Agonists of stimulator of interferon genes STING

Abstract

Disclosed herein are compounds of Formula (I), pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof: the compounds are useful as agonists of stimulator of interferon genes (STING), e.g., in methods of treating tumors.

Description

[0001] This application claims the priority benefit of U.S. Provisional Patent Application No. 62 / 706,683, filed September 2, 2020, which is incorporated herein as fully set forth herein. Background Technology

[0002] The cGAS-STING signaling pathway plays a crucial role in the innate immune response initiated by mammalian host cells to eliminate various DNA and RNA viruses (Q. Chen, L. Sun, ZJ Chen, Nat. Immunol. 17, 1142-1149 (2016); MH Christensen, SR Paludan, Cell. Mol. Immunol. 14, 4-13 (2017)). STING (interferon gene stimulator) is an endoplasmic reticulum (ER) resident signaling protein, partially localized to the mitochondrial-associated membrane, and is widely expressed in both immune and non-immune cell types. STING also serves as a direct link between inflammation and a variety of physiological processes, including: monitoring of micronuclei in cases of DNA damage (KJ Mackenzie et al., Nature 548, 461-465 (2017); SM Harding et al., Nature 548, 466-470 (2017)), age-related inflammation (De Cecco et al., Nature 566, 73-78 (2019)), mitochondrial DNA-associated inflammatory phenotypes (DASliter et al., Nature 561, 258-262 (2018)), and microbiome-dependent gut homeostasis (MCCCanesso et al., Mucosal Immunol. 11, 820-834 (2018)). STING is an endoplasmic reticulum signaling protein, partially localized to the mitochondrial-associated membrane, and is widely expressed in both immune and non-immune cell types. STING binds to cyclic dinucleotides (CDNs)—including 2′,3′-cyclic GMP-AMP (2′,3′-cGAMP) produced by cGAS in response to cytosol DNA (L. Sun, J. Wu, F. Du, X. Chen, ZJ Chen, Science 339, 786-791 (2013))—and scaffold function rapidly induces type I interferon (IFN) and pro-inflammatory cytokines in a TBK1-IRF3-dependent manner (H. Ishikawa, Z. Ma, GN Barber, Nature 461, 788-792 (2009); H. Ishikawa, GN Barber, Nature 455, 674-678 (2008)).

[0003] STING has been shown to play an important role in antitumor immunity. For example, efficient tumor-induced T cell activation requires STING pathway-dependent IFN-β expression and STING expression in dendritic cells (MBFuertes et al., J.Exp.Med.208, 2005-2016 (2011); SRWoo et al., Immunity41, 830-842 (2014)).

[0004] The initial STING agonist small molecules were synthesized as derivatives of natural CDN ligands. However, due to their poor stability, CDN-based agonist administration has been limited to intratumoral delivery. Although intratumoral delivery of CDN agonists has consistently demonstrated tumor regression in established homology models (Corrales et al., Cell Rep. 11, 1018-1030 (2015); KESivick et al., Cell Rep. 29, 785-789 (2019)), the success rate of intratumoral CDN administration in humans is inconsistent.

[0005] Activation of the STING pathway has also been shown to contribute significantly to the antitumor effects of radiation and chemotherapy (Harding et al. (2017), C. Vanpouille-Box et al., Nat. Commun. 8, 15618 (2017); C. Pantelidou et al., Cancer Discov. 9, 722-737 (2019)). Summary of the Invention

[0006] In several embodiments, this disclosure provides agonists of interferon gene stimulators (STING) that can be used to treat tumors. According to several embodiments, the agonist is a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Rings B and C are independently selected from Het, equation (a), and equation (b):

[0009]

[0010] Each ring A is arbitrarily divided by 1 to 4 Rs A The substitution is made independently of: 5- or 6-membered monocyclic heteroaryl groups containing 1 to 3 heteroatoms selected from O, S and N, and 8- to 10-membered bicyclic heteroaryl groups containing 1 to 6 heteroatoms selected from O, S and N.

[0011] Het is a heteroatom consisting of 1 to 6 heteroatoms selected from O, S, and N, optionally bounded by 1 to 4 R atoms. A Replaces 8- to 10-membered bicyclic heteroaryl groups.

[0012] X is N, S, -N = C(R) 1 - or -C(R) 3 )=C(R 3 )-.

[0013] W is -N = or -C(R) 3 = ).

[0014] Y 1 Selected from -O-, -CR4R5-, and -(CH2). L1 -O-、-(CH2) L1 -S(O) 0-2 -(where L1 is an integer selected from 1, 2, 3, 4, and 5); and -(CH2) L1 -N(R L )-(where R L Selected from benzyl, C1-C6-alkyl, H, optionally substituted with 1 or 2 methoxy groups.

[0015] Y 2 Selected from -O-, -CR4R5-, and -O-(CH2). L1 -、-S(O) 0-2 -(CH2) L1 _(where L1 is an integer selected from 1, 2, 3, 4, and 5); and -N(R L )-(CH2) L1 -(where R) L Is it H or C? 12 -C6-alkyl).

[0016] The subscript m is an integer selected from 0, 1, 2, 3, 4, 5, and 6.

[0017] The subscript n is an integer selected from 0, 1, and 2.

[0018] The subscripts x and y are integers independently selected from 0 and 1, where Y is 0 when m is 0 and each of x and y is 1. 1 and Y 2 Not simultaneously -O-.

[0019] Each R 1 and R 3Independently selected from: H, halogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C6-alkoxy, cyano, C1-C6-haloalkyl, and 3- to 10-membered heterocyclic groups (wherein 1 to 4 heterocyclic alkyl members are independently selected from N, O, and S), wherein any alkyl, alkenyl, alkoxy, or heterocyclic group is optionally surrounded by 1 to 4 R... A replace.

[0020] R 2 Selected from -C(O)OR, -(C1-C6-alkyl)C(O)OR, C1-C6-haloalkyl, -P(O)(OR)2, -C(O)NHR, halogen, -CN, C3-C6-cycloalkenyl, 3-membered to 10-membered heterocyclic groups (wherein 1 to 4 heterocyclic alkyl members are independently selected from N, O and S) and 5-membered to 10-membered heteroaryl groups (wherein 1 to 4 heteroaryl members are independently selected from N, O and S), wherein any alkyl, cycloalkenyl, heterocyclic or heteroaryl group is optionally surrounded by 1 to 4 R A replace.

[0021] R is selected from: H; C1-C6-alkyl, optionally substituted by: -((C1-C6-alkyl)OC(O)OC1-C6-alkyl), -OP(O)(OH)2, -OC(O)(C1-C6-alkyl)-OP(O)(OH)2, -NH2, -CH(NH2)COOH or 3- to 10-membered heterocyclic groups (wherein 1 to 4 heterocyclic alkyl members are independently selected from N, O and S); and -(C1-C6-alkyl)(C6-C 10 -Aryl).

[0022] Each R 4 and R 5 Independently selected from H, halogens, C1-C6-alkyl, and C3-C7-cycloalkyl. In some embodiments, any two R atoms bonded to the same carbon atom... 4 and R 5 Together with the carbon atoms they bond to, this indicates that they are optionally bound by 1 to 3 R atoms. A Substituted C3-C5-cycloalkyl groups, or those representing C2-C6-alkenyl groups. In other embodiments, R groups not bonded to the same carbon atom... 4 and R 5 Any two, together with the carbon atoms they are bonded to, represent optionally bounded by 1 to 3 R atoms. A Substituted C3-C7-cycloalkyl groups.

[0023] Each R A Examples are independently selected from H, halogen, -CN, -hydroxyl, oxo, C1-C6-alkyl, C1-C6-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl, NH2, -S(O).0-2 -(C1-C6-alkyl), -S(O) 0-2 -(C6-C 10 -aryl), -C(O)(C1-C6-alkyl), -C(O)(C1-C6-alkyl)COOH, -C(O)(C1-C6-alkyl)C(O)(C1-C6-alkoxy), -C(O)N(H or C1-C6-alkyl)2, -C(O)(C3-C 14 -cycloalkyl), -C3-C 14 -cycloalkyl, -(C1-C6-alkyl)(C3-C6-alkyl) 14 -cycloalkyl), C6-C 10 -aryl, 3- to 14-membered heterocyclic alkyl and -(C1-C6-alkyl)-(3- to 14-membered heterocyclic alkyl) (wherein 1 to 4 heterocyclic alkyl members are independently selected from N, O and S), and 5- to 10-membered heteroaryl (wherein 1 to 4 heteroaryl members are independently selected from N, O and S), which are optionally substituted with C1-C6-alkyl.

[0024] More specifically, in the illustrative embodiments, the compounds or pharmaceutically acceptable salts thereof according to this disclosure include any specific compounds shown in Table 1 or Table 2 below.

[0025] This disclosure also provides pharmaceutical compositions in several embodiments comprising the compounds disclosed herein or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers.

[0026] In one embodiment, this disclosure also provides: a method for stimulating the expression of an interferon gene, comprising administering to a patient an effective amount of an agonist of an interferon gene stimulator (STING), said agonist comprising a compound as described herein; and a method for treating a tumor in a patient, comprising administering to a patient an effective amount of an agonist of an interferon gene stimulator (STING), said agonist comprising a compound of formula (I).

[0027] In several embodiments, the method of treating tumors also includes administering an effective dose of the compound disclosed herein by oral or intratumoral administration or both.

[0028] In several embodiments, the method of treating tumors further includes administering an effective amount of a compound as disclosed herein, wherein administration includes administering the compound to the patient as an antibody-drug conjugate or in a liposomal formulation.

[0029] In several embodiments, the method of treating tumors further includes administering an effective amount of a compound as disclosed herein, which also includes administering an effective dose of an immune checkpoint-targeting drug. For example, the immune checkpoint-targeting drug may be an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA-4 antibody, or an anti-4-1BB antibody.

[0030] In several embodiments, the method of treating tumors also includes administering an effective amount of a compound as disclosed herein, and also includes administering ionizing radiation or an anticancer drug. Detailed Implementation

[0031] There is considerable interest in developing STING pathway agonists for various immuno-oncology applications. Most notably, STING pathway agonists, as part of combination therapies involving immune checkpoint-targeting drugs, have significant potential applications in patients who have failed to respond to checkpoint blockade alone. Therefore, systemic STING activators are not only valuable for the treatment of cancer and infectious diseases, but also serve as pharmacological probes enabling mechanistic studies in the context of STING-dependent antitumor immunity and multiple STING-dependent biological processes. This disclosure provides STING agonist compounds and pharmaceutically acceptable salts, pharmaceutical compositions thereof, and methods of administration to address these and other needs.

[0032] This disclosure relates in part to a nonnucleotide small molecule STING agonist, the activity of which was established through preliminary assays involving the human THP-1 cell line carrying five copies of an IRF-inducible reporter with an IFN signaling response element. Counter screening was used to eliminate luciferase artifacts to ensure cross-species reactivity and pathway selectivity in humans and rodents; this counter screening involved alternative reporter constructs, rodent cell-based assays, and cGAS and STING knockout cell lines. Biochemical assays were used to identify the specific targets of the identified hits, involving cGAS enzymatic activity and STING protein binding assays.

[0033] definition

[0034] Use standard abbreviations for chemical groups known in the art, such as Me = methyl, Et = ethyl, i-Pr = isopropyl, Bu = butyl, t-Bu = tert-butyl, Ph = phenyl, Bn = benzyl, Ac = acetyl, Bz = benzoyl, etc.

[0035] "Alkyl" refers to a straight-chain or branched hydrocarbon group containing 1 to 20 carbon atoms. For example, an alkyl group can have 1 to 10 carbon atoms or 1 to 6 carbon atoms. Exemplary alkyl groups include straight-chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc., and also include branched isomers of straight-chain alkyl groups, such as, but not limited to, those described above.

[0036] -CH(CH3)2, -CH(CH3)(CH2CH3), -CH(CH2CH3)2, -C(CH3)3, -C(CH2CH3)3, -CH2CH(CH3)2, -CH2CH(CH3)(CH2CH3), -CH2CH(CH2CH3)2, -CH2C(CH3)3, -CH2C(CH2CH3)3, -CH(CH3)CH(CH3)(CH2CH3), -CH2CH2CH(CH3)2, -CH2CH2C(CH3)3, -CH2CH2C(CH2CH3)3, -CH(CH3)CH2CH(CH3)2, -CH(CH3)CH(CH3)CH(CH3)2, etc. Therefore, alkyl groups include primary alkyl groups, secondary alkyl groups, and tertiary alkyl groups. The alkyl group may be unsubstituted or optionally substituted with one or more substituents as described herein.

[0037] The phrase “substituted alkyl” refers to an alkyl group that is substituted at one or more positions (e.g., 1, 2, 3, 4, 5, or even 6 positions), with the substituents attached at any available atom to produce a stable compound, wherein the substitutions are as described herein. “Optionally substituted alkyl” refers to an alkyl group or a substituted alkyl group.

[0038] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing 2 to about 20 carbon atoms (e.g., 2 to 6 carbon atoms) and having 1 to 3, 1 to 2, or at least one carbon-carbon double bond. The alkenyl group may be unsubstituted or optionally substituted with one or more substituents as described herein.

[0039] "Substituted alkenyl" refers to an alkenyl group that is substituted at one or more positions (e.g., 1, 2, 3, 4, 5, or even 6 positions), whereby these substituents are connected at any available atom to produce a stable compound, wherein the substitutions are as described herein. "Optionally substituted alkenyl" refers to an alkenyl group or a substituted alkenyl group.

[0040] "Alkyne" or "alkynyl group" refers to a straight-chain or branched unsaturated hydrocarbon having the indicated number of carbon atoms and at least one triple bond. Examples of (C2-C8) alkynyl groups include, but are not limited to, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne, 3-heptyne, 1-octyne, 2-octyne, 3-octyne, and 4-octyne. The alkynyl group may be unsubstituted or substituted with one or more substituents as described herein.

[0041] "Substituted alkynyl" refers to an alkynyl group that is substituted at one or more positions (e.g., 1, 2, 3, 4, 5, or even 6 positions), whereby these substituents are attached at any available atom to produce a stable compound, wherein the substitution is as described herein. "Optionally substituted alkynyl" refers to an alkynyl group or a substituted alkynyl group.

[0042] The term "alkoxy" or "alkoxyl" refers to an -O-alkyl group having a specified number of carbon atoms. For example, (C1-C6)-alkoxy groups include -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-butyl, -O-sec-butyl, -O-tert-butyl, -O-pentyl, -O-isopentyl, -O-neopentyl, -O-hexyl, -O-isohexyl, and -O-neohexyl.

[0043] Unless otherwise stated, the terms “halogenated” or “halogen” or “halide” mean, either by themselves or as part of another substituent, a fluorine, chlorine, bromine or iodine atom, preferably fluorine, chlorine or bromine.

[0044] "Halogenated alkyl" includes: monohalogenated alkyl; polyhalogenated alkyl, wherein all halogen atoms may be the same or different; and perhalogenated alkyl, wherein all hydrogen atoms are substituted with the same or different halogen atoms, such as fluorine and / or chlorine atoms. Some examples of halogenated alkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, etc.

[0045] Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms in their rings. As is known in the art, aromatic compounds are polyunsaturated cyclic systems containing 4n+2π electrons (where n is an integer). Therefore, aryl groups include, but are not limited to: phenyl, azulel, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrene, triphenylenyl, pyrene, and tetraphenyl. alkyl, biphenyl, anthracene, and naphthyl (see, for example, Lang's Handbook of Chemistry (Dean, JA, ed) 13) thed. Table 7-2

[1985] ). In some embodiments, the aryl group contains a specified number of carbon atoms, or if no number of carbon atoms is specified, it contains up to 14 carbon atoms, such as C6-C. 14 -Aryl. As defined above, aryl groups can be unsubstituted or substituted. Representative substituted aryl groups can be monosubstituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl groups or 2- to 8-substituted naphthyl groups, which can be substituted by carbon or non-carbon groups such as those listed above.

[0046] The term "heteroatom" refers to N, O, and S atoms. Compounds of this disclosure containing N or S atoms can be selectively oxidized to the corresponding N-oxides, sulfoxides, or sulfone compounds.

[0047] A heterocyclic group, or the term "heterocyclic group," includes aromatic and non-aromatic cyclic compounds containing three or more ring members, one or more of which are heteroatoms, such as, but not limited to, N, O, and S. Thus, a heterocyclic group can be a cycloheteroalkyl group, a heteroaryl group, or, if polycyclic, any combination thereof. In some embodiments, a heterocyclic group comprises 3 to about 20 ring members, while other such groups have 3 to about 14 ring members. A heterocyclic group represented as a C2-heterocyclic group can be a 5-ring having two carbon atoms and three heteroatoms, a 6-ring having two carbon atoms and four heteroatoms, etc. Similarly, a C4-heterocyclic group can be a 5-ring having one heteroatom, a 6-ring having two heteroatoms, etc. The sum of the number of carbon atoms and the number of heteroatoms equals the total number of ring atoms. The size of the ring can also be expressed by the total number of atoms in the ring, counting both carbon and non-carbon ring atoms, for example, 3- to 10-membered heterocyclic groups. The heterocyclic ring can also contain one or more double bonds. A heteroaryl ring is one embodiment of a heterocyclic group. The term "heterocyclic group" includes fused-ring substances, including those comprising fused aromatic and non-aromatic groups. For example, both the dioxolane ring and the benzodioxolane ring system (methylene dioxophenyl ring system) are heterocyclic groups as understood herein. The term also includes polycyclic systems containing one or more heteroatoms, such as bicyclic and tricyclic systems, such as, but not limited to, quinine rings.

[0048] "Optionally substituted heterocyclic alkyl" means a heterocyclic alkyl group substituted with 1 to 3 substituents (e.g., 1, 2 or 3 substituents), which are connected at any available atom to produce a stable compound, wherein the substituents are as described herein.

[0049] A heteroaryl group is a heterocyclic aromatic ring compound containing five or more ring members, one or more of which are heteroatoms, such as, but not limited to, N, O, and S; for example, a heteroaryl ring can have from 5 to about 8 to 12 ring members, such as 5-membered to 10-membered heteroaryls. Some bicyclic heteroaryl rings can have 8 to 10 ring members. A heteroaryl group is a variety of heterocyclic groups with aromatic electronic structures, which are polyunsaturated cyclic systems containing 4n + 2π electrons, where n is an integer. A heteroaryl group represented as C2-heteroaryl can be a 5-ring (i.e., a 5-membered ring) with two carbon atoms and three heteroatoms, a 6-ring (i.e., a 6-membered ring) with two carbon atoms and four heteroatoms, etc. Similarly, a C4-heteroaryl group can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, etc. The sum of the number of carbon atoms and the number of heteroatoms equals the total number of ring atoms. Heteroaryl groups are also intended to include oxidized S or N, such as sulfinyl, sulfonyl, and N oxides of tertiary cyclic nitrogen. The carbon or heteroatom is the linking point of the heteroaryl ring structure, resulting in a stable compound. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, quinoxalinyl, indoleazinyl, benzo[b]thiopheneyl, quinazolinyl, purine, indole, quinolinyl, pyrimidinyl, pyrroleyl, pyrazolyl, and others. azole group, thiazolyl group, thiophene group, iso azole group, Oxathiadiazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazolyl, furanyl, benzofuranyl, and indoleyl.

