YU CHENGUANG (US)
WANG JIE (US)
CHATTERJEE ARNAB K (US)
ALBERO ANA MARIA GAMO (US)
GUPTA ANIL (US)
TAMIYA JUNKO (US)
SCHULTZ PETER G (US)
JOHNSON KRISTEN (US)
CHU ALAN (US)
CHIN EMILY (US)
LAIRSON LUKE L (US)
WO2016016728A2 | 2016-02-04 | |||
WO2009058348A1 | 2009-05-07 | |||
WO2019165032A1 | 2019-08-29 |
US20090239982A1 | 2009-09-24 | |||
JPH1143488A | 1999-02-16 | |||
EP0032242A1 | 1981-07-22 | |||
JPS55147279A | 1980-11-17 | |||
EP0987257A1 | 2000-03-22 | |||
EP1820795A1 | 2007-08-22 | |||
GB2563642A | 2018-12-26 | |||
US20170146519A1 | 2017-05-25 | |||
US201962889669P | 2019-08-21 |
LI ET AL: "Preparation of novel antibacterial agents. Replacement of the central aromatic ring with heterocycles", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, PERGAMON, AMSTERDAM, NL, vol. 17, no. 8, 30 March 2007 (2007-03-30), pages 2347 - 2350, XP022009263, ISSN: 0960-894X, DOI: 10.1016/J.BMCL.2006.12.055
OSAMU KIRINO ET AL: "Fungicidal activity of N-benzoylanthranilates and related compounds: Struct ure-activity study of fungicidal N-benzoylanthranilates. Part I", AGRICULTURAL AND BIOLOGICAL CHEMISTRY, AGRICULTURAL CHEMICAL SOCIETY OF JAPAN, JP, vol. 44, no. 9, January 1980 (1980-01-01), pages 2143 - 2147, XP008085362, ISSN: 0002-1369, [retrieved on 20081127], DOI: 10.1271/BBB1961.44.2143
KALUSA A ET AL: "An efficient synthesis of 2,3-diaryl (3H)-quinazolin-4-ones via imidoyl chlorides", TETRAHEDRON LETTERS, ELSEVIER LTD, AMSTERDAM, NL, vol. 49, no. 41, 6 October 2008 (2008-10-06), pages 5840 - 5842, XP024339927, ISSN: 0040-4039, [retrieved on 20080722], DOI: 10.1016/J.TETLET.2008.07.091
FABIS F ET AL: "An expedient route to aromatic pyrrolo[2,1-c][1,4]benzodiazepines and a study of their reactivity", TETRAHEDRON LETTERS, ELSEVIER LTD, AMSTERDAM, NL, vol. 42, no. 31, 30 July 2001 (2001-07-30), pages 5183 - 5185, XP004254109, ISSN: 0040-4039, DOI: 10.1016/S0040-4039(01)00957-1
WANG YI-LIN ET AL: "Synthesis, crystal structures and biological evaluation of three ternary copper(II) complexes with fluorinated anthranilic acid derivatives", TRANSITION METAL CHEMISTRY, CHAPMAN & HALL, GB, vol. 41, no. 8, 21 September 2016 (2016-09-21), pages 897 - 907, XP036080678, ISSN: 0340-4285, [retrieved on 20160921], DOI: 10.1007/S11243-016-0092-X
DANYANG LI ET AL: "Synthesis of Benzyl Esters via Functionalization of Multiple C-H Bonds by Palladium Catalysis", ORGANIC LETTERS, vol. 17, no. 21, 13 October 2015 (2015-10-13), US, pages 5300 - 5303, XP055738057, ISSN: 1523-7060, DOI: 10.1021/acs.orglett.5b02518
DEFILIPPIS ET AL: "Characterization of a novel human-specific STING agonist that elicits antiviral activity against emerging alphaviruses", PLOS PATHOGENS, vol. 11, no. 12, January 2015 (2015-01-01), pages 1 - 30, XP055510920
LIT ET AL.: "Salt Selection for Basic Drugs", INT J. PHARM., vol. 33, 1986, pages 201 - 217
"THE PRACTICE OF MEDICINAL CHEMISTRY", 1996, ACADEMIC PRESS, pages: 203
CORRALES LGLICKMAN LHMCWHIRTER SMKANNE DBSIVICK KEKATIBAH GEWOO SRLEMMENS EBANDA TLEONG JJ: "Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity", CELL REP., vol. 11, 2015, pages 1018 - 30
DENG, L. ET AL.: "STTNG-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors", IMMUNITY, vol. 41, 2014, pages 843, XP055426473, DOI: 10.1016/j.immuni.2014.10.019
CORRALES LMATSON VFLOOD BSPRANGER SGAJEWSKI TF.: "Innate immune signaling and regulation in cancer immunotherapy", CELL RES., vol. 27, 2017, pages 96 - 108, XP055642436, DOI: 10.1038/cr.2016.149
CORRALES LMC HIRTER SMDUBENSKY TW JRGAJEWSKI TF.: "The host STING pathway at the interface of cancer and immunity", J CLIN INVEST., vol. 126, 2016, pages 2404 - 11, XP002790392, DOI: 10.1172/JCI86892
MACKENZIE, K.F.: "cGAS surveillance of micronuclei links genome instability to innate immunity", NATURE, vol. 548, 2017, pages 461
WANG, W. ET AL.: "Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer", CELL, vol. 165, 2016, pages 1092 - 1105, XP029552279, DOI: 10.1016/j.cell.2016.04.009
CHARLOTTE E. ARIYAN ET AL., ROBUST ANTITUMOR RESPONSES RESULT FROM LOCAL CHEMOTHERAPY AND CTLA-4 BLOCKADE, 16 January 2018 (2018-01-16), Retrieved from the Internet
CHUNG KIL SONG, CHEMOTHERAPY ENHANCES CD8+ T CELL-MEDIATED ANTITUMOR IMMUNITY INDUCED BY VACCINATION WITH VACCINIA VIRUS, vol. 15, no. 8, August 2007 (2007-08-01), Retrieved from the Internet
AGER, CR ET AL., CANCER IMMUNOL RES, vol. 5, no. 8, 2017, pages 676
FU, J. ET AL., SET TRANSL MED., vol. 7, no. 283, 15 April 2015 (2015-04-15), pages 283ra52
WANG, H. ET AL., PNAS, vol. 114, no. 7, 14 February 2017 (2017-02-14), pages 1637 - 1642
OUYANG, S.SONG, X.WANG, Y.RU, H.SHAW, N.JIANG, Y.NIU, F.ZHU, Y.QIU, W.PARVATIYAR, K. ET AL.: "Structural analysis of the STING adaptor protein reveals a hydrophobic dimer interface and mode of cyclic di-GMP binding", IMMUNITY, vol. 36, 2012, pages 1073 - 1086, XP028502133, DOI: 10.1016/j.immuni.2012.03.019
"The Science of Synthesis", vol. 1-8, 2001, THIEME PUBLISHERS
