WO2020118206A1 - Butenolide antagonists of the cgas/sting pathway - Google Patents

Abstract

A series of butenolide heterodimers were found to be inhibitors of the cGAS/STING pathway. They can be useful in therapies for treating a range of chronic interferon (IFN) associated autoimmune diseases and inflammation.

Description

BUTENOLIDE ANTAGONISTS OF THE CGAS/STING PATHWAY

[0001] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62/776,306, filed on December 6, 2018, which is incorporated herein by reference in its entirety .

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant number

CHE1352587 awarded by The National Science Foundation. The government has certain rights in the invention.

BACKGROUND

[0003] The cGAS/STING pathway is an evolutionarily conserved innate immune response, which serves as a key cytosolic sensor of nucleic acids in response to viral infection. The cGAS/STING pathway becomes activated upon detection of viral double- stranded DNA (dsDNA) in the cytosol, resulting in production of type I interferons.

Hyperactivation of type I interferon signaling is associated with the initiation of an adaptive immune response against self-antigens that is characteristic of diverse autoimmune diseases, including Systemic Lupus Erythematosus (SLE). De-repression of the cGAS/STING pathway through genetic deficiency of the suppressor protein TREX1, or other cytoplasmic exonucleases such as DNASE II, induces disease in mice that recapitulates many

characteristics of human lupus. Antibodies specific for dsDNA have also been identified in patients with SLE, suggesting that lack of clearance of dsDNA is associated with disease pathology. Notably, genetic deletion of cGAS expression rescues autoimmune symptoms of TrexJ ¹ mice (D Gao, et al . PN.AS, 2015).

[0004] Loss-of- function mutations in the TREX1 gene are associated with several auto- inflammatory diseases, such as familial Chilblain lupus, Aicardi-Goutieres syndrome, and autosomal dominant retinal vasculopathy. Small molecule antagonists of the cGAS/STING pathway could thus serve as potential therapies for a range of chronic interferon (IFN)- associated autoimmune diseases, including SLE, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, Sjogren syndrome, dermatomyositis, polymysitis, systemic sclerosis, familial chilblain lupus, or Aicardi-Goutieres syndrome, and may further impact major unmet needs in inflammation, such as inflammatory bowel disease, etc. SUMMARY

[0005] The present disclosure is directed, in various embodiments, to compounds that are effective antagonists of the cGAS/STING pathway, and which can be useful for treatment of medical conditions involving this pathway. The compounds of the present disclosure belong to a unique class of butenolide heterodimers that present a highly three-dimensional scaffold for biologi cal testing, that more closely resemble natural products than do many of the usual compounds found in libraries of synthetic compounds used for high-throughput screening.

[0006] Accordingly, in various embodiments, the present disclosure provides a compound of formula (I-A), or pharmaceutically acceptable salt thereof:

[0007] In formula (I-A):

R¹ is H or (Cl-C6)alkyl;

R² is H, (CI-C6)alkyl, (Cl-C6)alkoxyl, or (C6-C10)aryl that is optionally substituted with one or more substituents selected from -CN, -hydroxy, halo, (Cl-C6)alkyl, and (Cl- C6)alkoxyl;

R³ is H, (Cl -C6)alkyl, (C2-C6)alkenyl, (C3-C7)cycloalkyl;

R⁴ is (Cl-C6)alkyl or (Cl-C6)alkoxyl; and

R⁵ is (Cl-C6)alkyl or (C6-C10)aryl that is optionally substituted with one or more

substituents selected from -CN, -hydroxy, halo, (Cl-C6)alkyl, and (Cl-C6)alkoxyl.

[0008] R¹ and R² together with the atoms to which they are bonded can form a 5- to 7~ membered cycloalkenyl ring; and R² and R⁵ together with the atoms to wiiich they are bonded can form a 5- to 7-membered cycloalkyl ring.

[0009] It should be understood that, notwithstanding the provisions of formula (I-A), the compound of formula (I-A) is not of formula

[0010] In additional embodiments, the compound of formula (I-A) is a compound of formula (I)

wherein

R¹ is H or (C l~C4)alkyl;

R² is H, (Cl-C4)alkyl, (Cl-C4)alkoxyl, or substituted or unsubstituted aryl;

R is H, (Cl-C6(alkyl), (C2-C6)alkenyl, (C3-C7)cycloalkyl;

R⁴ is (Cl-C4)alkyl or (Cl-C4)alkoxyl;

R⁵ is (Cl-C4)alkyl, substituted or unsubstituted aryl;

provided that R^f and R² together with the atoms to which they are bonded can form a 5- to 7-membered cycloalkenyl ring; and

R ^' and R³ together with the atoms to which they are bonded can form a 5- to 7- membered cycloalkyl ring; and

provided that the compound of formula (I) is not of formula

compound 12.

