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

专利摘要:
Compounds having a structure according to formula (I) or (II) wherein R1, R2, R3, R4, Ar and X1 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 the targeted release to the organ or tissue of the planned action.
公开号:BR112020003108A2
申请号:R112020003108-9
申请日:2018-08-16
公开日:2020-08-04
发明作者:Liqi He；Sanjeev Gangwar；Yam B. POUDEL；Prasanna Sivaprakasam
申请人:Bristol-Myers Squibb Company；
IPC主号:

专利说明:

[0001] [0001] This application claims the benefit under 35 U.S.C. §119 (e) of US Serial Provisional Application No. 62 / 546.222, filed on August 16, 2017; whose disclosure is hereby incorporated by reference. BACKGROUND OF THE INVENTION
[0002] [0002] This disclosure relates to Toll type 7 ("TLR7") receptor agonists and their conjugates, and methods for the preparation and use of such agonists and their conjugates.
[0003] [0003] Toll-like receptors ("TLRs") are cell surface receptors that recognize molecular patterns associated with pathogens ("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. Human beings have 11 TLRs called TLR1 to TLR11.
[0004] [0004] 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 real pathogens , by stimulating the immune response.
[0005] [0005] TLR7 recognizes the 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 ssR-NA40 (Berghöfer et al. 2007) and one for guanosine (Zhang et al. 2016).
[0006] [0006] TLR7 can bind and be activated by synthetic guanosine type agonists, such as imiquimod, resiquimod and gardiquimod,
[0007] [0007] Synthetic TLR7 agonists based on a molecular scaffold of pteridinone are also known, as exemplified by vesatolimod (Desai et al. 2015), which has been in phase 2 clinical trials. The potency of vesatolimod is reported to be 100X less than that of the corresponding purine-8-one compound, as measured by IFN-α induction (Roethle et al. 2013).
[0008] [0008] Other synthetic TLR7 agonists are based on a purine-like scaffold, often according to formula (A): where R, R 'and R "are structural variables, with R" typically containing one unsubstituted or substituted aromatic or heteroaromatic ring.
[0009] [0009] Disclosures 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: Akin-bobuyi et al. 2015b and 2016; Barberis et al. 2012; Carson et al. 2014;
[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 disclose TLR7 modulators in which the two rings of a purine portion are measured by a macrocycle:
[0012] [0012] A TLR7 agonist can be conjugated to a partner molecule, which can be, for example, a phospholipid, a polyl (ethylene glycol) ("PEG") or another TLR (usually TLR2). Exemplary disclosures 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 was also disclosed: 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 scaffold have also been disclosed. See Cortez et al. 2017a and 2017b, McGowan et al. 2017 and Li et al. 2018.
[0014] [0014] Jensen et al. 2015 disclose 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;
[0016] [0016] TLR7 agonists based on a 5H-pyrrolo [3,2-d] pyrimidine scaffold have also been disclosed. See Cortez et al. 2017a and 2017b, McGowan et al. 2017 and Li et al. 2018.
[0017] [0017] The complete citations for the documents cited in this invention by the first author or inventor and yearbooks are listed at the end of this specification. BRIEF SUMMARY OF THE INVENTION
[0018] [0018] In one aspect, this specification provides a compound that has a structure according to formula (I) or (II) 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; X is, independently for each of its occurrences, CR2 or N; R2 is, independently for each of its occurrences, H, C 1- C3 alkyl, halo, O (C1-C3 alkyl), CN or NO2; R3 is O, S, NH or N (C1-C3 alkyl); Ar is,,, or; where one of Y, Y 'and Y ”is selected from –O-, -S-, -NH- and –N (C1-C3 alkyl) - and the other two from Y, Y' and Y” are selected de = N- and = CR2-; R4 is H, C1-C3 alkyl, halo, O (C1-C3 alkyl), CN, NO2, or (CH2) xR5, where the subscript x is 1, 2, 3 or 4; and
[0019] [0019] The compounds according to formulas (I) and (II) have activity as TLR7 agonists and some of them can be conjugated for the targeted release to a target tissue or organ of the planned action. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] [0020] Figures 1, Figure 2, Figure 3 and Figure 7 show schemes for the preparation of the compounds of this disclosure.
[0021] [0021] Figures 4 and Figure 5 show schemes for the binding of ligands to the compounds of this invention, making them suitable for conjugation.
[0022] [0022] Figure 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 fragments (ie, "antigen-binding part") or their single chain variants.
[0024] [0024] "Antigen binding fragment" and "antigen binding part" of an antibody (or simply "antibody part" or "antibody fragment") means one or more fragments of an antibody that retain 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 full-length antibody, such as (i) a Fab fragment, a modern fragment consisting of the VL, VH, CL and CH1 domains ; (ii) an F (ab ') 2 fragment, a divalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) a Fab 'fragment, which is essentially a Fab with part of the hinge 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 branch of an antibody, (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 domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, through a synthetic linker that allows them to be produced as a single chain protein in which the VL and VH regions pair to form monovalent molecules (known as single-chain Fv or scFv); see, for example, Bird et al. (1988) Science
[0025] [0025] Unless otherwise specified - for example, by reference to linear numbering in a SEQ ID NO listing: - references to the numbering of amino acid positions in a variable region of heavy or light chain of an antibody (VH or VL) are according to the Kabat system (Kabat et al., "Sequences of proteins of immunological logic, 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 presented in Kabat. See Lazar et al., US 2008/0248028 A1, the disclosure of which is hereby incorporated by reference, for examples of such use. In addition, the ImMunoGeneTics Information System (IMGT) provides on its web page a table entitled " IMGT Scientific Chart: Correspondence between C Numberings "which shows the correspondence between your system numbering system, 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 having different antigen specificities (for example, an isolated antibody that specifically binds to antigen X is substantially free of antibodies that specifically bind to antigens in addition to 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 X antigens (non-human).
[0027] [0027] "Monoclonal antibody" or "monoclonal antibody composition" means a preparation of antibody molecules of single molecular composition, which has a specificity and binding affinity unique to a specific epitope.
[0028] [0028] "Human antibody" means an antibody having variable regions in which both structural and 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 random or site-specific mutagenesis in vitro or via 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 framework sequences.