WE CLAIM: 1. A compound of formula (IA) or formula (II): wherein X is S, -N=C(R1)-, or -C(R1)=C(R1)-; each R1 is independently H, F, Cl, C1-C6-alkyl, ethenyl or ethynyl (either of which can be substituted), cyano, alkoxyl, or haloalkyl; R2 is selected from the group consisting of -C(O)OR, -C(O)NH(C1-C6-alkyl) (wherein the alkyl is optionally substituted), optionally substituted C3-C6- cycloalkenyl, and 3- to 10-membered heterocyclyl; R is selected from the group consisting of H, alkyl optionally substituted with - (C1-C6-alkyl)OC(O)OC1-C6-alkyl) or 3- to 10-membered hetero -cyclyl, and benzyl, wherein the benzyl can be unsubstituted or substituted with methoxyl or with an acid or ester isostere; Ring A is a 5- or 6-membered heteroaryl comprising 1, 2, or 3 N atoms, unsubstituted or substituted with 1, 2, or 3 groups independently selected from the group consisting of NH2, NH-benzyl (wherein the benzyl is unsubstituted or is substituted with methoxyl, cyano, alkylnitrile, haloalkyl, hydroxymethyl, aminomethyl, aminopropyl, carboxamido, or alkoxy), wherein a wavy line indicates a position of bonding; or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, wherein the compound of formula (IA) is of formula (I): wherein X is S, -N=C(R1)-, or -C(R1)=C(R1)-; each R1 is independently H, F, Cl, ethenyl or ethynyl (either of which can be substituted), cyano, alkoxyl, or haloalkyl; and R is H, alkyl, or benzyl, wherein the benzyl can be unsubstituted or substituted with methoxyl or with an acid or ester isostere. 3. The compound according to claim 1, wherein the compound is of formula (II). 4. The compound according to any one of claims 1 to 3, wherein ring A comprises any one of pyridazinyl, triazolyl, pyrimidinyl, and pyridinyl, any of which can be unsubstituted or substituted. 5. The compound according to claim 1, wherein the compound is one selected from the following table: 6. The compound according to claim 1, wherein the compound is one selected from the following table: 7. A method of stimulating expression of interferon genes in a human patient, comprising administering to the patient an effective dose of a compound or pharmaceutically acceptable salt therefore according to any one of claims 1 to 6. 8. A method of treating a tumor in a patient, comprising administering to the patient an effective dose of a compound or pharmaceutically acceptable salt therefore according to any one of claims 1 to 6. 9. The method according to claim 7 or 8, wherein the administering comprises oral or intratumoral administration, or both. 10. The method according to claim 7 or 8, wherein administering comprises administering the compound to the patient as an antibody-drug conjugate or in a liposomal formulation. 11. The method according to claim 7 or 8, further comprising administering an effective dose of an immune-checkpoint targeting drug. 12. The method according to claim 11, wherein the immune-checkpoint targeting drug comprises an anti-PD-L1 antibody, anti-PD-1 antibody, anti- CTLA-4 antibody, or an anti-4-1BB antibody. 13. The method according to claim 7 or 8, further comprising administering ionizing radiation or anticancer drugs. 14. A pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. 15. A compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 for use in a method of stimulating expression of interferon genes in a human patient. 16. A compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 for use in a method of treating a tumor in a patient. 17. The compound for use according to claim 15 or 16, wherein the compound is administered to the patient by oral or intratumoral administration, or both. |
and the like, wherein R has the same definition as R A as defined herein. See, e.g., THE PRACTICE OF MEDICINAL CHEMISTRY (Academic Press: New York, 1996), at page 203. [0040] Some compounds described herein can have asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms. A compound as described herein can be in the form of an optical isomer or a diastereomer. Accordingly, the disclosure encompasses compounds and their uses as described herein in the form of their optical isomers, diastereoisomers and mixtures thereof, including a racemic mixture. Optical isomers of the compounds of the disclosure can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, simulated moving bed technology or via chemical separation of stereoisomers through the employment of optically active resolving agents. [0041] Unless otherwise indicated, the term "stereoisomer" means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. Thus, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises greater 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 greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound, or greater than about 99% by weight of one stereoisomer of the compound and less than about 1% by weight of the other stereoisomers of the compound. The stereoisomer as described above can be viewed as composition comprising two stereoisomers that are present in their respective weight percentages described herein. [0042] If there is a discrepancy between a depicted structure and a name given to that structure, then the depicted structure controls. Additionally, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it. In some cases, however, where more than one chiral center exists, the structures and names may be represented as single enantiomers to help describe the relative stereochemistry. Those skilled in the art of organic synthesis will know if the compounds are prepared as single enantiomers from the methods used to prepare them. [0043] As used herein, and unless otherwise specified to the contrary, the term "compound" is inclusive in that it encompasses a compound or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof. Thus, for instance, a compound of formula (I), formula (IA), or formula (II) includes a pharmaceutically acceptable salt of a tautomer of the compound. [0044] COMPOUNDS [0045] The present disclosure provides in various embodiments a compound of formula (IA) or formula (II), or a pharmaceutically acceptable salt thereof: [0046] In formula (IA), X is S, -N=C(R 1 )-, or -C(R 1 )=C(R 1 )-. [0047] In some embodiments, the compound is a compound of formula (IA). In other embodiments, the compound is a compound of formula (II). [0048] The present disclosure provides in various embodiments, optionally in combination with any other embodiment