[0011] The present disclosure also provides in another embodiment a pharmaceutical composition comprising a compound as described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0012] In other embodiments, the present disclosure provides a method of inhibiting the cGAS/STING pathway in a patient, in a pathway-specific manner at non-toxic concentrations, comprising administering to the patient an effective dose of a compound of the present disclosure

[0013] In another embodiment, the present disclosure provides a method of treating a medical condition in a patient wherein inhibition of the cGAS/STING is medically indicated, comprising administering to the patient an effective dose of a compound of the present disclosure. More specifically, the medical condition treatable using an effective dose of a compound of the present disclosure can comprise chronic interferon (IFN)-associated autoimmune diseases, including SLE, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, Sjogren syndrome, dermatomyositis, polymysitis, systemic sclerosis, familial chilblain lupus, or Aicardi-Goutieres syndrome, and may further impact major unmet needs in inflammation, such as inflammatory bowel disease.

[0014] In further embodiments, the present disclosure provides a method of synthesis of a compound of formula (I) or formula (I-A), comprising contacting a conjugate base of nucleophile of formula (II)

and an electrophile of formula (III)

in an aprotic solvent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figs, 1A-G. Identification of butenolide heterodimer-based cGAS-STING pathway inhibitors from a cell-based chemical screen (a) Schematic representation of the cGAS/STING pathway (b) Flowchart of assays used in a screening pipeline (c) Structure of identified screening hit (compound 13) and a representative inactive analog (compound 12). (d) IRF-inducible luciferase activity was measured in human THP-1 cells co-treated with viral dsDNA oligonucleotide and either compound 12 or compound 13 [15 mM] or vehicle (DMSO). (e) Cell viability w_'as assessed using cell titer glo assay under the same treatment conditions to negate false-positive hits due to cytotoxicity (f) Pathway specificity was evaluated by measuring IRF-luciferase activity in response to co-treatment with downstream activator, !FN-B, and compound 12 and compound 13. (g) Gene expression analysis of an ISRE-driven target gene, CXCL10 , was evaluated in response to treatment with 13 and viral dsDNA or IFN-B. All data are the mean ± s.d.. *P < 0.05, **P < 0.01, ***P < 0.001, as analyzed by two-tailed Student’s t-test.

[0016] Figs. 2A-E. Vicinal fully-substituted stereocenters from butenolide

heterodimeri zation (a) Variation of nucl eophile (b) Variation of electrophile (e)

Heterospecificity: neither reactant is self-reactive (d) Proof-of-principle for extension to other coupling partners (e) Unreactive electrophilic partners.

[0017] Figs. 3A-B. (a) IRF-inducible luciferase activity was measured in human THP-1 cells co-treated with viral dsDNA oligonucleotide and indicated compound [20 mM] or vehicle

(DMSO). Cell viability was assessed using cell titer glo assay under the same treatment conditions to negate false-positive hits due to cytotoxicity (b) IRF-inducible luciferase activity was measured in human THP-1 cells co-treated with viral dsDNA oligonucleotide and indicated compound [20 mM] or vehicle (DMSO). Cell viability was assessed using cell titer glo assay under the same treatment conditions to negate false-positive hits due to cytotoxicity.

DETAILED DESCRIPTION

[0018] We developed a high-throughput screening platform to identify novel antagonists of cGAS/STING signaling. This has been established using a primary assay involving a human THP-1 cell line carrying an IRF-inducible reporter with 5 copies of the IFN signaling response element. Counter screens, involving alternative reporter constructs, rodent cell- based assays, as well as cGAS and STING knock-out cell lines, are used to eliminate luciferase artifacts and ensure human-rodent cross species reactivity, as well as pathway selectivity. Using this phenotypic pathway-targeted cell-based screening platform we screened -400K compounds, followed by primary' hit confirmation in triplicate and cell viability counter screening. Cross species reactivity was determined using rodent cell-based secondary assays, and pathway specificity was confirmed using various pathway specific stimulating dsDNA sequences. This led to the identification of a new' class of butenolide heterodimer small molecules that dose dependently inhibit the cGAS/STING pathway, in a pathway-specific manner at non-toxic concentrations.