[0029] [0029] "Human monoclonal antibody" means an antibody that has a unique binding specificity, which has variable regions in which both structural 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 moiety
[0031] [0031] "Alkyl" means a saturated aliphatic portion, with the same convention for designating the number of carbon atoms being applicable. 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 component having at least one carbon-carbon double bond, with the same convention for designating the number of carbon atoms being applicable. By way of illustration, the C2-C4 alkenyl portions 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 that has at least one carbon-carbon triple bond, with the same convention for designating the number of carbon atoms being applicable. By way of illustration, the C2-C4 alkynyl groups include ethynyl (acetylenyl),
[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 some cycloalkyls, 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 of its rings, 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 they can optionally be oxidized and N can optionally be quaternized. The preferred cycle-life portions consist of a ring, with a size of 5 to 6 members. Likewise, "heterocycloalkyl", "heterocycloalkenyl" and "heterocycloalkyl" mean a cycloalkyl, cycloalkenyl or cycloalkynyl moiety, respectively, in which at least one of its rings has been modified in this way. Exemplary heterocycloaliphatic moieties include aziridinyl, azetidinyl, 1,3-dioxanyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrothiopyranyl sulfone, tetrahydrin sulfone, morpholine, tetrahydrin, sulphonine, sulphonine, sulphonine, sulfate, morphine. of thiomorpholinyl, thiomorpholinyl sulfone, 1,3-dioxolanyl, tetrahydro-1,1-dioxothienyl, 1,4-dioxanyl, tietanyl, and the like. "He-
[0036] [0036] "Alkoxy", "aryloxy", "alkylthio" and "arylthio" mean –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, wherein 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 linked together (as in biphenyl) and can be fused or linked to non-aromatic rings (as in indanyl or cyclohexylphenyl). By means 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 that has a mono-, bi- or tricyclic ring system (preferably 5 to 7 membered monocyclic), where each ring has 3 to 7 carbon atoms and at least one ring it is an aromatic ring containing from 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 aromatic ring containing heteroatom can be fused with other types of rings (as in benzofuranil or tetrahydroisoquinolyl) or directly linked to other types of rings (as in phenylpyridyl or 2-cyclopentylpyridyl). As an additional illustration, the heteroaryl moieties include pyrrolyl, furanyl, thiophenyl (thienyl), imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl,
[0040] [0040] Where it is indicated that a portion can be substituted, such as by using the phrases "unsubstituted or substituted" or "optionally substituted" as in "C1-C5 unsubstituted or substituted alkyl" or "optionally heteroaryl substituted ", that portion may have one or more independently selected substituents, preferably one to five in number, more preferably one or two in number. Substituents and substitution patterns can be selected by a person of practical skill in the technique, taking into account the portion to which the substituent is attached, to provide compounds that are chemically stable and that can be synthesized by known techniques in the specialty, as well as the methods presented here. Where a portion is identified as being "unsubstituted or substituted" or "optionally substituted", in a preferred embodiment, that 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 portion, heterocycloaliphatic, biaryl, etc., as the case may be, with an open (unsatisfied) value 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., as the case may be, replaced by an alkyl, alkenyl, etc., as the case may be, for example, as in methylphenyl (tolyl) or allylcyclohexyl. "Hydro-
[0042] [0042] For example, permitted substituents include, but are not limited to, (especially methyl or ethyl), alkenyl (especially allyl), alkynyl, aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic, halo (especially fluorine), haloalkyl (especially trifluoro-romethyl), hydroxyl, hydroxyalkyl (especially hydroxyethyl), cyano, nitro, alkoxy, -O (hydroxyalkyl), -O (haloalkyl) (especially -OCF3), -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), -S (= O) alkyl, -SO2 (alkyl), -SO2NH2, -SO2NH (alkyl), -SO2N (alkyl) 2, and the like.
[0043] [0043] Where the portion being substituted is an aliphatic portion, preferred substituents are aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic, halo, hydroxyl, cyano, nitro, alkoxy, -O (hydroxyalkyl), -O (haloalkyl), - O (cycloalkyl), -O (heterocycloalkyl), -O (aryl), alkylthio, arylthio, = O, = NH, = N (alkyl), = NOH, = NO (alkyl), -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,
[0044] [0044] 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, -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), -S (= O) alkyl, -SO2 (alkyl), -SO2NH2, -SO2NH (alkyl), and -SO2N (alkyl) 2. The most preferred substituents are alkyl, alkenyl, halo, haloalkyl, hydroxyl, hydroxyalkyl, cyano, nitro, alkoxy, -O (hydroxyalkyl), -C (= O) (alkyl), -C (= O) H, -CO2H, -C (= O) NHOH, -C (= O) O (alkyl), -C (= O) O (hydroxyalkyl), -C (= O) NH2,
[0045] [0045] Where a range is mentioned, as in "C1-C5 alkyl" or "5 to 10%", this range includes the end points of the range, as in C1 and C5 in the first case and 5% and 10% in the second case.
[0046] [0046] Unless specific 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 through the use of stereochemical designation nomenclature), all stereoisomers are included within the scope of the invention, as pure compounds, as well as mixtures thereof. Unless otherwise indicated, enantiomers, diastereomers, individual geometric isomers and their combinations and mixtures are all included by this invention.
[0047] [0047] Those skilled in the art will observe that the compounds can 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 invention and that the Structural formulas cover such tautomeric, resonant or zwitterionic forms.
[0048] [0048] "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 included
[0049] [0049] "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 sulfate, hydrobromide, tartrate, mesylate, maleate, citrate, phosphate, acetate, pamoate (embonate), iodhydrate, nitrate, hydrochloride, lactate, methyl sulfate, fumarate, benzoate, succinate, mesylate, lactobionate, suberate, tosylate, and the like. Where a compound has one or more acidic groups, the salt can be a salt such as a calcium salt, potassium salt, magnesium salt, meglumine salt, ammonium salt, zinc salt, piperazine salt, tromethamine salt , 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 within the scope of this invention.
[0050] [0050] 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 of or that R is in the formula that R is signi. stay
[0051] [0051] In the formulas of this specification, a bond that crosses an aromatic ring between two carbons of the same means that the group attached to the bond can be located in any of the positions of the aromatic ring made available by the removal of the hydrogen that is implicitly over there. By way of illustration, the formula
[0052] [0052] Generally, tautomeric structures were presented here in the form of enol, as a matter of consistency and convenience.
[0053] [0053] R1 in formulas (I) and (II) preferably is n-BuO, n-BuNH, EtO, MeO or MeOCH2CH2O; more preferably n-BuO or Me-OCH2CH2O; and most preferably n-BuO.
[0054] [0054] In formulas (I) and (II), preferably each R2 is H.
[0055] [0055] In formulas (I) and (II), preferably R3 is O.
[0056] [0056] In one embodiment, a compound according to formula (I) is represented by formula (I '), where each X' is independently CH or N and R1, Ar and R4 are as defined above as to formulas (I) / (II):
[0057] [0057] In one embodiment, a compound according to formula (II) is represented by formula (II '), in which each X' is independent
[0058] [0058] In one embodiment, a compound according to formula (I) is represented by formula (Ia), where x, R1 and R5 are as defined above with respect to formulas (I) / (II):
[0059] [0059] In one embodiment, a compound according to formula (I) is represented by formula (Ib), where x, R 1 and R5 are as defined above with respect to formulas (I) / (II) :
[0060] [0060] In one embodiment, a compound according to formula (I) is represented by formula (Ic), where x, R1 and R5 are as defined above with respect to formulas (I) / (II):
[0061] [0061] In a modality, a compound according to formula (I) is represented by the formula (Id), where x, R 1 and R5 are as defined above regarding formulas (I) / (II) :
[0062] [0062] In one embodiment, a compound according to formula (I) is represented by formula (Ie), where x, R1 and R5 are as defined above with respect to formulas (I) / (II):
[0063] [0063] In a modality, a compound according to formula (I) is represented by the formula (If), where x, R1 and R5 are as defined above regarding formulas (I) / (II):
[0064] [0064] In one embodiment, a compound according to formula (I) is represented by formula (Ig), where x, R 1 and R5 are as defined above as for formulas (I) / (II) :
[0065] [0065] In one embodiment, a compound according to formula (II) is represented by formula (IIa), where x, R1 and R5 are as defined above in relation to formulas (I) / (II) :
[0066] [0066] In formulas (Ia), (Ib), (Ic), (Id), (Ie), (If) and (IIa), preferably R1 is n-BuO and the subscript x is 1.
[0067] [0067] In formulas (I), (I '), (II), (II'), (Ia), (Ib), (Ic), (Id), (Ie), (If) and (IIa) (in the first four formulas in the case where R4 is (CH2) xR5 with x preferably being 1), R5 is preferably OH, Cl,,,,,,,,,,,,,,,,,,,,,, ,, or.
[0068] [0068] Preferably, in formulas (I) and (II), not more than two X's, and more preferably not more than one X, in any given aromatic ring is N.
[0069] [0069] Preferably, in formulas (I '), (II'), (Ia), (Ib), (IIa) and (IId), no more than two X's, and more preferably no more than one X , in any aromatic ring provided is N.
[0070] [0070] Examples of compounds according to formula (Ia) include:,,,,,,, and.