described herein, a compound of formula (IA) that is a compound of formula (I) or a pharmaceutically acceptable salt thereof: [0049] X is S, -N=C(R 1 )-, or -C(R 1 )=C(R 1 )-. Each R 1 is independently H, F, Cl, C 1 -C 6 -alkyl, ethenyl or ethynyl (either of which can be substituted), cyano, alkoxyl, or haloalkyl. [0050] R 2 is selected from the group consisting of -C(O)OR, -C(O)NH(C 1 -C 6 - alkyl) (wherein the alkyl is optionally substituted), optionally substituted C 3 -C 6 - cycloalkenyl, and 3- to 10-membered heterocyclyl. For example, in some embodiments optionally in combination with any other embodiment described herein, R 2 is -C(O)OR. [0051] R is selected from the group consisting of H, alkyl optionally substituted with "((C 1 -C 6 -alkyl)OC(O)OC 1 -C 6 -alkyl) or 3- to 10-membered heterocyclyl, and benzyl, wherein the benzyl can be unsubstituted or substituted with methoxyl or with an acid or ester isostere. In various embodiments, R is H, alkyl, or benzyl, wherein the benzyl can be unsubstituted or substituted with methoxyl or with an acid or ester isostere. [0052] Ring A is a 5- or 6-membered heteroaryl comprising 1, 2, or 3 N atoms, unsubstituted or substituted with 1, 2, or 3 groups independently selected from the group consisting of NH 2 , NH-benzyl (wherein the benzyl is unsubstituted or is substituted with methoxyl, cyano, alkylnitrile, haloalkyl, hydroxymethyl, aminomethyl, aminopropyl, carboxamido, or alkoxy), wherein a wavy line indicates a position of bonding. [0053] In various embodiments, ring A comprises any one of pyridazinyl, triazolyl, pyrimidinyl, and pyridinyl, any of which can be unsubstituted or substituted as described herein. [0054] In further embodiments, the present disclosure provides specific examples of compounds, and their pharmaceutically acceptable salts, as set forth in Table 1 below. The compounds are presented with activity scores deriving, in part, from an ISG-LUC activation assay as described herein, and physico- chemical characterizing data. [0055] Table 1: Specific Compounds and Activity Scores. Activity scores are based upon potency and efficacy data (+ = EC 50 > 20,000 nM; ++ = active but less potent and efficacious than reference compound ( EC 50 > 1000 nM); +++ = activity comparable to reference compound (EC 50 < 3000 nM); ++++ = more potent and/or efficacious than reference compound (EC 50 < 900 nM)).
[0056] Related documents [1] Corrales L, Glickman LH, McWhirter SM, Kanne DB, Sivick KE, Katibah GE, Woo SR, Lemmens E, 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. [2] Deng, L. et al. (2014) STING-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors, Immunity.41: 843. [3] Corrales L, Matson V, Flood B, Spranger S, Gajewski TF. (2017) Innate immune signaling and regulation in cancer immunotherapy. Cell Res.27: 96-108. [4] Corrales L, McWhirter SM, Dubensky TW Jr, Gajewski TF. (2016) The host STING pathway at the interface of cancer and immunity. J Clin Invest.126: 2404-11. [0057] METHODS OF USE [0058] The present disclosure also provides in an embodiment a method of stimulating expression of interferon genes in a human patient. The method comprises administering to the patient an effective dose of a compound or pharmaceutically acceptable salt thereof as described herein. [0059] In another embodiment, the present disclosure provides a method of treating a tumor in a patient. The method comprises administering to the patient an effective dose of a compound or pharmaceutically acceptable salt thereof. [0060] With respect to combination therapies comprising administration of a compound of the present disclosure and an immune-checkpoint targeting drug, or as combination therapies for the potentiation of ionizing radiation-based and existing chemotherapies therapeutic approaches, such as DNA-damage-based chemotherapies, the STING agonists of the present disclosure can complement and potentiate the effects of these known therapeutic approaches. This is based on recent papers indicating the critical role of STING-dependent micronuclei- mediated tumor clearance using these approaches, see for example: [5] Mackenzie, K.F., et all, (2017), cGAS surveillance of micronuclei links genome instability to innate immunity, Nature, 548, 461. [6] Wang, W. et al., (2016), Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer, Cell, 165, 1092"1105. [7] Charlotte E. Ariyan, et al., January 16, 2018; DOI: 10.1158/2326-6066, Robust antitumor responses result from local chemotherapy and CTLA-4 blockade, cancerimmunolres.aacrjournals.org on January 31, 2018. [8] Chung Kil Song, et al., www.moleculartherapy.org vol.15 no.8 aug. 2007, Chemotherapy Enhances CD8+ T Cell-mediated Antitumor Immunity Induced by Vaccination With Vaccinia Virus. [0061] Compounds of the present disclosure can be used in therapeutic combinations with administration of an effective dose of an immune-checkpoint targeting drug. For example, the immune-checkpoint targeting drug can be an anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, or an anti-4- 1BB antibody. See, for example: [9] Ager, CR, et al., (2017) Cancer Immunol Res; 5(8), 676. [10] Fu, J. et al. (2015) Sci Transl Med.2015 April 15; 7(283): 283ra52. doi:10.1126/scitranslmed.aaa4306. [11] Wang, H., et al. (2017) PNAS, February 14, 2017, vol.114, no.7, 1637"1642. [0062] PHARMACEUTICAL COMPOSITION [0063] The present disclosure provides in another embodiment a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof as described herein in combination with a pharmaceutically acceptable carrier or excipient. [0064] Compositions of the present disclosure can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. [0065] Suitable