6-amino-7,9-dihydro-8h-purin-8-one derivatives as toll 7 immunostimulatory receptor (tlr7) agonists

专利摘要:
The present invention relates to compounds having a structure according to formula (I) or (II) where R1, R2, and Ar are as defined herein, are agonists for the Toll 7 type receptor (TLR7) and can be used as adjuvants to stimulate the immune system. Some of these compounds can be used in conjugates for targeted release to the organ or tissue of the intended action.
公开号:BR112020002953A2
申请号:R112020002953-0
申请日:2018-08-16
公开日:2020-08-11
发明作者:Liqi He；Sanjeev Gangwar；Shoshana L. Posy；Yam B. POUDEL；Prasanna Sivaprakasam
申请人:Bristol-Myers Squibb Company；
IPC主号:

专利说明:

[001] [001] This application claims the benefit under 35 U.S.C. §119 (e) of US Provisional Application No. Ser. 62 / 546.211, filed on August 16, 2017; whose description is incorporated herein by reference. BACKGROUND OF THE INVENTION
[002] [002] The present invention relates to Toll 7 Receptor agonists ("TLR7") and their conjugates, and methods for the preparation and use of such agonists and their conjugates.
[003] [003] Toll-like receptors ("TLRs") are cell surface receptors that recognize pathogen-associated molecular patterns ("PAMPs"). The activation of a TLR by binding a corresponding PAMP signals potential infection by a pathogen and stimulates the immune system to fight the infection. Humans have 11 TLRs, called TLR1 to TLR11.
[004] [004] The activation of a TLR - with TLR7 being the most studied - by an agonist can have an adjuvant effect on the action of vaccines and immunotherapy agents in the treatment of a variety of conditions in addition to infection by a real pathogen, stimulating the response immune.
[005] [005] TLR7 recognizes PAMPs associated with single-stranded RNA viruses. Its activation induces the secretion of Type I interferons, such as IFNα and IFNβ (Lund et al. 2004). It has two binding sites, one for single-stranded RNA ligands, such as ssRNA40 (Berghöfer et al. 2007) and one for guanosine (Zhang et al. 2016).
[006] [006] TLR7 can bind and be activated by synthetic agonists of the guanosine type, such as imiquimod, resiquimod and gardiquimod,
[007] [007] Synthetic TLR7 agonists based on a molecular structure of pteridinone are also known, as exemplified by vesatolimode (Desai et al. 2015), who was in Phase 2 clinical trials. The potency of vesatolimod is reported to be 100X less than that of the corresponding purine-8-one compound, measured by IFN-α induction (Roethle et al. 2013).
[008] [008] Other synthetic TLR7 agonists are based on a purine-like structure, often according to formula A: whereR, R ', and R "are structural variables, with R" typically containing an unsubstituted aromatic or heteroaromatic ring or substituted.
[009] [009] Descriptions of bioactive molecules having a type of purine and their uses in the treatment of conditions such as fibrosis, inflammatory disorders, cancer or pathogenic infections include: Akinbobuyi et al. 2015b and 2016; Barberis et al. 2012; Carson et al. 2014; Ding et al. 2016, 2017a, and 2017b; Graupe et al. 2015; Hashimoto et al. 2009;
[0010] [0010] The group R "can be pyridyl: Bonfanti et al. 2015a and 2015b; Halcomb et al. 2015; Hirota et al. 2000; Isobe et al. 2000, 2002, 2004, 2006, 2009a, 2011, and 2012; Kasibhatla et al. 2007; Koga-Yamakawa et al. 2013; Musmuca et al. 2009; Nakamura 2012; Ogita et al. 2007; and Yu et al. 2013.
[0011] [0011] Bonfanti et al. 2015b describes TLR7 modulators in which the two rings of a purine portion are covered by a macrocycle: NH2 N N OH
[0012] [0012] A TLR7 agonist can be conjugated to a partner molecule, which can be, for example, a phospholipid, a poly (ethylene glycol) ("PEG") or another TLR (usually TLR2). Exemplary descriptions include: Carson et al. 2013, 2015, and 2016, Chan et al. 2009 and 2011, Lioux et al. 2016, Maj et al. 2015, Ban et al. 2017; Vernejoul et al. 2014, and Zurawski et al. 2012. Conjugation with an antibody has also been described: Akinbobuyi et al. 2013 and 2015a, and Gadd et al. 2015. A frequent conjugation site is in the R "group of formula A.
[0013] [0013] TLR7 agonists based on a 5H-pyrrolo [3,2-d] pyrimidine structure have also been described. See, Cortez et al. 2017a and 2017b, McGowan et al. 2017, and Li et al. 2018.
[0014] [0014] Jensen et al. 2015 describes the use of cationic lipid vehicles for the release of TLR7 agonists.
[0015] [0015] Some TLR7 agonists, including resiquimod, are double TLR7 / TLR8 agonists. See, for example, Beesu et al. 2017; Lioux et al. 2016; and Vernejoul et al. 2014.
[0016] [0016] TLR7 agonists based on a 5H-pyrrolo [3,2-d] pyrimidine structure have also been described. See, Cortez et al. 2017a and 2017b, McGowan et al. 2017, and Li et al. 2018.
[0017] [0017] Complete citations for the documents cited here by the first author or inventor and year are listed at the end of this specification. BRIEF SUMMARY OF THE INVENTION
[0018] [0018] In one aspect, this specification provides a compound having a structure according to formula I or II wherein R1 is (C1-C5 alkyl) O, (C1-C2 alkyl) O (CH2) 2-3O, ( C1-C5 alkyl) C (= O) O, (C1-C5 alkyl) NH, (C1-C2 alkyl) O (CH2) 2-3NH, or (C1-C5 alkyl) C (= O) NH; R2 is, independently for each occurrence thereof, H, C1-C3 alkyl, halo, O (C1-C3 alkyl), CN, or NO2; X is, independently for each occurrence of the same, CR2 or N; and Ar is a 5-membered heteroaromatic portion selected from pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, 1 , 2,4-oxa-diazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-oxadiazolyl, 1,2,5-thiadiazolyl, 1 , 2,3,4-oxatiazolyl, and 1,2,3,4-thiatriazoylil.
[0019] [0019] The compounds according to formula I and II have activity as TLR7 agonists and some of them can be conjugated for targeted release to a target tissue or organ of the intended action. BRIEF DESCRIPTION OF THE DRAWING (S)
[0020] [0020] FIG. 1, FIG. 2, FIG. 3 and FIG. 7 show schemes for the preparation of compounds of this description.
[0021] [0021] FIG. 4 and FIG. 5 show schemes for the preparation of agonist-ligand compounds.
[0022] [0022] FIG. 6 is a representative graph showing the TLR7 agonism activity of a compound of this invention. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS
[0023] [0023] "Antibody" means whole antibodies and any antigen binding fragment (i.e., "antigen binding portion") or its single chain variants. A complete antibody is a protein that comprises at least two heavy (H) and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region comprising three domains, CH1, CH2 and CH3. Each light chain comprises a variable region of the light chain (VL or Vk) and a constant region of the light chain comprising a single domain, CL. The VH and VL regions can also be subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved structure regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from the amino terminal to the carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions contain a binding domain that interacts with an antigen. The constant regions can mediate the binding of the antibody to tissues or host factors, including several cells of the immune system (for example, effector cells) and the first component (Clq) of the classic complement system. An antibody is said to "specifically bind" to an X antigen if the antibody binds to X antigen with a KD of 5 x 10-8 M or less, more preferably 1 x 10-8 M or less, more preferably 6 x 10-9 M or less, more preferably 3 x 10-9 M or less, even more preferably 2 x 10-9 M or less. The antibody can be chimeric, humanized or, preferably, human. The heavy chain constant region can be designed to affect the type or extent of glycosylation, to extend the half-life of the antibody, to enhance or reduce interactions with effector cells or the complement system, or to modulate some other property. Engineering can be performed by replacing, adding or deleting one or more amino acids or by replacing a domain with a domain of another type of immunoglobulin or a combination of the above.
[0024] [0024] "Antigen binding fragment" and "antigen binding portion" of an antibody (or simply "antibody portion" or "antibody fragment") means one or more fragments of an antibody that maintain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a life-sized antibody, such as (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F (ab ') 2 fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a Fab 'fragment, which is essentially a Fab with part of the articulation region (see, for example, Abbas et al., Cellular and Molecular Immunology, 6th Ed., Saunders Elsevier 2007); (iv) an Fd fragment consisting of the VH and CH1 domains; (v) an Fv fragment consisting of the VL and VH domains of a single antibody subdivision, (vi) a dAb fragment (Ward et al., (1989) Nature 341: 544- 546)), which consists of a VH domain ; (vii) an isolated complementarity determining region (CDR); and (viii) a nanobody, a variable region of the heavy chain containing a single variable domain and two constant domains. The preferred antigen-binding fragments are Fab, F (ab ') 2, Fab', Fv and Fd fragments. In addition, although the two Fv fragment domains, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH pair to form monovalent molecules (known as single-chain Fv, or scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also included in the term "antigen binding portion" of an antibody.
[0025] [0025] Unless otherwise indicated - for example, by reference to linear numbering in a SEQ ID NO listing: - references to the numbering of amino acid positions in a heavy or light chain variable region of an antibody (VH or VL) are in accordance with the Kabat system (Kabat et al., "Sequences of proteins of immunological interest, 5th ed., Pub. No. 91-3242, US Dept. Health & Human Services, NIH, Bethesda, Md., 1991, hereinafter "Kabat") and references to the numbering of amino acid positions in a constant region of the heavy or light chain of an antibody (CH1, CH2, CH3 or CL) are in accordance with the EU index, as established in Kabat. See, Lazar et al., US 2008/0248028 A1, the description of which is incorporated here by reference, for examples of such use. In addition, the ImMunoGeneTics Information System (IMGT) provides on its website a table entitled "IMGT Scientific Chart: Correspondence between C Numberings "showing the correspondence between your num system eration, EU numbering and Kabat numbering for the heavy chain constant region.