[0071] [0071] Examples of compounds according to formula (Ib) include:
[0072] [0072] Examples of compounds according to formula (Ic) include:,,
[0073] [0073] Examples of compounds according to the formula (Id) include:,,,,
[0074] [0074] Examples of compounds according to formula (Ie) include:,,,,
[0075] [0075] Examples of compounds according to formula (If) included:,,,,
[0076] [0076] An example of a compound according to the formula (Ig) is:
[0077] [0077] Examples of compounds according to formula (IIa) include:
[0078] [0078] Table A presents biological activity data for the compounds disclosed here. One data set refers to the agonism activity of TLR7 using the HEK-Blue ™ TLR7 reporter assay, as described below. Another data set 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 (CAS Reg. No. 226906-84-9) are also shown.
[0079] [0079] The TLR7 agonists disclosed herein can be released at the site of the intended action through localized administration or through targeted release in a conjugate with a targeting moiety. Preferably, the targeting portion is an antibody or its antigen-binding portion and its antigen is found at the location of the intended action, for example, an antigen associated with the tumor if the intended site of action is a tumor (cannula). cer). Preferably, the tumor-associated antigen is expressed or overexpressed exclusively by the cancer cell, compared to a normal cell. The tumor-associated antigen may be
[0080] [0080] 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 portion of targeting, D is an agonist of this invention and - (XD) aC (XZ) b- are collectively referred to as a "linker portion" or "linker" because they link Z and D. Within the linker, 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 m subscript 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.
[0081] [0081] By binding to a target tissue or cell where its antigen or receptor is located, Z directs the conjugate over there. 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. Likewise, D is normally biologically inactive (or significantly less active) in its conjugated state, thus reducing nonspecific and unintended effects.
[0082] [0082] As reflected by the subscript m, each Z can combine with more than one D, depending on the number of sites that Z has available for conjugation and the experimental conditions employed. Those skilled in the art will observe that, although 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
[0083] [0083] Preferably, the targeting portion Z is an antibody. For convenience and brevity and not by way of limitation, the discussion detailed in this specification on Z and its conjugates is written in the context of being an antibody, but those skilled in the art will understand that other types of Z can be conjugated, changed tatis mutandis. For example, conjugates with folic acid as the targeting moiety can target cells with the folate receptor on their surfaces (Leamon et al., Cancer Res. 2008, 68 (23), 9839). For the same reasons, the debate detailed in this specification is written primarily in terms of a 1: 1 ratio from Z to D (m = 1).
[0084] [0084] The 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., muzzle), 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 disclosed in Korman et al., US 8,609,816 B2 (2013; B7H4, also known as 08E; in particular antibodies 2A7, 1G11 and 2F9); 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 8,481,683 B2 (2013; CD22; in particular antibodies 12C5, 19A3, 16F7 and 23C6); Keler et al., US
[0085] [0085] 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 organism, an antibody domain (dAb), a nanobody, a single body, a DARPin, anticalin, a versacebody, a duocalin, a lipocalin or an avimer.
[0086] [0086] Any one 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, dis-
[0087] [0087] Most antibodies have several lysine residues, which can be conjugated through their ε-amino groups through amide, urea, thiourea or carbamate bonds.
[0088] [0088] 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.
[0089] [0089] Typically, although antibodies have cysteine residues, they lack 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 Bio-technol. 2003, 21, 778. Alternatively, a cysteine having a free –SH group can be introduced by mutating the antibody, which replaces one cysteine with another amino acid or inserts one into the polypeptide chain. See, for example, Eigenbrot et al., US 7,521,541 B2 (2009); Chilkoti et al., Bioconjugate Chem. 1994, 5, 504; Urnovitz et al., US 4,698,420 (1987); Stimmel et al., J. Biol. Chem. 2000, 275, 30445; Bam et al., US 7,311,902 B2 (2007); Kuan et al., J. Biol. Chem. 1994, 269, 7610; Poon et al., J. Biol. Chem. 1995, 270, 8571; Junutula et al., Nature Biotechnology 2008, 26, 925 and Rajpal et al., US Provisional Application No. 62/270245, filed December 21, 2015. In yet another approach, a cysteine is added to the C-terminus of the chain heavy and light. See, for example, Liu et al., US 8,865,875 B2 (2014); Cumber et al., J. Immunol. 1992, 149, 120; King et al, Cancer Res. 1994, 54, 6176; Li et al., Bioconjugate Chem. 2002, 13, 985; Yang et al., Protein Engineering 2003, 16, 761; and Olafson et al., Protein Engineering Design & Selection 2004, 17, 21. The disclosures of the documents cited in this paragraph are hereby incorporated by reference. Binders and Their Portions
[0090] [0090] As noted above, the linker comprises up to three elements: a cleavable group C and optional spacers XZ and XD.
[0091] [0091] Group C is cleavable under physiological conditions. Preferably, it is relatively stable while the conjugate is circulating in the blood, but it is easily cleaved when the conjugate reaches its intended site of action.
[0092] A preferred group C is a peptide that is selectively cleaved by a protease within the target cell, as opposed to a protease in the serum. Typically, the peptide comprises from 1 to 20 amino acids, preferably from 1 to 6 amino acids, more preferably from 2 to 3 amino acids. Amino acids can be natural and / or unnatural α-amino acids. Natural amino acids are those encoded by the genetic code, as well as their derived amino acids, for example, hydroxyproline, y-carboxyglutamate, citrulline and O-phosphoserine. In this specification, the term "amino acid" also includes amino acid analogues and mimetics. Analogs are compounds having the same general H2N (R) CHCO2H structure as a natural amino acid, except that the R group is not found among the natural amino acids. Examples of analogs include homoserine, norleucine, methionine sulfoxide and methyl methionine sulfonium. An amino acid mimetic is a compound that has a different structure from the general chemical structure of an α-amino acid, but works similarly to one. The amino acid can be from the "L" stereochemistry of
[0093] [0093] Preferably, C contains an amino acid sequence which 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 disclosures are hereby incorporated by reference.
[0094] [0094] A group C can be selected so that it is cleaved by a protease present in the extracellular matrix around a cancer, for example, a protease released by nearby dying cancer cells or a protease associated with the secreted tumor cancer cells. 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 Beebington 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.
[0095] [0095] For conjugates designed to be in 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 B cleavable peptides exempl - Plares 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 direction of N to C, 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 disclosures are incorporated by reference.
[0096] [0096] Another enzyme that can be used to cleave peptidyl ligands is legumaine, a lysosomal protease from cysteine that preferably cleaves in Ala-Ala-Asn.
[0097] [0097] 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.
[0098] [0098] The preparation and design of cleavable groups C consisting of a single amino acid is disclosed in Chen et al., US 8,664,407 B2 (2014), the disclosure of which is incorporated herein by reference.
[0099] [0099] Group C can be linked directly to Z or D; that is, the XZ or XD spacers, as the case may be missing.
[00100] [00100] When present, the XZ spacer provides spatial separation between C and Z, so that the former does not interfere sterically with the antigen binding by the latter or the latter sterically interferes with the cleavage of the former. In addition, the XZ spacer can be used to provide greater solubility or reduced aggregation properties to the conjugates. An XZ spacer can comprise one or more modular segments, which can be mounted in any number of combinations. Examples of suitable segments for an XZ spacer are:
[00101] [00101] 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.
[00102] [00102] Spacers XZ and / or XD, where 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.
