H 2s-releasing mesalamine analogs

Abstract

The present application is directed to H2S-releasing mesalamine analogs of Formula I, and pharmaceutically acceptable salts, solvates and / or prodrugs thereof. The application also includes pharmaceutical compositions comprising these compounds and methods of treating diseases, disorders or conditions that benefit from treatment with mesalamine using these compounds (I).

Description

TITLE: H2S-RELEASING MESALAMINE ANALOGS FIELD

[0001] The present application is in the field of mesalamine compounds that have been modified to include an H2S releasing moiety, to compositions comprising such compounds and to their use, for example, as therapeutics. BACKGROUND

[0002] Mesalamine, also known as mesalazine or 5-aminosalicylic acid (5-ASA), is a medication used to treat inflammatory bowel disease, including ulcerative colitis and Crohn's disease. It is generally used for mildly to moderately severe disease. It is taken by mouth or rectally.

[0003] Hydrogen sulfide (H2S) has been shown to be produced in the GI tract and to contribute to gastrointestinal mucosal defense and the healing of gastrointestinal ulcers. In the intestine, H2S modulates epithelial secretion and promotes resolution of colitis. H2S inhibits leukocyte adherence to the vascular endothelium and appears to play an important role in the regulation of systemic blood pressure. Endogenous H2S and H2S donors have also been reported to have a potential role in protecting against viral infections. H2S has also been observed to exert anti-inflammatory and analgesic effects. (Wang, FASEB J.2002, 16:1792- 1798; Wang, Gastroenterology, 2005, 129:1210-1224; Li H et al. J. Virol.2015; 89:5557-5568; Bazhanov N et al. Sci. Rep. 2017; 7:41029; Yang G. Am. J. Physiol. Cell Physiol. 2020; 318:C244-C249; Citi V. et al. Brit. J. Pharmacol. 2020; 177:4931-4941; Kim JZJ Et al. 2020, Journal of Translational Medicine, volume 18, Article number: 257).

[0004] Inflammatory bowel disease (IBD), which include ulcerative colitis (UC) and Crohn’s disease, is recognized as an autoimmune disorder characterized by chronic inflammation of the digestive tract. Although the pathophysiology of IBD is not yet fully understood, genetic, gut microbial and environmental factors are believed to be involved with the pathophysiology of IBD. In addition to conventional therapies, e.g., corticosteroids and 5- aminosalicylates, a variety of novel medications have been developed to treat IBD, including macromolecular antibodies, such as anti-tumor necrosis factor (TNF)-α and anti-integrin antibodies. IBD remains a problematic disease as episodic relapses necessitate further rounds of clinical treatment. Several approved therapies for IBD can have side effects, such as opportunistic infection and infusion reaction and therefore, there is a need for new treatments for IBD that would improve clinical outcomes and limit side effects. - 1 -SUMMARY

[0005] The present application discloses mesalamine analogues, modified to include an H2S releasing moiety, which provide improved anti-inflammatory properties and reduced risk for causing gastric damage compared to administration of an equivalent dose of mesalamine.

[0006] Accordingly, the present application includes A compound of Formula I, or a pharmaceutically acceptable salt, solvate and / or prodrug thereof:wherein: R1is selected from H, C1-6alkyl, C(X1)R8, C(X1)X2R8, S(O)R8and SO2R8; X1and X2are independently selected from O, NH, NC1-4alkyl and S; R2is selected from H and C1-6alkyl; R3is selected from H, halo and C1-4alkyl; R4is selected from H and C1-4alkyl; R5is selected from H, C1-6alkyl, OC1-6alkyl and OH; R6is selected from C1-10alkyl, C1-6alkyleneC3-8cycloalkyl, C1-6alkyleneC3-8heterocycloalkyl, C1-6alkyleneC6-10aryl and C1-6alkyleneC6-10heteroaryl, wherein the alkyl is optionally interrupted by one to three heteromoieties independently selected from O, NH, NHC1-4alkyl and S and further optionally substituted by one to three of NH2, =O and =S; R7is selected from H and C1-6alkyl; or R5and R7are joined to form a 7 membered ring; R8is selected from H, C1-10alkyl, C3-8cycloalkyl, C3-8heterocycloalkyl, C6-10aryl and C5-10heteroaryl, wherein the alkyl is optionally substituted with one or more halo; and all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. - 2 -

[0007] The present application also includes a pharmaceutical composition comprising a compound of the application or a pharmaceutically acceptable salt, solvate and / or prodrug thereof, and a pharmaceutically acceptable carrier.

[0008] The present application also includes a method for treating diseases, disorders or conditions that benefit from treatment with mesalamine, the method comprising administering an effective amount of a compound of the application to a subject in need thereof.

[0009] In some embodiments, the disease, disorder or condition that benefits from treatment with mesalamine is an inflammatory condition. In some embodiments, the inflammatory condition is an inflammatory condition of the gastrointestinal (GI) tract. In some embodiments, the inflammatory condition of the GI tract is selected from inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS).

[0010] In some embodiments, the administration of the compound reduced gastric damage compared to administration of an equivalent dose of mesalamine and / or increases anti-inflammatory effects compared to administration of an equivalent dose of mesalamine and / or enhances the healing and recovery processes compared to administration of an equivalent dose of mesalamine.

[0011] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the application, are given by way of illustration only and the scope of the claims should not be limited by these embodiments but should be given the broadest interpretation consistent with the description as a whole. DRAWINGS

[0012] The embodiments of the application will now be described in greater detail with reference to the attached drawings in which:

[0013] Figure 1 shows effects of exemplary compound [(S)-I-6, 67 mpk, BID and (S)- I-22, 77 mpk, BID] administration compared to various controls in a TNBS-induced colitis model in mice: A) body weight loss over time (days), and B) disease activity index (DAI) score over time (days). Data were expressed as the mean ± standard error of mean, ANOVA, *p<0.05， **p<0.01，***p<0.005，****p<0.0001, versus vehicle. - 3 -

[0014] Figure 2 shows efficacy of exemplary compounds of the application [(S)-I-6, 67 mpk, BID and (S)-I-22, 77 mpk, BID] evaluated by A) colon density, B) colon length and C) colon weight in a TNBS-induced colitis model in mice. The data was expressed as the mean ± standard error of mean, ANOVA, *p<0.05， **p<0.01，***p<0.005，****p<0.0001, versus vehicle.

[0015] Figure 3 shows effects of exemplary compound [(S)-I-3, 69.25 mpk, BID] administration compared to various controls in a TNBS-induced colitis model in mice: A) body weight loss over time (days), and B) disease activity index (DAI) score over time (days). Data were expressed as the mean ± standard error of mean, ANOVA, *p<0.05， **p<0.01， ***p<0.005，****p<0.0001, versus vehicle.

[0016] Figure 4 shows efficacy of exemplary compound of the application [(S)-I-3, 69.25 mpk, BID] evaluated by A) colon density, B) colon length and C) colon weight in a TNBS- induced colitis model in mice. The data was expressed as the mean ± standard error of mean, ANOVA, *p<0.05， **p<0.01，***p<0.005，****p<0.0001, versus vehicle.

[0017] Figure 5 shows effects of exemplary compound [(S)-I-7, 92 mpk, BID, (S)-I-6, 66 mpk, BID and (S)-I-25, 67 mpk, BID] administration compared to various controls in a TNBS-induced colitis model in rats evaluated by A) body weight loss over time (days), and B) DAI score over time (days). Data were expressed as the mean ± standard error of mean, ANOVA, *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, versus vehicle.

[0018] Figure 6 shows efficacy of exemplary compounds of the application [(S)-I-7, 92 mpk, BID, (S)-I-6, 66 mpk, BID and (S)-I-25, 67 mpk, BID] evaluated by A) colon density, B) colon length and C) colon weight in a TNBS-induced colitis model in rats. The data was expressed as the mean ± standard error of mean, ANOVA, *p<0.05， **p<0.01，***p<0.005 ，****p<0.0001, versus vehicle.

[0019] Figure 7 shows results of myeloperoxidase peroxidation activity in units per gram (mU / g) of wet tissue for exemplary compounds of the application [(S)-I-7, 92 mpk, BID, (S)-I-6, 66 mpk, BID and (S)-I-25, 67 mpk, BID] from aTNBS-induced colitis model in rats. The data was expressed as the mean ± standard error of mean, ANOVA, *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, versus vehicle. DETAILED DESCRIPTION I. Definitions

[0020] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present - 4 -application herein described for which they are suitable as would be understood by a person skilled in the art.

[0021] The term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives.

[0022] The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0023] The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0024] Terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0025] As used in this application, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise.

[0026] In embodiments comprising an “additional” or “second” component, the second component as used herein is chemically different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0027] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present.

[0028] The term “compound(s) of the application” or “compound(s) of the present application”, and the like, as used herein refers to a compound of Formula I or a salt, solvate and / or prodrug thereof. - 5 -

[0029] The term “composition(s) of the application” or “composition(s) of the present application, and the like, as used herein refers to a composition comprising one or more compounds of the application and a carrier.

[0030] The term “suitable” as used herein means that the selection of the particular compound or conditions would depend on the specific synthetic manipulation to be performed, the identity of the molecule(s) to be transformed and / or the specific use for the compound, but the selection would be well within the skill of a person trained in the art.

[0031] The present description refers to a number of chemical terms and abbreviations used by those skilled in the art. Nevertheless, definitions of selected terms are provided for clarity and consistency.

[0032] The term “protecting group” or “PG” and the like as used herein refers to a chemical moiety which protects or masks a reactive portion of a molecule to prevent side reactions in those reactive portions of the molecule, while manipulating or reacting a different portion of the molecule. After the manipulation or reaction is complete, the protecting group is removed under conditions that do not degrade or decompose the remaining portions of the molecule. The selection of a suitable protecting group can be made by a person skilled in the art. Many conventional protecting groups are known in the art, for example as described in “Protective Groups in Organic Chemistry” McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W. and Wuts, P.G.M., “Protective Groups in Organic Synthesis”, John Wiley & Sons, 3rd Edition, 1999 and in Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas).