[0050] "Substituted heteroaryl" is a heteroaryl group that is independently substituted by one or more substituents (e.g., 1, 2, 3, 4, or 5 substituents, or 1, 2, or 3 substituents, or 1 substituent) unless otherwise specified, the substituents being linked at any available atom to produce a stable compound, wherein the substituents are as described herein. "Optionally substituted heteroaryl" refers to a heteroaryl or a substituted heteroaryl group.

[0051] Cycloalkyl groups are groups comprising one or more carbon rings, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the cycloalkyl group may have 3 to about 8 to 12 ring members, while in other embodiments, the number of ring carbon atoms is 3 to 4, 5, 6, or 7. Cycloalkyl groups also include polycyclic cycloalkyl groups, such as, but not limited to, norbornel, adamantyl, bornel, camphenyl, isocamphenyl, and caretenyl, and fused rings, such as, but not limited to, decalinyl. Cycloalkyl groups also include rings substituted with straight-chain or branched alkyl groups as defined above.

[0052] Cycloalkenyl groups include cycloalkyl groups having at least one double bond between two carbon atoms. Thus, for example, cycloalkenyl groups include, but are not limited to, cyclohexenyl, cyclopentenyl, and cyclohexadienyl. Cycloalkenyl groups may have 3 to about 8 to 12 ring members, while in other embodiments, the number of ring carbon atoms is 3 to 5, 6, or 7. Cycloalkyl groups also include polycyclic cycloalkyl groups, such as, but not limited to, norbornel, adamantyl, bornel, camphenyl, isocamphenyl, and caretenyl, and fused rings, such as, but not limited to, decalinyl, provided they contain at least one double bond in the ring. Cycloalkenyl groups also include rings substituted with straight-chain or branched alkyl groups as defined above.

[0053] The term "oxo" refers to the =O atom bonded to an atom that is part of a saturated or unsaturated part. Thus, for example, the =O atom can be bonded to carbon, sulfur, or nitrogen atoms that are part of a cyclic or acyclic part.

[0054] One or more optional substituents on any group described herein are independently selected from R A OR A Halogen, -N=NR A NR A R B -(C1-C6-alkyl)NR A R B -C(O)OR A -C(O)NR A R B -OC(O)R A and -CN. R A and R B Independently selected from H, -CN, -hydroxyl, oxo, C1-C6-alkyl, C1-C6-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl, NH2, -S(O) 0-2 -(C1-C6-alkyl), -S(O) 0-2 -(C6-C 10 -aryl), -C(O)(C1-C6-alkyl), -C(O)(C3-C 14 -Carbocyclic group), -C3-C 14 -Carbocyclic group, -(C1-C6-alkyl)(C3-C6-alkyl) 14 -Carbocyclic group), C6-C 10 -aryl, 3- to 14-membered heterocyclic alkyl and -(C1-C6-alkyl)-(3- to 14-membered heterocyclic alkyl) (wherein 1 to 4 heterocyclic alkyl members are independently selected from N, O and S), and 5- to 10-membered heteroaryl (wherein 1 to 4 heteroaryl members are independently selected from N, O and S). R A and R BEach alkyl, alkoxy, alkenyl, alkynyl, aryl, carbocyclic, heterocyclic, and heteroaryl moiety is optionally substituted by one or more substituents selected from the following: hydroxyl, halogen, -NR'2 (wherein each R' is independently selected from C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C6-C... 10 -aryl, 3- to 14-membered heterocyclic alkyl and -(C1-C6-alkyl)-(3- to 14-membered heterocyclic alkyl) (where 1 to 4 ring members are independently selected from N, O and S), and 5- to 10-membered heteroaryl (where 1 to 4 heteroaryl members are independently selected from N, O and S), -NHC(O) (OC1-C6-alkyl), -NO2, -CN, oxo, -C(O)OH, -C(O)O (C1-C6-alkyl), -C1-C6-alkyl (C1-C6-alkoxy), -C(O)NH2, C1-C6-alkyl, -C(O)C1-C6-alkyl, -OC1-C6-alkyl, -Si(C1-C6-alkyl)3, -S(O) 0-2 -(C1-C6-alkyl), C6-C 10 -aryl, -(C1-C6-alkyl)(C s -C 10 -aryl), 3- to 14-membered heterocyclic alkyl and -(C1-C6-alkyl)-(3- to 14-membered heterocycles) (where 1 to 4 heterocyclic members are independently selected from N, O and S), and -O(C6-C 14 -aryl). Each of the above alkyl, alkenyl, aryl and heterocyclic alkyl groups is optionally substituted with one or more substituents selected from hydroxyl, -OC1-C6-alkyl, halogen, -NH2, -(C1-C6-alkyl)NH2, C(O)OH, CN and oxo.

[0055] The compounds described herein can exist in a variety of isomeric forms, including configurational isomers, geometric isomers, and conformational isomers, including, for example, cis or trans conformations. The compounds can also exist in one or more tautomeric forms, including single tautomers and mixtures of tautomers. The term "isomer" is intended to cover all isomeric forms of the compounds of this disclosure (including tautomeric forms of the compounds). The compounds of this disclosure can also exist in open-chain or cyclized forms. In some cases, one or more cyclized forms can be generated by dehydration. The specific composition of the open-chain and cyclized forms can depend on how the compound is isolated, stored, or administered. For example, the compound may exist primarily in an open-chain form under acidic conditions and may be cyclized under neutral conditions. All forms are included in this disclosure.

[0056] The substituent -CO2H can be replaced by the following bioisosteric substitutes:

[0057]

[0058] etc., where R has the same characteristics as defined in this paper. A The same limitation applies. See, for example, THE PRACTICE OF MEDICINAL CHEMISTRY (Academic Press: New York, 1996), page 203.

[0059] Some of the compounds described herein may have an asymmetric center and thus exist in different enantiomers and diastereomers. Compounds as described herein may be in the form of optical isomers or diastereomers. Therefore, this disclosure covers compounds as described herein in their optical isomers, diastereomers, and mixtures thereof (including racemic mixtures), and their uses. Optical isomers of the compounds of this disclosure can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, simulated moving bed techniques, or by chemical separation of stereoisomers using optically active resolving agents.

[0060] Unless otherwise stated, the term "stereoisomer" means a stereoisomer of a compound that is substantially free of other stereoisomers of the compound. Thus, a stereoisomerically pure compound having one chiral center will be substantially free of its opposite enantiomers. A stereoisomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereoisomerically pure compound contains more than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example, more than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, or more than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, or more than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound, or more than about 99% by weight of one stereoisomer of the compound and less than about 1% by weight of other stereoisomers of the compound. The stereoisomers described above can be considered as a composition comprising two stereoisomers present in their respective weight percentages as described herein.

[0061] If there is a difference between the illustrated structure and its given name, the illustrated structure shall prevail. Furthermore, if the stereochemistry of a structure or part thereof is not indicated by, for example, bold or dashed lines, the structure or part thereof should be interpreted as encompassing all its stereoisomers. However, in some cases where more than one chiral center is present, the structure and name may be represented as a single enantiomer to aid in describing the relative stereochemistry. Those skilled in the art of organic synthesis will know whether a compound is prepared as a single enantiomer by the method used to prepare it.

[0062] As used herein, and unless otherwise stated to the contrary, the term "compound" is inclusive because it encompasses a compound or its pharmaceutically acceptable salts, stereoisomers, and / or tautomers. Thus, for example, a compound of this disclosure includes pharmaceutically acceptable salts of tautomers of that compound.

[0063] The term “medicinal salt” refers to a non-toxic inorganic or organic acid and / or base addition salt, see, for example, Lit, et al., Salt Selection for Basic Drugs (1986), Int J. Pharm., 33, 201-217, which is incorporated herein by reference. Representative pharmaceutically usable salts include, for example, alkali metal salts, alkaline earth salts, ammonium salts, water-soluble and water-insoluble salts, such as acetates, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonates, benzoates, bicarbonates, bisulfates, tartrates, borates, bromides, butyrates, calcium, calcium edetate, camphor sulfonate, carbonates, chlorides, citrates, clavulariate, dihydrochlorides, edetates, ethanedisulfonates, estolates, esylates, fiunarates, gluconates, glutamates, glycolyllarsanilates, hexafluorophosphates, hexylresorcinol salts, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthaleneate, and iodides. This includes isothiocyanates, lactates, lactobionates, laurates, malates, maleates, mandelates, methanesulfonates, methyl bromide, methyl nitrates, methyl sulfates, mucates, naphthalenesulfonates, nitrates, N-methylglucosamine ammonium salts, 3-hydroxy-2-naphthoate, oleates, oxalates, palmitates, 1,1-methylene-bis-2-hydroxy-3-naphthoate (einbonate), pantothenates, phosphates / bisphosphonates, picrates, polygalacturonic acids, propionates, p-toluenesulfonates, salicylates, stearates, hypoacetates, succinates, sulfates, sulfosalicates, suramates, tannins, tartrates, teoclates, toluenesulfonates, triethiodide, and valerates. It also contains amino acid salts, such as cysteine ​​salts. Medicinal salts may have more than one charged atom in their structure. In this case, the medicinal salt may have multiple counterions. Therefore, a medicinal salt may have one or more charged atoms and / or one or more counterions.

[0064] As used herein, "treatment" means the relief of symptoms associated with a disorder or disease, or the inhibition of further progression or worsening of such symptoms, or the prevention or treatment of the disease or disorder, or the cure of the disease or disorder. Similarly, as used herein, an "effective amount" or "therapeutic effective amount" of a compound of this disclosure means an amount of the compound that completely or partially relieves symptoms associated with a disorder or condition, or stops or slows further progression or worsening of such symptoms, or prevents or provides prevention of the disorder or condition. For example, a "therapeutic effective amount" means an amount that is effective at the necessary dose and for the necessary time period to achieve the desired therapeutic outcome. A therapeutically effective amount is also an amount in which the therapeutically beneficial effect of the compound of this disclosure outweighs any toxic or harmful effects.

[0065] When used to describe treatment for an individual suffering from a condition, the term "effective amount" refers to the amount or concentration of the compound of this disclosure that effectively activates or otherwise acts on STING (wherein STING is involved in the condition) in the individual's tissues, wherein such activation or other action occurs to a degree sufficient to produce a beneficial therapeutic effect. Furthermore, the therapeutically effective amount of the compounds described herein means the amount by which the therapeutic agent, alone or in combination with other treatments, provides a therapeutic benefit in the treatment or prevention of the disease. When used in conjunction with the compounds described herein, the term may cover amounts that improve the overall treatment of the disease, alleviate or prevent symptoms or causes of the disease, or enhance or synergize with the therapeutic efficacy of additional therapeutic agents.

[0066] Generally, the initial therapeutically effective dose of the compound described herein or its pharmaceutically acceptable salts is in the range of about 0.01 to about 200 mg / kg patient body weight or about 0.1 to about 20 mg / kg patient body weight per day, with a typical initial range of about 0.3 to about 15 mg / kg / day. Oral unit dosage forms (e.g., tablets and capsules) may contain about 0.1 mg to about 1000 mg of the compound or its pharmaceutically acceptable salts. In another embodiment, such a dosage form contains about 50 mg to about 500 mg of the compound or its pharmaceutically acceptable salts. In another embodiment, such a dosage form contains about 25 mg to about 200 mg of the compound or its pharmaceutically acceptable salts. In another embodiment, such a dosage form contains about 10 mg to about 100 mg of the compound or its pharmaceutically acceptable salts. In yet another embodiment, such a dosage form contains about 5 mg to about 50 mg of the compound or its pharmaceutically acceptable salts. In any of the above embodiments, the dosage form may be administered once or twice daily.

[0067] The term "patient" or "object" includes animals such as humans, cattle, horses, sheep, lambs, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs. According to some embodiments, the animal is a mammal, such as a non-primate or primate (e.g., monkeys and humans). In one embodiment, the patient is a person, such as a human infant, child, adolescent, or adult. In this disclosure, the terms "patient" and "object" are used interchangeably.

[0068] compound

[0069] This disclosure provides, in several embodiments, compounds of formula (I) or pharmaceutically acceptable salts thereof:

[0070]

[0071] Rings B and C are independently selected from Het, equation (a), and equation (b):

[0072]

[0073] Each ring A is arbitrarily divided by 1 to 4 Rs A The substitution is made independently of: 5- or 6-membered monocyclic heteroaryl groups containing 1 to 3 heteroatoms selected from O, S and N, and 8- to 10-membered bicyclic heteroaryl groups containing 1 to 6 heteroatoms selected from O, S and N.

[0074] Het is a heteroatom consisting of 1 to 6 heteroatoms selected from O, S, and N, optionally bounded by 1 to 4 R atoms. A Replaces 8- to 10-membered bicyclic heteroaryl groups.

[0075] X is N, S, -N = C(R) 1 - or -C(R) 3 )=C(R 3 )-.

[0076] W is -N = or -C(R) 3 = ).

[0077] Y 1 Selected from -O-, -CR 4 R5-、-(CH2) L1 -O-、-(CH2) L1 -S(O) 0-2 -(where L1 is an integer selected from 1, 2, 3, 4, and 5); and -(CH2) L1 -N(R L )-(where R L Selected from benzyl, C1-C6-alkyl, H, optionally substituted with 1 or 2 methoxy groups.

[0078] Y 2 Selected from -O-, -CR4 R5-、-O-(CH2) L1 -、-S(O) 0-2 -(CH2) L1 -(where L1 is an integer selected from 1, 2, 3, 4, and 5); and -N(R L )-(CH2) L1 -(where R) L Is it H or C? 12 -C6-alkyl).

[0079] The subscript m is an integer selected from 0, 1, 2, 3, 4, 5, and 6.

[0080] The subscript n is an integer selected from 0, 1, and 2.

[0081] The subscripts x and y are integers independently selected from 0 and 1, where Y is 0 when m is 0 and each of x and y is 1. 1 and Y 2 Not simultaneously -O-.

[0082] Each R 1 and R 3 Independently selected from: H, halogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C6-alkoxy, cyano, C1-C6-haloalkyl, and 3- to 10-membered heterocyclic groups (wherein 1 to 4 heterocyclic alkyl members are independently selected from N, O, and S), wherein any alkyl, alkenyl, alkoxy, or heterocyclic group is optionally surrounded by 1 to 4 R... A replace.

[0083] R 2 Selected from -C(O)OR, -(C1-C6-alkyl)C(O)OR, C1-C6-haloalkyl, -P(O)(OR)2, -C(O)NHR, halogen, -CN, C3-C6-cycloalkenyl, 3-membered to 10-membered heterocyclic groups (wherein 1 to 4 heterocyclic alkyl members are independently selected from N, O and S) and 5-membered to 10-membered heteroaryl groups (wherein 1 to 4 heteroaryl members are independently selected from N, O and S), wherein any alkyl, cycloalkenyl, heterocyclic or heteroaryl group is optionally surrounded by 1 to 4 R A replace.

[0084] R is selected from: H; C1-C6-alkyl, optionally substituted by: -((C1-C6-alkyl)OC(O)OC1-C6-alkyl), -OP(O)(OH)2, -OC(O)(C1-C6-alkyl)-OP(O)(OH)2, -NH2, -CH(NH2)COOH or 3- to 10-membered heterocyclic groups (wherein 1 to 4 heterocyclic alkyl members are independently selected from N, O and S); and -(C1-C6-alkyl)(C6-C 10 -Aryl).

[0085] Each R 4 and R 5 Independently selected from H, halogens, C1-C6-alkyl, and C3-C7-cycloalkyl. In some embodiments, any two R atoms bonded to the same carbon atom... 4 and R 5 Together with the carbon atoms they bond to, this indicates that they are optionally bound by 1 to 3 R atoms. A Substituted C3-C5-cycloalkyl groups, or those representing C2-C6-alkenyl groups. The following substructures illustrate the unit -(CR 4 R 5 ) m - These implementation plans:

[0086]

[0087] In other implementations, R does not bind to the same carbon atom. 4 and R 5 Any two, together with the carbon atoms they are bonded to, represent optionally bounded by 1 to 3 R atoms. A Substituted C3-C7-cycloalkyl groups. The following substructures illustrate the unit -(CR 4 R 5 ) m - These implementation plans:

[0088]

[0089] Each R A Examples are independently selected from H, halogen, -CN, -hydroxyl, oxo, C1-C6-alkyl, C1-C6-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl, NH2, -S(O). 0-2 -(C1-C6-alkyl), -S(O) 0-2 -(C6-C 10 -aryl), -C(O)(C1-C6-alkyl), -C(O)(C1-C6-alkyl)COOH, -C(O)(C1-C6-alkyl)C(O)(C1-C6-alkoxy), -C(O)N(H or C1-C6-alkyl)2, -C(O)(C3-C 14 -cycloalkyl), -C3-C 14 -cycloalkyl, -(C1-C6-alkyl)(C3-C6-alkyl) 14 -cycloalkyl), C6-C 10-aryl, 3- to 14-membered heterocyclic alkyl and -(C1-C6-alkyl)-(3- to 14-membered heterocyclic alkyl) (wherein 1 to 4 heterocyclic alkyl members are independently selected from N, O and S), and 5- to 10-membered heteroaryl (wherein 1 to 4 heteroaryl members are independently selected from N, O and S), optionally C 1- C6-alkyl substitution.