[0026] [0026] An "isolated antibody" means an antibody that is substantially free of other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to antigen X is substantially free of antibodies that specifically bind to antigens other than antigen X). An isolated antibody that specifically binds to antigen X may, however, cross-react with other antigens, such as molecules of antigen X from other species. In certain embodiments, an isolated antibody specifically binds to the human X antigen and does not cross-react with other (non-human) X antigens. In addition, an isolated antibody can be substantially free of other cellular material and / or chemicals.
[0027] [0027] "Monoclonal antibody" or "monoclonal antibody composition" means a preparation of antibody molecules of unique molecular composition, which exhibits a specificity and single-binding affinity for a specific epitope.
[0028] [0028] "Human antibody" means an antibody having variable regions in which both the structure and the CDR regions (and the constant region, if present) are derived from human germline immunoglobulin sequences. Human antibodies can include further modifications, including natural or synthetic modifications. Human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by randomized or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "human antibody" does not include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human structure sequences.
[0029] [0029] "Human monoclonal antibody" means an antibody that exhibits a unique binding specificity, which has variable regions in which the structure and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a transgenic non-human animal, for example, a transgenic mouse, having a genome comprising a human heavy chain transgene and a chain transgene light fused to an immortalized cell.
[0030] [0030] "Aliphatic" means a non-aromatic hydrocarbon, straight or branched, saturated or unsaturated, having the specified number of carbon atoms (for example, as in "C3 aliphatic", "C1-5 aliphatic" , "C1-C5 aliphatic" or "C1 to C5 aliphatic", the last three sentences being synonymous with an aliphatic portion having 1 to 5 carbon atoms) or, where the number of carbon atoms is not explicitly specified, from 1 to 4 carbon atoms (2 to 4 carbons in the case of unsaturated aliphatic portions). A similar understanding is applied to the number of carbons in other types, such as in C2-4 alkene, C4-C7 cycloaliphatic, etc. In a similar vein, a term like "(CH2) 1-3" should be understood as a shortcut to the subscript being 1, 2 or 3, so that this term represents CH2, CH2CH2 and CH2CH2CH2.
[0031] [0031] "Alkyl" means a saturated aliphatic portion, with the same convention for designating the number of applicable carbon atoms. By way of illustration, the C1-C4 alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, t-butyl, 1-butyl, 2-butyl and the like. "Alkylene" means a divalent counterpart of an alkyl group, such as CH2CH2, CH2CH2CH2 and CH2CH2CH2CH2.
[0032] [0032] "Alkenyl" means an aliphatic moiety having at least one carbon-carbon double bond, with the same convention for designating the number of applicable carbon atoms. By way of illustration, the C2-C4 alkenyl moieties include, but are not limited to, ethenyl (vinyl), 2-propenyl (ally or prop-2-enyl), cis-1-propenyl, trans-1-propenyl, E - (or Z-) 2-butenyl, 3-butenyl, 1,3-butadienyl (but- 1,3-dienyl) and the like.
[0033] [0033] "Alquinyl" means an aliphatic moiety having at least one carbon-carbon triple bond, with the same convention for designating the number of applicable carbon atoms. By way of illustration, C2-C4 alkynyl groups include ethynyl (acetylenyl), propargyl (prop-2-inyl), 1-propynyl, but-2-inyl and the like.
[0034] [0034] "Cycloaliphatic" means a non-aromatic, saturated or unsaturated hydrocarbon moiety, having 1 to 3 rings, each ring having 3 to 8 (preferably 3 to 6) carbon atoms. "Cycloalkyl" means a cycloaliphatic portion in which each ring is saturated. "Cycloalkenyl" means a cycloaliphatic moiety in which at least one ring has at least one carbon-carbon double bond. "Cycloalkynyl" means a cycloaliphatic moiety in which at least one ring has at least one carbon-carbon triple bond. By way of illustration, the cycloaliphatic moieties include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl and adamantyl. The preferred cycloaliphatic moieties are cycloalkyl, especially cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. "Cycloalkylene" means a divalent counterpart to a cycloalkyl group.
[0035] [0035] "Heterocycloaliphatic" means a cycloaliphatic portion in which, in at least one ring thereof, up to three (preferably 1 to 2) carbons have been replaced by a heteroatom independently selected from N, O or S, where N and S can optionally be oxidized and N can optionally be quaternized. The preferred cycloaliphatic moieties consist of a ring, ranging in size from 5 to 6 members. Similarly, "heterocycloalkyl", "heterocycloalkenyl", and "heterocycloalkyl" means a cycloalkyl, cycloalkenyl, or cycloalkynyl moiety, respectively, in which at least one ring thereof has been modified. Exemplary heterocycloaliphatic portions include aziridinyl, azetidinyl, 1,3-dioxanyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrothiopyrinyl sulfone, triforphine, triforphine, chloroform , 1,3-dioxolanyl, tetrahydro-1,1-dioxothienyl, 1,4-dioxanyl, tietanyl, and the like. "Heterocycloalkylene" means a divalent counterpart of a heterocycloalkyl group.
[0036] [0036] "Alkoxy", "aryloxy", "alkylthio", and "arylthio" means -O (alkyl), -O (aryl), -S (alkyl), and -S (aryl), respectively. Examples are methoxy, phenoxy, methylthio, and phenylthio, respectively.
[0037] [0037] "Halogen" or "halo" means fluorine, chlorine, bromine or iodine, unless a more restricted meaning is indicated. +
[0038] [0038] "Aryl" means a hydrocarbon moiety having a mono-, bi- or tricyclic (preferably monocyclic) ring system in which each ring has 3 to 7 carbon atoms and at least one ring is aromatic. The rings in the ring system can be fused together (as in naphthyl) or bonded to each other (as in biphenyl) and can be fused or linked to non-aromatic rings (as in indanyl or cyclohexylphenyl). By way of another illustration, the aryl portions include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthracenyl, and acenaphthyl. "Arylene" means a divalent counterpart of an aryl group, for example, 1,2-phenylene, 1,3-phenylene, or 1,4-phenylene.
[0039] [0039] "Heteroaryl" means a portion having a mono-, bi- or tricyclic ring system (preferably 5-7 membered monocyclic) in which each ring has 3 to 7 carbon atoms and at least one ring is a ring aromatic containing 1 to 4 heteroatoms independently selected from N, O or S, where N and S can optionally be oxidized and N can optionally be quaternized. Such at least one heteroatom containing aromatic ring can be fused to other types of rings (as in benzofuranyl or tetrahydroisoquinolyl) or directly linked to other types of rings (as in phenylpyridyl or 2-cyclopentylpyridyl). By way of another illustration, the heteroaryl moieties include pyrrolyl, furanyl, thiophenyl (thienyl), imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl, N-oxopyridyl, pyrimazinin, pyrimazinine , quinolinyl, isoquinolinyl, quinazolinyl, cinolinyl, quinozalinyl, naphthyridinyl, benzofuranyl, indolyl, benzothiophenyl, oxadiazolyl, thiadiazolyl, phenothiazolyl, benzimidazolyl, benzotriazolyl, dibenzofuranyl, dibenzofuranyl, carbazolyl, carbazolyl, carbazol. "Heteroarylene" means a divalent counterpart of a heteroaryl group.
[0040] [0040] Where it is indicated that a portion can be substituted, such as by using "unsubstituted or substituted" or "optionally substituted" phrases as in "C1-C5 unsubstituted or substituted alkyl" or "optionally substituted heteroaryl", such The portion may have one or more substituents independently selected, preferably one to five in number, more preferably one or two in number. Substituents and substitution patterns can be selected by someone of ordinary skill in the art, taking into account the portion to which the substituent is attached, to provide compounds that are chemically stable and that can be synthesized by techniques known in the art, as well as methods mentioned here. Where a portion is identified as being "unsubstituted or substituted" or "optionally substituted", in a preferred embodiment, such portion is unsubstituted.
[0041] [0041] "Arylalkyl", "(heterocycloaliphatic) alkyl", "arylalkenyl", "arylalkynyl", "biarylalkyl", and the like means an alkyl, alkenyl, or alkynyl portion, as appropriate, replaced by an aryl, heterocycloaliphatic portion, biaryl, etc., as the case may be, with an open (unsatisfied) valence in the alkyl, alkenyl, or alkynyl portion, for example, as in benzyl, phenethyl, N-imidazoylethyl, N-morpholinoethyl, and the like. Conversely, "alkylaryl", "alkenylcycloalkyl", and the like means an aryl, cycloalkyl, etc. portion, as appropriate, replaced by an alkyl, alkenyl portion, etc., as appropriate, for example, as in methylphenyl ( tolyl) or allylcyclohexyl. "Hydroxyalkyl", "haloalkyl", "alkylaryl", "cyanoaryl", and the like means an alkyl, aryl, etc. portion, as appropriate, replaced by one or more identified substituents (hydroxyl, halo, etc., as appropriate) ).
