding" refer to the binding of an antibody to a predetermined antigen. Typically, the antibody binds with an affinity of at least about 1x107 M-1, and binds to the predetermined antigen with an affinity that is at least twice that of its affinity for binding to a non-specific antigen (eg, BSA , casein) different from the predetermined antigen or a closely related antigen.
[0426]
[0427] The term "monoclonal antibody", as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible mutations that occur. naturally they may be present in smaller amounts. Monoclonal antibodies are very specific, targeting a single antigenic site. The "monoclonal" modifier indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not considered as a necessary production of the antibody by any particular procedure.
[0428]
[0429] The term "monoclonal antibodies" specifically includes "chimeric" antibodies in which a portion of the heavy chain and / or the light chain is identical to / or homologous with the corresponding sequence of antibodies derived from a particular species or belonging to a class or subclass of specific antibody, while the rest (s) of the chain (s) is identical to / or homologous with the corresponding sequences derived from other species or belonging to another class or subclass of antibody, as well as fragments of said antibodies , provided they have the desired biological activity.
[0430]
[0431] As used herein, "H (C) -" refers to trastuzumab (trade name 1 HERCEPTIN®) which is a monoclonal antibody that interferes with the HER2 / neu receptor, bound through one of its cystines to a compound of the invention. As used herein, "H (K) -" refers to trastuzumab which is a monoclonal antibody that interferes with the HER2 / neu receptor, linked through one of its lysines to the compound of the invention.
[0432]
[0433] An "intact antibody" is one that comprises a variable region of antigen binding as well as a constant domain of the light chain (Cl) and constant domains of the heavy chain, Ch ¹ , Ch ² , Ch ³ and Ch ⁴ , as Suitable for antibody class. The constant domains can be constant domains of native sequences (for example, constant domains of human native sequences) or amino acid sequence variants thereof.
[0434] An intact antibody may have one or more "effector functions", which refer to those biological activities attributable to the Fc region (eg, a native sequence Fc region or a variant amino acid sequence Fc region) of an antibody. Examples of antibody effector functions include complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis.
[0435]
[0436] An "antibody fragment" comprises a portion of an intact antibody, which preferably comprises binding to the antigen or the variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') ² , and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from of antibody fragment (s), a fragment (s) produced by a Fab expression library, or epitope binding fragments of any of the foregoing that immunospecifically bind to a target antigen (eg, an antigen of cancer cell, a viral antigen or a microbial antigen).
[0437]
[0438] The term "variable" in the context of an antibody refers to certain portions of the variable domains of the antibody that differ widely in sequence and are used in the binding and specificity of each particular antibody by its specific antigen. This variability is concentrated in three segments called "hypervariable regions" in the variable domains of the light chain and heavy chain. The most highly conserved portions of the variable domains are called the framework regions (FR). The variable domains of the native heavy and light chains each comprise four FRs connected by three hypervariable regions.
[0439]
[0440] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues of a "complementarity determining region" or "CDR" (eg, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the domain variable light chain and 31-35 (H1), 50-65 (H2) and 95-102 (L3) in the heavy chain variable domain; Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those remains from a "hypervariable loop" (for example, residues 26-32 (L1), 50-52 (L2) and 91- 96 (L3) in the variable domain of the light chain and 26 32 (H1), 53-55 (142) and 96-101 (H3) in the variable domain of the heavy chain; Chotia and Lesk, 1987, J. Mol Biol.
[0441] 196: 901-917). FR moieties are those moieties of the variable domain other than the moieties of the hypervariable region as defined herein.
[0442]
[0443] A "single chain Fv" or "scFv antibody fragment" comprises the V.sub.H and V.sub.L domains of an antibody, in which these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the V.sub.H and V.sub.L domains that allows the scFv to form the desired structure for antigen binding. For a scFv review, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
[0444] The term "diabody" refers to small antibody fragments with two antigen binding sites, whose fragments comprise a heavy chain variable domain (Vh) connected to a variable light chain domain (Vl) in the same polypeptide chain. . Using a linker that is too short to allow for pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described more fully in, for example, EP 0 404 097; WO 93/11161; and Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448.
[0445]
[0446] The "humanized" forms of non-human antibodies (eg rodents) are chimeric antibodies that contain the minimum sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which the remains of a hypervariable region of the receptor are replaced by residues of a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or a non-human primate that has the desired specificity, affinity, and capacity desired. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced by the corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine the antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the hypervariable loops corresponds to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody will also comprise optionally at least a portion of a constant region of immunoglobulin (Fc), usually of a human immunoglobulin. For additional details, see Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2: 593-596.
[0447]
[0448] As used herein, "isolated" means separated from other components of (a) a natural source, such as a plant or animal cell or a cell culture, or (b) an organosynthetic chemical reaction mixture. As used herein, "purified" means that when isolated, the isolate contains at least 95%, and in another aspect at least 98%, of a compound (eg, a conjugate) by weight of the isolated.
[0449] An "isolated" antibody is one that has been identified or separated and / or recovered from a component of its natural environment. Contaminating components of their natural environment are materials that would interfere with the diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones and other protein or non-protein solutes. In preferred embodiments, the antibody will be purified (1) to more than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain the minus 15 residues in the N-terminus sequence or the internal amino acid sequence through the use of a rotary cup sequencer, or (3) up to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, staining silver. The isolated antibody includes the antibody in situ in recombinant cells because at least one component of the natural environment of the antibody will not be present. Ordinarily, however, the isolated antibody will be prepared by at least one purification step.
[0450]
[0451] An antibody that "induces apoptosis" is one that induces programmed cell sampling as determined by attachment of annexin V, DNA fragmentation, cell contraction, endoplasmic reticulum dilation, cell fragmentation, and / or membrane vesicle formation (called apoptotic bodies). The cell is a tumor cell, for example, a breast cell, an ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreas or bladder cell. Various procedures are available to evaluate cellular events associated with apoptosis. For example, translocation of phosphatidyl serine (PS) can be measured by binding annexin; DNA fragmentation can be assessed through the unwinding of ladder-like DNA; and the condensation of cell chromatin together with DNA fragmentation can be evaluated by an increase in hypodiploid cells.
[0452]
[0453] The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce tumor size; inhibits (i.e., delays to some extent and preferably stops) the infiltration of cancer cells in peripheral organs; inhibits (that is, delays to some extent and preferably stops) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with cancer. To the extent that the drug can inhibit the growth and / or destruction of existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer therapy, efficacy, for example, can be measured by evaluating the disease progression time (TTP) and / or by determining the response rate (RR).
[0454]
[0455] The term "substantial quantity" refers to a majority, that is to say greater than 50% of a population, of a mixture or a sample.
[0456]
[0457] The term "intracellular metabolite" refers to a compound resulting from a metabolic process or reaction inside a cell of an antibody-drug conjugate (ADC). The metabolic process or reaction may be an enzymatic process such as proteolytic cleavage of a peptide linker from the ADC. Intracellular metabolites include, but are not limited to, antibodies and free drugs that have undergone intracellular excision after entry, diffusion, uptake or transport into a cell.
[0458]
[0459] The terms "intracellularly cleaved" and "intracellular cleavage" refer to a metabolic process or reaction inside a cell in an ADC or similar, whereby the covalent bond, for example, the linker, between the rest of the drug and the antibody is broken, resulting in the free drug, or other metabolite of the dissociated conjugate of the antibody inside the cell. The cleaved residues of the ADC are therefore intracellular metabolites.
[0460] The term "bioavailability" refers to the systemic availability (ie, blood plasma levels) of a given amount of a drug administered to a patient. Bioavailability is an absolute term that indicates the measurement of time (index) and the total amount (extent) of the drug that reaches the general circulation of each administered dosage form.
[0461]
[0462] The term "cytotoxic activity" refers to a destruction of cells, a cytostatic or antiproliferative effect of an ADC or an intracellular metabolite of said ADC. Cytotoxic activity can be expressed as the value of IC ⁵⁰ , which is the concentration (molar or mass) per unit volume at which half of the cells survive.
[0463]
[0464] A "disorder" is any medical condition that would benefit from treatment with a drug or an antibody-drug conjugate. This includes chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include benign and malignant cancers; leukemia and lymphoid neoplasms, neuronal disorders, glial, astrocital, hypothalamic and other glandular, macrophage, epithelial, stromal and blastocellic; and inflammatory, angiogenic and immunological disorders.
[0465]
[0466] The terms "cancer" and "cancerous" refer to or describe the ailment or physiological disorder in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancer cells.
[0467] Examples of a "patient" include, but are not limited to, a human being, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and bird. In an illustrative embodiment, the patient is a human being.
[0468] The terms "treat" or "treatment," unless the context indicates otherwise, refer to therapeutic and prophylactic treatment measures.
[0469] unwanted physiological disorder, such as the development or spread of cancer. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, symptom relief, decreased disease extension, stabilized state (i.e., no worsening) of disease, delay or slowdown. of disease progression, improvement or palliation of disease status, and remission (either partial or total), whether detectable or undetectable. "Treatment" may also mean prolongation of survival compared to expected survival if no treatment is received. Those who need treatment include those who already have the ailment or disorder as well as those prone to the ailment or disorder.
[0470]
[0471] In the context of cancer, the term "treat" includes any or all of inhibiting the growth of tumor cells, cancer cells, or a tumor; inhibit the replication of tumor cells or cancer cells, decrease the overall tumor burden or decrease the number of cancer cells, and improve one or more symptoms associated with the disease.
[0472]
[0473] In the context of an autoimmune disease, the term "treat" includes any or all of inhibiting the replication of cells associated with an autoimmune disease state including, but not limited to, cells that produce an autoimmune antibody, decreasing the burden of Autoimmune antibody and improve one or more symptoms of an autoimmune disease.
[0474]
[0475] In the context of an infectious disease, the term "treat" includes any or all of: inhibiting the growth, multiplication or replication of the pathogen that causes the infectious disease and improving one or more symptoms of an infectious disease.
[0476]
[0477] The term "leaflet" is used to refer to instructions usually included in commercial packages of therapeutic products, which contain information about the symptoms, use, dosage, administration, contraindications and / or warnings that refer to the use of such therapeutic products. .
[0478]
[0479] As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably and all such designations include progeny. The words "transformants" and "transformed cells" include the cell and the primary subject cultures or the progeny derived therefrom with respect to the number of transfers. It is also understood that the entire progeny may not be precisely identical in the DNA content, due to deliberate or inadvertent mutations. Mutant offspring that have the same biological function or activity as screened for the originally transformed cell are included. When different designations are proposed, this will be evident depending on the context.
[0480]
[0481] As used herein, CBI refers to 1,2,9,9a-tetrahydro-4H-benzo [e] cyclopropa [c] indole-4-one, or to a substituted or derivatized form thereof. CBI can also refer to the dry form of ^c B ⁱ , or dry-CBI, which is also known as 1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-ol, or a form (or forms) substituted or derivatized thereof.
[0482]
[0483] As used herein, CPI refers to 1,2,8,8a-tetrahydrocyclopropa [c] pyrrolo [3,2-e] indole-4 (5H) -one or a form thereof substituted or derivatized . CPI can also refer to the dry form of CPI, or dry-CPI, which is also known as 8- (chloromethyl) -1-methyl-3,6,7,8-tetrahydropyrrolo [3,2-e] indole- 4-ol, or a form (or forms) substituted or derivatized thereof.
[0484]
[0485] Unless otherwise indicated, the term "alkyl" by itself or as part of another term refers to a saturated straight or branched chain hydrocarbon having the indicated number of carbon atoms (for example, "C- alkyl iCa "refers to an alkyl group having 1 to 8 carbon atoms). Alkyl groups typically comprise 1 to 20 carbon atoms, preferably 1 to 8 carbon atoms, and more preferably 1 to 4 carbon atoms. When the amount of carbon atoms is not indicated, the alkyl group has 1 to 8 carbon atoms. Representative linear chain Ci-Ca alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl; while branched Ci-Ca alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tent-butyl, -isopentyl and -2-methylbutyl; unsaturated C ² -C ⁸ alkyls include, but are not limited to, vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2- methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1 -pentinyl, 2-pentinyl and 3- methyl-1-butynyl. Here, "alkyl" refers to unsubstituted and substituted moieties as described above.
[0486] Unless otherwise indicated, "alkylene," by itself or as part of another term, refers to a saturated linear or branched chain or cyclic hydrocarbon radical of the indicated number of carbon atoms, typically 1-18 atoms. carbon, and having two centers of monovalent radicals derived by removing two hydrogen atoms of the same or two different carbon atoms from an original alkane.) Alkylene groups typically comprise 1 to 18 carbon atoms, preferably of 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms, and most preferably 1 to 4 carbon atoms. Typical alkylene radicals include, but are not limited to: methylene (-CH ² -), 1,2-ethylene -CH ² CH ² -), 1,3-propylene (-CH ² CH ² CH ² -), 1 , 4-butylene (-CH ² CH ² CH ² CH ² -) and the like. A "C ¹ -C ¹⁰ " straight chain alkylene is a linear chain hydrocarbon group of the formula - (CH ² ) ^1-10 -. Examples of a C ¹ -C ¹⁰ alkylene include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene and decalene. Here, "alkylene" refers to unsubstituted and substituted moieties as described above.
[0487]
[0488] Unless otherwise indicated, the term "heteroalkyl," by itself or together with another term, means, unless otherwise indicated, a stable linear or branched chain hydrocarbon, or combinations thereof, fully saturated. or containing 1 to 3 degrees of unsaturation, which consists of the indicated number of carbon atoms and one to three heteroatoms selected from the group consisting of O, N, Si and S, and where the nitrogen and sulfur atoms they can be optionally oxidized and the nitrogen heteroatom can be optionally quaternized. The heteroatom or heteroatoms O, N and S may be placed in any interior position of the heteroalkyl group. The heteroatom Si can be placed in any position of the heteroalkyl group, including the position in which the alkyl group is attached to the rest of the molecule. Up to two heteroatoms can be consecutive. Heteroalkyl groups typically comprise 1 to 15 carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to 8 carbon atoms, and most preferably 1 to 4 carbon atoms. Here, "heteroalkyl" refers to unsubstituted and substituted moieties as described above.
[0489]
[0490] Unless otherwise indicated, the term "heteroalkylene" by itself or as part of another substituent means a divalent group derived from heteroalkyl (as discussed above). For heteroalkylene groups, heteroatoms can also occupy either or both ends of the chain. Here, "heteroalkylene" refers to unsubstituted and substituted moieties as described above.
[0491]
[0492] Unless otherwise indicated, "aryl", by itself or a part of another term, means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical of 5-20, preferably 5-14 or 6-14, derived by the removal of a hydrogen atom from a single carbon atom from a precursor aromatic ring system. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl and the like. A substituted carbocyclic aromatic group (for example, an aryl group) may be substituted with one or more, preferably 1 to 5, of the following groups: C ¹ -C ⁸ alkyl, -O- (C ¹ -C ⁸ alkyl) , -C (O) R ⁹ , -OC (O) R ⁹ , -C (O) OR ⁹ , -C (O) NH ² , -C (O) NHR ', -C (O) N (R' ) ² , -NHC (O) R ', -S (O) ² R', -S (O) R ', -OH, halogen, -N ³ , -NH ² , -NH (R ⁹ ), -N (R ⁹ ) ² and -CN; wherein each R ⁹ is independently selected from -H, C ¹ -C ⁸ alkyl and unsubstituted aryl. In some embodiments, a substituted carbocyclic aromatic group may additionally include one or more of: -NHC (= NH) NH ² , -NHCONH ² , -S (= O) ² R ⁹ and -SR ⁹ . "Arylene" is the corresponding divalent moiety.
[0493]
[0494] "Substituted alkyl" (or "substituted alkyl", "substituted heteroalkyl", or "substituted heteroalkylene") means one of the relevant alkyl or alkyl containing groups as discussed above in which one or more hydrogen atoms have been substituted, each independently, with a substituent. Typical substituents include, but are not limited to, -X, -R ¹⁰ , -O-, -OR ¹⁰ , -SR ¹⁰ , -S-, -NR ¹⁰² , -NR ¹⁰³ , = NR ¹⁰ , -CX ³ , -CN, -OCN, -SCN, -N = C = O, -NCS, -NO, -NO ² , = N ² , -N ³ , -NR ¹⁰ C (= O) R ¹⁰ R ¹⁰ , - C (= O) NR ¹⁰² , -SO ³ -, -SO ³ H, -S (= O) ² R ¹⁰ , -OS (= O) ² OR ¹⁰ , -S (= O) ² NR ¹⁰ , -S (= O) R ¹⁰ , -OP (= O) (OR ¹⁰ ) ² , -P (= O) (OR ¹⁰ ) ² , -PO ³² -, PO ³ H ² , -AsO ² H ² , -C ( = O) R ¹⁰ , -C (= O) X, -C (= S) R ¹⁰ , -CO ² R ¹⁰ , -CO ² -, -C (= S) OR ¹⁰ , -C (= O) SR ¹⁰ , -C (= S) SR ¹⁰ , -C (= O) NR ¹⁰² , -C (= S) NR ¹⁰² , or -C (= NR ¹⁰ ) NR ¹⁰² , in which each X is independently a halogen: -F, -CI, -Br or -I; and each R ¹⁰ is independently -H, C ¹ -C ²⁰ alkyl, heteroalkyl C ¹ -C ^20, aryl C ⁶ -C ²⁰ heterocyclyl ^C1 - ^C10, a protecting group or prodrug moiety. The aryl, alkylene and heteroalkylene groups as described above may also be similarly substituted.
[0495]
[0496] Unless otherwise indicated, "aralkyl" by itself or part of another term, means an alkyl group, as defined above, substituted with an aryl group, as defined above.
[0497]
[0498] Unless otherwise indicated, "C ³ -C ¹⁰ heterocycle," by itself or as part of another term, refers to a monocyclic, bicyclic or substituted or unsubstituted monovalent monocyclic, bicyclic or tricyclic aromatic or non-aromatic ring system having 2 to 10, 2 to 14, or 2-20 carbon atoms, preferably 3 to 8, carbon atoms (also referred to as ring members) and one to four ring members of heteroatoms independently selected from N, O , P or S, and derivatives by the removal of a hydrogen atom from a ring atom from a parental ring system. One or more of the atoms of N, C or S in the heterocyclyl can be oxidized. The ring that includes the heteroatom may be aromatic or non-aromatic. Aromatic heterocycles are sometimes referred to herein as heteroaryls. Unless otherwise indicated, the heterocyclyl is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Representative examples of a C ² -C ¹⁰ heterocyclyl include, but are not limited to, tetrahydrofuranyl, oxetanyl, pyranyl, pyrrolidinyl, piperidinyl, piperazinyl, benzofuranyl, benzothiophene, benzothiazolyl, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiophene), furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl moieties such as 1,2,3,4-tetraolinyl, pyridimyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, tetrazolyl, epoxide, oxetane and BODIPY (substituted or unsubstituted). A heterocyclyl C ² -C ¹⁰ may be substituted with up to seven groups including, but not limited to , alkyl C ¹ -C ⁸ heteroalkyl C ¹ -C ⁸ -alkyl, -OR ^11, aryl, -C (O) R ¹¹ , -OC (O) R ¹¹ , -C (O) OR ¹¹ , -C (O) NH ² , -C (O) NHR ¹¹ , -C (O) N (R ¹¹ ) ² , -NHC (O) R ¹¹ , -S (= O) ² R ¹¹ , -S (O) R ¹¹ , halogen, -N ³ , -NH ² , -NH (R ¹¹ ), -N (R ¹¹ ) ² and -CN; wherein each R ^11, C ¹ -C ⁸ alkyl, C ¹ -C ⁸ heteroalkyl is independently selected from -H and aryl. In some embodiments, a substituted heterocyclyl may also include one or more of: -NHC (= NH) NH ² , -NHCONH ² , -S (= O) ² R ¹¹ and -SR ¹¹ . Heterocycle or heterocycle C ² -C ¹⁰ is the corresponding divalent moiety. Divalent heterocycles are sometimes referred to herein as heteroarylene or C ² -C ¹⁰ heteroarylene.
[0499]
[0500] As mentioned above, aromatic heterocycles are sometimes referred to herein as heteroaryl, and preferably contain 5-14, 6-14, or 6-20 carbon atoms in addition to the heteroatoms. The heteroaryls can be monocyclic, bicyclic or tricyclic ring systems. Representative heteroaryls include, but are not limited to, tetrazolyl, oxadiazolyl, pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzoimidazolyl, thiazolyl, benzothiazolyl, pyrothiazolyl, pyrothiazolyl, pyrothiazolyl , pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, quinazolinyl, pyrimidyl, azepinyl, oxepinyl, and quinoxalinyl. Heteroaryls are optionally substituted. Typical substituents include, but are not limited to, -X, -R ^h , -O-, -OR ^h , -SR ^h , -S-, -NR ^h2 , -NR ^h3 , = NR ^h , -CX ³ , -CN, -OCN, -SCN, -N = C = O, -NCS, -NO, -NO ² , = N ² , -N ³ , -NR ^h C (= O) R ^h , -C ( = O) NR ^h2 , -SO ³ -, -SO ³ H, -S (= O) ² R ^h , -OS (= O) ² OR ^h , -S (= O) ² NR ^h , -S (= O) R ^h , -OP (= O) (OR ^h ) ² , -P (= O) (OR ^h ) ² , -PO ³² -, PO ³ H ² , -AsO ² H ² , -C (= O ) R ^h , -C (= O) X, -C (= S) R ^h , -CO ² R ^h , -CO ² -, -C (= S) OR ^h , -C (= O) SR ^h , -C (= S) SR ^h , -C (= O) NR ^h2 , -C (= S) NR ^h2 , -C (= NR) NR ^h2 , C ¹ -C ²⁰ heteroalkyl, C ⁶ -C ²⁰ aryl, C ³ -C ⁸ heterocyclyl, a protecting group or a prodrug moiety, in which each X is independently a halogen: -F, -CI, -Br or -I; and each R ^h is independently -H or C ¹ -C ⁶ alkyl. Divalent aromatic heterocycles are sometimes referred to herein as C ¹ -C ¹⁰ heteroarylenes or heteroarylenes.
[0501]
[0502] Unless otherwise indicated, "heteroaralkyl" by itself or as part of another term, means an alkyl group, as defined above, substituted with an aromatic heterocyclyl group, as defined above. Heteroaralkyl is the corresponding divalent moiety .
[0503]
[0504] Unless otherwise indicated, "C ³ -C ⁸ carbocyclyl" by itself or as part of another term, is a monovalent saturated or unsaturated monocyclic monocyclic or bicyclic carbocyclic ring of 3, 4, 5, 6, 7 or 8-member derived by removing a hydrogen atom from a ring atom from a parental ring system. Representative C ³ -C ⁸ carbocyclyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1 , 3,5-cycloheptatrienyl, cyclooctyl, cyclooctadienyl, bicyclo (1.1.1.) Pentane, and bicyclo (2.2.2.) Octane. A carbocyclyl group C ³ -C ⁸ may be unsubstituted or substituted with up to seven groups including, but not limited to , alkyl C ¹ -C ⁸ heteroalkyl C ¹ -C ⁸ -alkyl, -OR11, aryl, -C (O ) R11, -OC (O) R11, -C (O) OR11, -C (O) NH ² , -C (O) NHR11, -C (O) N (R11) ² , -NHC (O) R11, -S (= O) ² R11, -S (= O) R11, -OH, -halogen, -N ³ , -NH ² , -NH (R11), -N (R11) ² and -CN; wherein each R11, C ¹ -C ⁸ alkyl, C ¹ -C ⁸ heteroalkyl is independently selected from -H and aryl. "Carbocycle C ³ -C ⁸ " is the corresponding divalent moiety.
[0505]
[0506] As used herein, an azido substituent refers to -N = N = N; a cyanate substituent refers to -O-CN; a thiocyanate substituent refers to -S-CN; an isocyanate substituent refers to -N = C = O; and a thioisocyanate substituent refers to -S-N = C = O.
[0507]
[0508] The term "chiral" refers to molecules that have the property of non-superposition of the mirror image partner, while the term "aquiral" refers to molecules that can be superimposed on their mirror image partners.
[0509]
[0510] The term "stereoisomers" refers to compounds that have an identical chemical constitution, but differ with respect to the arrangement of atoms or groups in space.
[0511]
[0512] "Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of each other. Diastereomers have different physical properties, for example, melting points, boiling points, spectral properties and reactivities. Mixtures of diastereomers can be separated in high resolution analytical procedures, such as electrophoresis and chromatography.
[0513]
[0514] "Glycosyl" refers to the structure:
[0515] or substituted forms thereof, for example including substituted reference structures for structural forms such as:
[0516]
[0517]
[0518]
[0519]
[0520] and many others.
[0521] The definitions and stereochemical conventions used herein are generally based on S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms, McGraw-Hill Book Company, New York (1984); and Eliel and Wilen, Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York (1994). Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of polarized light. In the description of an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule around its chiral center (s). The prefixes d and l or (+) and (-) are used to designate the direction of rotation of the plane of light polarized by the compound, where (-) or 1 means that the compound is levógiro. A compound preset with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A specific stereoisomer can also be called an enantiomer, and a mixture of said isomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is called racemic mixture or racemate, which can occur when there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity. As used herein, "-PABA-" or "PABA" refers to p-aminobenzoic acid and residues derived therefrom, for example the structure:
[0522]
[0523]
[0524]
[0525] or variants thereof.
[0526] As used herein, "-PABC-" or "PABC" refers to p-aminobenzyloxycarbonyl and moieties thereof, for example the structure:
[0527]
[0528]
[0529]
[0530] or variants thereof.
[0531] An "amino acid derivative" includes an amino acid having covalent substitutions or modifications of a parent amino acid, such as, for example, by alkylation, glycosylation, acetylation, phosphorylation and the like. Also included in the definition of "derivative" are, for example, one or more analogs of an amino acid with substituted bonds, as well as other modifications known in the art.
[0532] A "natural amino acid" refers to arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine , unless the context indicates otherwise.
[0533] "Protective group" refers to a moiety that when attached to a reactive group in a molecule masks, reduces or prevents this reactivity. Examples of protective groups can be found in TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York, 1999, and Harrison and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8 (John Wiley and Sons, 1971-1996). Representative hydroxy protecting groups include acyl groups, benzyl and trityl ethers, tetrahydropyranyl ethers, ethers of trialqulsilyl and allyl. Representative amino protecting groups include, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), ferc-butoxycarbonyl (Boc), trimethyl silyl (TMS), 2-trimethylsilyl ethanesulfonyl (SES), trityl and substituted trityl, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC), nitroveratriloxycarbonyl (NVOC), and the like.
[0534]
[0535] Examples of "hydroxyl protecting group" include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ethers, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl, t-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate ester, substituted acetate esters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.
[0536]
[0537] "Outgoing group" refers to a functional group that may be substituted by another functional group. Such leaving groups are well known in the art and examples include, but are not limited to, a halide (for example, chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate.
[0538]
[0539] The term "pharmaceutically acceptable salt", as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a compound. The compound normally contains at least one amino group, and accordingly, acid addition salts can be formed with this amino group. Illustrative salts include, but are not limited to, salts of sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, Tanate, pantothenate, bitartrate, ascorbate, succinate, maleate, malate, gentisinate, fumarate, gluconate, glucuronate, sucrate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e. 1.1 ' -methylene-bis- (2-hydroxy-3-naphthoate)). A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or another counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge of the precursor compound. In addition, a pharmaceutically acceptable salt may have more than one atom charged in its structure. Cases in which multiple charged atoms are part of the pharmaceutically acceptable salt may have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / one or more counterions.
[0540]
[0541] "Pharmaceutically acceptable solvate" or "solvate" refers to an association of one or more solvent molecules and a compound or conjugate of the invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine.
[0542]
[0543] The terms "load" or "drug load" or "payload" represent or refer to the average number of loads ("load" and "payload" are used interchangeably herein with "drug" and "drugs") by antibody in an ADC molecule. The drug load can be comprised of 1 to 20 drugs per antibody. This is sometimes called DAR, or the drug-to-antibody ratio. The compositions of the ADCs described herein typically have DARs of between 1-20, and in certain embodiments of 1-8, 2-8, 2-6, 2-5 and 2-4. Typical DAR values are 2, 4, 6 and 8. The average number of drugs per antibody, or DAR value, can be characterized by conventional fabrics such as UV / visible spectroscopy, mass spectrometry, ELISA assay, and HPLC. The quantitative DAR value can also be determined. In some cases, the separation, purification, and characterization of homogeneous ADCs having a specific DAR value can be achieved by means such as reverse phase HPLC or electrophoresis. DAR may be limited by the number of binding sites in the antibody. For example, when the binding is carried out in a cysteine thiol, an antibody may have only one or more thiol groups of cysteine, or it may have only one or more thiol groups sufficiently reactive through which a unit of linker In some embodiments, the cysteine thiol is a thiol group of a cysteine residue that forms an inter-chain disulfide bond. In some embodiments, the cysteine thiol is a thiol group of a cysteine residue that does not form an inter-chain disulfide bond. Normally, less than the theoretical maximum of drug residues is conjugated with an antibody during a conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with a linker or linker intermediate. Only the most reactive lysine groups can react with a reactive linker reagent.
[0544]
[0545] In general, the antibodies do not contain many, if any, contain thiol group of free cysteine and reagent that can bind to a drug via a linker. Most of the thiol cysteine residues in the antibodies exist as disulfide bridges and should be reduced with a reducing agent such as dithiothreitol (DTT). The antibody can be subjected to denaturation conditions to reveal reactive nucleophilic groups such as lysine or cysteine. The loading (drug / antibody ratio) of an ADC can be controlled in several different ways, including: (i) limiting the molar excess of the drug-linker with respect to the antibody, (ii) limiting the time or temperature of the reaction of conjugation, and (iii) partial or limiting reducing conditions for thiol modification of cysteine. When more than one nucleophile group reacts with a drug-linker, then, the resulting product is a mixture of ADC a distribution of one or more drug residues per antibody. The average number of drugs per antibody can be calculated from the mixture by, for example, double ELISA antibody assay, specific for the antibody and specific for the drug. Individual a Dc can be identified in the mixture by mass spectroscopy, and separated by HPLC, for example, hydrophobic interaction chromatography.
[0546] Below is a list of abbreviations and definitions that may not be defined or otherwise described in this application: DMSO (refers to dimethylsulfoxide), HRMS (refers to high mass spectrometry resolution), DAD (refers to diode detection matrix), TFA (refers to 2,2,2-trifluoroacetic acid or trifluoroacetic acid), TFF (refers to tangential flow filtration), EtOH (refers to ethanol ), MW (refers to molecular weight), HPLC (refers to high performance liquid chromatography), prep HPLC (refers to preparative high performance liquid chromatography), etc. (refers to and so on), trityl (refers to 1,1 ', 1 "-ethane-1,1,1-trityltribbenzene), THF (refers to tetrahydrofuran), NHS (refers to 1-Hydroxy-2 , 5-pyrrolidinedione), Cbz (refers to carboxybenzyl), eq. (Refers to equivalent), n-BuLi (refers to n-butyllithium), OAc (refers to acetate), MeOH (refers to methanol) , i-Pr (refers to isopropyl or propan-2-yl), NMM (refers to 4-methylmorpholine), and "-" (in a table refers to data not available at this time).
[0547]
[0548] It is understood that the divalent moieties and substituents used herein refer to said moieties or linked substituents linked in either direction or in both directions. For example, the remainder -C (O) NR- (in the definition of LB1, and elsewhere) means that it carries -C (O) NR- as well as -NRC (O) -, the remainder -C (O ) is C ¹ -C6alquil- carrying means -C (O) C ¹ -C ⁶ alkyl as well as C (O) -C ¹ -C ⁶ etcetera. More generally, a description of a non-symmetrical divalent moiety joined on its "left" and "right" sides is understood to carry the rest that is presented (left side of the rest attached on the left side as written, the right side of the remainder joined on the right side as written) and the inverse of the remainder presented (left side of the remainder attached on the right side as written, right side of the remainder attached on the left side as written).
[0549]
[0550] The terms "bound" and "absent" are both used herein to describe a variable that does not include an atom or atoms. Therefore, when a divalent variant is "absent" it is understood that it means that adjacent remains are joined together. For example, if LB2 is absent it is understood that LB1 can bind to LB3; or if LB1 and LB2 are absent it is understood that LA can join LB3. Similarly, if a divalent variable is defined as being "bound", this is understood to mean that there are no atoms present and adjacent moieties join together. Therefore, for example, when the variable "D" is defined as being a bond, it will be appreciated that the adjacent carbon atoms D (in the structure that defines T2) join together. It is understood that an absent monovalent variable is a pair of hydrogens or electrons capable of additionally binding covalently.
[0551]
[0552] The antibody unit (A, Ab or AB)
[0553]
[0554] As mentioned above, the term "antibody" (or "A", "Ab" or "AB") is used herein in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments that exhibit the desired biological activity. In addition, although certain aspects of the invention described herein refer to antibody and drug conjugates, it is further envisioned that the antibody portion of the conjugate can be substituted with anything that specifically binds or associates reactively or complexes with a receptor. , antigen or other receptor moiety associated with a population of given target cells. For example, instead of containing an antibody, the conjugates of the invention could contain a targeting molecule that binds to, complexes with, or reacts with a receptor, antigen or other receptor moiety of a population of cells that is sought to be therapeutically or otherwise biologically modified. Examples of such molecules include smaller molecular weight proteins, polypeptides or peptides, lectins, glycoproteins, non-peptides, vitamins, nutrient transporting molecules (such as, but not limited to, transferrin), or any other molecule or substances of cell binding In certain aspects, the antibody or other of said targeting molecules act to deliver a drug to the specific target cell population with which the antibody or other targeting molecule interacts.
[0555]
[0556] In another aspect, the present invention relates to an antibody and drug conjugate compound of Formulas IIIA or IIIB in which the AB antibody is selected from: trastuzumab, trastuzumab mutants (eg, trastuzumab mutants disclosed herein or in international patent application PCT / IB2012 / 056234), oregovomab, edrecolomab, cetuximab, a humanized monoclonal antibody against the vitronectin receptor (ayp ³ ), alemtuzumab, antibodies directed against HLA-DR include a humanized antibody directed against HLA- DR for the treatment of non-Hodgkin lymphoma, 131I Lym-1, antibodies directed against HLA-Dr10 include a murine antibody directed against HLA-Dr10 for the treatment of non-Hodgkin lymphoma, antibodies directed against cd33, antibodies directed against CD22 include a humanized mAb directed against CD22 for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma, labetuzumab, beva cizumab, ibritumomab tiuxetán, ofatumumab, panitumumab, rituximab, tositumomab, ipilimumab, and gemtuzumab.
[0557]
[0558] Heteroatoms that may be present in an antibody unit include sulfur (in one embodiment, from a sulfhydryl group of an antibody), oxygen (in one embodiment, from a carbonyl, carboxyl or hydroxyl group of an antibody) and nitrogen ( in one embodiment, from a primary or secondary amino group of an antibody). These heteroatoms may be present in the antibody in the natural state of the antibody, for example a naturally occurring antibody, or they may be introduced into the antibody by chemical modification.
[0559]
[0560] In one embodiment, an antibody unit has a sulfhydryl group and the antibody unit is linked by the sulfur atom of the sulfhydryl group.
[0561]
[0562] In another embodiment, the antibody has lysine residues that can react with activated esters (said esters they include, but are not limited to, N-hydroxysuccinimide, pentafluorophenyl, and p-nitrophenyl esters) and thus form an amide bond consisting of the nitrogen atom of the antibody unit and a carbonyl.
[0563]
[0564] In yet another aspect, the antibody unit has one or more lysine moieties that may be chemically modified to introduce one or more sulfhydryl groups. Reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut Reagent).
[0565]
[0566] In another embodiment, the antibody unit may have one or more carbohydrate groups that may be chemically modified to have one or more sulfhydryl groups.
[0567]
[0568] In yet another embodiment, the antibody unit may have one or more carbohydrate groups that can be oxidized to provide an aldehyde group (see, for example, Laguzza, et al., 1989, J. Med. Chem. 32 (3): 548 -55). The corresponding aldehyde can form a bond with a reactive site such as, for example, hydrazine and hydroxylamine. Other protocols for protein modification for drug binding or association are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002).
[0569]
[0570] When the conjugates comprise proteins, polypeptides, or non-immunoreactive peptide units instead of an antibody, non-immunoreactive proteins, polypeptides, or peptide units include, but are not limited to, transferin, epidermal growth factors ("EGF") , bombesin, gastrin, gastrin releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors ("TOP"), such as TGF-a and TGF-p, vaccinia growth factor ("VGF"), insulin and insulin-like growth factors I and II, somatostatin, lectins and apoproteins from low-density lipoproteins.
[0571]
[0572] Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Useful monoclonal antibodies are homogeneous populations of antibodies directed against a particular antigenic determinant (e.g., a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical compound, nucleic acid, or fragments thereof). A monoclonal antibody (mAb) directed against an antigen of interest can be prepared using any technique known in the art that provides for the production of antibody molecules by continuous continuous cell lines.
[0573]
[0574] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric monoclonal antibodies. Human monoclonal antibodies can be prepared by numerous techniques known in the art (eg, Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80: 7308-7312; Kozbor et al., 1983, Immunology Today 4: 72-79; and Olsson et al., 1982, Meth. Enzymol. 92: 3-16).
[0575]
[0576] The antibody may also be a bispecific antibody. Methods for preparing bispecific antibodies are known in the art and are described below.
[0577]
[0578] The antibody may be a functionally active, derived or analogous fragment of an antibody that immunospecifically binds to target cells (e.g., cancer cell antigens, viral antigens, or microbial antigens) or other antibodies that bind to cells or to the tumor matrix. . In this regard, "functionally active" means that the fragment, derivative or analog is capable of stimulating anti-idiotype antibodies that recognize the same antigen as the antibody from which the fragment is derived, the derivative or analog is derived recognized. Specifically, in an illustrative embodiment, the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deleting the framework sequences and the CDRs that are at the C-terminus of the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind to the antigen, synthetic peptides containing the CDR sequences can be used in antigen binding assays by any binding assay procedure known in the art (e.g., the BIA nuclear assay ) (for the location of the CDR sequences, see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md .; Kabat E et al., 1980, J. Immunology 125 (3): 961-969).
[0579]
[0580] Other useful antibodies include antibody fragments such as, but not limited to, F (ab ') ² fragments, Fab fragments, Fvs, single chain antibodies, diabody, triabody, tetrabody, scFv, scFv-FV, or any other molecule with the same specificity as the antibody. Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, which comprise human and non-human portions, which can be prepared using standardized recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which different portions of different animal species are derived, such as, for example, those that have a variable region derived from monoclonal regions of a murine and from constant regions of human immunoglobulin. (See, for example, U.S. Patent No. 4,816,567; and U.S. Patent No. 4,816,397). Humanized antibodies are antibody molecules from non-human species that have one or more complementarity determining regions (CDRs) from non-human species and a framework region from a human immunoglobulin molecule. (See, for example, U.S. Patent No. 5,585,089). Said chimeric monoclonal antibodies and Humanized can be produced by recombinant DNA techniques known in the art, using, for example, the methods described in International Publication No. WO 87/02671; European Patent Publication EP 0184 187; European Patent Publication EP 0171 496; European Patent Publication EP 0173494; International Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Publication No. 012 023; Berter et al., 1988, Science 240: 1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al., 1987, J. Immunol. 139: 3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al., 1987, Cancer. Res. 47: 999-1005; Wood et al., 1985, Nature 314: 446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80: 1553-1559; Morrison, 1985, Science 229: 1202-1207; Oi et al., 1986, BioTechniques 4: 214; U.S. Patent No. 5,225,539; Jones et al., 1986, Nature 321: 552-525; Verhoeyan et al., 1988, Science 239: 1534; and Beidler et al., 1988, J. Immunol. 141: 4053-4060.
[0581]
[0582] Completely human antibodies are completely desirable and can be produced using transgenic mice that are unable to express the endogenous genes of the immunoglobulin heavy and light chains, but that express the human heavy and light chain genes. Transgenic mice are normally immunized with a selected antigen, for example, all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes housed by the transgenic mice are rearranged during the differentiation of B lymphocytes and subsequently subjected to class change and somatic mutation. Therefore, using said technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For a general description of this technology to produce human antibodies, see Lonberg and Huszar, 1995, Int. Rev. Immunol. 13: 65-93. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and the protocols for producing such antibodies, see, for example, US Pat. No. 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806. Other human antibodies can be obtained commercially from, for example, Abgenix, Inc. (now Amgen, Freemont, Calif.) And Medarex (Princeton, N.J.).
[0583]
[0584] Completely human antibodies that recognize a selected epitope can be generated using a technique called "guided selection." In this approach a selected non-human monoclonal antibody, for example, a mouse antibody, is used to guide the selection of a completely human antibody that recognizes the same epitope. (See, for example, Jespers et al., 1994, Biotechnology 12: 899-903). Human antibodies can also be produced using various techniques known in the art, including phage libraries (see, for example, Hoogenboom and Winter, 1991, J. Mol. Biol. 227: 381; Marks et al., 1991, J. Mol. Biol. 222: 581; Quan and Carter, 2002, The rise of monoclonal antibodies as therapeutics, in Anti-IgE and Allergic Disease, Jardieu and Fick, eds., Marcel Dekker, New York, NY, Chapter 20, pp. 427- 469).
[0585]
[0586] In other embodiments, the antibody is a fusion protein of an antibody, or a functionally active fragment thereof, for example in which the antibody is fused by a covalent bond (eg, a peptide bond), at any of the N-terminus. or the C-terminus of an amino acid sequence of another protein (or portion thereof, preferably at least a 10, 20 or 50 amino acid portion of the protein) that is not of an antibody. Preferably, the antibody or fragment thereof covalently binds to the other protein at the N-terminus of the constant domain.
[0587]
[0588] The antibodies include analogs and derivatives that are either modified, that is, by the covalent bond of any type of molecule provided that said covalent bond allows the antibody to retain its antigen-binding immunospecificity. For example, but not limited to, the derivatives and analogs of the antibodies include those that have been further modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, binding. to a unit of cellular antibody or other protein, etc. Any of numerous chemical modifications can be made by known techniques that include, but are not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. In addition, the analog or derivative may contain one or more unnatural amino acids.
[0589]
[0590] Antibodies may have modifications (eg, substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, the antibodies may have modifications in the amino acid residues identified as being involved in the interaction between the domain directed against Fc and the FcRn receptor (see, for example, International Publication No. WO 97/34631).
[0591]
[0592] Immunospecific antibodies to the antigen of a cancer cell can be obtained commercially or produced by any method known to a person skilled in the art such as, for example, chemical synthesis or recombinant expression techniques. Immunospecific antibodies encoding the nucleotide sequence of a cancer cell antigen can be obtained, for example, from the GenBank database or from a database of this type, bibliographic publications, or by routine cloning and sequencing.
[0593]
[0594] In a specific embodiment, known antibodies can be used for the treatment of cancer. Immunospecific antibodies to the antigen of a cancer cell can be obtained commercially or produced by any method known to a person skilled in the art such as, for example, recombinant expression techniques. Immunospecific antibodies encoding the nucleotide sequence of a nucleus can be obtained. cancer cell antigen, for example, from the GenBank database or from a database of this type, literature publications, or by routine cloning and sequencing. Examples of antibodies available for the treatment of cancer include, but are not limited to, OVAREX which is a murine antibody for the treatment of ovarian cancer; PANOREX (Glaxo Wellcome, NC) which is a murine IgG ^2a antibody for the treatment of colorectal cancer; Cetuximab ERBITUX (Imclone Systems Inc., NY) which is an IgG chimeric antibody directed against EGFR IgG for the treatment of epidermal growth factor positive cancers, such as head and neck cancer; Vitaxin (MedImmune, Inc., MD) which is a humanized antibody for the treatment of sarcoma; CAMPATH I / H (Leukosite, MA) which is a humanized IgG ¹ antibody for the treatment of chronic lymphocytic leukemia (LLC); SMART ID10 (Protein Design Labs, Inc., CA) which is a humanized antibody directed against HLA-DR for the treatment of non-Hodgkin lymphoma; ONCOLYM (Techniclone, Inc., CA) which is a radiolabeled murine antibody directed against HLA-Dr10 for the treatment of non-Hodgkin lymphoma; ALLOMUNE (BioTransplant, CA) which is a humanized mAb directed against CD2 for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma; and CEACIDE (Immunomedics, NJ) which is a humanized antibody directed against CEA for the treatment of colorectal cancer.
[0595]
[0596] In attempts to discover effective cell targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or tumor-associated polypeptides that are specifically expressed on the surface of one or more particular types of cancer cells compared to one or more normal non-cancerous cells. Often, such tumor-associated polypeptides are expressed more abundantly on the surface of cancer cells compared to on the surface of non-cancerous cells. The identification of said polypeptides associated with cell surface antigens has provided an increased ability to specifically target cancer cells for destruction by antibody-based therapies.
[0597]
[0598] The linker unit (L)
[0599]
[0600] A linker (sometimes referred to as "[linker]" herein) is a bifunctional compound that can be used to bind a drug and an antibody to form an antibody and drug conjugate (ADC). Such conjugates are useful, for example, in the formation of immunoconjugates directed against tumor associated antigens. Such conjugates allow selective administration of cytotoxic drugs to tumor cells.
[0601]
[0602] In an ADC the linker serves to bind the payload to the antibody.
[0603]
[0604] In one aspect, a second section of the linker unit having a second reactive site is introduced, for example, an electrophilic group that is reactive to a nucleophile group present in an antibody unit (for example, an antibody). Useful nucleophilic groups in an antibody include, but are not limited to, sulfhydryl, hydroxyl and amino groups. The heteroatom of the nucleophilic group of an antibody is reactive to an electrophilic group in a linker unit and forms a covalent bond to a linker unit. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. The electrophilic group provides a convenient site for antibody binding.
[0605]
[0606] In another embodiment, a linker unit has a reactive site that has a nucleophilic group that is reactive to an electrophilic group present in an antibody. Useful electrophilic groups in an antibody include, but are not limited to, carbonyl aldehyde and ketone groups. The heteroatom of a nucleophilic group of a linker unit can react with an electrophilic group in an antibody and forms a covalent bond with the antibody. Useful nucleophilic groups in a linker unit include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group in an antibody provides a convenient site for binding to a linker unit.
[0607]
[0608] Aminofunctional groups are also useful reactive sites for a linker unit for a linker unit because they can react with carboxylic acid, or activated esters of a compound to form an amide bond. Typically, the peptide-based compounds of the invention can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis procedure (see, for example, Schroder and Lubke, "The Peptides", volume 1, pp. 76-136, 1965, Academic Press) which is well known in the field of peptide chemistry.
[0609]
[0610] In the context of the invention, specifically, but not limited to linker components such as L ⁱ , L ² (including L ^2A , L ^2B and L ^2C ) and L ³ , the expression "selected from one or more of" or "one or more of" indicates that multiple components, which may be the same or different, are or may be arranged sequentially. Thus, for example, L ³ can be -C ^1-6 alkyl-, -NR- or the other individually related components, but also -Ci -6-Nr alkyl-, or any other combination of 2 or more related components.
[0611]
[0612] In another embodiment, a linker unit has a reactive site that can react with antibody nucleophiles, such as cysteines. The reactive site is comprised of a heterocycle that is substituted with a sulfone. The sulfone is then replaced by the antibody nucleophiles (i.e. cysteine) and the newly formed bond between the antibody and the heterocycle connects the antibody with the linker. See document WO 2014/144878.
[0613]
[0614] Synthesis of compounds and antibody conjugates with drugs thereof
[0615]
[0616] The compounds and conjugates of the invention can be prepared using the synthetic procedures outlined below in the illustration. As described in more detail below, the compounds and conjugates of the invention can be prepared using a section of a linker unit having a reactive site for binding to the compound. In one aspect, a second section of the linker unit having a second reactive site is introduced, for example, an electrophilic group that is reactive to a nucleophile group present in an antibody unit (for example, an antibody). Useful nucleophilic groups in an antibody include, but are not limited to, sulfhydryl, hydroxyl and amino groups. The heteroatom of the nucleophilic group of an antibody is reactive to an electrophilic group in a linker unit and forms a covalent bond to a linker unit. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. The electrophilic group provides a convenient site for antibody binding.
[0617]
[0618] In another embodiment, a linker unit has a reactive site that has a nucleophilic group that is reactive to an electrophilic group present in an antibody. Useful electrophilic groups in an antibody include, but are not limited to, carbonyl aldehyde and ketone groups. The heteroatom of a nucleophilic group of a linker unit can react with an electrophilic group in an antibody and forms a covalent bond with the antibody. Useful nucleophilic groups in a linker unit include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group in an antibody provides a convenient site for binding to a linker unit.
[0619]
[0620] Aminofunctional groups are also useful reactive sites for a linker unit for a linker unit because they can react with carboxylic acid, or activated esters of a compound to form an amide bond. Typically, the peptide-based compounds of the invention can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis procedure (see, for example, Schroder and Lubke, "The Peptides", volume 1, pp. 76-136, 1965, Academic Press) which is well known in the field of peptide chemistry.
[0621]
[0622] As described in more detail below, the conjugates can be prepared using a linker section having a reactive site for binding to a compound of the invention and introducing another section of the linker unit having a reactive site for a antibody. In one aspect, the linker unit has a reactive site that has an electrophilic group that is reactive with a nucleophilic group present in an antibody unit, such as an antibody. The electrophilic group provides a convenient site for antibody binding. Useful nucleophilic groups in an antibody include, but are not limited to, sulfhydryl, hydroxyl and amino groups. The heteroatom of the nucleophilic group of an antibody is reactive to an electrophilic group in a linker unit and forms a covalent bond to a linker unit. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups.
[0623]
[0624] In another embodiment, the linker unit has a reactive site that has a nucleophile group that is reactive with an electrophilic group present in an antibody unit. The electrophilic group in an antibody provides a convenient site for binding to a linker unit. Useful electrophilic groups in an antibody include, but are not limited to, carbonyl aldehyde and ketone groups. The heteroatom of a nucleophilic group of a linker unit can react with an electrophilic group in an antibody and forms a covalent bond with the antibody. Useful nucleophilic groups in a linker unit include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
[0625]
[0626] Conjugation with transglutaminase
[0627]
[0628] In certain embodiments, a compound of the invention can be covalently crosslinked with a genetically engineered polypeptide containing Fc or containing Fab with a label containing acyl donor glutamine (eg, peptide tags containing Gln or Q tags) or a endogenous reactively prepared glutamine (i.e., the ability to form a covalent bond as an acyl donor in the presence of an amine and a transglutaminase) by genetic engineering of polypeptides (eg, by deletion, insertion, substitution, mutation of an amino acid or any combination thereof in the polypeptide), in the presence of transglutaminase, with the proviso that the compound of the invention comprises an amine donor agent (for example, a small molecule that comprises or is linked to a reactive amine ), thus forming a stable and homogeneous population of a polypeptide conjugate di Genetically engineered containing Fc with the amine donor agent that is specifically conjugated at the site with the Fc-containing or Fab-containing polypeptide via the label containing acyl donor glutamine or exposed / accessible / reactive endogenous glutamine . For example, the compounds of the invention can be conjugated as described in the international patent application with serial number PCT / IB2011 / 054899. In certain embodiments, to facilitate the conjugation of the compound of the invention with a genetically engineered polypeptide containing Fc or containing Fab with a label containing acyl donor glutamine or an endogenous glutamine reactively prepared by genetic engineering of polypeptides in presence of transglutaminase, Z is NH ² .
[0629]
[0630] Conjugation with the constant region of the Kappa domain of the human light chain
[0631]
[0632] In certain embodiments, a compound of the invention can covalently bind to the secondary chain of K ¹⁸⁸ of the constant region of the kappa domain of the human light chain (CLk) (full light chain numbering according to Kabat). For example, the compounds of the invention can be conjugated as described in the US patent application with serial number 13 / 180,204. In certain embodiments, to facilitate conjugation to K188 CLk, Z is
[0633]
[0634]
[0635]
[0636]
[0637] R ⁷ is independently selected each time it is present from the group consisting of F, Cl, I, Br, NO ² , CN and CF ³ ; and h is 1,2, 3, 4 or 5.
[0638]
[0639] In certain embodiments, the invention provides a composition comprising a compound of the invention covalently conjugated with an antibody (or antigen binding portion thereof), wherein at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the compound of the invention in the composition is conjugated to the antibody or antigen-binding portion thereof at K ¹⁸⁸ CLk.
[0640]
[0641] In certain embodiments, the compounds of the invention may be conjugated to the combination site of a catalytic antibody, such as antibodies against aldolase, or antigen binding portion thereof. Aldolase antibodies contain portions of the combination site that, when not compromised (for example, by conjugation), catalyze an aldol addition reaction between an aliphatic ketone donor and an aldehyde acceptor. The contents of US Patent Application Publication No. US 2006/205670, in particular pages 78-118 describe the linkers, and paragraphs [0153] - [0233] describe the antibodies, the useful fragments, the variants and modifications thereof, h38C2, combination sites and complementarity determining regions (CDR), and related antibody technology. The term "combination site" includes the CDRs and adjacent framework moieties that are involved in antigen binding.
[0642] Compositions and administration procedures
[0643]
[0644] In other embodiments, another aspect of the invention relates to pharmaceutical compositions that include an effective amount of a compound of the invention and / or antibody and drug conjugate thereof and a pharmaceutically acceptable carrier or carrier. In certain embodiments, the compositions are suitable for veterinary or human administration.
[0645]
[0646] The current pharmaceutical compositions may be in any form that allows the composition to be administered to a patient. For example, the composition may be in the form of a solid or a liquid. Typical routes of administration include, without limitation, parenteral, ocular and intratumoral. Parenteral administration includes subcutaneous, intravenous injections, intramuscular or intrasternal injection or infusion techniques. In one aspect, the compositions are administered parenterally. In a specific embodiment, the compositions are administered intravenously.
[0647]
[0648] The pharmaceutical compositions may be formulated so as to allow a compound of the invention and / or an antibody and drug conjugate thereof to be bioavailable upon administration of the composition to a patient. The compositions may be in the form of one or more dosage units, where, for example, a tablet may be an individual unit dose, and a container of a compound of the invention and / or an antibody and drug conjugate thereof in the form Liquid may contain a plurality of unit doses.
[0649]
[0650] The materials used in the preparation of the pharmaceutical compositions may not be toxic in the amounts used. It will be apparent to a person normally skilled in the art that the optimal dosage of the active ingredient or principles in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of animal (eg, human), the particular form of the compound of the invention and / or the antibody and drug conjugate thereof, the method of administration, and the composition employed.
[0651]
[0652] The pharmaceutically acceptable carrier or carrier may be solid or in the form of particles, so that the compositions are, for example, in the form of tablets or powder. The one or more conveyors may be liquids. In addition, the one or more conveyors may be in the form of particles.
[0653]
[0654] The composition may be in the form of a liquid, for example, a solution, emulsion or suspension. In a composition for administration by injection one or more of a surfactant may also be included, preservative, wetting agent, dispersing agent, suspending agent, a buffer, stabilizer and isotonic agent.
[0655]
[0656] Liquid compositions, whether solutions, suspensions or other similar form, may also include one or more of the following: sterile diluents such as water for injection, saline, preferred physiological saline, Ringer's solution, isotonic sodium chloride, oils fixed, such as monoglycerides or synthetic diglycerides that can serve as solvent or suspension medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, phosphates or amino acids and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral composition can be introduced into a blister, a disposable syringe or a multi-dose vial made of glass, plastic or other material. Physiological saline is an illustrative adjuvant. An injectable composition is preferably sterile.
[0657]
[0658] The amount of a compound of the invention and / or antibody and drug conjugate thereof that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be used to help identify optimal dosage ranges. The precise dose to be used in the compositions will also depend on the route of administration, and the severity of the disease or disorder, and should be decided according to the criteria of the specialist in charge of the treatment and the circumstances of the patient.
[0659] The compositions comprise an effective amount of a compound of the invention and / or antibody and drug conjugate thereof so that a suitable dosage is obtained. Typically, this amount is at least about 0.01% of a compound of the invention and / or antibody and drug conjugate thereof by weight of the composition. In an illustrative embodiment, pharmaceutical compositions are prepared so that a parenteral unit dose comprises from about 0.01% to about 2% by weight of the amount of a compound of the invention and / or antibody and drug conjugate thereof.
[0660]
[0661] For intravenous administration, the composition may comprise from about 0.01 to about 100 mg of a compound of the invention and / or antibody and drug conjugate thereof per kg of the patient's body weight. In one aspect, the composition may include from about 1 to about 100 mg of a compound of the invention and / or antibody and drug conjugate thereof per kg of the patient's body weight. In another aspect, the amount administered will be in the range of about 0.1 to about 25 mg / kg body weight of a compound of the invention and / or antibody and drug conjugate thereof.
[0662]
[0663] In general, the dosage of a compound of the invention and / or antibody and drug conjugate thereof administered to a patient is typically from about 0.01 mg / kg to about 20 mg / kg of the patient's body weight. In one aspect, the dosage administered to a patient is from about 0.01 mg / kg to about 10 mg / kg of the patient's body weight. In another aspect, the dosage administered to a patient is between about 0.1 mg / kg and about 10 mg / kg of the patient's body weight. In yet another aspect, the dosage administered to a patient is between about 0.1 mg / kg and about 5 mg / kg of the patient's body weight. In another additional aspect, the dosage administered to a patient is between about 0.1 mg / kg and about 3 mg / kg of the patient's body weight. In yet another aspect, the dosage administered is between about 1 mg / kg to about 3 mg / kg of the patient's body weight.
[0664]
[0665] A compound of the invention and / or antibody and drug conjugate thereof can be administered by any convenient route, for example, by infusion or bolus injection. Administration can be systemic or local. Several delivery systems are known, for example, encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer a compound of the invention and / or antibody and drug conjugate thereof. In certain embodiments, more than one compound of the invention and / or antibody and drug conjugate thereof are administered to a patient.
[0666]
[0667] In specific embodiments, it may be desirable to administer one or more compounds of the invention and / or antibody and drug conjugates thereof locally to the area that needs treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery; topical application, for example, together with a wound dressing after surgery; by injection; through a catheter; or by means of an implant, the implant being a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration may be by direct injection into the site (or anterior site) of a cancer, tumor or neoplastic or preneoplastic tissue. In another embodiment, administration can be by direct injection into the site (or previous site) of a manifestation of an autoimmune disease.
[0668]
[0669] In yet another embodiment, the compound of the invention and / or antibody and drug conjugate thereof can be delivered in a controlled release system, such as, but not limited to, a pump or different polymeric materials can be used. In yet another embodiment, a controlled release system may be located near the target of the compound of the invention and / or the antibody and drug conjugate thereof, for example, the liver, therefore requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, cited above, vol. 2, pp. 115-138 (1984)). Other controlled release systems described in the Langer review (Science 249: 1527-1533 (1990)) can be used.
[0670]
[0671] The term "carrier" refers to a diluent, adjuvant or excipient, with which a compound or an antibody and drug conjugate thereof is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of origin in petroleum, of animal, vegetable or synthetic origin. The transporters can be saline, and the like. In addition, auxiliary, stabilizing and other agents can be used. In one embodiment, when administered to a patient, the compound or conjugate and pharmaceutically acceptable carriers are sterile. Water is an illustrative transporter when the compound or conjugate is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, especially for injectable solutions. The present compositions, if desired, may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
[0672]
[0673] The present compositions may take the form of solutions, agglomerates, powders, continuous release formulations, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
[0674]
[0675] In one embodiment, the compound of the invention and / or the antibody and drug conjugate are formulated according to routine procedures as pharmaceutical compositions adapted for intravenous administration to animals, especially humans. Typically, transporters or vehicles for intravenous administration are sterile buffered aqueous solutions. If necessary, the compositions may also include a solubilizing agent. Compositions for intravenous administration may optionally comprise a local anesthetic such as lignocaine to decrease pain at the injection site. In general, the ingredients are supplied either separately or mixed together in unit dose form, for example, as a dry lyophilized powder or water-free concentrate in a tightly sealed container such as a blister or sachet that indicates the amount of principle active. When a compound of the invention and / or antibody and drug conjugate thereof are to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When a compound of the invention and / or antibody and drug conjugate thereof are administered by injection, a blister of sterile water or saline may be provided so that the components are mixed before administration.
[0676]
[0677] The composition may include several materials that modify the physiological form of a solid or liquid pharmaceutical form. For example, the composition may include materials that constitute a coating wrap around the active ingredients. The materials form the coating envelope are normally inert, and can be selected from, for example, sugar, lacquer, and other enteric coating agents. As an alternative, the active substances can be included in a gelatin capsule.
[0678]
[0679] Whether in solid or liquid form, the present compositions may include a pharmacological agent used in the treatment of cancer.
[0680]
[0681] Therapeutic uses of antibody compounds and conjugates with drugs thereof
[0682]
[0683] Another aspect of the invention relates to the compounds of the invention and to antibody conjugates with drugs thereof for use in the treatment of cancer.
[0684]
[0685] The compounds of the invention and / or antibody conjugates with drugs thereof are useful for inhibiting the multiplication of a tumor cell or a cancer cell, causing apoptosis in a tumor or cancer cell, or for treating cancer in a patient. . The compounds of the invention and / or antibody conjugates with drugs thereof can therefore be used in a variety of scenarios for the treatment of animal cancers. Such conjugates can be used to administer a compound of the invention to a tumor cell or cancer cell. Without intending to be bound by theory, in one embodiment, the conjugate antibody binds or is associated with a cancer cell associated antigen or a tumor cell associated antigen, and the conjugate can be captured (internalized) inside a cell tumor or a cancer cell through receptor-mediated endocytosis or other internalization mechanism. The antigen can bind to a tumor cell or cancer cell or it can be an extracellular matrix protein associated with the tumor cell or cancer cell. In certain embodiments, once inside the cell, one or more specific peptide sequences are enzymatically or hydrolytically cleaved by one or more proteases associated with tumor cells or associated with cancer cells, resulting in the release of a compound of the invention. from the conjugate. The compound released from the invention is then free to migrate in the cell and induces cytotoxic or cytostatic activities. The conjugate can also be cleaved by an intracellular protease to release a compound of the invention. In an alternative embodiment, the compound of the invention is cleaved from the conjugate outside the tumor cell or cancer cell, and the compound of the invention subsequently penetrates the cell.
[0686]
[0687] In certain embodiments, the conjugates provide a targeting of the drug against tumor or cancer specific to the conjugation, thereby reducing the general toxicity of the compounds of the invention.
[0688] In another embodiment, the antibody unit binds to the tumor cell or cancer cell.
[0689]
[0690] In another embodiment, the antibody unit binds to a tumor cell antigen or cancer cell that is on the surface of the tumor cell or the cancer cell.
[0691]
[0692] In another embodiment, the antibody unit binds to a tumor cell or cancer cell antigen that is in an extracellular matrix protein associated with the tumor cell or the cancer cell.
[0693]
[0694] The specificity of the antibody unit for a particular tumor cell or cancer cell may be important in determining those tumors or cancers that are treated most effectively.
[0695]
[0696] Particular types of cancers that can be treated with a compound of the invention and / or an antibody and drug conjugate thereof, include, but are not limited to, carcinomas of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, skin, stomach, and testicles; and blood-borne cancers that include, but are not limited to, leukemias and lymphomas.
[0697]
[0698] Multimodal therapy for cancer. Cancers, which include, but are not limited to, a tumor, metastasis, or other disease or disorder characterized by uncontrolled cell growth, can be treated or inhibited by the administration of the compound of the invention and / or antibody and drug conjugate of the same.
[0699]
[0700] In other embodiments, a compound of the invention and / or an antibody and drug conjugate thereof is provided for use in the treatment of cancer together with a chemotherapeutic agent. In one embodiment the chemotherapeutic agent is one with which the cancer has not been found to be refractory. In another embodiment, the chemotherapeutic agent is one with which cancer has been found to be refractory. A compound of the invention and / or a drug conjugate antibody thereof can be administered to a patient who has also undergone surgery as a treatment for cancer.
[0701]
[0702] In some embodiments, a compound of the invention and / or the antibody and drug conjugate thereof is provided for use in the treatment of cancer together with an additional treatment, such as radiotherapy. In a specific embodiment, the compound of the invention and / or the antibody and drug conjugate thereof are administered simultaneously with the chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiotherapy is administered before or after the administration of a compound of the invention and / or antibody and drug conjugates thereof.
[0703]
[0704] A chemotherapeutic agent can be administered during a series of sessions. Any one or a combination of the chemotherapeutic agents, such as the chemotherapeutic agents of the reference medical treatment, can be administered.
[0705]
[0706] Additionally, a compound of the invention and / or an antibody and drug conjugate thereof is provided for use in the treatment of cancer in which chemotherapy or radiotherapy has proven or may prove to be too toxic, for example, results in unacceptable or unbearable side effects, for the subject being treated as an alternative to chemotherapy or radiotherapy. The patient being treated can, optionally, be treated with another cancer treatment such as surgery, radiotherapy or chemotherapy, depending on which treatment is considered acceptable or bearable.
[0707]
[0708] The compounds of the invention and / or the antibody and drug conjugates thereof can also be used in an in vitro or ex vivo manner, such as in the treatment of certain cancers, which include, but are not limited to leukemia and lymphomas, Such treatment involves autologous cytoblast transplants. This may involve a multistage process in which the autologous hemocytoblasts of the animal are collected and cleaned of all cancer cells, then the remaining bone marrow cell population is eradicated by administering a high dose of a compound of the invention. and / or antibody and drug conjugate thereof with or without high dose radiotherapy attached and the cytoblast graft is subsequently infused into the animal. Palliative care is provided below while restoring bone marrow function and recovering the patient. The invention is described in more detail in the following examples and reference examples.
[0709]
[0710] Illustration of payloads and linker-payloads
[0711]
[0712]
[0713] Compound 2 is a commercially known compound, see PCT international application, 2005112919, 01 Dec 2005
[0714]
[0715]
[0716]
[0717]
[0718] Preparation of (S) -1- (chloromethyl) -5-hydroxy-1,2-dihydro-3H-benzo [e] indole-3-carboxylic acid ferc-butyl (3): A solution of (S) -5- (benzyloxy) -1- (chloromethyl) -1,2-dihydro-3H-benzo [e] indole-3-carboxylic acid ferc-butyl 4 g, 9 mmol) in THF (250 ml) Pd-C (0 , 7 g) at 40 ° C. An aqueous solution of HCOONH4 (9.5 ml, 25%) was then added portionwise and the reaction mixture was stirred at 40 ° C for 1 hour. The reaction mixture was filtered and the filtrate was concentrated to dryness. The resulting residue was dissolved in ethyl acetate (250 ml) and washed with H ² O (20 ml), dried with Na ² SO ⁴ , concentrated to dryness to give 3 as a gray solid (2 , 9 g, 90%).
[0719]
[0720] Preparation of (S) -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-ol (4): To a round bottom flask containing 3 (820 mg, 2.46 mmol ), 4M HCI in dioxane (36 ml, 140 mmol) was added. The reaction was allowed to stir at room temperature. The reaction was reduced and then placed under vacuum (belt pump) to provide 4 (684 mg, 100%) as a gray solid.
[0721]
[0722]
[0723]
[0724]
[0725] Preparation of (S) -5-acetoxy-1- (chloromethyl) -1,2-dihydro-3H-benzo [e] indole-3-carboxylic acid ferc-butyl (5): Acetyl chloride (0.1 ml, 1.4 mmol) to a solution of (S) -1- (chloromethyl) -5-hydroxy-1H-benzo [e] indole-3 (2H)-ferc-butyl carboxylate [3] (230 mg, 0.7 mmol) in CH ² O ² (6 ml) at 0 ° C, followed by pyridine (0.11 ml, 1.4 mmol). The mixture was stirred at 0 ° C for 2 min, and then at room temperature for 1 h. The mixture was concentrated, and the residue was treated with EtOAc and water, extracted with EtOAc. The combined organic phases were washed with water and brine, dried with MgSO4. The solvent was removed in vacuo to give 5 as a light yellow solid (235 mg, 91%). LC-MS (Protocol B): m / z 398.3 [MH].
[0726]
[0727] Preparation of (S) -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-yl acetate (6): To a round bottom flask containing (S) -5-acetoxy- 1- (chloromethyl) -1H-benzo [e] indole-3 (2H) -carb-butyl carboxylate (375 mg, 0.998 mM), 10 ml of 4M HCl in dioxane (40 mM) was added. The reaction was allowed to stir at room temperature and then the solvent was removed in vacuo to afford 6 (312 mg, 100%).
[0728]
[0729]
[0730] Preparation of (1S) -1- (doromethyl) -5-hydroxy-8-methyl-1,6-dihydropyrrolo [3,2-e] indole-3 (2H) tert-butyl carboxylate (8). To a stirring solution of 7 (see J. Am. Chem. Soc. 1987, 109, 6837-6838) (12.2 g, 28.6 mmol) in 200 ml of THF at 0 ° C, palladium was added to the 10% by weight on carbon (4 g) followed by a slow dropwise addition of 30 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 ° C for ~ 90 minutes. The reaction was diluted with ether followed by the addition of sodium sulfate. The mixture was filtered through a thin pad of celite, which was washed with ether twice. The organic layers were combined and then reduced before being placed under vacuum provided 7 (9.65 g, quantitative) as a light gray solid. LC-MS (Protocol B): m / z 337.2 [M + H] +, retention time = 1.81 minutes.
[0731]
[0732] Preparation of ( S) -8- ( chloromethyl) -1-methyl-3,6,7,8-tetrahydropyrrolo [3,2-e] indole-4-ol
[0733]
[0734]
[0735]
[0736]
[0737] Step 1: Synthesis of (1S) -5- (acetyloxy) -1- (chloromethyl) -8-methyl-1,6-dihydro - pyrrolo [3,2-e] indole-3 (2H) -carboxylate tert - butyl ( 188).
[0738]
[0739] To a stirring solution of 43 (1.99 g, 5.91 mmol) in 30 ml of dichloromethane at 0 ° C, acetyl chloride (0.462 ml, 6.50 mmol) was added followed immediately by pyridine (0.714 ml, 8.86 mmol). The reaction was allowed to stir at 0 ° C for ~ 10 minutes. The reaction was reduced on silica. Next, silica gel chromatography was performed (gradient: 0% -15% acetone in heptans). Suitable test tubes were concentrated and placed under high vacuum to yield 10 (2.13 g, 95%) as a light brown solid. LC-MS (Protocol B): m / z 401.1 [M + Na] +, retention time = 1.93 minutes.
[0740]
[0741] Stage 2: Synthesis of (8S) -8- (chloromethyl) -1-methyl-3,6,7,8-tetrahydropyrrolo [3,2-e] indole-4-yl acetate, hydrochloric acid salt (189) .
[0742]
[0743] To a round bottom flask containing 10 (606 mg, 1.60 mmol), 4M HCl in dioxane (24 mL, 96 mmol) was added. The reaction was allowed to stir at room temperature for 90 minutes. The reaction was reduced and then placed in vacuo yielding 11 (589 mg, quantitative) as a light green solid. CL-Em (Protocol B): m / z 279.1 [M + H] +, retention time = 0.72 minutes.
[0744]
[0745] General procedure A: To a stirring solution of the monoacid or diacid, in THF, dichloromethane, or a mixture of both at 0 ° C, oxalyl chloride (1-2.5 equiv.) Was added followed by a catalytic amount of DMF . The reaction was allowed to stir at 0 ° C for several minutes before allowing to warm to room temperature, and then stirred at room temperature for 30 minutes to several hours. Then, the reaction was concentrated in vacuo. In some cases the raw material was azeotropically distilled after one to several times with heptane, or other relevant solvent or solvents. The crude material was then dried under high vacuum before being used in the next step.
[0746]