[0019] In a first round of screening, four butenolide heterodimers, including compound 13, were identified as hits from a cGAS/STING pathway-targeted cell-based phenotypic chemical screen of -250,000 compounds. The cyclic GMP-AMP synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway is an evolutionarily conserved pattern recognition mechanism, which serves as a sensor for cytosolic nucleic acid derived from invading pathogens. In response to double stranded DNA, cGAS becomes activated to generate the cyclic dinucleotide cyclic GMP-AMP (cGAMP), which is recognized by STING and serves to initiate TBKl/IRF3-dependent interferon-stimulated gene (ISG) expression (Fig. la), thereby stimulating innate and adaptive immune response mechanisms. Mislocalized cytosolic self-DNA also leads to aberrant cGAS activation, which is thought to play a causative role in diverse type I interferonopathy-related autoimmunity disorders. For example, activation of the cGAS/STING pathway through genetic deficiency of the suppressor protein TREX1, or other cytoplasmic exonucleases such as DNASE II, induces disease in mice that recapitulates many of the characteristics of systemic lupus erythematosus (SEE). Antibodies specific for dsDNA have also been identified in patients with SEE, suggesting that lack of clearance of dsDNA is associated with disease pathology. Thus, pharmacologic inhibition of the cGAS/STING pathway represents an attractive potential therapeutic strategy for the treatment of diverse autoimmunity disorders. The ability of 13 to inhibit cGAS/STING pathway activation was identified from a screen involving human THPl cells, harboring an IRF-inducible reporter construct (THPl-ISRE-Luciferase), transiently transfected with viral dsDNA Secondary assays, based on cell viability and direct versus secondary pathway selectivity (i.e. IFN l stimulation), were used to identify pathway- selective non-toxic hits (Fig. lb). Importantly, the physiological relevance of 13 was demonstrated by its observed ability to reduce mRNA levels of CXCL10 , in key indicator ISG expression, in dsDNA-stimulated THPl cells at non-toxic concentrations in a cGAS- STING pathway selective manner (Fig. le-g). Preliminary SAR analysis involving 21 butenolide dimers (see Table 1, below), revealed a clear trend consistent with specific site binding, and also enabled the identification of an inactive analog (12), which served as a negative control for the chemotype. Rapid identifi cation of 13 as a lead for inhibition of the cGAS/STING pathway substantiates in a compelling way the value of attached-ring motifs, which we have now shown to be accessible through stereoselective coupling.

Definitions

[0020] "Alkyl" refers to straight or branched chain hydrocarbyl including from 1 to about 20 carbon atoms. For instance, an alkyl can have from 1 to 10 carbon atoms or 1 to 6 carbon atoms. Exemplary alkyl includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecy!, dodecyl, and the like, and also includes branched chain isomers of straight chain alkyl groups, for example without limitation, -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(C H2CH3)3, -CH(CH3)CH(CH3)(CH2CH3), -CH2CH2CH(CH3)2, -CH2CH2CH(CH3)(CH2 CH3), -CH2CH2CH(CH2CH3)2, -CH2CH2C(CH3)3, -CH2CH2C(CH2CH3)3, -CH(CH3)C H2CH(CH3)2, -CH(CH3)CH(CH3)CH(CH3)2, and the like. Thus, alkyl groups include primary alkyl groups, secondary alkyl groups, and tertiary' alkyl groups. An alkyl group can be unsubstituted or optionally substituted with one or more substituents as described herein below.

[0021] Each of the terms“halogen,”“halide,” and“halo” refers to -F or fluoro, -Cl or ch!oro, -Br or brorno, or -I or iodo.

[0022] The ter “alkenyl” refers to straight or branched chain hydrocarbyl groups including from 2 to about 20 carbon atoms having 1-3, 1-2, or at least one carbon to carbon double bond. An alkenyl group can be unsubstituted or optionally substituted with one or more substituents as described herein below.

[0023] The term“alkoxy” or“alkoxyl” refers to an -O-alkyl group having the indicated number of carbon atoms. For example, a (Ci-Ce)alkoxy group includes -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-butyl, -O-sec-butyl, -0-/er/-butyl, -O-pentyl, -O-isopentyl, -O- neopentyl, -O-hexyl, -O-isohexyl, and -O-neohexyl.

[0024] The term“cycloaikyl” refers to a saturated carbocyclic monocyclic, bicyclic, tricyclic, or polycyclic, 3- to 14-membered ring system, such as a 3-, 4-, or 5- to 7-membered ring system. The cycloalkyl may be attached via any atom or can be fused to another ring, such as aryl or other ring. Representative examples of cycloalkyl include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, cyclopropenyl.

[0025] The term“cycloaikenyl” refers to a partially saturated cycloalkyl as defined herein, such as a 3-, 4-, or 5- to 7-membered ring system. Representative examples of cycloal enyl include, but are not limited to cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0026] “Aryl” when used alone or as part of another term means a carbocyclic aromatic group whether or not fused having the number of carbon atoms designated or if no number is designated, up to 14 carbon atoms, such as a Ce-Cn-aiyl. Particular aryl groups are phenyl, naphthyl, biphenyl, phenant.hr enyl, naphthacenyl, and the like (see e.g. Lang’s Handbook of Chemistry (Dean, J. A., ed) 13^th ed. Table 7-2 [1985]). A particular aryl is phenyl “Aryl” can be optionally fused with a carbocyclyi ring, as herein defined. An aryl group can be unsubstituted or optionally substituted with one or more substituents as described herein.

[0027] The term“nitrile” or“cyano” can be used interchangeably and refer to a ~CN group which is bound to a carbon atom of a heteroaryl ring, aryl ring and a heterocycloalkyl ring.

[0028] A“hydroxyl” or“hydroxy” refers to an -OH group.