[0033] The term “inert organic solvent” as used herein refers to a solvent that is generally considered as non-reactive with the functional groups that are present in the compounds to be combined together in any given reaction so that it does not interfere with or inhibit the desired synthetic transformation. Organic solvents are typically non-polar and dissolve compounds that are non soluble in aqueous solutions.

[0034] The term “alkyl” as used herein, whether it is used alone or as part of another group, means straight or branched chain, saturated alkyl groups. The number of carbon atoms that are possible in the referenced alkyl group are indicated by the prefix “Cn1-n2”. For example, the term C1-10alkyl means an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0035] The term “alkylene”, whether it is used alone or as part of another group, means straight or branched chain, saturated alkylene group, that is, a saturated carbon chain that contains substituents on two of its ends. The number of carbon atoms that are possible in the - 6 -referenced alkylene group are indicated by the prefix “Cn1-n2”. For example, the termC2-6alkylene means an alkylene group having 2, 3, 4, 5 or 6 carbon atoms.

[0036] The term “cycloalkyl,” as used herein, whether it is used alone or as part of another group, means a saturated carbocyclic group containing from 3 to 20 carbon atoms and one or more rings. The number of carbon atoms that are possible in the referenced cycloalkyl group are indicated by the numerical prefix “Cn1-n2”. For example, the term C3-10cycloalkyl means a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0037] The term “aryl” as used herein, whether it is used alone or as part of another group, refers to carbocyclic groups containing at least one aromatic ring and contains either 6 to 20 carbon atoms.

[0038] The term “heterocycloalkyl” as used herein, whether it is used alone or as part of another group, refers to cyclic groups containing at least one non-aromatic ring containing from 3 to 20 atoms in which one or more of the atoms are a heteroatom selected from O, S and N and the remaining atoms are C. Heterocycloalkyl groups are either saturated or unsaturated (i.e. contain one or more double bonds). When a heterocycloalkyl group contains the prefix Cn1-n2 this prefix indicates the number of carbon atoms in the corresponding carbocyclic group, in which one or more, suitably 1 to 5, of the ring atoms is replaced with a heteroatom as selected from O, S and N and the remaining atoms are C. Heterocycloalkyl groups are optionally benzofused.

[0039] The term “heteroaryl” as used herein, whether it is used alone or as part of another group, refers to cyclic groups containing at least one heteroaromatic ring containing 5-20 atoms in which one or more of the atoms are a heteroatom selected from O, S and N and the remaining atoms are C. When a heteroaryl group contains the prefix Cn1-n2 this prefix indicates the number of carbon atoms in the corresponding carbocyclic group, in which one or more, suitably 1 to 5, of the ring atoms is replaced with a heteroatom as defined above. Heteroaryl groups are optionally benzofused.

[0040] All cyclic groups, including aryl, heteroaryl, heterocycloalkyl and cycloalkyl groups, contain one or more than one ring (i.e. are polycyclic). When a cyclic group contains more than one ring, the rings may be fused, bridged, spirofused or linked by a bond.

[0041] The term “benzofused” as used herein refers to a polycyclic group in which a benzene ring is fused with another ring.

[0042] A first ring being “fused” with a second ring means the first ring and the second ring share two adjacent atoms there between. - 7 -

[0043] A first ring being “bridged” with a second ring means the first ring and the second ring share two non-adjacent atoms there between.

[0044] A first ring being “spirofused” with a second ring means the first ring and the second ring share one atom there between.

[0045] The term “fluorosubstituted” refers to the substitution of one or more, including all, available hydrogens in a referenced group with fluoro.

[0046] The terms “halo” or “halogen” as used herein, whether it is used alone or as part of another group, refers to a halogen atom and includes fluoro, chloro, bromo and iodo.

[0047] The term “available”, as in “available hydrogen atoms” or “available atoms” refers to atoms that would be known to a person skilled in the art to be capable of replacement by a substituent.

[0048] The term “subject” as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans. Thus, the methods and uses of the present application are applicable to both human therapy and veterinary applications.

[0049] The term “pharmaceutically acceptable” means compatible with the treatment of subjects, for example humans.

[0050] The term “pharmaceutically acceptable carrier” means a non-toxic solvent, dispersant, excipient, adjuvant or other material which is mixed with the active ingredient in order to permit the formation of a pharmaceutical composition, i.e., a dosage form capable of administration to a subject.

[0051] The term “pharmaceutically acceptable salt” means either an acid addition salt or a base addition salt which is suitable for, or compatible with the treatment of subjects.

[0052] The term “solvate” as used herein means a compound, or a salt and / or prodrug of a compound, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered.

[0053] The term “prodrug” as used herein means a compound, or salt and / or solvate of a compound, that, after administration, is converted into an active drug.

[0054] The term “treating” or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the - 8 -reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. “Treating” and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. “Treating” and “treatment” as used herein also include prophylactic treatment. For example, a subject with early cancer can be treated to prevent progression, or alternatively a subject in remission can be treated with a compound or composition of the application to prevent recurrence. Treatment methods comprise administering to a subject a therapeutically effective amount of one or more of the compounds of the application and optionally consist of a single administration, or alternatively comprise a series of administrations.

[0055] As used herein, the term “effective amount” or “therapeutically effective amount” means an amount of a compound, or one or more compounds, of the application that is effective, at dosages and for periods of time necessary to achieve the desired result.

[0056] The term “administered” as used herein means administration of a therapeutically effective amount of a compound, or one or more compounds, or a composition of the application to a cell either in cell culture or in a subject. II. Compounds and Compositions of the Application

[0057] The present application includes a compound of Formula I, or a pharmaceutically acceptable salt, solvate and / or prodrug thereof:I wherein: R1is selected from H, C1-6alkyl, C(X1)R8, C(X1)X2R8, S(O)R8and SO2R8; X1and X2are independently selected from O, NH, NC1-4alkyl and S; R2is selected from H and C1-6alkyl; R3is selected from H, halo and C1-4alkyl; R4is selected from H and C1-4alkyl; R5is selected from H, C1-6alkyl, OC1-6alkyl and OH; - 9 -R6is selected from C1-10alkyl, C1-6alkyleneC3-8cycloalkyl, C1-6alkyleneC3-8heterocycloalkyl, C1-6alkyleneC6-10aryl and C1-6alkyleneC6-10heteroaryl, wherein the alkyl is optionally interrupted by one to three heteromoieties independently selected from O, NH, NHC1-4alkyl and S and further optionally substituted by one to three of NH2, =O and =S; R7is selected from H and C1-6alkyl; or R5and R7are joined to form a 7 membered ring; R8is selected from H, C1-10alkyl, C3-8cycloalkyl, C3-8heterocycloalkyl, C6-10aryl and C5-10heteroaryl, wherein the alkyl is optionally substituted with one or more halo; and all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom.

[0058] In some embodiments, R1is selected from H, C1-6alkyl, C(O)R8, C(S)R8, C(NC1-4alkyl)R8, C(O)OR8, C(O)NHR8, C(S)OR8, C(NC1-4alkyl)OR8, C(S)NHR8, C(NC1-4alkyl)NHR8, S(O)R8and SO2R8, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R1is selected from H, C1-4alkyl, C(O)R8, C(O)OR8, C(O)NHR8, C(S)OR8, C(S)NHR8and SO2R8, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom.

[0059] In some embodiments, R1is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, C(CH3)3, CH(CH3)CH2CH3and CH2CH(CH3)2, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R1is H or CH3.

[0060] In some embodiments, R8is selected from H, C1-6alkyl, C3-6cycloalkyl, C3-6heterocycloalkyl, phenyl and C6heteroaryl, wherein the alkyl is optionally substituted with one or more halo and all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R8is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, C(CH3)3, CH(CH3)CH2CH3and CH2CH(CH3)2and phenyl, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R8is selected from H, CH3, CF3, CH2CH3, C(CH3)3and phenyl.

[0061] In some embodiments, R2is selected from H and C1-4alkyl, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R2is selected from H, CH3and CH2CH3. In some embodiments, R2is selected from H and CH3. In some embodiments, R2is H.

[0062] In some embodiments, R1and R2are both H. - 10 -

[0063] In some embodiments, R3is selected from H, halo and C1-3alkyl, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R3is selected from H, Cl, F, CH3, CF3and CH2CH3. In some embodiments, R3is H.

[0064] In some embodiments, R3, when present is located ortho to the OR7group and the compound of Formula I has the following structure: , or a pharmaceutically acceptag thereof, wherein R1, R2, R3, R4, R5, R6and R7are as defined for Formula I.

[0065] In some embodiments, R3is H and the compound of Formula I has the following structure: ,or a pharmaceutically acceptable salt, solvate and / or prodrug thereof, wherein R1, R2, R4, R5, R6and R7are as defined for Formula I.

[0066] In some embodiments, R4is selected from H and C1-4alkyl, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R4is selected from H and C1-3alkyl, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R4is selected from H, CH3and CH2CH3. In some embodiments, R4is H.

[0067] In some embodiments, R5is selected from H, C1-5alkyl, OC1-5alkyl and OH, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R5is selected from H, C1-4alkyl, OC1-4alkyl and OH, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R5is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, C(CH3)3, CH(CH3)CH2CH3, CH2CH(CH3)2, OCH3, OCH2CH3, - 11 -OCH2CH2CH3, OCH(CH3)2, OCH2CH2CH2CH3, OC(CH3)3, OCH(CH3)CH2CH3, OCH2CH(CH3)2and OH, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R5is OCH3or OCF3. In some embodiments, R5is OCH3. In some embodiments, R5is OH.