oral compositions as described herein include without limitation tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, syrups or elixirs. [0066] The compositions of the present disclosure that are suitable for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions. For instance, liquid formulations of the compounds of the present disclosure contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically palatable preparations of the compound or a pharmaceutically acceptable salt thereof. [0067] For tablet compositions, the compound or a pharmaceutically acceptable salt thereof in admixture with non-toxic pharmaceutically acceptable excipients is used for the manufacture of tablets. Examples of such excipients include without limitation inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known coating techniques to delay disintegration and absorption in the gastrointestinal tract and thereby to provide a sustained therapeutic action over a desired time period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. [0068] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. [0069] For aqueous suspensions, the compound or a pharmaceutically acceptable salt thereof is admixed with excipients suitable for maintaining a stable suspension. Examples of such excipients include without limitation are sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia. [0070] Oral suspensions can also contain dispersing or wetting agents, such as naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin. [0071] Oily suspensions may be formulated by suspending the compound or a pharmaceutically acceptable salt thereof in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. [0072] Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid. [0073] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the compound or a pharmaceutically acceptable salt thereof in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. [0074] Pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monoleate, and condensation reaction products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monoleate. The emulsions may also contain sweetening and flavoring agents. [0075] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable, an aqueous suspension or an oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer!s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. [0076] The compound the compound or a pharmaceutically acceptable salt thereof may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the compound with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the compound. Exemplary excipients include cocoa butter and polyethylene glycols. [0077] Compositions for parenteral administrations are administered in a sterile medium. Depending on the vehicle used and concentration the concentration of the compound or a pharmaceutically acceptable salt thereof in the formulation, the parenteral formulation can either be a suspension or a solution containing dissolved compound. Adjuvants such as local anesthetics, preservatives and buffering agents can also be added to parenteral compositions. [0078] EXAMPLES [0079] The following non-limiting examples are additional embodiments for illustrating the present disclosure. [0080] Tissue culture. Wild-type (cat. no. thpl-isg) and STING KO (cat. no. thpd-kostg) THP-1-Lucia ISG cells were purchased from Invivogen and maintained in growth media consisting of RPMI 1640, 2mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum (FBS), 1,000 units/ml penicillin, 1,000 mg/ml streptomycin, 0.25 mg/ml Amphotericin B, and 100 mg/ml zeocin unless otherwise stated. [0081] Type 1 interferon stimuli. Poly(dA:dT) and 2'3 -cGAMP were purchased from invivogen and resuspended according to manufacturer!s instructions. [0082] ISRE-luciferase assay. THP-1 Lucia ISG cells were resuspended in low-serum growth media (2% FBS) at a density of 5 x 10 5 cells/ml and treated with test article or vehicle (DMSO).50 mL of cells were seeded into each well of a 384-well white greiner plates and incubated for 24 hours. To evaluate expression of the luciferase reporter, 30 ml of Quanti-luc (Invivogen) detection reagent was added to each well and luminescence was read using an Envision plate reader (Perkin Elmer) set with an integration time of 0.1 seconds. [0083] Viability assay. Cells were resuspended in low-serum growth media at a density of 5 x 10 5 cells/ml and treated with test article or vehicle (DMSO).50 mL of cells were seeded into each well of a 384-well white greiner plates and incubated for 24 hours. To evaluate expression of the luciferase reporter, 30 ml of CellTiter-Glo (Promega) detection reagent was added to each well and luminescence was detected \using an Envision Plate Reader set with an integration time of 0.1 seconds. [0084] Western Blot. Cells were solubilized in 1X protein lysis buffer (25 mM HEPES, pH 7.4, 300 mM NaCl, 1.5 mM MgCl 2 , 1 mM EGTA, 1% P-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate) with freshly added protease and phosphatase inhibitors (Cell Signaling). Western blotting was performed using Bolt TM 4-12% Bis-Tris gels and Bolt TM mini transfer system following the manufacturer!s instructions (ThermoFisher Scientific). STING and m-tubulin antibodies were purchased from Cell Signaling diluted in 5% BSA, 1X TBS-T buffer (Table 3). Anti-rabbit HRP antibody was diluted in 5% non-fat dried milk, 1X TBS-T buffer and luminescence signal was imaged using a ChemiDoc Imager (BioRad). [0085] Semi-quantitative real-time PCR (qPCR). THP-1 cells were resuspended in low-serum growth media at a density of 5 x 10 5 cells/ml and treated with test article or vehicle (DMSO).2.5 mL of cells were seeded into