[0042] [0042] For example, permitted substituents include, but are not limited to, alkyl (especially, methyl or ethyl), alkenyl (especially, allyl), alkynyl, aryl, heteroaryl, cycloaliphatic, hetero-cycloaliphatic, halo (especially, fluorine ), haloalkyl (especially, trifluoromethyl), hydroxyl, hydroxyalkyl (especially, hydroxyethyl), cyano, nitro, alkoxy, -O (hydroxyalkyl), -O (haloalkyl) (especially, -OCF3), -O (cycloalkyl), -O (cycloalkyl), -O (heterocycloalkyl), -O (aryl), alkylthio, arylthio, = O, = NH, = N (alkyl), = NOH, = NO (alkyl), -C (= O) (alkyl), -C (= O ) H, -CO2H, -C (= O) NHOH, -C (= O) O (alkyl), -C (= O) O (hydroxyalkyl), -C (= O) NH2, -C (= O) NH (alkyl), -C (= O) N (alkyl) 2, -OC (= O) (alkyl), -OC (= O) (hydroxyalkyl), -OC (= O) O (alkyl), -OC (= O) O (hydroxyalkyl), -OC (= O) NH2, -OC (= O) NH (alkyl), -OC (= O) N (alkyl) 2, azido, -NH2, -NH (alkyl) , -N (alkyl) 2, -NH (aryl), -NH (hydroxyalkyl), -NHC (= O) (alkyl), -NHC (= O) H, -NHC (= O) NH2, -NHC (= O) NH (alkyl), -NHC (= O) N (alkyl) 2, -NHC (= NH) NH2, -OSO2 (alkyl), -SH, -S (alkyl), -S (aryl), -S (cycloalkyl),
[0043] [0043] Where the portion being replaced is a cycloaliphatic, heterocycloaliphatic, aryl or heteroaryl portion, preferred substituents are alkyl, alkenyl, alkynyl, halo, haloalkyl, hydroxyl, hydroxyalkyl, cyano, nitro, alkoxy, -O (hydroxyalkyl), -O (haloalkyl), -O (aryl), -O (cycloalkyl), -O (heterocycloalkyl), alkylthio, arylthio, -C (= O) (alkyl), -C (= O) H, -CO2H, - C (= O) NHOH, -C (= O) O (alkyl), -C (= O) O (hydroxyalkyl), -C (= O) NH2, -C (= O) NH (alkyl), -C (= O) N (alkyl) 2,
[0044] [0044] Where a range is declared, as in "C1-C5 alkyl" or "5 to 10%", such range includes the end points of the range, as in C1 and C5 in the first instance and 5% and 10% in the second instance.
[0045] [0045] Unless determined stereoisomers are specifically indicated (for example, by a bold or dashed bond in a relevant stereocenter in a structural formula, by representing a double bond as having an E or Z configuration in a structural formula or using stereochemical designation nomenclature), all stereoisomers are included in the scope of the invention, as pure compounds, as well as mixtures thereof. Unless otherwise indicated, individual enantiomers, diastereomers, geometric isomers and combinations and mixtures thereof are all covered by this invention.
[0046] [0046] Those skilled in the art will appreciate that the compounds may have tautomeric forms (for example, keto and enol forms), resonance forms and zwitterionic forms that are equivalent to those represented in the structural formulas used in this document and that the structural formulas cover such forms tautomeric, resonant or zwitterionic.
[0047] "Pharmaceutically acceptable ester" means an ester that hydrolyzes in vivo (for example, in the human body) to produce the parent compound or a salt thereof or that has an activity per se similar to that of the parent compound. Suitable esters include C1-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl, especially methyl, ethyl or n-propyl.
[0048] [0048] "Pharmaceutically acceptable salt" means a salt of a compound suitable for pharmaceutical formulation. Where a compound has one or more basic groups, the salt may be an acid addition salt, such as a sulphate, hydrobromide, tartrate, mesylate, maleate, citrate, phosphate, acetate, pamoate (embonate), hydroiodide, nitrate, hydrochloride , lactate, methylsulfate, fumarate, benzoate, succinate, mesylate, lactobionate, suberate, tosylate, and the like. Where a compound has one or more acidic groups, the salt may be a salt, such as a calcium salt, potassium salt, magnesium salt, meglumine salt, ammonium salt, zinc salt, piperazine salt, salt of tromethamine, lithium salt, choline salt, diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodium salt, tetramethylammonium salt, and the like. Polymorphic crystalline forms and solvates are also included in the scope of this invention.
[0049] [0049] In the formulas of this specification, a wavy line () across a bond or an asterisk (*) at the end of the bond indicates a covalent bond site. For example, a statement that R is or that R is in the formula means.
[0050] [0050] In the formulas of this specification, a bond that traverses an aromatic ring between two carbons of the same means that the group attached to the bond can be located in any of the aromatic ring positions made available by removing the hydrogen that is implicitly there. By way of illustration, the formula represents,, or.
[0051] [0051] Generally, tautomeric structures have been converted here into the form of enol, as a matter of consistency and convenience.
[0052] [0052] Those skilled in the art will understand that they could also have been processed in the equivalent keto form and the two equivalent tautomers. TLR7 AGONISTS
[0053] [0053] In formulas I / II, an Ar portion can optionally be replaced. Substituents can be, for example, C1-C3 alkyl, C3-C3 cycloalkyl (CH2) 1-3R3, (CH2) 1-3C (= O) (CH2) 0-3R3, where R3 is halo, OH, CN, NH2 , NH (C1-C5 alkyl), N (C1-C5 alkyl) 2, NH (C3-C6 cycloalkyl), NH (C4-C8 bicycloalkyl), NH (C6-C10 spirocycloalkyl), N (C3-C6 cycloalkyl) 2 , NH (CH2) 1-3 (aryl), N ((CH2) 1-3 (aryl)) 2, a cyclic amine moiety having the structure, a 6-membered aromatic or heteroaromatic moiety or a 5-membered heteroaromatic moiety; wherein an alkyl, cycloalkyl, bicycloalkyl, spiro-cycloalkyl, cyclic amine, 6-membered aromatic or heteroaromatic or 5-membered heteroaromatic moiety is optionally substituted by one or more substituents selected from OH, halo, CN, (C1-C3 alkyl) , O (C1-C3 alkyl), C (= O) (Me), SO2 (C1-C3 alkyl), C (= O) (Et), NH2, NH (Me), N (Me) 2, NH ( Et), N (Et) 2, and N (C1-C3 alkyl) 2; and a cycloalkyl, bicycloalkyl, spirocycloalkyl, or cyclic amine moiety may have a CH2 group substituted by O, S, NH, N (C1-C3 alkyl), or N (Boc). Those skilled in the art will appreciate that, depending on whether the substituent is attached to a carbon or a nitrogen of Ar, R1 in the formula is preferably n-BuO, n-BuNH, EtO, MeO, or MeOCH2CH2O; more preferably n-BuO or MeOCH2CH2O; and even more preferably n-BuO.
[0054] [0054] In formula I, preferably each R2 is H.
[0055] [0055] In one embodiment, a compound according to formula I is represented by formula I ', where each X' is independently CH or N and R1 and Ar are as defined above in relation to formula I:
[0056] [0056] In one embodiment, a compound according to formula II is represented by formula II ', where each X' is independently CH or N and R1 and Ar are as defined above in relation to formula II
[0057] [0057] In one embodiment, a compound according to formula I is represented by formula Ia where R1 is as defined above in relation to formula I, each X 'is independently CH or N and R4 is as defined below.
[0058] [0058] In one embodiment, a compound according to formula I is represented by formula Ib where R1 is as defined above in relation to formula I, each X 'is independently CH or N and R4 is as defined below
[0059] [0059] In one embodiment, a compound according to formula I is represented by formula Ic where R1 is as defined above in relation to formula I, each X 'is independently CH or N, and R4 is as defined down here.
[0060] [0060] In formulas Ia, Ib, and Ic, R4 is H, C1-C3 alkyl, C3-C3 cycloalkyl (CH2) 1-3R3, (CH2) 1-3C (= O) (CH2) 0-3R3, where R3 is halo, OH, CN, NH2, NH (C1-C5 alkyl), N (C1-C5 alkyl) 2, NH (C3-C6 cycloalkyl), NH (C4-C8 bicycloalkyl), NH (C6-C10 spirocycloalkyl) , N (C3-C6 cycloalkyl) 2, NH (CH2) 1-3 (aryl), N ((CH2) 1-3 (aryl)) 2, a cyclic amine moiety having the structure, an aromatic or heteroaromatic moiety of 6 limbs or a heteroaromatic portion of 5 limbs; wherein an alkyl, cycloalkyl, bicycloalkyl, spirocycloalkyl, cyclic amine, 6-membered aromatic or heteroaromatic, or 5-membered heteroaromatic moiety is optionally substituted with one or more substituents selected from OH, halo, CN, (C1-C3 alkyl), O (C1-C3 alkyl), C (= O) (Me), SO2 (C1-C3 alkyl), C (= O) (Et), NH2, NH (Me), N (Me) 2, NH (Et ), N (Et) 2, and N (C1-C3 alkyl) 2; and a cycloalkyl, bicycloalkyl, spirocycloalkyl, or cyclic amine moiety may have a CH2 group substituted by O, S, NH, N (C1-C3 alkyl), or N (Boc).
[0061] [0061] In formulas Ia, Ib, and Ic, preferably R1 is n-BuO.
[0062] [0062] In formulas Ia, Ib, and Ic R4 it is preferably
[0063] [0063] Examples of compounds according to formula Ia include:,,,,,, and.
[0064] [0064] Examples of compounds according to formula Ib include: NH2 (Ib-01) NH2 (Ib-02) N N N N
[0065] [0065] An example of a compound according to formula Ic is:.
[0066] [0066] Table A presents biological activity data for compounds described here. One data set refers to TLR7 agonism activity using the HEK-Blue ™ TLR7 reporter assay, as described below. Another set of data refers to the induction of interleukin 6 (IL-6), a cytokine that plays an important role in the TLR7 pathway. For comparison, the activities of resiquimod, vesatolimod, gardiquimod and compound B are also presented (CAS Reg. 226906-84-9).