[0747] General procedure B: To a solution under stirring of the amine (2-2.5 equiv.) In THF, dichloromethane, or a mixture of both at 0 ° C (or in some cases another relevant solvent or solvents), the chloride of acid, or acid dichloride was added followed by pyridine (3-6 equiv.), triethylamine (3-6 equiv.), or other relevant base (3-6 equiv.). The reaction was allowed to stir at 0 ° C for several seconds to several minutes before allowing to warm to room temperature, and then stirred at room temperature for 10 minutes to several hours. Then, the reaction was concentrated in vacuo. In some cases the raw material was azeotropically distilled after one to several times with heptane, or other relevant solvent or solvents. In most cases, the crude material was then purified by a described procedure such as silica gel chromatography or C18 inverted phase chromatography at intermediate pressure.
[0748]
[0749] Preparation of Reference Example (S) -furan-2,5-diylbis (((S) -1- (chloromethyl) -5-hydroxy-1H-benzo [e] indole-3 (2H) -yl) methanone) ( 13)
[0750]
[0751]
[0752] 4 was dissolved in DMF (0.75 ml), pyridine (13 pl) and a solution of 12 (8 mg) in DMF (0.2 ml, 0.168 mmol) were added, and the resulting solution was stirred at room temperature for one night. The mixture was diluted with DCM, and washed with water and brine, and dried with MgSO4. The crude product was purified by flash chromatography on silica gel (D ^c M / MOH = 0-10%) to give product 13 as a green solid (8 mg, 30%). LC-MS: m / z 587.4 [MH], retention time = 1.0 min. 1H NMR (400 MHz, DMSO-de), 5.10.50 (s), 8.14 (d), 7.95 (s), 7.88 (d), 7.55 (t), 7.50 (s), 7.40 (t), 4.78 (m), 4.58 (d), 4.25 (s), 4.02 (d), 3.92 (m).
[0753]
[0754] Preparation of Reference Example (S) - ((1R, 3S) -cyclohexane-1,3-diyl) bis (((S) -1- (chloromethyl) -5-hydroxy-1H-benzo [e] indole-3 (2H) -yl) methanone) (16)
[0755]
[0756]
[0757]
[0758]
[0759] Stage 1: Cis-cyclohexane-1,3-dicarboxylic acid (14, 10 mg, 0.058 mmol) was dissolved in THF (2 ml), oxalyl chloride (2 M in CH ² CU 0.09 ml, 0, 17 mmol) and Dm F (2 drops) at 0 ° C. The mixture was stirred at 0 ° C for 5 min, then at room temperature for 1 h. It was concentrated in vacuo to give the corresponding acid chloride 15 as a cream-colored solid, which was used in the next step without further purification.
[0760]
[0761] Step 2: The above compound 15 was dissolved in DMF (2 ml) at 0 ° C, (S) -1- (chloromethyl) -2,3-dihydro-1 H -benzo [e] indole-5-ol was added as HCl salt (4.25 mg, 0.093 mmol), followed by pyridine (0.029 ml, 0.36 mmol). The mixture was stirred at room temperature overnight. DMF was removed under reduced pressure, and the residue was purified by ISCO using MeOH / DCM (0-20%) to give the product 16 as a dark blue solid (8.5 mg, 31%). LC-MS: m / z 603.4 [MH], retention time = 1.03 min. 1H NMR (400 MHz, DMSO-de), 5 10.36 (s), 8.09 (d), 8.03 (s), 7.80 (t), 7.53 (t), 7.33 (t), 4.44 (m), 4.33 (d), 4.18 (s), 4.02 (m), 3.85 (m), 2.88 (m), 2.04 - 1.90 (m), 1.74 (q), 1.52 -1.45 (m).
[0762]
[0763] Preparation of Reference Example (S) -pyridin-2,6-diylbis (((S) -1- (chloromethyl) -5-hydroxy-1H-benzo [e] indole-3 (2H) -yl) methanone) ( 18)
[0764]
[0765]
[0766]
[0767]
[0768] (S) -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-ol (4) (13.5 mg, HCl salt, 0.05 mmol) was dissolved in DMF (2 ml), and pyridine (8 mg, 0.10 mmol) was added, followed by 2,6-pyridinedicarbonyl dichloride (8.5 mg, 0.025 mmol). The mixture was stirred at room temperature for 2 h. The crude product was purified by ISCO using MeOH / DCM (0-10%) to give the product as a green solid, which was washed with MeOH to give product 18 as a gray solid ( 10 mg, 67%). LC-MS: m / z 598.1 [MH], retention time = 1.0 min. 1H NMR (400 MHz, DMSO-de), 5 10.51 (s), 8.29 (t), 8.13 (d), 8.02 (s), 7.82 (d), 7.52 (t), 7.38 (t), 4.63 (s), 4.19-4.10 (m), 3.96 (m), 3.84 (m).
[0769]
[0770] Preparation of Reference Example (S) -1,3-phenylenebis (((S) -1- (chloromethyl) -5-hydroxy-1H-benzo [e] indole-3 (2H) -yl) methanone) [20]
[0771]
[0772]
[0773] (S) -1- (doromethyl) -2,3-dihydro-1H-benzo [e] indole-5-ol (4) (27 mg, HCl salt, 0.1 mmol) was dissolved in DMF (2 ml) , and was added (0.024 ml, 0.29 mmol), followed by isophthalic acid chloride (19.10 mg, 0.05 mmol). The mixture was stirred at room temperature overnight. The solvent was removed, and the residue was purified by ISCO using MeOH / DCM (0-10%) to give the product as a gray solid (20 mg, 68%). ^c L-MS (Protocol B): m / z 597.2 [MH], retention time = 0.99 min. 1H NMR (400 MHz, DMSO-de), 5 10.47 (s), 8.13 (d), 7.96 (s), 7.84 (d), 7.72 (t), 7.52 (t), 7.37 (t), 4.44 (s), 4.08 (s), 3.97 (s), 3.86 (s).
[0774]
[0775] Preparation of Reference Example (S) -3,3'-thiobis (1 - ((S) -1- (chloromethyl) -5-hydroxy-1H-benzo [e] indole-3 (2H) -yl) propan- 1-one) (23)
[0776]
[0777]
[0778]
[0779]
[0780] Stage 1: 3,3'-thiodipropanoic acid (21.8 mg, 0.04 mmol) was dissolved in THF (2 ml), oxalyl chloride (2 M in CH ² Cl ² , 0.4 ml, 0 was added , 2 mmol) and DMF (2 drops) at 0 ° C. The mixture was stirred at 0 ° C for 5 min, then at room temperature for 1 h. It was concentrated in vacuo to give the corresponding acid chloride 22 that was used in the next step without further purification.
[0781] Step 2: The above compound 22 was dissolved in DMF (2 ml) at 0 ° C, (S) -1- (chloromethyl) -2,3-dihydro-1 H -benzo [e] indole-5-ol salt was added HCl (6) (25 mg, 0.09 mmol), followed by pyridine (0.022 ml, 0.27 mmol). The mixture was stirred at room temperature overnight. DMF was removed under reduced pressure, and the residue was purified by ISCO using MeOH / DCM (0-10%) to give product 23 as a cream-colored solid (15 mg, 50%). LC-MS (Protocol B): m / z 609.1 [MH], retention time = 1.0 min. 1H NMR (400 MHz, DMSOC), 510.36 (s), 8.09 (d), 7.99 (s), 7.79 (d), 7.50 (t), 7.33 (t) , 4.37 (m), 4.19 (m), 3.99 (d), 3.82 (m), 2.90 - 2.82 (m).
[0782]
[0783] Preparation of Reference Example (S) -pyridin-3,5-diylbis (((S) -1- (chloromethyl) -5-hydroxy-1H-benzo [e] indole-3 (2H) -yl) methanone) ( 26)
[0784]
[0785]
[0786]
[0787]
[0788] Stage 1; To the pyridine-3,5-dicarboxylic acid (24.7 mg, 0.04 mmol) was added 2 ml of DCM, followed by 2M oxalyl chloride (0.2 ml, 0.4 mmol) and DMF (2 drops ). The clear solution was stirred at room temperature for 2 h and concentrated to give the corresponding acid chloride 25 as a yellow solid.
[0789]
[0790] Stage 2; The above solid 25 was dissolved in DMF (0.2 ml), and the solution was added to a solution of (S) -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5- ol HCl salt (4) (25 mg, 0.09 mmol) in DMF (1 ml), followed by pyridine (0.02 ml, 0.25 mmol). The mixture was stirred at room temperature overnight. The solvent was removed in vacuo, and the residue was purified using ISCO (MeOH / DCM = 0-10%) to give the product 26 as a gray solid (20 mg, 80%). LC-MS (Protocol B): m / z 598.1 [MH], retention time = 0.95 min. 1H NMR (400 MHz, DMSO-de), 5 10.55 (s), 9.00 (s), 8.2 (s), 7.97 (s), 7.84 (d), 7.53 (t), 7.36 (t), 4.50 (s), 4.10 (s), 3.98 (s), 3.86 (s).
[0791]
[0792] Preparation of Reference Example (S) -thiophene-2,5-diylbis (((S) -1- (chloromethyl) -5-hydroxy-1H-benzo [e] indole-3 (2H) -yl) methanone) ( 29) and (S) -5- (1- (chloromethyl) -5-hydroxy-2,3-dihydro-1 H -benzo [e] indole-3-carbonyl) thiophene-2-carboxylic acid (30)
[0793]
[0794] (S) -1- (doromethyl) -2,3-dihydro-1H-benzo [e] indole-5-ol [4] (102 mg, HCl salt, 0.38 mmol) was dissolved in DMA (2 ml) , and pyridine (0.061 ml, 0.76 mmol) was added, followed by thiophene-2,5-dicarbonyl dichloride (27, 40 mg, 0.19 mmol). The mixture was stirred at room temperature for 2 h. The crude product was purified by Gilson HPLC (0.02% TFA) to give two products: (S) -thiophene-2,5-diylbis (((S) -1- (chloromethyl) -5-hydroxy-1H -benzo [e] indole-3 (2H) -yl) methanone) (29) in the form of a yellow solid (60 mg, 52%). LC-MS (Protocol B): m / z 603.0 [MH], retention time = 1.99 min. 1H NMR (400 MHz, DMSO-d ^e ), S 10.48 (s), 8.14 (d), 7.86 (m), 7.55 (t), 7.40 (t), 4, 78 (t), 4.44 (d), 4.23 (s), 4.03 (d), 3.91 (m).
[0795] (S) -5- (1- (chloromethyl) -5-hydroxy-2,3-dihydro-1H-benzo [e] indole-3-carbonyl) thiophene-2-carboxylic acid (30) as a solid of green color (23 mg, 31%). LC-MS (Protocol B): m / z 388.1 [MH], retention time = 0.82 min. 1H NMR (400 MHz, MeOD-d ⁴ ), S 8.23 (d), 7.82 (m), 7.71 (s), 7.55 (t), 7.40 (t), 4, 64 (m), 4.53 (d), 4.15 (t), 4.01 (dd), 3.74 (m). Preparation of Reference Example (S) - (1H-pyrrole-2,5-diyl) bis (((S) -1- (chloromethyl) -5-hydroxy-1h-benzo [e] indole-3 (2H) - il) methanone) (32)
[0796]
[0797]
[0798]
[0799] DIPEA (33 mg, 0.25 mmol) was added to a solution of 1H-pyrrole-2,5-dicarboxylic acid (31, 10 mg, 0.064 mmol) in DMF (1.5 ml), followed by (s) - 1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-ol [4] (38 mg, sa1HCl, 0.14 mmol) and COMU (82 mg, 0.19 mmol), and The mixture was stirred at room temperature overnight. The crude product was purified by Gilson HPLC (ACN / water, 0.02% TFA) to give product 32 as a yellow solid (5 mg, 10%). LC-MS (Protocol B): m / z 586.3 [MH], retention time = 2.04 min. 1H NMR (400 MHz, DMSO-d6), S 11.66 (s), 10.44 (s), 8.13 (d), 7.92 (s), 7.86 (d), 7.55 (t), 7.38 (t), 5.76 (s), 4.71 (t), 4.44 (d), 4.22 (s), 4.03 (d), 3.88 ( m). Preparation of Reference Example (5) -thiophene-2,4-diylbis (((5) -1- (chloromethyl) -5-hydroxy-1 H -benzo [e] indole-3 (2 H) -yl) methanone) ( 35)
[0800]
[0801]
[0802]
[0803] Step 1: 2,4-thiophenedicarboxylic acid (33, 100 mg, 0.58 mmol) was dissolved in THF (5 ml), cooled to 0 ° C with an ice bath. Oxalyl chloride (0.75 ml, 2 M in CH2Cl2, 1.5 mmol) was added, followed by 2 drops of DMF. The resulting mixture was allowed to warm to room temperature, and stirred for 1 h. Some white precipitate can be observed during this period. The mixture was concentrated in vacuo to give thiophene-2,4-dicarbonyl dichloride (34) as a cream-colored solid (122 mg, 100%).
[0804] Stage 2: (S) -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-ol [4] (81 mg, sa1HCl, 0.3 mmol) was dissolved in THF (3 ml), and Et ³ N (0.125 ml, 0.9 mmol) was added at 0C, followed by a solution of thiophene-2,4-dicarbonyl dichloride (24, 31.4 mg, 0.15 mmol) in CH2Cl2 (1 ml). The mixture was stirred at 0 ° C for 5 min, and then stirred at room temperature for 2 h. The reaction mixture was reduced, and the residue was treated with MeOH, and the resulting yellow solid was collected by filtration to give the crude product. The crude product was purified by Gilson HPLC (ACN / water, 0.02 TFA) to give the product as a yellow solid (40 mg, 44%). LC-MS (Protocol B): m / z 603.3 [MH], retention time = 1.96 min. 1H NMR (400 MHz, DMSO-de), S 10.46 (d), 8.41 (s), 8.13 (d), 8.05 (s), 7.87 (t), 7.54 (t), 7.39 (m), 4.81 (t), 4.61 (s), 4.46 (d), 4.21 (m), 4.18 (m), 4.00 ( m), 3.98-3.86 (m).
[0805] Preparation of Reference Example (S) - (1-methyl-1H-pyrrole-2,5-diyl) bis (((S) -1- (chloromethyl) -5-hydroxy-1H-benzo [e] indole-3 (2H) -yl) methanone) (38)
[0806]
[0807]
[0808] Step 1: 1-methyl-1H-pyrrole-2,5-dicarboxylic acid (36.20 mg, 0.12 mmol) was dissolved in THF (2 ml), oxalyl chloride (2 M in CH ² CU 0) was added , 18 ml, 0.35 mmol) and DMF (2 drops) at 0 ° C. The mixture was stirred at 0 ° C for 5 min, then at room temperature for 1 h. It was concentrated in vacuo to give the corresponding acid chloride 37 as a cream-colored solid, which was used in the next step without further purification.
[0809]
[0810] Step 2: The above compound 37 was dissolved in THF (2 ml) at 0 ° C, (S) -1- (chloromethyl) -2,3-dihydro-1 H -benzo [e] indole-5-ol salt was added HCl [4] (65 mg, 0.24 mmol), followed by Et ³ N (0.1 ml, 0.71 mmol). The mixture was stirred at 0 ° C for 5 min, then at room temperature for 1 h. The mixture was concentrated in vacuo, and the residue was purified by Gilson HPLC (0.02% TFA) to give product 38 as a cream-colored solid (31 mg, 44%). LC-MS: m / z 600.5 [MH], retention time = 1.04 min. 1H NMR (400 MHz, DMSO-de), 510.44 (s), 8.13 (d), 7.84 (d), 7.75 (s), 7.53 (t), 7.38 ( t), 6.78 (s), 4.60 (t), 4.30 (d), 4.08 (s), 4.02 (d), 3.9 (s), 3.87 (d ).
[0811]
[0812] Preparation of Reference Example 3-Amino-1,5-bis - ((S) -1-chloromethyl-5-hydroxy-1,2-dihydro-enzo [e] indole-3-yl) -pentane-1,5 -diona (40).
[0813]
[0814]
[0815]
[0816]
[0817] Stage 1: To a round bottom flask purged with N ² , containing 3- (9H-Fluoren-9-ylmethoxycarbonylamino) -pentanedioic acid (918 mg, 2.48 mmol) in 20 ml of anhydrous dichloromethane was added oxalyl chloride (5.22 mmol, 0.469 ml). To this solution was added 1 drop of N, N-dimethylformamide. The reaction mixture was stirred for 3 hours and concentrated in vacuo to give a crude residue. The residue was taken up in dichloromethane (10 ml) and added dropwise to a round bottom flask containing (2) (1610 mg, 4.97 mmol) in 25 ml of dichloromethane and triethylamine (2.08 ml). The crude reaction mixture was concentrated in vacuo and recaptured in 25 ml of dichloromethane and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3 x), water (3 x), and brine (2 x). The organic layer was dried with sodium sulfate, filtered and the filtrate was concentrated to give a crude solid. Next, silica gel chromatography was performed (gradient: 0% -100% ethyl acetate in heptanes) yielding (39) (2.103 g, 86%) as a light yellow solid. LC-MS (Protocol B): m / z 982 [M + H ⁺ ], retention time = 2.81 minutes.
[0818]
[0819] Step 2: A stirring solution of 39, 9H-fluoren-9-ylmethyl ester of {3 - ((S) -5-benzyloxy-1-chloromethyl-1,2-dihydro-benzo [e] indole-3- il) -1- [2 - ((S) -5-benzyloxy-1-chloromethyl-1,2-dihydro-benzo [e] indole-3-yl) -2-oxo-ethyl] -3-oxopropyl} - carbamic, (92 mg, 0.104 mmol) in 10 ml of tetrahydrofuran in a nitrogen atmosphere was cooled to 0 C using an ice bath. Next, 10% palladium by weight on activated carbon (16 mg, 0.15 mmol) was added followed by a slow dropwise addition of 1 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 C for 5 hours. Then, the reaction was filtered through a layer of celite and the filtrate was then concentrated in vacuo. The crude residue was taken up in dichloromethane and washed with water. The dichloromethane layer was concentrated and 2 ml of 1M HCI (aq) was added and concentrated. The residue was taken up in ethyl acetate and the solids were filtered to give 40 as a white solid (52 mg, 51%). LC-MS (Protocol B): m / z 578 [M + H ⁺ ], retention time = 1.42 minutes.
[0820]
[0821] Preparation of the Reference Example 3- (3-Amino-phenyl) -1,5-bis - ((S) -1-chloromethyl-5-hydroxy-1,2-dihydro-benzo [e] indole 3-yl) -pentane-1,5 -diona (44)
[0822]
[0823]
[0824]
[0825]
[0826] Step 1: To a round bottom flask purged with N ² , containing 3- (3-Nitro-phenyl) -pentanedioic acid (3,330 mg, 1.30 mmol) in 15 ml of anhydrous dichloromethane was added oxalyl chloride (2 , 6 mmol, 0.24 ml). To this solution was added 1 drop of N, N-dimethylformamide. The reaction mixture was stirred for 3 hours and concentrated in vacuo to provide 42 as a white solid (378 mg, 1.30 mmol, quantitative).
[0827]
[0828] Step 2: To a round bottom flask containing 2 (124 mg, 0.344 mmol) in 15 ml of dichloromethane was added 3- (3-Nitro-phenyl) -pentanedioyl dichloride (42) (42 mg, 0.172 mmol). Triethylamine (0.08 ml) was added and the system was stirred for 1 hour at room temperature. The crude reaction mixture was concentrated in vacuo and recaptured in 25 ml of dichloromethane and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3 x), water (3 x), and brine (2 x). The organic layer was dried with sodium sulfate, filtered and the filtrate was concentrated to give a crude solid. The crude solid was collected in 10% MeOH in EtOAc and the white solid was filtered to give the desired product 43 (120 mg, 0.172 mmol, 80%). LC-MS (Protocol B): m / z 864 [M + H +], retention time = 2.75 minutes.
[0829]
[0830] Step 3: A stirring solution of 43 (85 mg, 0.098 mmol) in 10 ml of tetrahydrofuran in a nitrogen atmosphere was cooled to 0 C using an ice bath. Then 10% palladium by weight on activated carbon (16 mg, 0.15 mmol) was added followed by a slow dropwise addition of 2 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 C for 5 hours. Then, the reaction was filtered through a layer of celite and the filtrate was then concentrated in vacuo. The crude residue was taken up in dichloromethane and washed with water. The dichloromethane layer was concentrated and 2 ml of 1M HCI (aq) was added and concentrated. The residue was taken up in ethyl acetate and the solids filtered to provide (44) as a white solid. (35 mg, 52%). LC-MS: m / z 654 [M + H +], retention time = 1.93 minutes.
[0831]
[0832] Preparation of Reference Example 3- (4-Amino-phenyl) -1,5-bis - ((S) -1-chloromethyl-5-hydroxy-1,2-dihydro-benzo [e] indole-3-yl) -pentane-1,5-diona 48
[0833]
[0834]
[0835]
[0836]
[0837] Step 1: To a round bottom flask purged with N ² , containing 3- (4-Nitro-phenyl) -pentanedioic acid (45, 110 mg, 0.434 mmol) in 5 ml of anhydrous DCM was added oxalyl chloride (0.911 mmol, 0.082 ml). To this solution was added 1 drop of N, N-dimethylformamide. The reaction mixture was stirred for 3 hours and concentrated in vacuo. to provide (46) in the form of a white solid (125 mg, 0.434 mmol, quantitative). LCMS, captured in methanol: m / z 282.0 [M + H +, for the bismetanolysis product]. Retention time = 1.38 minutes. (7) (Commercial and known in the literature: Tetrahedron, 63 (39), 9741-9745; 2007
[0838]
[0839] Step 2: To a round bottom flask containing 2 (111 mg, 0.344 mmol) in 15 ml of dichloromethane was added 3- (4-Nitro-phenyl) -pentanedioyl dichloride (46) (50 mg, 0.172 mmol). Triethylamine (0.144 ml) was added and the system was stirred for 1 hour at room temperature. The crude reaction mixture was concentrated in vacuo and recaptured in 25 ml of dichloromethane and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3 x), water (3 x), and brine (2 x). The organic layer was dried with sodium sulfate, filtered and the filtrate was concentrated to give a crude solid. The crude solid was collected in 10% MeOH in EtOAc and the white solid was filtered to give the desired product (47) (101 mg, 0.115 mmol, 68%). LC-MS: m / z 864 [M + H +], retention time = 2.72 minutes.
[0840]
[0841] Stage 3: (10). A stirring solution of (47), 3- (4-nitrophenyl) -1,5-bis - ((S) -1-chloromethyl-5-hydroxy-1,2-dihydrobenzo [e] indole-3-yl) -pentane-1,5-dione (90 mg, 0.1 mmol) in 10 ml of tetrahydrofuran in a nitrogen atmosphere was cooled to 0 C using an ice bath. Next, 10% palladium by weight on activated carbon (17 mg, 0.16 mmol) was added followed by a slow dropwise addition of 1 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 C for 5 hours. Then, the reaction was filtered through a layer of celite and the filtrate was then concentrated in vacuo. The crude residue was taken up in dichloromethane and washed with water. The dichloromethane layer was concentrated and 2 ml of 1M HCI (aq) was added and concentrated. The residue was taken up in ethyl acetate and the solids filtered to provide 48 as a white solid. (44 mg, 61%). LC-MS: m / z 654 [M + H +], retention time = 1.73 minutes.
[0842]
[0843] Preparation of Reference Example (S) -3- {2- [2 - ((S) -5-acetoxy-1-chloromethyl-1,2-dihydro-benzo [e] indole-3-yl) -2-oxoethylamino ] -acetyl} -1-chloromethyl-2,3-dihydro-1H-benzo [e] indole-5-l acetic acid ester (53).
[0844]
[0845]
[0846]
[0847]
[0848] Stage 1: To a round bottom flask purged with N ² , containing 3- [carboxymethyl- (9H-fluoren-9-ylmethoxycarbonyl) -amino] -acetic acid (49, 300 mg, 0.844 mmol) in 5 ml of DCM anhydrous oxalyl chloride (1.94 mmol, 0.175 ml) was added. To this solution was added 1 drop of N, N-dimethylformamide. The reaction mixture was stirred for 3 hours and concentrated in vacuo to provide (50) as a white solid (330 mg, 0.844 mmol, quantitative). LCMS, captured in methanol: m / z 384.0 [M + H +, for the bismetanolysis product]. Retention time = 1.91 minutes.
[0849]
[0850] Step 2: To a round bottom flask containing 2 (76 mg, 0.21 mmol) in 5 ml of dichloromethane, 50 (41 mg, 0.105 mmol) was added. Triethylamine (0.088 ml) was added and the system was stirred for 1 hour at room temperature. The crude reaction mixture was concentrated in vacuo and recaptured in 25 ml of dichloromethane and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3 x), water (3 x), and brine (2 x). The organic layer was dried with sodium sulfate, filtered and the filtrate was concentrated to give a crude solid. Next, silica gel chromatography was performed (gradient: 0% -75% ethyl acetate in heptanes) yielding (51) (91 mg, 90%) as a light white solid. LC-MS: m / z 966 [M + H +], retention time = 2.91 minutes.
[0851]
[0852] Step 3: A stirring solution of 51 (40 mg, 0.041 mmol0) in 10 ml of tetrahydrofuran in a nitrogen atmosphere was cooled to 0 C using an ice bath. Next, 10% palladium by weight on activated carbon (10 mg, 0.09 mmol) was added followed by a slow dropwise addition of 1 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 C for 5 hours. Then, the reaction was filtered through a layer of celite and the filtrate was then concentrated in vacuo. The crude residue was taken up in dichloromethane and acetyl chloride (1 ml) was added and the reaction was then concentrated in vacuo. The residue was recaptured in 15 ml of dichloromethane and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3 x), water (3 x), and brine (2 x). The organic layer was dried with sodium sulfate, filtered and the filtrate was concentrated to give a solid. raw. Next, silica gel chromatography was performed (gradient: 0% -100% ethyl acetate in heptane) yielding (52) (27 mg, 76%) as a white solid. LC-MS: m / z 870 [M + H +], retention time = 2.51 minutes.
[0853]
[0854] Step 4: To a round bottom flask equipped with a stir bar containing 52 (25 mg, 0.29 mmol), 5 ml of dichloromethane and 5 ml of diethyl amine were added. The solution was stirred for 3 hours. The reaction mixture was concentrated in vacuo and captured in 50% dichloromethane in heptane and concentrated again in vacuo. This was repeated 3 times. The crude solid was collected in 50% tetrahydrofuran and 1 M HCI (aq.). The white solid was collected in ether and filtered to provide (15) as a white solid (14 mg, 70%). LC-MS: m / z 648 [M + H +], retention time = 1.78 minutes.
[0855]
[0856] Preparation of Reference Example 3- (4-Amino-phenyl) -N, N-bis- [2 - ((S) -1-chloromethyl-5-hydroxy-1,2-dihydrobenzo [e] indole-3-yl ) -2-oxoethyl] -propionamide (56)
[0857]
[0858]
[0859]
[0860]
[0861] Step 1: To a round bottom flask provided with a stir bar containing 51 (300 mg, 0.310 mmol), 5 ml of dichloromethane and 5 ml of diethyl amine were added. The solution was stirred for 3 hours. The reaction mixture was concentrated in vacuo and captured in 50% dichloromethane in heptane and concentrated again in vacuo. This was repeated 3 times to provide (54) as a white solid. (216 mg, 93%). LC-MS: m / z 744 [M + H +], retention time = 2.26 minutes.
[0862]
[0863] Stage 2: To a round bottom flask purged with N ² , containing 54 (100 mg, 0.134 mmol) in 5 ml of anhydrous dichloromethane was added 3- [4- (9H-Fluoren-9-ylmethoxycarbonylamino) -phenyl] -propionic (52 mg, 0.134 mmol). To this solution was added (dimethylamino) -W hexafluorophosphate, W-dimethyl (3 H- [1,2,3] triazolo [4,5-b] pyridin-3-yloxy) methaniminium (52 mg, 0.134 mmol) and triethylamine (0.05 ml). The reaction mixture was stirred for 3 hours and concentrated in vacuo to give a crude residue. The residue was recaptured in 25 ml of dichloromethane and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3 x), water (3 x), and brine (2 x). The organic layer was dried with sodium sulfate, filtered and the filtrate was concentrated to give a crude solid. Next, silica gel chromatography was performed (gradient: 0% -100% ethyl acetate in heptanes) yielding (55) (130 mg, 87%) as a light yellow solid. LC-MS: m / z 1113 [M + H +], retention time = 2,771 minutes.
[0864]
[0865] Step 3: A stirring solution of 55 (115 mg, 0.103 mmol) in 10 ml of tetrahydrofuran in a nitrogen atmosphere was cooled to 0 C using an ice bath. Next, 10% palladium by weight on activated carbon (10 mg, 0.1 mmol) was added followed by a slow dropwise addition of 1 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 C for 5 hours. Then, the reaction was filtered through a layer of celite and the filtrate was then concentrated in vacuo. The crude residue was taken up in dichloromethane and washed with water. The dichloromethane layer was concentrated and 2 ml of 1M HCI (aq) was added and concentrated. The residue was taken up in ethyl acetate and the solids filtered to provide (56) as a white solid. (26 mg, 34%). LC-MS: m / z 711 [M + H +], retention time = 1.6 minutes.
[0866]
[0867] Preparation of Reference Example 9H-fluoren-9-ylmethyl ester of [(S) -1 - ((S) -1-Chloromethyl-5-hydroxy-1,2-dihydrobenzo [e] indole-3-carbonyl) - 4 - ((S) -1-chloromethyl-5-hydroxy-1,2-dihydro-benzo [e] indole-3-yl) -4-oxo-butyl] -carbamic 60
[0868]
[0869]
[0870] Stage 1: To a round bottom flask purged with N ² , containing (S) -2- (9H-Fluoren-9-ylmethoxycarbonylamino) -pentanedioic acid 57 (400 mg, 1.08 mmol) in 15 ml of anhydrous dichloromethane oxalyl chloride (2.27 mmol, 0.205 ml) was added. To this solution was added 1 drop of N, N-dimethylformamide. The reaction mixture was stirred for 3 hours and concentrated in vacuo to give a crude residue 58. The residue was taken up in dichloromethane (10 ml) and added dropwise to a round bottom flask containing 2 (700 mg , 2.17 mmol) in 10 ml of dichloromethane and triethylamine (0.905 ml). The crude reaction mixture was concentrated in vacuo and recaptured in 25 ml of dichloromethane and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3 x), water (3 x), and brine (2 x). The organic layer was dried with sodium sulfate, filtered and the filtrate was concentrated to give a crude solid. Next, silica gel chromatography was performed (gradient: 0% -100% ethyl acetate in heptanes) yielding (59), (260 mg, 24%) as a white solid. LC-MS: m / z 980 [M + H +], retention time = 2.84 minutes.
[0871]
[0872] Step 2: A stirring solution of (59), (250 mg, 0.255 mmol) in 10 ml of tetrahydrofuran in a nitrogen atmosphere was cooled to 0 C using an ice bath. Next, 10% palladium by weight on activated carbon (64 mg, 12.8 mmol) was added followed by a slow dropwise addition of 2.1 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 C. for 30 min. Then, the reaction was filtered through a layer of celite and the filtrate was then concentrated in vacuo. The crude residue was taken up in dichloromethane and washed with water. Next, silica gel chromatography was performed (gradient: 0% -100% ethyl acetate in heptanes) yielding (60) (121 mg, 59%) as a light yellow solid. LC-MS: m / z 800 [M + H +], retention time = 2.25 minutes.
[0873]
[0874] Preparation of Reference Example [3 - ((S) -1-Chloromethyl-5-hydroxy-1,2-dihydro-benzo [e] indole-3-yl) -3-oxo-propyl] -amide of acid (S ) -1-Chloromethyl-5-hydroxy-1,2-dihydro-benzo [e] indole-3-carboxylic (31). A stirring solution of (65)
[0875]
[0876]
[0877]
[0878]
[0879] Step 1: To a round bottom flask containing (2) (200 mg, 0.555 mmol) in dichloromethane (10 ml), 3-isocyanate-propionic acid methyl ester 61 (79 mg, 0.555 mmol) was added dropwise and triethylamine (0.5 ml). The reaction was stirred for 3 hours. The crude reaction mixture was concentrated in vacuo and recaptured in 25 ml of dichloromethane and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3 x), water (3 x), and brine (2 x). The organic layer was dried with sodium sulfate, filtered and the filtrate was concentrated to give a crude solid. Next, silica gel chromatography was performed (gradient: 0% -100% ethyl acetate in heptanes) yielding (62) (0.231 mg, 89%) as a light yellow solid. LC-MS: m / z 467 [M + H +], retention time = 2.11 minutes. NMR yes
[0880]
[0881] Step 2: To a round bottom flask equipped with a stir bar containing 62 (230 mg, 0.493 mmol), 5 ml of 1M HCl (aq) in 5 ml of tetrahydrofuran was added. The solution was stirred for 3 hours at 70 ° C. The reaction mixture was concentrated in vacuo and captured in 50% dichloromethane in heptane and concentrated in vacuo. This was repeated 3 times to provide (63) (180 mg, 83%) as a white solid after concentrating. LC-MS: m / z 439 [M + H +], retention time = 1.83 minutes.
[0882]
[0883] Step 3: To a round bottom flask purged with N2, containing (63) (110 mg, 0.250 mmol) in 5 ml of anhydrous DCM was added oxalyl chloride (0.250 mmol, 0.02 ml). To this solution was added 1 drop of N, N-dimethylformamide. The reaction mixture was stirred for 3 hours and concentrated in vacuo to give a crude residue. The residue was taken up in dichloromethane (10 ml) and added dropwise to a round bottom flask containing 2 (90 mg, 0.250 mmol) in 10 ml of dichloromethane and triethylamine (0.5 ml). The crude reaction mixture was concentrated in vacuo and recaptured in 15 ml of dichloromethane and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3 x), water (3 x), and brine (2 x). The organic layer was dried with sodium sulfate, filtered and the filtrate was concentrated to give a crude solid. Next, silica gel chromatography was performed (gradient: 0% -100% ethyl acetate in heptanes) yielding (64) (80 g, 43%) as a light yellow solid. LC-MS: m / z 744 [M + H +], retention time = 2.60 minutes.
[0884]
[0885] Step 4: 64 (75 mg, 0.100 mmol) in 10 ml of tetrahydrofuran in a nitrogen atmosphere was cooled to 0 C using an ice bath. Next, 10% palladium by weight on activated carbon (25 mg, 0.24 mmol) was added followed by a slow dropwise addition of 1 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 C for 5 hours. Then, the reaction was filtered through a layer of celite and the filtrate was then concentrated in vacuo. The crude residue was taken up in dichloromethane and washed with water. The dichloromethane layer was concentrated and 2 ml of 1M HCI (aq) was added and concentrated. The residue was taken up in ethyl acetate and the solids filtered to provide (65 as a white solid. (15 mg, 26%). LC-MS: m / z 564 [M + H +], time of retention = 1.88 minutes.
[0886]
[0887] Preparation of the Reference Example (1S, 1'S) -3,3 '- (1H-pyrrol-2,5-dicarbonyl) bis (1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole diacetate -5,3-diyl) [68]
[0888]
[0889]
[0890]
[0891]
[0892] Step 1: 1H-pyrrole-2,5-dicarboxylic acid (32.50 mg, 0.3 mmol) was dissolved in THF (5 ml) at 0 ° C, oxalyl chloride (0.4 ml, 2 M was added in CH2Cl2, 0.8 mmol), followed by 2 drops of DMF. The mixture was stirred at 0 ° C for 5 min, then at room temperature for 2 h. It was concentrated in vacuo to give 1H-pyrrole-2,5-dicarbonyl dichloride (33) as a yellow solid, which was used in the next step without further purification.
[0893] Step 2: It was dissolved in THF (12 ml) at 0 ° C, 1 H -pyrrole-2,5-dicarbonyl dichloride (33, from step 2) was added, followed by Et ³ N (0.28 ml). The mixture was stirred at 0 ° C for 5 min, at room temperature for 3 h. The mixture was concentrated in vacuo, and the residue was treated with MeOH to give a gray solid. The solid was collected by filtration to give the crude product as a gray solid. The crude product was purified by Gilson HPLC (ACN / water, 0.02% TFA) to give the pure diacetate product of (1S, 1'S) -3.3 '- (1H-pyrrole-2,5-dicarbonyl ) bis (1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5,3-diyl) as an off-white solid (34, 60 mg, 30%). LC-MS: m / z 670.4 [MH], retention time = 2.20 min. 1H NMR (400 MHz, DMSO-de), 5 11.77 (s), 8.17 (s), 8.06 (d), 7.92 (d), 7.65 (t), 7.52 (t), 4.80 (t), 4.5 (d), 4.41 (s), 4.10 (d), 4.02 (m), 2.10 (s).
[0894]
[0895] Preparation of the Reference Example (1S, 1'5) -3,3 '- (thiazol-2,5-dicarbonyl) bis (1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole diacetate -5,3-diyl) [71]
[0896]
[0897]
[0898] Step 1: Dimethyl thiazol-2,5-dicarboxylate (35, 348 mg, 1.5 mmol) was dissolved in THF (10 mL), a solution of LiOHH2O (383 mg, 9.0 mmol) in water ( 5 ml) at 0 ° C. The mixture was stirred at 0 ° C for 30 min, then at room temperature for 4 h. It was concentrated in vacuo to remove THF, and the residue was acidified by the addition of a 1M aqueous HCI solution at pH approximately 4-5. The resulting solid was collected by filtration to give thiazol-2,5-dicarboxylic acid as a white solid (63 mg, 24%). The thiazol-2,5-dicarboxylic acid (20 mg, 0.12 mmol) was dissolved in THF (2 ml), oxalyl chloride (0.18 ml, 2M in DCM) was added at 0 ° C, followed by 2 drops of d Mf. The mixture was stirred at 0 ° C for 5 min, then at room temperature for 1 h. It was concentrated in vacuo to give the corresponding acid chloride 70 as a white solid.
[0899]
[0900] Step 2: The yellow solid 5 was suspended in THF (3 ml), the acid chloride of step 2 was added, followed by EfeN (0.05 ml, 0.4 mmol) at 0 ° C. The mixture was stirred at 0 ° C for 5 min, and then at room temperature for 1 h. The reaction mixture was concentrated in vacuo, and the residue was purified by Gilson HPLC to give the desired compound 71 as a yellow solid (3.6 mg, 3.9%). LC-MS: m / z 688.5 [MH], retention time = 2.27 min. 1H NMR (400 MHz, DMSO-de), 58.81 (s), 8.41 (s), 8.27 (s), 8.16 (m), 8.04 (m), 7.74 ( m), 7.64 (m), 5.25 (d), 4.94 (q), 4.53 (m), 4.21-4.08 (m), 2.63 (s).
[0901]
[0902] Preparation of Reference Example (S) -3- [5 - ((S) -5-acetoxy-1-chloromethyl-1,2-dihydro-benzo [e] indole-3-carbonyl) -1-methyl-1H- pyrazol-3-carbonyl] -1-chloromethyl-2,3-dihydro-1H-benzo [e] indole-5-yl acetic acid ester (74).
[0903]
[0904]
[0905]
[0906]
[0907] To a round bottom flask purged with N2, containing 1-methyl-1H-pyrazol-3,5-dicarboxylic acid 38 (20 mg, 0.12 mmol) in 5 ml of anhydrous dichloromethane was added oxalyl chloride (0.248 mmol , 0.022 ml). To this solution was added 1 drop of N, N-dimethylformamide. The reaction mixture was stirred for 3 hours and concentrated in vacuo to give a crude residue 73. 73 was taken up in dichloromethane (10 ml) and added dropwise to a round bottom flask containing (S) -1 -chloromethyl-2,3-dihydro-1H-benzo [e] indole-5-yl acetic acid ester (5.73 mg, 0.236 mmol) in 5 ml of dichloromethane and triethylamine (2.08 ml). The crude reaction mixture was concentrated in vacuo and recaptured in 25 ml of dichloromethane and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3 x), water (3 x), and brine (2 x). The organic layer was dried with sodium sulfate, filtered and the filtrate was concentrated to give a crude solid. Next, silica gel chromatography was performed (gradient: 0% -100% ethyl acetate in heptanes) yielding (74) (12 mg, 15%) as a light yellow solid. LC-MS: m / z 6852 [M + H +], retention time = 2.21 minutes.
[0908]
[0909] Preparation of Reference Example 7-azabicyclo [2.2.1] heptane-1,4-diylbis [carbonyl (1S) -1- (chloromethyl) -1,2-dihydro-3H-benzo [e] indole-3, diacetate, 5-diyl] 79
[0910]
[0911]
[0912]
[0913]
[0914] Step 1: A mixture of 7-benzyl 1,4-dimethyl 7-azabicyclo [2.2.1] heptane-1,4,7-tricarboxylate (3.20 g, 9.21 mmol) [repare as described in Chem. Eur. J. 2012, 18, 1127-1141] in the presence of Pd / C (10%, 1000 mg) was hydrogenated at the pressure of a balloon at room temperature for ~ 2 hours. The reaction was filtered through a layer of celite and the cake was washed with a solution of 40 ml of methanol and 40 ml of dichloromethane. The organic extracts were combined and concentrated in vacuo to provide a light yellow solid. To a stirring solution of this crude solid in 40 ml of acetone at 0 ° C, NaHCO ³ aq was added. (1 M, 65 ml, 64.6 mmol) followed by the dropwise addition of Fmoc-CI (3.34 g, 12.9 mmol) as a solution in 40 ml of acetone. The reaction was diluted with 100 ml of water and extracted with ethyl acetate (100 ml, 3x). The organic layers were combined with water, brine, dried over sodium sulfate and concentrated in vacuo. Next, silica gel chromatography was performed (gradient: 12.5% to 17% ethyl acetate in petroleum ether). Suitable test tubes were combined and concentrated in vacuo to provide a white solid. The crude material was then suspended in aq HCI (3 M, 60 ml) and 80 ml of dioxane. The reaction was heated to reflux and then allowed to stir under reflux for ~ 16 hours. Then, the reaction was concentrated in vacuo to remove most of the dioxane. Then, the aqueous phase was extracted with ethyl acetate (100 ml, 2x). The organic layers were combined, washed with brine, dried over sodium sulfate and concentrated in vacuo. Next, silica gel chromatography was performed (gradient: 8.3% 25% methanol in dichloromethane). Suitable test tubes were combined and concentrated in vacuo and then re-purified by preparative HPLC (method M, using a gradient from 50% B to 80% B for 30 minutes, then 95% for 5 minutes) to provide 76 (400 mg, 12%, 3 steps) in the form of a white solid. 1H NMR (400 MHz, DMSO-d ^e ): 87.81-7.79 (d, 2H), 7.72-7.71 (d, 2H), 7.42-7.38 (m, 2H) , 7.34-7.31 (m, 2H), 4.35-4.33-7.33 (d, 2H), 4.22-4.19 (m, 1H), 2.28-2, 26 (d, 4H), 1.93-1.91 (d, 2H).
[0915]
[0916] Stage 2: Following general procedure A using 76 (90 mg, 0.40 mmol), oxalyl chloride (0.033 ml, 0.39 mmol), THF (8 ml) and 1 drop of DMF, 77 was prepared as an off-white solid (79 mg, quant.). The crude product 77 was used immediately as is in the next step.
[0917]
[0918] Step 3: Following general procedure B using 6 (103 mg, 0.331 mmol), 77 (70 mg, 0.16 mmol), pyridine (0.051 ml, 0.63 mmol) and THF (12 ml) the material was prepared in stupid. The reaction was concentrated in vacuo, dissolved in DMSO and injected into a 12 g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 30% to 95% acetonitrile in water with 0.02% TFA in each phase). Suitable test tubes were concentrated using a genevac yielding 78 (23 mg, 16%) as a light brown solid. LC-MS (Protocol B): m / z 922.0 [M + H] +, retention time = 2.59 minutes.
[0919]
[0920] Step 4: To a stirring solution of 78 (17.9 mg, 0.019 mmol) in 1.0 ml of DMF, DMAP (47.4 mg, 0.388 mmol) was added. The reaction was allowed to stir at room temperature for ~ 60 minutes. The crude reaction was injected into a C18 pre-column of 5 g (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 30% to 95% acetonitrile in water with 0.02% TFA in each phase). Suitable test tubes were concentrated using a genevac yielding 79 (6.1 mg, 39%) as a light brown solid. LC-MS (Protocol B): m / z 700.1 [M + H] +, retention time = 1.47 minutes. 1H NMR (400 MHz, DMSO-d ^e ): 8 10.26-10.17 (m, 2H), 8.28-8.24 (m, 2H), 8.11-8.06 (d, 2H ), 8.00-7.95 (d, 2H), 7.71-7.64 (t, 2H), 7.60 7.53 (t, 2H), 4.56-4.37 (m, 6H), 4.18-4.05 (m, 4H), 2.83-2.59 (m, 8H), 2.49-2.37 (m, 6H).
[0921]
[0922] Preparation of (1S, 4S) -bicyclo [2.1.1] hexane-1,4-diylbis [carbonyl (1S) -1- (chloromethyl) -1,2-dihydro-3H-benzo [e] indole-3 diacetate , 5-diyl] 82.
[0923]
[0924]
[0925]
[0926]
[0927] Step 1: Following general procedure A using bicyclo [2.1.1] hexane-1,4-dicarboxylic acid 80 (30 mg, 0.18 mmol), oxalyl chloride (0.0303 ml, 0.353 mmol), THF (4 ml) and 1 drop of DMF, 81 was prepared as an off-white solid (39 mg, quant.). The crude product 81 was used immediately as is in the next step.
[0928]
[0929] Step 2: Following the general procedure B using 6 (106 mg, 0.338 mmol), 81 (35 mg, 0.17 mmol), pyridine (0.0545 ml, 0.676 mmol) and THF (8 ml), and purification using chromatography In the inverted phase C18 intermediate pressure (gradient: 10% to 75% acetonitrile in water with 0.02% TFA in each phase), 82 (52 mg, 45%) was produced as a white solid. LC-MS (Protocol B): m / z 685.2 [M + H] +, retention time = 2.16 minutes. 1H NMR (400 MHz, DMSO-d ^e ): 88.23 (s, 2H), 8.03-7.99 (d, 2H), 7.92-7.87 (d, 2H), 7.63 -7.57 (t, 2H), 7.51 7.44 (t, 2H), 4.47-4.25 (m, 6H), 4.13-3.98 (m, 4H), 2, 47 (s, 6H), 2.27-2.07 (m, 8H).
[0930]
[0931] Preparation of bicyclo diacetate [2.2.2] octane-1,4-diylbis [carbonyl (1S) -1- (chloromethyl) -8-methyl-1,6-dihydropyrrolo [3,2-e] indole-3,5 (2H) -diyl] 85.
[0932]
[0933]
[0934] Stage 1: Following general procedure A using bicido acid [2.2.2] octane-1,4-dicarboxylic 83 (16 mg, 0.081 mmol), oxalyl chloride (0.015 ml, 0.17 mmol), THF (5 ml) and 1 drop of DMF, 84 was prepared as an off-white solid (19 mg, quant.). The crude product 84 was used immediately as is in the next step.
[0935]
[0936] Step 2: Following general procedure B using 189 (50.9 mg, 0.145 mmol), 84 (17.0 mg, 0.0723 mmol), pyridine (0.0233 ml, 0.289 mmol) and THF (4 ml), and purification using intermediate pressure C18 inverted phase chromatography (gradient: 10% to 75% acetonitrile in water with 0.02% TFA in each phase), 85 (21.6 mg, 32%) was produced as a solid white. LC-MS (Protocol B): m / z 719.3 [M + H] +, retention time = 2.27 minutes. 1H NMR (400 MHz, DMSO-de): 5 11.00 (s, 2H), 7.79 (s, 2H), 7.19 (s, 2H), 4.68-4.62 (m, 2H ), 4.27-4.19 (m, 2H), 4.06-3.94 (m, 4H), 3.65-3.57 (m, 2H), 2.42-2.32 (m , 12H), 2.12-1.96 (m, 12H).
[0937]
[0938] Preparation of bicyclo diacetate [2.2.1] heptane-1,4-diylbis [carbonyl (1S) -1- (chloromethyl) -8-methyl-1,6-dihydropyrrolo [3,2-e] indole-3,5 (2H) -diyl] 88.
[0939]
[0940]
[0941]
[0942]
[0943] Stage 1: Following the general procedure A using bicyclo [2.2.1] heptane-1,4-dicarboxylic acid 86 (16 mg, 0.087 mmol), oxalyl chloride (0.016 ml, 0.18 mmol), THF (5 ml) and 1 drop of DMF, 87 was prepared as an off-white solid (19 mg, 99%). The crude product 87 was used immediately as is in the next step.
[0944]
[0945] Step 2: Following general procedure B using 189 (54.1 mg, 0.154 mmol), 87 (17.0 mg, 0.0769 mmol), pyridine (0.0248 ml, 0.308 mmol) and THF (4 ml), and purification using intermediate pressure C18 inverted phase chromatography (gradient: 10% to 75% acetonitrile in water with 0.02% TFA in each phase), 88 (13.6 mg, 19%) was produced as a light brown solid. LC-MS (Protocol B): m / z 705.3 [M + H] +, retention time = 2.32 minutes. 1H NMR (400 MHz, DMSO-de): 5 11.02 (s, 2H), 7.82 (s, 2H), 7.19 (s, 2H), 4.50-4.45 (d, 2H ), 4.26 4.16 (m, 2H), 4.10-4.02 (m, 2H), 3.98-3.92 (m, 2H), 3.65-3.58 (m, 2H), 2.41-2.33 (m, 12H), 2.22-2.03 (m, 10H).
[0946] Preparation of bicyclo diacetate [1.1.1] pentane-1,3-diylbis [carbonyl (1S) -1- (chloromethyl) -8-methyl-1,6-dihydropyrrolo [3,2-e] indole-3,5 (2H) -diyl] 91.
[0947]
[0948]
[0949]
[0950]
[0951] Step 1: Following general procedure A using bicyclo [1.1.1] pentane-1,3-dicarboxylic acid 89 (31 mg, 0.20 mmol), oxalyl chloride (0.025 ml, 0.40 mmol), THF (8 ml) and 1 drop of DMF, 90 was prepared as an off-white solid (40 mg, quant.). The crude product 90 was used immediately as is in the next step.
[0952]
[0953] Stage 2: Following general procedure B using 189 (142 mg, 0.404 mmol), 90 (39 mg, 0.20 mmol), pyridine (0.065 ml, 0.81 mmol) and THF (12 ml), and purification using chromatography In the inverted phase C18 intermediate pressure (gradient: 10% to 75% acetonitrile in water with 0.02% TFA in each phase), 91 (45.5 mg, 30%) was produced as a gray solid Clear. LC-MS (Protocol B): m / z 677.2 [M + H] +, retention time = 1.89 minutes. 1H NMR (400 MHz, DMSO-de): 5 11.04 (s, 2H), 7.78 (s, 2H), 7.20 (s, 2H), 4.47-4.39 (m, 2H ), 4.36 4.26 (m, 2H), 4.18-4.08 (m, 2H), 4.03-3.94 (m, 2H), 3.77-3.66 (m, 2H) , 2.56 (s, 6H), 2.41-2.31 (m, 12H).
[0954]
[0955] Preparation of (8S) -6 - [(3 - {[(1S) -5- (acetyloxy) -1- (chloromethyl) -8-methyl-1,6-dihydropyrrolo [3,2-e] indole) acetate 3 (2H) -yl] carbonyl} bicyclo [1.1.1] pent-1-yl) acetyl] -8- (chloromethyl) -1-methyl-3,6,7,8-tetrahydropyrrolo [3,2-e] indole-4-il 97.
[0956]
[0957]
[0958]
[0959]
[0960] Step 1: Following the general procedure A using 3- (2-tert-butoxy-2-oxoethyl) bicyclo [1.1.1] pentane-1-carboxylic acid 92 [repare as described in Bioorg. Med. Chem. 2009, 17, 242-250], (90 mg, 0.40 mmol), oxalyl chloride (0.041 ml, 0.477 mmol), THF (8 ml) and 1 drop of DMF, 93 was prepared as of an off-white solid (103 mg, quant.). The crude product 93 was used immediately as is in the next step.
[0961]
[0962] Stage 2: Following general procedure B using 11 (141 mg, 0.40 mmol), 93 (98 mg, 0.40 mmol), triethylamine (0.168 ml, 1.20 mmol) and THF (30 ml), and purification using silica gel chromatography (Gradient: 0% to 35 % acetone in heptane), 94 (188 mg, 96%) was produced as an off-white solid. LC-MS (Protocol B): m / z 487.2 [M + H] +, retention time = 2.04 minutes.
[0963]
[0964] Step 3: To a stirring solution of 94 (184 mg, 0.378 mmol), in 8 ml of dichloromethane, TFA (4.0 ml, 52 mmol) was added. The reaction was allowed to stir at room temperature for ~ 45 minutes. The reaction was concentrated in vacuo and placed under high vacuum yielding 95 (164 mg, 80%) as a light gray solid, which was used in the next step without purification. LC-MS (Protocol B): m / z 431.7 [M + H] +, retention time = 1.39 minutes.
[0965]
[0966] Step 4: Following the general procedure A using 95 (55 mg, 0.101 mmol), oxalyl chloride (0.0104 ml, 0.121 mmol), THF (3 ml), dichloromethane (1 ml) and 1 drop of DMF, 96 se prepared in the form of an off-white solid (46 mg, quant.). The crude product 96 was used immediately as is in the next step.
[0967] Step 5: Following general procedure B using 11 (31.3 mg, 0.089 mmol), 96 (40 mg, 0.089 mmol), pyridine (0.0215 ml, 0.267 mmol) and THF (8.0 ml), and purification using intermediate pressure C18 inverted phase chromatography (gradient: 10% to 70% acetonitrile in water with 0.02% TFA in each phase), 97 (10.1 mg, 12%) was produced as a solid of light gray color LC-MS (Protocol B): m / z 691.3 [M + H] +, retention time = 1.93 minutes. 1H NMR (400 MHz, DMSO-de): 5 11.02 (s, 2H), 7.86-7.72 (d, 2H), 7.19 (s, 2H), 4.43-4.36 (m, 1H), 4.28-4.14 (m, 3H), 4.13-4.05 (m, 2H), 3.96-3.89 (m, 2H), 3.68-3 , 60 (m, 2H), 2.89-2.82 (m, 2H), 2.73-2.66 (m, 2H), 2.40-2.30 (m, 12H), 2.24 -2.15 (m, 6H).
[0968]
[0969] Preparation of (1S) -8-amino-5- (benzyloxy) -1- (chloromethyl) -1,2-dihydro-3H-benzo [e] indole-3-carboxylate of tert-butyl 99 and tert-butyl (1R ) -8-amino-5- (benzyloxy) -1- (chloromethyl) -1,2-dihydro-3H-benzo [e] indole-3-carboxylic acid tert-butyl 98.
[0970]
[0971]
[0972] 98 8-amino-5- (benzyloxy) -1- (chloromethyl) -1,2-dihydro-3H-benzo [e] indole-3-carboxylic acid tere-butyl [repared using the chemistry described in J. Med Chem. 2012, 55, 5878-5886] was separated using supercritical fluid chromatography (method L1). Peak 1 was concentrated in vacuo, provided 99 (385 mg) that was arbitrarily assigned to (S). LC-MS (Protocol B): m / z 439.1 [M + H] +, retention time = 2.34 minutes. Peak 2 was concentrated in vacuo, provided 100 (401 mg) that was arbitrarily assigned to (S). LC-MS (Protocol B): m / z 439.1 [M + H] +, retention time = 2.34 minutes.
[0973]
[0974] Preparation of (1R) -8- (acetylamino) -5- (acetyloxy) -1- (chloromethyl) -1,2-dihydro-3H-benzo [e] indole-3-carboxylate terebutyl 102
[0975]
[0976]
[0977]
[0978]
[0979] Step 1: To a stirring solution of 99 (60 mg, 0.14 mmol) in 6 ml of dichloromethane at 0 ° C, acetyl chloride (0.015 ml, 0.206 mmol) was added followed by triethylamine (0.029 ml, 0.206 mmol). The reaction was allowed to stir at 0 ° C for ~ 1 minute and then allowed to warm to room temperature. The reaction was allowed to stir at room temperature for ~ 25 minutes. The reaction was diluted with dichloromethane and then transferred to a separatory funnel. The organic layer was separated, and then washed with 1N HCI, and then water. The organic layer was dried with sodium sulfate, filtered and then concentrated in vacuo, yielding an orange solid. To a stirring solution of the crude material in 4 ml of THF at 0 ° C, 10% Pd by weight on carbon (45 mg) was added followed by a solution of ac ammonium formate. 25% (0.3 ml). The reaction was allowed to stir at 0 ° C for ~ 4 hours. The reaction was diluted with THF and ether. Sodium sulfate was added, and the reaction was filtered through a small pad of celite. The organic layers were concentrated in vacuo and placed under high vacuum producing a light brown solid. To a stirring solution of the crude material in 6 ml of dichloromethane at 0 ° C, acetyl chloride (0.015 ml, 0.211 mmol) was added followed by pyridine (0.017 ml, 0.211 mmol). The reaction was allowed to stir at 0 ° C for ~ 1 minute and then allowed to warm to room temperature. The reaction was allowed to stir at room temperature for ~ 25 minutes. The reaction was concentrated in vacuo. Next, silica gel chromatography was performed (gradient: 0% to 45% acetone in heptane). Suitable test tubes were combined and concentrated in vacuo yielding 101 (49 mg, 80%, 3 steps) as an off-white solid. LC-MS (Protocol B): m / z 455.9 [M + Na] +23, retention time = 2.05 minutes.
[0980]
[0981] Step 2: To a round bottom flask containing 101 (45 mg, 0.10 mmol), 4M HCl in dioxane (6.0 mL, 24 mmol) was added. The reaction was allowed to stir at room temperature for ~ 2 hours. The reaction was concentrated in vacuo and placed under high vacuum yielding 102 (42 mg, quant.) As a dark brown solid. LC-MS (Protocol B): m / z 333.0 [M + H] +, retention time = 1.65 minutes.
[0982]
[0983] Preparation of (1S) -8- (acetylamino) -5- (acetyloxy) -1- (chloromethyl) -1,2-dihydro-3H-benzo [e] indole-3-carboxylate terebutyl 103
[0984]
[0985]
[0986] Step 1: To a stirring solution of 99 (65 mg, 0.15 mmol) in 6 ml of dichloromethane at 0 ° C, acetyl chloride (0.016 ml, 0.22 mmol) was added followed by triethylamine (0.031 ml, 0.22 mmol). The reaction was allowed to stir at 0 ° C for ~ 1 minute and then allowed to warm to room temperature. The reaction was allowed to stir at room temperature for ~ 25 minutes. The reaction was diluted with dichloromethane and then transferred to a separatory funnel. The organic layer was separated, and then washed with 1N HCI, and then water. The organic layer was dried with sodium sulfate, filtered and then concentrated in vacuo, yielding an orange solid. To a stirring solution of the crude material in 4 ml of THF at 0 ° C, 10% Pd by weight on carbon (45 mg) was added followed by a solution of ac ammonium formate. 25% (0.5 ml). The reaction was allowed to stir at 0 ° C for ~ 4 hours. The reaction was diluted with THF and ether. Sodium sulfate was added, and the reaction was filtered through a small pad of celite. The organic layers were concentrated in vacuo and placed under high vacuum producing a light brown solid. To a stirring solution of the crude material in 8 ml of dichloromethane at 0 ° C, acetyl chloride (0.015 ml, 0.21 mmol) was added followed by pyridine (0.017 ml, 0.21 mmol). The reaction was allowed to stir at 0 ° C for ~ 1 minute and then allowed to warm to room temperature. The reaction was allowed to stir at room temperature for ~ 25 minutes. The reaction was concentrated in vacuo. Next, silica gel chromatography was performed (gradient: 0% to 25% acetone in heptane). Suitable test tubes were combined and concentrated in vacuo yielding 103 (39.1 mg, 63%, 3 steps) as a white solid. LC-MS (Protocol B): m / z 455.0 [M + Na] +23, retention time = 2.00 minutes.
[0987]
[0988] Step 2: To a round bottom flask containing 103 (37 mg, 0.085 mmol), 4M HCl in dioxane (4.0 mL, 16 mmol) was added. The reaction was allowed to stir at room temperature for ~ 2 hours. The reaction was concentrated in vacuo and placed under high vacuum yielding 104 (34 mg, quant.) As a green solid. LC-MS (Protocol B): m / z 333.0 [M + H] +, retention time = 1.41 minutes.
[0989]
[0990] Preparation of (1S) -3 - {[3- (chlorocarbonyl) bicyclo [1.1.1] pent-1-yl] carbonyl} -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole- 5-yl 107.
[0991]
[0992]
[0993]
[0994]
[0995] Step 1: Following the general procedure A using 3- (tert-butoxycarbonyl) bicyclo [1.1.1] pentane-1-carboxylic acid 105 (212 mg, 1.0 mmol), oxalyl chloride (0.094 ml, 1, 10 mmol), t Hf (3 ml), dichloromethane (6 m) and 1 drop of DMF, 105 was prepared as an off-white solid (235 mg, quant.). The crude product 105 was used immediately as is in the next step.
[0996]
[0997] Stage 2: Following general procedure B using 11 (311 mg, 0.997 mmol), 105 (230 mg, 0.997 mmol), triethylamine (0.292 ml, 2.09 mmol) and THF (20 ml), and purification using gel chromatography of silica (Gradient: 10% to 75% acetone in heptane). Suitable test tubes were combined and concentrated in vacuo yielding a white solid. To a stirring solution of the crude material in 10 ml of dichloromethane, TFA (5.0 ml, 65 mmol) was added. The reaction was allowed to stir at room temperature for ~ 90 minutes. The reaction was concentrated in vacuo. The material was dissolved with dichloromethane, transferred to a separatory funnel, and then washed with 1 N aq. HCl, brine, and water. The organic layer was dried with sodium sulfate, filtered and then concentrated in vacuo. before putting on high vacuum producing a white solid. Using this raw material and following the general procedure A with oxalyl chloride (0.010 ml, 0.121 mmol), THF (4 ml), dichloromethane (2 ml) and 1 drop of DMF, 107 was prepared as a colored solid white (52 mg, 49%, 3 stages). The crude product 107 was used immediately as is in the next step.
[0998]
[0999] Preparation of (1R) -8- (acetylamino) -3 - [(3 - {[(1S) -5- (acetyloxy) -1- (chloromethyl) -1,2-dihydro-3H-benzo [e]] acetate indole-3-yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -1- (chloromethyl) -1,2-dihydro-3H-benzo [e] indole-5-yl 108
[1000]
[1001]
[1002] Following general procedure B using 107 (21 mg, 0.057 mmol), 102 (24.6 mg, 0.057 mmol), triethylamine (0.024 ml, 0.171 mmol) and THF (6 ml), and purification by preparative HPLC (procedure H1) 108 (5.8 mg, 14%) was produced as an off-white solid. LC-MS (Protocol B): m / z 728.1 [M + H] +, retention time = 2.12 minutes.
[1003]
[1004] Preparation of (1S) -3 - [(3 - {[(1S) -8- (acetylamino) -5- (acetyloxy) -1- (chloromethyl) -1,2-dihydro-3H-benzo [e] acetate) acetate indole-3-yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-yl 109
[1005]
[1006]
[1007]
[1008]
[1009] Following general procedure B using 107 (29.4 mg, 0.068 mmol), 104 (25 mg, 0.068 mmol), triethylamine (0.028 ml, 0.028 mmol) and THF (8 ml), and purification using C18 inverted phase chromatography of intermediate pressure (gradient: 10% to 75% acetonitrile in water with 0.02% TFA in each phase), 109 (11.8 mg, 24%) was produced as a white solid. LC-MS (Protocol B): m / z 728.0 [M + H] +, retention time = 2.13 minutes. 1H NMR (400 MHz, DM SO d): 510.25 (s, 1H), 8.27 (s, 1H), 8.20 (s, 1H), 8.06 (s, 1H), 8.04 -7.98 (m, 1H), 7.92-7.80 (m, 2H), 7.63-7.55 (m, 2H), 7.50-7.45 (m, 1H), 4 , 56-4.33 (m, 5H), 4.29-4.17 (m, 1H), 4.16-3.94 (m, 4H), 2.62 (s, 6H), 2.48 -2.43 (m, 6H), 2.11 (s, 3H).
[1010]
[1011] Preparation of Reference Example (S) -3- [5 - ((S) -5-amino-1-chloromethyl-1,2-dihydro-benzo [e] indole-3-carbonyl) -thiophene-2-carbonyl] -1-chloromethyl-2,3-dihydro-1H-benzo [e] indole-5-yl acetic acid ester 115
[1012]
[1013]
[1014]
[1015]
[1016] Step 1: To a round bottom flask purged with N ² , containing thiophene-2,5-dicarboxylic acid mono-tert-butyl ester (152 mg, 0.66 mmol) in 5 ml of anhydrous DCM was added chloride of oxalyl (0.66 mmol, 0.066 ml). To this solution was added 1 drop of N, N-dimethylformamide. The reaction mixture was stirred for 3 hours and concentrated in vacuo to obtain a crude residue. The residue was then added to a round bottom flask containing 110 (200 mg, 0.66 mmol) in 15 ml of anhydrous dichloromethane. The reaction was stirred for 2 hours. The residue was diluted with 15 ml of dichloromethane and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3x), water (3 x), and brine (2 x). The organic layer was dried with sodium sulfate, filtered and concentrated to give a crude solid. Next, silica gel chromatography was performed (gradient: 0% -100% ethyl acetate in heptane) yielding 111 (185 mg, 58%) as a yellow solid. LC-MS: m / z 473 [M + H +], retention time = 2.25 minutes.
[1017]
[1018] Step 2 to 111, 10 ml of 25% trifluoroacetic acid in dichloromethane was added. The reaction was stirred for 30 min. The crude reaction mixture was concentrated in vacuo to provide 112 as a yellow solid. LC-MS: m / z 416 [M + H +], retention time = 1.65 minutes.
[1019]
[1020] Step 3: To a round bottom flask purged with N ² , containing 112 (100 mg, 0.24 mmol) in 5 ml of anhydrous DCM was added oxalyl chloride (0.24 mmol, 0.02 ml). To this solution was added 1 drop of N, N-dimethylformamide. The reaction mixture was stirred for 3 hours and concentrated in vacuo to provide 113 as a yellow solid (100 mg, 0.24 mmol, quantitative). LCMS, captured in methanol: m / z 282.0 [M + H +, for the methanolysis product]. Retention time = 1.95 minutes.
[1021]
[1022] Step 4: To a round bottom flask containing 5.28 mg, 0.092 mmol) in 5 ml of dichloromethane was added 113 (40 mg, 0.092 mmol). Triethylamine (0.088 ml) was added and the system was stirred for 1 hour at room temperature. The crude reaction mixture was concentrated in vacuo and recaptured in 25 ml of dichloromethane and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3x), water (3 x), and brine (2 x). The organic layer was dried with sodium sulfate, filtered and the filtrate was concentrated to give a crude solid. Next, silica gel chromatography was performed (gradient: 0% -100% ethyl acetate in heptane) yielding 114 (40 mg, 64%) as a yellow solid. LC-MS: m / z 674 [M + H +], retention time = 2.25 minutes.
[1023]
[1024] Step 5 To a Parr flask containing 114 (30 mg, 0.044 mmol) in 15 ml of anhydrous tetrahydrofuran was added platinum oxide (5 mg, 0.02 mmol). The system was covered with a rubber septum and hydrogenation was carried out with H ^{2 at} 50 Psi for 3 hours. After 3 hours, the Parr flask was purged with N ² and the crude reaction was filtered through a bed of celite using ethyl acetate. The filtrate containing the desired crude product was then concentrated. Next, silica gel chromatography was performed (gradient: 0% -100% ethyl acetate in heptanes) yielding 115 (15 mg, 50%) as a yellow solid. LC-MS: m / z 644 [M + H +], retention time = 2.06 minutes.
[1025]
[1026] Preparation of Reference Example ((S) -1-Chloromethyl-5-hydroxy-1,2-dihydro-benzo [e] indole-3-ylH5 - ((S) -1-chloromethyl-5-hydroxy-8-methyl -1,6-dihydro-2H-pyrrolo [3,2-e] indole-3-carbonyl) -thiophene-2-yl] -methanone 117.
[1027]
[1028]
[1029]
[1030]
[1031] In a round bottom flask provided with a stirring bar containing 11 (34 mg, 0.1 mmol), N, N-dimethylformamide (5 mL) was captured and added dropwise to a round bottom flask containing acid 5 - ((S) -5-Acetoxy-1-chloromethyl-1,2-dihydro-benzo [e] indole-3-carbonyl) -thiophene-2-carboxylic acid (42, 44 mg, 0.1 mmol), 3 - (ethyliminomethyleneamino) -W, W-dimethylpropan-1-amine (59 mg, 0.3 mmol) and sodium bicarbonate (36 mg, 0.4 mmol) in 5 ml of W, W-dimethylformamide). The reaction was stirred for 30 min. 3 ml of 1M HCI (aq.) Was added and the crude reaction mixture was concentrated in vacuo. Next, inverted phase chromatography (gradient: acetonitrile in water 0% -65%) was performed to produce 117 (15 mg, 24%) as a white solid. LC-MS: m / z 604 [M-H +], retention time = 1.93 minutes
[1032]
[1033] Preparation of Reference Example (S) -1- (chloromethyl) -3- (5 - ((S) -1- (chloromethyl) -5-hydroxy-2,3-dihydro-1H-benzo [e] indole carbonate -3-carbonyl) thiophene-2-carbonyl) -2,3-dihydro-1H-benzo [e] indole-5-yl methyl 119
[1034]
[1035]
[1036]
[1037]
[1038] A solution of 4-nitrophenyl chloroformate (11 mg, 0.054 mmol) in THF (1 ml) was added to a solution of 29 (27 mg, 0.045 mmol) in THF (3 ml) and DIPEA (0.032 ml, 0.18 mmol) at 0 ° C. The mixture was stirred at 0 ° C for 2 h, and stirred at room temperature overnight. LCMS showed that the corresponding mono-PNP carbonate 118 had been formed. To the reaction mixture, methanol (1 ml) was added. After stirring for 5 min, it was concentrated in vacuo, and the residue was purified by Gilson HPLC (CAN / water, 0.02% TFA) to give product 119 as a white solid (5 mg, twenty %). LC-MS: m / z 660.7 [MH], retention time = 1.06 min. 1 H NMR (400 MHz, CDCla), 68.53 (d), 7.80 (m), 7.72 (d), 7.45 (m), 7.34 (m), 4.72 (m), 4.62 (d), 4.30 (m), 4.11 (t), 4.04 (s), 3.86 (d), 3.71 (d), 3.47 (t), 3 , 24 (m).
[1039]
[1040] Preparation of Reference Example (S) -1- (chloromethyl) -3- (5 - ((S) -1- (chloromethyl) -5-hydroxy-2,3-dihydro-1H-benzo [e] indole-3 -carbonyl) thiophene-2-carbonyl) -2,3-dihydro-1H-benzo [e] indole-5-yl (2- (dimethylamino) ethyl) carbamate 123
[1041]
[1042]
[1043] Step 1: A solution of 4-nitrophenyl chloroformate (164 mg, 0.78 mmol) in THF (1 ml) was added to a solution of 3 (200 mg, 0.60 mmol) in THF (6 ml) and DIPEA (0.315 ml, 1.8 mmol) at 0 ° C, and the mixture was stirred at room temperature for 2 h. It was concentrated and the residue was treated with EA and water, extracted with EA, washed with water and brine. It was dried with MgSO ⁴ , the solvent was removed in vacuo to give PNP carbonate 120 in yellow form (300 mg, 100%). LC-MS: m / z 399.0 [MH], retention time = 2.37 min.
[1044]
[1045] Step 2: W, W-dimethylethylenediamine (35 mg, 0.4 mmol) was added to a solution of the above PNP carbonate 120 (100 mg, 0.2 mmol) in DMA (3 ml), followed by lutidine (0, 07 ml, 0.6 mmol) and HOAt (14 mg, 0.1 mmol). The mixture was stirred at room temperature for 4 h. The mixture was subjected to purification by Gilson (ACN / water, 0.02% TFA) to give the carbamate (S) -1- (chloromethyl) -5 - (((2- (dimethylamino) ethyl) carbamoyl) oxy) -1H-benzo [e] indole-3 (2H) tere-butyl carboxylate 121 in the form of a yellow glass (86 mg, 77%). LC-MS: m / z 448.1 [MH], retention time = 0.70 min.
[1046]
[1047] Step 3: The above compound 121 (38 mg, 0.067 mmol) was treated with TFA (0.5 ml) and CH ² Cl ² (2 ml) for 2 h, then concentrated in vacuo to give the corresponding unprotected amine 122 which It was dissolved in DMA (3 ml). To this solution, (S) -5- (1- (chloromethyl) -5-hydroxy-2,3-dihydro-1H-benzo [e] indole-3-carbonyl) thiophene-2-carboxylic acid was added [58] (26 mg, 0.067 mmol), followed by EDCI (27 mg, 0.14 mmol), and the mixture was stirred at room temperature overnight. The crude product was purified by Gilson HPLC (ACN / water, 0.02% TFA) to give 123 (4.5 mg, 8%). LC-MS: m / z 717.4 [MH], retention time = 1.38 min. 1 H NMR (400 MHz, CDCla), 8 8.24 (d), 8.0 (d), 7.75 (d), 7.64 (s), 7.55 - 7.34 (m), 4, 62 (m), 4.13 (t), 4.05 (t), 3.94 (t), 3.64 (t), 3.57-3.45 (m), 3.33 (s) , 3.25 (s), 2.89 (s).
[1048]
[1049] Preparation of Reference Example (S) -1- (chloromethyl) -3- (5 - ((S) -1- (chloromethyl) -5-hydroxy-2,3-dihydro-1H-benzo [e] indole-3 -carbonyl) thiophene-2-carbonyl) -2,3-dihydro-1H-benzo [e] indole-5-yl methyl (2- (methylamino) ethyl) carbamate 126
[1050]
[1051]
[1052]
[1053]
[1054] Step 1: To the above solution of 120, N, N, N-trimethylethylenediamine (222 mg, 0.28 mmol) was added, followed by lutidine (0.37 ml, 3.2 mmol) and HOAt (29 mg, 0.2 mmol). The mixture was stirred at room temperature for 1 h. The mixture was concentrated, and the residue was diluted with ethyl acetate, washed with brine, dried with MgSO4. The crude reaction product was purified by ISCO using MeOH / DCM (0-20%) to give 124 as a white foam (245 mg, 50%). LC-MS: m / z 462.2 [MH], retention time = 1.45 min.
[1055]
[1056] Step 2: The above compound 124 (40 mg, 0.087 mmol) was treated with previously cooled TFA (1 ml) at 0 ° C for 10 min. TFA was removed in vacuo to give the corresponding unprotected amine 125, which was dissolved in DMF (3 ml). To this solution, (S) -5- (1- (chloromethyl) -5-hydroxy-2,3-dihydro-1H-benzo [e] indole-3-carbonyl) thiophene-2-carboxylic acid was added [58] (34 mg, 0.087 mmol), followed by EDCI (35 mg, 0.17 mmol), and the mixture was stirred at room temperature overnight. The crude product was purified by Gilson HPLC (ACN / water, 0.02% TFA) to give product 126 as a white solid (25 mg, 39%). LC-MS: m / z 731.1 [MH], retention time = 1.71 min. 1H NMR (400 MHz, DMSO-de), 510.49 (s), 8.26 (s), 8.14 (d), 7.98 (d), 7.88 (d), 7.66 ( t), 7.77 (t), 7.40 (t), 4.89 (t), 4.78 (t), 4.55 (d), 4.43 (d), 4.23 (s ), 4.08-3.91 (m), 3.73 (s), 3.50 (s), 3.40 (s), 3.26 (s), 2.89 (m).
[1057]
[1058] Preparation of bicyclo [1.1.1] pentane-1,3-diylbis {[(1S) -5-amino-1- (chloromethyl) -1,2-dihydro-3H-benzo [e] indole-3-yl] methanone } 130
[1059]
[1060]
[1061]
[1062]
[1063] Step 1: To a round bottom flask equipped with a stir bar with fluorenylmethyloxycarbonyl chloride (560 mg, 2.1 mmol) 5 ml of anhydrous DCM was added and the system was purged with nitrogen. 127 (800 mg, 2.1 mmol) was added followed by TEA (0.3 ml, 2.1 mmol). The system was left under stirring for 5 hours. The crude reaction mixture was taken up in ethyl acetate and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3x), water (3x), sodium bicarbonate and brine (2x). The organic layer was dried with sodium sulfate, filtered and the filtrate was concentrated to give a crude residue. The crude product was purified by silica gel chromatography (Gradient: 0% 100% ethyl acetate in heptane) to give 128 as a yellow solid (1.096 g, 91%). LC-MS (Protocol B): m / z 455 [M-Boc] +, retention time = 2.58 minutes.
[1064]
[1065] Step 2: 15 ml of 25% TFA in DCM was added to a round bottom flask equipped with a stir bar containing 128 (1000 mg, 1.96 mmol). The solution was stirred for 30 min. The reaction mixture was concentrated in vacuo and captured in 50% DCM and heptane and concentrated in vacuo. This was repeated 3 times (to remove excess TFA) to give a white solid after concentration. This white solid was added to a stirred solution of bicyclo dichloride [1.1.1] pentane-1,3-dicarbonyl 90 in 10 ml of anhydrous DCM. The reaction was stirred for 1 hour and concentrated to give a crude glass. The crude reaction mixture was taken up in ethyl acetate and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3x), water (3x), sodium bicarbonate and brine (2x). The organic layer was dried with sodium sulfate, filtered and the filtrate was concentrated to give a crude residue. The crude product was purified by silica gel chromatography (Gradient: 0% 100% ethyl acetate in heptane) to give 129 as a yellow solid (250 mg, 12%). LC-MS (Protocol B): m / z 1030.7 [M- ^h ] -, retention time = 2.29 minutes.
[1066]
[1067] Stage 3: To a round bottom flask fitted with a stir bar containing bis (9H-fluoren-9-ylmethyl) (bicyclo [1.1.1] pentane-1,3-diylbis {carbonyl [(1S) -1- (chloromethyl ) -1,2-dihydro-3H-benzo [e] indole-3,5-diyl]}) biscarbamate 129 (20 mg, 0.19 mmol) 10 ml of DCM in 1: 1 DEA was added. The solution was stirred for 3 hours. The reaction mixture was concentrated in vacuo and captured in 50% DCM in heptane and concentrated in vacuo. This was repeated 3 times (to remove excess DEA) to give a white solid after concentrating. The crude product was purified by silica gel chromatography (Gradient: 0% 10% methanol in DCM) to give 130 as a yellow solid (4 mg, 30%). LC-MS (Protocol B): m / z 585.1 [M + H] +, retention time = 1.99 minutes.
[1068] Preparation of (1S) -1- (chloromethyl) -3 - [(4 - {[(1S) -1- (chloromethyl) -5-hydroxy-1,2-dihydro-3H-benzo [e] indole acetate) 3-yl] carbonyl} bicido [2.2.1] hept-1-yl) carbonyl] -2,3-dihydro-1 H -benzo [e] indole-5-yl 134.
[1069]
[1070]
[1071]
[1072]
[1073] Step 1: Following the general procedure A using 4- (methoxycarbonyl) bicido acid [2.2.1] heptane-1-carboxylic acid 131 (75 mg, 0.38 mmol), oxalyl chloride (0.032 ml, 0.378 mmol), THF ( 1.5 ml), dichloromethane (1.5) and 1 drop of DMF, was prepared132 in the form of a white oil and a solid mixture (85 mg, quant.). The crude product 132 was used immediately as is in the next step.
[1074]
[1075] Step 2: Following general procedure B using 2 (125 mg, 0.346 mmol), 132 (75 mg, 0.35 mmol), pyridine (0.112 ml, 1.38 mmol), dichloromethane (2 ml) and THF (6 ml ), and purification using silica gel chromatography (Gradient: 25% 0% acetone in heptane) The appropriate test tubes were combined and concentrated in vacuo to yield a white solid. To a stirring solution of the crude material in 6 ml of THF, lithium hydroxide (52.9 mg, 2.21 mmol) dissolved in 1.5 ml of water was added. The reaction was allowed to stir at room temperature for ~ 3.5 hours. The filtrate was concentrated to a smaller volume, transferred to a separatory funnel, and diluted with dichloromethane. The reaction was washed with 1 N HCl. The ac layer. washed once with dichloromethane. The organic layers were combined, washed with brine, water, dried over sodium sulfate, filtered and then concentrated in vacuo before placing them under high vacuum. Following general procedure A using raw material, oxalyl chloride (0.024 ml, 0.281 mmol), THF (4.0 ml), dichloromethane (4.0 ml) and 1 drop of DMF, was prepared133 in the form of an oil of white color and a solid mixture (85 mg, quant.). The crude product 133 was used immediately as is in the next step.
[1076]
[1077] Step 3: Following general procedure B using 19a (79.9 mg, 0.256 mmol), 133 (130 mg, 0.256 mmol), pyridine (0.103 ml, 1.28 mmol) and THF (6 ml) a solid was produced raw pink after concentrating this reaction in vacuo. To a stirring solution of the crude material in 3 ml of DMF and 1 ml of THF at 0 ° C, 10% Pd by weight on carbon (100 mg) was added followed by a solution of ac ammonium formate. 25% (0.4 ml). The reaction was allowed to stir at 0 ° C for ~ 90 minutes. The reaction was filtered through a bed of C18 which was washed with a solution of 70% / 30% acetonitrile and water with 0.02% TFA in each phase. The material was reduced using a genevac yielding 134 (54 mg, 32%, 2 steps) as a light gray solid. LC-MS (Protocol B): m / z 657.1 [M + H] +, retention time = 2.10 minutes. 1H NMR (400 MHz, DMSO-de): 5 10.32 (s, 1H), 8.24 (s, 1H), 8.11-8.07 (s, 1H), 8.02-7.96 (m, 2H), 7.91-7.86 (d, 1H), 7.83-7.78 (d, 1H), 7.63-7.57 (m, 1H), 7.52-7 , 45 (m, 2H), 7.36-7.30 (m, 1H), 4.54-4.38 (m, 3H), 4.35-4.27 (m, 2H), 4.16 -4.05 (m, 2H), 4.02-3.90 (m, 2H), 3.80-3.73 (m, 1H), 2.47 (s, 3H), 2.26-2 , 03 (m, 10H).
[1078]
[1079] Preparation of (3bR, 4aS, 3b'R, 4a'S) -6.6 '- (bicyclo [1.1.1] pentane-1,3-diyldicarbonyl) bis (3-methyl-4,4a, 5,6-tetrahydrocyclopropa [ c] pyrrolo [3,2-e] indole-8 (1H) -one).
[1080]
[1081]
[1082]
[1083]
[1084] To a mixture of 109 (21 mg, 0.026 mmol) in 2 ml of acetonitrile, triethylamine (0.40 ml, 2.9 mmol) was added followed by 0.4 ml of water. The reaction was allowed to stir at room temperature for ~ 40 minutes. The reaction was concentrated in vacuo. Next, silica gel chromatography was performed (gradient: 0% to 10% methanol in dichloromethane). Suitable test tubes were combined and concentrated in vacuo yielding 135 (1.6 mg, 12%) as a light brown solid. LC-MS (Protocol B): m / z 521.3 [M + H] +, retention time = 1.28 minutes. 1H NMR (400 MHz, DMSO-cfe): 5 11.47 (s, 2H), 6.85 (s, 4H), 4.29-4.22 (m, 2H), 4.17-4.10 (m, 2H), 3.19-3.09 (m, 2H), 1.97 (s, 6H), 1.93-1.87 (m, 2H), 1.27-1.22 (m , 2H).
[1085] Preparation of Reference Example (1aS, 9bR, 1a'S, 9b'R) -3,3 '- (has-2,5-diyldicarbonyl) bis (1,1a, 2,3-tetrahydro-5H-benzo [e] cyclopropa [c] indole-5-one).
[1086]
[1087]
[1088]
[1089]
[1090] To a mixture of 57 (44 mg, 0.073 mmol) in 3 ml of acetonitrile, triethylamine (0.40 ml, 2.9 mmol) was added followed by 0.4 ml of water. The reaction was allowed to stir at room temperature for ~ 40 minutes. The reaction was concentrated in vacuo. Next, silica gel chromatography was performed (gradient: 0% to 5% methanol in dichloromethane). Suitable test tubes were combined and concentrated in vacuo yielding 136 (16.3 mg, 39%) as a light brown solid. LC-MS (Protocol B): m / z 531.1 [M + H] +, retention time = 1.55 minutes. 1H NMR (400 MHz, DMSO-de): 58.05-8.01 (d, 2H), 7.80 (s, 2H), 7.65-7.59 (t, 2H), 7.48- 7.43 (t, 2H), 7.28 7.23 (d, 2H), 6.76 (s, 2H), 4.57-4.51 (m, 2H), 4.34-4.26 (m, 2H), 1.85-1.76 (m, 4H).
[1091]
[1092] Preparation of 2,3,4-tri-O-acetyl-beta-D-glucopyranosiduronate of (1S) -1- (chloromethyl) -3 - [(3 - {[(1S) -1- (chloromethyl) -5- hydroxy-1,2-dihydro-3H-benzo [e] indol-3-yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -2,3-dihydro-1H-benzo [e] indole -5-yl methyl 141.
[1093]
[1094]
[1095]
[1096]
[1097] 140 (464 mg, 0.6 mmol) was dissolved in DCM (4 ml), TFA (2 ml) was added, and the mixture was sealed tightly for 2 h. The mixture was concentrated in vacuo to give the corresponding acid LC-MS (Protocol B) was dissolved: 688.0 [M + H] +, retention time 0.98 min. It was dissolved in THF (8 ml), cooled to 0 ° C, oxalyl chloride (0.9 ml, 2 M in DCM) was added, followed by 2 drops of DMF. The mixture was stirred at 0 ° C for 5 min, then at rt for 50 min. It was concentrated in vacuo to give the corresponding acid chloride.
[1098]
[1099] LC-MS: 702.1 (1.05 min at Larry, the peak of the corresponding ester Me);
[1100] 4 was dissolved in THF (10 ml), cooled to 0 ° C, the above acid chloride was added, followed by Et3N (0.5 ml, 4.0 mmol). The mixture was stirred at 0 ° C for 30 min. The mixture was diluted with EA, washed with water and brine, dried with MgSO4. The solvent was removed under reduced pressure, and the residue was treated with MeOH. The resulting solid was collected by filtration to give 141 as a green solid (414 mg, 73.5%). LC-MS (Protocol B): 903.2 [M + H] +, retention time 1.11 min.
[1101]
[1102] (continuation)
[1103]
[1104] (continuation)
[1105]
[1106] (continuation)
[1107]
[1108] (continuation)
[1109]
[1110] (continuation)
[1111]
[1112] (continuation)
[1113]
[1114]
[1115] The names of the compounds in Table 1 are given below:
[1116]
[1117] (continuation)
[1118]
[1119] (continuation)
[1120]
[1121]
[1122]
[1123]
[1124] Preparation of Reference Example bis (2 - ((S) -1- (chloromethyl) -5- (phosphonooxy) -1H-benzo [e] indole-3 (2H) -yl) -2-oxoethyl) 4- carbamate ((23S, 26S) -1- (2,5-dioxo-2,5-dihydro-lH-pyrrol-1-yl) -23-isopropyl-2l, 24-dioxo-26- (3-ureidopropyl) -3 , 6,9,12,15,18-hexaoxa-22,25-diazaheptacosanamido) benzyl (186)
[1125]
[1126]
[1127]
[1128]
[1129] Step 1: A stirring solution of 51 (120 mg, 0.124 mmol) in 10 ml of tetrahydrofuran in a nitrogen atmosphere was cooled to 0 ° C using an ice bath. Next, 10% palladium by weight on activated carbon (106 mg, 0.298 mmol) was added followed by a slow dropwise addition of 1 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 C for 5 hours. Then, the reaction was filtered through a layer of celite and the filtrate was then concentrated in vacuo. Next, silica gel chromatography was performed (gradient: 0% -100% ethyl acetate in heptanes) yielding 182 (35 mg, 36%) as a light yellow solid. LC-MS: m / z 786 [M + H +], retention time = 2.22 minutes.
[1130]
[1131] Step 2: To a stirring solution of 182 (274 mg, 0.348 mmol) in 10 ml of THF and 10 ml of acetonitrile, Carbon tetrachloride (2.04 ml, 21.0 mmol) and Hunig's base (1.12 ml, 6.45 mmol) were added dibenzyl phosphite (0.9 ml, 4.32 mmol) and DMAP (catalytic). The reaction was allowed to stir at room temperature for ~ 20 minutes. The crude reaction mixture was concentrated in vacuo and then silica gel chromatography (gradient: 0% -100% ethyl acetate in heptanes) was performed yielding 183 (239 mg, 52%) as a yellow solid. Clear. LC-MS: m / z 1308 [M + H +], retention time = 2.70 minutes.
[1132]
[1133] Step 3: To a round bottom flask provided with a stir bar containing 183 (200 mg, 0.153 mmol) 5 ml of dichloromethane and 5 ml of diethyl amine were added. The solution was stirred for 3 hours. The reaction mixture was concentrated in vacuo and captured in 50% dichloromethane in heptane and concentrated again in vacuo. This was repeated 3 times. The crude residue was taken up in 10 ml of 25% trifluoroacetic acid in dichloromethane followed by thiophenol (1 ml). The reaction was stirred at room temperature for two days. The crude reaction mixture was concentrated in vacuo and then silica gel chromatography (gradient: 0% -100% ethyl acetate in heptanes) was performed yielding 184 (60 mg, 47%) as a yellow solid. Clear. LC-MS: m / z 724 [M + H +], retention time = 1.02 minutes.
[1134]
[1135] Step 4: To a round bottom flask containing 184 (75 mg, 0.1 mmol), 10 ml DMA was added and the system was purged with N ² . To this solution under stirring was added 185 (99 mg, 0.104 mmol) followed by HOAt (416 mg, 0.104 mmol) and Hunig's base (1 drop). The system was stirred at 45 ° C for 3 hours. The crude reaction mixture was concentrated in vacuo and then reversed phase chromatography was performed yielding 186 (34 mg, 21%) as a white solid. LC-MS: m / z 1546 [M + H +], retention time = 1.23 minutes.
[1136] Preparation of (S) -3- (5- (chlorocarbonyl) thiophene-2-carbonyl) -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-yl 191 acetate
[1137]
[1138]
[1139]
[1140]
[1141] Step 1: To a stirring solution of 5- (tert-butoxycarbonyl) thiophene-2-carboxylic acid (187) in 20 ml of THF at 0 ° C, oxalyl chloride (0.677 ml, 7.88 mmol) was added followed of 1 drop of DMF. The reaction was allowed to stir at room temperature at 0 ° C for ~ 1 minute and then allowed to warm to room temperature. The reaction was allowed to stir at room temperature for ~ 90 minutes. The reaction was reduced or placed under high vacuum to yield 188 (1.67 g, quant.) As a white solid. The raw material was then used immediately in the next step.
[1142]
[1143] Step 2: To a stirring solution of 6 (1.54 g, 4.93 mmol) in 25 ml of THF at 0 ° C, triethylamine (1.38 ml, 9.87 mmol) was added followed immediately by the addition of 188 (1.46 g, 5.92 mmol) dissolved in 25 ml of THF. The reaction was allowed to stir at 0 ° C for ~ 1 minute and then allowed to warm to room temperature while stirring. Then, the reaction was allowed to stir at room temperature for ~ 45 minutes. The reaction was reduced on silica. Next, silica gel chromatography was performed (gradient: 0% -100% acetone in heptans). Suitable test tubes were concentrated and placed under high vacuum to yield 189 (2.24 g, 94%) as a brown solid. LC-MS (Protocol B): m / z 486.3 [M + H] +, retention time = 2.19 minutes.
[1144]
[1145] Step 3: 189 (144 mg, 0.3 mmol) was treated with previously cooled TFA (3 ml) at 0 ° C for 30 min, then concentrated in vacuo. to give the corresponding acid 190. LC-MS: m / z 430.3 [MH], retention time = 1.59 min. 190 was dissolved in THF (3 ml), oxalyl chloride (0.2 ml, ² M in CH ² O ² , 0.4 mmol) was added at 0 ° C, followed by 2 drops of DMF (cat), the The mixture was stirred at 0 ° C for 5 min, and then at room temperature for 2 h. It was concentrated in vacuo to give 191 as a yellow solid.
[1146]
[1147] Preparation of (S) -dibenzyl (1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-yl) phosphate (193)
[1148]
[1149]
[1150] Step 1: To a stirring solution of 3 (889 mg, 2.66 mmol) in 20 ml of THF and 20 ml of acetonitrile, carbon tetrachloride (3.61 ml, 37.3 mmol) was added followed by base of Hunig (2.0 ml, 11.5 mmol), dibenzylphosphonate (3.65 ml, 16.5 mmol) and DMAP (65.1 mg, 0.533 mmol). The reaction was allowed to stir at room temperature for ~ 20 minutes. The reaction was concentrated to a smaller volume, diluted with a few ml of DMSO and then injected into a 25g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 85% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac producing 192 (839 mg, 53%) in the form of a transparent dark brown solid / oily mixture. LC-MS (Protocol B): m / z 595.3 [M + 2H] +, retention time = 2.47 minutes.
[1151]
[1152] Step 2: To a stirring solution of 192 (834 mg, 1.40 mmol) in 16 ml of dichloromethane, TFA (16 ml, 210 mmol) was added. The reaction was allowed to stir at room temperature for 1 minute and then reduced immediately before being placed under vacuum yielding 193 (701 mg, quant.) As an oily / solid green mixture. LC-MS (Protocol B): m / z 494.2 [M + H] +, retention time = 2.17 minutes.
[1153]
[1154] Preparation of Reference Example (1S) -3- (5 - ((1S) -5 - (((benzyloxy) (hydroxy) phosphoryl) oxy) -1- (chloromethyl) -2,3-dihydro-1H- acetate benzo [e] indole-3-carbonyl) thiophene-2-carbonyl) -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-yl [194] and (S) acetate - 3- (5 - ((S) -5 - ((bis (benzyloxy) phosphoryl) oxy) -1- (chloromethyl) -2,3-dihydro-1 H -benzo [e] indole-3-carbonyl) thiophene-2 -carbonyl) -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-yl [195]
[1155]
[1156]
[1157]
[1158]
[1159] 193 was dissolved in THF (3 ml) at 0 ° C, Et ³ N (0.165 ml, 1.2 mmol) was added, followed by a solution of 191 in THF (2 ml). The mixture was stirred at 0 ° C for 5 min, and room temperature for 2 h. The reaction mixture was concentrated in vacuo, and the residue was purified by Gilson HPLC (ACN / water, 0.02% TFA) to give two products 194 as a yellow solid (50 mg, 21%) . CL- ^and M: m / z 815.4 [MH], retention time = 0.96 min. 1H NMR (400 MHz, DMSO-cfe), 58.42 (s), 8.16 (s), 8.07 (d), 8.02 (d), 7.94 (d), 7.90 ( s), 7.64 (q), 7.54 (q), 7.10 - 7.29 (m), 5.14 (d), 4.86 (q), 4.52 (t), 4 , 42 (m), 4.11-4.00 (m) and 195 in the form of a green solid (50 mg, 19%). LC-MS: m / z 905.4 [MH], retention time = 2.43 min.
[1160]
[1161] Preparation of Reference Example of (1S) -1- (chloromethyl) -3 - [(5 - {[(1S) -1- (chloromethyl) -5- (phosphonooxy) -1,2-dihydro-3H-benzo carbonate [e] indole-3-yl] carbonyl} thiophene-2-yl) carbonyl] -2,3-dihydro-1 H -benzo [e] indol-5-yl 4-nitrophenyl (196).
[1162]
[1163]
[1164]
[1165]
[1166] To a stirring mixture of 195 (200 mg, 0.221 mmol) in 8 ml of methanol, 4M HCI in dioxane (8.0 ml, 230 mmol) was added. The reaction was allowed to stir at room temperature for ~ 20 minutes. The reaction was reduced. The crude material was collected in 8 ml of THF and 8 ml of dichloromethane. To this solution under stirring at 0 ° C, 4-nitrophenyl carbochloridate (86.3 mg, 0.428 mmol) was added followed by triethylamine (0.179 ml, 1.28 mmol). The reaction was allowed to stir at 0 ° C for ~ 1 minute and then allowed to warm to room temperature while stirring. The reaction was allowed to stir at room temperature for ~ 20 minutes. The reaction was reduced and then put to high vacuum. To a stirred mixture of the crude material in 10 ml of dichloromethane, a solution of TFA (5 ml, 70 mmol) in 10 ml of dichloromethane was added followed by thiophenol (0.107 ml, 1.04 mmol). The reaction was allowed to stir at room temperature for ~ 6-7 hours. The reaction was concentrated to a smaller volume, diluted with a few ml of DMSO and then injected into a 25g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 60% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac yielding 196 (71 mg , 40%) in the form of a yellow solid. LC-MS (Protocol B): m / z 848.3 [M + H] +, retention time = 1.78 minutes. 1H NMR (400 MHz, DMSO-de) 88.51 (sa), 8.35-8.41 (m), 8.09-8.15 (m), 8.00, 7.97-8.02 (d ), 7.87-7.93 (m), 7.80-7.86 (m), 7.67-7.73 (m), 7.58-7.65 (m), 7.50- 7.55 (m), 4.80-4.93 (m), 4.42-4.58 (m), 4.31-4.37 (m), 3.96-4.15 (m) .
[1167]
[1168] Preparation of Reference Example 4 - ((S) -2 - ((S) -2 - ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -5-ureidopentanamido) benzyl (2 - ((((((S) -1- (chloromethyl) -3- (5 - ((S) -1- (chloromethyl) -5- (phosphonooxy) -2,3-dihydro-1H-benzo [e] indole-3-carbonyl) thiophene-2 -carbonyl) -2,3-dihydro-1H-benzo [e] indole-5-yl) oxy) carbonyl) (2-methoxyethyl) amino) ethyl) (methyl) carbamate [198]
[1169]
[1170]
[1171]
[1172]
[1173] 196 (15 mg, 0.018 mmol) was dissolved in DMF (1 ml), a solution of 197 (17 mg, 0.023 mmol) in DMF (1 ml) was added, followed by DIPEA (0.013 ml, 0.072 mmol) and lutidine ( 0.008 ml, 0.072 mmol), HoAt (2.6 mg). The mixture was stirred at rt for 30 min. The reaction was completed in 30 min observed by LCMS. The crude product was purified by Gilson HPLC (0.02% TFA) to give product 198 as a light yellow solid (13 mg, 53%). LC-MS: m / z 1346.8 [MH], retention time = 1.77 min. 1H NMR (400 MHz, DMSO-de), 88.39 (s), 8.15 (d), 8.01 (m), 7.89 (m), 7.63 (m), 7.53 ( m), 7.45-7.23 (m), 6.73 (d), 5.98 (s), 5.07-4.95 (m), 4.84 (t), 4.51 ( m), 4.49-4.60 (m), 4.08-3.95 (m), 3.84-3.63 (m), 3.00-2.89 (m), 1.68 - 1.59 (m), 0.85 (m).
[1174]
[1175] Preparation of Reference Example (2 - ((((((5) -1- (chloromethyl) -3- (5 - ((5) -1- (chloromethyl) -5- (phosphonooxy)) -2,3-dihydro- 1H-benzo [e] indole-3-carbonyl) thiophene-2-carbonyl) -2,3-dihydro-1H-benzo [e] indole-5-yl) oxy) carbonyl) (2-methoxyethyl) amino) ethyl) (methyl) carbamate of 4 - ((26S, 29S) -1-bromo-26-isopropyl-2,24,27-trioxo-29- (3-ureidopropyl) -6,9,12,15,18,21- hexaoxa-3,25,28-triazatriacontanamide) benzyl [201]
[1176]
[1177]
[1178]
[1179]
[1180] Step 1: 198 (13 mg, 0.01 mmol) was treated with previously cooled TFA (0 ° C, 2 ml) for 2 min, and concentrated in vacuo to give product 199 as a yellow solid ( 14 mg, TFA salt, 100%). LC-MS: m / z 1247.9 [MH], retention time = 1.57 min. 1H NMR (400 MHz, DMF-dz), 8 10.13 (s), 8.65 (d), 8.45 (s), 8.17 (d), 7.95 - 7.85 (m) , 7.65 -7.22 (m), 5.04-4.97 (m), 4.81 (dd), 4.56 (s), 4.33 (d), 4.07-3, 94 (m), 3.73-3.64 (m), 3.50 (s), 3.55-3.09 (m), 2.95-2.85 (m), 2.21 (dd ), 1.76 (m), 1.62 (m), 1.46 (s), 0.99 (m).
[1181]
[1182] Step 2: 199 (5 mg, 0.004 mmol) was added to a solution of 1-bromo-2-oxo-6,9,12,15,18,21-hexaoxa-3-azatetracosan-24-perfluorophenyl 200 (oato) 3.8 mg, 0.006 mmol) in DMF (0.5 ml), followed by DIPEA (0.003 ml, 0.016 mmol). The mixture was stirred at room temperature for 1 h. The crude product was purified by Gilson HPLC using ACN / water (0.02% t Fa) to give product 201 as a yellow solid (3 mg, 40%). LC-MS: m / z 1704.0 [MH], retention time = 1.61 min. 1H NMR (400 MHz, DMSO-de), 89.88 (s), 8.30 (s), 8.24 (s), 8.06 (m), 7.91 (m), 7.81 ( m), 7.54 (m), 7.47 (m), 7.43-7.13 (m), 5.91 (s), 4.98-4.85 (m), 4.76 ( m), 4.43 (m), 4.30 (s), 4.14 (m), 4.00 - 3.90 (m), 3.52 (m), 3.16 (m), 2 , 92 -2.86 (m), 2.31 -2.25 (m), 1.90 (s), 1.52 (s), 1.34 (s), 1.32 (m), 0 , 78 (m).
[1183] Preparation of Reference Example (2 - ((((((S) -1- (chloromethyl) -3- (5 - ((S) -1- (chloromethyl)) -5- (phosphonooxy) -2,3-dihydro- 1H-benzo [e] indole-3-carbonyl) thiophene-2-carbonyl) -2,3-dihydro-1H-benzo [e] indole-5-yl) oxy) carbonyl) (2-methoxyethyl) amino) ethyl) (methyl) carbamate of 4 - ((23S, 26S) -1- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -23-isopropyl-21,24-dioxo-26- (3-ureidopropyl) -3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosanamido) benzyl [206]