[00103] [00103] The linker can perform other functions besides the covalent binding of the antibody and the drug. For example, the linker may contain a poly (ethylene glycol) ("PEG") group. Since the conjugation step typically involves coupling a drug-ligand to an antibody in an aqueous medium, a PEG group can greatly increase the aqueous solubility of the drug-ligand. Likewise, a group
[00104] [00104] 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 that results in the self-immolating portion that comes off from Z or D, as appropriate. 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 a self-immolating portion is desirable in the case of the XD spacer because, if, after cleavage of the conjugate, the XD spacer or a part of it remains attached to D, the biological activity of D may be impaired. The use of a self-immolating portion is especially desirable where the cleavable group C is a polypeptide, in which case the self-immolating portion is typically located adjacent to it, in order to prevent D from interfering sterically or electronically in the peptide cleavage. deos.
[00105] [00105] The exemplary self-immolating portions (i) to (v) attached to a hydroxyl or amino group of D are shown below:
[00106] [00106] The self-immolating portion is the structure between the dotted lines a and b (or dotted lines b and c), with adjacent structural features shown to provide context.
[00107] [00107] In other words, the cleavage of a first chemical bond in a part of a self-immolating group initiates a sequence of steps that results in the cleavage of a second chemical bond - one that connects to the self-immolating group to the drug - in a different part the self-immolation group, thus releasing the drug.
[00108] [00108] In some cases, self-immolating groups can be used together, as shown by the structure (vii). In this case, the cleavage on the dotted line c activates the auto-immolation of the portion between the dotted lines b and c through an elimination reaction of 1.6, followed by the auto-immolation of the portion between the dotted lines a and b through a cyclization-elimination reaction. For additional disclosures about the autoimmune portions, see Carl et al., J. Med. Chem. 1981, 24, 479; Carl et al., WO 81/01145 (1981); Dubowchik et al., Pharmacology & Therapeutics 1999, 83, 67; Firestone et al., US 6,214,345 B1 (2001); Toki et al., J. Org. Chem. 2002, 67, 1866; Doronina et al., Nature Biotechnology 2003, 21, 778 (erratum, p. 941); Boyd et al., US 7,691,962 B2; Boyd et al., US 2008/0279868 A1; Sufi et al., WO 2008/083312 A2; Feng, US 7,375,078 B2; Jeffrey et al., US 8,039,273; and Senter et al., US 2003/0096743 A1; whose disclosures are incorporated by reference.
[00109] [00109] In another mode, Z and D are connected by a non-cleavable link, 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
[00110] [00110] TLR7 agonist conjugates disclosed herein are preferably prepared by first 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-ligand compound represented by formula (V):
[00111] [00111] Preferably the reactive functional group -R31 is -NH2, -OH, -CO2H, -SH, maleimido, cyclooctin, azido (-N3), hydroxylamino (-ONH2) or N-hydroxysuccinimide. Especially preferred functional groups -R31 are: or
[00112] [00112] A –OH group can be esterified with a carboxy group in the antibody, for example, in a side chain of aspartic or glutamic acid.
[00113] [00113] A -CO2H group can be esterified with an -OH group or amidated with an amino group (for example, on a lysine side chain) in the antibody.
[00114] [00114] An N-hydroxysuccinimide group is functionally an activated carboxyl group and can be conveniently amidated by reaction with an amino group (for example, from lysine).
[00115] [00115] 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.
[00116] [00116] Where 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-pyridylditium) 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 another accepting group of nucleophiles to effect the conjugation. The mechanism is illustrated below with 2-iminothiolane.
[00117] [00117] Typically, a thiol level of two to three thiols per antibody is achieved. For a representative procedure, see Cong et al., US 8,980,824 B2 (2015), whose disclosure is incorporated herein by reference.
[00118] [00118] In an inverse arrangement, a Z antibody can be modified with N-succinimidyl 4- (maleimidomethyl) -cyclohexanecarboxylate ("SMCC") or its sulphonated sulfo-SMCC variant, both of which are available from Sigma-Aldrich , to introduce a maleimide group. Then, the conjugation can be carried out with a drug-linker compound having a -SH group on the linker.
[00119] [00119] An alternative conjugation method employs copper-free "click chemistry", in which an azide group is added through a reinforced cyclooctin 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 disclosures 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 preferable cyclooctin group is dibenzocyclooctin (DIBO). Various reagents having a DIBO group are available from Invitrogen / Molecular Pro- bes, Eugene, Oregon. The reaction below illustrates the conjugation of the “click” chemistry in the case where the DIBO group is linked to the antibody (Ab): [drug] - [ligand] [drug] - [ligand] conjugate
[00120] [00120] Yet another conjugation technique involves introducing an unnatural amino acid into an antibody, with the unnatural amino acid providing functionality for conjugation to a group.
[00121] [00121] Yet another conjugation technique uses the enzyme transglutaminase (preferably bacterial transglutaminase from Streptoyces 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 alkyleneamino group (the amine donor), which can be, for example, the ε-amino group of a lysine or a 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: [linker] - [drug] antibody
[00122] [00122] 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 presented 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; then “Kabat”) of the heavy chain - glutamine 295 (Q295) nearby makes BTG susceptible. An antibody can be deglycosylated enzymatically through 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 mode, the antibody has an N297Q substitution. Those skilled in the art will note that deglycosylation through post-synthesis modification or the introduction of an N297A mutation generates two BTG-reactive glutamine residues per antibody (one per heavy chain, at position 295), while one antibody with an N297Q substitution will have four BTG-reactive glutamine residues (two per heavy chain, at positions 295 and 297).
[00123] [00123] An antibody can also become susceptible to conjugation
[00124] [00124] In a complementary approach, the specificity of the BTG substrate can be altered by varying its amino acid sequence, in such a way that it becomes able to react with glutamine 295 in an unmodified antibody, as taught in Rao-Naik et al., WO 2017/059158 A1 (2017).
[00125] [00125] Although the bacterial transglutaminase most commonly available is that of S. mobaraensis, the transglutaminase of other bacteria, having slightly different substrate specificities, can be considered, such as the transglutaminase of Streptoverticilium ladakanum (Hu et al ., US 2009/0318349 A1 (2009), US 2010/0099610 A1 (2010) and US 2010/0087371 A1 (2010)).
[00126] [00126] The TLR7 agonists of this disclosure having a primary or secondary alkylamine 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 22, which contains an enzymatically cleavable linker. FIG. 5 shows a scheme according to which compound 22 can be prepared.
[00127] [00127] An example of a TLR7-linker agonist compound that contains a non-enzymatically cleavable linker is compound 24. FIG. 6 shows a scheme for synthesizing compound 24.
[00128] [00128] Both compounds 22 and 24 contain a primary alkylamino group, which makes them receptive to conjugation with transglutamine. A suitable conjugation procedure is described in the examples below.
[00129] [00129] 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 recognition motif of Sortase A (typically LPXTG, where X is any natural amino acid) can be located in the Z linker and the nucleophilic accepting motif (typically GGG) can be the group R31 in formula (III), or vice versa. TLR7 Agonist Conjugates
[00130] [00130] By applying the techniques described above, TLR7 agonist conjugates such as those shown below, can be prepared: Conjugate 1
[00131] [00131] 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 a 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, 167.
[00132] [00132] Initially, PEGylation was applied to biological drugs. In 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 of biological products, attention has turned to its application in small molecule drugs. In addition to the benefits mentioned above, PEGylated small molecule drugs can have greater solubility and cause less toxic effects. Li et al. Prog. Polymer Sci. 2013, 38, 421.