each well of a 6-well plate and incubated for 24 hours. RNA was isolated using an RNeasy Plus Mini Kit (Qiagen) and 1 mg of purified RNA was reverse- transcribed into cDNA (VILO, cat. no.11755050, ThermoFisher Scientific). Gene expression was assessed using Taqman primers and probes listed in Table 4 with the Taqman Universal Mix II (cat. no.4440038, ThermoFisher) following manufacturer's instructions. Gene expression was normalized using the double delta Ct method and was reported as fold change in expression. [0086] STING Thermal Shift Assay (TSA). The c-terminal domains (CTD) of human and mouse STING were expressed and purified as detailed previously (Ouyang, S., Song, X., Wang, Y., Ru, H., Shaw, N., Jiang, Y., Niu, F., Zhu, Y., Qiu, W., Parvatiyar, K., et al. (2012). Structural analysis of the STING adaptor protein reveals a hydrophobic dimer interface and mode of cyclic di-GMP binding. Immunity 36, 1073-1086.). Test article or vehicle controls were added to diluted STING protein (0.22 mg/ml) in 1X Protein Thermal Shift Buffer provided in the Protein Thermal Shift Dye Kit (cat * 4461146, ThermoFisher Scientific). Thermal Shift dye was added and mixed prior to performing a melt curve following parameters outlined for the Dye kit. Melt temperatures (Tm) were calculated using the Derivative method using Protein Thermal Shift Software v1.3 (cat * 4466038, ThermoFisher Scientific). [0087] WT STING binding assay (Cisbio, Catalog " 64BDSTGPEH). An assay format was optimized to demonstrate binding of recombinant 6x His- tagged human STING protein labeled with Terbium Cryptate by the natural ligand, 2'3 cGAMP labeled with d2 (the acceptor). Upon proximity of the two dyes, the excitation of the donor by the flash lamp on the PHERAstar FSX plate reader triggers a Fluorescence Resonance Energy Transfer (FRET) towards the acceptor, which in turn fluoresces at 665 nm. To assess the ability of the synthetic small molecule STING ligands to bind to human STING, a competitive assay format was applied. A 10-point titration of each of the synthetic ligands in 5uL were transferred into a 384 well plate, followed by 20uL of assay buffer containing the 6x His-tagged human STING protein and labeled 2'3 cGAMP ligand and incubated for three hours at room temperature. The raw values obtained from the PHERAstar were used to calculate the reported IC50 values (the signal is inversely proportional to the binding of the synthetic ligand) through curve fitting in Genedata. The percent inhibition was calculated based upon the maximal amount of binding by synthetic compound versus the maximum binding of unlabeled 2'3 cGAMP which was used as a control in each assay. [0088] Table 2: Cell Signaling Antibodies [0089] Table 3: ThermoFisher Scientific Taqman Primers/Probe [0090] Compounds useful for carrying out a method of the present disclosure can be prepared according to the following procedures in conjunction with ordinary knowledge and skill in organic synthesis, substituting appropriate reagents as apparent to the practitioner. [0091] Experimental Procedures [0092] Abbreviations. The following abbreviations are used: tetrahydrofuran (THF), dichloromethane (DCM), N,N-dimethylformamide (DMF), dimethylacetamide (DMA), dimethylsulfoxide (DMSO), trifluoroacetic acid (TFA), triethylamine (TEA), diisopropylethylamine (DIPEA), (1-Cyano-2- ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbe nium hexafluorophosphate (COMU), 1-[bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, N-[(dimethylamino)-1H- 1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethana minium hexafluorophosphate N-oxide (HATU). [0093] General Examples for the Preparation of Compounds of the Present disclosure. The starting materials and intermediates for the compounds of this present disclosure may be prepared by the application or adaptation of the methods described below, their obvious chemical equivalents, or, for example, as described in literature such as The Science of Synthesis, Volumes 1-8. Editors E. M. Carreira et al. Thieme publishers (2001-2008). Details of reagent and reaction options are also available by structure and reaction searches using commercial computer search engines such as Scifinder (www.cas.org) or Reaxys (www.reaxys.com). [0094] PART I: PREPARATION OF INTERMEDIATES [0095] Scheme 1: synthesis of Intermediate-A: [0096] Step 1: Synthesis of ethyl 6-(pyridin-4-yl) pyridazine-3-carboxylate: To a argon-purged solution of ethyl 6-chloropyridazine-3-carboxylate (4.0 g, 21.4 mmol) was added 4-(tributylstannyl)pyridine (8.71 g, 23.65 mmol) in 1,4- dioxane (40 mL) and the resulting mixture was stirred at room temperature for 10 min before Pd(PPh3)4 (2.48 g, 2.15 mmol) was added. The reaction mixture was stirred at 110 o C for 16 hours. After completion, the reaction mixture was diluted with saturated aq. solution of NaHCO 3 (50 mL) solution and extracted with EtOAc (30 mL x 3), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure. The obtained residue was purified by column chromatography to afford ethyl 6-(pyridin-4-yl)pyridazine-3-carboxylate (2.5 g, 46% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6): d 8.84 (m, 2H), 8.58 (d, J = 8.8 Hz, 1H), 8.38 (d, J = 8.8 Hz, 1H), 8.21 (m, 2H), 4.48 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H). LC-MS (ESI+): m/z; 230.14 [M+H] + . [0097] Step 2: Synthesis of 6-(pyridin-4-yl) pyridazine-3-carboxylic acid (A): An aqueous solution of lithium hydroxide monohydrate (0.55 g, 13.1 mmol) in water (10 mL) was added to a solution of ethyl 6-(pyridin-4-yl)pyridazine-3- carboxylate (2.5 g, 10.9 mmol) in THF (10 mL) at 0 o C and the resulting mixture was stirred at room temperature for 5 hours. MeOH (10 mL) was added and the mixture was stirred at 60 o C for 1 h. After completion of the reaction, THF and MeOH were removed under reduced pressure and the aqueous layer was acidified with 2N HCl (pH-4). The obtained solid was filtered, washed with water and dried. Then, it was triturated with acetonitrile, filtered and the filter cake was dried to afford compound A (1.4 g, 53 % yield) as a pale brown solid. 