[0029] Compounds described herein can exist in various isomeric forms, including configurational, geometric, and conformational isomers, including, for example, eis- or trans- conformations. The compounds may also exist in one or more tautomeric forms, including both single tautomers and mixtures of tautomers, such as mixtures of keto and enol tautomers of the compounds. The term“isomer” is intended to encompass all isomeric forms of a compound of this disclosure, including tautomeric forms of the compound. The compounds of the present disclosure may also exist in open-chain or cyclized forms. In some cases, one or more of the cyclized forms may result from the loss of water. The specific composition of the open-chain and cyclized forms may be dependent on how the compound is isolated, stored or administered. For example, the compound may exist primarily in an open-chained form under acidic conditions but cyclize under neutral conditions. All forms are included in the disclosure. [0030] 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, diastereoi somers and mixtures thereof, including a racemic mixture. Optical isomers of the compound s 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.

[0031] 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.

[0032] If there is a discrepancy between a depicted structure and a name gi ven 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.

[0033] 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 or Formula I-A includes a pharmaceutically acceptable salt of a tautomer of the compound.

[0034] In this description, a“pharmaceutically acceptable salt” is a pharmaceutically acceptable, organic or inorganic acid or base salt of a compound described herein.

Representative pharmaceutically acceptable salts include, e.g., alkali metal salts, alkali earth salts, ammonium salts, water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-di sulfonate), benzenesulfonate, benzonate, bicarbonate, bi sulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, g!ycollylarsani!ate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methyl sulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt,

3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (l,l-methene-bis-2-hydroxy-3- naphthoate, einbonate), pantothenate, phosphate/diphosphate, pi crate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate,

sulfosa!icuiate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts.

A pharmaceutically acceptable salt can have more than one charged atom in its structure. In this instance the pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterions.

[0035] The terms“treat”,“treating” and“treatment” refer to the amelioration or eradication of a disease or symptoms associated with a disease. In certain embodiments, such terms refer to minimizing the spread or worsening of the disease resulting from the administration of one or more prophylactic or therapeutic agents to a patient with such a disease. [0036] The terms“prevent,”“preventing,” and“prevention” refer to the prevention of the onset, recurrence, or spread of the disease in a patient resulting from the administration of a prophylactic or therapeutic agent.

[0037] The term“effective amount” refers to an amount of a compound as described herein or other active ingredient sufficient to provide a therapeutic or prophylactic benefit in the treatment or prevention of a disease or to delay or minimize symptoms associated with a disease. Further, a therapeutically effective amount with respect to a compound as described herein means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or prevention of a disease. Used in connection with a compound as described herein, the term can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease, or enhances the therapeutic efficacy of or is synergistic with another therapeutic agent.

[0038] A“patient” or subject” includes an animal, such as a human, cow, horse, sheep, iamb, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig. In accordance with some embodiments, the animal is a mammal such as a non-primate and a primate (e.g., monkey and human). In one embodiment, a patient is a human, such as a human infant, child, adolescent or adult. In the present disclosure, the terms“patient” and“subject” are used interchangeably.

COMPOUNDS

[0039] As described generally above, the present disclosure provides compounds according to formula (I-A), or pharmaceutically acceptable salt thereof:

[0040] In formula (I-A), R¹ is H or (Cl-C6)alkyl.

[0041] R² is H, (Cl-C6)alkyl, (Cl-C6)alkoxyi, or (C6-C10)aryl that is optionally substituted with one or more substituents selected from -CN, -hydroxy, halo, (Cl~C6)alkyi, and (Cl-C6)alkoxyl. [0042] R³ is H, (Cl-C6)alkyl, (C2-C6)alkenyl, (C3-C7)cycloalkyl.

[0043] R⁴ is (Cl-C6)alkyl or (Cl-C6)alkoxyl.

[0044] R⁵ is (Cl-C6)alkyl or (C6-C10)aryl that is optionally substituted with one or more substituents selected from -CN, -hydroxy, halo, (Cl-C6)alkyl, and (Cl-C6)alkoxyl;

[0045] In some embodiments, R¹ and R² together with the atoms to which they are bonded can form a 5- to 7-membered cycloalkenyl ring

[0046] In other embodiments, R² and R³ together with the atoms to which they are bonded can form a 5- to 7-membered cycloalkyl ring.

[0047] The compound formula (I-A) does not include compound 12, which has the following structure:

[0048] In various embodiments, the compound of formula (I-A) is a compound of formula (I):

[0049] In formula (I), R¹ is H or (Cl-C4)alkyl; R² is H, (C!-C4)alkyl, (Cl-C4)alkoxyl, or substituted or un substituted aryl; R⁵ is H, (Cl-C6(alkyl), (C2-C6)alkenyl, (C3-C7)cycloalkyl; R⁴ is (Cl-C4)alkyl or (Cl-C4)alkoxyl; and R⁵ is (Cl-C4)alkyl, substituted or unsubstituted aryl. In some embodiments, R¹ and R² together with the atoms to which they are bonded can form a 5- to 7-membered cycloalkenyl ring. In other embodiments, R² and R^J together with the atoms to which they are bonded can form a 5- to 7-membered cycloalkyl ring. The compound formula (I) does not include compound 12 as described herein. [0050] In various embodiments, the disclosure provides specific examples of Formula I and Formula I-A compounds, and their pharmaceutically acceptable salts, and/or tautomers thereof as set forth in Table 1 below.