[0068] In some embodiments, R6is selected from C1-6alkyl, C1-4alkyleneC3-6cycloalkyl, C1-4alkyleneC3-6heterocycloalkyl, C1-4alkylenephenyl and C1-4alkyleneC6heteroaryl, wherein the alkyl is optionally interrupted by one to two heteromoieties independently selected from O, NH, NHC1-4alkyl and S and further optionally substituted by one or two of NH2, =O and =S, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R6is selected from C1-6alkyl, C1-4alkyleneC5-6cycloalkyl, C1-4alkyleneC5-6heterocycloalkyl, C1-4alkylenephenyl and C1-4alkyleneC6heteroaryl, wherein the alkyl is optionally interrupted by one heteromoiety selected from O and S and further optionally substituted by one of NH2, =O and / or =S, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R6is selected from C1-4alkyl, C1-3alkylenephenyl, wherein the alkyl is optionally interrupted by one heteromoiety selected from O and S and further optionally substituted by one of NH2and / or =S, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R6is selected from C1-4alkyl, C1alkylenephenyl, wherein the alkyl is optionally interrupted by one heteromoiety selected from O and S and further optionally substituted by one of NH2and / or =S, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R6is selected from CH3, CF3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, C(CH3)3, CH(CH3)CH2CH3, CH2CH(CH3)2, CH2CH2SCH3, CH2CH2OCH3, CH2CH2SCF3, CH2CH2OCF3and CH2phenyl. In some embodiments, R6is selected from CH3, CH(CH3)2, C(CH3)3, CH2CH2SCH3and CH2phenyl.

[0069] In some embodiments, R7is selected from H and C1-3alkyl, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R7is selected from H and C1-2alkyl, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. In some embodiments, R7is selected from H, CF3and CH3. In some embodiments, R7is H.

[0070] In some embodiments, R5and R7are joined to form a 7 membered ring and the compound of Formula I has the following structure: - 12 -or a pharmaceutically acceptablrug thereof, wherein R1, R2, R3, R4, and R6are as defined for Formula I.

[0071] A person skilled in the art would appreciate that the carbon to which R6is bonded is chiral. Therefore, in some embodiments, the carbon to which R6is bonded is racemic. In some embodiments, the stereochemistry at the carbon to which R6is bonded is S and the compound of Formula I has one of the following structures: or a pharmaceutiwherein R1, R2, R3, R4, R5, R6and R7are as defined for Formula I. In some embodiments, the stereochemistry at the carbon to which R6is bonded is R and the compound of Formula I has one of the following structures: or a pharmaceuticay p , p g , wherein R1, R2, R3, R4, R5, R6and R7are as defined for Formula I.

[0072] In some embodiments, the compounds of Formula I are selected from the compounds listed below, or a pharmaceutically acceptable salt, solvate and / or prodrug thereof: Compound ID Structure- 13 -(S)-I-1 (S)-I-2 (S) (S) (S) (S) (S) (S)- 14 -(S)-I-9- 15 -(S)-I-18

[0073] In some embodiments, the pharmaceutically acceptable salt is an acid addition salt or a base addition salt. The selection of a suitable salt may be made by a person skilled in the art. Suitable salts include acid addition salts that may, for example, be formed by mixing - 16 -a solution of a compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid, or benzoic acid. Additionally, acids that are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al, Camille G. (eds.) and Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley VCH; S. Berge et al, Journal of Pharmaceutical Sciences 197766(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33201-217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, D.C. on their website).

[0074] An acid addition salt suitable for, or compatible with, the treatment of subjects is any non-toxic organic or inorganic acid addition salt of any basic compound. Basic compounds that form an acid addition salt include, for example, compounds comprising an amine group. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric, nitric and phosphoric acids, as well as acidic metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids which form suitable salts include mono-, di- and tricarboxylic acids. Illustrative of such organic acids are, for example, acetic, trifluoroacetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, mandelic, salicylic, 2-phenoxybenzoic, p- toluenesulfonic acid and other sulfonic acids such as methanesulfonic acid, ethanesulfonic acid and 2-hydroxyethanesulfonic acid. In some embodiments, exemplary acid addition salts also include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates (“mesylates”), naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartarates, thiocyanates, toluenesulfonates (also known as tosylates) and the like. In some embodiments, the mono- or di-acid salts are formed and such salts exist in either a hydrated, solvated or substantially anhydrous form. In general, acid addition salts are more soluble in water and various hydrophilic organic solvents and generally demonstrate higher melting points in comparison to their free base forms. The selection criteria for the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts such as but not limited to oxalates may be used, for example in the isolation of compounds of the application for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0075] A base addition salt suitable for, or compatible with, the treatment of subjects is any non-toxic organic or inorganic base addition salt of any acidic compound. Acidic - 17 -compounds that form a basic addition salt include, for example, compounds comprising a carboxylic acid group. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide as well as ammonia. Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as isopropylamine, methylamine, trimethylamine, picoline, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. The selection of the appropriate salt may be useful, for example, so that an ester functionality, if any, elsewhere in a compound is not hydrolyzed. The selection criteria for the appropriate salt will be known to one skilled in the art. In some embodiments, exemplary basic salts also include ammonium salts, alkali metal salts such as sodium, lithium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as dicyclohexylamine, abutyl amine, choline and salts with amino acids such as arginine, lysine and the like. Basic nitrogen containing groups may be quarternized with agents such as lower alkyl halides (e.g., methyl, ethyl and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl and dibutyl sulfates), long chain halides (e.g., decyl, lauryl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides) and others. Compounds carrying an acidic moiety can be mixed with suitable pharmaceutically acceptable salts to provide, for example, alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts) and salts formed with suitable organic ligands such as quaternary ammonium salts. Also, in the case of an acid (-COOH) or alcohol group being present, pharmaceutically acceptable esters can be employed to modify the solubility or hydrolysis characteristics of the compound.

[0076] All such acid salts and base salts are intended to be pharmaceutically acceptable salts within the scope of the application and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the application. In addition, when a compound of the application contains both a basic moiety, such as, but not limited to an aliphatic primary, secondary, tertiary or cyclic amine, an aromatic or heteroaryl amine, pyridine or imidazole and an acidic moiety, such as, but not limited to tetrazole or carboxylic acid, zwitterions (“inner salts”) may be formed and are included within the terms “salt(s)” as used herein. It is understood that certain compounds of the application may exist - 18 -in zwitterionic form, having both anionic and cationic centers within the same compound and a net neutral charge. Such zwitterions are included within the application.

[0077] Solvates of compounds of the application include, for example, those made with solvents that are pharmaceutically acceptable. Examples of such solvents include water (resulting solvate is called a hydrate) and ethanol and the like. Suitable solvents are physiologically tolerable at the dosage administered.

[0078] Prodrugs of the compounds of the present application include, for example, conventional esters formed with available hydroxy, thiol, amino or carboxyl groups. Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (C1-C24) esters, acyloxymethyl esters, carbamates and amino acid esters.

[0079] It is understood and appreciated that in some embodiments, compounds of the present application may have at least one chiral center and therefore can exist as enantiomers and / or diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present application. It is to be further understood that while the stereochemistry of the compounds may be as shown in any given compound listed herein, such compounds may also contain certain amounts (for example, less than 20%, suitably less than 10%, more suitably less than 5%) of compounds of the present application having an alternate stereochemistry. It is intended that any optical isomers, as separated, pure or partially purified optical isomers or racemic mixtures thereof are included within the scope of the present application.

[0080] The compounds of the present application may further exist in varying amorphous and polymorphic forms and it is contemplated that any amorphous forms, polymorphs, or mixtures thereof, which form are included within the scope of the present application.

[0081] The compounds of the present application are suitably formulated in a conventional manner into compositions using one or more carriers. Accordingly, the present application also includes a composition comprising one or more compounds of the application and a carrier. The compounds of the application are suitably formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. Accordingly, the present application further includes a pharmaceutical composition comprising one or more compounds of the application and a pharmaceutically acceptable carrier. - 19 -

[0082] A compound of the application is suitably used on their own but will generally be administered in the form of a composition in which the one or more compounds of the application (the active ingredient) is in association with an acceptable carrier.

[0083] The compounds of the application may be administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. A compound of the application may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration and the pharmaceutical compositions formulated accordingly. Administration can be by means of a pump for periodic or continuous delivery. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington’s Pharmaceutical Sciences (2000 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

[0084] Parenteral administration includes intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary (for example, by use of an aerosol), intrathecal, rectal and topical (including the use of a patch or other transdermal delivery device) modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

[0085] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists.

[0086] A compound of the application may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the compound may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, caplets, pellets, granules, lozenges, chewing gum, powders, syrups, elixirs, wafers, aqueous solutions and suspensions, and the like. In the case of tablets, carriers that are used include lactose, corn starch, sodium citrate and salts of phosphoric acid. Pharmaceutically acceptable excipients include binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. In the case of tablets, capsules, caplets, pellets or granules for oral administration, pH sensitive enteric coatings, such as Eudragits™ - 20 -designed to control the release of active ingredients are optionally used. Oral dosage forms also include modified release, for example immediate release and timed-release, formulations. Examples of modified-release formulations include, for example, sustained-release (SR), extended-release (ER, XR, or XL), time-release or timed-release, controlled-release (CR), or continuous-release (CR or Contin), employed, for example, in the form of a coated tablet, an osmotic delivery device, a coated capsule, a microencapsulated microsphere, an agglomerated particle, e.g., as of molecular sieving type particles, or, a fine hollow permeable fiber bundle, or chopped hollow permeable fibers, agglomerated or held in a fibrous packet. Timed-release compositions can be formulated, e.g. liposomes or those wherein the active compound is protected with differentially degradable coatings, such as by microencapsulation, multiple coatings, etc. Liposome delivery systems include, for example, small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. For oral administration in a capsule form, useful carriers or diluents include lactose and dried corn starch.

[0087] Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they are suitably presented as a dry product for constitution with water or other suitable vehicle before use. When aqueous suspensions and / or emulsions are administered orally, the compound of the application is suitably suspended or dissolved in an oily phase that is combined with emulsifying and / or suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents may be added. Such liquid preparations for oral administration may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non- aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid). Useful diluents include lactose and high molecular weight polyethylene glycols.