[0067] [0067] The TLR7 agonists described herein can be released to the site of the intended action by localized administration or by targeted release in a conjugate with a targeting moiety. Preferably, the target portion is an antibody or antigen binding portion and its antigen is found in the location of the intended action, for example, an antigen associated with a tumor if the intended site of action is a tumor (cancer). Preferably, the tumor-associated antigen is expressed or suppressed exclusively by the cancer cell, as compared to a normal cell. The tumor-associated antigen may be located on the surface of the cancer cell or secreted by the cancer cell in its surroundings.
[0068] [0068] In one aspect, a conjugate is provided comprising the compound of this invention and a linker, represented by the formula IV [D (XD) to (C) c (XZ) b] mZ (IV) where Z is a targeting moiety , D is an agonist of this invention and (XD) aC (XZ) b- are collectively referred to as a "linking moiety" or "ligand" because they link Z and D. Within the ligand, C is a cleavable group designed to be cleaved at or near the site of the intended biological action of D; XD and XZ are spacer portions (or "spacers") that space D and C and C and Z, respectively; subscripts a, b and c are independently 0 or 1 (that is, the presence of XD, XZ and C is optional). The subscript m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (preferably 1, 2, 3 or 4). D, XD, C, XZ and Z are described in more detail below.
[0069] [0069] When binding to a target tissue or cell where its antigen or receptor is located, Z directs the conjugate. Cleavage of group C in the target tissue or cell releases D to exert its effect locally. In this way, the precise release of D is achieved at the site of the intended action, reducing the required dosage. In addition, D is normally biologically inactive (or significantly less active) in its conjugated state, thereby reducing off-target effects.
[0070] [0070] As reflected by the subscript m, each Z can conjugate with more than one D, depending on the number of sites that Z has available for conjugation and the experimental conditions used. Those skilled in the art will understand that, while each individual Z is conjugated to an integer number of Ds, a preparation of the conjugate can analyze a non-integer relationship from D to Z, reflecting a statistical average. This relationship is referred to as the substitution ratio ("SR") or the drug-antibody ratio ("DAR"). Z targeting portion
[0071] [0071] Preferably, the Z targeting portion is an antibody. For convenience and brevity and not by way of limitation, the detailed discussion in this specification on Z and its conjugates is written in the context of being an antibody, however, those skilled in the art will understand that other types of Z can be conjugated, mutatis mutandis. For example, conjugates with folic acid as the targeting portion can target cells with the folate receptor on their surfaces (Leamon et al., Cancer Res. 2008, 68 (23), 9839). For the same reasons, the detailed discussion in this specification is written primarily in terms of a 1: 1 ratio from Z to D (m = 1).
[0072] [0072] Antibodies that can be used in the conjugates of this invention include those that recognize the following antigens: mesothelin, prostate specific membrane antigen (PSMA), CD19, CD22, CD30, CD70, B7H3, B7H4 (also known as O8E) , protein tyrosine kinase 7 (PTK7), glipican-3, RG1, fucosyl-GM1, CTLA 4 and CD44. The antibody can be animal (e.g., murine), chimeric, humanized or, preferably, human. The antibody is preferably monoclonal, especially a human monoclonal antibody. The preparation of human monoclonal antibodies against some of the antigens mentioned above is described in Korman et al., US 8,609,816 B2 (2013; B7H4, likewise known as 08E; in particular 2A7, 1G11, and 2F9 antibodies); Rao-Naik et al., 8,097,703 B2 (2012; CD19; in particular 5G7, 13F1, 46E8, 21D4, 21D4a, 47G4, 27F3, and 3C10 antibodies); King et al., US
[0073] [0073] In addition to being an antibody, Z can also be an antibody fragment (such as Fab, Fab ', F (ab') 2, Fd or Fv) or antibody mimetic, such as an antibody, an antibody domain (dAb), a nanobody, a single body, a DARPin, anticalin, a version, a duocalin, a lipocalin or an avimer.
[0074] [0074] Any of several different reactive groups in Z can be a conjugation site, including ε-amino groups in lysine residues, pendant carbohydrate moieties, carboxylic acid groups in aspartic or glutamic acid side chains, cysteine disulfide groups -cysteine and cysteine thiol groups. For reviews of suitable antibody reactive groups for conjugation, see, for example, Garnett, Adv. Drug Delivery Rev. 2001, 53, 171-216 and Dubowchik and Walker, Pharmacology & Therapeutics 1999, 83, 67- 123, whose descriptions are hereby incorporated by reference.
[0075] [0075] Most antibodies have several lysine residues, which can be conjugated through their -amino groups through amide, urea, thiourea or carbamate bonds.
[0076] [0076] A thiol group (-SH) on the side chain of a cysteine can be used to form a conjugate by several methods. It can be used to form a disulfide bond between it and a thiol group in the linker. Another method is by adding Michael to a maleimide group in the linker.
[0077] [0077] Typically, although antibodies have cysteine residues, they do not have free thiol groups because all of their cysteines are involved in intra- or inter-chain disulfide bonds. To generate a free thiol group, a native disulfide group can be reduced. See, for example, Packard et al., Biochemistry 1986, 25, 3548; King et al., Cancer Res. 1994, 54, 6176; and Doronina et al., Nature Biotechnol. 2003, 21, 778. Alternatively, a cysteine having a free -SH group can be introduced by mutating the antibody, replacing one cysteine with another amino acid or inserting one into the polypeptide chain. See, for example, Eigenbrot et al., US
[0078] [0078] As noted above, the linker comprises up to three elements: a cleavable group C and optional spacers XZ and XD.
[0079] [0079] Group C is cleavable under physiological conditions. Preferably, it is relatively stable while the conjugate is circulating in the blood, however, it is rapidly cleaved when the conjugate reaches its intended site of action.
[0080] [0080] A preferred group C is a peptide that is selectively cleaved
[0081] [0081] Preferably, C contains an amino acid sequence that is a cleavage recognition sequence for a protease. Many cleavage recognition sequences are known in the art. See, for example, Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995); whose descriptions are hereby incorporated by reference.
[0082] [0082] A group C can be chosen so that it is cleaved by a protease present in the extracellular matrix in the vicinity of a cancer, for example, a protease released by nearby dying cancer cells or a protease associated with a tumor secreted by cells carcinogenic. Extracellular proteases associated with exemplary tumors are plasmin, matrix metalloproteases (MMP), thimet oligopeptidase (TOP) and CD10. See, for example, Trouet et al., US 7,402,556 B2 (2008); Dubois et al., US 7,425,541 B2 (2008); and Bebbington et al., US 6,897,034 B2 (2005). Cathepsin D, normally a lysosomal enzyme found within cells, is sometimes found in the vicinity of a tumor, possibly released by dying cancer cells.
[0083] [0083] For conjugates designed to be by an enzyme, C preferably comprises an amino acid sequence selected for cleavage by proteases, such as cathepsins B, C, D, H, L and S, especially cathepsin B. Cathepsin cleavable peptides Examples include Val-Ala, Val-Cit, Val-Lys, Lys-Val-Ala, Asp-Val-Ala, Val-Ala, Lys-Val-Cit, Ala-Val-Cit, Val-Gly, Val-Gln , and Asp-Val-Cit. (Here, amino acid sequences are written in the N-to-C direction, as in H2N-AA2-AA1-CO2H, unless the context clearly indicates otherwise). See, Dubowchik et al., Biorg. Med. Chem. Lett. 1998, 8, 3341; Dubowchik et al., Bioorg. Med. Chem. Lett. 1998, 8, 3347; and Dubowchik et al., Bioconjugate Chem. 2002, 13, 855; whose descriptions are incorporated by reference.
[0084] [0084] Another enzyme that can be used for the cleavage of peptidyl ligands is legumaine, a lysosomal cysteine protease that cleaves preferentially in Ala-Ala-Asn.
[0085] [0085] In one embodiment, Group C is a peptide comprising a sequence of two amino acids -AA2-AA1- where AA1 is lysine, arginine or citrulline and AA2 is phenylalanine, valine, alanine, leucine or isoleucine. In another modality, C consists of a sequence of one to three amino acids, selected from the group consisting of Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu -Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Lys, Cit, Ser, and Glu. More preferably, it is a two to three amino acid peptide from the previous group.
[0086] [0086] The preparation and design of cleavable groups C consisting of a single amino acid is described in Chen et al., US 8,664,407 B2 (2014), the description of which is incorporated herein by reference.
[0087] [0087] Group C can be linked directly to Z or D; that is, the XZ or XD spacers, as appropriate, may be missing.
[0088] [0088] When present, the XZ spacer provides spatial separation between C and Z, so that the former does not interfere stereotypically with the antigen binding by the latter or the latter interferes sterically with the cleavage of the former. In addition, the XZ spacer can be used to impart greater solubility or decreased aggregation properties to the conjugates. An XZ spacer can comprise one or more modular segments, which can be assembled in any number of combinations. Examples of suitable segments for an XZ spacer are:,,,,,, and combinations thereof, where subscript g is 0 or 1 and subscript h is 1 to 24, preferably 2 to 4. These segments can be combined as as illustrated below:,, or.
[0089] [0089] The XD spacer, if present, provides spatial separation between C and D, so that the latter does not interfere sterically or electronically in the cleavage of the former. The XD spacer can also serve to introduce additional molecular weight and chemical functionality into a conjugate. Generally, additional mass and functionality will affect the serum half-life and other properties of the conjugate. Thus, through the careful selection of spacer groups, the serum half-life of a conjugate can be modulated. The XD spacer can also be assembled from modular segments, similar to the description above for the XZ spacer.
[0090] [0090] Spacers XZ and / or XD, when present, preferably provide a linear separation of 4 to 25 atoms, more preferably 4 to 20 atoms, between Z and C or D and C, respectively.