[00133] [00133] The compounds disclosed herein can be PEGylated. When a compound has an aliphatic hydroxyl or primary or secondary aliphatic amine, as in the case of compound Ia-01 or Ia-02 (arrows), it can be PEGylated by means of an ester, amide, carbonate or carbamate group with a PEG molecule containing
[00134] [00134] If desired, a TLR7 agonist disclosed herein can be PEGylated by means of an enzymatically cleavable linker comprising a self-immolating portion, to allow the release of the non-PEGylated agonist in a designed manner. In addition, PEGylation can be combined with conjugation to a protein such as an antibody, if the molecule containing PEG has a suitable functional group such as an amine, for binding to the protein. Protein can provide an additional therapeutic function or, if an antibody, it can provide a targeting function. These concepts are illustrated in the following reaction sequence, where TLR7-NH-R generically represents a TLR7 agonist:
[00135] [00135] In the reaction sequence above, the dipeptide valine-citrulline (Val-Cit) is cleavable by the enzyme cathepsin B, with a group of p-aminobenzyl oxycarbonyl (PABC) serving as a self-adhesive spacer. The functional group for the conjugation is an amine group, which is temporarily protected by an Fmoc group. The conjugation is effected by the enzyme transglutaminase, with a glutamine side chain (Gln) that acts as an acyl acceptor. The subscript x, which signifies the number of PEG repetition units, can vary widely, depending on the purpose of PEGuilation, as examined below. For some purposes, x may be relatively small, such as 2, 4, 8, 12 or 24. For other purposes, x is large, for example, between about 45 and about 910.
[00136] [00136] Those skilled in the art will understand that the sequence is illustrative and that other elements - peptide, autoimolation 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.
[00137] [00137] Where the compound lacks aliphatic hydroxyl or primary or secondary aliphatic amine, as in the case of compound 13 (FIG. 2), it can still be PEGylated in the aromatic amine (arrow). A method for PEGuilation in this position is disclosed by Zarraga, US 2017/0166384 A1 (2007), whose disclosure is incorporated by reference.
[00138] [00138] In some embodiments, it may be desirable to have several PEGylated agonists attached to a single molecule. For example, four PEGylated branches can be constructed in pentaerythritol (C (CH2OH) 4) and a TLR7 agonist can be attached to each PEGylated branch. See Gao et al., US 2013/0028857 A1 (2013), the disclosure of which is incorporated by reference.
[00139] [00139] To modulate the pharmacokinetics, it is generally preferable that the PEG portion has a formula weight between about 2 kDa (which corresponds to about 45 repeat units (CH2CH2O)) and between about 40 kDa (which corresponds to about 910 repeating units (CH2CH2O)), more preferably between about 5 kDa and about 20 kDa. That is, the range of the subscript x in the formulas above is about 45 to about 910. It should be understood that the PEG compositions are not 100% homogeneous, but, on the contrary, have a molecular weight distribution. Thus, a reference to, for example, "20kDa PEG" means PEG having an average molecular weight of 20 kDa.
[00140] [00140] 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
[00141] [00141] 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. Example 1 - Series Compounds (Ia) and (Ib)
[00142] [00142] This example and FIG. 1 refer to the synthesis of series compounds (Ia) and (Ib), with the compound (Ib-02) being used as an example.
[00143] [00143] COMPOUND 3. A suspension of methyl 4-hydroxybenzoate 2 (2 g, 13.15 mmol), 6-fluoronicotinaldehyde 1 (1.809 g, 14.46 mmol) and K2CO3 (1.998 g, 14.46 mmol) in DMF (26.3 ml) was stirred at 110 ° C for 4 h. LCMS indicated that the reaction was complete. After cooling, the reaction was suppressed with water and the resulting solid was collected by filtration and rinsed with water and dried in vacuo to produce compound 3, (3.30 g, 12.84 mmol, 95.1% yield) - to). LCMS ESI: calculated for C14H11NO4 = 258.1 (M + H +), observed 258.0 (M + H +), 1H NMR (400 MHz, CHLOROPHORUM-d) δ 10.01 (s, 1H), 8.63 ( d, J = 2.4 Hz, 1H), 8.23 (dd, J = 8.6, 2.4 Hz, 1H), 8.17 - 7.97 (m, 2H), 7.27 - 7 , 22 (m, 2H), 7.10 (d, J = 8.6 Hz, 1H), 3.93 (s, 3H).
[00144] [00144] COMPOUND 4. A solution of compound 3 (3.76 g, 14.62 mmol) in MeOH (100 ml) was treated with NaBH4 (0.553 g, 14.62 mmol) in portions at 0 ° C, then stirred for 10 min while the bath was still in effect. LCMS indicated that the reaction was complete. The reaction was suppressed by the slow addition of half saturated NH4Cl. The reaction mixture is stirred for 30 min at RT and extracted with EtOAc. The organic extracts were dried over Na2SO4, filtered and concentrated. The crude solid was slurried with water and collected by filtration and dried in vacuo to produce compound 4 (3.37 g, 13.00 mmol, 89% yield). LCMS ESI: calculated for C14H13NO4 = 260.1 (M + H +), observed 260.0 (M + H +). 1H
[00145] [00145] COMPOUND 5. A suspension of compound 4 (0.2 g, 0.771 mmol) in THF (3.86 ml) was treated with triphenylphosphine (0.223 g, 0.849 mmol), followed by N-bromosuccinimide (NBS, 0.165 g , 0.926 mmol). After stirring at RT for 60 min, the reaction was complete. The reaction was suppressed with water and the reaction mixture was extracted with EtOAc. The organic extracts were dried over Na2SO4, filtered, and concentrated. The crude product was purified on a 12 g silica column, eluted with 0 to 80% EtOAc / hexane to produce compound 5 (0.15 g, 0.466 mmol, 60 , 4% yield). LCMS ESI: calculated for C14H12BrNO3 = 322.0 (M + H +), observed 321.9 (M + H +). 1H NMR (400 MHz, CHLOROPHORMUM-d) δ 8.21 (d, J = 2.4 Hz, 1H), 8.15 - 8.01 (m, 2H), 7.80 (dd, J = 8, 5, 2.5 Hz, 1H), 7.23 - 7.13 (m, 2H), 6.97 (d, J = 8.6 Hz, 1H), 4.47 (s, 2H), 3, 92 (s, 3H).
[00146] [00146] COMPOUND 7. A suspension of 2-butoxy-8-methoxy-9H-purin-6-amine 6 (CAS Reg. No. 866268-31-7, in TFA, 562 mg, 1,600 mmol) and cesium carbonate (1.67 g, 5.12 mmol) in DMF (20 ml) was treated with compound 5 (567 mg, 1.760 mmol). The reaction mixture was stirred at RT for 3 h. LCMS indicated that the reaction was complete. The reaction was quenched with saturated NH4Cl and extracted with EtOAc. The organic extracts were dried over Na2SO4, filtered and concentrated. The crude product was purified on a 40 g silica column, eluting with 20% MeOH in DCM (0 to 40% gradient). The desired fractions were concentrated to produce compound 7 (478 mg, 0.999 mmol, 62.4% yield). LCMS ESI: calculated for C24H26N6O5 = 479.2 (M + H +), observed 479.1 (M + H +). 1H NMR (400 MHz, CHLOROPHORMUM-d) δ 8.25 (d, J = 2.2 Hz, 1H), 8.11 - 8.01 (m, 2H), 7.78 (dd, J = 8, 4, 2.4 Hz, 1H), 7.17 - 7.06 (m, 2H), 6.91 (d, J =
[00147] [00147] COMPOUND 8. A stirred solution of ester 7 (0.46 g, 0.934 mmol) in THF (10 ml) was dripped with LiAlH 4 (1.0 M in THF; 1.401 ml, 1.401 mmol) at 0 ° Ç. After stirring for 3 h. LCMS indicated that the reaction was complete. Na2SO4 • 10 H2O was added slowly. After stirring for 1 h, the solid was extracted by filtration and rinsed with MeOH. The filtrate was concentrated. The crude product was purified on a 40 g silica column, eluting with 20% MeOH in DCM (0 to 40% gradient). The desired fractions were concentrated to produce compound 8 (363 mg, 0.806 mmol, 86% yield). LCMS ESI: calculated for C24H26N6O5 = 451.2 (M + H +), observed 451.1 (M + H +). 1H NMR (400 MHz, CLO-ROFORRY-d) δ 8.22 (d, J = 2.2 Hz, 1H), 7.73 (dd, J = 8.5, 2.5 Hz, 1H), 7 , 40 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz, 1H), 5.82 - 5.39 (m, 1H), 5.04 (s, 2H), 4.70 (s, 2H), 4.33 (t, J = 6.7 Hz, 2H), 4.10 (s, 3H), 1.86 - 1.73 (m, 2H), 1.50 (dq, J = 15.0, 7.5 Hz, 2H), 1.01 - 0.87 (m, 3H).