[0088] It is also possible to freeze-dry the compounds of the application and use the lyophilizates obtained, for example, for the preparation of products for injection.

[0089] A compound of the application may also be administered parenterally. Solutions of a compound of the application can be prepared in water, suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations. For parenteral administration, sterile solutions of the compounds of the - 21 -application are usually prepared, and the pH of the solutions are suitably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic. For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or polyvinyl alcohol, preservatives such as sorbic acid, EDTA or benzyl chromium chloride, and the usual quantities of diluents or carriers. For pulmonary administration, diluents or carriers will be selected to be appropriate to allow the formation of an aerosol.

[0090] The compounds of the application may be formulated for parenteral administration by injection, including using conventional catheterization techniques or infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi- dose containers, with an added preservative. The compositions may take such forms as sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and / or dispersing agents. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. Alternatively, the compounds of the application are suitably in a sterile powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0091] Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders.

[0092] For intranasal administration or administration by inhalation, the compounds of the application are conveniently delivered in the form of a solution, dry powder formulation or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively, the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon. Suitable propellants include but are not limited to dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, heptafluoroalkanes, carbon dioxide or another suitable gas. In the case of a pressurized aerosol, the dosage unit is suitably determined by providing a valve to deliver a metered amount. The pressurized container or nebulizer may contain a solution or - 22 -suspension of the active compound. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the application and a suitable powder base such as lactose or starch. The aerosol dosage forms can also take the form of a pump-atomizer.

[0093] Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.

[0094] Suppository forms of the compounds of the application are useful for vaginal, urethral and rectal administrations. Such suppositories will generally be constructed of a mixture of substances that is solid at room temperature but melts at body temperature. The substances commonly used to create such vehicles include but are not limited to theobroma oil (also known as cocoa butter), glycerinated gelatin, other glycerides, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol. See, for example: Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing, Easton, PA, 1980, pp.1530-1533 for further discussion of suppository dosage forms.

[0095] Compounds of the application may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, compounds of the application may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.

[0096] In an embodiment, compounds of the application may be coupled with viral, non-viral or other vectors. Viral vectors may include retrovirus, lentivirus, adenovirus, herpesvirus, poxvirus, alphavirus, vaccinia virus or adeno-associated viruses. Non-viral vectors may include nanoparticles, cationic lipids, cationic polymers, metallic nanoparticles, nanorods, liposomes, micelles, microbubbles, cell-penetrating peptides, or lipospheres. Nanoparticles may include silica, lipid, carbohydrate, or other pharmaceutically acceptable polymers. - 23 -

[0097] In some embodiments, depending on the mode of administration, the pharmaceutical composition will comprise from about 0.05 wt% to about 99 wt% or about 0.10 wt% to about 70 wt%, of the compounds of the application, and from about 1 wt% to about 99.95 wt% or about 30 wt% to about 99.90 wt% of one or more pharmaceutically acceptable carriers, all percentages by weight being based on the total composition.

[0098] In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the compounds of the application are administered in a dose of about 0.01 mg / kg body weight to about 250 mg / kg body weight, about 0.1 mg / kg to about 230 mg / kg, about 1 mg / kg to about 210 mg / kg, about 30 mg / kg to about 200 mg / kg, about 50 mg / kg to about 180 mg / kg, about 60 mg / kg, to about 150 mg / kg, about 80 mg / kg to about 120 mg / kg once daily or twice daily.

[0099] In some embodiments, a compound of the application is administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present application provides a single unit dosage form comprising one or more compounds of the application, an additional therapeutic agent, and a pharmaceutically acceptable carrier.

[0100] In the above, the term “a compound” also includes embodiments wherein one or more compounds are referenced. III. Methods and Uses of the Application

[0101] Exemplary compounds of the application were tested in a trinitrobenzene sulfonic acid (TNBS)-induced colitis model in both rats and mice and were shown to reduce body weight loss, improve disease activity index (DAI) and to maintain colon weight, density and length compared to vehicle control. Exemplary compounds showed activity that was comparable to mesalamine and in the case of maintenance of colon weight, density and length, some exemplary compounds of the application showed improved activity compared with mesalamine. In the TNBS-induced colitis model in rats, exemplary compounds of the application also showed reduced myeloperoxidase (MPO) activity compared to vehicle control and comparable activity to mesalamine. Further results from the TNBS-induced colitis model in rats included an induction of beneficial effects in the levels of anti-inflammatory cytokines, including INF^, IL-2, IL-5, IL-12β, TNFα, IL-1β and IL-4 upon administration of exemplary compounds of the application. Finally, exemplary compounds of the application released H2S when incubated with Fasted State Simulated Intestinal Fluid (FaSSIF) and Phosphate Buffered Saline (PBS). While not wishing to be limited by theory, a short-lived increase in plasma H2S concentrations upon administration of compound of the application, which is - 24 -within the physiological range, may contribute to enhanced anti-inflammatory activity as well as a reduction in side effects, for example reduced gastric injury or damage.

[0102] Accordingly, compounds of the application, may, for example, be useful for the treatment of various diseases, disorders or conditions that benefit from treatment with mesalamine. Compounds of the application are H2S-releasing derivatives of mesalamine which have been shown to provide reduced levels of anti-inflammatory cytokines and reduced myeloperoxidase (MPO) activity, which is associated with inflammation, as well as to show good activity in the TNBS-induced colitis model in rats and mice. The H2S releasing properties of the compounds of the application are expected to provide compounds with improved anti- inflammatory properties and reduced risk for causing gastric damage. Therefore the compounds of the present application represent a safe and effective replacement for mesalamine.

[0103] In some embodiments, the present application includes a use of a compound of the application as a medicament.

[0104] The present application also includes a method for treating diseases, disorders or conditions that benefit from treatment with mesalamine, the method comprising administering an effective amount of a compound of the application to a subject in need thereof. The present application further includes a use of a compound of the application for treating diseases, disorders or conditions that benefit from treatment with mesalamine, a use of a compound of the application for preparation of a medicament for treating diseases, disorders or conditions that benefit from treatment with mesalamine, as well as a compound of the application for use to treat diseases, disorders or conditions that benefit from treatment with mesalamine.

[0105] In some embodiments, the compound of the application is useful in the treatment of an inflammatory condition. In some embodiments, the inflammatory condition is an inflammatory condition of the gastrointestinal (GI) tract.

[0106] Accordingly, the present application also includes a method of treating an inflammatory condition of the GI tract comprising administering an effective amount of a compound of the application to a subject in need thereof.

[0107] The present application also includes a use of a compound of the application for treatment of an inflammatory condition of the GI tract as well as a use of a compound of the application for the preparation of a medicament for treatment of an inflammatory condition of the GI tract. The application further includes a compound of the application for use in treating an inflammatory condition of the GI tract. - 25 -

[0108] In some embodiments, the inflammatory condition of the GI tract is selected from inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). In some embodiments, the IBD is ulcerative colitis or Crohn's Disease. In some embodiments, the IBD is ulcerative colitis. In some embodiments, the IBD is Crohn's Disease.

[0109] In some embodiments, administration of the compounds of the application reduces gastric damage compared to administration of an equivalent dose of mesalamine. In some embodiments, administration of the compounds of the application increases anti- inflammatory effects compared to administration of an equivalent dose of mesalamine. In some embodiments, administration of the compounds of the application enhances the healing and recovery processes of diseases, disorders or conditions that benefit from treatment with mesalamine, compared to administration of an equivalent dose of mesalamine. While not wishing to be limited by theory, the administration of compound of the application may induce beneficial cytokine effects and thereby enhance the healing and recovery processes of said diseases, disorders or conditions.

[0110] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0111] When used in combination with other agents or therapies, it is an embodiment that compounds of the application are administered contemporaneously with those agents or therapies. As used herein, “contemporaneous administration” of two substances or therapies to a subject means providing each of the two substances or therapies so that they are both biologically active in the individual at the same time. The exact details of the administration will depend on the pharmacokinetics of the two substances or therapies in the presence of each other, and can include administering the two substances or therapies within a few hours of each other, or even administering one substance or therapy within 24 hours of administration of the other, if the pharmacokinetics are suitable. Design of suitable dosing regimens is routine for one skilled in the art. In particular embodiments, the substances or therapies will be administered substantially simultaneously, i.e., within minutes of each other, or in a single composition in the case of administration of two substances. It is a further embodiment of the present application that a combination of agents or therapies is administered to a subject in a non-contemporaneous fashion.

[0112] Effective amounts may vary according to factors such as the disease state, age, sex and / or weight of the subject. The amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of condition, disease or disorder, the identity of the subject being treated, and the like, but can nevertheless - 26 -be routinely determined by one skilled in the art. The effective amount is one that following treatment therewith manifests as an improvement in or reduction of any disease symptom.

[0113] In the context of treating inflammation or diseases, disorders or conditions associated with inflammation, in particular inflammation of the GI tract, an effective amount is an amount that, for example, reduces inflammation, compared to inflammation without administration of a compound of the application.

[0114] The dosage of a compound of the application can vary depending on many factors such as the pharmacodynamic properties of the compound, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment and the type of concurrent treatment, if any, and the clearance rate of the compound in the subject to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. Compounds of the application may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. Dosages will generally be selected to maintain a serum level of compounds from about 0.01 µg / cc to about 1000 µg / cc, or about 0.1 µg / cc to about 100 µg / cc. As a representative example, oral dosages of a compound of the application will range between about 1 mg per day to about 1000 mg per day for an adult, suitably about 10 mg, about 25 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600mg or about 650 mg of per day. For parenteral administration, a representative amount is from about 0.001 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 1 mg / kg or about 0.1 mg / kg to about 1 mg / kg will be administered. For oral administration, a representative amount is from about 0.001 mg / kg to about 10 mg / kg or about 0.1 mg / kg to about 10 mg / kg. For administration in suppository form, a representative amount is from about 0.1 mg / kg to about 10 mg / kg. In some embodiments, of the application, compositions are formulated for oral administration and the compounds are suitably in the form of tablets or suppositories containing 0.25, 0.5, 0.75, 1.0, 5.0, 10.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 75.0, 80.0, 90.0, 100.0, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg of active ingredient per tablet. Compounds of the application may be administered in a single daily, weekly or monthly dose or the total daily dose may be divided into two, three, four, five or six daily doses.