[1184]
[1185]
[1186]
[1187]
[1188] Step 1: 202 (227 mg, 0.52 mmol) was dissolved in CH ² CI ² (2 ml) and DMF (2 ml), PFP-O-TFA (0.19 ml, 1.05 mmol) was added and DIPEA (0.275 ml, 1.57 mmol). The mixture was stirred at room temperature for 2 h. It was concentrated in vacuo, and the residue was purified by Gilson HPLC (0.02% TFA) to give the corresponding PFP ester 203 as a yellow oil (34 mg, 11%). LC-MS: m / z 623.4 [M Na], retention time = 0.92 min.
[1189]
[1190] Step 2: 203 (3 mg, 0.005 mmol) was added to a solution of 199 (7 mg, 0.005 mmol) in DMF (0.3 ml), followed by DIPEA (0.005 ml, 0.03 mmol). The mixture was stirred at room temperature for 2 h. The reaction mixture was subjected to Gilson HPLC separation (0.02% TFA) to give product 204 as a yellow solid (4.6 mg, 60%). LC-MS: m / z 1664.1 [MH], retention time = 1.63 min. 1H NMR (400 MHz, DMSO-de), 58.39 (s), 8.14 (m), 8.10-7.99 (m), 7.63 (m), 7.55-7.5 (m), 7.48 (s), 7.02 (s), 6.52 (s), 5.99 (s), 5.07 -4.95 (m), 4.84 (t), 4.52 (t), 4.38 (s), 4.24 (t), 4.08-3.99 (m), 3.61 - 3.48 (m), 3.00 - 2.89 (m), 2.68 (s), 2.34 (s), 0.86 (dd).
[1191]
[1192] Preparation of Reference Example (2 - ((((((S) -1- (chloromethyl) -3- (5 - ((S) -1- (chloromethyl)) -5- (phosphonooxy) -2,3-dihydro- 1H-benzo [e] indole-3-carbonyl) thiophene-2-carbonyl) -2,3-dihydro-1H-benzo [e] indole-5-yl) oxy) carbonyl) (2-methoxyethyl) amino) ethyl) (methyl) carbamate of 4 - ((23S, 26S) -1-amino-23-isopropyl-21,24-dioxo-26- (3-ureidopropyl) -3,6,9,12,15,18-hexaoxa- 22,25-diazaheptacosanamido) benzyl [208]
[1193]
[1194]
[1195]
[1196]
[1197] Step 1: 205 (43 mg, 0.07 mmol) was dissolved in DMF (2 ml), PFP-O-TFA (0.026 ml, 0.14 mmol) was added, followed by DIPEA (0.038 ml, 0.21 mmol ). The mixture was stirred at room temperature for 2 h. The crude product was purified by Gilson HPLC (0.02% TFA) to give product 206 as a colorless oil (39 mg, 72%). LC-MS: m / z 742.2 [MH], retention time = 2.17 min.
[1198]
[1199] Step 2: 199 (7 mg, 0.005 mmol) was dissolved in DMF (0.6 ml), a solution of the above PFP ester 206 (3.7 mg, 0.005 mmol) in d Cm (0.1 ml) was added , followed by DIp Ea (0.005 ml, 0.03 mmol). The mixture was stirred at rt for 1 h. The crude product 207: LC-MS: m / z 1805.3 [M ^h ], retention time = 1.97 min.
[1200] Step 3: To the previous reaction mixture of 207, piperidine (0.02 ml, 0.2 mmol) was added, and the mixture was stirred at rt for 30 min. It was concentrated in vacuo, and the crude product was purified by Gilson HPLC (0.02% TFA) to give product 208 as a yellow solid (4.2 mg, TFA salt, 50%). LC-MS: m / z 1584.0 [MH], retention time = 1.54 min. 1H NMR (400 MHz, DMSO-cfe), 8 9.98 (s), 8.38 (s), 8.14 (m), 7.98 (m), 7.88 (m), 7.70 (s), 7.62 (m), 7.54 (m), 7.47 (m), 7.27 (m), 6.01 (s), 5.06-5.00 (m), 4.84 (m), 4.51 (m), 4.37 (m), 4.25 (m), 4.08 (m), 4.02 (m), 3.59 (m), 3 , 25 (m), 2.98 (m), 2.37 (m), 1.97 (s), 1.69 (s), 1.59 (s), 1.39 (m), 0, 86 (dd).
[1201]
[1202] Preparation of Reference Example (1S) -1- (chloromethyl) -3 - [(5 - {[(1S) -1- (chloromethyl) -5 - {[(4-nitrophenoxy) carbonyl] oxy} -1 , 2-dihydro-3H-benzo [e] indol-3-yl] carbonyl} thiophen-2-yl) carbonyl] -2,3-dihydro-1H-benzo [e] indole-5-yl (211).
[1203]
[1204]
[1205]
[1206]
[1207] Step 1: To a stirring mixture of 2 (425 mg, 1.31 mmol) in 5 ml of THF under a nitrogen atmosphere at 0 ° C, triethylamine (0.333 ml, 2.39 mmol) was added followed immediately by 191 ( 535 mg, 1.19 mmol) dissolved in 5 ml of THF. The reaction was allowed to stir at 0 ° C for 5 minutes and then allowed to warm to room temperature while stirring. The reaction was allowed to stir at room temperature for ~ 30 minutes. Then the reaction was reduced on silica. Next, silica gel chromatography was performed (gradient: 5% -80% acetone in heptans). Suitable test tubes were concentrated and placed under high vacuum to yield 209 (530 mg, 60%) as a yellow solid. LC-MS (Protocol B): m / z 735.1 [M + H] +, retention time = 2.48 minutes.
[1208]
[1209] Step 6: A stirring solution of 209 (610 mg, 0.829 mmol) in 15 ml of THF under a nitrogen atmosphere was cooled to 0 ° C using an ice bath. Next, 10% palladium by weight on activated carbon (203 mg) was added followed by a slow dropwise addition of 2 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 ° C for 12-24 hours. The reaction was diluted with ether followed by the addition of sodium sulfate. The reaction was filtered through celite, and the celite was washed twice with ether. The organic layers were combined and then reduced. The residue was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 80% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac yielding 210 (206 mg , 44%) in the form of a yellow solid. LC-MS (Protocol B): m / z 645.0 [M + H] +, retention time = 2.08 minutes. 1H NMR (400 MHz, DMSO) 8 10.49 (sa), 8.13-8.18 (d), 8.05-8.10 (d), 7.93-7.97 (d), 7 , 83-7.91 (m), 7.63-7.69 (t), 7.53-7.58 (m), 7.38-7.43 (m), 4.83-4.92 (m), 4.74-4.82 (m), 4.50-4.55 (d), 4.39-4.47 (m), 4.20-4.27 (m), 4, 01-4.15, 3.88-3.96 (m), 3.57-3.68 (m), 1.74-1.80, 1.36-1.39 (m).
[1210]
[1211] Step 7: To a stirring solution of 210 (195 mg, 0.302 mmol) in 12 ml of dichloromethane and 8 ml of THF at 0 ° C, 4-nitrophenyl carbonochloridate (122 mg, 0.604 mmol) was added followed by triethylamine ( 0.168 ml, 1.21 mmol). The reaction was allowed to stir at 0 ° C for 5 minutes, and then allowed to warm to room temperature while stirring. The reaction was allowed to stir at room temperature for ~ 30 minutes. The reaction was reduced. The residue was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 85% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac yielding 211 (240 mg , 98%) in the form of a yellow solid. LC-MS (Protocol B): m / z 810.3 [M + H] +, retention time = 2.35 minutes.
[1212]
[1213] Preparation of Reference Example N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] -L-valyl-N- [4 - ({[{2- [ ({[(1S) -3 - [(5 - {[(1S) -5- (acetyloxy) -1- (chloromethyl) -1,2-dihydro-3H-benzo [e] indole-3-yl] carbonyl } thiophen-2-yl) carbonyl] -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indol-5-yl] oxy} carbonyl) (methyl) amino] ethyl} (methyl) carbamoyl] oxy} methyl) phenyl] -N-5-carbamoyl-L-ornithinamide (215)
[1214]
[1215]
[1216] Step 1: To a stirring solution of 212 (750 mg, 1.02 mmol) and 213 methyl [2- (methylamino) ethyl] tert- butyl carbamate (192 mg, 1.02 mmol) in 6 ml of DMA, 2-6-lutidine (0.236 ml, 2.03 mmol) was added followed by Hunig's base (0.354 ml, 2.03 mmol) and HOAT (69.1 mg, 0.5 mmol). The reaction was allowed to stir at room temperature for ~ 40 minutes. The reaction was injected into a 25 g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase) and then purified by intermediate pressure C18 inverted phase chromatography (gradient: 5% to 45% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac yielding 214 (663 mg, 83%) as a white solid. LC-MS (Protocol B): m / z 787.3 [M + H] +, retention time = 1.45 minutes.
[1217]
[1218] Step 2: To a stirring mixture of 214 (40.9 mg, 0.052 mmol) in 2 ml of dichloromethane, TFA (1 ml, 10 mmol) was added. The reaction was allowed to stir at room temperature for ~ 40 minutes. The reaction was reduced and then placed under high vacuum. The crude material was collected in 2 ml of DMA and Hunig's base (0.03 ml, 0.17 mmol) was added to this stirring solution followed by 2,6-Lutidine (0.02 ml, 0.17 mmol) , HOAT (5.9 mg, 0.043 mmol) and then n 211 (35 mg, 0.043 mmol) dissolved in 1 ml of DMA. The reaction was allowed to stir at room temperature for ~ 40 minutes. The reaction was injected into a C18 pre-column of 5 g (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase) and then purified by intermediate pressure C18 inverted phase chromatography (gradient: 5% to 60% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac yielding 215 (14.1 mg, 24%) as a yellow solid. LC-MS (Protocol B): m / z 1359.3 [M + 3H] +, retention time = 2.01 minutes. HR-MS: m / z 1359.4549 [M + 3H] +.
[1219]
[1220] Preparation of N- [1- (2,5-dioxo-2,5-dihydro-1 H -pyrrol-1-yl) -21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl ] -L-Valyl-N-5-carbamoyl-N- {4 - [({methyl [2- (methylamino) ethyl] carbamoyl} oxy) methyl] phenyl} -L-ornithinamide (215).
[1221]
[1222]
[1223] Stage 1: To a round bottom flask containing 1- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -3,6,9,12,15,18-hexaoxahenicosan- 21-oico, 216 (628 mg, 1.45 mmol), 20 ml of dichloromethane, 2 ml of DMF, HATU (501 mg, 1.32 mmol) and Hunig's base (0.92 ml, 5.3) were added mmol). The reaction was allowed to stir at room temperature for 2 minutes before the addition of L-Valyl-N-5-carbamoyl-N- [4- (hydroxymethyl) phenyl] -L-ornithinamide, 217 (500 mg, 1.32 mmol). The reaction was allowed to stir at room temperature for ~ 90 minutes before being quenched through the addition of TFA. The reaction was concentrated to a smaller volume, diluted with a few ml of DMSO and then injected into a 25g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 40% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac yielding 218 (514 mg , 49%) in the form of a transparent solid. LC-MS (Protocol B): m / z 795.5 [M + H] +, retention time = 1.01 minutes.
[1224]
[1225] Step 2: To a stirring solution of 218 (210mg, 0.264mmol) and bis (4-nitrophenyl) carbonate (161 mg, 0.528 mmol) in 4 ml of DMF, Hunig's base (0.096 ml, 0.554 mmol) was added . The reaction was allowed to stir at room temperature for ~ 2 hours. The reaction was injected into a 25g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 55% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac yielding 219 (180 mg , 71%) in the form of a solid. LC-MS (Protocol B): m / z 960.5 [M + H] +, retention time = 1.48 minutes.
[1226]
[1227] Step 3: To a stirring solution of 219 (640 mg, 0.667 mmol) and 213 [repared as described in J. Med. Chem. 1992, 33, 559-567] (127 mg, 0.674 mmol) in 6 ml of DMA, 2,6-lutidine (0.154 ml, 1.33 mmol) was added followed by Hunig's base (0.232 ml, 1.33 mmol) and HOAT (9.1 mg, 0.67 mmol). The reaction was allowed to stir at room temperature for ~ 15 minutes. The reaction was injected into a 25g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 40% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac yielding 220 (564 mg , 84%) in the form of a cerulean white solid. LC-MS (Protocol B): m / z 1009.7 [M + H] +, retention time = 1.43 minutes.
[1228]
[1229] Step 4: To a stirring mixture of 220 (470 mg, 0.466 mmol) in 6 ml of dichloromethane, TFA (3.0 ml, 40 mmol) was added. The reaction was allowed to stir at room temperature for ~ 10 minutes. The reaction was reduced. The residue was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 30% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac yielding 221 (326 mg , 68%) in the form of a white oily / solid mixture. LC-MS (Protocol B): m / z 909.8 [M + H] +, retention time = 0.91 minutes.
[1230]
[1231] Reference Example Preparation N- [1- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) -21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl] - L-Valyl-N- [4 - ({[{2 - [({[(1S) -3 - [(5 - {[(1S) -5- (acetyloxy) -1- (chloromethyl) -1.2 -dihydro-3H-benzo [e] indole-3-yl] carbonyl} thiophene-2-yl) carbonyl] -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5
[1232] il] oxy} carbonyl) (methyl) amino] ethyl} (methyl) carbamoyl] oxy} methyl) phenyl] -N-5-carbamoyl-L-omitinamide (222).
[1233]
[1234]
[1235]
[1236]
[1237] To a stirring mixture of 221 (50.1 mg, 0.05 mmol) in 1 ml of DMA and to this solution under stirring, Hunig's base (0.03 ml, 0.172 mmol) was added followed by 2,6-Lutidine (0.02 ml, 0.172 mmol), HOAT (5.9 mg, 0.043 mmol) and 211 (35 mg, 0.043 mmol) dissolved in 1 ml of DMA. The reaction was allowed to stir at room temperature for ~ 40 minutes. The reaction was injected into a 5 g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 60% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac yielding 222 (15, 4 mg, 23%) in the form of a yellow / white solid. LC-MS (Protocol B): m / z 1580.4 [M + 2H] +, retention time = 1.95 minutes. HRMS: m / z 790.7923 [M + 2H] +.
[1238]
[1239] Preparation of Reference Example N- [1- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -21-oxo-3,6,9,12,15,18-hexaoxahenicosan- 21-yl] -L-Valyl-N ~ 5 ~ -carbamoyl-N- [4 - ({[(2 - {[({(1S) -1- (chloromethyl)) -3 - [(5 - ([( 1S) -1- (chloromethyl) -5- (phosphonooxy) -1,2-dihydro-3H-benzo [e] indole-3-yl] carbonyl} thiophene-2-yl) carbonyl] -2,3-dihydro- 1H-benzo [e] indole-5-yl} oxy) carbonyl] (methyl) amino} ethyl) (methyl) carbamoyl] oxy} methyl) phenyl] -L-ornithinamide (223)
[1240]
[1241]
[1242]
[1243]
[1244] Step 1: To a stirring solution of 196 (29.8 mg, 0.035 mmol) in 0.5 ml of DMA, 221 (17.3 mg, 0.019 mmol) was added as a solution in 1.5 ml of DMA followed by Hunig base addition (0.024 ml, 0.14 mmol), 2,6-Lutidine (0.016 ml, 0.14 mmol) and HOAT (4.8 mg, 0.035 mmol). The reaction was allowed to stir at room temperature for ~ 20 minutes. The reaction was injected into a 5 g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 75% acetonitrile in water with 0.02% TFA in each phase), followed by purification by preparative HPLC (procedure B) and the tubes Suitable assays were concentrated using a genevac yielding 222 (22.6 mg, 40%) as a colored solid. yellow. LC-MS (Protocol B): m / z 1619.9 [M + 3H] +, retention time = 1.62 minutes. HPLC (Protocol D): retention time = 9.339 minutes.
[1245]
[1246] Preparation of methyl 3- (chlorocarbonyl) bicyclo [1.1.1] pentane-1-carboxylate (225).
[1247]
[1248]
[1249]
[1250]
[1251] To a stirring solution of 224 in 12 ml of THF at 0 ° C, oxalyl chloride (0.381 ml, 4.44 mmol) was added followed by 1 drop of DMF. The reaction was allowed to stir at 0 ° C for ~ 1 minute and then allowed to warm to room temperature while stirring. The reaction was allowed to stir at room temperature for ~ 30 minutes. The reaction was reduced and then placed under high vacuum yielding 225 (701 mg, quantitative) as a white solid.
[1252]
[1253] Preparation of the trifluoroacetic acid carbonate salt of (8S) -8- (chloromethyl) -6 - [(3 - {[(1S) -1- (chloromethyl) -8-methyl-5- (phosphonooxy) -1, 6-dihydropyrrolo [3,2-e] indole-3 (2H) -yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -1-methyl-3,6,7,8-tetrahydropyrrolo [ 3,2-e] indole-4-yl 4-nitrophenyl 230.
[1254]
[1255]
[1256]
[1257]
[1258] Step 1: To a stirring solution of 8 (4.5 g, 13.4 mmol) in 80 ml of THF and 80 ml of acetonitrile, carbon tetrachloride (18.1 ml, 187 mmol) was added followed by base of Hunig (9.31 ml, 53.4 mmol), dibenzyl phosphite (17.7 ml, 80.2 mmol) and DMAP (326 mg, 2.67 mmol). The reaction was allowed to stir at room temperature for ~ 10 minutes. The reaction was reduced on silica. Next, silica gel chromatography was performed (gradient: 0% -20% acetone in heptans). Suitable test tubes were concentrated and placed under high vacuum to yield 226 (6.04 g, 76%) as a light yellow solid. LC-MS (Protocol B): m / z 614.3 [M + NH] +, retention time = 2.38 minutes.
[1259]
[1260] Step 2: To a stirring solution of 226 (2.15 g, 3.60 mmol) in 24 ml of dichloromethane, TFA (24 ml, 310 mmol) was added. The reaction was allowed to stir at room temperature for ~ 60 seconds, immediately reduced, and then placed under vacuum (belt pump). To a stirring solution of the crude material (2.59 g, 3.57 mmol) in 15 ml of THF at 0 ° C, triethylamine (1.49 ml, 10.7 mmol) was added followed immediately by 225 (674 mg, 3.57 mmol) dissolved in 15 ml of THF. The reaction was allowed to stir at 0 ° C for ~ 5 minutes and then allowed to warm to room temperature while stirring. The reaction was allowed to stir at room temperature for ~ 20 minutes. The reaction was reduced on silica. Next, silica gel chromatography was performed (gradient: 0% -30% acetone in heptans). Suitable test tubes were concentrated and placed under high vacuum to yield 227 (920 mg, 40%, 2 steps) as a white solid. LC-MS (Protocol B): m / z 649.2 [M + H] +, retention time = 2.04 minutes.
[1261]
[1262] Step 3: To a stirring solution of 227 (895 mg, 1.38 mmol) in 16 ml of THF, lithium hydroxide (330 mg, 13.8 mmol) dissolved in 4 ml of water was added. The reaction was allowed to stir at room temperature for ~ 90 minutes. Dichloromethane was added followed by an aqueous solution of 1N HCI. The material was transferred to a separatory funnel. The organic layer was separated and the aqueous layer was washed twice with dichloromethane. The organic layers were combined, washed once with brine, water, dried over sodium sulfate, filtered and then it was reduced before being placed under high vacuum. The crude material was collected in 15 ml of DMA and 5 ml of dichloromethane then cooled to 0 ° C. To this solution under stirring at 0 ° C, oxalyl chloride (0.135 ml, 1.63 mmol) was added followed by 1 drop of DMF. The reaction was allowed to warm to room temperature and then allowed to stir at room temperature for ~ 60 minutes. The reaction was reduced and then placed under high vacuum yielding 228 (820 mg, 91%, 2 steps) as a light brown solid. The raw material was used as is in the next stage.
[1263]
[1264] Step 4: To a stirring solution of 11 (527 mg, 1.50 mmol) in 12 ml of THF at 0 ° C, triethylamine (0.348 ml, 2.50 mmol) was added followed immediately by 228 (816 mg, 1 , 25 mmol) dissolved in 12 ml of THF. The reaction was allowed to stir at 0 ° C for ~ 5 minutes before allowing to warm to room temperature while stirring. The reaction was allowed to stir at room temperature for ~ 30 minutes. The reaction was reduced on silica. Next, silica gel chromatography was performed (gradient: 0% -45% acetone in heptans). Suitable test tubes were concentrated and placed under high vacuum to yield 229 (660 mg, 59%) as a white solid. LC-MS (Protocol B): m / z 895.3 [M + H] +, retention time = 2.21 minutes.
[1265]
[1266] Step 5: To a stirring solution of 229 (652 mg, 0.728 mmol) in 20 ml of methanol, 4M HCl in dioxane (20 ml, 80 mmol) was added. The reaction was allowed to stir at room temperature for ~ 24 minutes. The reaction was reduced and then placed under high vacuum. To a stirring solution of the crude material in 16 ml of dichloromethane and 16 ml of THF at 0 ° C, p-nitrophenyl chloroformate (191 mg, 0.946 mmol) was added followed immediately by triethylamine (0.508 ml, 3.64 mmol ). The reaction was allowed to stir at 0 ° C for ~ 5 minutes and then allowed to warm to room temperature while stirring. The reaction was allowed to stir at room temperature for ~ 10 minutes. The reaction was reduced. To a stirring solution of the crude material in 12 ml of dichloromethane, a solution of TFA (12 ml, 160 mmol) in 12 ml of dichloromethane was added followed by the addition of thiophenol (0.745 ml, 7.28 mmol) . The reaction was allowed to stir at room temperature for ~ 6 hours. The reaction was reduced. The crude material was diluted with a few milliliters of DMSO and then injected into a C18 25g pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 15% to 60% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac yielding 230 (267 mg , 34%, 3 stages) in the form of a light yellow solid. LC-MS (Protocol B): m / z 838.3 [M + H] +, retention time = 1.68 minutes.
[1267] Preparation of the trifluoroacetic acid salt of N- [1- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -21-oxo-3,6,9,12,15,18 -hexaoxahenicosan-21-yl] -L-valyl-N-5-carbamoyl-N- [4 - ({[(2 - {[({(8S) -8- (chloromethyl)) -6 - [(3- { [(1S) -1- (chloromethyl) -8-methyl-5- (phosphonooxy) -1,6-dihydropyrrolo [3,2-e] indole-3 (2H) -yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -1-methyl-3,6,7,8-tetrahydropyrrolo [3,2-e] indole-4-yl} oxy) carbonyl] (methyl) amino} ethyl) (methyl) carbamoyl ] oxy} methyl) phenyl] -L-ornithinamide (231).
[1268]
[1269]
[1270]
[1271]
[1272] Stage 1: To a 2 drachma vial (7.4 ml) containing 230 (90 mg, 0.11 mmol) and 215 (121 mg, 0.118 mmol), 3.0 ml of DMA was added followed by Hunig's base (0.0748 ml, 0.429 mmol), 2,6-Lutidine (0.0497 ml, 0.429 mmol) and HOAT (14.7 mg, 0.108 mmol). The reaction was allowed to stir at room temperature for ~ 15 minutes. The crude reaction was injected into a 12g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 45% acetonitrile in water with 0.02% TFA in each phase), followed by a second purification by procedure H and test tubes Suitable were concentrated using a genevac yielding 231 (117 mg, 60%) as a white solid. LC-MS (Protocol B): m / z 1607.8 [M + H] +, retention time = 1.60 minutes.
[1273]
[1274] Preparation of the trifluoroacetic acid salt of N-2-acetyl-N-6- (tert-butoxycarbonyl) -L-lysyl-L-valyl-N-5-carbamoyl-N- {4 - [({methyl [2- (methylamino) ethyl] carbamoyl} oxy) methyl] phenyl} -L-ornithinamide (236).
[1275]
[1276]
[1277] Step 1: To a stirring solution of compound 213 (16.0 g, 85.0 mmol) and Hunig's base (23 g, 178 mmol) in 450 ml of THF at 0 ° C, Fmoc-CI (22 g, 85.0 mmol) dropwise as a solution in 450 ml of THF. The mixture was stirred at 0 ° C for 10 minutes. The reaction was allowed to stir at room temperature overnight. The reaction was diluted with ethyl acetate and then washed with NH ⁴ Cl (aq.) And brine. The organic layer was separated, dried with Na ² SO ⁴ and reduced. The residue was purified by chromatography on silica (gradient: 2.5% -50% ethyl acetate in petroleum ether). Suitable test tubes were concentrated. The material was dissolved in 150 ml of ethyl acetate followed by the addition of 150 ml of HCl in ethyl acetate. The reaction was allowed to stir at room temperature overnight. The reaction was concentrated and 300 ml of MTBE was added. The resulting precipitate was collected by filtration to provide 232 (10.4 g, 42%, 2 steps) as a white solid. 1H NMR (400 MHz, DMSO-de), 88.89 (a, 2H), 7.91 (d, 2H), 7.66 (d, 2H) 7.42 (m, 2H), 7.36 ( m, 2H), 4.34 (m, 3H), 3.51 (m, 1H), 3.04 (m, 1H), 2.85 (s, 3H), 2.72 (m, 1H), 2.32 (m, 1 H).
[1278]
[1279] Step 2: To a solution of 217 (481 mg, 1.27 mmol) in 10 ml of DMF, 233 (366 mg, 1.27 mmol), HATu (660 mg, 1.65 mmol) and Hunig's base were added (0.302 ml, 1.6 mmol). The reaction was allowed to stir at room temperature for ~ 30 minutes. The reaction was diluted with ethyl acetate which caused the solids to precipitate. This suspension was allowed to stir for ~ 30 minutes. The solids were collected by filtration, rinsed with fresh ethyl acetate and dried under high vacuum to obtain 234 (797 mg, 97%) as a brown solid. LC-MS (Protocol B): m / z 650.3 [M + H] +, retention time = 0.64 minutes.
[1280]
[1281] Step 3: To a solution of compound 234 (18.5 g, 28.5 mmol) in DMF (500 ml), bis (4-nitrophenyl) carbonate (9.54 g, 31.4 mmol) was added followed by Hunig base (5.5 g, 42.8 mmol). The reaction was allowed to stir at room temperature for ~ 12 hours. The reaction was concentrated. The residue was purified by silica chromatography (gradient: 1% -10% methanol in dichloromethane to provide 235 (6.9 g, 29.7%) as a white solid. 1 H NMR (400 MHz, DMSO-de) , 88.30 (d, 2H), 8.12 (d, 1H), 8.01 (d, 1H), 7.70 (d, 1H), 7.64 (d, 2H), 7.56 ( d, 2H), 7.40 (d, 2H), 6.78 (m, 1H), 5.98 (m, 1H), 5.43 (s, 2H), 5.24 (s, 2H), 4.49 (m, 1H), 4.19 (m, 2H), 2.86 (m, 4H), 1.99 (m, 1H), 1.60 (m, 3H), 1.36 (m , 16H), 0.82 (m, 6H).
[1282]
[1283] Step 4: To a stirring solution of 235 (500 mg, 0.605 mmol) and 232 (210 mg, 0.605 mmol) in 3.0 ml of DMA, Hunig's base (0.316 ml, 1.82 mmol) was added. The reaction was allowed to stir at room temperature for ~ 30 minutes. Then piperidine (0.598 ml, 6.05 mmol) was added to the reaction. The reaction was allowed to stir at room temperature for an additional ~ 15 minutes. The crude reaction was injected into a 12g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 35% acetonitrile in water with 0.02% TFA in each phase). Suitable test tubes were concentrated using a genevac yielding 236 (475 mg, 89%, 2 steps) as a transparent white solid. LC-MS (Protocol B): m / z 764.4 [M + H] +, retention time = 1.03 minutes.
[1284]
[1285] Preparation of the trifluoroacetic acid salt of N ~ 2 ~ -acetyl-L-lysyl-L-valyl-N-5-carbamoyl-N- [4 - ({[(2 - {[({(8 (S)) -8- (chloromethyl) -6 - [(3 - {[(1S) -1- (chloromethyl) -8-methyl-5- (phosphonooxy) -1,6-dihydropyrrolo [3,2-e] indole-3 (2H) -yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -1-methyl-3,6,7,8-tetrahydropyrrolo [3,2-e] indole-4-yl} oxy) carbonyl] (methyl) amino} ethyl) (methyl) carbamoyl] oxy} methyl) phenyl] -L-ornithinamide (237).
[1286]
[1287]
[1288] To a 2 drachma vial (7.4 ml) containing 230 (100 mg, 0.119 mmol) and 236 (115 mg, 0.131 mmol), DMF (2.0 ml) was added followed by Hunig's base (0.0831 ml, 0.477 mmol), 2,6-Lutidine (0.0552 ml, 0.477 mmol) and HOAT (16.2 mg, 0.119 mmol). The reaction was allowed to stir at room temperature for ~ 10 minutes. The reaction was reduced. Dichloromethane (2 ml) was added to the crude sample. To this mixture under stirring was added TFA (1.0 ml, 13 mmol). The reaction was allowed to stir at room temperature for ~ 30 minutes. The reaction was reduced. The crude material was dissolved in DMSO and injected into a 12 g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 10% to 50% acetonitrile in water with 0.02% TFA in each phase), followed by a second purification by procedure G and test tubes Suitable were concentrated using a genevac yielding 237 (55.8 mg, 27%) as a white solid. LC-MS (Protocol B): m / z 1362.8 [M + H] +, retention time = 1.44 minutes. 1H NMR (400 MHz, DMSO-de): S 10.96-10.83 (m), 10.06-9.97 (m), 8.16 7.97 (m), 7.87-7, 66 (m), 7.59-7.47 (m), 7.37-6.97 (m), 6.54 (s), 6.05 (s), 5.47 (s), 5, 12-4.96 (m), 4.45-3.91 (m), 3.74 2.83 (m), 2.76-2.68 (m), 2.59-2.52 (m ), 2.39-2.32 (m), 2.02-1.93 (m), 1.83 (s), 1.71-1.21 (m), 0.88-0.77 ( m).
[1289]
[1290] Preparation of 3 - {[2 - ({[(2 - {[({(1S) -1- (chloromethyl)) -3 - [(3 - {[(1S) -1- (chloromethyl) -5- (phosphonooxy) ) -1,2-dihydro-3H-benzo [e] indole-3-yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -2,3-dihydro-1H-benzo [e] indole -5-yl} oxy) carbonyl] (methyl) amino} ethyl) (methyl) carbamoyl] oxy} methyl) phenyl] disulfanyl} -N- [6- (2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl) hexanoyl] -L-alanine (244)
[1291]
[1292]
[1293]
[1294]
[1295] Step 1: To a stirring mixture of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-cysteine 238 (17.9 g, 52.1 mmol) in dry ethanol (360 ml) at 0 ° C acetic acid (2.41 g, 40.1 mmol) was added. Then a solution of [2- (pyridin-2-yldisulfanyl) phenyl] methanol 239 (10 g, 40,104 mmol) in dry ethanol (200 ml) was added to the reaction mixture at 0 ° C. The mixture was stirred at room temperature for 20 minutes. The reaction mixture was concentrated in vacuo yielding a yellow oil. The residue was purified by preparative HPLC (method M) to produce a yellow gum (3.5 g). To a stirring solution of this crude material (2.5 g, 5.191 mmol) in dry dichloromethane (100 ml) at 0 ° C, bis (4-nitrophenyl) carbonate (1.9 g, 6.23) was added mmol) followed by Hunig's base (805 mg, 6.23 mmol). The mixture was stirred at 0 ° C for% hour, and then allowed to warm to room temperature. The reaction was allowed to stir at room temperature for ~ 23 hours. The reaction mixture was heated to 30 ° C and allowed to stir at 30 ° C for ~ 18 hours. The reaction was heated to 40 ° C and allowed to Stirring at 40 ° C for ~ 6 hours. The reaction mixture was washed with 1M HCl (20 ml x 2) and brine, dried over sodium sulfate and concentrated in vacuo to give the residue (3.89 g) as a yellow oil. The residue was purified by chromatography on silica gel (Gradient: 0% 4% methanol in dichloromethane) to yield a yellow solid (2.48 g). To a stirring solution of this crude material in THF (35 ml) at 0 ° C, 213 (635 mg, 3.37 mmol) was added followed by Hunig's base (793 mg, 6.14 mmol), 2, 6-lutidine (657 mg, 6.14 mmol) and HOAT (41.8 mg, 0.307 mmol). The reaction mixture was allowed to warm to room temperature and then allowed to stir at room temperature for 40 minutes. The reaction mixture was diluted with ethyl acetate (200 ml), washed with 1M HCl (30 ml, x2), and brine. The organic layer was dried with sodium sulfate and concentrated in vacuo to give the crude product (3.6 g) as a yellow oil. The crude product was purified by silica gel chromatography (Gradient: 0% 4% methanol in dichloromethane) to give the product (2.35 g) as a yellow gum. The product was then purified by preparative HPLC using (method M, using a gradient of 50% B to 80% B for 30 minutes, then 95% for 5 minutes). The mixture was concentrated in vacuo and extracted with ethyl acetate (100 ml, x3). The organic layers were combined, washed with brine, dried with sodium and concentrated in vacuo to give 240 (1.45 g, 7%, 3 steps) as a yellow gum. 1H NMR (400 MHz, DMSO-de): 87.91-7.89 (m, 3H), 7.74 7.72 (m, 3H), 7.44-7.31 (m, 7H), 5 , 14 (s, 2H), 4.34-4.24 (m, 4H), 3.31-3.29 (m, 3H), 3.10-3.09 (m, 1H), 3.04 -3.02 (m, 1H), 2.86-2.82 (d, 3H), 2.75-2.73 (m, 2H), 2.67-2.50 (m, 2H), 1 , 38-1.31 (m, 9H).
[1296]
[1297] Step 2: To a stirring solution of 240 (35 mg, 0.050 mmol) in 4 ml of dichloromethane, TFA (2 ml, 30 mmol) was added. The reaction was allowed to stir at room temperature for ~ 10 minutes. The reaction was concentrated in vacuo and placed under high vacuum to yield 24l as a white solid (40 mg, quant.). LC-MS (Protocol B): m / z 596.5 [M + H] +, retention time = 1.38 minutes.
[1298]
[1299] Stage 3: To a vial containing 241 (29.8 mg, 0.042 mmol) and 242 (1S) -1- (chloromethyl) -3 - [(3 - {[(1S) -1- (chloromethyl) - carbonate] 5- (phosphonooxy) -1,2-dihydro-3H-benzo [e] indole-3-yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -2,3-dihydro-1H-benzo [e] indole-5-yl 4-nitrophenyl [repared using the chemistry described in the preparation of 229], (35.0 mg, 0.042 mmol), 2.0 ml of Dm A was added followed immediately by Hunig's base ( 0.0293 ml, 0.168 mmol), 2,6-Lutidine (0.0195 ml, 0.168 mmol) and HOAT (5.72 mg, 0.042 mmol). The reaction was allowed to stir at room temperature for ~ 10 minutes. Then piperidine (0.30 ml, 3 mmol) was added to the reaction and the reaction was allowed to stir at room temperature for ~ 10 minutes. The crude reaction was injected into a 12g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 65% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac yielding 243 (30 mg , 60%) in the form of a gray solid. LC-MS (Protocol B): m / z 838.3 [M + 2H] +, retention time = 1.55 minutes.
[1300]
[1301] Step 4: To a stirring solution of 243 (20 mg, 0.017 mmol) and 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) pentafluorophenyl hexanoate (7.03 mg, 0.0186 mmol) in 1.5 ml of DMF, Hunig base (0.0118 ml, 0.0677 mmol) was added. The reaction was allowed to stir at room temperature for ~ 15 minutes. The crude reaction was injected into a C18 pre-column of 5 g (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 20% 70% acetonitrile in water with 0.02% TFA in each phase), followed by purification by preparative HPLC (method 11) and the tubes Suitable assays were concentrated using a genevac yielding 244 (0.8 mg, 4%) as a gray solid. LC-MS (Protocol D): m / z 630.8 [1/2 M + 1H] +, retention time = 10,786 minutes.
[1302]
[1303] Preparation of 3 - {[4 - ({[(2 - {[({(1S) -1- (chloromethyl)) -3 - [(3 - {[(1S) -1- (chloromethyl) -5- (phosphonooxy) ) -1,2-dihydro-3H-benzo [e] indole-3-yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -2,3-dihydro-1H-benzo [e] indole -5-yl} oxy) carbonyl] (methyl) amino} ethyl) (methyl) carbamoyl] oxy} methyl) phenyl] disulfanyl} -N- [6- (2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl) hexanoyl] -L-alanine 250
[1304]
[1305]
[1306] Step 1: To a stirring mixture of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-cysteine 238 (11.6 g, 33.7 mmol) in dry ethanol (230 ml) at 0 ° C acetic acid (1.93 g, 32.1 mmol) was added. A solution of [4- (pyridin-2-yldisulfanyl) fenM] methanol 245 (10 g, 40,104 mmol) in dry ethanol (160 ml) was then added to the reaction mixture at 0 ° C. The mixture was left at room temperature and then stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo yielding a yellow oil. The residue was purified by prep HPLC. (procedure M, using a gradient of 45% B to 75% B for 30 minutes, then 95% for 5 minutes) producing a yellow gum (8.5 g). To a stirring solution of this crude material (8.0 g, 16.61 mmol) in dry dichloromethane (320 ml) at 0 ° C, bis (4-nitrophenyl) carbonate (6.06 g, 19.9 mmol) followed by Hunig's base (2.58 g, 19.9 mmol). The mixture was stirred at 0 ° C for 10 minutes and then allowed to warm to room temperature. The reaction was allowed to stir at room temperature for ~ 15 hours. More bis (4-nitrophenyl) carbonate (1.52 g, 4.98 mmol) and Hunig base (644 mg, 4.98 mmol, 0.3 equiv.) Were added to the reaction mixture. The reaction was allowed to stir at room temperature for a further 2 hours. The reaction mixture was washed with 1M HCl (50 ml x 2) and brine, dried over sodium sulfate and concentrated in vacuo to give the residue (17.1 g) as a yellow oil. The residue was purified by chromatography on silica gel (Gradient: 0% 7% methanol in dichloromethane) to yield a yellow oil. To a stirring solution of this crude material in THF (103 ml) at 0 ° C, 171 (1.89 g, 10.0 mmol) was added followed by Hunig's base (2.36 g, 18.2 mmol ), 2,6-lutidine (1.96 g, 18.2 mmol) and HOAT (124 mg, 0.912 mmol). The reaction mixture was allowed to warm to room temperature and then allowed to stir at room temperature for 60 minutes. The reaction mixture was diluted with ethyl acetate (200 ml), washed with 1M HCl (30 ml, x2), and brine. The organic layer was dried with sodium sulfate and concentrated in vacuo to give the crude product (7.5 g) as a yellow oil. The crude product was purified by silica gel chromatography (Gradient: 0% 4% methanol in dichloromethane) to give the product (4.0 g) as a yellow gum. The product was then purified by (method M, using a gradient of 50% B to 80% B for 30 minutes, then 95% for 5 minutes). The mixture was concentrated in vacuo and extracted with ethyl acetate (100 ml, x3). The organic layers were combined, washed with brine, dried with sodium and concentrated in vacuo to give 246 (3.0 g, 13%, 3 steps) as a white solid. 1H NMR (400 MHz, DMSO-de): 87.89-7.87 (d, 2H), 7.71-7.70 (d, 2H), 7.55-7.52 (m, 2H), 7.50-7.41 (m, 2H), 7.39-7.30 (m, 4H), 4.97 (s, 2H), 4.30-4.22 (m, 4H), 3, 29 (a, 4H), 3.10-3.01 (m, 2H), 2.82-2.80 (d, 3H), 2.73 (s, 1H), 2.66 (s, 2H) , 1.32-1.30 (d, 9H).
[1307]
[1308] Step 2: To a stirring solution of 246 (499 mg, 0.717 mmol) in DMF 4.0, piperidine (1.13 ml, 11.5 mmol) was added. The reaction was allowed to stir at room temperature for ~ 5 minutes. The crude reaction was injected into a 12g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% 50% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac producing 3- { [4- (4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl) phenyl] disulfanyl} -L-alanine 247 (320 mg, 76 %) in the form of a gray solid. LC-MS (Protocol B): m / z 474.5 [M + H] +, retention time = 1.19 minutes.
[1309]
[1310] Step 3: To a stirring solution of 247 (140 mg, 0.238 mmol) and 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) pentafluorophenyl hexanoate (98.9 mg, 0.262 mmol), 2 ml of DMF was added followed immediately by Hunig's base (0.124 ml, 0.715 mmol). The reaction was allowed to stir at room temperature for ~ 5 minutes. The crude reaction was injected into a 12g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by chromatography on C18 medium pressure inverted phase (gradient: 10% to 70% acetonitrile in water with 0.02% TFA in each phase) and suitable test tubes were concentrated using a genevac producing N- [6- (2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] -3 - {[4- (4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4, 7-diazaundec-1-yl) phenyl] disulfanyl} -L-alanine 248 (56 mg, 35%) in the form of a transparent solid. LC-MS (Protocol B): m / z 667.3 [M + H] +, retention time = 1.71 minutes.
[1311]
[1312] Step 4: To a stirring solution of 248 (35 mg, 0.050 mmol) in 4 ml of dichloromethane, TFA (2 ml, 30 mmol) was added. The reaction was allowed to stir at room temperature for ~ 10 minutes. The reaction was concentrated in vacuo and placed under high vacuum to yield 249 as a white solid (40 mg, quant.).
[1313]
[1314] Step 4: To a vial containing 249 (18.0 mg, 0.0264 mmol) and 242 (22.0 mg, 0.0264 mmol), 1.6 ml of DMA was added followed immediately by Hunig's base (0 , 0184 ml, 0.106 mmol), 2,6-Lutidine (0.0123 ml, 0.106 mmol) and HOAT (3.60 mg, 0.0264 mmol). The reaction was allowed to stir at room temperature for ~ 10 minutes. The crude reaction was injected into a 12g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 10% to 60% acetonitrile in water with 0.02% TFA in each phase), followed by purification by preparative HPLC (procedure 12) and the tubes Suitable assays were concentrated using a genevac yielding 250 (16.7 mg, 50%) as a white solid. LC-MS (Protocol B): m / z 1261.4 [M + 3H] +, retention time = 1.71 minutes.
[1315]
[1316] Preparation of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] -L-valyl-N-5-carbamoyl-N- [4 - ({[( 2 - {[({(8S) -8- (chloromethyl) -6 - [(3 - {[(1S) -1- (chloromethyl) -8-methyl-5- (phosphonooxy) -1,6-dihydropyrrolo [ 3,2-e] indole-3 (2H) -yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -1-methyl-3,6,7,8-tetrahydropyrrolo [3,2- e] indole-4-yl} oxy) carbonyl] (methyl) amino} ethyl) (methyl) carbamoyl] oxy} methyl) phenyl] -L-ornithinamide 255.
[1317]
[1318]
[1319]
[1320]
[1321] Stage 1: N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-Valyl-N-5-carbamoyl-N- [4- (hydroxymethyl) phenyl] -L-omitinamide 251 (725 mg, 1.2 mmol) was dissolved in 6 ml of d Mf followed by sonication for ~ 10 minutes. A stir bar was then added and this solution was allowed to stir at room temperature. Then bis (4-nitrophenyl) carbonate (403 mg, 1.33 mmol) was added followed by Hunig's base (0.44 ml, 2.5 mmol). The reaction was allowed to stir at room temperature for ~ 5 hours. 213 (227 mg, 1.2 mmol) dissolved in 1 ml of DMF was added. The reaction was allowed to stir at room temperature for ~ 1 minute. The crude reaction was injected into a C18 pre-column of 24 g (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% to 60% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac producing N- [ (9H-fluoren-9-ylmethoxy) carbonyl] -L-valyl-N ~ 5 ~ -carbamoyl-N- [4- (4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa -4,7-diazaundec-1-yl) phenyl] -L-ornithinamide 252 (395 mg, 40%, 2 steps) in the form of a brown solid. LC-MS (Protocol B): m / z 816.7 [M + H] +, retention time = 1.88 minutes.
[1322] Step 2: To a stirring mixture of 252 (197 mg, 0.241 mmol) in 6 ml of dichloromethane, TFA (2 ml, 30 mmol) was added. The reaction was allowed to stir at room temperature for ~ 20 minutes. The reaction was concentrated in vacuo and placed under high vacuum yielding N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-valyl-N-5-carbamoyl-N- [4- (4,7,10, 10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl) phenyl] -L-ornithinamide 253 (210 mg, quant.) In the form of a white and brown solid Clear. LC-MS (Protocol B): m / z 716.7 [M + H] +, retention time = 1.27 minutes.
[1323]
[1324] Step 3: To a vial containing 230 (48 mg, 0.053 mmol) and 253 (52.4 mg, 0.063 mmol), 2.0 ml of DMA was added followed immediately by Hunig's base (0.036 ml, 0.211 mmol), 2,6-Lutidine (0.024 ml, 0.211 mmol) and HOAT (7.1 mg, 0.0525 mmol). The reaction was allowed to stir at room temperature for ~ 10 minutes. Piperidine (0.30 ml, 3 mmol) was then added and the reaction was allowed to stir at room temperature for ~ 10 minutes. The crude reaction was injected into a 12g C18 pre-column (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 10% to 50% acetonitrile in water with 0.02% TFA in each phase) and the appropriate test tubes were concentrated using a genevac yielding 254 (68 mg , 84%, 2 stages) in the form of a light gray solid. LC-MS (Protocol B): m / z 1193.5 [M + 2H] +, retention time = 1.46 minutes.
[1325]
[1326] Step 4: To a stirring solution of 254 (30 mg, 0.020 mmol) and 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) pentafluorophenyl hexanoate (8.11 mg, 0.0215 mmol) in 2.0 ml of DMF, Hunig's base (0.0136 ml, 0.0782 mmol) was added. The reaction was allowed to stir at room temperature for ~ 10 minutes. The crude reaction was injected into a C18 pre-column of 5 g (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% 50% acetonitrile in water with 0.02% TFA in each phase) followed by a second purification by preparative HPLC (method J1). Suitable test tubes were concentrated using a genevac yielding 255 (9.1 mg, 29%) as a light brown solid. LC-MS (Protocol B): m / z 1386.9 [M + 2H] +, retention time = 1.60 minutes.
[1327]
[1328] Preparation of N- (24-bromo-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatetracosan-1-oil) -L-Valyl-N-5-carbamoyl-N- [4 - ({[(2 - {[({(8S) -8- (chloromethyl) -6 - [(3 - {[(1S) -1- (chloromethyl) -8-methyl-5- (phosphonooxy) -1 , 6-dihydropyrrolo [3,2-e] indole-3 (2H) -yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -1-methyl-3,6,7,8-tetrahydropyrrolo [3,2-e] indole-4-yl} oxy) carbonyl] (methyl) amino} ethyl) (methyl) carbamoyl] oxy} methyl) phenyl] -L-ornithinamide 257.
[1329]
[1330]
[1331]
[1332]
[1333] To a stirring solution of 254 (30 mg, 0.020 mmol) and 1-bromo-2-oxo-6,9,12,15,18,21-hexaoxa-3-azatetracosan-24-pentafluorophenyl 255 6 (13) , 8 mg, 0.0215 mmol) [repared as described in WO2014 / 068443] in 2.0 ml of DMF, Hunig's base (0.0136 ml, 0.0782 mmol) was added. The reaction was allowed to stir at room temperature for ~ 40 minutes. The crude reaction was injected into a C18 pre-column of 5 g (which had previously been equilibrated with acetonitrile and then water, with 0.02% TFA in each phase). The material was purified by medium pressure C18 inverted phase chromatography (gradient: 5% 50% acetonitrile in water with 0.02% TFA in each phase) followed by a second purification by preparative HPLC (method K1). Suitable test tubes were concentrated using a genevac yielding 257 (10.8 mg, 26%) as a white solid. LC-MS (Protocol B): m / z 1649.7 [M + 3H] +, retention time = 1.53 minutes.
[1334]
[1335] Preparation of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-valyl-N - [(1S) -3 - [(3-carboxybicyclo [1.1.1] pent-1-yl) carbonyl] -1 - (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-yl] -L-alaninamide 261
[1336]
[1337]
[1338] Step 1: A stirring solution of 3 - {[(1S) -1- (chloromethyl) -5-nitro-1,2-dihydro-3H-benzo [e] indole-3-yl] carbonyl} bicide [1.1. 1] tere-butyl pentane-1-carboxylate 258 (prepared analogously to 189) (980 mg, 2.14 mmol) in 7 ml of THF under a nitrogen atmosphere was cooled to 0 C using an ice bath. Next, 10% palladium by weight on activated carbon (30 mg) was added followed by a slow dropwise addition of 2 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 C for 3 hours. After completion, the reaction mixture was filtered through a layer of celite and the filtrate was concentrated in vacuo. The crude products were purified by silica gel chromatography (Gradient: 0% to 10% MeOH in DCM) to give 3 - {[(1S) -5-amino-1- (chloromethyl) -1,2-dihydro- 3H-benzo [e] indole-3-yl] carbonyl} bicyclo [1.1.1] tere-butyl pentane-1-carboxylate 259 in the form of a yellow solid (905 mg, 98%). ^c L-MS (Protocol B): m / z 427 [M + H] +, retention time = 1.92 minutes.
[1339]
[1340] Step 2: To a stirring solution of 259 (900, 2.11 mmol) in 5 ml of anhydrous DCM was added (S) - (1-chloro-1-oxopropan-2-yl) carbamate of (9H-fluoren- 9-yl) methyl (695 mg, 2.11 mmol) followed by the dropwise addition of TEA (0.5 ml). The reaction was allowed to stir for 2 hours. After completion, the reaction mixture was concentrated in vacuo. The crude products were purified by silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane) to give 3 - {[(1S) -1- (chloromethyl) -5 - ({N - [( 9H-fluoren-9-ylmethoxy) carbonyl] -L-alanyl} amino) -1,2-dihydro-3H-benzo [e] indole-3-yl] carbonyl} bicyclo [1.1.1] pentane-1-carboxylate tere-butyl 260 in the form of a white solid (1,102 g, 73%). LC-MS (Protocol B): m / z 720 [M + H] +, retention time = 2.32 minutes.
[1341] Step 3: To a round bottom flask provided with stir bar containing 260 (1000 mg, 1.388 mmol) 15 ml of DCM 1: 1 in DEA was added. The solution was stirred for 3 hours. The reaction mixture was concentrated in vacuo and captured in 50% DCM in heptane and concentrated again in vacuo. This was repeated 3 times (to remove excess DEA) to give a white crude solid after concentrating. This white crude solid was added to a round bottom flask containing (((9H-fluoren-9-yl) methoxy) carbonyl) -L-valine (471 mg, 1.38 mmol) and HATU (350 mg , 1.38 mmol) in 10 ml of anhydrous DCM. TEA (0.5 ml) was then added and the reaction was stirred at room temperature for 3 hours. After completion, the reaction mixture was concentrated in vacuo. The crude products were purified by silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane) to give N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-valyl-N- [ (1S) -3 - {[3- (tere-butoxycarbonyl) bicyclo [1.1.1] pent-1-yl] carbonyl} -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole- 5-yl] -L-alaninamide 261 in the form of a white solid (1.005 g, 88%). LC-MS (Protocol B): m / z 819 [M + H] +, retention time = 2.31 minutes.
[1342]
[1343] Step 4: 10 ml of 25% TFA in DCM was added to a round bottom flask containing 261 (1000 mg, 1.22 mmol). The reaction was stirred for 3 hours. The solution was stirred for 3 hours. The reaction mixture was concentrated in vacuo and captured in 50% DCM and heptane and concentrated in vacuo. This was repeated 3 times (to remove excess TFA) to give 262 as a white solid after concentrating (920 mg, 98%). LC-MS (Protocol B): m / z 763 [M + H] +, retention time = 1.88 minutes.
[1344]
[1345] Preparation of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) hexanoyl] -L-valyl-N - {(1S) -1- (chloromethyl) -3- [(3 - {[(1S) -1- (chloromethyl) -8-methyl-5- (phosphonooxy) -1,6-dihydropyrrolo [3,2-e] indole-3 (2H) -yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -2,3-dihydro-1H-benzo [e] indole-5-yl} -L-alaninamide 266.
[1346]
[1347]
[1348] Stage 1: To a round bottom flask containing N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-valyl-N - [(1S) -3 - [(3-carboxycycle [1.1.1] pent -1-yl) carbonyl] -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-yl] -L-alaninamide 262 (580 mg, 0.76 mmol) in 5 ml of THF HATU (298 mg, 0.76 mmol) was added. The solution mixture was stirred at room temperature for 30 min. Then (1S) -5- (benzyloxy) -1- (chloromethyl) -8-methyl-1,2,3,6-tetrahydropyrrolo [3,2-e] indole 7 was added followed by 0.3 ml of base from Hunig. The reaction was stirred for 1 hour and concentrated to give a crude glass. The crude reaction mixture was purified by silica gel chromatography (Gradient: 0% to 10% MeOH in DCM) to give N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-Valyl-N- [ (1S) -3 - [(3 - {[(1S) -5 - {[bis (benzyloxy) phosphoryl] oxy} -1- (chloromethyl) -8-methyl-1,6-dihydropyrrolo [3,2-e ] indole-3 (2H) -yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-yl ] -L-alaninamide 263 in the form of a white solid (723 mg, 98%). LC-MS (Protocol B): m / z 1071 [M + H] +, retention time = 2.45 minutes.
[1349]
[1350] Step 2: A stirring solution of 263 (100 mg, 0.932 mmol) in 7 ml of THF under a nitrogen atmosphere was cooled to 0 C using an ice bath. Next, 10% palladium by weight on activated carbon (10 mg) was added followed by a slow dropwise addition of 0.5 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 C for 1 hour. After completion, the reaction mixture was filtered through a layer of celite and the filtrate was concentrated in vacuo. The crude products were purified by silica gel chromatography (Gradient: 0% to 10% MeOH in DCM) to give N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-valyl-N - {(1S ) -1- (chloromethyl) -3 - [(3 - {[(1S) -1- (chloromethyl) -5-hydroxy-8-methyl-1,6-dihydropyrrolo [3,2-e] indole-3 ( 2H) -yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -2,3-dihydro-1H-benzo [e] indole-5-yl} -L-alaninamide 264 in the form of a solid yellow (821 mg, 89%). %). LC-MS (Protocol B): m / z 981 [M + H] +, retention time = 2.16 minutes.
[1351]
[1352] Step 3: To a stirring solution of 264 (650 mg, 0.66 mmol) in 10 ml of THF and 10 ml of AcCN, carbon tetrachloride (2.04 ml, 21.0 mmol) was added followed by base of Hunig (1.12 ml, 6.45 mmol), dibenzyl phosphite (694 mg, 2.65 mmol) and DMAP (catalytic). The reaction was allowed to stir at room temperature for 20 minutes. The reaction was concentrated to give a crude glass. The crude reaction mixture was purified by silica gel chromatography (Gradient: 0% to 10% MeOH in DCM) to give N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-Valyl-N- [ (1S) -3 - [(3 - {[(1S) -5 - {[bis (benzyloxy) phosphoryl] oxy} -1- (chloromethyl) -8-methyl-1,6-dihydropyrrolo [3,2-e ] indole-3 (2H) -yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-yl ] -L-alaninamide 265 in the form of a white glass (502 mg, 66%). LC-MS (Protocol B): m / z 1243 [M + H] +, retention time = 2.46 minutes.
[1353]
[1354] Step 4: A stirring solution of 264 (100 mg, 0.932 mmol) in 7 ml of THF under a nitrogen atmosphere was cooled to 0 C using an ice bath. Next, 10% palladium by weight on activated carbon (10 mg) was added followed by a slow dropwise addition of 0.5 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 C for 1 hour. After completion, the reaction mixture was filtered through a layer of celite and the filtrate was concentrated in vacuo. The crude products were purified by silica gel chromatography (Gradient: 0% to 10% MeOH in DCM) to give L-Valyl-N - {(1S) -1- (chloromethyl) -3 - [(3 - {[(1S) -1- (chloromethyl) -8-methyl-5- (phosphonooxy) -1,6-dihydropyrrolo [3,2-e] indole-3 (2H) -yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -2,3-dihydro-1H benzo [e] indole-5-yl} -L-alaninamide 265 in the form of a yellow solid (25 mg, 18%). LC-MS (Protocol B): m / z 839 [M + H] +, retention time = 1.54 minutes.
[1355]
[1356] Step 5: To a round bottom flask equipped with a stir bar and 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) pentafluorophenyl hexanoate (18 mg, 0.046 mmol) was added 5 ml of anhydrous DCM and the system was purged with N2. To this solution were added 265 (40 mg, 0.046 mmol)) and TEA (0.05 ml). The system was left under stirring for 1 hour. The reaction was concentrated in vacuo and purified to provide 267 (20% 9 mg Procedure N), retention time = 15,462 minutes. LC-MS (Protocol B): m / z 1032 [M + H] +, retention time = 1.55 minutes. 1H NMR (400 MHz, DMSO-cfe) 5 11.34 (s, 1H), 9.89 (s, 1H), 8.43 (s, 1H), 8.20 (d, J = 6.8 Hz , 2H), 7.91 (dd, J = 14.4, 8.4 Hz, 3H), 7.85-7.74 (m, 2H), 7.49 (t, J = 7.7 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 6.96 (d, J = 25.4 Hz, 4H), 4.52 (t, J = 7.1 Hz, 1H) , 4.37 (dc, J = 22.0, 10.7 Hz, 4H), 4.18 (dt, J = 19.7, 8.5 Hz, 2H), 4.07-3.85 (m , 4H), 3.58 (t, J = 9.8 Hz, 1H), 3.43-3.12 (m, 34H), 2.71 (d, J = 8.2 Hz, 1H), 2 , 62 - 2.37 (m, 49H), 2.28 (s, 3H), 2.09 (ct, J = 14.0, 7.1 Hz, 3H), 1.98 - 1.86 (m , 1H), 1.39 (dt, J = 22.2, 7.2 Hz, 11H), 1.22 - 1.05 (m, 6H), 0.78 (dd, J = 9.7, 6 , 7 Hz, 10H).
[1357]
[1358] Preparation of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) hexanoyl] -L-valyl-N - {(1S) -1- (chloromethyl) -3- [(3 - {[(1S) -1- (chloromethyl) -5- (phosphonooxy) -1,2-dihydro-3H-benzo [e] indole-3-yl] carbonyl} bicyclo [1.1.1] pent- 1-yl) carbonyl] -2,3-dihydro-1H-benzo [e] indole-5-yl} -L-alaninamide 270
[1359]
[1360]
[1361]
[1362]
[1363] Step 1: 226 (214 mg, 0.36 mmol) was taken up in CH2Cl2 (2 ml) and TFA (0.5 ml) was added and after completing the deprotection the solvent was removed. To a round bottom flask purged with N2, containing 262 (200 mg, 0.26 mmol)) in 5 ml of anhydrous DCM was added oxalyl chloride (0.024 ml, 0.26 mmol). To this solution was added 1 drop of DMF and the system was stirred for 3 hours. The reaction was concentrated in vacuo. The residue was taken up in DCM and added to a round bottom flask containing 226 unprotected in 15 ml of DCM and TEA (0.144 ml). The reaction was stirred at room temperature for 2 hours. The crude reaction mixture was concentrated in vacuo and captured in 25 ml of DCM and transferred to a separatory funnel. The organic layer was washed with 1M HCl (3x), water (3 x), and brine (2 x). The organic layer dried with Na 2 SO ⁴ is filtered, and the filtrate was concentrated to give a crude solid. The crude products were purified by silica gel chromatography (Gradient: 0% to 10% MeOH in DCM) to give N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-Valyl-N - [(1S ) -3 - [(3 - {[(1S) -5 - {[bis (benzyloxy) phosphoryl] oxy} -1- (chloromethyl) -1,2-dihydro-3H-benzo [e] indole-3-yl ] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-5-yl] -L-alaninamide 268 in the form of a yellow solid (75 mg, 23%). LC-MS (Protocol B): m / z 1238 [M + H] +, retention time = 2.53 minutes.
[1364]
[1365] Step 2: A stirring solution of 268 (75 mg, 0.061 mmol) in 5 ml of THF in a nitrogen atmosphere was cooled to 0 C using an ice bath. Next, 10% palladium by weight on activated carbon (5 mg) was added followed by a slow dropwise addition of 0.5 ml of 25% ammonium formate in water. The reaction was allowed to stir at 0 C for 3 hours. After completion, the reaction mixture was filtered through a layer of celite and the filtrate was concentrated in vacuo. The crude product was taken up in ethyl acetate and the solids filtered to give L-Valyl-N - {(1S) -1- (chloromethyl) -3 - [(3 - {[(1S) -1- (chloromethyl ) -5- (phosphonooxy) -1,2-dihydro-3H-benzo [e] indole-3-yl] carbonyl} bicyclo [1.1.1] pent-1-yl) carbonyl] -2,3-dihydro-1H -benzo [e] indole-5-yl} -L-alanine 269 in the form of a light yellow solid (20 mg, 30%). %). C ^l -EM (Protocol B): m / z 838 [M + H] +, retention time = 1.27 minutes.
[1366]
[1367] Stage 3: To a round bottom flask equipped with a stir bar and 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) pentafluorophenyl hexanoate (9.0 mg, 0.024 mmol) 5 ml of anhydrous d Cm was added and the system was purged with N2. To this solution were added 269 (20 mg, 0.024 mmol)) and TEA (0.05 ml). The system was left under stirring for 1 hour. The reaction was concentrated in vacuo and purified by HPLC. Procedure N provided 270 (5 mg, 20%) retention time = 10,734 minutes. CL-E ^m (Protocol B): m / z 1031 [M + H] +, retention time = 1.54 minutes.
[1368] Preparation of Acid Reference Example (2S, 3S, 4S, 5R, 6S) -6 - (((S) -1- (chloromethyl) -3- (5 - ((S) -1- (chloromethyl) -5 - (((2 (( ((4 - ((23S, 26S) -1- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) -23-isopropyl-21,24-dioxo-26- (3- ureidopropyl) -3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido) benzyl) oxy) carbonyl) (methyl) amino) ethyl) (methyl) carbamoyl) oxy) -2, 3-dihydro-1H-benzo [e] indole-3-carbonyl) thiophene-2-carbonyl) -2,3-dihydro-1H-benzo [e] indole-5-yl) oxy) -3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic 278.
[1369]
[1370]
[1371]
[1372]
[1373] Step 1. (1S) -1- (Chloromethyl) -5-hydroxy-1,2-dihydro-3H-benzo [e] indole-3-carboxylic acid ferc-butyl 3 (683 mg, 2.05 mmol) was dissolved in DCM (70 ml), 4A MS (3.8 g, powder, <5 micro, activated) was added, and the mixture was stirred at room temperature for 30 min. To the reaction mixture, alpha-D-glucuronide methyl ester 2,3,4-triacetate 1-2,2,2-trichloroethaneimidate 271 (1178 mg, 2.45 mmol) was added and cooled to -15 ° C. A solution of BF ³ Et ² O (0.13 ml, 1.02 mmol) in DCM (10 ml) was added slowly, and the reaction mixture was stirred below -20 ° C for 1 h. To the mixture, a solution of BF ³ Et ² O (0.76 ml, 6 mmol) in DCM (10 ml) was added to remove the Boc group, and the reaction mixture was allowed to warm to rt for 2 h . The mixture was filtered through a layer of Celite, and the filtrate was concentrated to give a green (sticky) foam. 4M HCl (2 ml) was added, and concentrated again to give a green foam as a crude product 272, 1130 mg (94%), which was used in the next step without further purification.
[1374]
[1375] Step 2. Mono-tBu diacid thiophene ester 187 (189 mg, 0.83 mmol) was dissolved in THF (10 ml), cooled to 0C, and oxalyl chloride (2 M in DCM, 0.8 ml) was added , 1.6 mmol), followed by DMF (2 drops). The mixture was stirred at 0C for 5 min, and then at room temperature for 1 h. It was concentrated in vacuo to give the corresponding acid chloride as an off-white solid. The above solids were mixed with 272 (246 mg, 0.42 mmol) and treated with THF (10 ml) at 0C, followed by Et3N (0.29 ml, 2 mmol). The mixture was stirred at 0C for 5 min, and room temperature for 30 min. The mixture was concentrated, and the residue was purified by silica gel column chromatography using EA / Hep (50/50) to give the product as a yellow solid 273 (302 mg, 90%) LC-MS : 760.1.
[1376]
[1377] Step 3. 273 (790 mg, 1.04 mmol) was treated with TFA (2 ml) and DCM (4 ml) at rt for 1 h, concentrated to give a yellow solid. The solid was dissolved in THF (10 ml), cooled to 0C, oxalyl chloride (2 M in DCM, 1 ml, 2 mmol) was added, followed by DMF (1 drop). The mixture was stirred at 0C for 5 min, and then at rt for 1 h. It was concentrated to give the acid chloride as a yellow solid. 3 (118 mg, 1.56 mmol) was treated with 4M HCl (4 ml) for 1 h. it was concentrated in vacuo to give the Boc compound as a green solid. It was dissolved in THF (10 ml), a solution of the above acid chloride in THF (10 ml) was added at 0C, followed by the addition of Et3N (0.58 ml, 4.16 mmol), and the mixture was stirred at rt for 30 min. The mixture was diluted with EA, washed with water and brine, dried with MgSO4. It was concentrated in vacuo, and the residue was treated with MeOH, the resulting solid was collected by filtration to give the product as a yellow solid 274 (668 mg, 70%). LC-MS: 919.1 Step 4. 274 (576 mg, 0.63 mmol) was dissolved in THF (20 ml), cooled to 0 C, a solution of paranitrophenyl chloroformate (263 mg, 1.26 was added mmol) in DCM (2 ml), followed by Et3N (0.52 ml, 3.76 mmol). The mixture was stirred at 0C for 5 min, and then at rt for 2 h. LC-MS indicated completion of carbonate formation. 213 (354 mg, 1.88 mmol) in THF (2 ml) was added to the above mixture, and stirred at rt for 30 min. The mixture was diluted with EtOAc, washed with water and brine, and dried with MgSO4. It was concentrated in vacuo to give a solid residue, which was treated with MeOH to form precipitates. The resulting solid was collected by filtration to give the product as a yellow solid 275 (550 mg, 77%).
[1378] Step 5. 275 (550 mg, 0.48 mmol) was dissolved in THF / MeOH (1/1, 10 mL), cooled to 0C, a solution of UOHH ² O (206 mg, 4.8 mmol) was added in water (3 ml), and the mixture was stirred at 0C for 1 h. HOAc (300 mg) was added to neutralize the above solution, concentrated in vacuo. The residue was purified by Gilson HPLC (0.02 % TFA) to give the product as a yellow solid 276 (243 mg, 50%).
[1379]
[1380] Step 6. 276 (50 mg, 0.05 mmol) was treated with previously cooled TFA (2 ml) at 0C for 5 min, and concentrated in vacuo to give a Boc compound as a yellow solid. The above solid was dissolved in DMF (2 ml), 277 (48 mg, 0.05 mmol) was added, followed by lutidine (0.035 ml, 0.3 mmol), DIPEA (0.052 m, 0.3 mmol) and HOAt (7 mg, 0.05 mmol). The mixture was stirred at 30 ° C for 7 h. The crude product was subjected to Gislon HPLC (0.02 TFA) to give product 278 as a yellow solid 39 mg (45%). LC-MS: 1715.8 / 1737.8 (1.71 min at Larry); 1713.7 (-).
[1381]
[1382] Preparation of the acid Reference Example (2S, 3S, 4S, 5R, 6S) -6 - (((S) -3- (5 - ((S) -5 - (((2 - ((((4- ( (S) -2 - ((S) -2 - ((S) -2-acetamido-6-aminohexanamido) -3-methylbutanamido) -5-ureidopentanamido) benzyl) oxy) carbonyl) (methyl) amino) ethyl) ( methyl) carbamoyl) oxy) -1- (chloromethyl) -2,3-dihydro-1H-benzo [e] indole-3-carbonyl) thiophene-2-carbonyl) -1- (chloromethyl) -2,3-dihydro- 1H-benzo [e] indole-5-yl) oxy) -3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic 280
[1383]
[1384]
[1385]
[1386]
[1387] 276 was treated with TFA (2 ml) at 0C for 1 h. He concentrated in vacuo to give it. It was dissolved in DMF (2 ml), 279 (59 mg, 0.07 mmol) was added, followed by lutidine (0.033 ml, 0.29 mmol), DIPEA (0.051 ml, 0.29 mmol) and HOAt (7 mg , 0.05 mmol). The mixture was stirred at 30 C for 4 h. It was concentrated, and the residue was purified by Gilson HPLC (0.02% TFA) to give the product as a yellow solid 48 mg (62%). It was treated with previously cooled TFA (1.5 ml) for 5 min, then concentrated in vacuo to give the crude product as a yellow solid. The crude product was purified by Gilson HPLC (0.02% TFA) to give product 280 as a yellow powder after cryo drying (21 mg, 43%). LC-MS: 1470.6
[1388]
[1389] Preparation of Reference Example (S) -1- (chloromethyl) -3- (5 - ((S) -1- (chloromethyl) -5 - (((2S, 3R, 4S, 5R, 6R) -3.4 , 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) -2,3-dihydro-1H-benzo [e] indole-3-carbonyl) thiophene-2-carbonyl) -2, 3-dihydro-1H-benzo [e] indole-5-yl (4 - ((23S, 26S) -1- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) -23 -isopropyl-21,24-dioxo-26- (3-ureidopropyl) -3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido) benzyl) ethane-1,2-diylbis (methylcarbamate)
[1390]
[1391]
[1392] Step 1.3 (775 mg, 2.3 mmol) was dissolved in DCM (80 ml), 4A MS (6.2 g, power, <5 micro, activated) was added, and the mixture was stirred at room temperature for 30 min . To the reaction mixture, alpha-D-galactopane, 1-2,2,2-2,3,4,6-tetraacetate 281 trichloroethaneimidate (1260 mg, 2.3 mmol) was added and cooled to -15 ° C. A solution of BF ³ Et2O (0.144 ml, 1.2 mmol) in DCM (10 ml) was added slowly, and the reaction mixture was stirred at -15 ° C -20 ° C for 1 h. The reaction mixture was filtered through a layer of Celite, and the filtrate was concentrated. The crude product was purified by ISCO using MeOH / DCM (0-20%) to give the product as a 282 green solid (1400 mg, 91%).
[1393]
[1394] Step 2. Mono-tBu diacid thiophene ester 187 (300 mg, 1.3 mmol) was dissolved in THF (10 ml), cooled to C0C, and oxalyl chloride (2 M in DCM, 1 ml, 2 mmol) was added ), followed by DMF (2 drops). The mixture was stirred at 0C for 5 min, and then at room temperature for 1 h. It was concentrated in vacuo to give the corresponding acid chloride as a white solid. 282 (664 mg, 1 mmol) was treated with 4M HCl (4 ml) for 1 h at room temperature. It was concentrated in vacuo to give the green deBoc amine solid. The above solids were mixed with THF (10 ml) at 0C, Et ³ N (0.83 ml, 6 mmol) was added. The mixture was stirred at 0C for 5 min, and room temperature for 30 min. The mixture was diluted with EtOAc, washed with water and brine, dried with MgSO ⁴ . It was concentrated in vacuo, and the residue was treated with MeOH, and concentrated again to give a solid residue, which was recrystallized from MeOH. The resulting yellow solid was collected by filtration to give the product as a yellow solid 283 (500 mg, 65%).
[1395]
[1396] Step 3. 283 (200 mg, 0.26 mmol) was dissolved in THF (6 ml), oxalyl chloride (0.64 ml, 2M in DCM) was added at 0 ° C, followed by DMF (2 drops). The mixture was stirred at 0 ° C for 5 min, then at room temperature for 0.5 h. It was concentrated in vacuo to give the corresponding acid chloride as a yellow solid.
[1397] 3 (138 mg, 0.41 mmol) was treated with 4M HCl (1 ml in dioxane) for 2 h. It was concentrated in vacuo to give the deBoc amine in a green form. This was dissolved in THF (5 ml), the above acid chloride in THF (5 ml) was added at 0 ° C, followed by Et3N (0.23 ml, 1.55 mmol). The mixture was stirred at 0 ° C for 5 min, then at room temperature for 1 h. The mixture was diluted with EtOAc, washed with water and brine, dried over MgSO4. It was concentrated in vacuo to give a solid residue, which was treated with MeOH, and the resulting solid was collected by filtration and washed with ether to give the product as a yellow solid. The filtrate was concentrated, and purified by separation with Gilson HPLC using ACN / water (0.02% TFA) to give the product as a yellow solid 284 (200 mg, 83%).
[1398]
[1399] Step 4. 284 (68 mg, 0.073 mmol) was dissolved in THF (3 ml), cooled to 0 ° C, a solution of 4-nitrophenyl chloroformate (46 mg, 0.22 mmol) in DCM (0 , 6 ml), followed by Et3N (0.061 ml, 0.44 mmol). The mixture was stirred at 0 ° C for 5 min, and at room temperature for 1 h to provide 285. To the above reaction mixture was added N-Boc DMEDA (55 mg, 0.29 mmol) and stirred at room temperature for 1 h more. It was concentrated in vacuo, and the residue was purified by Gilson HPLC to give the product as a yellow solid 286 (65 mg, 78%).
[1400]
[1401] Step 5. 286 (10 mg, 0.009 mmol) was dissolved in MeOH (1 ml) at 0C, MeONa (0.054 ml, 0.5M in MeOH, 0.027 mmol) was added, and the mixture was stirred at 0C for 5 min. The mixture was neutralized with HOAc (0.4 ml, 0.1M in MeOH), and concentrated in vacuo to give the product as a yellow solid. TFA previously cooled (0.8 ml) was treated for 2 min, and concentrated in vacuo to give the compound of Bac as a ^yellow solid 287 ^{(8.3 mg, 90%).}
[1402] Step 6. 287 (8.3 mg, 0.008 mmol) was dissolved in DMF (1 ml), Malc-Peg6C2ValCitPABC (9.6 mg, 0.01 mmol) was added, followed by Lutidine (0.004 ml), DIPEA (0.006 ml) and HOAt (1.1 mg, 0.008 mmol). The mixture was stirred at room temperature for 4 h. The crude product was purified by Gilson HPLC (0.02% TFA) to give product 288 as a yellow solid (4 mg, 30%). 1H NMR (400 MHz, METHANOL-d ⁴ ) 0 = 8.42 (d, J = 8.2 Hz, 1H), 8.15 (d, J = 7.4 Hz, 1H), 7.95 (d , J = 8.2 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 8.6 Hz, 1H), 7.80 - 7.66 (m, 2H), 7.63-7.48 (m, 4H), 7.43 (sa, 3H), 7.23 (d, J = 7.8 Hz, 1H), 6.81 (s, 2H), 5.26-5.12 (m, 2H), 5.09 (d, J = 8.6 Hz, 1H), 4.71-4.54 (m, 4H), 4.49 (sa , 1H), 4.33-4.15 (m, 3H), 4.10-3.95 (m, 4H), 3.93-3.77 (m, 6H), 3.77-3.64 (m, 8H), 3.64-3.54 (m, 24H), 3.51 (sa, 1H), 3.23-3.03 (m, 5H), 3.03-2.95 (m , 2H), 2.60-2.51 (m, 2H), 2.13 (d, J = 7.0 Hz, 1H), 1.90 (sa, 1H), 1.72 (sa, 1H) , 1.57 (sa, 2H), 0.99 (t, J = 6.4 Hz, 6H). LC-MS: 1702.3 / 829.9 / 748.7
[1403]
[1404] Preparation of Reference Example ((S) -1- (chloromethyl) -3- (5 - ((S) -1- (chloromethyl) -5 - (((2S, 3R, 4S, 5R, 6R) -3, 4,5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) -2,3-dihydro-1H-benzo [e] indole-3-carbonyl) thiophene-2-carbonyl) -2 , 3-dihydro-1H-benzo [e] indole-5-yl) ethane-1,2-diylbis (methylcarbamate) of 4 - ((23S, 26S) -1-amino-23-isopropyl-21,24-dioxo -26- (3-ureidopropyl) -3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido) benzyl 289
[1405]
[1406]
[1407]
[1408]
[1409] Step 1. BocValCitPABC 287 (30.9 mg, 0.048 mmol) was added to a solution of 286 (32 mg, 0.032 mmol) in DMF (2 ml), followed by lutidine (0.015 ml), DIPEA (0.022 ml) and HOAt (4.4 mg). The mixture was stirred at rt for 5 h. The crude product was subjected to Gilson HPLC separation (0.02% TFA) to give the product as a yellow solid 288 (32 mg, 72%).
[1410]
[1411] Step 2. 288 (16 mg, 0.012 mmol) was treated with previously cooled TFA (1 ml) for 5 min, and concentrated in vacuo to give the compound of Bac as a yellow solid. The above solid was dissolved in DMF (0.5 ml), DIPEA (0.013 ml) was added, followed by a solution of 206 (12 mg, 0.016 mmol) in DCM (0.1 ml). The mixture was stirred at room temperature for 1 h. To the above solution was added piperidine (0.2 ml), and stirred for 30 min. It was concentrated in vacuo, the residue was purified by Gilson HPLC using ACN / water (0.02% TFA) to give the product 289 as a yellow solid (8 mg, 40%). 1H NMR (400 MHz, DMSO-cfe) 5 = 9.89 (sa, 1H), 8.35-8.21 (m, 1H), 8.15-8.00 (m, 2H), 7.96 (d , J = 7.8 Hz, 1H), 7.91-7.84 (m, 1H), 7.84-7.72 (m, 4H), 7.63 (sa, 2H), 7.58 - 7.50 (m, 3H), 7.50-7.44 (m, 2H), 7.40 (d, J = 6.2 Hz, 2H), 7.18 (sa, 2H), 5.90 (sa, 1H), 5.06-4.90 (m, 2H), 4.87 (d, J = 7.4 Hz, 1H), 4.83-4.68 (m, 2H), 4, 42 (t, J = 12.3 Hz, 2H), 4.32 (sa, 2H), 4.23 (sa, 1H), 4.19-4.11 (m, 1H), 4.10-3 , 95 (m, 3H), 3.95-3.80 (m, 2H), 3.77-3.62 (m, 3H), 3.60-3.46 (m, 15H), 3.15 (sa, 2H), 3.06 (sa, 1H), 2.89 (d, J = 5.1 Hz, 3H), 2.92 (d, J = 5.1 Hz, 3H), 2.86 - 2.74 (m, 3H), 2.35 - 2.21 (m, 1H), 1.96-1.82 (m, 1H), 1.61 (sa, 1H), 1.52 (sa , 1H), 1.43 -1.21 (m, 2H), 0.76 (d, J = 6.6 Hz, 3H), 0.79 (d, J = 6.2 Hz, 3H); 1622.2 [M + H] +;
[1412]
[1413] (continuation)
[1414]
[1415] (continuation)
[1416]
[1417] (continuation)
[1418]
[1419] (continuation)
[1420]
[1421] (continuation)
[1422]
[1423]
[1424] (continuation)
[1425]
[1426] (continuation)
[1427]
[1428]
[1429] The invention also provides the compounds described in Tables 5A and 5B.
[1430]
[1431] (continuation)
[1432]
[1433] (continuation)
[1434]
[1435]
[1436]
[1437]
[1438]
[1439]
[1440]
[1441] HPLC and CL-EM conditions used for analysis
[1442]
[1443] Protocol A: Column: Waters Acquity UPLC HSS T3, 2.1 mm x 50 mm, C18, 1.7 ^ m; Mobile phase A:: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B for 0.1 minute, 5% to 95% B for 0.9 minutes, 95% B for 0.1 minute; Flow rate: 1.25 ml / minute. Temperature: 60 ° C; Detection: 200-450 nm; Range of e M (+) 100-2000 Da; Injection volume: 5 ^ l; Instrument: Waters Acquity.
[1444]
[1445] Protocol B: Column: Waters Acquity UPLC HSS T3, 2.1 mm x 50 mm, C18, 1.7 ^ m; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B for 0.1 minute, 5% to 95% B for 2.5 minutes, 95% B for 0.35 minute; Flow rate: 1.25 ml / minute. Temperature: 60 ° C; Detection: 200-450 nm; MS interval (+) 100-2000 Da; Injection volume: 5 pl; Instrument: Waters Acquity.
[1446]
[1447] Protocol C: Column: Phenomenex Luna C18 (2), 150 x 3.0 mm, 5 pm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 50% B for 1.5 minutes, 50% to 100% B for 6.5 minutes, then 100% B for 3 minutes; Flow rate: 0.75 ml / minute. Temperature: 45 ° C; Detection: DAD 215 nm, 254 nm; MS interval (+) 150-2000 Da; Injection volume: 10 pl; Instrument: Agilent 1200 LCMS.
[1448]
[1449] Protocol D: Column: Phenomenex Luna C18 PFP (2), 150 x 3.0 mm, 5 pm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 0% to 5% B for 1.5 minutes, 5% to 100% B for 8.5 minutes, then B 100% for 2 minutes; Flow rate: 0.75 ml / minute. Temperature: not controlled; Detection: DAD 215 nm, 254 nm; MS interval (+) 150-2000 Da; Injection volume: 10 pl; Instrument: Agilent 1200 LCMS.
[1450]
[1451] Protocol E: Column: Phenomenex Luna C18 PFP (2), 150 x 3.0 mm, 5 pm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B for 1.5 minutes, 5% to 100% B for 8.5 minutes, then 100% B for 2 minutes; Flow rate: 0.75 ml / minute. Temperature: not controlled; Detection: DAD 215 nm, 254 nm; MS interval (+) 150-2000 Da; Injection volume: 10 pl; Instrument: Agilent 1200 LCMS.
[1452]
[1453] Protocol F: Column: Xtimate C18, 30 x 2.1 mm, 3 pm; Mobile phase A: 0.037% TFA in water (v / v); Mobile phase B: 0.037% TFA in acetonitrile (v / v); Gradient: 10% B for 0.1 minute, 10% to 80% B for 3 minutes, then B 80% for 0.1 minutes; Flow rate: 1.5 ml / minute. Temperature: 40 ° C; Detection: DAD 220 nm; MS interval (+) 100-1000 Da; Injection volume: 3 pl; Instrument: Shimadzu
[1454]
[1455] Protocol G: Column: Xtimate C18, 30 x 2.1 mm, 3 pm; Mobile phase A: 0.037% TFA in water (v / v); Mobile phase B: 0.037% TFA in acetonitrile (v / v); Gradient: 10% B for 0.1 minute, 10% to 80% B for 3 minutes, then B 80% for 0.1 minutes; Flow rate: 1.5 ml / minute. Temperature: 40 ° C; Detection: DAD 220 nm; MS interval (+) 100-1000 Da; Injection volume: 3 pl; Instrument: Shimadzu
[1456]
[1457] Protocol H: Column: Xtimate C18, 30 x 2.1 mm, 3 pm; Mobile phase A: 0.037% TFA in water (v / v); Mobile phase B: 0.037% TFA in acetonitrile (v / v); Gradient: 0% B for 0.1 minute, 0% to 60% B for 2 minutes, then B 60% for 0.1 minutes; Flow rate: 1.5 ml / minute. Temperature: 40 ° C; Detection: DAD 220 nm; MS interval (+) 100-1000 Da; Injection volume: 2 pl; Instrument: Shimadzu
[1458]
[1459] HPLC conditions used for purification
[1460]
[1461] Procedure A: Column: Phenomenex Luna C18 (2), 150 x 21.2 mm, 5 pm; Mobile phase A: 0.02% formic acid in water; Mobile phase B: 0.02% formic acid in acetonitrile; Gradient: 40% B for 1.5 minutes, 40% to 100% B for 8.5 minutes, 100% B for 0.5 minutes; Flow rate: 27 ml / minute; Detection: DAD 215 nm, 254 nm; MS interval (+) 150-2000 Da; Instrument: Waters FractionLynx.
[1462]
[1463] Procedure B: Column: Phenomenex Luna PFP (2), 150 x 21.2 mm, 5 pm; Mobile phase A: 0.02% formic acid in water; Mobile phase B: 0.02% formic acid in acetonitrile; Gradient: 30% B for 1.5 minute, 30% to 60% B for 8.5 minutes, B 60% to 100% B for 0.5 minutes, 100% B for 2 minutes; Flow rate: 27 ml / minute; Detection: DAD 215 nm, 254 nm; MS interval (+) 150-2000 Da; Instrument: Waters FractionLynx.
[1464]
[1465] Procedure C: Column: Phenomenex Synergi Polar RP, 150 x 21.2 mm, 4 pm; Mobile phase A: 0.02% formic acid in water; Mobile phase B: 0.02% formic acid in acetonitrile; Gradient: 20% B for 1.5 minute, 20% to 50% B for 8.5 minutes, 50% B to 100% B for 0.5 minutes, 100% B for 2 minutes; Flow rate: 27 ml / minute; Detection: DAD 210-360 nm; MS interval (+) 150-2000 Da; Instrument: Waters FractionLynx.
[1466]
[1467] Procedure D: Column: Xtimate C18, 30 x 2.1 mm, 3 pm; Mobile phase A: 0.2% TFA in water (v / v); Mobile phase B: 0.2% TFA in acetonitrile (v / v); Gradient: 25% B for 1.5 minutes, 25% to 50% B for 25 minutes, then 100% B for 5.0 minutes; Flow rate: 90 ml / minute. Temperature: not controlled; Detection: DAD 220 nm; MS interval (+) 100-1000 Da; Instrument: Shimadzu
[1468]
[1469] Procedure E: Column: LUNA C18, 250 x 50 mm, 10 pm; Mobile phase A: 0.2% TFA in water (v / v); Mobile phase B: 0.2% TFA in acetonitrile (v / v); Gradient: 25% B for 1.5 minutes, 25% to 55% B for 25 minutes, then 100% B for 5.0 minutes; Flow rate: 90 ml / minute. Temperature: not controlled; Detection: DAD 220 nm; MS interval (+) 100-1000 Da; Instrument: Shimadzu
[1470]
[1471] Procedure F: Column: Phenomenex Luna C18 (2), 250 x 50 mm, 10 pm; Mobile phase A: 0.2% TFA in water (v / v); Mobile phase B: 0.2% TFA in acetonitrile (v / v); Gradient: 35% to 65% of B for 30 minutes, then 100% B for 5.0 minutes; Flow rate: 90 ml / minute. Temperature: not controlled; Detection: DAD 220 nm; MS interval (+) 100-1000 Da; Instrument: Shimadzu
[1472]
[1473] Procedure G ^: Column: Phenomenex Luna C18 (2), 250 x 50 mm, 10 | jm; Mobile phase A: 0.2% TFA in water (v / v); Mobile phase B: 0.2% TFA in acetonitrile (v / v); Gradient: 10% B for 1.5 minutes, 10% B to 55% B for 8.5 minutes, 55% B to 100% B for 0.5 minutes, then hold with 100% B for 1.5 minutes; Flow rate: 27 ml / minute. Temperature: not controlled; Detection: DAD 210-360 nm; MS interval (+) 150-2000 Da; Instrument: 305 RP Waters Fractional Lynx LCMS Procedure H ^: Column: Phenomenex Luna C18 (2), 150 x 21.2 mm, 5 jm; Mobile phase A: 0.2% TFA in water (v / v); Mobile phase B: 0.2% TFA in acetonitrile (v / v); Gradient: 10% B for 1.5 minutes, 10% B to 75% B for 8.5 minutes, after 75% B to 100% B B for 2.0 minutes; Flow rate: 27 ml / minute. Temperature: not controlled; Detection: DAD 210 360 nm; MS interval (+) 150-2000 Da; Instrument: 305 RP Waters Fractional Lynx LCMS.
[1474]
[1475] Procedure H ^{1 :} Column: Phenomenex Luna C18 (2), 150 x 21.2 mm, 5 jm; Mobile phase A: 0.2% TFA in water (v / v); Mobile phase B: 0.2% TFA in acetonitrile (v / v); Gradient: 1% of B for 1.5 minutes, 1% B to 100% B for 8.5 minutes, then 100% B for 2.0 minutes; Flow rate: 27 ml / minute. Temperature: not controlled; Detection: DAD 210-360 nm; MS interval (+) 150-2000 Da; Instrument: 305 RP Waters Fractional Lynx LCMS.
[1476]
[1477] Procedure I ^{1 :} Column: Phenomenex Luna PFP (2), 150 x 21.2 mm, 5 jm; Mobile phase A: 0.02% TFA in water; Mobile phase B: 0.02% TFA in acetonitrile; Gradient: 40% B for 1.5 minutes, 40% to 100% B for 8.5 minutes, 100% B for 2.0 minutes; Flow rate: 27 ml / minute; Detection: DAD 215 nm, 254 nm; MS interval (+) 150-2000 Da; Instrument: 305 Waters FractionLynx LCMS.
[1478]
[1479] Procedure ^{1 2 :} Column: Phenomenex Luna PFP (2), 150 x 21.2 mm, 5 jm; Mobile phase A: 0.02% TFA in water; Mobile phase B: 0.02% TFA in acetonitrile; Gradient: 1% of B for 1.5 minutes, 1% to 100% of B for 8.5 minutes, 100% of B for 2.0 minutes; Flow rate: 27 ml / minute; Detection: DAD 215 nm, 254 nm; MS interval (+) 150-2000 Da; Instrument: 305 Waters FractionLynx LCMS.
[1480]
[1481] Procedure J ^{1 :} Column: Phenomenex Synergi Polar RP, 150 x 21.2 mm, 4 jm; Mobile phase A: 0.02% TFA in water; Mobile phase B: 0.02% TFA in acetonitrile; Gradient: 10% B for 1.5 minutes, 10% to 75% B for 8.5 minutes, 75% B to 100% B for 0.5 minutes, 100% B for 2 minutes; Flow rate: 27 ml / minute; Detection: DAD 210-360 nm; MS interval (+) 150-2000 Da; Instrument: Waters FractionLynx.
[1482] Procedure K ^{1 :} Column: Phenomenex Luna C18 (2), 250 x 50 mm, 10 jm; Mobile phase A: 0.2% TFA in water (v / v); Mobile phase B: 0.2% TFA in acetonitrile (v / v); Gradient: 1% of B for 1.5 minutes, 1% B to 75% B for 8.5 minutes, 75% B to 100% B for 0.5 minutes, then keep with 100% B for 1.5 minutes; Flow rate: 27 ml / minute. Temperature: not controlled; Detection: DAD 210-360 nm; MS interval (+) 150-2000 Da; Instrument: 305 RP Waters Fractional Lynx LCMS.
[1483]
[1484] Procedure L ^{1 :} Column: ChiralTech AD-H, 500 X 21.5 mm, 5 jm; Mobile phase A: CO ² (v / v); Mobile phase B: methanol (v / v); Gradient: Isocratic conditions 60% CO ² , 40% methanol; Flow rate: 36 ml / minute CO ² , 24 ml / minute methanol. 100 bar back pressure; Detection: DAD 210; Instrument: Thar 80 (Waters).
[1485]
[1486] Procedure M ^: Column: Phenomenex Synergi, 250 x 50 mm, 10 jm; Mobile phase A: 0.1% TFA in water; Mobile phase B: 0.1% TFA in acetonitrile; Gradient: 40% to 70% B for 30 minutes, then 95% B for 5.0 minutes; Flow rate: 80 ml / minute; Detection: DAD 220, 254 nm; MS interval (+) 100-1000 Da; Instrument: Shimadzu LC-20AP.
[1487]
[1488] Procedure N Column: Phenomenex Luna Fenilhexil 150 x 21.2 mm, 5 jm; Mobile phase A: 0.2% TFA in water (v / v); Mobile phase B: 0.2% TFA in acetonitrile (v / v); Gradient: 35% of B for 1.5 minutes, 35% B to 100% B for 18.5 minutes, then 100% B for 2.0 minutes; Flow rate: 27 ml / minute. Temperature: not controlled; Detection: DAD 210-360 nm; MS interval (+) 150-2000 Da; Instrument: 305 r P Waters Fractional Lynx LCMS.
[1489]
[1490] Illustration of antibody and drug conjugates
[1491]
[1492] Protocol A ^: General procedure for conjugation of the antibody with a linker-payload by internal disulfides
[1493]
[1494] IL13Ra2-AB08-v1.0 / 1.0-human IgG1 antibody [Pfizer, 12-13 mg / ml solution in saline solution buffered with Dulbecco phosphate (DPBS, Lonza, pH 7.4)] or VEGFR-1121B-IgG1 antibody human [Pfizer, 19.3 mg / ml solution in Dulbecco phosphate buffered saline (DPBS, Lonza, pH 7.4)] was reduced with the addition of 2.9-3 equivalents of tris hydrochloride (2- Carboxyethyl) phosphine (TCEP, 5 mM solution in DPBS). The reaction was incubated at 37 ° C for 1-1.25 h and then allowed to cool to room temperature. Conjugation was performed by adding 7 equivalents of linker-payload [10 mM solution in N, N-dimethylacetamide (DMA)]. Additional DMA was added to the reaction mixture to get 10-15% (v / v) of a total organic solvent component in the final reaction mixture. The reaction was incubated for 1 h at room temperature. For ADC 1-5, after 1 h at room temperature, the excess linker-payload was inactivated by the addition of 10 cysteine equivalents (solution 20 mM in DPBS). The inactivated reaction mixture was aged at room temperature for 15 minutes, and then at 4 ° C until purified. For ADC 6-14, after 1 h at room temperature, the reaction mixture was desalted by desalination columns of GE Sephadex gel and DPBS eluent (pH7.4), and then stored at 4 ° C until purified. The crude material was purified by molecular exclusion chromatography (SEC) using the GE AKTA Explorer system with the Ge Superdex 200 column (10/300 GL) and the DPBS eluent (pH 7.4).
[1495]
[1496] Protocol B: Column: Agilent Poroshell 300SB-C8, 75 x 2.1 mm, 2.6 ^ m; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: Initial conditions: 20% B to 45% B for 4 minutes; Flow rate: 1.0 ml / minute. Temperature: 60 ° C; Detection: 220 nm; MS interval (+) 400 2000 Da; Injection volume: 10 ^ l; Instrument: Agilent 1100 LC, Waters MicromassZQ MS. Deconvolution was performed with MaxEnt1.
[1497]
[1498] Protocol C: Column: GE Superdex 200 (5/150 GL); Mobile phase: Phosphate buffered saline solution (PBS, 1X, pH 7.4) with 2% acetonitrile; Isocratic; Flow rate: 0.25 ml / minute. Temperature: room temperature; Injection volume: 10 ^ l; Instrument: Agilent 1100 HPLC.
[1499]
[1500] Protocol D: Preparation of ADC transglutaminase, illustrated for the AcLys-vc-MMAD linker payload ("Localization materials: the conjugation site modulates the stability and pharmacokinetics of the antibody and drug conjugates", Chem Biol. 2013, 20, 161-7). For the conjugation of C16-HC and C16-LC with AcLys-vcMMAD, the antibody was adjusted to 5 mg / ml in buffer containing 25 mM Tris-HCl at pH 8.0, and 150 mM NaCl,
[1501]
[1502] AcLys-vc-MMAD was added to a 5-fold (C16-HC) as well as 10-fold (C16-LC) molar excess on the antibody and the enzymatic reaction was initiated by the addition of bacterial transglutaminase to 1 % (C16-HC) (p / v) or 2% (p / v) (C16-LC) (Ajinomoto Activa TI, Japan).
[1503]
[1504] After incubation with gentle agitation at 22 ° C (C16-HC) or 37 ° C (C16-LC) for 16 hours, the ADC was purified using MabSelect SuRe (GE Healthcare, Inc) using standard procedures.
[1505]
[1506] The invention also provides the compounds described in Tables 6A and 6B.
[1507]
[1508]
[1509] (continuation)
[1510]
[1511] (continuation)
[1512]
[1513] (continuation)
[1514]
[1515]
[1516] In the table above, "X and Y" indicate an antibody. The illustrated ADCs were conjugated with an IL13 antibody (IL13Ra2-AB08-v1.0 / 1.0-human IgG1 antibody) as denoted by X and the VEGF antibody VEGFR-1121B-hG1 as denoted by Y.
[1517]
[1518] (continuation)
[1519]
[1520] (continuation)
[1521]
[1522]
[1523] In the table above, "X and Y" indicate an antibody. The illustrated ADCs are conjugated with an antibody against IL13 (IL13Ra2-AB08-v1.0 / 1.0-human IgG1 antibody) as denoted by X and an antibody against VEGF VEGFR-1121B-hG1 as denoted by Y.
[1524] Illustrated ADC - Analytical Data
[1525]
[1526] (continuation)
[1527]
[1528]
[1529]
[1530]
[1531] Experimental procedures for the biological evaluation of payloads and conjugates of antibody and drug
[1532]
[1533] Cell lines
[1534]
[1535] Cancer cell lines were obtained from the ATCC (Manassas, VA). N87 (human gastric carcinoma derived from metastatic site in the liver). HL60 (leukemia), A375 cells (melanoma) and HUVEC (human umbilical vein endothelial cells) were grown in RPMI 1640 medium. All media were supplemented with 10% bovine fetal serum, 1% sodium pyruvate, and 1% L-glutamine (Invitrogen, Grand Island, NY). Human umbilical vein endothelial cells (HUVEC) from Lonza (Allendale, NJ) were obtained and maintained in EGM2 medium supplemented with EGM-2 SingleQuots (Lonza # CC-4176). All cells were kept in a humidified incubator (37 ° C, 5% CO2).
[1536]
[1537] Cytotoxicity test procedure for charges
[1538]
[1539] Cells were grown in 100 pl of medium in a 96-well plate. The cancer cell lines treated with the indicated compounds by adding 50 pl of 3X mother deposits in duplicate at 10 concentrations. The cells were incubated with compounds for four days, then 30 pl of CellTiter® 96 AQueous One MTS Solution (Promega catalog No. G3582) was added to the cells, 1.5 h at 37 ° C, then incubated measured absorbance at 490 nm in a Victor plate reader (Perkin Elmer, Waltham, MA). Relative cell viability was determined as a percentage of the untreated control wells. The IC50 values were calculated using the four-parameter logistic model No. 203 with XLfit v4.2 (IDBS, Guildford, Surry, United Kingdom).
[1540]
[1541] Cytotoxicity test procedure for ADCs
[1542]
[1543] Day 0: cells seeded in 100ul of complete medium in 96 transparent black bottom plates and O / N culture. Day 1: add 50 ul of 3X titrated test compounds to a final volume of 150 ul, and grow for 72 hours at 370 ° C, 5% CO2. Day 4: add 50 ul of Cell TiterGlo to all wells, vortex during 20 30 min, read with Victor 3 with a luminescent program. Data analysis: The % survival was calculated as 100 X (readings of each data point - average BKG) avg. of cells with control only - avg. from BKG. The following table provides IC50 data of the selected loads of the present invention.
[1544]
[1545] (continuation)
[1546]
[1547] (continuation)
[1548]
[1549]
[1550] The following table provides IC50 data for selected ADCs of the present invention.
[1551]
[1552]
[1553] The following drawing illustrates how the load is released after administration to the patient and after cleavage of the linker from the ADC, illustrated with a type of linker. various species were formed after the release of the linker that are interconverted in the biological environment. All species formed are claimed as part of the present invention and refer to the general formula F1-L1-T-L2-F2
[1554]
[1555]
[1556] A = autoimmolation of diaminoethane
[1557] B = h idra ^isis de fo s fa to
[1558] C = cyclopropane formation