[0090] In multiple implementation schemes:

[0091] Y 1 and Y 2 Independently selected from -O- and -CR4R5-;

[0092] Each R 1 and R 3 Independently selected from H, halogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C6-alkoxy, cyano, and C1-C6-haloalkyl, wherein any alkyl, alkenyl, alkynyl, or alkoxy group is optionally surrounded by 1 to 4 R groups. A replace;

[0093] R 2 Selected from -C(O)OR, -C(O)NHR, C3-C6-cycloalkenyl and 3- to 10-membered heterocyclic groups, wherein any alkyl, cycloalkenyl or heterocyclic group is optionally surrounded by 1 to 4 R groups. A replace;

[0094] R is selected from: H; C1-C6-alkyl, which is optionally substituted by: -((C1-C6-alkyl)OC(O)OC1-C6-alkyl) or 3- to 10-membered heterocyclic groups; and -(C1-C6-alkyl)(C6-C 10 -aryl);

[0095] Each R 4 and R 5 Independently selected from H, halogens, C1-C6-alkyl and C3-C7-cycloalkyl, wherein optionally, any two R atoms bonded to the same carbon atom 4 and R 5 Together with the carbon atoms they bond to, this indicates that they are optionally bound by 1 to 3 R atoms. A Substituted C3-C5-cycloalkyl; and

[0096] Optionally, R that does not bond with the same carbon atom 4 and R 5 Any two, together with the carbon atoms they are bonded to, represent optionally bounded by 1 to 3 R atoms. A Substituted C3-C7-cycloalkyl; and each R AIndependently selected from H, halogen, -CN, -hydroxyl, oxo, C1-C6-alkyl, C1-C6-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl, NH2, -S(O) 0-2 -(C1-C6-alkyl), -S(O) 0-2 -(C6-C 10 -aryl), -C(O)(C1-C6-alkyl), -C(O)(C1-C6-alkyl)COOH, -C(O)(C3-C 14 -cycloalkyl), -C3-C 14 -cycloalkyl, -(C1-C6-alkyl)(C3-C6-alkyl) 14 -cycloalkyl), C6-C 10 -aryl, 3- to 14-membered heterocyclic alkyl and -(C1-C6-alkyl)-(3- to 14-membered heterocyclic alkyl) (wherein 1 to 4 heterocyclic alkyl members are independently selected from N, O and S), and 5- to 10-membered heteroaryl (wherein 1 to 4 heteroaryl members are independently selected from N, O and S).

[0097] In some embodiments, optionally in combination with any other embodiments described herein, ring B is identical to ring C. In other embodiments, optionally in combination with any other embodiments described herein, ring B is different from ring C.

[0098] In illustrative embodiments where ring B differs from ring C, ring B conforms to formula (a), wherein ring A is a 5- or 6-membered monocyclic heteroaryl group containing 1 to 3 heteroatoms selected from O, S, and N. Examples of monocyclic heteroaryl groups of ring A are selected from pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, pyrroleyl, pyrazolyl, and others. azole group, thiazolyl group, thiophene group, iso azole group, Thiadiazolyl, isothiazolyl, tetrazolyl, imidazoleyl, triazolyl, furanyl. In some embodiments, the monocyclic heteroaryl group of ring A is pyridinyl, pyridazinyl, pyrazinyl, or pyrimidinyl. Within ring B, in these embodiments, ring A is optionally surrounded by 1 to 4 R groups. A Replacement. For example, ring A is replaced by 1 R. A Replace, R A It is a 5- to 10-membered heteroaryl group (of which 1 to 4 heteroaryl members are independently selected from N, O and S), such as tetrazolyl, imidazolyl or triazolyl.

[0099] In further conjunction with these embodiments, ring C is also of formula (a), wherein ring A is an 8- to 10-membered bicyclic heteroaryl group comprising 1 to 6 heteroatoms selected from O, S, and N, optionally separated by 1 to 4 R atoms. ASubstitution. Non-limiting examples of bicyclic heteroaryl rings include indolazidyl, benzothiophene, quinazolinyl, purine, indola, quinolinyl, tetrazo[1,5-b]pyridazinyl, [1,2,3]triazolo[1,5-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[4,3-a]pyrimidinyl, and imidazo[1,2-a]pyrimidinyl.

[0100] Other embodiments of this disclosure provide compounds of formula (I), wherein ring B and ring C are identical and each is of formula (a). In these embodiments, ring A is a 5- or 6-membered monocyclic heteroaryl group comprising 1 to 3 heteroatoms selected from O, S, and N, and ring A is optionally surrounded by 1 to 4 R atoms. A Substitution. Examples of monocyclic heteroaryl rings include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrroleyl, pyrazolyl, oxazolyl, thiazolyl, thiopheneyl, and isoaryl. azole group, Thiadiazolyl, isothiazolyl, tetrazolyl, imidazoleyl, triazolyl, and furanyl.

[0101] In other embodiments, ring B and ring C are identical and are of formula (a). In these embodiments, ring A is an 8- to 10-membered bicyclic heteroaryl group.

[0102] This disclosure also provides compounds of formula (I) in other embodiments, wherein B is optionally surrounded by 1 to 4 R groups. A The substituted Het, and the ring C is of formula (a). Illustrative examples of Het include indoleazinyl, benzothiopheneyl, quinazolinyl, purineyl, indoleyl, quinolinyl, tetrazo[1,5-b]pyridazinyl, [1,2,3]triazolo[1,5-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[4,3-a]pyrimidinyl, and imidazo[1,2-a]pyrimidinyl. In some embodiments, Het is substituted with 1 to 4 R groups selected from halogen, C1-C6-alkoxy, -C(O)(C1-C6-alkyl)COOH. A Optional substituted benzothiophene group. For example, in some embodiments, Het is a group that is:

[0103]

[0104] According to some implementation schemes, and optionally in combination with any other implementation scheme described herein, X is -C(R 3 )=C(R 3 - and W is -C(R) 3 = ).

[0105] In several implementation schemes, R 3Each instance is independently selected from H, halogens, and C1-C6-alkoxy groups.

[0106] In some other implementation schemes, R 2 It is -C(O)OR. For example, R is H or C1-C6-alkyl, such as methyl or ethyl.

[0107] In several implementations, x and y are 0 and 0, 0 and 1, 1 and 0, or 1 and 1, respectively. For example, in some implementations, each of x and y is 1, and Y is 1. 1 and Y 2 Each of them is either -O- or Y. 1 and Y 2 Each of them is -CR4R5-. In one implementation, each of x and y is 1, and Y is 1. 1 and Y 2 Each of them is -O-, and m is 4. In another implementation, Y 1 and Y 2 Each of these is -CR4R5-, each of x and y is 1, and m is 1. Consider all these combinations.

[0108] In several implementations, optionally in combination with any other implementation described herein, each R 1 Independently selected from H and halogens. For example, in some embodiments where ring B or ring C is of formula (a), R 1 It is H or a halogen. In some embodiments of formula (b), where ring B or ring C is , n can be 0, 1, or 2, and in each instance R 1 It is H or halogen.

[0109] Further embodiments of this disclosure are compounds of formula (I), wherein:

[0110] Ring B is of formula (a), wherein ring A is a 6-membered monocyclic heteroaryl group containing 1 to 3 heteroatoms selected from O, S and N, and is substituted by 5- to 10-membered heteroaryl groups (of which 1 to 4 heteroaryl members are independently selected from N, O and S).

[0111] Ring C is of formula (a), where ring A is an 8- to 10-membered bicyclic heteroaryl group;

[0112] X is -C(R) 3 )=C(R 3 )-, and W is -C(R 3 = ), where each R 3 Independently selected from H, halogens, and C1-C6-alkoxy groups;

[0113] R 1 It is H;

[0114] R 2 It is -C(O)OR and R is H or C1-C6-alkyl;

[0115] Each R 4 and R 5 It is H;

[0116] Each of x and y is 1; and

[0117] Y 1 and Y 2 Each of them is -O- and m is 4, or Y 1 and Y 2 Each of them is -CH2- and m is 1.

[0118] In other embodiments, this disclosure provides compounds of formula (I), wherein:

[0119] Each of rings B and C is of formula (a), wherein each ring A is a 6-membered monocyclic heteroaryl group containing 1 to 3 heteroatoms selected from O, S, and N, and is bounded by an R group that is a 5- to 10-membered heteroaryl group (where 1 to 4 heteroaryl members are independently selected from N, O, and S). A replace;

[0120] X is -C(R) 3 )=C(R 3 )-, and W is -C(R 3 = ), where each R 3 Independently selected from H and halogens;

[0121] R 1 It is H;

[0122] R 2 It is -C(O)OR and R is H;

[0123] Each of x and y is 1;

[0124] m is 0 or 1;

[0125] Y 1 It is -CR4R5- or -(CH2) L1 -N(R L )-;as well as

[0126] Y 2 It is -O- or -CR4R5-.

[0127] For example, in an illustrative embodiment, optionally in conjunction with any other embodiment described herein, each ring A is bounded by an R that is an imidazole group. A Substituted pyridazinyl group.

[0128] In other embodiments, this disclosure provides specific examples of compounds of formula (I) and their pharmaceutically acceptable salts as listed in Table 1 below. These compounds are presented with physicochemical characterization data.

[0129] Table 1: Examples of compounds of formula (I) and selected analytical data.

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167] Pharmaceutical Composition

[0168] In another embodiment, this disclosure provides a pharmaceutical composition comprising a compound as described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0169] The compositions disclosed herein can be administered orally, topically, parenterally, by inhalation or spraying, or rectally in dosage units. As used herein, the term parenterally includes subcutaneous injection, intravenous injection, intramuscular injection, intrasternal injection, or infusion techniques.

[0170] Suitable oral compositions as described herein include, but are not limited to, tablets, troche, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs.

[0171] The compositions of this disclosure suitable for oral use can be prepared according to any method known in the art for manufacturing pharmaceutical compositions. For example, liquid formulations of the compounds of this disclosure comprise one or more agents selected from sweeteners, flavoring agents, coloring agents, and preservatives to provide a pharmaceutically palatable formulation of the compound or a pharmaceutically acceptable salt thereof.

[0172] For tablet compositions, a compound or a pharmaceutically acceptable salt thereof, mixed with a non-toxic, pharmaceutically acceptable excipient, is used to manufacture the tablet. Examples of such excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrants such as corn starch or alginic acid; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. The tablets may be uncoated or may be coated using known coating techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing sustained therapeutic effect over the desired time period. For example, time-delaying substances such as glyceryl monostearate or glyceryl distearate may be used.

[0173] Preparations intended for oral use can also be formulated as hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin; or as soft gelatin capsules, wherein the active ingredient is mixed with an aqueous or oily medium, such as peanut oil, liquid paraffin, or olive oil.

[0174] For aqueous suspensions, the compound or its pharmaceutically acceptable salt is mixed with an excipient suitable for maintaining a stable suspension. Examples of such excipients include, but are not limited to, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, astragalus gum, and gum arabic.

[0175] Oral suspensions may also contain dispersants or wetting agents, such as naturally occurring phospholipids, such as lecithin, or condensation products of alkyl esters and fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide and long-chain fatty alcohols, such as heptadecanol, or condensation products of ethylene oxide and esters derived from fatty acids and hexitols, such as polyoxyethylene sorbitan oleate, or condensation products of ethylene oxide and esters derived from fatty acids and hexitol anhydrides, such as polyethylene dehydrated sorbitan oleate. Aqueous suspensions may also contain one or more preservatives, such as ethylparaben or n-propylparaben, one or more colorants, one or more flavoring agents, and one or more sweeteners, such as sucrose or saccharin.

[0176] Oily suspensions can be formulated by suspending a compound or its pharmaceutically acceptable salt in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or a mineral oil (e.g., liquid paraffin). The oily suspension may contain a thickener such as beeswax, anhydride, or cetyl alcohol.

[0177] Sweeteners, such as those shown above, and flavoring agents can be added to provide palatable oral formulations. These compositions can be preserved by adding antioxidants such as ascorbic acid.

[0178] Dispersible powders and granules suitable for preparing aqueous suspensions by adding water provide compounds or pharmaceutically acceptable salts thereof mixed with dispersants or wetting agents, suspending agents, and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweeteners, flavoring agents, and coloring agents, may also be present.

[0179] The pharmaceutical compositions disclosed herein may also be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin, or a mixture thereof. Suitable emulsifiers may be naturally occurring gums, such as gum arabic or astragalus gum; naturally occurring phospholipids, such as soybean lecithin; and esters or metaesters derived from fatty acids and hexitan anhydrides, such as sorbitan monooleate and condensation products of said metaesters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweeteners and flavoring agents.

[0180] Syrups and elixirs can be formulated with sweeteners such as glycerol, propylene glycol, sorbitol, or sucrose. Such formulations may also contain demulcents, preservatives, flavoring agents, and coloring agents. Pharmaceutical compositions can be in the form of sterile injectables, aqueous suspensions, or oil suspensions. The suspension can be formulated using suitable dispersants or wetting agents and suspending agents already mentioned above, according to known techniques. Sterile injectable formulations can also be sterile injectable solutions or suspensions in non-toxic, parenterically acceptable diluents or solvents, such as solutions in 1,3-butanediol. Water, Ringer's solution, and isotonic sodium chloride solution can be used among the available carriers and solvents. Additionally, sterile non-volatile oils are commonly used as solvents or suspension media. For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Furthermore, fatty acids such as oleic acid can be used to prepare injectables.

[0181] Compounds or their pharmaceutically acceptable salts can also be administered in suppository form for rectal use. These compositions can be prepared by mixing the compound with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the compound. Exemplary excipients include cocoa butter and polyethylene glycol.

[0182] Compositions intended for parenteral administration are administered in a sterile medium. Depending on the carrier and concentration of the compound or its pharmaceutically acceptable salt in the formulation, parenteral preparations can be suspensions or solutions containing dissolved compounds. Excipients such as local anesthetics, preservatives, and buffers may also be added to the parenteral composition.

[0183] How to use

[0184] This disclosure also provides, in one embodiment, a method for stimulating the expression of an interferon gene in a human patient. The method includes administering to the patient a therapeutically effective amount of the compound described herein or a pharmaceutically acceptable salt thereof. Based on exemplary data described herein, the compound of this disclosure is useful in this method as an agonist of STING. In one embodiment, administration is performed in vivo, or in another embodiment, in vitro.

[0185] In another embodiment, this disclosure provides a method of treating a tumor in a patient. The method comprises administering to the patient a therapeutically effective amount of the compound disclosed herein or a pharmaceutically acceptable salt thereof. In such cases, the role of STING, particularly its activation, has been recognized in antitumor immunology, for example in the following publications 1 to 4:

[0186] [1a] Corrales L, Glickmao LH, McWhirter SM, Kanne DB, Sivick KE, KatibahGE, Woo SR, LemmensE, Banda T, Leong JJ, Metchette K, Dubensky TW Jr, Gajewski TF (2015) Direct Activation of STING In the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity.Cell Rep.11:1018-30.

[0187] [1b]Chin,E.et al.(2020)Antitumor activity of a systemic STING-activating non-nucleotide eGAMP mimctic,Science.369:6506.

[0188] [1c]Pan,B.et al.(2020)An orally available non-nucleotide STINGagonist with antitumor activity,Science.369:6506.

[0189] [1d]Ramanjulu,J.et al.(2018)Design of amidobcnzimidazole STINGreceptor agonists with systemic activity,Nature.564:7736.

[0190] [2]Deng,L.et al.(2014)STING-Dependent Cytosolic DNA Sensing PromotesRadiation-Induced Type I Intcrferon-Dependent Antitumor Immunity inImmunogenic Tumors,Immunity,411:843.

[0191] [3]Corrales L,Mason V,Flood B,Sprangcr S,Gajewski TF、(2017)Innateimmune signaling and regulation in cancer immunotherapy.Cell Res.27:96-108.

[0192] [4]Corrales L,McWhirter SM,Dubensky TW Jr,Gajewski TF.(2016)The hostSTING pathway at the interface of cancer and immunity.J Clin Invest.126:2404-11.

[0193] In several embodiments, the methods described herein require combination therapy. For example, in embodiments optionally combined with any other embodiments described herein, the method further includes administration of an immune checkpoint-targeting drug. In other embodiments, the compounds described herein are administered synergistically with antitumor therapies requiring ionizing radiation and / or with existing chemotherapy therapies, such as DNA damage-based chemotherapy. The STING agonists of this disclosure can complement, enhance the potency of these known therapies, and / or amplify the adverse effects of these known therapies. Evidence illustrating the key role of STING-dependent micronucleus-mediated tumor clearance using these methods is illustrated, for example, in publications 5 through 8 below.

[0194] [5] Mackenzie, KF, et all, (2017), cGAS surveillance of micronucleilinks genome instability to innate immunity, Nature, 548, 461.

[0195] [6] Wang, W. et al-(2016), Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer, Cell, 165, 1092-1105.

[0196] [7] Charlotte E. Ariyan, et al., January 16, 2018; DOI: 10.1158 / 2326-6066, Robust antitumor responses result from local chemotherapy and CTLA-1 blockade, cancerinumunolres aacrjournals.org cn January 31, 2018.

[0197] [8] Chung Kil Song, et al., www.moleculartherapy.org vol.15 no.8aug.2007, Chemotherapy Enhances CD8+T Cell-mediated Antitumor Immunity Induced by Vaccinat:ion With Vaccinia Virus.

[0198] The compounds disclosed herein are also useful in the methods described herein, which further include administering an effective dose of an immune checkpoint-targeting agent. For example, in several embodiments, the immune checkpoint-targeting agent is an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA-4 antibody, or an anti-4-1BB antibody, as shown in publications 9 through 11 below:

[0199] [9]Ager, CR, et al., (2017) Cancer Immunol Res; 5(8), 676.

[0200]

[10] Fu, J. et al. (2015) Sci Transl Med.2015 April 15;7(283):283ra52.doi:10.1126 / scitranslmed.aaa4306.

[0201]

[11] Wang, H., et al. (2017) PNAS.February 14, 2017, vol 114, no.7, 1637-1642.

[0202] Example

[0203] The following non-limiting embodiments are further implementations used to illustrate the contents of this disclosure.

[0204] The compounds disclosed herein were prepared according to the following procedure, combined with general knowledge and skills of organic synthesis, and by substituting suitable reagents (as is obvious to a practitioner).

[0205] Experimental Procedure

[0206] Abbreviations. Use the following abbreviations: tetrahydrofuran (THF), dichloromethane (DCM), N,N-dimethylformamide (DMF), dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), trifluoroacetic acid (TFA), triethylamine (TEA), diisopropylethylamine (DIPEA), (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino-morpholino-carbon Hexafluorophosphate (COMU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-Oxide hexafluorophosphate, N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylformamide Hexafluorophosphate N-oxide (HATU), (2-biphenyl)dicyclohexylphosphine (CyJohnPhos), 1-propanephosphonic anhydride (T3P).

[0207] General embodiments for the preparation of the compounds of this disclosure. The starting materials and intermediates of the compounds of this disclosure may be prepared by application or modification of the methods described below, their obvious chemical equivalents, or, for example, as described in the literature such as *The Science of Synthesis*, Volumes 1-8, Editors E.M. Carreira et al., Thieme publishers (2001-2008). Details of reagents and reaction schemes may also be obtained by searching for structures and reactions using commercial computer search engines such as Scifinder (www.cas.org) or Reaxys (www.reaxys.com).

[0208] Part 1: Preparation of Intermediates

[0209] Option 1: Synthesis of intermediate A:

[0210]

[0211] Step 1: Synthesis of tetrazolo[1,5-b]pyridazine-6-carboxylic acid methyl ester: NaN3 (2.26 g, 34.8 mmol, 3.00 equivalent) was added to a solution of 6-chloropyridazine-3-carboxylic acid methyl ester (2.00 g, 11.6 mmol, 1.00 equivalent) in DMF (10 mL). The mixture was stirred at 80 °C for 4 hours. The residue was diluted with water (20 mL) and extracted with ethyl acetate (25 mL × 3). The combined organic layers were washed with water (25 mL × 3) and brine (25 mL × 2), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give the compound tetrazolo[1,5-b]pyridazine-6-carboxylic acid methyl ester (900 mg, 5.02 mmol, 43% yield, 99% purity) as a white solid.