[0091] [0091] The ligand can perform other functions, in addition to covalently binding the antibody and the drug. For example, the linker may contain a poly (ethylene glycol) ("PEG") group. Since the conjugation step typically involves the coupling of a drug-ligand to an antibody in an aqueous medium, a PEG group enhances the aqueous solubility of the drug-ligand. In addition, a PEG group can enhance solubility or reduce aggregation in the resulting ADC. Where a PEG group is present, it can be incorporated into the XD XZ spacer or both. The number of repeat units in a PEG group can be from 2 to 20, preferably between 4 and 10.
[0092] [0092] The spacer XZ or XD, or both, may comprise an auto-immolating portion. A self-immolating portion is a portion that (1) is linked to C and Z or D and (2) has a structure such that the cleavage of group C initiates a reaction sequence resulting in the self-immolating portion that detaches from Z or D, as the case. In other words, the reaction at a distal site of Z or D (cleavage of group C) causes the XZ-Z or XD-D bond to also break. The presence of an auto-immolating portion is desirable in the case of the XD spacer because, if, after cleavage of the conjugate, the XD spacer or a portion of it remains attached to D, the biological activity of D may be impaired. The use of an autoimmune moiety is especially desirable when the cleavable group C is a polypeptide, in which case the autoimmune moiety is typically located adjacent to it, in order to prevent D from interfering sterically or electronically with peptide cleavage.
[0093] [0093] Exemplary self-immolating portions i - v attached to a hydroxyl or amino group of D are shown below: (vii) a b c Me O The N
[0094] [0094] The self-immolating portion is the structure between dotted lines a and b (or dotted lines b and c), with adjacent structural features shown to provide context. The self-immolating moieties i and v are linked to a D-NH2 (that is, the conjugation is by means of an amino group), while the auto-immolating moieties ii, iii and iv are linked to a D-OH (that is, the conjugation is by hydroxyl or carboxyl group). Cleavage of the bond on the dotted line b by an enzyme - a peptidase in the case of structures i - ve a β-glucuronidase in the case of structure vi - initiates a sequence of auto-immolating reaction that results in the cleavage of the connection on the dotted line a and the consequent D-OH or D-NH2, as appropriate. By way of illustration, the autoimmune mechanisms for structures i and iv are shown below:
[0095] [0095] In other words, the cleavage of a first chemical bond in a part of an autoimmune group initiates a sequence of steps that result in the cleavage of a second chemical bond - the one that connects the autoimmune group to the drug - in a different part of auto-immolating group, thus releasing the drug.
[0096] [0096] In some cases, self-immolating groups can be used together, as shown in structure vii. In such a case, cleavage on the dotted line c triggers the self-immolation of the portion between the dotted lines b and c by a 1.6-elimination reaction, followed by auto-immolation of the portion between the dotted lines a and b by a cyclization-elimination reaction. For additional descriptions of autoimmune portions, see Carl et al., J. Med. Chem. 1981, 24, 479; Carl et al., WO 81/01145 (1981); Dubowchik et al., Pharmacology &
[0097] [0097] In another embodiment, Z and D are linked by a non-cleavable linker, that is, C is absent. D metabolism eventually reduces the ligand to a small attached portion that does not interfere with the biological activity of D. Conjugation Techniques
[0098] [0098] The conjugates of TLR7 agonists described herein are preferably made by preparing a compound comprising D and linker (XD) a (C) c (XZ) b (where XD, C, XZ, a, b , and c are as defined for formula II) to form the drug-linking compound represented by formula V: D- (XD) to (C) c (XZ) b-R31 (V) where R31 is a functional group suitable for reacting with a complementary functional group in Z to form the conjugate. Examples of suitable groups R31 include amino, azide, thiol, cyclooctin,,,,,,,, and; where R32 is Cl, Br, F, mesylate, or tosylate and R33 is Cl, Br, I, F, OH, -ON- succinimidyl, -O- (4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluo - rofenila. Chemistry generally usable for the preparation of suitable D- (XD) aC (XZ) b-R31 moieties is disclosed in Ng et al., US 7,087,600
[0099] [0099] Preferably reactive functional group -R31 is -NH2, -OH, -CO2H, -SH, maleimido, cyclooctin, azido (-N3), hydroxylamino (-ONH2) or N-hydroxysuccinimide. Especially preferred functional groups -R31 are: Legend of figures: - or = or
[00100] [00100] A -OH group can be esterified with a carboxy group in the antibody, for example, in an aspartic or glutamic acid side chain.
[00101] [00101] A -CO2H group can be esterified with an -OH group or amidated with an amino group (for example, in a lysine side chain) in the antibody.
[00102] [00102] An N-hydroxysuccinimide group is functionally an activated carboxyl group and can be conveniently amidated by reaction with an amino group (for example, lysine).
[00103] [00103] A maleimide group can be conjugated to a -SH group in the antibody (for example, from cysteine or from the chemical modification of the antibody to introduce a sulfhydryl functionality) in a Michael addition reaction.
[00104] [00104] When an antibody does not have an -SH cysteine available for conjugation, an ε-amino group on the side chain of a lysine residue can react with 2-iminothiolane or N-succinimidyl-3- (2-pyridyldithio) propionate (" SPDP ") to introduce a free thiol group (-SH) - creating a cysteine substitute, so to speak. The thiol group can react with a maleimide or other nucleophile-accepting group to effect conjugation. The mechanism is illustrated below with 2-iminothiolane.
[00105] [00105] Normally, a thiol level of two to three thiols per antibody is reached. For a representative procedure, see, Cong et al., US 8,980,824 B2 (2015), the description of which is incorporated herein by reference.
[00106] [00106] In a reverse arrangement, a Z antibody can be modified with N-succinimidyl 4- (maleimidomethyl) -cyclohexanecarboxylate ("SMCC") or its sulphonated sulfo-SMCC variant, both available from Sigma-Aldrich, to introduce a maleimide group. Then, conjugation can be performed with a drug-linker compound having a -SH group on the linker.
[00107] [00107] An alternative method of conjugation uses copper-free "click chemistry", in which an azide group adds through a tensioned cyclooctine to form a 1,2,3-triazole ring. See, for example, Agard et al., J. Amer. Chem. Soc. 2004, 126, 15046; Best, Biochemistry 2009, 48, 6571, the descriptions of which are incorporated herein by reference. Azide can be located in the antibody and cyclooctin in the drug-ligand moiety, or vice versa. A preferred cyclooctin group is dibenzocyclooctin (DIBO). Various reagents with a DIBO group are available from Invitrogen / Molecular Probes, Eugeno, Oregon. The reaction below illustrates the conjugation of the click chemistry in the case where the DIBO group is linked to the Ab antibody: Conjugate Legend of the figures: - [Drug] - [Ligand]
[00108] [00108] Yet another conjugation technique involves introducing an unnatural amino acid into an antibody, with the unnatural amino acid providing functionality for conjugation to a reactive functional group in the drug moiety. For example, the unnatural amino acid p-acetylphenylalanine can be incorporated into an antibody or other polypeptide, as taught in Tian et al., WO 2008/030612 A2 (2008). The ketone group in p-acetylphenylanine can be a conjugation site by forming an oxime with a hydroxylamino group in the linker-drug moiety. Alternatively, the unnatural amino acid p-azidophenylalanine can be incorporated into an antibody to provide an azide functional group for conjugation via click chemistry, as discussed above. Unnatural amino acids can also be incorporated into an antibody or other polypeptide using cellless methods, as taught in Goerke et al.,
[00109] [00109] Yet another conjugation technique uses the enzyme transglutaminase (preferably bacterial transglutaminase from Streptomyces mobaraensis or BTG), by Jeger et al., Angew. Chem. Int. Ed. 2010, 49, 9995. BTG forms an amide bond between the side-chain carboxamide of a glutamine (the amine acceptor) and an alkylene amino group (the amine donor), which can be, for example, the ε-amino group of a lysine or 5-amino-n-pentyl group. In a typical conjugation reaction, the glutamine residue is located in the antibody, while the alkyleneamino group is located in the linker-drug moiety, as shown below: Conjugated Antibody Legend of the figures: - [Ligand] - [Drug]
[00110] [00110] The positioning of a glutamine residue in a polypeptide chain has a great effect on its susceptibility to BTG-mediated transamidation. None of the glutamine residues in an antibody are normally substrates for BTG. However, if the antibody is deglycosylated - the glycosylation site is asparagine 297 (N297; EU index numbering, as established in Kabat et al., "Sequences of proteins of immunological interest", 5th ed., Pub. No 91-3242, US Dept. Health & Human Services, NIH, Bethesda, Md., 1991; hereinafter "Kabat") of the heavy chain - the nearby glutamine 295 (Q295) makes it susceptible to BTG. An antibody can be deglycosylated enzymatically by treatment with PNGase F (peptide-N-glycosidase F). Alternatively, an antibody can be synthesized free of glycoside by introducing an N297A mutation into the constant region, to eliminate the N297 glycosylation site. In addition, it has been shown that an N297Q substitution not only eliminates glycosylation, but also introduces a second glutamine residue (at position 297) which is also an amine acceptor. Thus, in one embodiment, the antibody is deglycosylated. In another embodiment, the antibody has an N297Q substitution. Those skilled in the art will appreciate that deglycosylation by post-synthesis modification or by introducing an N297A mutation generates two BTG-reactive glutamine residues per antibody (one per heavy chain, at position 295), while an antibody with an N297Q substitution will have four glutamine residues reactive to BTG (two per heavy chain, in positions 295 and 297).
[00111] [00111] An antibody can also be made susceptible to BTG-mediated conjugation by introducing into it a peptide containing glutamine, or "marker", as taught, for example, in Pons et al., US 2013/0230543 A1 (2013) and Rao-Naik et al., WO 2016/144608 A1.