[0051] Table L Examples of Formula 1 and Formula I-A Compounds

PHARMACEUTICAL COMPOSITION

[0052] The disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds according to Formula I, Formula I-A, or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof in admixture with a pharmaceutically acceptable carrier. In some embodiments, the composition further contains, in accordance with accepted practices of pharmaceutical compounding, one or more additional therapeutic agents, pharmaceutically acceptable excipients, diluents, adjuvants, stabilizers, emulsifiers, preservatives, colorants, buffers, flavor imparting agents.

[0053] In one embodiment, the pharmaceutical composition comprises a compound selected from those illustrated in Table 1 or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof, and a pharmaceutically acceptable carrier.

[0054] The pharmaceutical composition of the present disclosure is formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for

consideration in this context include the particular disorder being treated, the particular subject being treated, the clinical condition of the subject, the cause of the disorder, the site of deliver_}' of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[0055] The“therapeutically effective amount” of a compound or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof that is administered is governed by such considerations, and is the minimum amount necessary to inhibit the cGAS/STING pathway. Such amount may be below the amount that is toxic to normal cells, or the subject as a whole. Generally, the initial therapeutically effective amount of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure that is administered is in the range of about 0.01 to about 200 mg/kg or about 0.1 to about 20 mg/kg of patient body weight per day, with the typical initial range being about 0.3 to about 15 mg/kg/ day. Oral unit dosage forms, such as tablets and capsules, may contain from about 0.1 mg to about 1000 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In another embodiment, such dosage forms contain from about 50 mg to about 500 rng of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In yet another embodiment, such dosage forms contain from about 25 mg to about 200 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In still another embodiment, such dosage forms contain from about 10 mg to about 100 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In a further embodiment, such dosage forms contain from about 5 mg to about 50 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In any of the foregoing embodiments the dosage form can be administered once a day or twice per day.

[0056] The compositions of the present disclosure can be administered orally, topically, parenteraliy, by inhalation or spray or rectaily in dosage unit formulations. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternai injection or infusion techniques.

[0057] 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. [0058] In another aspect, also encompassed are pharmaceutical compositions suitable for single unit dosages that comprise a compound of the disclosure or its pharmaceutically acceptable stereoisomer, salt, or tautomer and a pharmaceutically acceptable carrier.

[0059] 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.

[0060] For tablet compositions, a compound of the present disclosure 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.

[0061] 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.

[0062] For aqueous suspensions, a compound of the present disclosure 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.

[0063] 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.

[0064] Oily suspensions may be formulated by suspending a compound of the present disclosure 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.

[0065] 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.

[0066] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide a compound of the present di scl osure 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.

[0067] 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 polvoxvethvlene sorbitan monoleate. The emulsions mav also contain sweetening and flavoring agents.

[0068] 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.

[0069] The compounds described herein may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug 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 drug. Such materials are cocoa butter and polyethylene glycols.

[0070] Compositions for parenteral administrations are administered in a sterile medium. Depending on the vehicle used and concentration the concentration of the drug in the formulation, the parenteral formulation can either be a suspension or a solution containing dissolved drug. Adjuvants such as local anesthetics, preservatives and buffering agents can also be added to parenteral compositions.

EXAMPLES

[0071] The following non-limiting examples are additional embodiments for illustrating the present disclosure. Synthetic Methods of Preparation of Butenolide Heterodimers

[0072] Here we show an unusual butenolide dimerization that exhibits high

heteroselectivity and high stereoselectivity. In support of the idea that natural product character leads to high hit rates, the products in our scope table find a target in the cyclic GMP-AMP Synthase (cGAS)/ Stimulator of Interferon Genes (STING) pathway,

outcompeting -250,000 other compounds in a high-throughput screen. This study

demonstrates the value of substituted attached-ring motifs in synthetic compound

collections— value not based only on scarcity alone but shown here through lead

identification. More importantly, we show proof-of-principle that stereoselective

heterocoupling of hindered cyclic building blocks may prove a general phenomenon.

[0073] Recent total syntheses of jiadefenolide and 0-debenzoyltashironin featured a stereoselective Michael addition that was unusual in its fusion of different ring systems at the most-substituted positions. Whereas the y-selective additions of butenolide anions or siloxyfurans to electrophiles were precedented, the stereoselective merger of such hindered systems to form vicinal fully-substituted carbons was not. Yet this reaction occurred quickly even at -100 °C.