[0212] 1 H-NMR (400MHz, DMSO-d6) δ8.95 (d, J=9.6Hz, 1H), 8.25 (d, J=9.2Hz, 1H), 4.03 (s, 3H).

[0213] Step 2: Synthesis of tetrazolo[1,5-b]pyridazine-6-carboxylic acid (A): A solution of LiOH·H₂O (632 mg, 15.1 mmol, 3.00 equivalent) in H₂O (4 mL) was added to a solution of tetrazolo[1,5-b]pyridazine-6-carboxylic acid methyl ester (900 mg, 5.02 mmol, 1.00 equivalent) in THF (4 mL). After stirring at 25 °C for 1 h, the mixture was neutralized with 6 M HCl. The precipitate was filtered and the filter cake was dried under reduced pressure to give intermediate A (700 mg, 4.24 mmol, 84% yield, 99% purity) as a white solid.

[0214] 1 H NMR (400MHz, DMSO-d6) δ14.69 (s, 1H), 8.91 (d, J=9.6Hz, 1H), 8.222 (d, J=9.2Hz, 1H).

[0215] Option 2: Synthesis of intermediate B:

[0216]

[0217] Synthesis of 6-(1H-imidazol-1-yl)pyridazine-3-carboxylic acid (B): K₂CO₃ (940 mg, 6.8 mmol) was added to a suspension of imidazole (0.4 g, 5.8 mmol) and methyl 6-chloropyridazine-3-carboxylic acid (1 g, 5.8 mmol) in anhydrous DMF (10 mL), and the reaction mixture was stirred at 120 °C for 3 h. The reaction was monitored by LCMS. After the reaction was complete, 2.5 M LiOH aqueous solution (2.8 mL, 6.96 mmol) was added to the reaction mixture, and the mixture was stirred at 60 °C for 1 h. The reaction was monitored by LCMS. After the reaction was complete, the reaction mixture was acidified with 1 M HCl aqueous solution, and the resulting precipitate was filtered and washed with water to give intermediate B (720 mg) as a grayish-white solid, which could be used in the next step without further purification.

[0218] LC-MS (ES(+): m / z 191.0 [M+H]) + .

[0219] Option 3: Synthesis of intermediate C:

[0220]

[0221] Step 1: Synthesis of ethyl 6-(1H-pyrazol-4-yl)pyridazine-3-carboxylate: Argon was purged through a solution of ethyl 6-chloropyridazine-3-carboxylate (5 g, 26.88 mmol) and pyrazol-4-boronic acid (4.51 g, 40.31 mmol), Na₂CO₃ (7.1 g, 67.2 mmol), in 1,4-dioxane (175 mL) and water (25 mL) for 10 minutes, followed by the addition of Pd(PPh₃)₄ (1.55 g, 1.34 mmol). The reaction mixture was stirred at 90 °C for 1 hour. After the reaction was complete, it was cooled to room temperature and diluted with EtOAc (250 mL). It was then washed with water (100 mL) and brine (100 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude substance was purified with silica gel by silica gel column chromatography to obtain 3.2 g of ethyl 6-(1H-pyrazol-4-yl)pyridazine-3-carboxylate as a grayish-white solid.

[0222] LC-MS(ESI+): m / z; 219.0[M+H] + .

[0223] Step 2: Synthesis of ethyl 6-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridazine-3-carboxylate: NaH (60% w / w) (0.422 g, 17.6 mmol) was added fractionally to a stirred solution of ethyl 6-(1H-pyrazol-4-yl)pyridazine-3-carboxylate (3.2 g, 14.67 mmol) in THF (64 mL) and DMF (30 mL) at 0 °C, and the mixture was stirred for 10 min. SEM-Cl (2.93 g, 17.61 mmol) was added, and the reaction mixture was stirred at 0 °C for 30 min. The mixture was then quenched with 10% citric acid solution, and the resulting solid was filtered, washed with water (5 mL × 2), and dried. The residue was purified by silica gel column chromatography using 0 to 5% methanol in dichloromethane as the eluent to give 2.65 g of ethyl 6-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridazine-3-carboxylate as a grayish-white solid.

[0224] LC-MS(ESI+): m / z; 349.1[M+H] + .

[0225] Step 3: Synthesis of 6-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridazine-3-carboxylic acid (C): At 0 °C, an aqueous solution of lithium hydroxide monohydrate (0.382 g, 9.13 mmol, in 3 mL of water) was added to a solution of ethyl 6-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridazine-3-carboxylic acid (2.65 g, 7.61 mmol) in THF (9 mL), and the reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was diluted with water (10 mL) and washed with EtOAc (30 mL × 2). The aqueous layer was acidified with 2N HCl solution (pH = 4), and the solid was filtered, washed with water (2 mL × 2), and dried to give 1.1 g of intermediate C as a grayish-white solid.

[0226] 1 H NMR (400MHz., DMSO-d6) δ13.62 (s, 1H), 8.78 (s, 1H), 8.33 (s, 1H), 8.18-8.13 (m, 2H), 5. 51 (s, 2H), 3.61 (t, J=8.0Hz, 2H), 0.87 (d, J=8.0Hz, 2H), 0.04 (s, 9H). LC-MS (ESI+): m / z 321.0[M+H] + .

[0227] Option 4: Synthesis of intermediates D and E:

[0228]

[0229] Step 1: Synthesis of methyl 4-allyl-5-fluoro-2-nitrobenzoate (D): Allyltributyltin (30.96 g, 93.50 mmol, 1.3 equivalent) was added to a stirred solution of methyl 4-bromo-5-fluoro-2-nitrobenzoate (20 g, 71.92 mmol, 1 equivalent) in toluene (200 mL) at room temperature. The reaction mixture was purged with argon for 20 min. Pd(PPh3)4 (1.67 g, 1.44 mmol, 0.02 equivalent) was added to the mixture at room temperature and stirred overnight at 110 °C. After the reaction was complete, the reaction mixture was cooled to room temperature and diluted with cold water (200 mL). The resulting aqueous solution was stirred with an aqueous solution of 1 M potassium fluoride (KF) for 30 min and extracted with ethyl acetate (2 × 300 mL). The combined organic layers were dried over anhydrous Na2SO4 and evaporated to give the crude product. The crude material was purified by silica gel column chromatography using 2 to 3% ethyl acetate in hexane to give pure intermediate D (15.1 g, 87.76%) as a brown liquid.

[0230] 1 H-NMR (400MHz, DMSO-d6) δ7.87 (d, J=6Hz, 1H), 7.41 (d, J=8.4Hz, 1H), 6.05-5.95 (m, 1H), 5.27-5.18 (m, 2H), 3.99 (s, 3H), 3.53 (d, J=6.4, 2H).

[0231] Step 2: Synthesis of methyl 4-(2,3-dihydroxypropyl)-5-fluoro-2-nitrobenzene: A solution of 0.02 M osmium tetroxide (OsO4) and N-methylmorpholine N-oxide (NMO) (2.45 g, 20.92 mmol, 1 equivalent) in tert-butanol (21 mL, 0.42 mmol, 0.02 equivalent) was added to an intermediate D (5 g, 20.92 mmol, 1 equivalent) in THF (100 mL) and water (20 mL) at room temperature. The reaction mixture was stirred at room temperature for 12 hours and monitored by TLC. After the reaction was complete, the reaction mixture was diluted with cold water (300 mL). The aqueous layer was extracted with ethyl acetate (2 × 150 mL). The combined organic layers were dried over anhydrous Na2SO4 and evaporated to give the crude product. The crude material was purified by silica gel column chromatography using 4% MeOH in DCM as the eluent to give pure methyl 4-(2,3-dihydroxypropyl)-5-fluoro-2-nitrobenzoate as a solid (3.1 g, 54.28% yield).

[0232] 1 H-NMR (400MHz, DMSO-d6) δ8.12 (d, J=6.5Hz, 1H), 7.72 (d, J=9.6Hz, 1H), 4.85 (d, 1H), 4.7 5 (t, 1H), 3.91 (s, 3H), 3.68 (m, 1H), 3.48 (m, 1H); 3.33 (m, 1H); 2.96 (m, 1H); 2.66 (m, 1H).

[0233] Step 3: Synthesis of methyl 5-fluoro-4-(2-hydroxyethyl)-2-nitrobenzene (E): Sodium periodate (2.91 g, 13.62 mmol, 1.2 equivalent) was added to a solution of intermediate C (3.1 g, 11.35 mmol, 1 equivalent) in MeOH (90 mL) and water (90 mL). The reaction mixture was stirred at 0 °C for 1 h and monitored by TLC. Then, sodium borohydride (0.52 g, 13.62 mmol, 1.2 equivalent) was added and stirred at room temperature for 1 h. After the reaction was complete, the reactants were diluted with cold water (300 mL). The aqueous solution was extracted with 10% MeOH in DCM (2 × 150 mL), and the combined organic layers were dried over Na₂SO₄ and evaporated to give the crude product. The crude material was purified by silica gel column chromatography with a gradient of 2 to 3% MeOH in DCM to give pure intermediate E (2.7 g, 97.85%) as a solid.

[0234] 1 H-NMR (400MHz, DMSO-d6) δ8.18 (d, J=6.4Hz, 1H), 7.76 (d, J=6.4Hz, 1H), 5.75 (m, 1H), 4.66 (d, J=6.4Hz, 2H), 3.86 (t, J=11.2Hz, 2H), 3.38 (s, 3H).

[0235] Option 5: Synthesis of intermediates F and G:

[0236]

[0237] Step 1: Synthesis of methyl 2-amino-5-bromo-4-chlorobenzoate: K₂CO₃ (16.0 g, 116 mmol, 2 equivalents) was added to a solution of CH₃I (16.4 g, 116 mmol, 7.23 mL, 2 equivalents) in DMF (200 mL). The mixture was stirred at 25 °C for 3 hours. The reaction mixture was filtered and slowly poured into water to filter out the solid, which was then washed with ethyl acetate (100 mL) and brine (50 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give methyl 2-amino-5-bromo-4-chlorobenzoate (22.2 g, crude) as a yellow solid. The crude product was used for the next step without further purification. MS-ESI: [M+H] observed at m / z 265.9 + .

[0238] Step 2: Synthesis of methyl 2-amino-5-bromo-4-chlorobenzoate: DMAP (9.36 g, 76.6 mmol, 1 equivalent) was added to a solution of Boc₂O (66.9 g, 306 mmol, 70.4 mL, 4 equivalents) and methyl 2-amino-5-bromo-4-chlorobenzoate (22.2 g, 76.6 mmol, 1 equivalent) in CH₂Cl₂ (200 mL). The mixture was stirred at 25 °C for 3 hours. The reaction solution was quenched with water (100 mL) and extracted with ethyl acetate (200 mL × 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude material was purified by rapid silica gel chromatography with a gradient of 0 to 25% ethyl acetate / petroleum ether to give methyl 2-amino-5-bromo-4-chlorobenzoate as a white solid (4.08 g, 8.81 mmol, 15% yield).

[0239] 1 H NMR (400MHz, DMSO-d6) δ8.20 (s, 1H), 7.84 (s, 1H), 3.80 (s, 3H), 1.33 (s, 18H).

[0240] Step 3: Synthesis of methyl 5-allyl-2-(bis(tert-butoxycarbonyl)amino)-4-chlorobenzoate (F): A mixture of Pd(dppf)Cl2 (629 mg, 0.860 mmol, 0.1 equivalent), K2CO3 (3.57 g, 25.8 mmol, 3 equivalent), potassium allyl trifluoroborate (2.55 g, 17.2 mmol, 2 equivalent), and methyl 2-amino-5-bromo-4-chlorobenzoate (4 g, 8.61 mmol, 1 equivalent) in dioxane (60 mL) and water (6 mL) was degassed and purged three times with N2. The mixture was then stirred at 80 °C for 12 hours under N2 atmosphere. The reaction mixture was partitioned between water (100 mL) and ethyl acetate (80 mL). The organic phase was separated, washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude material was purified by rapid silica gel chromatography with a gradient of 0 to 5% ethyl acetate / petroleum ether to give intermediate F (1.28 g, 3.01 mmol, 34% yield) as a yellow oil.

[0241] 1 H NMR (400MHz, CDCl3) δ7.89 (s, 1H), 7.23 (s, 1H), 6.01-5.92 (m, 1H), 5.17-5.13 (m, 1H), 5.08-5.03 (m, 1H), 3.87 (s, 3H), 3.54 (d, J=6.4Hz, 2H), 1.40 (s, 18H).

[0242] Step 4: Synthesis of methyl 2-(bis(tert-butoxycarbonyl)amino)-4-chloro-5-(2-hydroxyethyl)benzoate (G): A mixture of methyl 5-allyl-2-[bis(tert-butoxycarbonyl)amino]-4-chlorobenzoate (1.28 g, 3.01 mmol, 1 equivalent) in CH2Cl2 (20 mL) and EtOH (2 mL) was ozonated with ozone (15 psi) at -50 °C. The mixture was then heated to 20 °C, and NaBH4 (227 mg, 6.01 mmol, 2 equivalents) was added to the mixture. The mixture was stirred at 20 °C for 2 hours. The mixture was carefully acidified with 10% HCl aqueous solution (30 mL), concentrated under reduced pressure, and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude material was purified by rapid silica gel chromatography with a gradient of 0 to 40% ethyl acetate / petroleum ether to give intermediate G as a white solid (500 mg, 1.11 mmol, 37% yield, 95% purity).

[0243] 1 H NMR (400MHz, DMSO-d6) δ=7.90 (s, 1H), 7.49 (s, 1H), 4.79 (t, J=5.2Hz, 1H); 3.66-3.61 (m, 2H), 2.91 (t, J=6.4Hz, 2H), 1.34 (s, 18H).

[0244] Part Two: Preparation of Example Compounds

[0245] All compounds disclosed herein were prepared using the procedures illustrated below.

[0246] Example 1

[0247] Scheme 6: Synthesis of Compound 1:

[0248]

[0249] Step 1: Synthesis of methyl 4-(4-bromobutoxy)-2-nitrobenzoate: K₂CO₃ (630 mg, 4.57 mmol, 3 equivalents) was added to a solution of 1,4-dibromobutane (1.64 g, 7.61 mmol, 917 μL, 5 equivalents) and methyl 4-hydroxy-2-nitrobenzoate (300 mg, 1.52 mmol, 1 equivalent) in DMF (10 mL). The mixture was then stirred at 25 °C for 3 hours. The reaction mixture was diluted with ethyl acetate (10 mL) and washed with water (10 mL × 3). The combined organic layers were then washed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated. The crude substance was purified by silica gel column chromatography to give methyl 4-(4-bromobutoxy)-2-nitrobenzoate (400 mg, 1.2 mmol, 79% yield) as a white solid.

[0250] 1 H NMR (400MHz, CDCl3) δ7.79 (d, J=8.8Hz, 1H), 7.24 (d, J=2.4Hz, 1H), 7.10 (dd, J=8.8, 2.4Hz, 1H), 4.10 (t, J=6.0Hz, 2H), 3.89 (s, 3H), 3.50 (t, J=6.4Hz, 2H), 2.13-2.06 (m, 2H), 2.04-1.96 (m, 2H).

[0251] Step 2: Synthesis of methyl 5-fluoro-4-(4-(4-(methoxycarbonyl)-3-nitrophenoxy)butoxy)-2-nitrobenzoate: K₂CO₃ (499 mg, 3.61 mmol, 3 equivalents) was added to a solution of methyl 5-fluoro-4-hydroxy-2-nitrobenzoate (259 mg, 1.2 mmol, 1 equivalent) and methyl 4-(4-bromobutoxy)-2-nitrobenzoate (400 mg, 1.2 mmol, 1 equivalent) in DMF (6 mL), and the mixture was stirred at 50 °C for 12 hours. After the reaction was complete, the reaction mixture was poured into ethyl acetate (10 mL), and the mixture was subsequently washed with water (10 mL × 3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated. The crude material was purified by silica gel column chromatography to give methyl 5-fluoro-4-[4-(4-methoxycarbonyl-3-nitro-phenoxy)butoxy]-2-nitro-benzoate as a yellow solid (380 mg, 0.814 mmol, 67% yield).

[0252] 1H NMR (400MHz, DMSO-d6) δ7.89 (d, J=7.2Hz, 1H), 7.86 (d, J=8.8Hz, 1H), 7.80 (d, J=10.8Hz, 1H), 7.54 (d, J=2.4Hz, 1H), 7 .31 (dd, J=8.8, 2.4Hz, 1H), 4.30 (t, J=5.6Hz, 2H), 4.21 (t, J=5.6Hz, 2H), 3.82 (s, 3H), 3.80 (s, 3H), 1.93-1.91 (m, 4H).

[0253] Step 3: Synthesis of methyl 2-amino-4-(4-(3-amino-4-(methoxycarbonyl)phenoxy)butoxy)-5-fluorobenzoate: NH4Cl (436 mg, 8.15 mmol, 10 equivalents) and Fe (227 mg, 4.07 mmol, 5 equivalents) were added to a solution of methyl 5-fluoro-4-[4-(4-methoxycarbonyl-3-nitro-phenoxy)butoxy]-2-nitro-benzoate (380 mg, 0.814 mmol, 1 equivalent) in MeOH (8 mL), and the mixture was stirred at 60 °C for 3 hours. After the reaction was complete, the reaction mixture was diluted with DCM (20 mL), filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography to give methyl 2-amino-4-[4-(3-amino-4-methoxycarbonyl-phenoxy)butoxy]-5-fluorobenzoate as a yellow solid (220 mg, 0.541 mmol, 66% yield).

[0254] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.80 (br d, J = 8.8 Hz, 1H), 7.55 (d, J = 12.4 Hz, 1H), 6.30–6.09 (m, 3H), 4.12–4.02 (m, 4H), 3.85 (s, 6H), 2.01–1.99 (m, 4H). MS-ESI; m / z 407.0 Observed [M+H] + .