[00112] [00112] In a complementary method, the specificity of the BTG substrate can be changed by varying its amino acid sequence, so that it becomes able to react with glutamine 295 in an unmodified antibody, as taught in Rao-
[00113] [00113] Although the most commonly available bacterial transglutaminase is that of S. mobaraensis, transglutaminase from other bacteria, having slightly different substrate specificities, can be considered, such as the Streptoverticillium ladakanum transglutaminase (Hu et al., US 2009/0318349 A1 (2009), US 2010/0099610 A1 (2010), and US 2010/0087371 A1 (2010)).
[00114] [00114] The TLR7 agonists of this description having a primary or secondary alkyl amine are particularly suitable for use in conjugates, as the secondary amine provides a functional group for ligand binding. An example of such a TLR7 agonist-linker compound is compound 11, which contains an enzymatically cleavable linker. FIG. 4 shows a scheme according to which compound 11 can be prepared. NH2 NH2 N NH N
[00115] [00115] An example of a TLR7 agonist-linker compound that contains a non-enzymatically cleavable linker is compound 13. FIG. 5 shows a scheme for synthesizing compound 13.
[00116] [00116] Both compounds 11 and 13 contain a primary alkylamino group, making them susceptible to conjugation with transglutaminase.
[00117] [00117] Conjugation can also be performed using the enzyme Sortase A, as taught in Levary et al., PLoS One 2011, 6 (4), e18342; Proft, Biotechnol. Lett. 2010, 32, 1-10; Ploegh et al., WO 2010/087994 A2 (2010); and Mao et al., WO 2005/051976 A2 (2005). The Sortase A recognition motif (typically LPXTG, where X is any natural amino acid) can be located in the Z linker and the nucleophilic acceptor motif (usually GGG) can be the R31 group in formula III or vice versa. TLR7 Agonist Conjugates
[00118] [00118] Applying the techniques described above, TLR7 agonist conjugates such as those shown below can be prepared: NH2 NH2 N NH N
[00119] [00119] The attachment of a poly (ethylene glycol) (PEG) chain to a drug ("PEGylation") can improve the pharmacokinetic properties of the latter. The drug's circulation half-life is increased, sometimes above an order of magnitude, concomitantly reducing the dosage necessary to achieve the desired therapeutic effect. PEGylation can also decrease the metabolic degradation of a drug and reduce its immunogenicity. For a review, see Kolate et al., J. Controlled Release 2014, 192,
[00120] [00120] Initially, PEGylation was applied to biological drugs. By 2016, more than ten PEGylated biological products had been approved. Turecek et al., J. Pharmaceutical Sci. 2016, 105, 460. More recently, stimulated by the successful application of the concept in biological products, attention has turned to its application in small molecule drugs. In addition to the benefits mentioned above, PEGylated small molecule drugs may have greater solubility and cause less toxic effects. Li et al. Prog. Polymer Sci. 2013, 38, 421.
[00121] [00121] The compounds described herein can be PEGylated. Where a compound has an aliphatic hydroxyl or a primary or secondary aliphatic amine, as in the case of compound Ia-01 or Ia-02 (arrows), it can be PEGylated by an ester, amide, carbonate or carbamate group with a PEG molecule containing carboxy using conventional techniques such as dicyclohexylcarbodiimide (DCC), HATU, N-hydroxysuccinimide esters, and the like. Several other methods for the PEGylation of pharmaceutical molecules are described in Alconcel et al., Polymer Chem. 2011, 2, 1442, the description of which is incorporated herein by reference.
[00122] [00122] If desired, a TLR7 agonist described here can be
[00123] [00123] In the above reaction sequence, the dipeptide valine-citrulline (Val-Cit) is cleavable by the enzyme cathepsin B, with a p-amino-benzyl oxycarbonyl group (PABC) serving as a self-immolating spacer. The functional group for the conjugation is an amine group, which is temporarily protected by an Fmoc group. The conjugation is carried out by the enzyme transglutaminase, with a side chain of glutamine (Gln) acting as an acyl acceptor. The subscript x, denoting the number of PEG repeat units, can vary widely, depending on the purpose of PEGylation, as discussed below. For some purposes, x can be relatively small, such as 2, 4, 8, 12 or
[00124] [00124] Those skilled in the art will understand that the sequence is illustrative and that other elements - peptide, auto-immolating group, conjugation method, length of PEG, etc. - can be used, as is well known in the art. They will also understand that, while the above sequence combines PEGylation and conjugation, PEGylation does not require conjugation and vice versa.
[00125] [00125] Where the compound does not have aliphatic hydroxyl or primary or secondary aliphatic amine, as in the case of compound 3 (FIG. 1), it can still be PEGylated in the aromatic amine (arrow). A method for PEGilaçaõ in this position is described by Zarraga, US 2017/0166384 A1 (2007), the description of which is incorporated by reference.
[00126] [00126] In some embodiments, it may be desirable to have several PEGylated agonists attached to a single molecule. For example, four PEGylated subdivisions can be constructed of pentaerythritol (C (CH2OH) 4) and a TLR7 agonist can be attached to each PEGylated subdivision. See Gao et al., US 2013/0028857 A1 (2013), the description of which is incorporated by reference.
[00127] [00127] To modulate the pharmacokinetics, it is generally preferred that the PEG portion has a formula weight between about 2 kDa (corresponding to about 45 repeat units of - (CH2CH2O) - and between about 40 kDa (corresponding to about 910 repeat units of - (CH2CH2O) -, more preferably between about 5 kDa and about 20 kDa. That is, the range of the subscript x in the above formulas is about 45 to about 910. It should be understood that PEG compositions are not 100% homogeneous, however, on the contrary, they exhibit a molecular weight distribution, so a reference to, for example, "20kDa PEG" means PEG having an average molecular weight of 20 kDa.
[00128] [00128] PEGylation can also be used to improve the solubility of an agonist. In such cases, a shorter PEG chain can be used, for example, comprising 2, 4, 8, 12 or 24 repeat units. EXAMPLES
[00129] [00129] The practice of this invention can be further understood by reference to the following examples, which are provided by way of illustration and not by way of limitation.
[00130] [00130] This example and FIGs. 1 and 7 refer to the synthesis of compounds according to formula Ia.
[00131] [00131] Referring first to FIG. 1: nitrogen was bubbled through a sealable tube containing 9- (4-bromobenzyl) -2-butoxy-8-methoxy-9H-purin-6-amine 1 (prepared according to WO 2011/049815 A1, 400mg , 0.985 mmol), ethyl 2- (3- (4,4,5,5-tetramethyl-1,3,2-dioxabo-rolan-2-yl) -1H-pyrazol-1-yl) acetate 2 (359 mg , 1.280 mmol) and K2CO3 (476 mg, 3.45 mmol) in dioxane (12 ml) and water (3 ml) for 2 minutes. Tetracis (triphenylphosphine) palladium (0) (114 mg, 0.098 mmol) was added and N2 was bubbled through for another 1 min. The tube was then sealed and stirred at 80 ° C for 2.5 hours, after which the reaction was complete. On cooling, EtOAc (20 m) was added and the solid was filtered through a CELITE ™ pad. The two layers of filtrate were separated. The aqueous layer was extracted again with EtOAc twice. The combined organic extracts were dried over Na2SO4, filtered and concentrated. The crude product was purified on a 12 g silica column, eluted with 20% MeOH in dichloromethane (DCM, 0-30% gradient). The desired fractions were concentrated to produce ester 3 (168 mg, 0.350 mmol, 35.6% yield). LCMS ESI: calculated for C24H29N7O4 = 480.2 (M + H +), found 480.1 (M + H +).
[00132] [00132] The aqueous layer of the filtrate was acidified with 1.0 N HCl to pH 4 and extracted with 20% MeOH in DCM (3x15 ml). The combined organic extracts were dried over Na2SO4, filtered and concentrated to produce acid 4 (97 mg, 0.215 mmol, 21.82% yield). LCMS ESI: calculated for C22H25N7O4 = 452.2 (M + H +), found 452.1 (M + H +).
[00133] [00133] A stirred solution of acid 4 (50 mg, 0.111 mmol) in N, N-dimethylformamide (1.5 mL) was treated with tert-butyl (2-aminoethyl)
[00134] [00134] Referring now to FIG. 7: a suspension of 2-butoxy-8-methoxy-9H-purin-6-amine 14 (CAS Reg. No. 866268-31-7, TFA salt, 500 mg, 1.423 mmol) and cesium carbonate (1.484 mg, 4.55 mmol) in DMF (10 mL) 2- (4- (bromomethyl) phenyl) -4,4,5,5-tetramethyl-1,3,2-dioxaborolane 15 (CAS Reg. No. 138500-85-3, 507 mg, 1.708 mmol). The reaction mixture was stirred at room temperature for 3 h, after which the reaction was completed with two main products. The reaction was quenched with saturated NH4Cl. The resulting solid was collected by filtration and dried to produce a mixture of compound 17 (600 mg, 0.242 mmol, 17.03% yield) and compound 16 (600 mg, 0.926 mmol, 65.1% yield). LCMS ESI (compound 17): calculated for C17H23BN5O4 = 372.2 (M + H +), found 372.2 (M + H +). LCMS ESI (compound 16): calculated for
[00135] [00135] To a mixture of compound 17 (600 mg, 0.242 mmol,) and compound 16 (600 mg, 0.926 mmol,) was added 2- (4-bromo-1H-pyrazol-1-yl) ethan-1-ol 18 (CAS Reg. No. 214614-81-0, 336 mg, 1.757 mmol) and K2CO3 (607 mg, 4.39 mmol) in dioxane (8 mL) and water (2,000 mL). Nitrogen gas was bubbled through the reaction mixture for 2 min. Then, the adduct of PdCl2 (dppf) -CH2Cl2 (154 mg, 0.188 mmol) was added. Nitrogen was bubbled through for another 1 min. The reaction vessel was sealed and its contents were stirred at 80 ° C for 5 h. The reaction mixture was cooled, diluted with ethyl acetate and filtered. The filtrate was dried over Na2SO4 and concentrated. The crude product was purified by an ISCO silica column (40 g), eluted with 20% MeOH in DCM, DCM gradient 0 to 40%. The desired fractions were concentrated to produce compound 19 (247 mg, 0.565 mmol, 45.0% yield). LCMS ESI: calculated for C23H27N7O3 = 438.2 (M + H +), found 438.1 (M + H +). 1H NMR (400 MHz, METANOL-d4) δ 8.00 (s, 1H), 7.84 (s, 1H), 7.53 (d, J = 8.1 Hz, 2H), 7.32 (d , J = 8.1 Hz, 2H), 5.13 (s, 2H), 4.34 (t, J = 6.6 Hz, 2H), 4.26 (s, 2H), 4.14 (s , 3H), 3.92 (t, J = 5.4 Hz, 2H), 1.84 - 1.65 (m, 2H), 1.59 - 1.37 (m, 2H), 1.00 ( t, J = 7.5 Hz, 3H).