[0074] Instead, the reaction proved very general. Shown in Figures 2a/b are products of direct, attached-ring coupling between prochiral‘electron-rich’ butenolide nucleophiles and electron-deficient butenolide electrophiles. Heterodimerization to form these hindered attached-rings was diastereoselective and high yielding despite significant steric repulsion between vicinal fully-substituted carbon centers. Variation of nucleophile structure (Fig. 2a) showed that even coupling partners bearing large alkyl groups at the site of bond formation— isopropyl, cyclopropyl, and cyclopentyl— surmounted the barrier to reaction. Strained bi cyclic nucleophiles also underwent attached-ring coupling, enabling the rapid generation of saturated, polycyclic systems— characteristic of natural product chemical space. A range of electrophiles (Fig. 2b) were interrogated and also found to form attached-ring products, with a focus on electronically differentiated b-aryl butenolides. Mutually exclusive reactivity was observed between‘nucleophilic’ and‘electrophilic’ butenolides. As shown in Figure 2c, both nucleophilic butenolide 5 and electrophilic butenolide 6 were completely unreactive with themselves, in contrast to unhindered butenolides, which homodimerize. [0075] Formation of these hindered, fully-substituted carbons between attached-rings occurred under irreversible, basic conditions and apparently at an extremely high rate, i.e. fast enough to outcompete proton transfer from the significantly more acidic partner 6. To underscore this point, fast-quenching experiments indicated bond formation was complete within 5 s under the standard conditions and even competed with proton transfer from 2 equiv. HCl premixed with electrophile. The fast rate for C-C bond formation permitted formation of attached-ring product 7 in water / THF mixtures, despite use of a lithium enolate. Only competition against 10 equiv. of HCl completely suppressed product formation at -78 °C, which allowed us to accurately define the reaction rate by rapid quench experiments.

[0076] The value of the small library displayed in Figure 2 derives from its orthogonality in chemical space to existing synthetic compound collections. Unlike many synthetic libraries, the butenolide heterodimers have a large number of stereocenters per total heavy atom (15% for model 7), low aromatic ring content, high Fsp³, high ring content, high oxygen content and low nitrogen content. The PMI metric used to evaluate library shape diversity distinguishes the butenolide heterodimers from the ChEMBL database of bioactive molecules developed by the European Bioinformatics Institute. Compounds in the ChEMBL database are largely linear or flat, typical of combinatorial synthetic collections, whereas the heterodimers populate diverse shape space, including highly spherical motifs. As a result, the butenolide dimers of Figure 2 were collected by the California Institute for Biomedical Research (Calibr) and added to high throughput screens for therapeutically relevant targets.

[0077] Synthetic Procedures

[0078] Lithium diisopropylamide (LDA) preparation. A 13 x 100 mm test tube fitted with a Teflon stir bar and rubber lined screw cap septum was flame dried, backfilled with argon, and charged with 2.95 mL of anhydrous THF. Next, freshly distilled diisopropylamine (2,14 mmol, 0.300 mL, 1.1 equiv) was added and the resulting solution was cooled to -78 °C using a dry ice/acetone bath. After allowing the solution to cool for 10 minutes, w-BuLi (1.94 mmol, 0.712 mL of a 2.73 M solution, 1.0 equiv) was added dropwise over 5 minutes. The resulting 0.49 M solution of LDA was allowed to stir at -78 °C for 1 hour before use.

[0079] General Procedure A

[0080] The title compound was prepared by heating a mixture of a-hydroxy ketone (5 mmol) and 2,2,6-trimethyl-4H-l,3-dioxin-4-one (5.1 mmol) in anhydrous toluene (12.5 mL) at 120 °C. The reaction was monitored by TLC and, upon completion, concentrated in vacou. The crude material can be used in the next step without purification (however, higher yields were obtained using purified material). Next, the crude material was transferred to a separatory funnel using a minimal amount of organic solvent (EtaO) followed by addition of 15% NaOH (ratio: (1 mmol) 2-oxopropyl 3-oxobutanoate: (25.0 mL) 15% NaOH solution). The resulting mixture was shaken vigorously for 2 minutes (carefully vent every 10-15 seconds). 6 M HCi was then carefully added until pH ^:::: 0. The solution was shaken vigorously for an additional 60 seconds (carefully vent every 5-10 seconds). Organic solvent (EhaO) was added and the mixture was extracted three times (Caution: care should be taken when extracting, vent often.) The combined organics were dried over MgSCri, concentrated in vacou , and purified via column chromatography.

[0081] General Procedure B

[0082] To a flame-dried flask equipped with a magnetic stir bar was added a-bromo ketone (1.0 mmol). After purging the flask with argon, dry DMF (10 mL) and 3-ethoxy-3- oxopropanoic acid (1.0 mmol) were added. Then, DB!J (3.0 mmol) was added dropwise at 25 °C and the reaction mixture was stirred for 16 hours at 25 ^C'C. The reaction mixture was diluted with saturated NH Cl (aq.) and extracted with EtOAc. The combined organic layers were washed with H2O and brine, dried over MgSCfi, concentrated in vacuo , and purified via column chromatography.