[0255] Step 4: Synthesis of methyl 5-fluoro-4-(4-(4-(methoxycarbonyl)-3-(tetrazo[1,5-b]pyridazine-6-carboxamido)phenoxy)butoxy)-2-(tetrazo[1,5-b]pyridazine-6-carboxamido)benzoate: POCl3 (226 mg, 1.17 mmol, 137 μL, 6 equivalents) was added to a solution of intermediate A (102 mg, 0.615 mmol, 2.5 equivalents) and methyl 2-amino-4-[4-(3-amino-4-methoxycarbonyl-phenoxy)butoxy]-5-fluorobenzoate (100 mg, 0.246 mmol, 1 equivalent) in pyridine (1 mL) at 0 °C, and the mixture was then stirred at 25 °C for 2 hours. The reaction mixture was poured into water (20 mL), the mixture was filtered, and the filter cake was collected. The crude product was ground with water (2 mL) for 5 minutes at 25 °C to give methyl 5-fluoro-4-[4-[4-methoxycarbonyl-3-(tetrazol[1,5-b]pyridazin-6-carbamate)phenoxy]butoxy]-2-(tetrazol[1,5-b]pyridazin-6-carbamate)benzoate as a yellow solid (80 mg, 0.114 mmol, 46% yield).

[0256] 1 ¹H NMR (400MHz, DMSO-d⁶) δ 12.95–12.84 (m, 1H), 12.77 (br s, 1H), 9.07–8.88 (m, 2H), 8.77–8.56 (m, 1H), 8.45–8.26 (m, 3H), 8.04 (br d, J = 8.4Hz, 1H), 7.78 (br d, J = 11.2Hz, 1H), 6.96–6.83 (m, 1H), 4.35–4.17 (m, 4H), 4.00–3.90 (m, 6H), 2.05–1.96 (m, 4H). MS-ESI: m / z 701.1 Observed [M+H] + .

[0257] Step 5: Synthesis of 4-(4-(4-carboxy-3-(tetrazol[1,5-b]pyridazine-6-carboxamido)phenoxy)butoxy)-5-fluoro-2-(tetrazol[1,5-b]pyridazine-6-carboxamido)benzoic acid (1): LiCl·H2O (130 mg, 2.06 mmol, 24 equivalents) was added to a solution of methyl 5-fluoro-4-[4-[4-methoxycarbonyl-3-(tetrazol[1,5-b]pyridazine-6-carbonylamino)phenoxy]butoxy]-2-(tetrazol[1,5-b]pyridazine-96-carbonylamino)benzoate (60 mg, 0.086 mmol, 1 equivalent) in DMSO (1 mL), and the mixture was stirred at 150 °C for 4 hours. Water (0.3 mL) was added to the reaction mixture, and the mixture was filtered, and the filter cake was collected. The crude product was ground with water (2 mL) at 25 °C for 5 minutes to give compound 1 as a yellow solid (43 mg, 0.064 mmol, 74% yield).

[0258] 1 ¹H NMR (400MHz, DMSO-d⁶) δ 13.71 (s, 2H), 8.97 (d, J = 9.4Hz, 2H), 8.64 (d, J = 8.0Hz, 1H), 8.49–8.27 (m, 3H), 8.04 (d, J = 8.7Hz, 1H), 7.77 (d, J = 12.0Hz, 1H), 6.87 (d, J = 8.9Hz, 1H), 4.37–4.15 (m, 4H), 2.14–1.90 (m, 4H), MS-ESI: m / z 673.2. [M+H] observed. +

[0259] The same procedure used to synthesize compound 1 was used to synthesize compounds 19, 25, 28, 30, 32, 49, 58, 69, 81 and 203.

[0260] Example 2

[0261] Scheme 7: Synthesis of compound 2-Li:

[0262]

[0263] Step 1: Synthesis of methyl 2-amino-5-fluoro-4-hydroxybenzoate: Fe powder (2.05 g, 37.19 mmol, 4 equivalents) was added to a stirred solution of methyl 5-fluoro-4-hydroxy-2-nitrobenzoate (2 g, 9.30 mmol, 1 equivalent) in acetic acid (20 mL) at room temperature, and the mixture was heated at 80 °C for 2 hours. After the reaction was complete, the reaction mixture was poured into cold water (300 mL). The resulting aqueous solution was extracted with ethyl acetate (2 × 300 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and evaporated to give a crude product. The crude product was purified by silica gel column chromatography with a gradient of 15 to 20% ethyl acetate in hexane to give pure methyl 2-amino-5-fluoro-4-hydroxybenzoate as a solid (700 mg, 41% yield).

[0264] 1 H-NMR (400MHz, DMSO-d6) 10.54 (s, 1H), 7.36 (d, J=12.4Hz, 1H), 6.53 (s, 2H), 6.30 (d, J=7.6Hz, 1H), 3.73 (s, 3H).

[0265] Step 2: Synthesis of methyl 2-amino-5-fluoro-4-(2-fluoro-4-(methoxycarbonyl)-5-nitrophenylethoxy)benzoate: Ph3P (1.51 g, 5.76 mmol, 2 equivalents) was added to a solution of intermediate E (0.7 g, 2.88 mmol, 1 equivalent) and methyl 2-amino-5-fluoro-4-hydroxybenzoate (0.53 g, 2.88 mmol, 1 equivalent) in toluene (7 mL). Diethyl azodicarboxylate (DEAD) (1 g, 5.76 mmol, 2 equivalents) was added to the solution at 55 °C, and the mixture was stirred at the same temperature for 5 hours. After the reaction was complete, the reaction mixture was poured into cold water (500 mL). The resulting aqueous solution was extracted with ethyl acetate (2 × 200 mL). The combined organic layers were dried over anhydrous Na2SO4 and evaporated to give the crude product. The crude material was purified by silica gel column chromatography using 20% ​​ethyl acetate in hexane as the eluent to give pure methyl 2-amino-5-fluoro-4-(2-fluoro-4-(methoxycarbonyl)-5-nitrophenylethoxy)benzoate as a solid (650 mg, 55% yield).

[0266] 1¹H-NMR (400MHz, DMSO-d⁶) δ 8.29 (d, J = 6.0Hz, 1H), 7.80 (d, J = 9.1Hz, 1H), 7.37 (d, J = 12.4Hz, 1H), 6.63 (s, 2H), 6.50 (d, J = 7.6Hz, 1H), 4.31 (t, J = 6.3Hz, 2H), 3.87 (s, 3H), 3.75 (s, 3H), 3.34–3.22 (m, 2H), MS-ESI: m / z 410.87 Observed [M+H] + .

[0267] Step 3: Synthesis of methyl 5-fluoro-4-(2-(2-fluoro-4-(methoxycarbonyl)-5-(tetrazo[1,5-b]pyridazine-6-carboxamido)phenoxy)ethyl)-2-nitrobenzoate: POCl3 (0.9 g, 0.55 mL, 5.85 mmol, 4 equivalents) was added dropwise to a solution of intermediate A (0.6 g, 3.66 mmol, 2.5 equivalents) and methyl 2-amino-5-fluoro-4-(2-fluoro-4-(methoxycarbonyl)-5-nitrophenylethoxy)benzoate (0.6 g, 1.46 mmol, 1 equivalent) in pyridine (6 mL) at 0 °C and stirred at room temperature for 1.5 h. After the reaction was complete, the reaction mixture was poured into cold water (50 mL) and stirred for 10 min. The solid was filtered and washed with 1 N HCl solution to remove excess pyridine from the solid. The crude material was purified by silica gel column chromatography using 2% methanol in DCM as eluent to give pure methyl 5-fluoro-4-(2-(2-fluoro-4-(methoxycarbonyl)-5-(tetraazolo[1,5-b]pyridazine-6-carboxamido)phenoxy)ethyl)-2-nitrobenzene as a solid (0.325 g, 40% yield). MS-ESI: m / z 558.3 ​​observed [M+H] + .

[0268] Step 4: Synthesis of methyl 2-amino-5-fluoro-4-(2-(2-fluoro-4-(methoxycarbonyl)-5-(tetraazolo[1,5-b]pyridazine-6-carboxamido)phenoxy)ethyl)benzoate: Acetic acid (5 mL) was added to a stirred solution of methyl 5-fluoro-4-(2-(2-fluoro-4-(methoxycarbonyl)-5-(tetraazolo[1,5-b]pyridazine-6-carboxamido)phenoxy)ethyl)-2-nitrobenzoate (0.325 g, 0.58 mmol, 1 equivalent) in MeOH (5 mL) and THF (5 mL) at room temperature, followed by the addition of Fe powder (0.19 g, 3.50 mmol, 6 equivalents) and heating at 85 °C for 1 hour. After the reaction was complete, the reaction mixture was poured into cold water (50 mL) to give a solid. The resulting solid was filtered and thoroughly dried to give pure methyl 2-amino-5-fluoro-4-(2-(2-fluoro-4-(methoxycarbonyl)-5-(tetrazo[1,5-b]pyridazine-6-carbamate)phenoxy)ethyl)benzoate as a solid (250 mg, 81.30% yield).

[0269] 1 ¹H NMR (400MHz, DMSO-d⁶) δ 3.12 (d, J = 7.6Hz, 2H), 379 (s, 3H), 3.97 (s, 3H), 4.43 (t, J = 6.5Hz, 2H), 6.57 (s, 2H), 6.82 (d, J = 6.4Hz, 1H), 7.41 (d, J = 10.8Hz, 1H), 7.88 (d, J = 11.5Hz, 1H), 8.41 (d, J = 9.2Hz, 1H), 8.64 (d, J = 8.1Hz, 1H), 9.06 (d, J = 9.1Hz, 1H), 12.83 (s, 1H); MS-ESI: m / z 5279 observed [M+H] + .

[0270] Step 5: Synthesis of methyl 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluoro-4-(2-(2-fluoro-4-(methoxycarbonyl)-5-(tetrazo[1,5-b]pyridazin-6-carboxamido)phenoxy)ethyl)benzoate: At room temperature, a 50% solution (1.5 mL, 2.37 mmol, 5 equivalents) of DIPEA (0.43 g, 0.58 mL, 3.32 mmol, 7 equivalents) and T3P (in ethyl acetate) was added to a stirred solution of intermediate B (0.11 g, 0.57 mmol, 1.2 equivalents) in DCE (5 mL). Methyl 2-amino-5-fluoro-4-(2-(2-fluoro-4-(methoxycarbonyl)-5-(tetraazolo[1,5-b]pyridazine-6-carboxamido)phenoxy)ethyl)benzoate (0.25 g, 0.47 mmol, 1 equivalent) was added. The reaction mixture was heated overnight at 80 to 90 °C. After the reaction was complete, the reaction mixture was concentrated directly under vacuum. The crude product was purified by silica gel column chromatography using 2 to 3% MeOH in DCM as eluent to give the pure desired product (0.185 g, 56% yield).

[0271] 1 ¹H NMR (400 MHz, DMSO-d⁶) δ 3.19 (s, 2H), 3.96 (s, 6H), 4.54 (s, 2H), 7.29 (s, 1H), 7.85 (t, J = 11.2 Hz, 2H), 8.24 (s, 1H), 8.39 (d, J = 9.6 Hz, 1H), 8.51 (d, J = 18.3 Hz, 2H), 8.64 (d, J = 7.9 Hz, 1H), 8.84 (s, 1H), 8.95 (s, 1H), 9.04 (d, J = 9.6 J Hz, 1H), 12.81 (s, 1H), 12.9 (s, 1H); MS-ESI: m / z 700.2 Observed [M+H] + .

[0272] Step 6: Synthesis of 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-(2-(4-carboxy-2-fluoro-5-(tetrazol[1,5-b]pyridazin-6-carboxamido)phenoxy)ethyl)-5-fluorobenzoic acid (2): TEA (0.27 g, 0.37 mL, 2.64 mmol, 10 equivalents) was added to a solution of methyl 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluoro-4-(2-(2-fluoro-4-(methoxycarbonyl)-5-(tetrazol[1,5-b]pyridazin-6-carboxamido)phenoxy)ethyl)benzoate (0.185 g, 0.26 mmol, 1 equivalent) in ACN (5 mL) and water (5 mL). The reaction mixture was stirred in a microwave oven at 120 °C for 2 hours. After the reaction was complete, the reaction mixture was concentrated under vacuum. The crude substance was purified by preparative HPLC to give compound 2 (110 mg, 62% yield). MS-ESI: m / z 672.2 [M+H]+ observed.

[0273] Step 7: Synthesis of lithium 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-(2-(4-carboxy-2-fluoro-5-(tetraazolo[1,5-b]pyridazin-6-carboxamido)phenoxy)ethyl)-5-fluorobenzoate (2-Li): LiOH·H₂O (13.8 mg, 0.33 mmol, 2 equivalents) was added to a suspension of compound 2 (110 mg, 0.16 mmol, 1 equivalent) in water (6 mL). The resulting clear solution was then filtered to remove any insoluble particles and lyophilized to obtain 2-Li (100 mg, 91% yield).

[0274] 1 H NMR (400MHz, DMSO) δ16.69 (s, 1H), 15.77 (s, 1H), 8.95 (d, J=9.6Hz, 1H), 8.85 (d, J=7.2Hz, 1H), 8.80 (s, 1H), 8.64 (d, J=8.0Hz, 1H), 8.47 (d, J=8.8Hz, 1H), 841 (d, J =9.2Hz, 1H), 8.36 (d, J = 9.6Hz, 1H), 8.21 (s, 1H), 7.77 (d, J = 11.6Hz, 1H), 7.27 (s, 1H), 4.35 (t, J = 6.8Hz, 2H), 3.21 (t, J = 6.0Hz, 2H). MS-ES1: m / z 672.14 observed [M+H] + .

[0275] Procedures similar to those used for synthesizing compound 2 are used to synthesize compounds such as: 20, 22, 67, 97 to 100, 24, 63, 44, 60, 196, 62, 211 to 214, 64, 72 to 77, 82, 85 to 89, 126, 83, 91, 92, 95, 57, 102, 104 to 107, 109 to 118, 135 to 137, 158, 159, 184, 192, 205, 207, and 218.

[0276] Example 3

[0277] Scheme 8: Synthesis of compounds 3-Mg and 173:

[0278]

[0279] Step 1: Synthesis of dimethyl 4,4′-(prop-1-en-1,3-diyl)(E)-bis(2-amino-5-fluorobenzoate): TEA (13.43 mL, 95.50 mmol, 2.5 equivalences) was added to a solution of methyl 2-amino-4-bromo-5-fluorobenzoate (9.48 g, 38.23 mmol, 1 equivalent) and intermediate D (8 g, 38.23 mmol, 1 equivalent) in 1,4-dioxane (80 mL) at room temperature. The reaction mixture was purged with argon for 30 min. Pd(OAc)₂ (0.43 g, 1.91 mmol, 0.05 equivalent) and CyJohnPhos (1.34 g, 3.82 mmol, 0.1 equivalent) were added to the mixture at room temperature, and the resulting mixture was stirred at 110 °C for 16 h. After the reaction was complete, the reaction mixture was cooled to room temperature and diluted with cold water (750 mL). The aqueous layer was extracted with ethyl acetate (3 × 500 mL), and the combined organic layers were dried over anhydrous Na₂SO₄ and evaporated to give a crude product. The crude product was purified by silica gel column chromatography using 15% ethyl acetate in hexane as eluent to give pure dimethyl 4,4′-(prop-1-en-1,3-diyl)(E)-bis(2-amino-5-fluorobenzoate) as a solid (3.8 g, 26.41% yield).

[0280] 1 ¹H NMR (400 MHz, DMSO-d⁶) δ 7.41–7.38 (m, 2H), 6.96 (d, J = 6.7 Hz, 1H), 6.72 (d, J = 6.6 Hz, 1H), 6.57–6.45 (m, 6H), 3.79 (s, 6H), 3.54 (d, J = 5.8 Hz, 2H). MS-ESI: m / z 377.0 observed [M+H] + .

[0281] Step 2: Synthesis of dimethyl 4,4′-(propane-1,3-diyl)bis(2-amino-5-fluorobenzoate): 1.9 g of 10% Pd / C catalyst (50% humidity) was added to a solution of dimethyl 4,4′-(prop-1-en-1,3-diyl)(E)-bis(2-amino-5-fluorobenzoate) (3.8 g, 10.09 mmol, 1 equivalent) in MeOH (60 mL) and THF (60 mL) at room temperature. The reaction mixture was purged with hydrogen for 5 hours. After the reaction was complete, the reaction mixture was filtered through a diatomaceous earth bed and washed with 10% MeOH in DCM. The filtrate was concentrated under vacuum to give crude dimethyl 4,4′-(propane-1,3-diyl)bis(2-amino-5-fluorobenzoate) (3.6 g, 94.23%), which was used in the next step without further purification.

[0282] 1 ¹H NMR (400 MHz, DMSO-d⁶) δ 7.36 (d, J = 11.0 Hz, 2H), 6.69 (d, J = 6.7 Hz, 2H), 6.51 (s, 4H), 3.79 (s, 6H), 2.58 (t, J = 7.7 Hz, 4H), 1.83–1.79 (m, 2H). MS-ESI: m / z 379.0 observed [M+H] + .

[0283] Step 3: Synthesis of dimethyl 4,4′-(propane-1,3-diyl)bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoate: At room temperature, a 50% solution (5.04 mL, 7.93 mmol, 6 equivalents) of DIPEA (1.84 mL, 10.57 mmol, 8 equivalents) and T3P (in ethyl acetate) was added to a stirred solution of intermediate B (0.55 g, 2.91 mmol, 2.2 equivalents) in DCE (7 mL). Dimethyl 4,4′-(propane-1,3-diyl)bis(2-amino-5-fluorobenzoate) (0.5 g, 1.32 mmol, 1 equivalent) was added to the solution at room temperature. The reaction mixture was heated overnight at 80–90 °C. After the reaction was complete, the reaction mixture was concentrated directly under reduced pressure to give the crude product. A cold, saturated solution of NaHCO3 was added and the mixture was stirred at room temperature for 15 minutes. The resulting precipitate was collected by filtration, washed with water, and dried to give a brown solid, which was further purified by grinding with methanol (2 × 10 mL) and ethyl acetate (10 mL) to give pure dimethyl 4,4′-(propane-1,3-diyl)bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoate as a solid (0.75 g, 79% yield). MS-ESI: m / z 723.2 Observed [M+H] + .

[0284] Step 4: Synthesis of 4,4′-(propane-1,3-diyl)bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoic acid (3): TEA (2.91 mL, 20.76 mmol, 10 equivalents) was added to a solution of dimethyl 4,4′-(propane-1,3-diyl)bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoate (1.5 g, 2.07 mmol, 1 equivalent) in ACN (7.5 mL) and water (7.5 mL) at room temperature. The reaction mixture was stirred at 115 to 120 °C for 3 hours (in a sealed tube). After the reaction was complete, the reaction mixture was evaporated under reduced pressure. Water (20 mL) was added to the resulting solid, and it was acidified to pH 2.0 using 1 N HCl solution. The precipitate was collected by filtration, washed with water and dried to give a brown solid, which was further purified by grinding with methanol (3 × 10 mL) to give compound 3 (650 mg, 45% yield).