[00136] [00136] To a stirred suspension of compound 19 (57 mg, 0.130 mmol) in THF (0.5 mL) was added HCl (1.0 N in water, 1 mL, 0.130 mmol). The reaction mixture was stirred at 60 ° C for 3 h, after which the reaction was complete. Upon cooling, the white solid precipitate was collected by filtration and rinsed with water and air-dried to produce compound Ia-01 (37 mg, 0.086 mmol, 65.7% yield). C22H25N7O3 = 424.2 (M + H +), found 423.9 (M + H +). 1H NMR (400 MHz, METANOL-d4) δ 8.20 - 7.94 (m, 1H), 7.89 (br s, 1H), 7.55 (br d, J = 7.7 Hz, 2H) , 7.43 (br d, J = 7.9 Hz, 2H), 5.05 (s, 2H), 4.55 (t, J = 6.3 Hz, 2H), 4.25 (br d, J = 1.8 Hz, 2H), 4.08 - 3.59 (m, 2H), 1.87 - 1.69 (m,
[00137] [00137] A stirred suspension of compound Ia-01 (341 mg, 0.805 mmol) in THF (10 mL) was treated with thionyl chloride (1.175 mL, 16.10 mmol) and then stirred at room temperature for 4 minutes. H. The thionyl chloride was removed azeotropically with DCM (3X). The residue was then purified on an ISCO silica column (24 g), eluted with 20% MeOH in DCM, gradient from 0 to 45% DCM. The desired fractions were concentrated to produce compound Ia-07 (200 mg, 0.453 mmol, 56.2% yield). LCMS ESI: calculated for C22H24ClN7O2 = 442.2 (M + H +), found 442.2 (M + H +). 1H NMR (500 MHz, DMSO-d6) δ 9.99 (br s, 1H), 8.21 (s, 1H), 7.91 (s, 1H), 7.53 (d, J = 8.1 Hz, 2H), 7.30 (br d, J = 8.1 Hz, 2H), 6.46 (br s, 2H), 4.85 (s, 2H), 4.51 - 4.39 (m , 2H), 4.20 - 4.10 (m, 2H), 4.02 (br t, J = 5.8 Hz, 2H), 1.68 - 1.58 (m, 2H), 1.44 - 1.31 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H).
[00138] [00138] To a suspension of compound Ia-07 (87 mg, 0.1969 mmol) in DMF (5 mL) was added cyclobutanamine (0.5 mL, 5.9 mmol). The reaction mixture was stirred at 60 ° C for 18 h. After cooling, the excess amine was removed with a rotary evaporator. The reaction mixture was diluted with DMSO (15 ml). The insoluble material was filtered. Purification by C18 chromatography (Accu Prep), eluting with 0.05% TFA in acetonitrile: 0.05% TFA in water with a 0-40% gradient. The desired fractions were concentrated to 1/3 volume, frozen and lyophilized to produce compound Ia-04 (47 mg, 0.079 mmol, 40.1% yield). LCMS ESI: calculated for C26H32N8O2 = 477.3 (M + H +), found 477.2 (M + H +). 1H NMR (400 MHz, METANOL-d4) δ 7.87 (s, 1H), 7.72 (s, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.28 (d , J = 8.1 Hz, 2H), 4.88 (s, 2H), 4.19 (t, J = 6.6 Hz, 2H), 4.13 (t, J = 6.5 Hz, 2H ), 3.14 (br t, J = 7.7 Hz, 1H), 2.87 (t, J = 6.4 Hz, 2H), 2.18 - 2.06 (m, 2H), 1, 71 - 1.56 (m, 6H), 1.46 - 1.30 (m, 2H), 0.86 (t, J = 7.4
[00139] [00139] Other compounds according to formula Ia were prepared analogously. Their analytical data is provided in Table B. Table B - Additional Compounds Ia Mass spectrum Calculated Compound (M + H +) Found (M + H +) Ia-01 422.2 (MH) 422.2 (MH) Ia-03 435 , 2 435.2 Ia-05 438.2 438.2 Ia-06 492.2 492.3
[00140] [00140] In an illustration of PEGylation, the DCC-mediated esterification of compound Ia-01 with HO2C (CH2CH2O) 37CH3 produces PEGylated Example 2 - Synthesis of compounds of formula Ib
[00141] [00141] This example and FIG. 2 refer to the synthesis of compounds according to formula Ib.
[00142] [00142] Compound 6 was prepared from compound 1 and compound 5 (CAS Reg. 1266480-56-1) using a procedure similar to that described for compound 3. LCMS ESI: calculated for C27H34N6O4S = 539.2 (M + H +), found 539.1 (M + H +). 1H NMR (400 MHz, METANOL-d4) δ 7.59 (d, J = 8.4 Hz, 2H), 7.47 (s, 1H), 7.34 (d, J = 8.4 Hz, 2H ), 7.28 (s, 1H), 5.14 (s, 2H), 4.41 (s, 2H), 4.34 (t, J = 6.6 Hz, 2H), 4.14 (s , 3H), 1.84 - 1.65 (m, 2H), 1.56 - 1.40 (m, 11H), 1.00 (t, J = 7.4 Hz, 3H).
[00143] [00143] A stirred solution of compound 6 (45 mg, 0.084 mmol) in THF (1 ml) was treated with HCl (1.0 N in water, 1.0 ml). After stirring at 70 ° C for 5 h, the reaction was complete. The reaction mixture was concentrated. The crude product was purified on 15.5 g of the C18 Aq column and eluted with 0.05% TFA in acetonitrile: 0.05% TFA in H2O (0-50% gradient) to produce the compound (Ib-01 ) as the trifluoroacetate salt (31.1 mg, 0.057 mmol, 67.7% yield). LCMS ESI: calculated for C27H34N6O4S = 425.2 (M + H +), found 425.1 (M + H +). 1H NMR (400 MHz, METANOL-d4) δ 7.58 (d, J = 1.3 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.45 (s, 1H ), 7.32 (d, J = 8.1 Hz, 2H), 4.89 (s, 2H), 4.23 (s, 2H), 4.18 (t, J = 6.6 Hz, 2H ), 1.71 - 1.47 (m, 2H), 1.43 - 1.16 (m, 2H), 0.85 (t, J = 7.4 Hz, 3H).
[00144] [00144] Compound Ib-02 was prepared analogously. LCMS ESI: calculated = 426.2 (M + H +), found 426.2 (M + H +). Example 3 - Synthesis of compounds of formula Ic
[00145] [00145] This example and FIG. 3 refer to the synthesis of compounds according to formula Ic.
[00146] [00146] Compound 9 was prepared from compound 7 (prepared according to WO 2011/049815 A1) and compound 8 (CAS Reg. 1092351-92-2) using a procedure similar to that described for compound 3. LCMS ESI: calculated for C23H32BN5O4 = 452.3 (M-H +), found 452.1 (M-H +).
[00147] [00147] The compound (Ic-01) was prepared from compound 9, using a procedure similar to that described for compound (Ib-01). LCMS ESI: calculated for C20H22N6O4 = 411.2 (M + H +), found 411.2 (M + H +). 1H NMR (500 MHz, DMSO-d6) δ 7.98 - 7.79 (m, 3H), 7.43 (br d, J = 8.2 Hz, 2H), 6.35 (s, 2H), 4.93 (s, 2H), 4.43 (br s, 2H), 4.15 (t, J = 6.6 Hz, 2H), 1.67 - 1.49 (m, 2H), 1, 42 - 1.28 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H). Example 4 - TLR7 Agonist Activity Assay
[00148] [00148] This example describes a method for evaluating the TLR7 agonist activity of the compounds described in this specification.
[00149] [00149] Blue human embryonic kidney cells projected
[00150] [00150] FIG. 6 is a representative graph showing the data thus obtained for compound Ib-02. Example 5 - Transglutaminase-Mediated Conjugation
[00151] [00151] The following procedure can be used for transglutaminase-mediated conjugation of agonist-ligand compounds, in which the ligand has an amine group that can act as an amine donor. The antibody can be one that has a transglutaminase-reactive glutamine, for example, one with an N297A or N297Q substitution. Conjugation is performed by recombinant bacterial transglutaminase with an antibody: enzyme molar ratio of 5: 1. Conjugation is performed using standard protocols in 50 mM Tris buffer, pH 8.0, incubated overnight at 37 ° C. The resulting conjugate is purified on a protein A column, pre-equilibrated with 50 mM Tris, pH 8.0. The conjugate is eluted with 0.1 M sodium citrate buffer, pH 3.5. The eluted fractions are neutralized with 1M Tris pH 9.0. The conjugate can be formulated in 20 mg / ml Sorbitol, 10 mg / ml Glycine, pH 5.0. Example 6 - Interleukin Induction Assay 6
[00152] [00152] This example describes a method for testing the induction of interleukin 6 by compounds described in this specification.