[00148] [00148] COMPOUND Ib-01. A suspension of compound 8 (363 mg, 0.806 mmol) in THF (10 ml) was treated with HCl (1.0 M in water, 2 ml, 2000 mmol). After stirring at 60 ° C for 4 h, LCMS indicated that the reaction was carried out. After cooling, a white solid was precipitated. It was collected by filtration, rinsed with water and dried in vacuo to produce compound Ib-01 (263.6 mg, 0.598 mmol, 74.2% yield). LCMS ESI: calculated for C22H24N6O4 = 437.2 (M + H +), observed 437.1 (M + H +). 1H NMR (400 MHz, METANOL-d4) δ 8.28 (d, J = 2.4 Hz, 1H), 7.94 (dd, J = 8.6, 2.4 Hz, 1H), 7.42 (d, J = 8.6 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.6 Hz, 1H), 5.06 (s , 2H), 4.63 (s, 2H), 4.54 (t, J = 6.5 Hz, 2H), 1.99 - 1.61 (m, 2H), 1.64 -
[00149] [00149] COMPOUND Ib-02. A solution of compound Ib-01 (100 mg, 0.229 mmol) in THF (10 ml) was treated with thionyl chloride (0.334 ml, 4.58 mmol) in RT and stirred for 3 h, after which the reaction was completed by LCMS. The excess thionyl was removed in an azeotropic manner with DCM. The resulting chloromethyl compound was directly transferred to the next step without purification.
[00150] [00150] A stirred solution of the chloromethyl compound (15 mg, 0.033 mmol) in DMF (0.5 ml) was treated with methyl amine (2.0 M in THF, 0.165 ml, 0.330 mmol) and stirred at RT during night, after which the reaction was completed. The reaction mixture was diluted with 50% MeOH in DMF and the precipitated solid was extracted by filtration. The crude filtrate was purified by means of preparative LC / MS with the following conditions: Column: XBridge C18, 19 x 200 mm, 5 μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10 mM ammonium acetate; Mobile Phase B: 95: 5 acetonitrile: water with 10 mM ammonium acetate; Gradient: 7 to 47% B over 20 minutes, then a 4-minute hold at 100% B; Flow: 20 ml / min. The fractions containing the desired product were combined and dried by means of centrifugal evaporation to produce compound Ib-02 (3.7 mg, 7.08 µmol, 21.47% yield). LCMS ESI: calculated for C23H27N7O3 = 450.2 (M + H +), observed 450.2 (M + H +). 1H NMR (500 MHz, DMSO-d6) δ 8.15 (s, 1H), 7.80 (dd, J = 8.4, 2.0 Hz, 1H), 7.37 (d, J = 8, 2 Hz, 2H), 7.07 (d, J = 8.3 Hz, 2H), 6.97 (d, J = 8.5 Hz, 1H), 6.36 (s, 2H), 4.86 (s, 2H), 4.18 (t, J = 6.6 Hz, 2H), 3.77 (s, 2H), 2.37 (s, 3H), 1.71 - 1.56 (m, 2H), 1.47 - 1.34 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H).
[00151] [00151] Following the preceding procedures in a general way, additional compounds in the series (Ib) were prepared, as shown in Table B below, using alternative amines.
[00152] [00152] Those skilled in the art will note that other compounds can be prepared following the procedures above, mutatis mutandis. For example, the compound can be prepared by using as a phenol starting material in place of methyl 4-hydroxybenzoate in the scheme of FIG. 1.
[00153] [00153] The compound can be prepared by completely reducing the methyl ester group in compound 7 of FIG. 1.
[00154] [00154] The compounds in the series Ia were produced using 4-fluorobenzaldehyde instead of 6-fluoronicotinaaldehyde in the scheme of FIG. 1 and generally following the procedures described above.
[00155] [00155] The compounds (Ia) thus prepared are listed in Table C. Table C - Compounds (Ia) Number of the Mass Spectrum put Amine Expected Mass Observed Mass (M + H) Ia- 01 n / a 434.2 (MH) 434.2 (MH)
[00156] [00156] This example and FIG. 2 refer to the synthesis of compounds from the series (Ic), (Id), (Ie) and (IIa), with the compound (Id-02) being used as an example.
[00157] [00157] With one exception, the steps in FIG. 2 are parallel to those of FIG. 1 and the previous example and procedures described above can be applied, mutatis mutandis, in this example. The only exception is that a benzyl chloride compound 12 is prepared in the third step, instead of a benzyl bromide compound 5 (in FIG. 1). The detailed procedure for this step is given below: To a stirred solution of compound 11 (1.28 g, 4.92 mmol) in DCM (20 ml) was added DIPEA (0.988 ml, 5.66 mmol), followed by Ms-Cl (0.422 ml, 5.41 mmol) at 0 ° C, followed by stirring at RT for 16 h, after which the reaction was complete. The reaction was suppressed with water and extracted with DCM three times. The combined organic extracts were dried over Na2SO4, filtered and concentrated to produce compound 12 (1.35 g, 4.84 mmol, 98% yield), which was carried on to the next step without purification.
[00158] [00158] Table D shows the compounds of the series (Id) prepared following the procedure of FIG. 2 and using the amines indicated in the final step. Table D - Compounds (Id) Mass Spectrum Amine Number Expected Mass- Observer Mass (M + H) Compound (M + H) (Id-01) 493.3 493.1 (Id-02) 491, 2 490.9 (Id-03) 521.3 521.3 (Id-04) 495.2 495.1 (Id-05) 481.2 481.3 (Id-06) 501.2 501.2 (Id -07) 507.2 506.9 (Id-08) 520.3 520.0
[00159] [00159] The compounds of the series (Ic) were prepared in general following the procedures of FIG. 2 for series compounds (Id), except that 4-hydroxybenzaldehyde and methyl-6-fluoropicolinate were used as starting materials, instead of 6-fluoronicotinaldehyde 1 and methyl 5-hydroxypicolinate 9.
[00160] [00160] Table E shows the compounds of the series (Ic) prepared in this way. Table E - Compounds (Ic) Mass Spectrum Amine Number Expected Mass - Observed Mass Compound from (M + H) to (M + H) (Ic-01) 492.2 492.3 (Ic-02) 490, 2 490.3 (Ic-03) 492.2 492.3 (Ic-04) 520.3 520.1 (Ic-05) 480.2 480.3 (Ic-06) 494.2 494.3 (Ic -07) 489.2 489.2 (Ic-08) 505.3 505.2
[00161] [00161] The compounds of the series (Ie) were prepared in general following the procedures of FIG. 2 for series compounds (Id), except that methyl 3,4-difluorobenzaldehyde and 4-hydroxybenzoate were used as starting materials, instead of methyl 6-fluoronicotinaldehyde 1 and methyl 5-hydroxypicolinate 9.