[0115] In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition is formulated for rectal administration. in some embodiments, the compounds of the application are administered in a dose of about 0.01 mg / kg body weight to about 250 mg / kg body weight, about 0.1 mg / kg to about 230 mg / kg, about 1 mg / kg to about 210 mg / kg, about 30 mg / kg to about 200 mg / kg, - 27 -about 50 mg / kg to about 180 mg / kg, about 60 mg / kg, to about 150 mg / kg, about 80 mg / kg to about 120 mg / kg and values therebetween in a single daily, weekly or monthly dose or the total daily dose may be divided into two, three, four, five or six daily doses.

[0116] In some embodiments, a compound of the application is administered at least once a week. However, in another embodiment, a compounds is administered to the subject from about one time per two weeks, three weeks or one month. In another embodiment, a compound of the application is administered about one time per week to about once daily. In another embodiment, a compound of the application is administered 2, 3, 4, 5 or 6 times daily. The length of the treatment period depends on a variety of factors, such as the severity of the disease, disorder or condition, the age of the subject, the concentration and / or the activity of the a compound of the application, and / or a combination thereof. It will also be appreciated that the effective dosage of the compound used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration is required. For example, the compounds are administered to the subject in an amount and for duration sufficient to treat the subject.

[0117] All references to “a compound” above, also include embodiments where one or more compounds are administered or used. IV. Methods of Preparing the Compounds of the Application

[0118] Compounds of the application can be prepared by various synthetic processes. The choice of particular structural features and / or substituents may influence the selection of one process over another. The selection of a particular process to prepare a given compound of the application is within the purview of the person of skill in the art. Some starting materials for preparing compounds of the application are available from commercial chemical sources. Other starting materials, for example as described below, are readily prepared from available precursors using straightforward transformations that are well known in the art. In the Schemes below showing the preparation of compounds of the application, all variables are as defined in Formula I, unless otherwise stated.

[0119] In some embodiments, where R5and R7are joined to form a 7 membered ring, a compound of Formula I is prepared as shown in Scheme 1, where R1-R7are as defined in Formula I and Pg is a suitable protecting group. - 28 -

[0120] Step a in Scheme 1 is an amide coupling which may be achieved using a variety of conditions known to those skilled in the art, for example, by activating the carboxylic acid with common coupling reagents (for example 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), propylphosphonic anhydride (T3P) or 1,1'-carbonyldiimidazole (CDI)) prior to reaction with the corresponding amine with a suitable non-nucleophilic base (for example triethylamine, N,N-diisopropylethylamine (DIPEA) or 4-dimethylaminopyridine (DMAP)) in an suitable polar aprotic solvent (for example dichloromethane (DCM), dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), tetrahydrofuran (THF) or EtOAc) with heating if necessary. Alternatively, the carboxylic acid may first be converted to an acyl halide (using for example thionyl chloride, oxalyl chloride, PCl3or PCl5) prior to reaction with the corresponding amine with a suitable non-nucleophilic base (for example triethylamine, DIPEA or DMAP) in a suitable polar aprotic solvent (for example DCM, DMF, NMP, THF or 1,4-dioxane) with heating if required.

[0121] Step b in Scheme 1 is an ester hydrolysis which may be achieved using a variety of conditions known to those skilled in the art, for example, by use of a suitable base (for example lithium hydroxide, sodium hydroxide or potassium hydroxide) in a suitable solvent (for example water, THF, 1,4-dioxane, methanol, ethanol or mixtures thereof) with heating if required.

[0122] Step c in Scheme 1 is an intramolecular ester coupling / cyclization which may be achieved using a variety of conditions known to those skilled in the art, for example, by activating the carboxylic acid with common coupling reagents (for example EDC, HOBt, HATU, T3P or CDI) prior to reaction with the corresponding amine with a suitable non-nucleophilic - 29 -base (for example triethylamine, DIPEA or DMAP) in a suitable polar aprotic solvent (for example DCM, CHCl3, DMF, NMP, THF or EtOAc) with heating if necessary. Alternatively, the carboxylic acid may first be converted to an acyl halide (using for example thionyl chloride, oxalyl chloride, PCl3or PCl5) prior to reaction with the corresponding amine with a suitable non-nucleophilic base (for example triethylamine, DIPEA or DMAP) in a suitable polar aprotic solvent (for example DCM, DMF, NMP, THF or 1,4-dioxane) with heating if required.

[0123] Step d in Scheme 1 is a thiolation reaction which may be achieved using a variety of thionating conditions known to those skilled in the art, for example, using reagents Phosphorus pentasulfide (P4S10), Lawesson's reagent (LR), elemental sulfur (S8), hexamethyldisilathiane, a combination of P4S10and hexamethyldisiloxane in a suitable solvent (for example, THF, MeCN, toluene or xylenes) with heating if required.

[0124] Step e in Scheme 1 is a deprotection which may be achieved using a variety of deprotecting conditions known to those skilled in the art depending on the identity of Pg. For example, when Pg is a t-butoxycarbonyl (t-BOC) protecting group, removal may achieved using a suitable acid (for example, hydrochloric acid or trifluoroacetic acid) in a suitable solvent (for example, DCM, MeOH, 1,4-dioxane, EtOAc or diethyl ether). Removal of Pg provides compounds of Formula I, wherein R1is H. Compounds of Formula I, wherein R1is other than H, are provided by reacting a compound of Formula I, wherein R1is H under standard nucleophilic substitution reaction conditions with a compound of the Formula R1-Lg, wherein Lg is a suitable leaving group, such as halo.

[0125] In some embodiments, where R5and R7are not joined to form a 7 membered ring, a compound of Formula I is prepared as shown in Scheme 2, where R1-R7are as defined in Formula I and Pg is a suitable protecting group.- 30 -Scheme 2

[0126] Step a in Scheme 2 is an amide coupling which may be achieved using a variety of conditions known to those skilled in the art, for example, by activating the carboxylic acid with common coupling reagents (for example 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), propylphosphonic anhydride (T3P) or 1,1'-carbonyldiimidazole (CDI)) prior to reaction with the corresponding amine with a suitable non-nucleophilic base (for example triethylamine, N,N-diisopropylethylamine (DIPEA) or 4-dimethylaminopyridine (DMAP)) in an suitable polar aprotic solvent (for example dichloromethane (DCM), dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), tetrahydrofuran (THF) or EtOAc) with heating if necessary. Alternatively, the carboxylic acid may first be converted to an acyl halide (using for example thionyl chloride, oxalyl chloride, PCl3or PCl5) prior to reaction with the corresponding amine with a suitable non-nucleophilic base (for example triethylamine, DIPEA or DMAP) in a suitable polar aprotic solvent (for example DCM, DMF, NMP, THF or 1,4-dioxane) with heating if required.

[0127] Step b in Scheme 2 is a thiolation reaction which may be achieved using a variety of thionating conditions known to those skilled in the art, for example, using reagents Phosphorus pentasulfide (P4S10), Lawesson's reagent (LR), elemental sulfur (S8), hexamethyldisilathiane, a combination of P4S10and hexamethyldisiloxane in a suitable solvent (for example, THF, MeCN, toluene or xylenes) with heating if required.

[0128] Step e in Scheme 2 is a deprotection which may be achieved using a variety of deprotecting conditions known to those skilled in the art depending on the identity of Pg. For example, when Pg is a t-butoxycarbonyl (t-BOC) protecting group, removal may achieved using a suitable acid (for example, hydrochloric acid or trifluoroacetic acid) in a suitable solvent (for example, DCM, MeOH, 1,4-dioxane, EtOAc or diethyl ether). Removal of Pg provides compounds of Formula I, wherein R1is H. Compounds of Formula I, wherein R1is other than H, are provided by reacting a compound of Formula I, wherein R1is H under standard nucleophilic substitution reaction conditions with a compound of the Formula R1-Lg, wherein Lg is a suitable leaving group, such as halo.

[0129] Generally, the reactions described above are performed in a suitable inert organic solvent and at temperatures and for times that will optimize the yield of the desired compounds. Examples of suitable inert organic solvents include, but are not limited to, dimethylformamide (DMF), 1,4-dioxane, methylene chloride, chloroform, tetrahydrofuran (THF), toluene, and the like. - 31 -

[0130] Salts of the compound of Formula I, or a pharmaceutically acceptable prodrug and / or solvate thereof, are generally formed by dissolving the neutral compound in an inert organic solvent and adding either the desired acid or base and isolating the resulting salt by either filtration or other known means.

[0131] The formation of solvates of the compound of Formula I, or a pharmaceutically acceptable salt and / or prodrug thereof, will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions. The selection of suitable conditions to form a particular solvate can be made by a person skilled in the art.

[0132] Prodrugs may be, for example, conventional esters formed with available hydroxy, thiol, amino or carboxyl groups. For example, available hydroxy or amino groups may be acylated using an activated acid in the presence of a base, and optionally, in inert solvent (e.g. an acid chloride in pyridine).