[0083] General Procedure C

[0084] To a solution of hydroxy -furan-2(5H)-one or unsubstituted maleic anhydride (1 .00 mmol) in THF (16.6 mL) and water (0 694 mL) (THF/H2O 24: 1) was added sodium borohydride (3.00 mmol) in portions at 0 °C. The solution was stirred at 0 °C for 2 hours and then the reaction mixture was quenched by addition of 1 M HC1 (6 mL). The mixture was extracted with EtOAc, washed with brine, dried over MgS04, concentrated in vacuo, and purified via column chromatography.

[0085] General procedure D

[0086] Substituted maleic anhydride (8 00 mmol) was added to a flame dried flask and dissolved in anhydrous THF (50 mL). The solution was cooled to -78 °C and MeLi (1.6M in THF, 8 mmol, 2.5 mL) was added drop wise over 15 minutes. The reaction was monitored by TLC, quenched with saturated ammonium chloride (20 mL), and extracted with EtOAc. The combined organics were washed with brine, dried over MgSCL, concentrated in vacuo, and purified via column chromatography.

[0088] The title compound was prepared by adding 2-(diethoxyphosphoryl)acetic acid (1.00 mmol) to a mixture of phenacyl bromide derivative (1.00 mmol) in DMF (10 mL). The reaction mixture was cooled to 0 °C and DBU (3 mmol) was added. The mixture was stirred for 30 minutes at 0 °C then warmed to 25 °C and allowed to stir an additional 30 minutes.

The reaction was quenched with 1 M HC1 (20 mL), extracted with EtOAc, dried over MgSOu concentrated in vacou, and purified via column chromatography. [0089] General Procedure F

[0090] A 13 x 100 mm test tube fited with a Teflon stir bar and rubber lined screw cap septum was flame dried and backfilled with argon. The flask was then cooled to -78 °C and charged with LDA (0.1 1 mmol, 0.224 mL, l . leq). Next, nucleophilic butenolide (0.11 mmol, 1.1 eq) was dissolved in 0.388 mL of anhydrous THF and added dropwise (ca. 5 minutes) to the stirring LDA solution. After 1 hour of stirring, electrophilic butenolide (0.10 mmol, 1.0 eq), dissolved in 0.388 mL of anhydrous THF, was added dropwise (ca. 5 minutes). After stirring for J hour the reaction was quenched with sat. LC!, and extracted with EtOAc (3x 3 mL). The combined organics were washed with brine, dried with MgSCifi, and concentrated in vacuo. Compounds were purified via preparative TLC.

[0091] General Procedure G

[0092] A 13 x 100 mm test tube fitted with a Teflon stir bar and rubber lined screw cap septum was flame dried and backfilled with argon. The flask was then cooled to -78 °C and charged with LDA (0.12 mmol, 0.224 mL, l . leq). Next, nucleophilic butenolide (0.11 mmol, 1.1 eq) was dissolved in 0.270 mL of anhydrous THF and added dropwise (ca. 5 minutes) to the stirring LDA solution. After 1 hour of stirring, electrophilic butenolide (0.10 mmol, 1.0 eq) and DMPU (0.200 mL), dissolved in 0.270 mL of anhydrous THF, were added dropwise (ca. 5 minutes). After stirring for 1 hour the reaction was quenched with sat. MiUCl, and extracted with EtOAc (3x 3 mL). The combined organics were washed with brine, dried with MgSOu and concentrated in vacuo. Compounds were purified via preparative TLC (EtiO) [0093] Lucia ISG Reporter Assay. THP-l Lucia ISG cells (cat. no. thpl-isg, Invivogen) were transiently transfected with viral dsDNA VacV70:LyoVec (cat. no. tlrl-vav70c, Invivogen) and treated with compounds or vehicle control (DMSO, 0.2% v/v). Following 24 h, luminescence was measured using an Envision plate reader (Perkin Elmer) set with an integration time of 0.1 seconds. Luminescence signals for test article samples were normalized to vehicle-treated samples and reported as relative light units (RLU).

[0094] Quantitative RT-PCR Assay. THP-l Lucia ISG cells (cat. no. thpl-isg,

Invivogen) were transiently transfected with viral dsDNA VacV70:LyoVec (cat. no. tlrl- vav70c, Invivogen) and treated with compounds or vehicle control (DMSO, 0.2% v/v). Following 24 h, RNA was extracted from THP-l cells and reverse-transcribed into cDNA. Gene expression was assessed using Taqman primers and probes with the Taqman Universal Mix II (cat. no. 4440038, ThermoFisher) following manufacturer’s instructions. Gene expression was normalized using the delta-delta Ct method and w¾s reported as fold change in expression. Results are presented in Table 2 below (+++ EC50 <20 uM; ++ EC5Q ==^: 20 uM; + EC50 > 20 uM (but active); - inactive).