[0285] 1¹H NMR (400MHz, m DMSO-d⁶) δ 9.66 (s, 2H), 8.79 (d, J = 9.0Hz, 2H), 8.60 (d⁻¹, J = 6.3Hz, 2H), 8.37 (d, J = 9.1H₂, 2H), 8.29 (t, J = 1.9Hz, 2H), 7.90 (d, J = 9.6Hz, 2H), 7.75–7.69 (m, 2H), 2.91 (t, J = 7.8Hz, 4H), 2.14 (d, J = 9.5Hz, 2H). MS-ESI: m / z 695.1 Observed [M+H] + .

[0286] Step 5: Synthesis of magnesium 4,4′-(propane-1,3-diyl)bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoate (3-Mg): 100 mg of compound 3 and 18.57 mg of Mg(OH)₂ (2.1 equivalents) were suspended in 10 mL of 1:1 MeOH-water. The suspension was then subjected to a heat-cooling cycle (60 °C to 5 °C) in a Thermomixer for 24 hours.

[0287] Thermomixer conditions:

[0288] Step 1: 60℃, 6 hours, 850 rpm, heating rate: 1℃ / minute

[0289] Step 2: 5℃, 6 hours, 850 rpm, cooling rate: 0.1℃ / min

[0290] Step 3: 60℃, 6 hours, 850rpm

[0291] Step 4: 5℃, 6 hours, 850rpm

[0292] After the reaction, the white solid was collected by centrifugation and dried at room temperature for 24 hours to obtain 3-Mg.

[0293] 1 H NMR (400MHz, DMSO-d6) δ8.75 (d, J=7.2Hz, 4H), 8.44 (d, J=9.2Hz, 2H), 8.38 (d, J=9.1Hz, 2H), 8.16 (t, J=1.5Hz, 2H), 7.75 (d, J=10.9Hz, 2H) ， 7.28–7.19 (m, 2H), 2.75 (t, J = 7.7 Hz, 4H), 1.96 (t, J = 7.7 Hz, 2H), MS-ESI: m / z 695.44 observed [M+H]+.

[0294] Procedures similar to those used to synthesize compound 3 were used to synthesize compounds such as: 13 to 15, 29, 48, 51 to 56, 61, 65, 66, 68, 70, 71, 119, 134, 148, 172, 174, 161, 164, 165, 170, 180, 187, 194, 199, 201, 202, 219, 78, 80, 59, 182, and 127.

[0295] Step 6: Synthesis of 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-(3-(5-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-(ethoxycarbonyl)-2-fluorophenyl)propyl)-5-fluorobenzoic acid (173): Iodoethane (0.034 g, 0.216 mmol, 1 equivalent) was added to a solution of K2CO3 (0.045 g, 0.324 mmol, 1 equivalent) and compound 3 (0.15 g, 0.216 mmol, 1 equivalent) in anhydrous DMF (1.5 mL) at room temperature. The reaction mixture was then stirred at 80 °C for 4 hours. After the reaction was complete, the reaction mixture was diluted with cold water (10 mL). The aqueous layer was extracted with ethyl acetate (3 × 10 mL), and the combined organic layers were dried over Na2SO4 and evaporated to give the crude product. The crude substance was purified by preparative HPLC to obtain pure [M+H] at 173 (1.5 mg) MS-ESI: m / z 723.2. + .

[0296] A procedure similar to that used to synthesize compound 173 was used to synthesize compounds such as 47 and 62. A similar method was also used to prepare compounds 224 to 234.

[0297] Example 4

[0298] Scheme 9: Synthesis of compound 4-Li:

[0299]

[0300] Step 1: Synthesis of methyl 5-fluoro-4-(2-fluoro-4-(methoxycarbonyl)-5-(6-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridazin-3-carboxamido)phenethoxy)-2-(6-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridazin-3-carboxamido)benzoate: At room temperature, a 50% solution of DIPEA (0.46 g, 0.62 mL, 3.55 mmol, 9 equivalents) and T3P (in ethyl acetate) (1.5 g, 2.37 mmol, 6 equivalents) was added to a stirred solution of C (0.32 g, 0.99 mmol, 2.5 equivalents) in DCE (7 mL). Methyl 2-amino-4-(5-amino-2-fluoro-4-(methoxycarbonyl)phenethoxy)-5-fluorobenzoate (0.15 g, 0.39 mmol, 1 equivalent) was added to the mixture at room temperature. The reaction mixture was heated overnight at 80 to 90 °C. After the reaction was complete, the reaction mixture was concentrated directly under vacuum. The crude product was poured into cold water to allow the residue to fall out. The crude material was filtered and purified by silica gel column chromatography using 60% ethyl acetate in hexane as eluent to give pure methyl 5-fluoro-4-(2-fluoro-4-(methoxycarbonyl)-5-(6-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridazin-3-carboxamido)phenylethoxy)-2-(6-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridazin-3-carboxamido)benzoate (0.23 g, 59.20% yield). MS-ESI: m / z 986.0 observed [M+H] + .

[0301] Step 2: Synthesis of methyl 5-fluoro-4-(2-fluoro-4-(methoxycarbonyl)-5-(6-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridazin-3-carboxamido)phenethoxy)-2-(6-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridazin-3-carboxamido)benzoate: at room temperature, to methyl 5-fluoro-4-(2-fluoro-4-(methoxycarbonyl)-5-(6-(1- A solution of methyl benzoate (0.150 g, 0.20 mmol, 1 equivalent) in ACN (7.5 mL) and water (7.5 mL) was mixed with TEA (0.2 g, 2.03 mmol, 10 equivalents). The reaction mixture was stirred at 120 °C under microwave irradiation for 4 hours. After the reaction was complete, the reaction mixture was distilled and the residue was ground with ethyl acetate to give pure methyl 5-fluoro-4-(2-fluoro-4-(methoxycarbonyl)-5-(6-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridazin-3-carboxamido)phenethoxy)-2-(6-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyridazin-3-carboxamido)benzoate (105 mg, 72.05% yield).

[0302] 1 ¹H NMR (400 MHz, DMSO-d⁶) δ 15.17 (s, 2H), 10.1 (s, 2H), 8.88–8.74 (m, 4H), 8.36 (s, 2H), 8.35–8.18 (m, 4H), 7.76–7.73 (t, J = 12.8 Hz, 2H), 5.53 (s, 4H), 4.37 (s, 2H), 3.62 (t, J = 8.0 Hz, 4H), 3.09 (s, 2H), 0.88 (t, J = 8.0 Hz, 4H), 0.0 (s, 18H); MS-ESI: m / z 958.4 [M+H] observed. + .

[0303] Step 3: Synthesis of 2-(6-(1H-pyrazol-4-yl)pyridazin-3-carboxamido)-4-(5-(6-(1H-pyrazol-4-yl)pyridazin-3-carboxamido)-4-carboxy-2-fluorophenylethoxy)-5-fluorobenzoic acid (4): 5-fluoro-4-(2-fluoro-4-(methoxycarbonyl)-5-(6-(1-((2-(trimethylsilyl)ethoxy)methyl) Methyl benzoate (0.105 g, 0.11 mmol, 1 equivalent) was added to a stirred solution of methyl benzoate (0.105 g, 0.11 mmol, 1 equivalent) in DCM (4 mL) with TFA (50 mg, 0.44 mmol, 4 equivalents). The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was concentrated directly under vacuum. The crude material was milled with water (5 mL). The residue was purified by preparative HPLC to give compound 4 (26 mg, 34.02% yield). MS-ESI: m / z 697.2 observed [M+H] + .

[0304] Step 4: Synthesis of lithium 2-(6-(1H-pyrazol-4-yl)pyridazin-3-carboxamido)-4-(5-(6-(1H-pyrazol-4-yl)pyridazin-3-carboxamido)-4-carboxy-2-fluorophenylethoxy)-5-fluorobenzoate (4): LiOH·H2O (3.3 mg, 0.08 mmol, 2.1 equivalents) was added to a suspension of 4 (26 mg, 0.04 mmol, 1 equivalent) in water (6 mL), and the resulting clear solution was filtered to remove any insoluble particles. The solution was lyophilized to obtain compound 4-Li (26 mg).

[0305] 1 ¹H NMR (500MHz, DMSO) δ 9.15 (t, J = 6.5Hz, 1H), 8.82 (d, J = 7.0Hz, 1H), 8.70 (dd, J = 8.2, 4.2Hz, 1H), 8.57 (d, J = 3.4Hz, 1H), 8.36–8.05 (m, 6H), 7.73 (d, J = 11.6Hz, 2H), 5.50–5.38 (m, 2H), 4.31 (t, J = 7.0Hz, 2H). MS-ESI: m / z 697.16 observed [M+H]. + .

[0306] The same procedure used to synthesize compound 4 was used to synthesize compounds 123, 125, 129, 131, 133, 141 to 144, 150, 152 to 154, 157, 159, 162, 163, 166, 167, 175, 178, 179, 181, 183, 186, 195, 197, 198, 200, 208, 209, 216, 217 and 238.

[0307] Example 5

[0308] Scheme 10: Synthesis of compound 5-Li:

[0309]

[0310] Step 1: Synthesis of dimethyl 4,4′-(butane-1,3-diylbis(oxy))bis(5-fluoro-2-nitrobenzene): K₂CO₃ (1.28 g, 9.30 mmol, 2 equivalents) and 1,3-dibromobutane (0.5 g, 2.33 mmol, 0.5 equivalents) were added to a solution of methyl 5-fluoro-4-hydroxy-2-nitrobenzene (1 g, 4.65 mmol, 1 equivalent) in DMF (10 mL) at room temperature. The resulting solution was stirred at 50 °C for 16 hours. After the reaction was complete, the reaction mixture was cooled at room temperature and diluted with water (30 mL). The aqueous layer was extracted with ethyl acetate (2 × 50 mL), and the combined organic layers were dried over anhydrous Na₂SO₄ and evaporated to give the crude product. The crude material was purified by silica gel column chromatography using 15% ethyl acetate in hexane as the eluent to give pure dimethyl 4,4′-(butane-1,3-diylbis(oxy))bis(5-fluoro-2-nitrobenzene ester) (0.6 g, 27%) as a solid.

[0311] 1 ¹H NMR (400 MHz, DMSO-d⁶) δ 1.42 (d, J = 6.0 Hz, 3H), 2.76 (s, 1H), 2.92 (s, 1H), 3.84 (s, 6H), 4.38 (d, J = 4.3 Hz, 2H), 4.97 (d, J = 6.1 Hz, 1H), 7.81 (d, J = 10.8 Hz, 2H), 7.93 (dd, J = 9.4, 7.2 Hz, 2H), MS-ESI: [M+18] observed at m / z 502. + .

[0312] Step 2: Synthesis of dimethyl 4,4′-(butane-1,3-diylbis(oxy))bis(2-amino-5-fluorobenzoate): At room temperature, 0.2 g of a 10% Pd / C catalyst (50% humidity) was added to a solution of dimethyl 4,4′-(butane-1,3-diylbis(oxy))bis(5-fluoro-2-nitrobenzene) (0.6 g, 1.23 mmol, 1 equivalent) in MeOH (10 mL) and THF (10 mL). The reaction mixture was purged with hydrogen for 1 hour. After the reaction was complete, the reaction mixture was filtered through a diatomaceous earth bed and washed with 10% MeOH in DCM solution. The filtrate was concentrated under vacuum to give crude dimethyl 4,4′-(butane-1,3-diylbis(oxy))bis(2-amino-5-fluorobenzoate) (0.45 g, 86%), which was used in the next step without further purification. MS-ESI: [M+H] observed at m / z 425 + .

[0313] Step 3: Synthesis of dimethyl 4,4′-(butane-1,3-diylbis(oxy))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoate: At room temperature, a 50% solution (12.02 mL, 18.86 mmol, 8 equivalents) of DIPEA (1.46 g, 2.03 mL, 11.31 mmol, 12 equivalents) and T3P (in ethyl acetate) was added to a stirred solution of intermediate B (0.45 g, 2.35 mmol, 2.5 equivalents) in DCE (8 mL). Dimethyl 4,4′-(butane-1,3-diylbis(oxy))bis(2-amino-5-fluorobenzoate) (0.4 g, 0.94 mmol, 1 equivalent) was then added to the solution at room temperature. The reaction mixture was heated overnight at 80 to 90 °C. After the reaction was complete, the reaction mixture was subsequently concentrated directly under vacuum. The crude material was purified by silica gel column chromatography with a gradient of 1.5% to 2% MeOH in DCM to give pure dimethyl 4,4′-(butane-1,3-diylbis(oxy))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoate as a solid (0.15 g, 20.7% yield). MS-ESI: m / z 769 observed [M+H] + .

[0314] Step 4: Synthesis of 4,4′-(butane-1,3-diylbis(oxy))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoic acid (5): TEA (0.27 mL, 1.95 mmol, 10 equivalents) was added to a solution of dimethyl 4,4′-(butane-1,3-diylbis(oxy))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoate (150 mg, 0.2 mmol, 1 equivalent) in a 50% mixture of ACN:water (15 mL). The reaction mixture was heated in a microwave at 120 °C for 4 hours. After the reaction was complete, the reaction mixture was directly purified by preparative HPLC to give pure compound 5 (30 mg, 20.76% yield). MS-ESI: m / z 741.2 observed [M+H] + .

[0315] Step 5: Synthesis of lithium 4,4′-(butane-1,3-diylbis(oxy))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoate (5-Li): LiOH·H₂O (3.5 mg, 0.09 mmol, 2.1 equivalents) was added to a suspension of compound 5 (30 mg, 0.04 mmol, 1 equivalent) in water (6 mL), and the resulting clear solution was subsequently filtered to remove any insoluble particles. The resulting solution was lyophilized to obtain 5-Li (27 mg, 90% yield).

[0316] 1 ¹H NMR (400MHz, DMSO-d⁶) δ 16.08 (s, 1H), 16.05 (s, 1H), 8.78 (s, 2H), 8.73–8.68 (m, 2H), 8.48–8.45 (m, 2H), 8.40 (d, J = 8.8Hz, 2H), 8.19 (s, 2H), 7.75 (dd, J = 12.4, 4.4Hz, 2H), 7.25 (s, 2H), 4.80–4.61 (m, 1H), 4.28–4.26 (m, 2H), 2.34–2.28 (m, 2H), 1.45–1.43 (m, 4H). MS-ESI: m / z 741.2. Observed [M+H] + .

[0317] The same procedure used to synthesize compound 5 was used to synthesize compounds 11, 12, 16, 17, 21, 23, 34, 36, 37, 38, 42, 43, 45, 50, 138, 139, 168, 185, 206 and 220.

[0318] Example 6

[0319] Scheme 11: Synthesis of compound 6-Li:

[0320]

[0321] Step 1: Synthesis of methyl 2-(bis(tert-butoxycarbonyl)amino)-4-chloro-5-(2-(2-methoxy-4-(methoxycarbonyl)-5-nitrophenoxy)ethyl)benzoate: DIAD (352 mg, 1.74 mmol, 0.339 mL, 1.5 equivalent) and PPh3 (457 mg, 1.74 mmol, 1.5 equivalent) were added to a solution of methyl 2-[bis(tert-butoxycarbonyl)amino]-4-chloro-5-(2-hydroxyethyl)benzoate (500 mg, 1.16 mmol, 1 equivalent) and methyl 4-hydroxy-5-methoxy-2-nitrobenzoate (264 mg, 1.16 mmol, 1 equivalent) in THF (10 mL). The reaction mixture was stirred at 20 °C for 12 hours. The reaction mixture was then partitioned between water (20 mL) and ethyl acetate (20 mL). The organic phase was separated, washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by rapid silica gel chromatography with a gradient of 0 to 60% ethyl acetate / petroleum ether to give methyl 2-[bis(tert-butoxycarbonyl)amino]-4-chloro-5-[2-(2-methoxy-4-methoxycarbonyl-5-nitro-phenoxy)ethyl]benzoate as a white solid (700 mg, 1.05 mmol, 90% yield). MS-ESI: m / z 439.1 observed [M+H] + .

[0322] Step 2: Synthesis of methyl 2-amino-4-(4-(bis(tert-butoxycarbonyl)amino)-2-chloro-5-(methoxycarbonyl)phenethoxy)-5-methoxybenzoate: Fe (305 mg, 5.48 mmol, 5 equivalents) and NH4Cl (585 mg, 10.95 mmol, 10 equivalents) were added to a solution of methyl 2-[bis(tert-butoxycarbonyl)amino]-4-chloro-5-[2-(2-methoxy-4-methoxycarbonyl-5-nitro-phenoxy)ethyl]benzoate (700 mg, 1.10 mmol, 1 equivalent) in MeOH (10 mL). The reaction mixture was stirred at 60 °C for 12 hours. The reaction mixture was filtered, and the filtrate was concentrated under vacuum. The residue was diluted with ethyl acetate (15 mL) and extracted with water (15 mL × 3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give methyl 2-amino-4-(4-(bis(tert-butoxycarbonyl)amino)-2-chloro-5-(methoxycarbonyl)phenethoxy)-5-methoxybenzoate (540 mg, crude product) as a brown oil. This crude product was used in the next step without further purification.

[0323] Step 3: Synthesis of methyl 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-(4-(bis(tert-butoxycarbonyl)amino)-2-chloro-5-(methoxycarbonyl)phenethoxy)-5-methoxy-benzoate: T3P (4.18 g, 6.57 mmol, 3.91 mL, 1 equivalent) and DIPEA (1.59 g, 12.31 mmol, 2.14 mL, 15 equivalent) were added to a solution of methyl 5-[2-(5-amino-2-methoxy-4-methoxycarbonyl-phenoxy)ethyl]-2-[bis(tert-butoxycarbonyl)amino]-4-chloro-benzoate (500 mg, 0.820 mmol, 1 equivalent) and intermediate B (234 mg, 1.23 mmol, 1.5 equivalent) in DMF (10 mL). The mixture was stirred at 80 °C for 12 hours. Add water (15 mL) and stir the resulting mixture at 25 °C for another 30 minutes. Purify the crude material by rapid silica gel chromatography with a gradient of 0 to 100% ethyl acetate / petroleum ether to give methyl 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-(4-(bis(tert-butoxycarbonyl)amino)-2-chloro-5-(methoxycarbonyl)phenethoxy)-5-methoxybenzoate (480 mg, 74% yield) as a brown solid.

[0324] Step 4: Synthesis of methyl 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-(4-amino-2-chloro-5-(methoxycarbonyl)phenethoxy)-5-methoxybenzoate: TFA (7.70 g, 67.5 mmol, 5.00 mL, 109 equivalents) was added to a solution of methyl 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-(4-(bis(tert-butoxycarbonyl)amino)-2-chloro-5-(methoxycarbonyl)phenethoxy)-5-methoxybenzoate (480 mg, 0.614 mmol, 1 equivalent) in CH2Cl2 (5 mL). The mixture was stirred at 20 °C for 2 hours. The reaction mixture was concentrated under reduced pressure and washed with CH2Cl2 (5 mL × 3) to obtain the residue. The crude product was ground with ethyl acetate to give methyl 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-(4-amino-2-chloro-5-(methoxy-carbonyl)phenethoxy)-5-methoxybenzoate (210 mg, 53% yield) as a gray solid. MS-ESI: m / z 581.2 [M+H] observed. + .