[0133] Throughout the processes described herein it is to be understood that, where appropriate, suitable protecting groups will be added to, and subsequently removed from, the various reactants and intermediates in a manner that will be readily understood by one skilled in the art. Conventional procedures for using such protecting groups as well as examples of suitable protecting groups are described, for example, in “Protective Groups in Organic Synthesis”, T.W. Green, P.G.M. Wuts, Wiley-Interscience, New York, (1999). It is also to be understood that a transformation of a group or substituent into another group or substituent by chemical manipulation can be conducted on any intermediate or final product on the synthetic path toward the final product, in which the possible type of transformation is limited only by inherent incompatibility of other functionalities carried by the molecule at that stage to the conditions or reagents employed in the transformation. Such inherent incompatibilities, and ways to circumvent them by carrying out appropriate transformations and synthetic steps in a suitable order, will be readily understood to one skilled in the art. Examples of transformations are given herein, and it is to be understood that the described transformations are not limited only to the generic groups or substituents for which the transformations are exemplified. References and descriptions of other suitable transformations are given in “Comprehensive Organic Transformations – A Guide to Functional Group Preparations” R.C. Larock, VHC Publishers, Inc. (1989). References and descriptions of other suitable reactions are described in textbooks of organic chemistry, for example, “Advanced Organic Chemistry”, March, 4th ed. McGraw Hill (1992) or, “Organic Synthesis”, Smith, McGraw Hill, (1994). Techniques for purification of intermediates and final products include, for example, straight - 32 -and reversed phase chromatography on column or rotating plate, recrystallisation, distillation and liquid-liquid or solid-liquid extraction, which will be readily understood by one skilled in the art.

[0134] The products of the processes of the application may be isolated according to known methods, for example, the compounds may be isolated by evaporation of the solvent, by filtration, centrifugation, and / or chromatography or other suitable method.

[0135] One skilled in the art will recognize that where a reaction step of the present application is carried out in a variety of solvents or solvent systems, said reaction step may also be carried out in a mixture of the suitable solvents or solvent systems.

[0136] The following non-limiting examples are illustrative of the present application: EXAMPLES General Methods

[0137] Unless otherwise noted, chemicals were purchased from Sigma-Aldrich Canada Ltd, Oakville, ON, Canada or Combi-Blocks, Inc., San Diego, CA, USA, or Ambeed, Inc., IL, USA, or Oakwood Products, Inc., SC, USA, or Enamine Ltd, USA. All solvents were HPLC grade and used as such or treated as specified for each case.

[0138] Flash column chromatography (FCC) was carried out using silica gel grade 60, mesh size 230–400 or C18 silica 100 Å 30 µm. When required, flash chromatographic purifications were performed on Biotage Isolera Prime systems (wavelength 254 and 280 nm) using pre-packed Biotage® or InnoflashTMflash cartridges.

[0139] Nuclear magnetic resonance spectra were recorded on Bruker 400-MHz Avance spectrometer (400 MHz for1H and 100 MHz for13C). Chemical shifts (δ) are reported in parts per million (ppm) after calibration to residual isotopic solvent or relative to tetramethylsilane. Coupling constants (J) are reported in Hz and reported to one decimal place. The splitting pattern for NMR spectra are denoted as follows: s (singlet), br (broad), d (doublet), t (triplet), m (multiplet), dd (doublet of doublet).

[0140] Mass spectrometry data were obtained on an Agilent 1100 series HPLC system equipped with an autosampler, binary pump, degasser, and a UV detector connected directly to a mass detector (Agilent 6110 Quadrupole LC / MS mass spectrometer) with an electrospray ionization (ESI) source. Chromatographic separations were carried out at room temperature using an Eclipse XDB-C18 column (5 μm particle size silica, 150 mm × 4.6 mm I.D.). The mobile phase consisted of a linear gradient of: H2O (0.1% formic acid) –CH3CN (0.1% formic acid) from 50 : 50 to 0 : 100 in 10 min and a flow rate of 1.0 mL min−1. Data acquisition was - 33 -carried out in positive or negative polarity mode, and data processing was carried out with Agilent Chemstation Software. Example 1A: (3S)-7-amino-3-methyl-5-thioxo-3,4-dihydro-1,4-benzoxazepin-2-one: (S)-I-1 S

[0141] A mixture of 5-(tert-butoxycarbonylamino)-2-hydroxy-benzoic acid (1.5 g, 5.92 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.70 g, 8.88 mmol), hydroxybenzotriazole (1.20 g, 8.88 mmol), triethylamine (1.20 g, 11.85 mmol, 1.65 mL) in dichloromethane (DCM) (20 mL) was stirred at rt for 1 h. L-alanine methyl ester hydrochloride (830 mg, 5.92 mmol) was added and the mixture was stirred at rt for 18 h. The mixture was concentrated and purified by flash column chromatography (ethyl acetate (EtOAc)-hexane) to give methyl (2S)-2-[[5-(tert-butoxycarbonylamino)-2-hydroxy-benzoyl]amino]propanoate (1.1 g, 55% yield).1H NMR (400 MHz, Chloroform-d): ^ 11.87 (1H, s), 7.74 (1H, s), 7.18 (1H, dd, J = 8.9, 2.5 Hz), 6.94 (1H, s), 6.94 (1H, d, J = 8.9 Hz), 6.37 (1H, s), 4.82-4.84 (1H, m), 3.83 (3H, s), 1.56 (3H, d, J = 7.2 Hz), 1.55 (9H, s). ESI-MS [M+H]+: 339.0. Step b:

[0142] A mixture of methyl (2S)-2-[[5-(tert-butoxycarbonylamino)-2-hydroxy- benzoyl]amino]propanoate (1.1 g, 3.25 mmol) and NaOH 1N (32 mL) in MeOH (100 mL) was stirred at rt for 2 h. The mixture was concentrated, diluted with HCl 1N and extracted with EtOAc. The organic extract was dried over Na2SO4, filtered and concentrated to give (2S)-2- [[5-(tert-butoxycarbonylamino)-2-hydroxy-benzoyl]amino]propanoic acid (1.0 g, 98% yield), which was used for the next step without further purification. ESI-MS [M+H]+: 325.0. Step c: - 34 -

[0143] To a stirred solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (910 mg, 4.76 mmol), hydroxybenzotriazole (640 mg, 4.76 mmol) in DCM (500 mL) at rt was added a solution of (2S)-2-[[5-(tert-butoxycarbonylamino)-2-hydroxy- benzoyl]amino]propanoic acid (1.0 g, 3.18 mmol) in DCM (500 mL). The reaction was stirred at rt for 18 h and concentrated. The crude was purified by flash column chromatography (EtOAc-hexane) to give tert-butyl N-[(3S)-3-methyl-2,5-dioxo-3,4-dihydro-1,4-benzoxazepin- 7-yl]carbamate (600 mg, 62% yield).1H NMR (400 MHz, Chloroform-d): ^ 7.81 (1H, d, J = 9.0 Hz), 7.77 (1H, d, J = 2.7 Hz), 7.19 (1H, d, J = 8.9 Hz), 6.71 (1H, s), 6.38 (1H, s), 4.13-4.08 (1H, m), 1.59 (3H, d, J = 6.7 Hz), 1.56 (9H, s). ESI-MS [M-H]-: 305.0. Step d:

[0144] A mixture of tert-butyl N-[(3S)-3-methyl-2,5-dioxo-3,4-dihydro-1,4- benzoxazepin-7-yl]carbamate (150 mg, 0.49 mmol), Lawesson’s reagent (200 mg, 0.49 mmol) in tetrahydrofuran (THF) (3 mL) in a sealed vial was stirred at 65 °C under nitrogen. After 1 h, the mixture was concentrated and purified by flash column chromatography (EtOAc- hexane) to give tert-butyl N-[(3S)-3-methyl-2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7- yl]carbamate (130 mg, 82% yield).1H NMR (400 MHz, Chloroform-d): ^ 8.01-7.96 (2H, m), 7.79 (1H, s), 7.14 (1H, d, J = 8.9 Hz), 6.71 (1H, s), 4.30-4.27 (1H, m), 1.63 (3H, d, J = 6.7 Hz), 1.55 (10H, s). ESI-MS [M+H]+: 323.0. step e:

[0145] To a solution of tert-butyl N-[(3S)-3-methyl-2-oxo-5-thioxo-3,4-dihydro-1,4- benzoxazepin-7-yl]carbamate (120 mg, 0.37 mmol) in DCM (3 mL) at rt was added trifluoroacetic acid (1 mL, 12.98 mmol). The mixture was stirred for 1 h and concentrated under rotary evaporator. The crude mixture was purified by flash column chromatography (EtOAc-hexane) to give (3S)-7-amino-3-methyl-5-thioxo-3,4-dihydro-1,4- benzoxazepin-2-one: (S)-I-1 (52 mg, 60% yield) as a yellow solid.1H NMR (400 MHz, Chloroform-d): ^ 7.82 (1H, s), 7.44 (1H, d, J = 2.9 Hz), 6.99 (1H, d, J = 8.7 Hz), 6.86 (1H, dd, J = 8.7, 2.9 Hz), 4.34-4.28 (1H, m), 1.62 (4H, d, J = 6.8 Hz). ESI-MS [M+H]+: 222.9. Example 1B: (3S)-7-amino-3-benzyl-5-thioxo-3,4-dihydro-1,4-benzoxazepin-2-one: (S)-I-2 - 35 -

[0146] To a mixture of 5-[1,1-di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzoic acid (1.0 g, 3.95 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 g, 5.92 mmol), hydroxybenzotriazole (800 mg, 5.92 mmol), triethylamine (1.1 Ml, 7.90 mmol) in DCM (20 Ml) was added L-phenylalanine methyl ester hydrochloride (850 mg, 3.95 mmol) and the mixture was stirred at rt for 20 h. The mixture was concentrated and purified by flash column chromatography (EtOAc-hexane) to give methyl (2S)-2-[[5-[1,1- di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzoyl]amino]-3-phenyl-propanoate (840 mg, 50% yield) as a white solid.1H NMR (400 MHz, Chloroform-d): ^ 11.72 (1H, s), 7.59 (1H, s), 7.38-7.19 (6H, m), 6.93 (1H, d, J = 8.9 Hz), 6.81 (1H, d, J = 7.6 Hz), 6.34 (1H, s), 5.06-5.01 (1H, m), 3.79 (3H, s), 3.31-3.22 (2H, m), 1.54 (9H, s). ESI-MS [M+H]+: 415.2. Step b:

[0147] A mixture of methyl (2S)-2-[[5-[1,1-di(methyl)ethoxycarbonylamino]-2- oxidanyl-benzoyl]amino]-3-phenyl-propanoate (840 mg, 2.03 mmol) and NaOH 1N (20 Ml) in methanol (60 Ml) was stirred at rt for 4 h. The mixture was concentrated, diluted with HCl 1N and extracted with EtOAc. The organic extract was dried over Na2SO4, filtered and concentrated to give (2S)-2-[[5-[1,1-di(methyl)ethoxycarbonylamino]-2-oxidanyl- benzoyl]amino]-3-phenyl-propanoic acid (800 mg, 99% yield). ESI-MS [M+H]+: 400.9. Step c:

[0148] To a stirred solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (570 mg, 3.0 mmol), hydroxybenzotriazole (405 mg, 3.0 mmol) in DCM (400 Ml) at rt was added a solution of (2S)-2-[[5-[1,1-di(methyl)ethoxycarbonylamino]-2-oxidanyl- benzoyl]amino]-3-phenyl-propanoic acid (800 mg, 2.0 mmol) in DCM (400 Ml). The reaction - 36 -was stirred at rt for 20 h and concentrated under rotary evaporator. The crude was purified by flash column chromatography (EtOAc-hexane) to give 1,1-di(methyl)ethyl N-[(3S)-3-benzyl-5- oxidanylidene-2-oxo-3,4-dihydro-1,4-benzoxazepin-7-yl]carbamate (510 mg, 67% yield) as a white solid.1H NMR (400 MHz, Chloroform-d) δ 7.81 – 7.69 (m, 2H), 7.37 – 7.29 (m, 3H), 7.27 – 7.23 (m, 2H), 7.18 (d, J = 8.8 Hz, 1H), 6.69 (s, 1H), 6.36 (d, J = 4.9 Hz, 1H), 4.21 (dt, J = 8.3, 5.7 Hz, 1H), 3.43 (dd, J = 14.6, 6.1 Hz, 1H), 3.11 (dd, J = 14.6, 8.3 Hz, 1H), 1.54 (s, 9H). ESI-MS [M+H]+: 381.1. Step d:

[0149] A mixture of 1,1-di(methyl)ethyl N-[(3S)-3-benzyl-5-oxidanylidene-2-oxo-3,4- dihydro-1,4-benzoxazepin-7-yl]carbamate (0.78 mmol), Lawesson's reagent (320 mg, 0.78 mmol) in anhydrous THF (5 Ml) in a sealed vial was stirred at 65 °C under nitrogen. After 1 h, the mixture was concentrated and purified by flash column chromatography (EtOAc-hexane) to give tert-butyl N-[(3S)-3-benzyl-2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7- yl]carbamate (240 mg, 77% yield) as a yellow solid.1H NMR (400 MHz, Chloroform-d) δ 8.04 – 7.90 (m, 2H), 7.73 (d, J = 8.8 Hz, 1H), 7.38 – 7.19 (m, 5H), 7.09 (d, J = 8.9 Hz, 1H), 6.64 (s, 1H), 4.37 (q, J = 6.7 Hz, 1H), 3.44 (dd, J = 14.6, 6.6 Hz, 1H), 3.17 (dd, J = 14.6, 7.7 Hz, 1H), 1.52 (s, 9H). ESI-MS [M+H]+: 399.2. Step e:

[0150] A solution of 1,1-di(methyl)ethyl N-[(3S)-3-benzyl-2-oxo-5-sulfanylidene-3,4- dihydro-1,4-benzoxazepin-7-yl]carbamate (160 mg, 0.40 mmol) in trifluoroacetic acid (1.0 Ml, 12.98 mmol) and anhydrous DCM (1.0 Ml) was stirred at rt for 1.5 h. The mixture was concentrated and purified by flash column chromatography (EtOAc-hexane) to give (3S)-7- amino-3-benzyl-5-thioxo-3,4-dihydro-1,4-benzoxazepin-2-one: (S)-I-2 (33 mg, 28% yield) as a yellow solid.

[0151] 1H NMR (400 MHz, Chloroform-d) δ 7.86 (s, 1H), 7.38 – 7.24 (m, 6H), 6.95 (d, J = 8.7 Hz, 1H), 6.82 (dd, J = 8.7, 2.9 Hz, 1H), 4.39 (dt, J = 7.8, 6.2 Hz, 1H), 3.44 (dd, J = 14.7, 6.5 Hz, 1H), 3.15 (dd, J = 14.7, 7.8 Hz, 1H). ESI-MS [M+H]+: 298.9. Example 1C: (3S)-7-azanyl-3-(1-methylethyl)-5-sulfanylidene-3,4-dihydro-1,4-benzoxazepin- 2-one: (S)-I-3 and (3S)-7-amino-3-isopropyl-5-thioxo-3,4-dihydro-1,4-benzoxazepin-2- one;2,2,2-trifluoroacetic acid: (S)-I-7 and 1,1-di(methyl)ethyl N-[(3S)-3-(1-methylethyl)-2-oxo- 5-sulfanylidene-3,4-dihydro-1,4-benzoxazepin-7-yl]carbamate: (S)-I-8 - 37 -

[0152] To a mixture of 5-[1,1-di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzoic acid (1.0 g, 3.95 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (1.14 g, 5.92 mmol), hydroxybenzotriazole (HOBt) (800 mg, 5.92 mmol), triethylamine (1.1 mL, 7.90 mmol) in DCM (20 mL) was added L-valine methyl ester hydrochloride (660 mg, 3.95 mmol) and the mixture was stirred at rt for 20 h. The mixture was concentrated and purified by flash column chromatography using EtOAc-hexane to give methyl (2S)-2-[[5-[1,1- di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzoyl]amino]-3-methyl-butanoate (520 mg, 36% yield). ).1H NMR (400 MHz, Chloroform-d): ^11.8 (1H, s), 7.65 (1H, s), 7.29-7.26 (1H, m), 6.95 (1H, d, J = 9.0 Hz), 6.87 (1H, d, J = 8.5 Hz), 6.38 (1H, s), 4.73 (1H, dd, J = 8.5, 5.0 Hz), 3.82 (3H, s), 2.35-2.26 (1H, m), 1.55 (9H, s), 1.04 (6H, dd, J = 7.0, 1.5 Hz). ESI-MS [M+H]+: 367.1. Step b:

[0153] A mixture of methyl (2S)-2-[[5-[1,1-di(methyl)ethoxycarbonylamino]-2-oxidanyl- benzoyl]amino]-3-methyl-butanoate (520 mg, 1.42 mmol) in NaOH 1N (15 mL) and methanol (50 mL) was stirred at rt for 2 h. Additional NaOH 1N (15 mL) was added and reaction was stirred for another 3 h. The mixture was concentrated, diluted with HCl 1N and extracted with EtOAc. The organic extract was dried over Na2SO4, filtered and concentrated to give (2S)-2- - 38 -[[5-[1,1-di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzoyl]amino]-3-methyl-butanoic acid (520 mg, 98% yield). ESI-MS [M+H]+: 353.0. Step c:

[0154] To a stirred solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (425 mg, 2.21 mmol), hydroxybenzotriazole (300 mg, 2.21 mmol) in DCM (400 mL) at rt was added (2S)-2-[[5-[1,1-di(methyl)ethoxycarbonylamino]-2-oxidanyl- benzoyl]amino]-3-methyl-butanoic acid (520 mg, 1.48 mmol). The reaction was stirred at rt for 18 h and concentrated under rotary evaporator. The crude was purified by flash column chromatography using EtOAc-hexane to give 1,1-di(methyl)ethyl N-[(3S)-3-(1-methylethyl)-5- oxidanylidene-2-oxo-3,4-dihydro-1,4-benzoxazepin-7-yl]carbamate (140 mg, 28% yield).1H NMR (400 MHz, Chloroform-d): ^ 7.79-7.76 (2H, m), 7.19 (1H, d, J = 9.0 Hz), 6.68 (1H, s), 6.26 (1H, br), 3.66 (1H, dd, J = 8.0, 5.0 Hz), 2.36-2.28 (1H, m), 1.56 (9H, s), 1.13 (6H, dd, J = 8.0, 7.0 Hz). ESI-MS [M+H]+: 335.2. Step d: (S)-I-8

[0155] A mixture of 1,1-di(methyl)ethyl N-[(3S)-3-(1-methylethyl)-5-oxidanylidene-2- oxo-3,4-dihydro-1,4-benzoxazepin-7-yl]carbamate (140 mg, 0.42 mmol) , Lawesson's reagent (170 mg, 0.42 mmol) in anhydrous THF (2 mL) was stirred at 65 °C in a sealed vial. After 1.5 h, the mixture was concentrated and purified by flash column chromatography using EtOAc- hexane to give 1,1-di(methyl)ethyl N-[(3S)-3-(1-methylethyl)-2-oxo-5-sulfanylidene-3,4- dihydro-1,4-benzoxazepin-7-yl]carbamate (110 mg, 74% yield).