权利要求:
Claims (1)
[0001]
1. A compound of Formula (I):
F1-L1-T-L2-F2 (I)
or a pharmaceutically acceptable salt thereof, in which:
each of F1 and F2 is independently selected from ring systems A, B, C and D:

stema e an o
(Ring D system);
each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ -alkenyl , C ² -C ⁶ -alkynyl , C ² -C ^6, halo, hydroxy, alkoxy, ^-NH2, -NH ( C ^V C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ² , -NO ² , -C ⁶ -C ¹⁴ -aryl and C ⁶ -C ¹⁴ -heteroaryl, in which two or more Rs are optionally linked to forming a ring or rings, and wherein said -C ⁶ -C ¹⁴ -aryl and -C ⁶ -C ¹⁴ -aryl are optionally substituted with 1 to 5 substituents independently selected from -C ¹ -C ¹⁰ alkyl, -C-alkoxy ¹ -C ¹⁰ -halo, ^C1 - ^C10 alkylthio, -trifluoromethyl, -NH ^2, -NH (C ¹ -C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ^2, -C ¹ -C ¹⁰ -N (C ¹ -C ⁸ alkyl) ² , -C ¹ -C ³ alkylthio, -NO ² or -C ¹ -C ¹⁰ heterocyclyl, for each ring system in which R appears;
each V ¹ is independently a link, O, N (R) or S, for each ring system in which V ¹ appears; each V ² is independently O, N (R) or S, for each ring system in which V ² appears;
each of W ¹ and W ² is independently H, or -C ¹ -C ⁵ alkyl, for each ring system in which W ¹ and W ² appear;
each X is independently -OH, -O-acyl, azido, halo, cyanate, thiocyanate, isocyanate, thioisocyanate, or