[0325] Step 5: Synthesis of methyl 2-(6-(IH-imidazol-1-yl)pyridazin-3-carboxamido)-4-(2-chloro-5-(methoxycarbonyl)-4-(tetrazo[1,5-b]pyridazin-6-carboxamido)phenethoxy)-5-methoxybenzoate: To 2-amino-4-chloro-5-[2-[5-[(6-imidazol-1-ylpyridazin-3-carbonyl)amino]-2-methoxy-4-methoxy-carbonyl A solution of methyl benzoate [phenoxy]ethyl benzoate (210 mg, 0.361 mmol, 1.0 equivalent) and intermediate A (89.5 mg, 0.542 mmol, 1.5 equivalent) in DMF (4 mL) was supplemented with T3P (1.84 g, 2.89 mmol, 1.72 mL, 8 equivalent) and DIPEA (700 mg, 5.42 mmol, 0.944 mL, 15 equivalent). The reaction mixture was stirred at 80 °C for 12 hours. Ethyl acetate (20 mL) was added to the reaction mixture and stirred at 25 °C for 30 minutes. The mixture was filtered, and the filter cake was washed with water (15 mL), acetonitrile (5 mL × 3), ethyl acetate (5 mL × 3), and petroleum ether (5 mL × 3), and dried under reduced pressure to give methyl 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-(2-chloro-5-(methoxycarbonyl)-4-(tetraazolo[1,5-b]pyridazin-6-carboxamido)phenethoxy)-5-methoxybenzoate (180 mg, 66% yield) as a pale yellow solid. MS-ESI: m / z 728.1 Observed [M+H + .

[0326] Step 6: Synthesis of 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-(5-carboxy-2-chloro-4-(tetrazol[1,5-b]pyridazin-6-carboxamido)phenethoxy)-5-methoxybenzoic acid (6): Et3N (3.64 g, 35.9 mmol, 5 mL, 153 equivalents) was added to a solution of methyl 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-(2-chloro-5-(methoxycarbonyl)-4-(tetrazol[1,5-b]pyridazin-6-carboxamido)phenethoxy)-5-methoxybenzoate (170 mg, 0.233 mmol, 1 equivalent) in acetonitrile (5 mL) and water (5 mL). The mixture was stirred at 120 °C for 4 hours. The reaction mixture was concentrated under reduced pressure. The crude material was purified by preparative HPLC to give compound 6 (20 mg, 10% yield) as a yellow solid.

[0327] 1 H NMR (400MHz, DMSO-d6) δ = 8.95 (d, J = 9.6Hz, 1H), 8.82 (s, 1H), 8.78 (s, 1H), 8.63 (s, 1H), 8.50 (d, J = 9.2Hz ， 1H), 8.42 (d, J = 9.2 Hz, 1H), 8.35 (d, J = 9.6 Hz, 1H), 8.19 (s, 1H), 8.16 (s, 1H), 7.62 (s, 1H), 7.26 (s, 1H), 4.26 (t, J = 7.6 Hz, 2H), 3.78 (s, 3H), 3.25 (t, J = 7.2 Hz, 2H). MS-ESI: m / z. 700.2 Observed [M+H] +

[0328] Step 7: Synthesis of lithium 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-(5-carboxy-2-chloro-4-(tetraazolo[1,5-b]pyridazin-6-carboxamido)phenethoxy)-5-methoxybenzoate (6-Li): LiOH (0.02M, 2.86mL, 2 equivalents) was added to a solution of compound 6 (20 mg, 0.028 mmol, 1 equivalent) in water (3 mL) and acetonitrile (3 mL). The mixture was stirred at 20 °C for 0.5 h. The reaction mixture was lyophilized to give compound 6-Li.

[0329] 1¹H NMR (400MHz, DMSO-d⁶) δ 15.59 (s, 1H), 8.94 (d, J = 9.6Hz, 1H), 8.81 (s, 1H), 8.76 (s, 1H), 8.59 (s, 1H), 8.45 (d, J = 9.2Hz, 1H), 8.38 (d, J = 8.8Hz, 1H), 8.35 (d, J = 9.6Hz, 1H), 8.17 (s, 2H), 767 (s, 1H), 7.25 (s, 1H), 4.21 (t, J = 7.2Hz, 2H), 3.76 (s, 3H), 3.23 (t, J = 7.2Hz, 2H). MS-ESI: m / z 700.2. Observed [M+H] + .

[0330] The same procedure used to synthesize compound 6 was used to synthesize compounds 84, 90, 93, 94, 96, 101, 103, 108, 128, 130, 145, 147, 156, 169, 176, 177, 188 to 190, 193, 204, 222 and 237.

[0331] Example 7

[0332] Scheme 12: Synthesis of compound 7-Li:

[0333]

[0334] Step 1: Synthesis of methyl 4-(bromomethyl)-5-fluoro-2-nitrobenzoate: PPh3 (13.7 g, 52.3 mmol, 2 equivalents) was added to a solution of methyl 5-fluoro-4-(hydroxymethyl)-2-nitrobenzoate (6 g, 26.1 mmol, 1 equivalent) in DCM (100 mL) at 0 °C, followed by the addition of CBr4 (17.3 g, 52.3 mmol, 2 equivalents). The reaction mixture was stirred at 0 °C for 0.5 h. After the reaction was complete, water (60 mL) was added to the reaction mixture, and the mixture was extracted with DCM (40 mL × 3). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude material was purified by rapid silica gel chromatography with a gradient of 0 to 20% ethyl acetate / petroleum ether to give methyl 4-(bromomethyl)-5-fluoro-2-nitrobenzoate (6.6 g, 73% yield) as a brown solid.

[0335] 1 H NMR (400MHz, DMSO-d6) δ8.45 (d, J=6.4Hz, 1H), 7.85 (d, J=10.4Hz, 1H), 4.80 (s, 2H), 3.88 (s, 3H).

[0336] Step 2: Synthesis of methyl 4-((acetylthio)methyl)-5-fluoro-2-nitrobenzoate: K₂CO₃ (2.84 g, 20.5 mmol, 2 equivalents) and ethanethioic acid (938 mg, 12.3 mmol, 0.876 mL, 1.2 equivalents) were slowly added to a solution of methyl 4-(bromomethyl)-5-fluoro-2-nitrobenzoate (3 g, 10.2 mmol, 1 equivalent) in THF (30 mL). The reaction mixture was then stirred at 20 °C for 0.5 h. After the reaction was complete, the reaction mixture was added to water (20 mL) and extracted with ethyl acetate (30 mL × 2). The combined phases were then dried and concentrated under reduced pressure. The crude material was purified by rapid silica gel chromatography with a gradient of 0 to 20% ethyl acetate / petroleum ether to give methyl 4-(acetylthiomethyl)-5-fluoro-2-nitrobenzoate (2.2 g, 71% yield) as a yellow oil.

[0337] 1 H NMR. (400MHz, CDCl3) δ8.05 (d, J=6.0Hz, 1H), 7.4 (d, J=8.8Hz, 1H), 4.18 (d, J=0.8Hz, 2H), 3.94 (s, 3H), 2.40 (s, 3H).

[0338] Step 3: Synthesis of dimethyl 4,4′-(thiobis(methylene))bis(5-fluoro-2-nitrobenzoate): K₂CO₃ (402 mg, 2.91 mmol, 0.5 equivalence) was added to a solution of methyl 4-(bromomethyl)-5-fluoro-2-nitrobenzoate (1.7 g, 5.82 mmol, 1 equivalent) in DMF (8 mL) and MeOH (8 mL). The reaction mixture was stirred at 25 °C for 20 min. After the reaction was complete, water (20 mL) was added to the reaction mixture, and the mixture was subsequently extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude material was purified by rapid silica gel chromatography with a gradient of 0 to 20% ethyl acetate / petroleum ether to give dimethyl 4,4′-(thiobis(methylene))bis(5-fluoro-2-nitrobenzene) as a yellow solid (910 mg, 33% yield).

[0339] 1¹H NMR (400 MHz, CDCl₃) δ 8.00 (d, J = 6.0 Hz, 2H), 7.40 (d, J = 8.8 Hz, 2H), 3.96 (s, 6H), 3.79 (s, 4H). MS-ESI: m / z 474.0 Observed [M+H] + .

[0340] Step 4: Synthesis of dimethyl 4,4′-(sulfinylbis(methylene))bis(5-fluoro-2-nitrobenzene): m-CPBA (66.7 mg, 0.329 mmol, 1 equivalent) was added to a mixture of dimethyl 4,4′-(thiobis(methylene))bis(5-fluoro-2-nitrobenzene) (150 mg, 0.329 mmol, 1 equivalent) in DCM (10 mL) at 0 °C, and the reaction mixture was subsequently stirred at 0 °C for 2 h. After the reaction was complete, the reaction mixture was quenched with an aqueous solution of NaHCO3 (20 mL) and extracted with DCM (10 mL × 3). The combined organic layers were dried, filtered, and concentrated under reduced pressure to give dimethyl 4,4′-(sulfinylbis(methylene))bis(5-fluoro-2-nitrobenzene) (210 mg, crude product) as a white solid. This crude product was used directly in the next step without further purification. MS-ESI: [M+H] observed at m / z 473.0 + .

[0341] Step 5: Synthesis of dimethyl 4,4′-(sulfinylbis(methylene))bis(2-amino-5-fluorobenzoate): Fe (77.0 mg, 1.38 mmol, 5 equivalents) and NH4Cl (147 mg, 2.76 mmol, 10 equivalents) were added to a mixture of methyl 5-fluoro-4-[(2-fluoro-4-methoxycarbonyl-5-nitro-phenyl)methylsulfinylmethyl]-2-nitrobenzoate (210 mg, 0.276 mmol, 62% purity, 1 equivalent) in MeOH (10 mL). The mixture was stirred at 50 °C for 5 hours. The reaction mixture was filtered and concentrated under reduced pressure. The crude material was purified by preparative TLC (SiO2, petroleum ether / ethyl acetate = 1 / 1) to obtain dimethyl 4,4′-(sulfinylbis(methylene))bis(2-amino-5-fluorobenzoate) as a white solid (30.0 mg, 26% yield). MS-ESI: m / z 413.3 Observed [M+H] + .

[0342] Step 6: Synthesis of dimethyl 4,4′-(sulfinylbis(methylene))bis(2-(6-(1H-imidazol-1-yl)pyridazine-3-carboxamido)-5-fluorobenzoate: T3P (123 mg, 0.194 mmol, 4 equivalents) and DIPEA (37.6 mg, 0.291 mmol, 0.051 mL, 6 equivalents) were added to a mixture of intermediate B (36.9 mg, 0.194 mmol, 4 equivalents, 50% purity) and DIPEA (37.6 mg, 0.291 mmol, 0.051 mL, 6 equivalents) in DMF (1 mL). The mixture was stirred at 80 °C for 12 hours. After the reaction was complete, the reaction mixture was diluted with ethyl acetate (4 mL) and filtered. The filter cake was then added to saturated Na₂CO₃ (5 mL) and stirred at 20 °C for 10 minutes. The mixture was filtered, and the filter cake was washed with ethyl acetate (1 mL), acetonitrile (1 mL), and PE (1 mL) to give dimethyl 4,4′-(sulfinylbis(methylene))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoate (18.0 mg, crude product) as a white solid. This crude product was used in the next step without further purification.

[0343] 1 H NMR (400MHz, DMSO-d6) δ12.98 (s, 2H), 10.28 (s, 2H), 8.94 (d, J = 6.8Hz, 2H), 8.78-8.60 (m, 6H), 7.97 (s , 2H), 7.81 (d, J=10.0Hz, 2H), 4.55 (d, J=12.8Hz, 2H), 4.32 (d, J=12.8Hz, 2H), 3.90 (s, 6H).MS-ESI: m / z

[0344] 757.2 Observed [M+H] + .

[0345] Step 7: Synthesis of 4,4′-(sulfinylbis(methylene))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoic acid (7): Et3N (13.4 mg, 0.132 mmol, 0.018 mL, 10 equivalents) was added to a mixture of dimethyl 4,4′-(sulfinylbis(methylene))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoate (10.0 mg, 0.013 mmol, 1 equivalent) in ACN (0.5 mL) and H2O (0.5 mL), and the reaction mixture was stirred at 120 °C for 1 hour. The reaction mixture was then concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative HPLC to give compound 7 (8.00 mg, 83% yield) as a white solid. MS-ESI: [M+H] observed at m / z 729.2 + .

[0346] Step 8: Synthesis of lithium 4,4′-(sulfinylbis(methylene))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoate (7-Li): LiOH·H2O (0.02M, 1.10mL, 2 equivalents) was added to a suspension of compound 7 (8.00 mg, 0.011 mmol, 1 equivalent) in H2O (1 mL), and the reaction mixture was stirred at 20 °C for 0.5 h. The reaction mixture was then lyophilized to obtain compound 7-Li (8.00 mg, 0.011 mmol) as a white solid.

[0347] 1 H NMR (400MHz, DMSO-d6) δ15.72 (s, 2H), 8.87-8.82 (m, 2H), 8.77 (s, 2H), 8, 48-8 .36 (m, 4H), 8, 19 (s, 2H), 7.78 (d, J = 12.8Hz, 2H), 725 (s, 2H), 4.38 (d, J = 13.2Hz ， 2H), 4.18(s, 2H).LCMS[ESI, M+1]: 729.2

[0348] A procedure similar to that used to synthesize compound 7 was used to synthesize compounds 124, 132, 143, 149, 151, and 155.

[0349] Example 8

[0350] Scheme 13: Synthesis of Compound 8:

[0351]

[0352] Step 1: Synthesis of methyl 2-(bis(tert-butoxycarbonyl)amino)-4-bromo-5-fluorobenzoate: Di-tert-butyl dicarbonate (1.11 mL, 4.84 mmol, 1 equivalent) and DMAP (12 mg, 0.40 mmol, 0.1 equivalent) were added to a stirred solution of methyl 2-amino-4-bromo-5-fluorobenzoate (1.0 g, 4.03 mmol, 1 equivalent) in THF (10 mL) at 0 °C. The reaction mixture was stirred at 70 °C for 4 hours. After the reaction was complete, the solvent was removed under reduced pressure, diluted with water (100 mL), and extracted with ethyl acetate (3 × 300 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to give the crude product. The crude material was then purified by rapid chromatography with a gradient of 2 to 3% ethyl acetate in petroleum ether to give methyl 2-(bis(tert-butoxycarbonyl)amino)-4-bromo-5-fluorobenzoate (1.4 g, 74% yield) as a grayish-white solid. MS-ESI: m / z 470.54 observed [M+Na] + .

[0353] Step 2: Synthesis of methyl 2-(bis(tert-butoxycarbonyl)amino)-5-fluoro-4-vinylbenzoate: Vinyltributyltinane (6.61 g, 20.86 mmol, 1.1 equivalent) was added to a stirred solution of methyl 2-(bis(tert-butoxycarbonyl)amino)-4-bromo-5-fluorobenzoate (8.5 g, 18.96 mmol, 1 equivalent) in toluene (85 mL). The resulting mixture was deoxygenated by purging with argon for 15 min, followed by the addition of Pd(PPh3)4 (0.44 g, 0.38 mmol, 0.02 equivalent), and the mixture was stirred at 110 °C for 16 h. After the reaction was complete, the reaction mixture was concentrated under reduced pressure, diluted with water (100 mL), and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure to give the crude product. The crude residue was then purified by rapid chromatography with a gradient of 2 to 3% EtOAc in petroleum ether to give methyl 2-(bis(tert-butoxycarbonyl)amino)-5-fluoro-4-vinylbenzoate (5.6 g, 75% yield) as a pale yellow solid. MS-ESI: m / z 418.21 observed [M+Na] + .

[0354] Step 3: Synthesis of methyl 2-(bis(tert-butoxycarbonyl)amino)-5-fluoro-4-carboxymethyl benzoate: Ozone gas was purged from a stirred solution of methyl 2-(bis(tert-butoxycarbonyl)amino)-5-fluoro-4-vinylbenzoate (5.6 g, 14.16 mmol, 1 equivalent) in MeOH (14 mL) and DCM (42 mL) for 45 min at room temperature. After the reaction was complete, the reaction mixture was concentrated under reduced pressure to give methyl 2-(bis(tert-butoxycarbonyl)amino)-5-fluoro-4-carboxymethyl benzoate (4.7 g, 89% yield) as a grayish-white solid. MS-ESI: m / z 420.18 observed [M+Na] + .

[0355] Step 4: Synthesis of methyl 4-(((4-((l1-oxoalkyl)carbonyl)-5-(bis(tert-butoxycarbonyl)amino)-2-fluorobenzyl)(methyl)amino)methyl)-2-(bis(tert-butoxycarbonyl)amino)-5-fluorobenzoate: Methylamine hydrochloride (0.17 g, 2.52 mmol, 1 equivalent) was added to a stirred solution of methyl 2-(bis(tert-butoxycarbonyl)amino)-5-fluoro-4-carboxymethyl benzoate (2.0 g, 5.03 mmol, 2 equivalents) in DCM (20 mL) at 0 °C, followed by the addition of STAB (2.13 g, 10.07 mmol, 4.0 equivalents), and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was diluted with water (50 mL) and extracted with DCM (3 × 70 mL). The combined organic layers were dried over anhydrous Na2SO4 and evaporated under reduced pressure to give the crude product. The crude residue was then purified by rapid chromatography with a gradient of 25 to 30% EtOAc in petroleum ether to give methyl 4-(((4-((l1-oxoalkyl)carbonyl)-5-(bis(tert-butoxycarbonyl)amino)-2-fluorobenzyl)(methyl)amino)methyl)-2-(bis(tert-butoxycarbonyl)amino)-5-fluorobenzoate (0.65 g, 33% yield) as a colorless gel. MS-ESI: m / z 794.65, observed [M+H]+.

[0356] Step 5: Synthesis of dimethyl 4,4′-((methylazonidyl)bis(methylene))bis(2-amino-5-fluorobenzoate): TFA (3 mL) was added to a stirred solution of methyl 4-(((4-((l1-oxoalkyl)carbonyl)-5-(bis(tert-butoxycarbonyl)amino)-2-fluorobenzyl)(methyl)amino)methyl)-2-(bis(tert-butoxycarbonyl)amino)-5-fluorobenzoate (0.65 g, 0.82 mmol, 1 equivalent) in DCM (3 mL) at 0 °C, and the reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was concentrated under reduced pressure to give the crude product. The crude residue was then purified by rapid chromatography with a gradient of 25 to 30% EtOAc in petroleum ether to give dimethyl 4,4′-((methylazonidyl)bis(methylene))bis(2-amino-5-fluorobenzoate) as a light brown gel (0.3 g, 96% yield). MS-ESI: m / z 380.08 observed [M+H] + .