[00162] [00162] Table F shows the compounds of the series (Ie) prepared in this way. Table F - Compounds (Ie) Mass Spectrum Amine Number Expected Mass Observed Mass Compound of (M + H) (M + H) (Ie-01) 509.3 509.1 (Ie-02) 507.2 507 , 1 (Ie-03) 493.2 493.1 (Ie-04) 517.2 517.3 (Ie-05) 511.3 511.0 (Ie-06) 497.2 497.3 (Ie-07 ) 559.2 559.1 (Ie-08) 537.3 537.1 (Ie-09) 523.2 523.3 (Ie-10) 536.3 536.0
[00163] [00163] The compounds of the series (IIa) were prepared generally following the procedures of FIG. 2 for serial compounds (Id), except that methyl 3-fluorobenzaldehyde and 3-hydroxybenzoate were used as starting materials, instead of methyl 6-fluoronicotinaldehyde 1 and methyl 5-hydroxypicolinate 9.
[00164] [00164] Table G shows the compounds of the series (IIa) prepared in this way. Table G - Compounds (IIa) Mass Spectrum Amine Number Expected Mass - Observed Mass (M + H) of (M + H) (IIa-01) 449.2 449.3 (IIa-02) 489, 2 489.3 (IIa-03) 491.2 491.3 (IIa-04) 519.3 519.4 (IIa-05) 479.2 479.3 (IIa-06) 493.2 493.3 (IIa -07) 488.2 488.3 (IIa-08) 541.2 541.4 (IIa-09) 525.2 525.3 (IIa-10) 504.3 504.3 (IIa-11) 526.2 526.4 Example 3 - Series compounds (If)
[00165] [00165] This example and FIG. 3 refer to the synthesis of
[00166] [00166] Compounds 6 and 16 were coupled with Cs2CO3 to provide compound 17.
[00167] [00167] A mixture of compound 17 (1.05 g, 2.58 mmol), methyl 4-aminobenzoate 18 (0.468 g, 3.09 mmol), Pd2 adduct (dba) 3 CHCl3 (0.264 g, 0.258 mmol) ), BINAP (0.321 g, 0.516 mmol) and Cs2CO3 (2.52 g, 7.73 mmol) in dioxane (15 ml) was bubbled with N2 for 5 min, then sealed, stirred at 100 ° C for 5 h after what the reaction has been completed. The reaction mixture was diluted with ethyl acetate and the solid was removed by filtration. The filtrate was concentrated. The crude product was purified on a 12 g ISCO silica column, eluting with 20% MeOH in DCM (gradient DCM 0 to 20%). The desired fractions were concentrated to produce compound 19 (342.7 mg, 0.718 mmol, 27.8%).
[00168] [00168] Generally following the previous procedures and the schematic of FIG. 3, compound 19 was used to prepare compounds from the series (If) using the amines shown in Table H. Table H - Compounds (If) Mass Spectrum Amine Number Expected Mass Observed Mass Compound (M + H) (M + H ) (If-01) 491.3 491.0 (If-02) 479.2 479.0 (If-03) 519.3 519.0 (If-04) 489.3 489.4 (If-05) 499.2 499.1
[00169] [00169] This example and FIG. 7 refer to the preparation of the Ig-01 compound.
[00170] [00170] A mixture of compound 25 (CAS Reg. No. 1394947-77-3, 2.36 g, 12.81 mmol) and compound 1 (1.923 g, 15.38 mmol) in DMF (25.6 ml) it was treated with K2CO3 (2.125 g, 15.38 mmol) and then stirred at 50 ° C overnight. The reaction was quenched with water and stirred for 1 h. The resulting cream colored solid was collected by filtration and dried in the open air to yield compound 26 (3.51 g, 12.13 mmol, 95% yield). 1H NMR (400 MHz, CHLOROPHORM-d) δ 10.03 (s, 1H), 8.69 (d, J = 2.0 Hz, 1H), 8.36 (d, J = 0.7 Hz, 1H ), 8.21 (dd, J = 8.6, 2.4 Hz, 1H), 7.81 (d, J = 0.7 Hz, 1H), 7.09 (d, J = 8.8 Hz , 1H), 1.70 - 1.65 (m, 9H).
[00171] [00171] To a stirred mixture of compound 26 (1.0 g, 3.46 mmol) in DCM (5 ml) was added TFA (2.66 ml, 34.6 mmol), after stirring at RT for 5 h, the reaction mixture was concentrated to dryness. The crude product was then absorbed in DCM and carefully neutralized
[00172] [00172] A mixture of compound 27 (243 mg, 1.285 mmol) in DMF (5 ml) was treated with K2CO3 (231 mg, 1.670 mmol), followed by compound 28 (461 mg, 1.927 mmol). The reaction mixture was stirred at 90 ° C overnight. The reaction mixture was suppressed with water and extracted with ethyl acetate (3x). The combined organic extracts were dried over Na2SO4, filtered and concentrated. The crude product was purified by column on ISCO silica (24 g), eluted with ethyl acetate / hexanes, 0 to 80% gradient. The desired fractions were concentrated to produce compound 29 (230 mg, 0.662 mmol, 51.5% yield). LCMS ESI: calculated for C17H26N3O3Si = 348.2 (M + H +), observed 348.2 (M + H +). 1H NMR (400 MHz, CHLOROPHORMUM-d) δ 10.01 (s, 1H), 8.65 (d, J = 2.0 Hz, 1H), 8.20 - 8.16 (m, 1H), 7 , 79 (s, 1H), 7.61 (s, 1H), 7.06 (d, J = 8.6 Hz, 1H), 4.28 (t, J = 5.2 Hz, 2H), 3 .99 (t, J = 5.1 Hz, 2H), 0.87 (s, 6H), 0.00 (d, J = 2.9 Hz, 9H).
[00173] [00173] A solution of compound 29 (100 mg, 0.288 mmol) in MeOH (3 ml) was treated with NaBH4 (10.89 mg, 0.288 mmol) and then stirred at RT for 2 h. The reaction mixture was suppressed with water and extracted with ethyl acetate (3x). The combined organic extracts were dried over Na2SO4, filtered and concentrated. The crude product was purified by column on ISCO silica (24 g), eluted with ethyl acetate / hexanes, 0 to 100% gradient. The desired fractions were concentrated to produce compound 30 (70 mg, 0.200 mmol,
[00174] [00174] Compound 30 (110 mg, 0.315 mmol) in DCM (3 ml) was treated, with stirring, with Hunig's base (0.060 ml, 0.346 mmol), followed by Ms-Cl (0.026 ml, 0.330 mmol ) slowly at 0 ° C, followed by stirring at RT for 14 h. The reaction mixture was suppressed with water and extracted with DCM (3x). The combined organic extracts were dried over Na2SO4, filtered and concentrated to produce compound 31 (110 mg, 0.353 mmol, 94% yield), which was directly transferred to the next step without further purification. LCMS ESI: calculated for C17H27ClN3O2Si = 368.1 (M + H +), observed 368.2 (M + H +). 1H NMR (400 MHz, CHLOROPHORUM-d) δ 8.21 (d, J = 2.0 Hz, 1H), 7.80 (dd, J = 8.6, 2.6 Hz, 1H), 7.74 (s, 1H), 7.63 (s, 1H), 7.00 (d, J = 8.4 Hz, 1H), 4.57 (s, 2H), 4.37 - 4.28 (m, 2H), 4.05 (dd, J = 5.5, 4.2 Hz, 2H), 0.92 (s, 9H), 0.10 (s, 6H).