for each ring system in which X appears;
each Y is independently selected from the group consisting of H, -C ¹ -C ⁶ alkyl -R ^A , -C (O) R ^a , -C (S) R ^a , -C (O) OR ^a , -S (O) ² OR ^a , -C (O) N (R ^a ) ² , -C (S) N (R ^a ) ² , glycosyl, -NO ² and -PO (OR ^a ) ² , for each ring system in which Y appears, in which each R ^A is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ aryl, aralkyl, - C ¹ -C ¹⁰ heterocyclyl, C ³ -C ⁸ -carbocyclyl and -C ¹ -C ²⁰ alkyl (R) ² , wherein said -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl alkyl, -aryl C ⁶ -C ¹⁴ , aralkyl, -heterocyclyl C ¹ -C ¹⁰ , -carbocyclyl C ³ -C ⁸ and -C ¹ -C ²⁰ N (R ^{) 2} alkyl are optionally substituted with 1 to 3 substituents independently selected from R;
each Z is independently selected from the group consisting of H, and -C ¹ -C ⁸ alkyl and in which each of said C ¹ -C ⁸ alkyl is optionally substituted with 1 to 3 substituents independently selected from R, for each ring system in which Z appears;
each of L1 and L2 is independent a direct link;
T is selected from:
-C (A1) X1-T2-X1C (B1) -,
where T2 is:

in which each X1 is independently a bond, in which each of A1 and B1 are independently = O, in which each of gyj are independently 0 and m is 1, and in which D is a bicycles (1.1.1. ) pentane or Cuban, wherein said bicyclo (1.1.1.) pentane or Cuban is optionally substituted with -RE, -C (O) RE, -C (O) ORE, -N (RE) ² , -N (R) C (O) RE or -N (R) C (O) ORE, and D is additionally optionally substituted with 1 to 2 R, and
wherein each RE is independently selected from the group consisting of H, -C1-C8 alkyl, -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ -aryl, -aralkyl, -C ¹ -C ¹⁰ -heterocyclyl, - C ³ -C ⁸ carbocyclyl, -C (O) -C ¹ -C ⁸ alkyl, -C (O) N (CVC ⁸ alkyl) ² , and
-C (O) -halo, and in which each RE is optionally substituted with 1 to 3 substituents independently selected from R.
2. A compound of Formula (IIA):
L-P (IIA)
or a pharmaceutically acceptable salt thereof, in which:
P is:
F1-L1-T-L2-F2
in which:
each of F1 and F2 is independently selected from ring systems A, B, C and D:
(Ring system A)

(Ring B system)
(Ring C system)
(Ring D system);
each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ -alkenyl , C ² -C ⁶ -alkynyl , C ² -C ^6, halo, hydroxy, alkoxy, ^-NH2, -NH ( C ¹ -C ^8), -N (C ¹ -C ⁸ alkyl) ^{2, -NO2,} -aryl C ⁶ -C ¹⁴ -heteroaryl C ⁶ -C and ^14, in which two or more R are optionally joined to form a ring or rings, and wherein said -C ⁶ -C ¹⁴ -aryl and -C ⁶ -C ¹⁴ -heteroaryl are optionally substituted with 1 to 5 substituents independently selected from -C ¹ -C ¹⁰ alkyl, -alkoxy ^C1 - ^C10, -halo, ^C1 - ^C10 alkylthio, -trifluoromethyl, -NH ^2, -NH (C ¹ -C ⁸ alkyl), -N (C rC ^ ^8, -C ¹ -C ¹⁰ -N (C ¹ -C ⁸ alkyl) ² , -C ¹ -C ³ alkylthio, -NO ² or -C ¹ -C ¹⁰ heterocyclyl, for each ring system in which R appears;
each V1 is independently a link, O, N (R) or S, for each ring system in which V1 appears; each V2 is independently O, N (R) or S, for each ring system in which V2 appears;
each of W 1 and W2 is independently H, or -C ¹ -C ⁵ alkyl, for each ring system in which W 1 and W2 appear; each X is independently selected from -OH, -O-acyl, azido, halo, cyanate, thiocyanate, isocyanate, thioisocyanate, or