1H NMR (400 MHz, DMSO-d6): d 14.02 (s, 1H), 8.84 (m, 2H), 8.56 (d, J = 8.8 Hz, 1H), 8.36 (d, J = 8.8 Hz, 1H), 8.21 (m, 2H). LC-MS (ESI-): m/z; 200.11 [M- H]-. [0098] Scheme 2: synthesis of Intermediate-B: [0099] Step 1: Synthesis of ethyl 6-(1H-pyrazol-4-yl) pyridazine-3- carboxylate: Argon gas was purged through a solution of pyrazole-4-boronic acid (4.51 g, 40.31 mmol), Na 2 CO 3 (7.1 g, 67.2 mmol) and ethyl 6- chloropyridazine-3-carboxylate (5 g, 26.88 mmol) in 1, 4-dioxane (175 mL) and water (25 mL) for 10 mins before addition of Pd (PPh3)4 (1.55 g, 1.34 mmol). The reaction mixture was stirred at 90 o C for 1 h. After completion of the reaction, it was cooled to room temperature and diluted with EtOAc (250 mL). It was then washed with water (100 mL), brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography over silica gel to afford 3.2 g of ethyl 6- (1H-pyrazol-4-yl) pyridazine-3-carboxylate as an off-white solid. LC-MS (ESI+): m/z; 219.0 [M+H] + . [00100] Step 2: Synthesis of ethyl 6-(1-((2-(trimethylsilyl) ethoxy) methyl)-1H-pyrazol-4-yl)pyridazine-3-carboxylate: [00101] NaH (60% w/w) (0.422 g, 17.6 mmol) was added portion wise 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 o C and stirred for 10 mins. To this was added SEM-Cl (2.93 g, 17.61 mmol) and the reaction mixture was stirred at 0 o C for 30 min. It was then quenched with 10% citric acid solution and the solid thus obtained was filtered, washed with water (5 mL x 2) and dried. The residue was purified by column chromatography over silica gel (using 0-5% Methanol in Dichloromethane as an eluent) to afford 2.65 g of ethyl 6-(1-((2- (trimethylsilyl) ethoxy) methyl)-1H-pyrazol-4-yl)pyridazine-3-carboxylate as off white solid. LC-MS (ESI+): m/z; 349.1 [M-H] + . [00102] Step 3: Synthesis of 6-(1-((2-(trimethylsilyl)ethoxy)methyl)- 1H-pyrazol-4-yl)pyridazine-3-carboxylic acid (B): [00103] An aqueous solution of lithium hydroxide monohydrate (0.382 g, 9.13 mmol, in 3 mL water) was added to a solution of ethyl 6-(1-((2- (trimethylsilyl) ethoxy) methyl)-1H-pyrazol-4-yl)pyridazine-3-carboxylate (2.65 g, 7.61 mmol) in THF (9 mL) at 0 o C and stirred at room temperature for 2 h. After completion of the reaction, the reaction mixture was diluted with water (10 mL) and washed with EtOAc (30 mL x 2). The aqueous layer was acidified using 2N HCl (pH-4) solution and the solid thus obtained was filtered, washed with water (2 mL x 2) and dried to afford 1.1 g of B as an off-white solid. 1 H NMR (400 MHz, DMSO-d 6 ) d 13.62 (s, 1H), 8.78 (s, 1H), 8.33 (s, 1H), 8.18- 8.13 (m, 2H), 5.51 (s, 2H), 3.61 (t, J = 8.0 Hz, 2H), 0.87 (d, J = 8.0 Hz, 2H), 0.04 (s, 9H). LC-MS (ESI+): m/z 321.0 [M+H] + . [00104] Scheme 3: synthesis of Intermediate-C: [00105] Step 1: Synthesis of methyl 6- ((trimethylsilyl)ethynyl)pyridazine-3-carboxylate: To a solution of methyl 6- chloropyridazine-3-carboxylate (1 g, 5.79 mmol) in THF (10 mL) was added ethynyl(trimethyl)silane (4.0 mL, 29.0 mmol), Pd(PPh 3 ) 2 Cl 2 (407 mg, 0.58 mmol), CuI (221 mg, 1.2 mmol) and Et3N (0.807 mL, 5.79 mmol), and the resulting mixture was stirred at 25 °C for 1 hour. After completion of the reaction, the mixture was filtered through a pad of silica-gel and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (PE/EtOAc) to afford methyl 6- ((trimethylsilyl)ethynyl)pyridazine-3-carboxylate (500 mg, 37% yield) as a yellow solid. [00106] Step 2: Synthesis of methyl 6-ethynylpyridazine-3- carboxylate: To a solution of methyl 6-((trimethylsilyl)ethynyl)pyridazine-3- carboxylate (500 mg, 2.13 mmol) in THF (10 mL) was added TBAF (1M in THF, 4.27 mL, 4.27 mmol), the reaction mixture was stirred at room temperature for 1 hour. After completion, the reaction mixture was poured into H 2 O (50 mL) and extracted with DCM (30 mL × 3). The combined organic layers were washed with brine (50 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by column chromatography (PE/EtOAc) to afford methyl 6-ethynylpyridazine-3-carboxylate (260 mg, 75% yield) as a brown solid. [00107] Step 3: Synthesis of methyl 6-(1-(4-methoxybenzyl)-1H-1,2,3- triazol-4-yl)pyridazine-3-carboxylate: To a solution of methyl 6- ethynylpyridazine-3-carboxylate (500 mg, 3.1 mmol) and 1-(azidomethyl)-4- methoxy-benzene (1.0 g, 6.2 mmol) in H 2 O (4 mL) and t-BuOH (16 mL) was added CuSO4 (98.4 mg, 0.62 mmol) and sodium ascorbate (489 mg, 2.5 mmol). The reaction mixture was purged with nitrogen and stirred at 40 °C for 2 h. After completion of the reaction, it was diluted with EtOAc (50 mL) and H 2 O (20 mL). The precipitate was filtered, and the filter cake was washed with DCM/MeOH 10/1 (500 mL). The filtrate was concentrated under reduced pressure to afford methyl 6-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4- yl)pyridazine-3-carboxylate (600 mg, 60 % yield) as a gray solid. LCMS (ESI+): m/z 325.9 [M+H] + . [00108] Step 4: Synthesis of lithium 6-(1-(4-methoxybenzyl)-1H-1,2,3- triazol-4-yl)pyridazine-3-carboxylate (C): To a solution of methyl 6-(1-(4- methoxybenzyl)-1H-1,2,3-triazol-4-yl)pyridazine-3-carboxylat e (250 mg, 0.77 mmol) in THF (2.5 mL) was added a solution of lithium hydroxide monohydrate (96.7 mg, 2.3 mmol) in water (2.5 mL) at 0 °C. After stirred at room temperature for 12 h, the precipitate was filtered, and the filter cake was dried under reduced pressure. The residue was triturated with acetonitrile and filtered to afford the acid C (70.0 mg, 29 % yield) as a gray solid. LCMS (ESI+): m/z 312 [M+H] + . [00109] PART II: PREPARATION OF EXAMPLE COMPOUNDS [00110] All compounds were prepared using the procedures exemplified below. [00111] Example 1: [00112] Scheme 4: Synthesis of Compound 1:
[00113] Step 1: Synthesis of methyl 5-fluoro-2-(6-(pyridin-4-yl) pyridazine-3-carboxamido)-4-((trimethylsilyl) ethynyl) benzoate: [00114] To a solution of intermediate C (1.4 g, 7.0 mmol) and DIPEA (6.17 mL, 34.8 mmol) in DCE (30 mL) was added T3P (50% in EtOAc) (13.29 mL, 20.89 mmol) at room temperature, followed by methyl 2-amino-5-fluoro-4- ((trimethylsilyl)ethynyl)benzoate (1.8 g, 7.0 mmol). The reaction mixture was stirred at 80 o C for 7 h. After completion of reaction, the volatiles were removed under reduced pressure and saturated aq. solution of NaHCO 3 (15 mL) was added. The obtained solid was filtered, washed with water and dried. The residue was purified by column chromatography (PE/EtOAc) to afford methyl 5-fluoro- 2-(6-(pyridin-4-yl) pyridazine-3-carboxamido)-4-((trimethylsilyl) ethynyl) benzoate (2.2 g, 70% yield) as a pale cream solid. 1 H NMR (400 MHz, DMSO- d6): d 12.96 (s, 1H), 8.98 " 8.84 (m, 1H), 8.69 (d, J = 8.8 Hz, 2H), 8.52 (m, 1H), 8.26 (m, 1H), 8.24 (m, 2H), 7.92 (m, 1H), 3.97 (s, 3H), 0.29 (s, 9H). LC-MS (ESI-): m/z; 447.28 [M-H]-. [00115] Step 2: Synthesis of methyl 4-ethynyl-5-fluoro-2-(6-(pyridin- 4-yl) pyridazine-3-carboxamido) benzoate: TBAF (1M in THF) (4.9 mL, 4.9 mmol) was added to a stirring solution of 5-fluoro-2-(6-(pyridin-4-yl) pyridazine-3-carboxamido)-4-((trimethylsilyl) ethynyl) benzoate (2.2 g, 4.90 mmol) in THF (22 mL) at 0 o C and the resulting mixture was stirred at room temperature for 30 min. After completion of the reaction, saturated aq. solution of NaHCO 3 (20 mL) was added. The solid was filtered, washed with water and dried. The obtained residue was purified by column chromatography (DCM/MeOH) to afford methyl 4-ethynyl-5-fluoro-2-(6-(pyridin-4-yl) pyridazine-3-carboxamido) benzoate (1.1 g, 60% yield) as pale orange solid. 1 H NMR (400 MHz, DMSO-d6): d 12.96 (s, 1H), 8.97 (d, J = 6.8 Hz, 1H), 8.86 " 8.84 (m, 2H), 8.68 (d, J = 8.8 Hz, 1H), 8.51 (d, J = 8.8 Hz, 1H), 8.26 " 8.24 (m, 2H), 7.93 (d, J =10.0 Hz, 1H), 4.87 (s, 1H), 3.97 (s, 3H). LCMS: m/z 377.2 [M+H] + . [00116] Step 3: Synthesis of lithium 4-ethynyl-5-fluoro-2-(6-(pyridin- 4-yl) pyridazine-3-carboxamido) benzoate (1): An aqueous solution of lithium hydroxide monohydrate (33.4 mg, 0.8 mmol) in water (2 mL) was added to a solution of methyl 4-ethynyl-5-fluoro-2-(6-(pyridin-4-yl) pyridazine-3- carboxamido) benzoate (200 mg, 0.5 mmol) in THF (4 mL) at 0 o C and the resulting mixture was stirred at room temperature for 2 hours. After completion of the reaction, the obtained solid was filtered, washed with water and dried. Then, it was triturated with acetonitrile, filtered and dried to afford compound 1 as lithium salt (99 mg, 54% yield) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6): d 8.93 (d, J = 6.8 Hz, 1H), 8.85 " 8.83 (m, 2H), 8.61 (d, J = 8.8 Hz, 1H), 8.43 (d, J = 8.8 Hz, 1H), 8.25 " 8.24 (m, 2H), 7.79 (d, J = 10.4 Hz, 1H), 4.52 (s, 1H). LC-MS (ESI+): m/z 363.2 [M+H] + . [00117] Procedures analogous to those for the synthesis of compound 1 were used for the synthesis of compounds 10, 13, 16, 19, 38, 44, 49, 52, 29, 31, 33, 46, 47, 77, 54, 53, 57, 58, 63, 66, 60, 55, 56, 46, 79, 66, 67, 68, 69, 70, 71, 73, 96, 97, 98, 99, 100, 101, 102, 103, 104, 107, 108, 10, 90, 82, 88 and 81 et al.
[00118] Example 2: [00119] Scheme 5: synthesis of compound 2: [00120] Step 1: Synthesis of tert-butyl ((3,6-dichloropyridazin-4- yl)methyl)carbamate: To a suspension of Boc-glycine (20.0 g, 114.2 mmol) in H 2 O (100 mL) was added 3,6-dichloropyridazine (10.0 g, 67.1 mmol) and silver nitrate (1.1 g, 6.7 mmol) and the resulting mixture was heated at 80 °C. To the reaction mixture was added, drop wise at 80 °C during 30 min, a solution of ammonium sulfate (27.6 g, 120.9 mmol) in H 2 O (40 mL). The reaction mixture was then stirred at 80 °C for additional 30 min. Then, it was cooled to room temperature, basified with conc. ammonium hydroxide (pH 10) and extracted with EtOAc (100 mL x 2). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by column chromatography (hexanes/EtOAc) to afford tert-butyl ((3,6-dichloropyridazin-4-yl)methyl)carbamate (15.0 g, 40% yield) as a light red thick oil. 1 H NMR (400 MHz, CDCl3): d 7.49 (s, 1H), 4.38 (d, J = 6.0 Hz, 2H), 1.49 (s, 9H). LC-MS (ESI-): m/z 278.1 [M-H]-. [00121] Step 2: Synthesis of tert-butyl ((6-chloro-3-(1H-imidazol-1- yl)pyridazin-4-yl)methyl)carbamate and tert-butyl ((3-chloro-6-(1H- imidazol-1-yl)pyridazin-4-yl)methyl)carbamate: To a solution of imidazole (5.9 g, 86.2 mmol) in THF (200 mL) was added NaH (60% in mineral oil) (3.5 g, 86.2 mmol) at 0 o C and the resulting mixture was stirred for 15 min. tert-Butyl ((3,6-dichloropyridazin-4-yl)methyl)carbamate (20.0 g, 72.1 mmol) was added and the reaction mixture was stirred at 60 o C for 2 h. After completion, the reaction was cooled to room temperature, diluted with water (200 mL), and extracted with EtOAc (200 mL x 3). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by column chromatography (PE/EtOAc), to obtain a mixture of the desired compounds (8.1 g, 36% yield) as a light brown solid, which were used in the next step as a mixture. LC-MS (ESI+): m/z r.t. = 1.24 min, 310.19 [M+H] + and r.t. = 1.28 min, 310.15 [M+H] + . [00122] Step 3: Synthesis of ethyl 5-(((tert- butoxycarbonyl)amino)methyl)-6-(1H-imidazol-1-yl)pyridazine- 3- carboxylate and ethyl 4-(((tert-butoxycarbonyl)amino)methyl) -6-(1H- imidazol-1-yl)pyridazine-3-carboxylate: To a solution of a mixture of compounds tert-butyl ((6-chloro-3-(1H-imidazol-1-yl)pyridazin-4- yl)methyl)carbamate and tert-butyl ((3-chloro-6-(1H-imidazol-1-yl)pyridazin-4- yl)methyl)carbamate (6.5 g, 21.0 mmol) in EtOH (97.5 mL) was added sodium acetate (3.4 g, 41.9 mmol) and the resulting mixture was purged with argon for 10 min. Then, Pd(dppf)Cl 2 (0.77 g, 1.0 mmol) was added and the reaction mixture was stirred under CO pressure (100 psi) at 90 °C for 24 h. Then it was cooled to room temperature and volatiles were evaporated under reduced pressure. Saturated aq. solution of NaHCO 3 (100 mL) was added and extracted with EtOAc (100 mL x 3). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH) to afford a mixture of ethyl 5-(((tert- butoxycarbonyl)amino)methyl)-6-(1H-imidazol-1-yl)pyridazine- 3-carboxylate and ethyl 4-(((tert-butoxycarbonyl)amino)methyl) -6-(1H-imidazol-1- yl)pyridazine-3-carboxylate (6.5 g, 89% yield) as a brown solid. LC-MS: m/z r.t. = 1.36 min, 348.4 [M+H] + and r.t. = 1.29 min, 348.3 [M+H] + . [00123] Step 4 and 5: Synthesis of methyl 2-(5-(((tert- butoxycarbonyl)amino)methyl)-6-(1H-imidazol-1-yl)pyridazine- 3- carboxamido)-4,5-difluorobenzoate and methyl 2-(4-(((tert- butoxycarbonyl)amino)methyl)-6-(1H-imidazol-1-yl)pyridazine- 3- carboxamido)-4,5 difluorobenzoate: An aqueous solution of lithium hydroxide monohydrate (0.32 g, 7.7 mmol) in water (12.5 mL) was added to a solution of ethyl 5-(((tert-butoxycarbonyl)amino)methyl)-6-(1H-imidazol-1-yl)p yridazine-3- carboxylate and ethyl 4-(((tert-butoxycarbonyl)amino)methyl) -6-(1H-imidazol- 1-yl)pyridazine-3-carboxylate (2.5 g, 7.2 mmol) in THF (25 mL) and the resulting mixture was stirred at room temperature for 30 min. After completion of the reaction, THF was removed under reduced pressure and the aqueous layer was acidified with 3N HCl (pH 4-5). Volatiles were removed by lyophilization to get a mixture of the corresponding carboxylic acids.The mixture was dissolved in DMF (41 mL), and methyl 4,5-difluoroanthranilate (3.2 g, 17.1 mmol) and DIPEA (7.38 mL, 42.40 mmol) were added. To the reaction mixture, HATU (4.9 g, 12.8 mmol) was added and the reaction mixture was stirred at 80 o C for 7 hours. After completion, the reaction mixture was cooled to room temperature, diluted with saturated aq. solution of NaHCO 3 (220 mL) and extracted with EtOAc (100 mL x 3). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH) to afford methyl 2-(5-(((tert-butoxycarbonyl)amino)methyl)-6- (1H-imidazol-1-yl)pyridazine-3-carboxamido)-4,5-difluoro benzoate (0.75 g, 21% yield) as a yellow solid and methyl 2-(4-(((tert- butoxycarbonyl)amino)methyl)-6-(1H-imidazol-1-yl)pyridazine- 3- carboxamido)-4,5-difluoro benzoate (0.11 g, 3% yield) as a fluffy light brown solid.5-substituted compound: 1 H NMR (400 MHz, DMSO-d 6 ): d 13.09 (s, 1H), 8.88 " 8.81 (m, 1H), 8.35 (s, 1H), 8.43 (s, 1H), 8.14 " 8.06 (m, 1H), 7.89 " 7.84 (m, 2H), 7.26 (s, 1H), 4.37 (d, J = 6.0 Hz, 2H), 3.95 (s, 3H), 1.40 (s, 9H). LC- MS (ESI+): m/z 489.69 [M+H] + .4-substituted compound: 1 H NMR (400 MHz, DMSO-d 6 ): d 12.99 (s, 1H), 8.84 " 8.77 (m, 2H), 8.15 " 8.07 (m, 3H), 7.36 (s, 1H), 7.29 (s, 1H), 4.79 (d, J = 5.6 Hz, 2H), 3.95 (s, 3H), 1.42 (s, 9H). LC-MS (ESI+): m/z 487.3 [M-H]-. [00124] Step 6: Synthesis of methyl 2-(4-(aminomethyl)-6-(1H- imidazol-1-yl)pyridazine-3-carboxamido)-4,5-difluorobenzoate 2: To a solution of 2-(4-(((tert-butoxycarbonyl)amino)methyl)-6-(1H-imidazol-1- yl)pyridazine-3-carboxamido)-4,5-difluoro benzoate (600 mg, 1.2 mmol) in DCM (0.5 mL) was added 4M HCl in dioxane (5 mL) and the reaction mixture was stirred at room temperature for 3 h. After completion of the reaction, volatiles were removed under reduced pressure and diethyl ether (10 mL) was added to the residue. The obtained solid was filtered and dried to afford compound 2 (HCl salt) (34 mg, 7% yield) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6): d 13.10 (s, 1H), 9.43 (s, 1H), 8.91 (s, 1H), 8.77 " 8.83 (m, 4H), 8.44 (s, 1H), 8.10 " 8.15 (m, 1H), 7.62 (s, 1H), 4.68 (t, J = 5.2 Hz, 2H), 3.96 (s, 3H). LC-MS (ESI+): m/z 389.2 [M+H] + . [00125] Compounds 7, 42, 43 and 74 were prepared by using procedures analogous to those for synthesizing compound 2. [00126] Example 3: [00127] Scheme 5: synthesis of compound 3: [00128] Synthesis of 7-ethynyl-6-fluoro-2-(6-(pyridin-4-yl)pyridazin-3- yl)-4H-benzo[d][1,3]oxazin-4-one (3): A suspension of compound 1 (36 mg, 0.1 mmol) in 0.5 mL of thionyl chloride was heated under reflux for 2h. Then, the excess thionyl was removed under vacuum.2 mL of anhydrous acetonitrile was added the solid and a solution of DIPEA (35 µL, 0.2 mmol) in 2 mL of anhydrous acetonitrile was added at room temperature. After stirring for 30 minutes, the obtained precipitate was isolated and washed with acetonitrile to give the product (28 mg, 80 % yield). 1 H NMR (400 MHz, DMSO) d 8.91-8.82 (m, 2H), 8.63 (q, J = 9.0 Hz, 2H), 8.30-8.20 (m, 2H), 8.13 (d, J = 8.6 Hz, 1H), 8.08 (d, J = 6.2 Hz, 1H), 5.04 (s, 1H). MS-ESI: m/z 345.46 observed (M+H) + [00129] Compounds 75, 80 and 93 were prepared by using a procedure analogous to that use for synthesizing compound 3. [00130] While the present disclosure has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements will be apparent to those skilled in the art without departing from the spirit and scope of the claims. [00131] All patents and publications referred to herein are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.
Next Patent: BICYCLIC AGONISTS OF STIMULATOR OF INTERFERON GENES STING