[00175] [00175] Compound 31 was then chosen for compound Ig-01, using the procedures of the preceding examples, mutatis mutandis. LCMS ESI: calculated for C20H25N8O4 = 441.2 (M + H +), observed 441.0 (M + H +). 1H NMR (500 MHz, DMSO-d6) δ 10.10 (s, 1H), 8.15 (d, J = 1.8 Hz, 1H), 7.81 (s, 1H), 7.76 (dd , J = 8.5, 2.4 Hz, 1H), 7.41 (s, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.48 (br s, 2H), 4.84 (s, 2H), 4.15 (t, J = 6.6 Hz, 2H), 4.08 (t, J = 5.6 Hz, 2H), 2.91 (br d, J = 6.4 Hz, 2H), 1.71 - 1.54 (m, 2H), 1.46 - 1.28 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H). Example 5 - TLR7 Agonist Activity Assay
[00176] [00176] This example describes a method for evaluating the TLR7 agonist activity of the compounds disclosed in this report described
[00177] [00177] Manipulated human embryonic kidney blue cells (HEK-Blue ™ TLR cells; Invivogen) that have a human embryonic alkaline phosphatase reporter transgene secreted by TLR7 (SEAP) were suspended in culture medium non-selective (DMEM with high glucose (Invitrogen), supplemented with 10% fetal bovine serum (Sigma)). TLR7 HEK-Blue ™ cells were added to each well of a 384 well tissue culture plate (15,000 cells per well) and incubated 16 to 18 hours at 37 ° C, 5% CO2. The compounds (100 nl) were dispensed into the reservoirs containing the TLR HEK-Blue ™ cells and the treated cells were incubated at 37 ° C, 5% CO2. After 18 h of treatment, ten microliters of freshly prepared Quanti-Blue ™ reagent (Invitrogen) were added to each well, incubated for 30 min (37 ° C, 5% CO2) and the measured SEAP levels used. - using an Envision plate reader (OD = 620 nm). The values of effective half-maximum concentration (EC50; concentration of the compound that induced a response midway between the baseline and the assay maximum) were calculated.
[00178] [00178] FIG. 6 is a representative graph showing the data thus obtained for the compound (Ib-02). Example 6 - Interleukin Induction Assay 6
[00179] [00179] This example describes a method for experimenting with the induction of interleukin 6 through the compounds disclosed in this descriptive report.
[00180] [00180] Compounds diluted in DMSO were transferred to individual reservoirs of a plate of 384 transparent V-bottom reservoirs from Matrix Technologies using ECHO acoustic liquid handling technology (25 nL per reservoir). Samples of human whole blood (25 µl) were added to each reservoir 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 RPMI 1640 medium (supplemented with L-glutamine) was then added to each reservoir (25 µl per reservoir) prior to the release of plasma from each sample by centrifugation (450 x g, 5 min, room temperature). The treated plasma samples (3 µl) were subsequently transferred to individual reservoirs of a white, shallow 384 reservoir 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. The data analysis software was used to determine the EC50 values of the compound on which the baseline was established using mean values of DMSO and 100% of established induction using reference values of compounds at the highest concentration tested. EC50s can be determined with software such as Graphpad Prism ™. Example 7 - Transglutaminase-Mediated Conjugation
[00181] [00181] The following procedure can be used for the 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 glutamine reactive to transglutaminase, for example, one with an N297A or N297Q substitution. Conjugation is performed by recombinant bacterial transglutaminase with a 5: 1 antibody: enzyme molar ratio. 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
[00182] [00182] 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 approaches to conjugation are known in the art and usable in the present invention.
[00183] [00183] The previous detailed description of the invention includes passages that are mainly or exclusively related to the particular parts or aspects of the invention. It should be understood that this is for clarity and convenience, that a particular feature may be relevant to more than just the passage in which it is disclosed and that the disclosure in this invention includes all appropriate combinations of information found in the different passages. - genes. Likewise, although the various figures and descriptions contained herein are related to the specific modalities of the invention, it should be understood that when a specific characteristic is disclosed in the context of a specific figure or modality, that characteristic can also be used, to the extent appropriate, in the context of another figure or modality, in combination with another characteristic, or in the invention in general.
[00184] [00184] Furthermore, although the present invention has been described in particular in terms of certain preferred embodiments, the invention is not limited to those preferred embodiments. On the contrary, the scope of the invention is defined by the appended claims. REFERENCES
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权利要求:
Claims (21)
[1]
1. Compound represented by the 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 of its occurrences, H, C1- C3 alkyl, halo, O (C1-C3 alkyl), CN, or NO2; X is, independently for each of its occurrences, CR2 or N; R3 is O, S, NH, or N (C1-C3 alkyl); Ar is,,, or; where one of Y, Y 'and Y ”is selected from –O-, -S-, -NH- and –N (C1-C3 alkyl) - and the other two from Y, Y' and Y” are selected de = N- and = CR2-; R4 is H, C1-C3 alkyl, halo, O (C1-C3 alkyl), CN, NO2, or (CH2) xR5, where the subscript x is 1, 2, 3, or 4; and R5 is H, 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 moiety aromatic or heteroaromatic 6-member or a hetero-romantic 5-membered portion;
wherein an alkyl, cycloalkyl, bicycloalkyl, spriocycloalkyl, cyclic amine, 6-membered aromatic or heteroaromatic or 5-membered heteroaromatic moiety is optionally substituted with one or more substitutes 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, esprioalkyl, or cyclic amine moiety may have a CH2 group substituted by O, S, NH, N (C1-C3 alkyl), or N (Boc).
[2]
2. A compound according to claim 1, represented by the formula (I ') characterized by the fact that each X' is independently CH or N.
[3]
3. Compound according to claim 2, characterized by the fact that R4 is CH2R5 and R5 is OH, Cl,,,,,,,,,,,,
,,,,,,,,, or.
[4]
4. Compound, according to claim 1, characterized by the fact that it is represented by the formula (Ia).
[5]
5. Compound according to claim 4, characterized by the fact that R1 is n-BuO, x is 1, and R5 is OH, Cl,,,,,,,,,,,,
,,,,,,,,, or.
[6]
6. Compound, according to claim 1, characterized by the fact that it is represented by the formula (Ib).
[7]
7. Compound according to claim 6, characterized by the fact that R1 is n-BuO, x is 1, and R5 is OH, Cl,,,,,,,,
,,,,,,,,,,, or.
[8]
8. Compound, according to claim 1, characterized by the fact that it is represented by the formula (Ic).
[9]
9. Compound according to claim 8, characterized by the fact that R1 is n-BuO, x is 1, and R5 is OH, Cl,,,,,,,,
,,,,,,,,,,, or.
[10]
10. Compound, according to claim 1, characterized by the fact that it is represented by the formula (Id).
[11]
11. Compound according to claim 10, characterized by the fact that R1 is n-BuO, x is 1, and R5 is OH, Cl,,,,,
,,,,,,,,,, or.
[12]
12. Compound, according to claim 1, characterized by the fact that it is represented by the formula (Ie).
[13]
13. A compound according to claim 12, characterized by the fact that R1 is n-BuO, x is 1, and R5 is OH, Cl,,,,,
,,,,,,,,,, or.
[14]
14. Compound, according to claim 1, characterized by the fact that it is represented by the formula (If).
[15]
15. A compound according to claim 14, characterized by the fact that R1 is n-BuO, x is 1, and R5 is OH, Cl,,,,,
,,,,,,,,,, or.
[16]
16. Compound according to claim 1, characterized by the fact that it is represented by the formula (IIa).
[17]
17. A compound according to claim 16, characterized by the fact that R1 is n-BuO, x is 1, and R5 is OH, Cl,
,,,,,,,,,,,,,,,.
[18]
18. Compound, according to claim 1, characterized by the fact that it has a structure represented by the formula (Ig):.
[19]
19. A compound according to claim 1, characterized by the fact that it is conjugated to an antibody.
[20]
20. 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.
[21]
21. Compound, according to claim 1, characterized by the fact that it is for use in the treatment of a condition receptive to treatment by activating the Toll-like Receiver 7.

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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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2021-11-23| 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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US201762546222P| true| 2017-08-16|2017-08-16|

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US62/546,222|2017-08-16|

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PCT/US2018/000246|WO2019035971A1|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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