for each ring system in which X appears;
each Y is independently selected from a link, H, -C (O) RA, -C (S) RA, -C (O) ORA, -S (O) ² ORA, -C (O) N (Ra) ² , -C (S) N (Ra) ² , glycosyl, -NO ² and -P (O) (OR ^{A) 2} for each ring system in which Y appears, in which each RA is independently selected from H, -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ -aryl, aralkyl, -C ¹ -C ¹⁰ heterocyclyl, -C ³ -C ⁸ -carbocyclyl, -C ¹ -C ²⁰ N -alkyl (R) ^2, -alkylene C ¹ -C ^20, C ¹ -C 8 -heteroalquileno, C ⁶ -C ¹⁴ arylene, aralkylene, -heterocycle ^C1 - ^C10, C ³ -C ⁸ -carbociclo alkyl and -C ¹ -C ²⁰ N (R) -, and RF where said RA is optionally substituted with 1 to 3 substituents independently selected from R and in which a Y is divalent and is linked to L,
RF is -N (R6) QN (R5) C (O) - and is attached to L in the carbonyl adjacent to N (R5), in which each of R5 and R6 is independently selected from the group consisting of H , -C ¹ -C ⁸ -alkyl, C ¹ -C ⁸ -heteroalquilo, C6-C ¹⁴ -aryl, -aralkyl, ^C1 - ^C10 heterocyclyl and carbocyclyl -C ³ -C ^8, or R5 or R6 join a carbon atom substituted on Q to form a C ¹ -C ¹⁰ heterocycle or C ⁶ -C ¹⁴ heteroaryl ring, or R 5 and R 6 join together to form a C ¹ -C ¹⁰ heterocyclic ring system or of -C ⁶ -C ¹⁴ heteroaryl, and where Q is -C ¹ -C ⁸ alkylene -, - C ¹ -C ⁸ -alkylene, - -C ⁶ -C ¹⁴ -arylene, - -C ¹ -arykylene- C ¹⁰ - or -C ³ -C ⁸ -carbocycle, in which each of Q, R5 and R6 are independently and optionally substituted with 1 to 3 substituents independently selected from R;
each Z is independently selected from the group consisting of H, and -C ¹ -C ⁸ alkyl and wherein each of said C ¹ -C ⁸ alkyl is optionally substituted with 1 to 3 substituents independently selected from R, for each ring system in which Z appears;
each of L1 and L2 is independent a direct link;
T is selected from:
-C (A1) X1-T2-X1 C (B1) -,
where T2 is:

in which each X ¹ is independently a bond, in which each of A ¹ and B ¹ are independently = O, in which each of gyj are independently 0 and m is 1, and in which D is a bicycles (1.1 .1.) Pentane or Cuban, where said bicyclo (1.1.1.) Pentane or Cuban is optionally substituted with -R ^E , -C (O) R ^E , -C (O) OR ^E , -N (R ^e ) ² , -N (R) C (O) R ^e or -N (R) C (O) OR ^e , and D is additionally optionally substituted with 1 to 2 R; Y
wherein each R ^E is independently selected from the group consisting of H, C ¹ -C ⁸ -alkyl, C ¹ -C ⁸ -heteroalquilo, -aryl, -aralkyl, ^C1 - ^C10 heterocyclyl, C -carbociclilo ³ -C ⁸ , -C (O) -C ¹ -C ⁸ alkyl, -C (O) N (C ¹ -C ⁸ alkyl) ² , and -C (O) -halo, and in which each R ^E it is optionally substituted with 1 to 3 substituents independently selected from R;
L is L ^a -L ^b - (L ^c ) ^1-3 , in which L ^A is selected from the group consisting of -halo, -N (R) ² , -CON (R) ² , -S-aryl optionally substituted with -NO ² or -CON (R) ² , -S-heteroaryl optionally substituted with -NO ² , alkyl-SO ² -heteroaryl, arylSO ² -heteroaryl-,