[00153] [00153] Compounds diluted in DMSO were transferred to individual wells of a 384 well plate with transparent V bottom from Matrix Technologies using ECHO acoustic liquid handling technology (25 nL per well). Human whole blood samples (25 uL) were added to each well using a CyBio FeliX liquid handling instrument. The plate was shaken on a plate shaker for three minutes before incubating the reaction mixtures at 37 ° C for 20 h. Basel's RPMI 1640 medium (supplemented with L-glutamine) was then added to each well (25 µl per well) before the plasma was released from each sample by centrifugation (450 x g, 5 min, room temperature). The treated plasma samples (3 uL) were subsequently transferred to individual wells of a white, shallow 384 well ProxiPlate (Perkin Elmer) using the FeliX liquid handling instrument and their interleukin 6 levels were measured using AlphaLISA technology, as described by the manufacturer, PerkinElmer. Data analysis software was used to determine the EC50 values of the compound on which the baseline was established using mean DMSO values and 100% established induction using reference compound values at the highest concentration tested. EC50s can be determined with software such as Graphpad Prism ™.
[00154] [00154] Those skilled in the art will understand that the conditions and methodologies in this example are illustrative and not limiting and that their variations or other methods for conjugation are known in the art and usable in the present invention.
[00155] [00155] The previous detailed description of the invention includes passages that are mainly or exclusively related to particular parts or aspects of the invention. It should be understood that this is for clarity and convenience, that a particular aspect may be relevant to more than just the passage in which it is disclosed and that the description here includes all the appropriate combinations of information found in the different passages. Likewise, although the various figures and descriptions contained herein are related to specific embodiments of the invention, it should be understood that where a specific aspect is described in the context of a particular figure or modality, that aspect can also be used, to the extent appropriate, in the context of another figure or modality, in combination with another aspect, or in the invention in general.
[00156] [00156] Furthermore, although the present invention has been particularly described in terms of certain preferred embodiments, the invention is not limited to such preferred embodiments. On the contrary, the scope of the invention is defined by the appended claims. REFERENCES
[00157] [00157] Complete citations for the following references cited in abbreviated form by the first author (or inventor) and the date earlier in this specification are provided below. Each of these references is incorporated by reference for all purposes. Akinbobuyi et al., ACS 2013 69th Southwest Regional Meet- ing, Abstract SWRM-70, "Synthesis and evaluation of purine-based toll- like receptor 7 agonists and their antibody conjugates". Akinbobuyi et al., ACS 2015 Joint Southeastern / Southwest
Regional Meeting, Abstract 392, "Synthesis of functionalized purine analogs for antibody conjugation" [2015a]. Akinbobuyi et al., Tetrahedron Lett. 2015, 56, 458, "Facile syntheses of functionalized toll-like receptor 7 agonists" [2015b]. Akinbobuyi et al., Bioorg.
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Ding et al., WO 2017/076346 A1 (2017) [2017b]. Gadd et al., Bioconjugate Chem. 2015, 26, 1743, "Targeted Activation of Toll-Like Receptors: Conjugation of a Toll-Like Receptor 7 Agonist to a Monoclonal Antibody Maintains Antigen Binding and Specification". Graupe et al., US 8,993,755 B2 (2015). Halcomb et al., US 9,161,934 B2 (2015). Hashimoto et al., US 2009/0118263 A1 (2009). Hirota et al., US 6,028,076 (2000). Holldack et al., US 2012/0083473 A1 (2012). Isobe et al., US 6,376,501 B1 (2002). Isobe et al., JP 2004137157 (2004). Isobe et al., J.
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Cancer 2013, 132 (3), 580, "Intratracheal and oral administration of SM-276001: A selective TLR7 agonist, leads to antitumor efficacy in primary and metastatic models of cancer". Li et al., US 9,902,730 B2 (2018). Lioux et al., US 9,295,732 B2 (2016). Lund et al., Proc.
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Toward the Comprehension of the Molecular Determinants through Ligand-Based Approaches ". Ogita et al., US 2007/0225303 A1 (2007). Peterson, Rachel; Honor Program Thesis," Synthesis of Sulfur and Amino-8- Substituted Adenine Derivatives as TLR7 Agonists ", Baylor University (2014). Pryde, US 7,642,350 B2 (2010). Roethle et al., J.
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权利要求:
Claims (14)
[1]
1. A compound having a structure according to formula (I) or (II) characterized by the fact that R1 is (C1-C5 alkyl) O, (C1-C2 alkyl) O (CH2) 2-3O, (C1- C5 alkyl) C (= O) O, (C1-C5 alkyl) NH, (C1-C2 alkyl) O (CH2) 2-3NH, or (C1-C5 alkyl) C (= O) NH; R2 is, independently for each occurrence thereof, H, C1-C3 alkyl, halo, O (C1-C3 alkyl), CN, or NO2; X is, independently for each occurrence of the same, CR2 or N; and Ar is a 5-membered heteroaromatic portion selected from pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, 1 , 2,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-oxaziazolyl, 1,2,5-thiadiazolyl , 1,2,3,4-oxatiazolyl, and 1,2,3,4-thiatriazolyl.
[2]
A compound according to claim 1, having a structure represented by the formula (I ') characterized by the fact that each X' is independently CH or N and Ar and R1 are as defined in claim 1.
[3]
A compound according to claim 1, having a structure represented by the formula (II ') characterized by the fact that each X' is independently CH or N and Ar and R1 are as defined in claim 1.
[4]
A compound according to claim 1, having a structure represented by the formula (Ia), (Ib), or (Ic) characterized by the fact that R1 is as defined in claim 1, each X 'is independently CH or N, and R4 is H, C1-C3 alkyl, C3-C3 cycloalkyl (CH2) 1-3R3, (CH2) 1-3C (= O) (CH2) 0-3R3, where R3 is halo, OH, CN, NH2, NH (C1-C5 alkyl), N (C1-C5 alkyl) 2, NH (C3-C6 cycloalkyl), NH (C4-C8 bicycloalkyl), NH (C6-C10 spirocycloalkyl), N (C3-C6 cycloalkyl) 2 , NH (CH2) 1-3 (aryl), N ((CH2) 1-3 (aryl)) 2, a cyclic amine portion having the structure, an aromatic or heteroaromatic portion of 6 members or a heteroaromatic portion of 5 members; where an alkyl, cycloalkyl, bicycloalkyl, spirocycloalkyl, cyclic amine, 6-membered aromatic or heteroaromatic, or 5-membered heteroaromatic portion is optionally substituted with one or more substituents selected from OH, halo, CN, ( C1-C3 alkyl), O (C1-C3 alkyl), C (= O) (Me), SO2 (C1-C3 alkyl), C (= O) (Et), NH2, NH (Me), N (Me) 2, NH (Et), N (Et) 2, and N (C1-C3 alkyl) 2; and a cycloalkyl, bicycloalkyl, spirocycloalkyl, or a cyclic amine moiety may have a CH2 group substituted by O, S, NH, N (C1-C3 alkyl), or N (Boc).
[5]
5. Compound according to claim 4, characterized by the fact that it has a structure represented by the formula (Ia): NH2 (Ia)
N N
OH R1 N N X 'X'
N N R4.
[6]
6. Composed according to claim 4, characterized by the fact that it has a structure represented by the formula (Ib):.
[7]
7. Composed according to claim 4, characterized by the fact that it has a structure represented by the formula (Ic):.
[8]
8. Compound according to claim 4, characterized by the fact that R4 is H2 N
O , , , ,
HN H
N
AT THE
NH,,, o.
[9]
9. Compound according to claim 1, characterized by the fact that it is conjugated to an antibody.
[10]
10. Compound according to claim 1, characterized by the fact that it is covalently linked to a poly (ethylene glycol) portion between 2 kDa and 40 kDa in size.
[11]
11. Composed according to claim 1, characterized by the fact that it is to treat a condition that can be treated by activating the Toll 7 Receiver.
[12]
12. Compound according to claim 4, characterized by the fact that it is conjugated to an antibody.
[13]
13. A compound according to claim 4, characterized by the fact that it is covalently attached to a poly (ethylene glycol) portion between 2 kDa and 40 kDa in size.
[14]
14. Composed according to claim 4, characterized by the fact that it is to treat a condition that can be treated by activating the Toll 7 Receiver.

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法律状态:
2021-10-19| B08F| Application dismissed because of non-payment of annual fees [chapter 8.6 patent gazette]|Free format text: REFERENTE A 3A ANUIDADE. |

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2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2022-02-08| B08K| Patent lapsed as no evidence of payment of the annual fee has been furnished to inpi [chapter 8.11 patent gazette]|Free format text: EM VIRTUDE DO ARQUIVAMENTO PUBLICADO NA RPI 2650 DE 19-10-2021 E CONSIDERANDO AUSENCIA DE MANIFESTACAO DENTRO DOS PRAZOS LEGAIS, INFORMO QUE CABE SER MANTIDO O ARQUIVAMENTO DO PEDIDO DE PATENTE, CONFORME O DISPOSTO NO ARTIGO 12, DA RESOLUCAO 113/2013. |

优先权:

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申请号 | 申请日 | 专利标题

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US201762546211P| true| 2017-08-16|2017-08-16|

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US62/546,211|2017-08-16|

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PCT/US2018/000245|WO2019035970A1|2017-08-16|2018-08-16|6-amino-7,9-dihydro-8h-purin-8-one derivatives as immunostimulant toll-like receptor 7agonists|

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