[0357] Step 6: Synthesis of dimethyl 4,4′-((methylazinidinediyl)bis(methylene))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoate: 6-(1H-imidazol-1-yl)pyridazin-3-carbonyl chloride (0.48 g, 2.29 mmol, 3.0 equivalent) was added to a stirred solution of DIPEA (1.06 mL, 6.10 mmol, 8.0 equivalent) and dimethyl 4,4′-((methylazinidinediyl)bis(methylene))bis(2-amino-5-fluorobenzoate) (0.3 g, 0.76 mmol, 1.0 equivalent) in ACN (3 mL), and the mixture was stirred at 80 °C for 2 hours. After the reaction was complete, the reaction mixture was diluted with water (50 mL), the precipitate was filtered off, and dried under vacuum. The crude product was then purified by rapid chromatography with a gradient of 2 to 5% MeOH in DCM to give dimethyl 4,4′-((methylazonidyl)bis(methylene))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoate as a grayish-white solid (115 mg, 12% yield). MS-ESI: m / z 738.70 observed [M+H] + .

[0358] Step 7: Synthesis of 4,4′-((methylazinidinediyl)bis(methylene))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoic acid (8): Et3N (0.38 mL, 2.71 mmol, 20 equivalences) was added to a stirred solution of dimethyl 4,4′-((methylazinidinediyl)bis(methylene))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-fluorobenzoic acid ester (100 mg, 0.14 mmol, 1.0 equivalences) in ACN (1 mL) and H2O (1 mL), and the mixture was heated at 120 °C for 1 hour using a microwave reactor. After the reaction was complete, the reaction mixture was concentrated under reduced pressure, and the crude residue was purified by preparative HPLC to give compound 8 (40 mg, 40% yield) as a grayish-white solid.

[0359] 1 ¹H NMR (400 MHz, DMSO-d⁶) δ 15.70 (s, 2H), 8.85 (d, J = 7.0 Hz, 2H), 8.77 (s, 2H), 8.45–8.30 (m, 4H), 8.18 (s, 2H), 7.71 (d, J = 10.8 Hz, 2H), 7.25 (s, 2H), 366 (s, 4H), 2.20 (s, 3H). MS-ESI: m / z 710.47 observed [M+H]. + .

[0360] A procedure similar to that used to synthesize compound 8 was used to synthesize compounds 235 and 236.

[0361] Example 9

[0362] Scheme 14: Synthesis of Compound 9:

[0363]

[0364] Step 1: Synthesis of methyl 2-amino-4-[4-(3-amino-2,6-difluoro-4-methoxycarbonyl-phenoxy)butoxy]-3,5-difluorobenzoate: K₂CO₃ (1.63 g, 11.8 mmol, 2.00 equivalent) was added to a solution of 1,4-dibromobutane (425 mg, 1.97 mmol, 238 oz, 1.00 equivalent) and methyl 2-amino-3,5-difluoro-4-hydroxybenzoate (800 mg, 3.94 mmol, 2.00 equivalent) in DMF (12.0 mL). After stirring at 50 °C for 3 hours, the reaction mixture was diluted with ethyl acetate (80.0 mL), washed with water (80 mL × 3), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to give the crude product. The crude material was purified by silica gel column chromatography to give methyl 2-amino-4-[4-(3-amino-2,6-difluoro-4-methoxycarbonyl-phenoxy)butoxy]-3,5-difluorobenzoate (756 mg, 83% yield) as a white solid.

[0365] 1 H NMR (400MHz, DMSO-d6) δ7.36 (dd, J=2.0, 12.4Hz, 2H), 6.47 (s, 4H), 4.38-4.17 (m, 4H), 3.80 (s, 6H), 1.88-1.81 (m, 4H). LCMS (ESI): m / z 461.1[M+H] + .

[0366] Step 2: Synthesis of 2-amino-4-[4-(3-amino-4-carboxy-2,6-difluoro-phenoxy)butoxy]+3,5-difluoro-benzoic acid: LiOH·H2O (274 mg, 6.52 mmol, 10.0 equivalent) was added to a solution of methyl 2-amino-4-[4-(3-amino-2,6-difluoro-4-methoxycarbonyl-phenoxy)butoxy]-3,5-difluoro-benzoate (300 mg, 0.652 mmol, 1.00 equivalent) in THF (1.50 mL), H2O (1.50 mL), and MeOH (1.50 mL). The mixture was stirred at 25 °C for 1 hour. The reaction mixture was quenched with a solution of HCl (0.1 N) at 0 °C to pH 7. The precipitate was filtered to give a white solid. The crude product was ground with ACN at 25°C to obtain 2-amino-4-[4-(3-amino-4-carboxy-2,6-difluoro-phenoxy)butoxy]-3,5-difluoro-benzoic acid (275 mg, crude product) as a white solid.

[0367] 1H NMR (400MHz, DMSO-d6) δ = 7.37 (dd, J = 2.0, 12.4Hz, 2H), 6.56 (br s, 4H), 4.12 (br s, 4H), 1.83 (br s, 4H), LCMS (ESI): m / z 433.1 [M+H] + .

[0368] Step 3: 7,7′-(butane-1,4-diylbis(oxy))bis(2-(6-(1H-imidazol-1-yl)pyridazin-3-yl)-6,8-difluoro-4H-benzo[d][1,3] Synthesis of azinon-4-one (3): DIPEA (419 mg, 3.24 mmol, 0.564 mL, 10.0 equivalent) and T3P (1.24 g, 1.94 mmol, 1.16 mL, 50% purity in ethyl acetate, 6.00 equivalent) were added to a solution of compound B (308 mg, 1.62 mmol, 5.00 equivalent) in DCE (8.00 mL). The mixture was stirred at 80 °C for 8 hours. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was washed with saturated NaHCO3 (5 mL) and water (4 mL) to give a gray solid. The crude product was ground with ACN at 25°C for 5 minutes, then filtered, and the filter cake was dried under vacuum to give compound 9 as a yellow solid (73.6 mg, 31% yield in two steps).

[0369] 1 ¹H NMR (400MHz, DMSO-d⁶) δ 8.79 (s, 2H), 8.64 (d, J = 9.2Hz, 2H), 8.42 (d, J = 9.2Hz, 2H), 8.19 (s, 2H), 7.98 (dd, J = 1.2, 10.4Hz, 2H), 7.26 (s, 2H), 4.52 (br s, 4H), 1.99 (br s, 4H). MS-ESI.mz 741.3 Observed [M+H] + .

[0370] A procedure similar to that used to synthesize compound 9 was used to synthesize compounds 40, 41, and 46.

[0371] Example 10

[0372] Scheme 15: Synthesis of compound 10-Li:

[0373]

[0374] Step 1: Synthesis of methyl 4-fluoro-5-(3-hydroxypropoxy)-2-nitrobenzene: K₂CP₃ (2.56 g, 1.86 mmol, 2 equivalents) and 3-bromopropane-1-ol (1.55 g, 1.12 mmol, 1.2 equivalents) were added to a solution of methyl 4-fluoro-5-hydroxy-2-nitrobenzene (2 g, 9.30 mmol, 1 equivalent) in DMF (20 mL) at room temperature. The resulting solution was stirred at 80 °C for 2 hours. After the reaction was complete, the reaction mixture was cooled at room temperature and diluted with water (50 mL). The aqueous layer was extracted with ethyl acetate (2 × 100 mL), and the combined organic layers were dried over anhydrous Na₂SO₄ and evaporated under reduced pressure to give the crude product. The crude material was purified by silica gel column chromatography using 30% ethyl acetate in hexane as the eluent to give pure methyl 4-fluoro-5-(3-hydroxypropoxy)-2-nitrobenzene as a solid (1.8 g, 71% yield).

[0375] 1 ¹H NMR (400MHz, DMSO-d⁶) δ 8.19 (d, J = 10.8 Hz, ¹H), 7.62 (d, J = 8.0 Hz, ¹H), 4.65 (t, J = 5.2 Hz, ¹H), 4.32 (t, J = 6.3 Hz, 2H), 3.87 (s, 3H), 3.59 (d, J = 5.9 Hz, 2H), 1.93 (p, J = 6.3 Hz, 2H). MS-ESI: m / z 273.0 Observed [M+H] +

[0376] Step 2: Methyl 5-(3-bromopropoxy)-4-fluoro-2-nitrobenzene: CBr4 (1.10 g, 9.89 mmol, 1.5 equivalent) and PPh3 (2.59 g, 9.89 mmol, 1.5 equivalent) were added to a solution of methyl 4-fluoro-5-(3-hydroxypropoxy)-2-nitrobenzene (1.80 g, 6.59 mmol, 1 equivalent) in DCM (18 mL) at room temperature. The resulting solution was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was diluted with water (50 mL). The aqueous layer was extracted with ethyl acetate (2 × 100 mL), and the combined organic layers were dried over anhydrous Na2SO4 and evaporated to give the crude product. The crude material was purified by silica gel column chromatography using 5% ethyl acetate in hexane as the eluent to give pure methyl 5-(3-bromopropoxy)-4-fluoro-2-nitrobenzoate as a solid (1 g, 45% yield).

[0377] 1¹H NMR (400MHz, DMSO-d⁶) δ 8.22 (dd, J = 10.8, 3.5Hz, 1H), 7.67 (d, J = 8.0Hz, 1H), 4.37 (t, J = 5.9Hz, 2H), 3.87 (s, 3H), 3.67 (t, J = 6.5Hz, 2H), 2.33 (s, J = 5.9Hz, 2H). MS-ESI: m / z 336.0 observed [M+H]. + .

[0378] Step 3: Synthesis of methyl 4-fluoro-5-(3-(2-fluoro-4-(methoxycarbonyl)-5-nitrophenoxy)propoxy)-2-nitrobenzoate: K₂CO₃ (1.28 g, 2.97 mmol, 2 equivalents) and methyl 5-(3-bromopropoxy)-4-fluoro-2-nitrobenzoate (0.5 g, 1.48 mmol, 1 equivalent) were added to a solution of methyl 5-fluoro-4-hydroxy-2-nitrobenzoate (0.384 g, 1.78 mmol, 1.2 equivalents) in ACN (5 mL) at room temperature. The resulting solution was stirred at 80 °C for 16 hours. After the reaction was complete, the reaction mixture was cooled to room temperature and diluted with water (25 mL). The aqueous layer was extracted with ethyl acetate (2 × 30 mL), and the combined organic layers were dried over anhydrous Na₂SO₄ and evaporated to give the crude product. The crude material was purified by silica gel column chromatography using 15% ethyl acetate in hexane as eluent to give pure methyl 4-fluoro-5-(3-(2-fluoro-4-(methoxycarbonyl)-5-nitrophenoxy)propoxy)-2-nitrobenzoate as a solid (0.35 g, 50.0% yield).

[0379] 1 ¹H NMR (400 MHz, DMSO-d⁶) δ 8.19 (dd, J = 10.8, 1.3 Hz, 1H), 8.05 (s, 1H), 7.90–7.97 (m, 1H), 7.82 (dd, J = 10.9, 1.3 Hz, 1H), 4.40 (q, J = 6.2 Hz, 4H), 3.84 (dd, J = 12.6, 1.4 Hz, 6H), 2.32 (s, 2H). MS-ESI: [M+H] observed at m / z 470.0. + .

[0380] Step 4: Synthesis of methyl 2-amino-5-(3-(5-amino-2-fluoro-4-(methoxycarbonyl)phenoxy)propoxy)-4-fluorobenzoate: At room temperature, 0.2 g of 10% Pd / C catalyst (50% humidity) was added to a solution of methyl 4-fluoro-5-(3-(2-fluoro-4-(methoxycarbonyl)-5-nitrophenoxy)propoxy)-2-nitrobenzoate (0.35 g, 0.74 mmol, 1 equivalent) in MeOH (7 mL) and THF (7 mL). The reaction mixture was purged with hydrogen for 1 hour. After the reaction was complete, the reaction mixture was filtered through a diatomaceous earth bed and washed with 10% MeOH in DCM solution. The filtrate was concentrated under vacuum to give crude methyl 2-amino-5-(3-(5-amino-2-fluoro-4-(methoxycarbonyl)phenoxy)propoxy)-4-fluorobenzoate (0.30 g, 98.2% yield), which was used directly for the next step without further purification. MS-ESI: m / z 410.0 [M+H] observed + .

[0381] Step 5: Synthesis of methyl 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-(3-(5-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-2-fluoro-4-(methoxycarbonyl)phenoxy)propoxy)-4-fluorobenzoate: At room temperature, a 50% solution of DIPEA (0.755 g, 5.85 mmol, 12 equivalents) and T3P (in ethyl acetate) (1.2 g, 3.902 mmol, 8 equivalents) was added to a stirred solution of intermediate B (0.203 g, 1.073 mmol, 2.2 equivalents) in DCE (3 ml). Methyl 2-amino-5-(3-(5-amino-2-fluoro-4-(methoxycarbonyl)phenoxy)propoxy)-4-fluorobenzoate (0.200 g, 0.487 mmol, 1 equivalent) was added to the mixture at room temperature. The reaction mixture was heated overnight at 80 to 90 °C. After the reaction was complete, the reaction mixture was concentrated directly under vacuum. The crude product was purified by silica gel column chromatography with a gradient of 1.5% to 2% MeOH in DCM to give pure methyl 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-(3-(5-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-2-fluoro-4-(methoxycarbonyl)phenoxy)propoxy)-4-fluorobenzoate (0.15 g, 41% yield) as a solid. MS-ESI: m / z 754.0 observed [M+H] + .

[0382] Step 6: Synthesis of 2-(6-(1H-imidazol-1-yl)pyridazine-3-carboxamido)-5-(3-(5-(6-(1H-imidazol-1-yl)pyridazine-3-carboxamido)-4-carboxy-2-fluorophenoxy)propoxy)-4-fluorobenzoic acid (10): Et3N (0.25 g, 1.98 mmol, 10 equivalents) was added to a solution of methyl 2-(6-(1H-imidazol-1-yl)pyridazine-3-carboxamido)-5-(3-(5-(6-(1H-imidazol-1-yl)pyridazine-3-carboxamido)-2-fluoro-4-(methoxycarbonyl)phenoxy)propoxy)-4-fluorobenzoate (0.15 g, 0.19 mmol, 1 equivalent) in CAN (7.5 mL) and water (7.5 mL) at room temperature. The reaction mixture was heated in a microwave at 120 °C for 5 hours. After the reaction was complete, the reaction mixture was purified directly by preparative HPLC without concentration to give compound 10 as a grayish-white solid (0.050 g, 35% yield). MS-ESI: m / z 726.17 observed [M+H] + .

[0383] Step 7: Synthesis of lithium 2-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-5-(3-(5-(6-(1H-imidazol-1-yl)pyridazin-3-carboxamido)-4-carboxy-2-fluorophenoxy)propoxy)-4-fluorobenzoate (10-Li): LiOH·H₂P (6 mg, 0.14 mmol, 2.1 equivalents) was added to a suspension of compound 10 (0.050 g, 0.07 mmol, 1 equivalent) in water (4 mL), and the resulting clear solution was filtered to remove any insoluble particles. The solution was lyophilized to obtain compound 10-Li (0.045 g).

[0384] 1 ¹H NMR (500MHz, DMSO-d⁶) δ 8.78 (s, 2H), 8.71 (d, J = 8.2Hz, 1H), 8.62 (d, J = 14.1Hz, 1H), 8.51–8.37 (m, 4H), 8.19 (s, 2H), 7.81 (dd, J = 50.9, 11.2Hz, 2H), 7.25 (s, 2H), 4.28 (d, J = 21.7Hz, 4H), 2.36 (s, 2H). MS-ESI: m / z 727.2 Observed [M+H] + .

[0385] A procedure similar to that used to synthesize compound 10 was used to synthesize compounds 26, 27, 31, 33 and 191.

[0386] Example 11: Bioactivity of the compound

[0387] ISRE-luciferase assay. THP-1 Lucia ISG cells were used at 5 × 10⁻⁶. 5 Cells were resuspended at a density of 1,000 cells / ml in low serum growth medium (2% FBS) and treated with either the test product or the carrier (DMSO). 50 μL of cells were seeded into each well of a 384-well white Greiner plate and incubated for 24 h. To evaluate the expression of the luciferase reporter gene, 30 μL of Quanti-luc (Invivogen) assay reagent was added to each well, and luminescence was read using an Envision plate reader (Perkin Elmer) with an integration time set to 0.1 seconds. For each cell type, the luminescence signal of the test product sample was normalized relative to the carrier-treated sample and reported in relative light units (RLU).

[0388] WT STING Binding Assay (Cisbio, Catalog No. 64BDSTGPEH). The assay was optimized to demonstrate the binding of the natural ligand, d2 (acceptor)-labeled 2′3′cGAMP, to recombinant 6×His-labeled human STING protein labeled with Terbium Cryptate. When the two dyes were brought close together, a flash lamp on a Pherastar FSX reader excited the donor, triggering fluorescence resonance energy transfer (FRET) toward the acceptor, which then emitted fluorescence at 665 nm. A competitive assay was used to evaluate the binding ability of the synthesized small-molecule STING ligands to human STING. Ten-point titrations of five volts of each synthetic ligand were transferred to 384-well plates, followed by a 20-volt assay buffer containing 6×His-labeled human STING protein and the labeled 2′3′cGAMP ligand, and incubated at room temperature for 3 hours. Raw values ​​obtained from Pherastar were used to calculate the reported IC50 using curve fitting from Genedata. 50 The value (signal is inversely proportional to the binding of the synthetic ligand). Percentage inhibition is calculated based on the maximum binding of the synthetic compound relative to the maximum binding of unlabeled 2′3′cGAMP, which is used as a control in each assay.

[0389] Table 2 lists the determination results of selected representative compounds of this disclosure. The results are scored as follows:

[0390]

[0391] Table 2. Results of ISRE-Luc and STING combined with HTRF assays.

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein said compound is selected from one of the following: 。 2. The following compound or its pharmaceutically acceptable salt: 。 3. The following compound or its pharmaceutically acceptable salt: 。 4. The following compound or its pharmaceutically acceptable salt: 。 5. The following compound or its pharmaceutically acceptable salt: 。 6. A pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier.

7. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 in a medicament used in a method for stimulating the expression of an interferon gene in a human patient, the method comprising administering the medicament to the patient.

8. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 in a method for preparing a medicament for treating a tumor in a patient, the method comprising administering the medicament to the patient.

9. The use according to claim 7 or 8, wherein administration comprises administering the compound to the patient as an antibody-drug conjugate or in a liposomal formulation.

Images