Lb is L ^B1 -L ^B2 -L ^B3 in which L ^B1 is absent or is one or more components selected from the group consisting of -C (O) -, -C (S) -, -C (O) NR- , -C (O) C ¹ -C ⁶ alkyl -, -C (O) NR C ¹ -C ⁶ alkyl,, -C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ^1-6 -, -C (O ) alkyl C ¹ -C NRC (O) -, -C (O) ^C1 - ^C6 alkyl - (OCH ² CH ^{2) 1-6} -, C ⁶ -C ^r - (OCH ² CH ^{2) 1 -6} -C (O) -, -C ¹ -C alkyl ^to -SS-C ¹ -C alkyl ^to NRC (O) CH ² -, -C ¹ -C alkyl ^to - (OCH ² CH ² ) ^1-6 NRC (O) CH ² -, -C (O) C ¹ -C alkyl ^to -NRC (O) -C ^1.6 alkyl -, -N = CR-phenyl-O-C ¹ -C ⁶ alkyl -, -N = CR-phenyl-O-C ¹ -C ⁶ -C (O) -, -C (O) -C ¹ -C ⁶ -alkyl (OCH ² CH ² ) ^1-6 NRC (O) -, -C ( O) C ¹ C ⁶ alkyl -phenyl (NR-C (O) C ¹ -C ⁶ alkyl) ^1-4 -, -C (O) C ^1- C ⁶ alkyl (OCH ² CH ² ) ^1-6 -NRC (O) -alkyl ^C1 - ^C6 -, -C ^C1 - ^C6 -, -S-, -C (O) -CH (NR-C (O) ^C1 - ^C6 alkyl) C ¹ -aiquil -C ⁶ - and (-CH ² -CH ² -O -K ²⁰ ,
in which L ^B2 is AA ^0-12 , in which a A is a natural amino acid, an unnatural amino acid or - (CR ¹⁵ ) ^or -SS- (CR ¹⁵ ) ^p where oyp are each independently an integer number of 1 to 20,
L ^B3 is -PABA-, -PABC- or is absent;
L ^C is absent or independently selected from the group consisting of -C ^1- C ⁶ alkylene -, -NRheterocyclyl C ³ -C ⁸ -NR-, -NR Carbocyclyl C ³ -C ⁸ -NR-, -NRalkyl C ¹ -C ⁶ NR-, -NRalkylene C ¹ -C ⁶ -, -S-, -NR-, -NRNR-, -O (CR ² ) ^1-4 SS (CR ² ) ^1-4 N (R) -, -NRalquilen C ¹ -C ⁶ -phenylene NR-, -NRphenylene C ¹ -C ⁶ SO ² NR-, -O-alkyl C ¹ -C ⁶ S-Salkyl C ¹ -C ⁶ C (COOR) NR-, -NRC (COOR) alkyl C ^1- C ⁶ S-Salquil C ^1. C ⁶ O-,

in which
Xa is CR or N,
XB is CH, CR (C (R) ² ) ¹ - ³ NR, CR (C (R) ² ) ¹ - ³ O, CR (C (R) ² ) ¹ - ³ C (O) NR, CR- ( C (R) ² ) ¹ - ³ C (O) NRNR,
CR (C (R) 2) 1-3SO2NR, CR (C (R) 2) 1-3NRNR, CR (C (R) 2) 1-3NRC (O) or N,
each XC is R,
each XD is - (CH ² ) i - ⁵ -, or is absent;
XE is O, S, C (R) ² , C (R) (C (R) 2) and -3-NR2 or NR and
Each XF is (C (R) ² ) ¹ - ³ -NR or C (R) ² - (C (R) ² ) ¹ - ³ -O.
3. A compound of Formula (IIIA):
AB- (L-P) i -20 (IIIA)
or a pharmaceutically acceptable salt thereof, in which:
AB is an antibody;
P is:
F1-L1-T-L2-F2
in which:
each of F1 and F2 is independently selected from ring systems A, B, C and D:

s ema e an o

each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ -alkenyl , C ² -C ⁶ -alkynyl , C ² -C ^6, halo, hydroxy, alkoxy, ^-NH2, -NH ( C ¹ -C ^8), -N (C ¹ -C ⁸ alkyl) ^{2, -NO2,} -aryl C ⁶ -C ¹⁴ -heteroaryl C ⁶ -C and ^14, in which two or more R are optionally joined to form a ring or rings, and wherein said -C ⁶ -C ¹⁴ -aryl and -C ⁶ -C ¹⁴ -heteroaryl are optionally substituted with 1 to 5 substituents independently selected from -C ¹ -C ¹⁰ alkyl, -alkoxy C ¹ -C ¹⁰ , -halo, -C ¹ -C ¹⁰ -alkylthio, -trifluoromethyl, -NH ² , -NH (C ¹ -C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ² , -C alkyl ¹ -C ¹⁰ -N (C ¹ -C ⁸ alkyl) ² , -C ¹ -C ³ alkylthio, -NO ² or -C ¹ -C ¹⁰ heterocyclyl, for each ring system in which R appears;
each V ¹ is independently a link, O, N (R) or S, for each ring system in which V ¹ appears; each V ² is independently O, N (R) or S, for each ring system in which V ² appears;
W ¹ and W ² are each independently H, or -C ¹ -C ⁵ alkyl for each ring system in which W ¹ and W ² appear; each X is independently selected from -OH, -O-acyl, azido, halo, cyanate, thiocyanate, isocyanate, thioisocyanate, or

for each ring system in which X appears;
each Y is independently selected from a link, H, -C (O) RA, -C (S) RA, -C (O) ORA, -S (O) ² ORA, -C (O) N (Ra) ² , -C (S) N (Ra) ² , glycosyl, -NO ² and -P (O) (OR ^{A) 2} for each ring system in which Y appears, in which each RA is independently selected from H, -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ -aryl, aralkyl, -C ¹ -C ¹⁰ heterocyclyl, -C ³ -C ⁸ -carbocyclyl, -C ¹ -C ²⁰ N -alkyl (R) ² , C ¹ -C ²⁰ alkylene, C ¹ -C ⁸ -alkylene, C ⁶ -C ¹⁴ -arylene, aralkylene, C ¹ -C ¹⁰ heterocycle, C ³ -C ⁸ -carbocycle and alkyl-alkyl C ¹ -C ²⁰ N (R) -, and RF where said RA is optionally substituted with 1 to 3 substituents independently selected from R and in which a Y is divalent and is linked to L, RF is -N (R6) QN (R5) C (O) - and is attached to L in the carbonyl adjacent to N (R5), in which each of R5 and R6 is independently selected from the group consisting of H, -C ¹ -C ⁸ alkyl , -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ aryl , -aralkyl, -C ¹ -C ¹⁰ heterocyclyl and -C ³ -C ⁸ carbocyclyl, or R5 or R6 bind to a carbon atom substituted at Q to form a C ¹ -C ¹⁰ -heterocycle or -heteroaryl ring C ⁶ -C ¹⁴ , or R5 and R6 join together to form a C ¹ -C ¹⁰ -heterocyclic or C ⁶ -C ¹⁴ -heteroaryl ring system, and where Q is -C ¹ -C ⁸ alkylene -, -C ¹ -C ⁸ heteroalkylene -, -C ⁶ -C ¹⁴ -arylene, -aralkylene-, C ¹ -C ¹⁰ heterocycle - or -C ³ -C ⁸ -carbocycle, in which each of Q, R5 and R6 are independently and optionally substituted with 1 to 3 substituents independently selected from R;
each Z is independently selected from the group consisting of H, and each C ¹ -C ⁸ alkyl is optionally substituted with 1 to 3 substituents independently selected from R, for each ring system in which Z appears;
each of L1 and L2 is independent a direct link;
T is selected from:
-C (A1) X1-T2-X1C (B1) -,
where T2 is:

in which each X ¹ is independently a bond, in which each of A ¹ and B ¹ are independently = O, in which each of gyj are independently 0 and m is 1, and in which D is a bicycles (1.1 .1.) Pentane or Cuban, where said bicyclo (1.1.1.) Pentane or Cuban is optionally substituted with -R ^E , -C (O) R ^E , -C (O) OR ^E , -N (R ^E ) ² , -N (R) C (O) R ^E or -N (R) C (O) OR ^E , and D is additionally optionally substituted with 1 to 2 R; Y
wherein each R ^E is independently selected from the group consisting of H, C ¹ -C ⁸ -alkyl, C ¹ -C ⁸ -heteroalquilo, -aryl, -aralkyl, ^C1 - ^C10 heterocyclyl, C -carbociclilo ³ -C ⁸ , -C (O) -C ¹ -C ⁸ alkyl, -C (O) N (C ¹ -C ⁸ alkyl) ² , and -C (O) -halo, and in which each R ^E it is optionally substituted with 1 to 3 substituents independently selected from R;
L is La-Lb- (Lc) 1-3;
L ^A is selected from: a bond to AB, -NR- (bond to AB), alkyl-SO ² -heteroaryl, arylSO ² -heteroaryl-,

L ^B is L ^B1 -L ^B2 -L ^B3
in which L ^B1 is absent or is one or more components selected from the group consisting of -C (O) -, -C (S) -, -C (O) NR-, -C (O) C ¹ alkyl - C ⁶ -, -C (O) NR alkyl C ¹ -C ⁶ -, -alkyl C ¹ -C ⁶ - (OCH ² CH ² ) ^1-6 -, -C (O) C ¹ -C ⁶ alkyl NRC (O ) -, -C (O) C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ^1-6 -, -C ¹ -C alkyl ^to r (OCH ² CH ² ) ^1-6 -C (O) -, -C ¹ -C alkyl ^to rS-S-C ¹ -C ⁶ alkyl NRC (O) CH ² -, - C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ^1-6 NRC (O) CH ² -, -C (O) C ¹ -C ⁶ alkyl -NRC (O) -C ^1.6 alkyl -, -N = CR-phenyl-O-C ¹ -C ⁶ alkyl, - -N = CR-phenyl-O-C-alkyl ¹ -C ⁶ -C (O) -, -C (O) -C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ^1-6 NRC (O) -, -C (O) C ¹ alkyl ^. C ⁶ -phenyl (NR-C (O) C ¹ -C ⁶ alkyl) ^1-4 -, -C (O) C ^1- C ⁶ alkyl (OCH ² CH ² ) ^1-6 -NRC (O) -C ¹ alkyl -C ⁶ -, -C ¹ -C ⁶ -alkyl, -S-, -C (O) -CH (NR-C (O) alkyl C ¹ C ⁶ ) -C ¹ -C ⁶ alkyl - and (-CH ² -CH ² -O-) ^1-2o ;
L ^B2 is AA ^o-12 , in which AA is a natural amino acid, an unnatural amino acid or - (CR ¹⁵ ) ^or -SS- (CR ¹⁵ ) ^p where oyp are each independently an integer from 1 to 20,
L ^B3 is -PABA-, -PABC- or is absent,
L ^C is absent or independently selected from the group consisting of -C ¹ -C ⁶ alkylene -, -NR-heterocyclyl C ³ -C ⁸ -NR-, -NRcarbocyclyl C ³ -C ⁸ -NR-, -NRalkyl C ¹ -C ⁶ NR-, -NRalkylene C ¹ -C ⁶ -, -S-, -NR-, -NRNR-, -O (CR ² ) ^1-4 SS (CR ² ) ^1-4 N (R) - , -NRalkylene C ¹ -C ⁶ -phenyleneNR-, -NRfenilen C ¹ -C ⁶ SO ² NR-, -Olkyl C ¹ -C ⁶ S-Salkyl C ¹ -C ^to C (COOR) NR-, -NRC (COOR ) alkyl C ^1. C ^to S-Salquil C ^1. C ^to O-,

in which
X ^a is CR or N,
X ^B is CH, CR (C (R) ² ) ^1-3 NR, CR (C (R) ² ) ^1-3 O, CR (C (R) ² ) ^1-3 C (O) NR, CR- (C (R) ² ) ^1-3 C (O) NRNR,
CR (C (R) ² ) ^1-3 SO ² NR, CR (C (R) ² ) ^1-3 NRNR, CR (C (R) ² ) ^1-3 NRC (O) or N,
each X ^C is R;
each X ^D is - (CH ² ) ^{and -5} -, or is absent;
X ^E is O, S, C (R) ² , C (R) (C (R) ² ) ^{and -3} -NR ² or NR, and
each X ^F is (C (R) ² ) ^{i -3} -NR or C (R) ² - (C (R) ² ) ^i-3 -O.
4. A compound of Formula (IIB):
F 1 - L 1 -T -L 2 -F 2
L (IIB)
or a pharmaceutically acceptable salt thereof, in which:
each of F1 and F2 is independently selected from ring systems A, B, C and D:

(Ring system A)
(Ring B system)

(Ring C system)
(Ring D system);
each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ -alkenyl , C ² -C ⁶ -alkynyl , C ² -C ^6, halo, hydroxy, alkoxy, ^-NH2, -NH ( C ¹ -C ^8), -N (C ¹ -C ⁸ alkyl) ^{2, -NO2,} -aryl C ⁶ -C ¹⁴ -heteroaryl C ⁶ -C and ^14, in which two or more R are optionally joined to form a ring or rings, and wherein said -C ⁶ -C ¹⁴ -aryl and -C ⁶ -C ¹⁴ -heteroaryl are optionally substituted with 1 to 5 substituents independently selected from -C ¹ -C ¹⁰ alkyl, -alkoxy C ¹ -C ¹⁰ , -halo, -C ¹ -C ¹⁰ -alkylthio, -trifluoromethyl, -NH ² , -NH (C ¹ -C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ² , -C alkyl ¹ -C ¹⁰ -N (C ¹ -C ⁸ alkyl) ² , -C ¹ -C ³ alkylthio, -NO ² or -C ¹ -C ¹⁰ heterocyclyl, for each ring system in which R appears;
each V ¹ is independently a link, O, N (R) or S, for each ring system in which V ¹ appears; each V ² is independently O, N (R) or S, for each ring system in which V ² appears;
each of W ¹ and W ² is independently H, or -C ¹ -C ⁵ alkyl, for each ring system in which W ¹ and W ² appear; each X is independently -OH, -O-acyl, azido, halo, cyanate, thiocyanate, isocyanate, thioisocyanate, or

for each ring system in which X appears;
each Y is independently selected from the group consisting of H, -C ¹ -C ⁶ -R ^A -C (O) R ^A , -C (S) R ^A , -C (O) OR ^A , -S ( O) ² OR ^a , -C (O) N (R ^a ) ² , -C (S) N (R ^a ) ² , glycosyl, -NO ² and -PO (OR ^A ) ² , for each ring system in which Y appears, in which each R ^A is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ⁶ -C ¹⁴ -aryl, aralkyl, -heterocyclyl C ¹ -C ¹⁰ , -C ³ -C ⁸ -carbocyclyl and -C ¹ -C ²⁰ alkyl (R) ² , wherein said -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl alkyl, -C-aryl ^6- C ¹⁴ , aralkyl, -C ¹ -C ¹⁰ heterocyclyl, -C ³ -C ⁸ -carbocyclyl and -C ¹ -C ²⁰ N (R) ² alkyl are optionally substituted with 1 to 3 substituents independently selected from R;
each Z is independently selected from the group consisting of H, and -C ¹ -C ⁸ alkyl and wherein said C ¹ -C ⁸ alkyl, each is optionally substituted with 1 to 3 substituents independently selected from R, for each ring system in which Z appears;
each of L ¹ and L ² is independent a direct link;
T is selected from:
-C (A ¹ ) X ¹ -T ² -X ¹ C (B ¹ ) -,
where T ² is:

in which each X1 is independently a bond, in which each of A1 and B1 are independently = O in which each of gyj are independently 0 and m is 1, and in which D is a bicycles (1.1.1.) pentane or Cuban, where said bicyclo (1.1.1.) pentane or Cuban is substituted with a member of the group selected from N (RE) C (O) - where the carbonyl is attached to L, and -C (O) - where the carbonyl is attached to L, and additionally it is optionally substituted with 1 to 2 R;
where each RE is independently selected from the group consisting of H, -C ¹ -C ⁸ alkyl, -heteroalkyl C ¹ -C ⁸ , -aryl, -aralkyl, -heterocyclic C ¹ -C ¹⁰ , -C ³ -C ⁸ -carbocyclyl, -C (O) -C ¹ -C ⁸ alkyl, -C (O) N (C alkyl iC ⁸ ) ² , and -C (O) -halo, and in which each RE is optionally substituted with 1 to 3 substituents independently selected from R;
L is LA-LB (LC) ^{1 to 3;}
LA is selected from -halo, -N (R) ^2, -CON (R) ^2, -S-aryl optionally substituted by ^-NO2 or -CONR ^2, -S-heteroaryl optionally substituted with ^-NO2, alkyl-SO ^2- heteroaryl, arylSO ² -heteroaryl-,

L ^B is L ^B1 -L ^B2 -L ^B3
in which L ^B1 is absent or is one or more components selected from the group consisting of -C (O) -, -C (S) -, -C (O) NR-, -C (O) C ¹ alkyl - C ⁶ -, -C (O) ^C1 - ^C6 NRalquil -, C ¹ -C ^a -alkyl - (OCH ² CH ^{2) 1-a} -, -C (O) C ¹ -C alkyl NRC (O ) -, -C (O) C ¹ -C alkyl ^to - (OCH ² CH ² ) ^1-a -, -C ¹ -C alkyl ^to - (OCH ² CH ² ) ^1-a -C (O) -, C ¹ -C ^a -alkyl -SS-substituted C _1- C ^to NRC (O) CH ² -, C ¹ -C ^a -alkyl - (OCH ² CH ^{2) 1-NRC} (O) CH ² -, - C (O) C ¹ -C alkyl ^to -NRC (O) -C ^ ^a -, -N = CR-phenyl-O-C ¹ -C ^a alkyl -, -N = CR-phenyl-O-alkyl C -C ^{1 to} -C (O) -, -C (O) -C ¹ -C ^a - (OCH ² CH ^{2) 1-NRC} (O) -, -C (O) alkyl C ^1- C -phenyl (NR-C (O) C ¹ -C alkyl ^{a) 1-4} -, -C (O) alkyl C ^1- C (OCH ² CH ^{2) 1-a} -NRC (O) -C ¹ -C ^a -, C ¹ -C ^a -alkyl -, -S-, -C (O) -CH (NR-C (O) -C ¹ -C ^a) C ¹ -C ^a -alkyl - and ( -CH ² -CH ² -O-) ^1-2o ;
L ^B2 is AA ^0-12 , in which AA is a natural amino acid, an unnatural amino acid or - (CR ¹⁵ ) ^or -SS- (CR ¹⁵ ) ^p where o and ^p are each independently an integer from 1 to 20,
L ^B3 is -PABA-, -PABC- or is absent;
L ^C is absent or independently selected from the group consisting of -C ^1- C alkylene ^to -, -NR-heterocyclyl C ³ -C ⁸ -NR-, -NRcarbocyclyl C ³ -C ⁸ -NR-, -NRalkyl C ¹ -C ^to NR-, -NRalkylene C ¹ -C ^to -, -S-, -NR-, -NRNR-, -O (CR ² ) ^1-4 SS (CR ² ) ^1-4 N (R) - , C ¹ -C -NRalquilen ^to -fenilenoNR-, C ¹ -C -NRfenilen ^to SO ² NR-, -OC ¹ -C S-C ¹ -C Salquil C (COOR) NR-, -NRC (COOR ) C ^1- C alkyl ^to S-C ^1- C alkyl ^to O-,

in which
Xa is CR or N,
X ^B is CH, CR (C (R) ² ) ^1-3 NR, CR (C (R) ² ) ^1-3 O, CR (C (R) ² ) ^1-3 C (O) NR, CR- (C (R) ² ) ^1-3 C (O) NRNR, CR (C (R) ² ) ^1-3 SO ² NR, CR (C (R) ² ) ^1-3 NRNR, CR (C (R) ² ) ^1-3 NRC (O) or N;
each X ^C is R;
each X ^D is - (CH ² ) ^1-5 -, or is absent;
X ^E is O, S, C (R) ² , C (R) (C (R) ² ) ^1-3 -NR ² or NR, and
Each X ^F is (C (R) ² ) ^1-3 -NR or C (R) ² - (C (R) ² ) ^1-3 -O.
5. A compound of Formula (IIIB):

A pharmaceutically acceptable salt thereof, in which:
AB is an antibody;
each of F1 and F2 is independently selected from ring systems A, B, C and D:
(Ring system A)
(Ring B system)
(Ring C system)
(Ring D system);
each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ -alkenyl , C ² -C ⁶ -alkynyl , C ² -C ^6, halo, hydroxy, alkoxy, ^-NH2, -NH ( C ¹ -C ^8), -N (C ¹ -C ⁸ alkyl) ^{2, -NO2,} -aryl C ⁶ -C ¹⁴ -heteroaryl C ⁶ -C and ^14, in which two or more R are optionally joined to form a ring or rings, and wherein said C ⁶ -C ¹⁴ -aryl and -heteroaryl C ⁶ -C ¹⁴ optionally substituted with 1 to 5 substituents independently selected from alkyl ^C1 - ^C10, alkoxy C ¹ -C ¹⁰ -halo, ^C1 - ^C10 alkylthio, -trifluoromethyl, -NH ^2, -NH (C ¹ -C ⁸ alkyl), -N (C ¹ -C ⁸ alkyl) ^2, -C ¹ -C ¹⁰ -N (C ¹ -C ⁸ alkyl) ² , -C ¹ -C ³ alkylthio, -NO ² or -C ¹ -C ¹⁰ heterocyclyl, for each ring system in which R appears;
each V ¹ is independently a link, O, N (R) or S, for each ring system in which V ¹ appears; each V ² is independently O, N (R) or S, for each ring system in which V ² appears;
each of W ¹ and W ² is independently H, or -C ¹ -C ⁵ alkyl, for each ring system in which W ¹ and W ² appear; each X is independently -OH, -O-acyl, azido, halo, cyanate, thiocyanate, isocyanate, thioisocyanate, or

for each ring system in which X appears;
each Y is independently selected from the group consisting of H, -C ¹ -C ⁶ alkyl -RA -C (O) RA, -C (S) RA, C (O) OR ^A , -S (O) ² OR ^A , -C (O) N (R ^A ) ² , -C (S) N (R ^a ) ² , glycosyl, -NO ² and -PO (OR ^A ) ² , for each ring system in which Y appears, in which each R ^A is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl alkyl, -aryl C ⁶ -C ¹⁴ , aralkyl, -C ¹ -C ¹⁰ heterocyclyl, -C ³ -C ⁸ -carbocyclyl and -C ¹ -C ²⁰ alkyl N (R) ² , wherein said -C ¹ -C ²⁰ alkyl, - C ¹ -C ⁸ heteroalkyl, -C ⁶ -C ¹⁴ aryl, aralkyl, -C ¹ -C ¹⁰ heterocyclyl, C ³ -C ⁸ -carbocyclyl and -C ¹ -C ²⁰ N (R) ² alkyl are optionally substituted with de 1 to 3 substituents independently selected from R;
each Z is independently selected from the group consisting of H, and -C ¹ -C ⁸ alkyl and wherein each of said C ¹ -C ⁸ alkyl is optionally substituted with 1 to 3 substituents independently selected from R, for each ring system in which Z appears;
each of L ¹ and L ² is independent a direct link;
T is selected from:
-C (A ¹ ) X ¹ -T ² -X ¹ C (B ¹ ) -,
where T ² is:

in which each X ¹ is independently a bond, in which each of A ¹ and B ¹ are independently = O in which each of gyj are independently 0 and m is 1, and in which D is a bicycles (1.1. 1.) pentane or Cuban, wherein said bicyclo (1.1.1.) Pentane or Cuban is substituted with a member of the group selected from N (R ^E ) C (O) - where the carbonyl is attached to L, and -C ( O) - where the carbonyl is attached to L, and additionally is optionally substituted with 1 to 2 R;
wherein each R ^E is independently selected from the group consisting of H, C ¹ -C ⁸ -alkyl, C ¹ -C ⁸ -heteroalquilo, -aryl, -aralkyl, ^C1 - ^C10 heterocyclyl, -carbociclilo C ³ - C ⁸ , -C (O) -C ¹ -C ⁸ alkyl, -C (O) N (C ¹ -C ⁸ alkyl) ² and -C (O) -halo, and in which each R ^E is optionally substituted with 1 to 3 substituents independently selected from R;
L is La-Lb- (Lc) 1-3;
L ^A is selected from: a bond to AB, -NR- (bond to AB), alkyl-SO ² -heteroaryl, arylSO ² -heteroaryl-,

L ^B is L ^B1 -L ^B2 -L ^B3
in which L ^B1 is absent or is one or more components selected from the group consisting of -C (O) -, -C (S) -, -C (O) NR-, -C (O) C ¹ alkyl - C ⁶ -, -C (O) ^C1 - ^C6 NRalquil -, -C ¹ -C ⁶ -alkyl - (OCH ² CH ^{2) 1-6} -, -C (O) C _1- C ⁶ alkyl NRC (O ) -, -C (O) C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ^1-6 -, -C ¹ -C alkyl ^to r (OCH ² CH ² ) ^1-6 -C (O) -, -C ¹ -C ⁶ alkyl -SS-C ¹ -C ⁶ alkyl NRC (O) CH ² -, - C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ^1-6 NRC (O) CH ² -, - C (O) C ¹ -C ⁶ alkyl -NRC (O) -C ^1.6 alkyl -, -N = CR-phenyl-O-C ¹ alkyl ^. C ⁶ -, -N = CR-phenyl-O-C ¹ alkyl -C ⁶ -C (O) -, -C (O) -C ¹ -C ⁶ alkyl - (OCH ² CH ² ) ^1-6 NRC (O) -, -C (O) C ^1- C ⁶ alkyl - phenyl (NR-C (O) C ¹ -C ⁶ alkyl) ^1-4 -, -C (O) C ^1- C ⁶ alkyl (OCH ² CH ² ) ^1-6 -NRC (O) -C ¹ alkyl - C ⁶ -, -C ^C1 - ^C6 -, -S-, -C (O) -CH (NR-C (O) -alkyl ^C1 - ^C6) -alkyl ^C1 - ^C6 - and (- CH ² -CH ² -O-) ^1-20 ;
L ^B2 is AA ^0-12 , in which AA is a natural amino acid, an unnatural amino acid or - (CR ¹⁵ ) ^or -SS- (CR ¹⁵ ) ^p where o and ^p are each independently an integer from 1 to 20,
L ^B3 is -PABA-, -PABC- or is absent;
L ^C is absent or independently selected from the group consisting of -C ¹ -C ⁶ alkylene -, -NR-heterocyclyl C ³ -C ⁸ -NR-, -NRcarbocyclyl C ³ -C ⁸ -NR-, -NRalkyl C ¹ -C ⁶ NR-, -NRalkylene C ¹ -C ⁶ -, -S-, -NR-, -NRNR-, -O (CR ² ) ^1-4 SS (CR ² ) ^1-4 N (R) - , -NRalkylene C ¹ -C ⁶ -phenyleneNR-, -NRfenilen C ¹ -C ⁶ SO ² NR-, -Oalkyl C ¹ -C ⁶ S-Salkyl C ¹ -C ⁶ C (COOR) NR-, -NRC (COOR ) C ^1- C ⁶ alkyl S-C ¹ alkyl ^. C ⁶ O-,

in which
Xa is CR or N,
X ^B is CH, CR (C (R) ² ) ^{i - a} NR, CR (C (R) ² ) ^{i - a} O, CR (C (R) ² ) ^1-3 C (O) NR, CR- (C (R) ² ) ^{i - a} C (O) NRNR,
CR (C (R) ² ) ^{i - a} SO ² NR, CR (C (R) ² ) ^1-a NRNR, CR (C (R) ² ) ^1-3 NRC (O) or N;
each X ^C is R;
each X ^D is - (CH ² ) ^1-5 -, or is absent;
X ^E is O, S, C (R) ² , C (R) (C (R) ² ) ^1-3 -NR ² or NR, and
Each X ^F is (C (R) ² ) ^1-3 -NR or C (R) ² - (C (R) ² ) ^1-3 -O.
6. The compound of claim 1, wherein:
each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl and -NH ² ;
each V ¹ is independently O or N (R) for each ring system in which V ¹ appears;
each V ² is independently O or N (R) for each ring system in which V ² appears;
each of W ¹ and W ² is independently H, or -C ¹ -C ⁵ alkyl, for each ring system in which W ¹ and W ² appear;
each X is independently halo, for each ring system in which X appears;
each Y is independently selected from the group consisting of H, -C (O) R ^A , -C (O) N (R ^A ) ² , glycosyl, -NO ² and -PO (OR ^a ) ² , for each system ring in which Y appears, in which each R ^A is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ³ -C ⁸ -carbocyclyl and - C ¹ -C ²⁰ alkyl (R) ² , wherein said -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ³ -C ⁸ -carbocyclyl and -C ¹ -C ²⁰ N -alkyl (R ) ² are optionally substituted with 1 to 3 substituents independently selected from R;
each of L ¹ and L ² is independent a direct link; Y
T is selected from:
-C (A ¹ ) X ¹ -T ² -X ¹ C (B ¹ ) -,
where T ² is:

in which each X ¹ is a link, in which each of A ¹ and B ¹ are independently = O, in which each of gyj are independently 0 and m is 1, and in which D is a bicycles (1.1. 1.) pentane or Cuban,
wherein said bicyclo (1.1.1.) pentane or Cuban is optionally substituted with -NH ² , -N (R) C (O) H or -N (R) C (O) OH.
7. The compound of any of claims 2-3, wherein:
each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl and -NH ² ;
each V ¹ is independently O or N (R) for each ring system in which V ¹ appears;
each V ² is independently O or N (R) for each ring system in which V ² appears;
W ¹ and W ² are each independently H, or -C ¹ -C ⁵ alkyl for each ring system in which W ¹ and W ² appear;
each X is independently halo, for each ring system in which X appears;
each Y is independently selected from a link, H, -C (O) R ^A , -C (S) R ^A , -C (O) OR ^A , -S (O) ² OR ^A , C (O) N (RA) ² , -C (S) N (Ra) ² , glycosyl, -NO ² and -P (O) (OR ^{A) 2} for each ring system in which Y appears, in the that each RA is independently selected from H, -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -Cb-Cm aryl, aralkyl, -C ¹ -C ¹⁰ -heterocyclyl, -C ³ -C ⁸ -carbocyclyl, - C ¹ -C ²⁰ N (R) ² alkyl, -C ¹ -C ²⁰ alkylene, C ¹ -C ⁸ -alkylene, C ⁶ -C ¹⁴ -arylene, aralkylene, -C ¹ -C ¹⁰ heterocycle, -C ³ -carbocycle -C ⁸ and C ¹ -C ²⁰ N alkyl (R) -, and RF where said RA is optionally substituted with 1 to 3 substituents independently selected from R and in which a Y is divalent and is linked to L,
RF is -N (R6) QN (R5) C (O) - and is attached to L in the carbonyl adjacent to N (R5), in which each of R5 and R6 is independently selected from the group consisting of H , -C ¹ -C ⁸ alkyl, and -C ¹ -C ⁸ heteroalkyl, or R5 or R6 binds with a Q-substituted carbon atom to form a C ¹ -C ¹⁰ heterocyclic ring or a C ⁶ heteroaryl ring -C ¹⁴ , or R5 and R6 join together to form a C ¹ -C ¹⁰ hetero-cyclic or hetero-C ⁶ -C ¹⁴ ring system, and where Q is -C ¹ -C ⁸ alkylene -, - arylene C ⁶ -C ¹⁴ - or carbocycle -C ³ -C ⁸ -, in which each of Q, R5 and R6 are independently and optionally substituted with 1 to 3 substituents independently selected from R;
each of L1 and L2 is independently selected from a direct link; Y
T is selected from:
-C (A1) X1-T2-X1C (B1) -,
where T2 is:

in which each X1 is a bond, in which each of A1 and B1 are independently = O, in which each of gyj are independently 0 and m is 1, and in which D is a bicycles (1.1.1.) pentane or Cuban, wherein said bicyclo (1.1.1.) pentane or Cuban is optionally substituted with -NH ² , -N (R) c (o) H or -N (R) C (O) OH.
8. The compound of any of claims 4-5, wherein:
each R is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl and -NH ² ;
each V1 is independently O or N (R) for each ring system in which V1 appears;
each V2 is independently O or N (R) for each ring system in which V2 appears;
each of W 1 and W2 is independently H, or -C ¹ -C ⁵ alkyl, for each ring system in which W 1 and W2 appear;
each X is independently halo, for each ring system in which X appears;
each Y is independently selected from the group consisting of H, -C (O) RA, -C (O) N (RA) ² , glycosyl, -NO ² and
-PO (ORa) ² , for each ring system in which Y appears, in which each RA is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ heteroalkyl, -C ³ -C ⁸ -carbocyclyl and -C ¹ -C ²⁰ alkyl (R) ² , wherein said -C ¹ -C ²⁰ alkyl, -C ¹ -C ⁸ -alkyl, -C ³ -C ⁸ -carbocyclyl and - C ¹ -C ²⁰ N (R ^{) 2} alkyl are optionally substituted with 1 to 3 substituents independently selected from R;
each of L1 and L2 is independent a direct link; Y
T is -C (A1) X1-T2-X1C (B1) -, where T2 is:

in which each X1 is a bond, in which each of A1 and B1 are independently = O, in which each of gyj are independently 0 and m is 1, and in which D is a bicycles (1.1.1.) pentane or Cuban, wherein said bicyclo (1.1.1.) pentane or Cuban is optionally substituted with -NH ² , -N (R) c (o) H or -N (R) C (O) OH.
9. The compound of any of claims 2 and 4, wherein:
LA is selected from the group consisting of -halo, -N (R) ² , -CON (R) ² , -S-aryl optionally substituted with -NO ² or -CON (R) ² , -S-heteroaryl optionally substituted with -NO ² , alkyl-SO ² -heteroaryl, arylSO ² -heteroaryl-, and

L ^b is l ^b1 -L ^B2 -L ^B3 in which L ^B1 is absent or is one or more components selected from the group consisting of -C (O) -, -C (S) -, -C (O) NR -, -C (O) C ¹ -C alkyl ^to -, -C (O) NR C ¹ -C ⁶ -, - C ^r C ⁶ alkyl - (OCH ² CH ² ) ^1-6 -, -C (O ) alkyl C ^- -C ^to NRC (O) -, -C (O) alkyl C ⁱ -C ^a - (OCH ² CH ² ) ^{i - a} -, - alkyl C ⁱ -C ^a - (OCH ² CH ² ) ^{i- to} -C (O) -, -alkyl C ⁱ -C ^to -SS -alkyl C ^- -C ^to NRC (O) CH ² , -alkyl C ⁱ -C ^to - (OCH ² CH ² ) ^{i - a} NRC (O) CH ² -, -C (O) C ¹ -C alkyl ^to -NRC (O) -C ⁱ alkyl ^{- a} -, -C (O) -C-alkyl ^- -C ^to - (OCH ² CH ² ) ^{i - a} NRC (O) -, -C (O) C ¹ alkyl ^. C ^a -phenyl (NR-C (O) alkyl C ^- -C ^a ) ^i-4 -, -C (O) alkyl C ^{i -} C ^a (OCH ² CH ² ) ^{i - a} -NRC (O) -alkyl C ⁱ -C ^a -, -alkyl C ⁱ -C ^a -, -S-, -C (O) -CH (NR-C (O) alkyl C ⁱ -C ^a ) -alkyl C ⁱ -C ^a -y (-CH ² -CH ² -O-) ^{i -20} , in which L ^B2 is AA ^0-12 , in which AA is a natural amino acid, an unnatural amino acid or - (CR ^{i 5} ) ^or -SS- (CR ^{i 5} ) ^p where each of oyp are independently an integer of ia 20, and L ^B3 is -PABA-, -PABC- or is absent; and L ^c is absent.
i0. The compound of any of claims 3 and 5, wherein:
L ^A is selected from: a bond to AB, -NR- (bond to AB), alkyl-SO ² -heteroaryl, arylSO ² -heteroaryl-,

L ^B is l ^bi -LB2-LB3 in which L ^Bi is absent or is one or more components selected from the group consisting of -C (O) -, -C (S) -, -C (O) NR-, -C (O) -C ⁱ alkyl C ^a -, -C (O) C ⁱ -C NRalquil ^a -, -C ⁱ -C ^a - (OCH ² CH ^{2) i - to} -, -C (O) alkyl C ^- -C ^to NRC (O) -, -C (O) alkyl C ⁱ -C ^to - (OCH ² CH ² ) ^{i - a} -, -alkyl C ⁱ -C ^to - (OCH ² CH ² ) ^{i - a} -C (O) -, -alkyl C ⁱ -C ^to -SS-alkyl C ^- -C ^to NRC (O) CH ² -, -alkyl C ⁱ -C ^to - (OCH ² CH ² ) ^{i - a} NRC (O) CH ² -, -C (O) C ¹ -C alkyl ^to -NRC (O) -C ⁱ alkyl ^{- a} -, -C (O) -C-alkyl ^- -C ^to - (OCH ² CH ² ) ^{i - a} NRC (O) -, -C (O) alkyl C ^{i -} C ^a -phenyl (NR-C (O) alkyl C ⁱ -C ^a ) ^i-4 -, -C (O) alkyl C ^{i -} C ^a (OCH ² CH ² ) ^{i - a} -NRC (O) -alkyl C ⁱ -C ^a -, -alkyl C ⁱ -C ^a -, -S-, -C (O) -CH (NR-C (O) C ⁱ -C alkyl ^a) -C ⁱ -C ^a - and (-CH ² -CH ² -O-) ^{i -20,} wherein LB2 is AA ^{or - i2,} where AA is a natural amino acid, and an unnatural amino acid or - (CRi5) ^or -SS- (CRi5) ^p where each of oyp are independently an integer of ia 20, and LB3 is -PAB A-, -PABC- or is absent; Y
L ^c is absent.
ii. The compound according to claim 2 or 3, wherein R ^F is selected from:

where q is i-iO, and each b is independently CR ^D , N, NR ^D , O or S; Y
wherein each R ^D is independently selected from the group consisting of H, C ⁱ -C ⁸ -alkyl, -C (O) -C ⁱ -C ^8, C ⁱ -C ⁸ -heteroalquilo, aryl C -C ^{i 4} , -aralkyl, -heterocyclyl C ⁱ -C ^io , -carbocyclyl C ³ -C ⁸ , -C (O) -O-alkyl C ⁱ -C ⁸ , -C (O) N (alkyl C ⁱ -C ⁸ ) ² , and -C (O) -halo, optionally substituted with R ^E.
12. The compound of claim i, wherein each V ¹ is O, for each ring system in which V ¹ appears; and each Y is independently selected from the group consisting of H, -C (O) N (R ^A ) ² , -C (S) N (R ^a ) ² , -PO (OR ^a ) ² or glycosyl for each system ring in which Y appears, in which each R ^A is independently selected from the group consisting of H, -C ¹ -C ²⁰ alkyl, and -C ¹ -C ²⁰ N (R) ² alkyl, and in the said -C ¹ -C ²⁰ -alkyl and C ⁱ -C N ^{the 2nd} (R) ² are optionally substituted with ai 3 substituents independently selected from R.
13. The compound according to any one of claims i-5, wherein one or more W is C ¹ -C ³ alkyl.
14. The compound according to any one of claims i-5, wherein one or more X is chlorine.
13O
15. The compound according to any one of claims 1-5, wherein a Y is H or -C (O) Ci-Cio alkyl.
16. The compound according to any one of claims 1-5, wherein one or more Z is H.
17. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from:


18. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from:

19. A linker-payload or an antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, comprising a radical of a compound of claim 17.
20. A compound according to claim 2, or a pharmaceutically acceptable salt thereof, selected from:
Ċ
5




21. A compound according to claim 2, or a pharmaceutically acceptable salt thereof, selected from:
Ċ
5



22. A compound according to claim 2, or a pharmaceutically acceptable salt thereof, selected from:





23. A compound according to claim 2, or a pharmaceutically acceptable salt thereof, selected from:


24. A compound according to claim 3, or a pharmaceutically acceptable salt thereof, selected from:


where X is an antibody against IL13 or Y is an antibody against VEGF.
25. A compound according to claim 3, or a pharmaceutically acceptable salt thereof, selected from:


where X is an antibody against IL13 or Y is an antibody against VEGF.
26. A compound according to claim 3, or a pharmaceutically acceptable salt thereof, selected from:


where X is an antibody against IL13 or Y is an antibody against VEGF.
27. A compound according to claim 3, or a pharmaceutically acceptable salt thereof, selected from:



where X is an antibody against IL13 or Y is an antibody against VEGF.
28. A pharmaceutical composition comprising a compound of any one of claims 1 to 27, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
29. A compound according to any one of claims 1 to 27, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 28, for use in the treatment of cancer.
30. A compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition for use according to claim 29, wherein said cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, cancer Endometrial, kidney cancer, bronchopulmonary cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreas cancer, skin cancer, stomach cancer (gastric), testicular cancer, leukemia and lymphomas.

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RU2016127553A|2018-03-01|

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EP3099692A1|2016-12-07|

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SG11201605437YA|2016-08-30|

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JP2017503847A|2017-02-02|

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RU2016127553A3|2018-03-01|

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HUE043427T2|2019-08-28|

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EP3099692B1|2019-03-13|

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US20180339060A1|2018-11-29|

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JP2018076307A|2018-05-17|

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SI3099692T1|2019-06-28|

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SA516371557B1|2019-10-07|

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KR20160111517A|2016-09-26|

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MX371147B|2020-01-20|

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PT3099692T|2019-05-24|

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ZA201604824B|2020-01-29|

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NZ722020A|2018-05-25|

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US10391182B2|2019-08-27|

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CN106459055A|2017-02-22|

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PE20161031A1|2016-10-26|

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AU2015208814B2|2018-11-01|

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PH12016501411B1|2016-08-31|

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JP6236540B2|2017-11-22|

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KR101871088B1|2018-06-25|

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US10086085B2|2018-10-02|

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IL246739A|2019-05-30|

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MX2016009717A|2016-09-22|

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DK3099692T5|2020-03-16|

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EP3521292A1|2019-08-07|

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TW201538169A|2015-10-16|

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RU2669807C2|2018-10-16|

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AU2015208814A1|2016-07-21|

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CA2937731A1|2015-07-30|

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CN106459055B|2019-11-01|

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US201461932118P| true| 2014-01-27|2014-01-27|

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US201462046685P| true| 2014-09-05|2014-09-05|

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PCT/IB2015/050280|WO2015110935A1|2014-01-27|2015-01-14|Bifunctional cytotoxic agents|

---