[0095] Table 2. ISRE-luciferase activities of compounds of the present disclosure.

Claims

WE CLAIM:

1. A compound of formula (I- A), or pharmaceutically acceptable salt thereof:

wherein

R¹ is H or (CI-C6)alkyl;

R² is H, (Cl -C6)alkyl, (Cl-C6)alkoxyl, or (C6-C10)aryl that is optionally substituted with one or more substituents selected from -CN, -hydroxy, halo, (Cl-C6)alkyl, and (Cl- C6)alkoxyl ;

R³ is H, (Cl-C6)alkyl, (C2-C6)alkenyl, (C3-C7)cycloalkyl;

R⁴ is (Cl-C6)aikyi or (Cl-C6)alkoxyl;

R⁵ is (Cl-C6)alkyl or (C6-C10)aryl that is optionally substituted with one or more

substituents selected from -CN, -hydroxy, halo, (Cl-C6)alkyl, and (Cl-C6)alkoxyl; wherein R¹ and R² together with the atoms to which they are bonded can form a 5- to 7- membered cycloalkenyl ring; and

wherein R² and R³ together with the atoms to which they are bonded can form a 5- to 7- membered cycloalkyl ring;

provided that the compound of formula (I-A) is not of formula

compound 12.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I-A) is a compound of formula (I);

wherein

R¹ is H or (Cl-C4)alkyl;

R² is H, (Cl-C4)alkyl, (Cl-C4)aikoxyl, or substituted or unsubstituted aryl;

R³ is H, (Cl-C6(alkyl), (C2-C6)alkenyl, (C3-C7)cycloalkyl;

R⁴ is (Cl -C4)alkyl or (Cl-C4)alkoxyl; and

R^s is (Cl-C4)alkyl, substituted or unsubstituted aryl

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt

4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof,

5. A pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof according to any one of claims 1 - 4 and a pharmaceutically acceptable carrier.

6. A method of inhibiting the cGAS/STING pathway in a patient, in a pathway-specific manner at non-toxic concentrations, comprising administering to the patient an effective dose of a compound or pharmaceutically acceptable salt thereof according to any one of claims 1 - 4

7. A method of treating a medical condition in a patient wherein i nhibition of the cGAS/STING is medically indicated, comprising administering to the patient an effective dose of a compound or pharmaceutically acceptable salt thereof according to any one of claims 1 - 4.

8. The method according to claim 7, wherein the medical condition is selected from the group consisting of chronic interferon (IFN)-associated autoimmune diseases and

inflammatory diseases.

9. The method according to claim 7 or 8, wherein the medical condition is a chronic interferon (IFN)-associated autoimmune disease selected from the group consisting of Systemic Lupus Erythematosus (SLE), type 1 diabetes, multiple sclerosis, rheumatoid arthritis, Sjogren syndrome, dermatomyositis, polymysitis, systemic sclerosis, familial Chilblain lupus, and Aicardi-Goutieres syndrome.

1(3. The method according to claim 7 or 8, wherein the medical condition is an inflammatory' disease.

1 1. The method according to any one of claims 7, 8, or 10, wherein the medical condition is inflammatory bowel disease.

12. A method of synthesis of a compound of formula (I- A):

wherein

R^! is H or (Cl-C6)alkyl;

R² is H, (Cl~C6)alkyl, (CI-C6)alkoxyl, or (C6-C10)aryl that is optionally substituted with one or more substituents selected from -CN, -hydroxy, halo, (Cl-C6)alkyl, and (Cl - C6)alkoxyl;

R³ is H, (Cl-C6)alkyl, (C2-C6)alkenyl, (C3-C7)cycloalkyl;

R⁴ is (Cl-C6)alkyl or (Cl-C6)alkoxyl;

R^s is (Cl-C6)alkyl or (C6~C10)aryl that is optionally substituted with one or more

substituents selected from -CN, -hydroxy, halo, (Cl-C6)alkyl, and (Cl-C6)aJkoxyl; wherein R¹ and R² together with the atoms to which they are bonded can form a 5- to 7- membered cycloalkenyl ring; and

wherein R² and R³ together with the atoms to which they are bonded can form a 5- to 7- membered cycloalkyl ring;

comprising contacting a conjugate base of the nucleophile according to formula (II)

with an electrophile according to formula (III)

in an aprotic solvent.

13. The method according to claim 12, wherein the compound of formula (I- A) is a compound of formula (I):

wherein

R¹ is H or (Cl-C4)alkyl;

R² is H, (Cl-C4)alkyl, (Cl-C4)alkoxyl, or substituted or unsubstituted aryl;

R³ is H, (Cl-C6(alkyl), (C2-C6)alkenyl, (C3-C7)cycloalkyl;

R⁴ is (Cl-C4)alkyl or (Cl-C4)alkoxyl; and

R^s is (Cl-C4)alkyl, substituted or unsubstituted aryl.