[0156] 1H NMR (400 MHz, Chloroform-d): ^ 7.99 (1H, d, J = 3.0 Hz), 7.98 (1H, s), 7.75 (1H, d, J = 9.0 Hz), 7.11 (1H, d, J = 9.0 Hz), 6.64 (1H, s), 3.79 (1H, dd, J = 8.0, 6.5 Hz), 2.35- 2.34 (1H, m), 1.52 (9H, s), 1.15 (3H, d, J = 7.0, 3.0 Hz), 1.11 (3H, d, J = 7.0, 3.0 Hz). ESI-MS [M+H]+: 351.0. Step e: (S)-I-3

[0157] To a solution of 1,1-di(methyl)ethyl N-[(3S)-3-(1-methylethyl)-2-oxo-5- sulfanylidene-3,4-dihydro-1,4-benzoxazepin-7-yl]carbamate (1.1 g, 3.14 mmol) in DCM (30 mL) at rt was added trifluoroacetic acid (10 mL) and the mixture was stirred for 2 h. The mixture was concentrated and purified by flash column chromatography using EtOAc-hexane. The obtained product was partitioned between aq. NaHCO3and DCM, the organic extract was dried over Na2SO4and concentrated to give (3S)-7-azanyl-3-(1-methylethyl)-5-sulfanylidene- 3,4-dihydro-1,4-benzoxazepin-2-one (55 mg, 55% yield) as a yellow solid. - 39 -

[0158] 1H NMR (400 MHz, Chloroform-d): ^ 7.95 (1H, s), 7.41 (1H, d, 3.0 Hz), 6.97 (1H, d, J = 9.0 Hz), 6.84 (1H, dd, J = 9.0, 3.0 Hz), 3.82 (1H, dd, J = 8.0, 6.0 Hz), 2.36-2.34 (1H, m), 1.15 (3H, d, J = 7.0 Hz), 1.11 (3H, d, J = 7.0 Hz). ESI-MS [M+H]+: 250.9. Step f: (S)-I-7

[0159] To a solution of tert-butyl (S)-(3-isopropyl-2-oxo-5-thioxo-2,3,4,5- tetrahydrobenzo[f][1,4]oxazepin-7-yl)carbamate (3.0 g, 8.56 mmol) in anhydrous DCM (30 mL) at 0 °C, trifluoroacetic acid (TFA) (15.72 mL, 205.462 mmol) was added dropwise and the mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated, the crude was triturated with diethyl ether (20 mL) and pentane (45 mL) to get (3S)-7-amino- 3-isopropyl-5-thioxo-3,4-dihydro-1,4-benzoxazepin-2-one;2,2,2-trifluoroacetic acid (1.48 g) as yellow solid.

[0160] 1H NMR (400 MHz, Chloroform-d) δ 7.94 (s, 1H), 7.41 (d, J = 2.8 Hz, 1H), 6.97 (d, J = 8.4 Hz, 2H), 6.84 (dd, J = 8.8 Hz, 2.8 Hz, 1H), 3.81 (t, J = 7.6 Hz, 6 Hz, 1H), 1.13 (dd, J = 16.8 Hz, 6.8 Hz, 6H). ESI-MS m / z 251.09 [M+H]+. Example 1D: (3S)-7-azanyl-3-(2-methylpropyl)-5-sulfanylidene-3,4-dihydro-1,4- benzoxazepin-2-one: (S)-I-4Step a:

[0161] To a mixture of 5-[1,1-di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzoic acid (2 g, 7.90 mmol) , 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.27 g, 11.85 mmol), hydroxybenzotriazole (1.60 g, 11.85 mmol) , triethylamine (1.60 g, 15.79 mmol, 2.20 mL) in DCM (40 mL) was added L-leucine methyl ester hydrochloride (1.43 g, 7.90 mmol) and the mixture was stirred at rt for 20 h. The mixture was concentrated and purified by flash column chromatography (EtOAc-hexane) to give methyl (2S)-2-[[5-[1,1- - 40 -di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzoyl]amino]-4-methyl-pentanoate (1.2 g, 40% yield) as a solid..1H NMR (400 MHz, Chloroform-d) δ 11.87 (s, 1H), 7.71 (s, 1H), 7.14 (dd, J = 8.9, 2.6 Hz, 1H), 7.00 – 6.85 (m, 2H), 6.39 (s, 1H), 4.82 (s, 1H), 3.78 (s, 3H), 3.48 (d, J = 5.0 Hz, 1H), 1.75 – 1.65 (m, 3H), 1.52 (s, 9H), 1.02 – 0.93 (m, 6H). ESI-MS [M+H]+: 380.7. Step b:

[0162] To a solution of methyl (2S)-2-[[5-[1,1-di(methyl)ethoxycarbonylamino]-2- oxidanyl-benzoyl]amino]-4-methyl-pentanoate (1.2 g, 3.15 mmol) in methanol (90 mL) was added NaOH 1N (47 mL) and the mixture was stirred at rt for 4 hr . The mixture was concentrated, diluted with HCl 1N and extracted with EtOAc. The organic extract was dried over Na2SO4, filtered and concentrated to give the (2S)-2-[[5-[1,1- di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzoyl]amino]-4-methyl-pentanoic acid (1.16 g, 98% yield).1H NMR (400 MHz, DMSO-d6) δ 12.68 (s, 1H), 11.42 (s, 1H), 9.11 (s, 1H), 8.82 (d, J = 7.7 Hz, 1H), 7.92 (d, J = 2.7 Hz, 1H), 7.46 – 7.28 (m, 1H), 6.83 (d, J = 8.8 Hz, 1H), 4.51-4.45 (m, 1H), 1.78 – 1.57 (m, 3H), 1.46 (s, 9H), 0.91 (dd, J = 10.2, 5.9 Hz, 6H). Step c:

[0163] To a stirred suspension of (2S)-2-[[5-[1,1-di(methyl)ethoxycarbonylamino]-2- oxidanyl-benzoyl]amino]-4-methyl-pentanoic acid (1.16 g, 3.15 mmol) in DCM (700 mL) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (900 mg, 4.73 mmol) , followed by hydroxybenzotriazole (640 mg, 4.73 mmol). The mixture was stirred at rt for 20 h and concentrated. The crude was purified by flash column chromatography (EtOAc-hexane) to give 1,1-di(methyl)ethyl N-[(3S)-3-(2-methylpropyl)-5-oxidanylidene-2-oxo-3,4-dihydro-1,4- benzoxazepin-7-yl]carbamate (800 mg, 73% yield) as a white solid.1H NMR (400 MHz, Chloroform-d) δ 7.86 – 7.71 (m, 2H), 7.16 (d, J = 8.9 Hz, 1H), 6.93 – 6.67 (m, 2H), 3.95-3.90 (m, 1H), 1.94-1.68 (m, 3H), 1.52 (s, 9H), 0.94 (d, J = 6.4 Hz, 3H), 0.88 (d, J = 6.3 Hz, 3H). ESI-MS [M+H]+: 348.9. Step d:

[0164] A mixture of 1,1-di(methyl)ethyl N-[(3S)-3-(2-methylpropyl)-5-oxidanylidene-2- oxo-3,4-dihydro-1,4-benzoxazepin-7-yl]carbamate (300 mg, 0.86 mmol) , Lawesson's reagent (350 mg, 0.86 mmol) in anhydrous THF (5 mL) in a sealed vial was stirred at 66 °C for 1.5 h. The mixture was concentrated and purified by flash column chromatography (EtOAc-hexane) to give 1,1-di(methyl)ethyl N-[(3S)-3-(2-methylpropyl)-2-oxo-5-sulfanylidene-3,4-dihydro-1,4- benzoxazepin-7-yl]carbamate (290 mg, 92% yield) as a yellow solid.1H NMR (400 MHz, Chloroform-d) δ 7.99 (d, J = 2.8 Hz, 1H), 7.92 (s, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.12 (d, J = 8.9 Hz, 1H), 6.66 (s, 1H), 4.17 – 4.06 (m, 1H), 1.97– 1.75 (m, 3H), 1.53 (s, 9H), 0.95 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.3 Hz, 3H). - 41 -Step e:

[0165] To a solution of 1,1-di(methyl)ethyl N-[(3S)-3-(2-methylpropyl)-2-oxo-5- sulfanylidene-3,4-dihydro-1,4-benzoxazepin-7-yl]carbamate (200 mg, 0.55 mmol) in DCM (3 mL) was added trifluoroacetic acid (1.0 mL, 12.98 mmol). After stirring at rt for 1.5 h, the mixture was concentrated and purified by flash column chromatography (EtOAc-hexane) to give (3S)-7-azanyl-3-(2-methylpropyl)-5-sulfanylidene-3,4-dihydro-1,4-benzoxazepin-2-one: (S)-I-4 (85 mg, 59% yield) as a yellow solid.1H NMR (400 MHz, Chloroform-d) δ 7.90 (s, 1H), 7.42 (d, J = 2.9 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 6.85 (dd, J = 8.7, 2.9 Hz, 1H), 4.15-4.10 (m, 1H), 1.95-1.75 (m, 3H), 0.96 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.4 Hz, 3H). ESI-MS [M+H]+: 265.2. Example 1E: (3S)-7-azanyl-3-(2-methylsulfanylethyl)-5-sulfanylidene-3,4-dihydro-1,4- benzoxazepin-2-one: (S)-I-5Step a:

[0166] To a mixture of 5-[1,1-di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzoic acid (2.0 g, 7.90 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.27 g, 11.85 mmol), hydroxybenzotriazole (1.60 g, 11.85 mmol), triethylamine (2.2 mL, 15.79 mmol) in DCM (40 mL) was added L-methionine methyl ester hydrochloride (1.58 g, 7.90 mmol) and the mixture was stirred at rt for 20 h. The mixture was concentrated and purified by flash column chromatography (EtOAc-hexane) to give methyl (2S)-2-[[5-[1,1- di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzoyl]amino]-4-methylsulfanyl-butanoate (1.55 g, 49% yield) as a solid.1H NMR (400 MHz, Chloroform-d) δ 11.83 (s, 1H), 7.76 (s, 1H), 7.45 (s, 1H), 7.14 (dd, J = 8.9, 2.6 Hz, 1H), 6.90 (d, J = 8.9 Hz, 1H), 6.38 (s, 1H), 4.90 (td, J = 7.4, 5.0 Hz, 1H), 3.80 (s, 3H), 2.65 – 2.56 (m, 2H), 2.32-2.23 (m, 1H), 2.21 – 2.08 (m, 4H), 1.51 (s, 9H). ESI-MS [M+H]+: 399.0. - 42 -Step b:

[0167] To a solution of methyl (2S)-2-[[5-[1,1-di(methyl)ethoxycarbonylamino]-2- oxidanyl-benzoyl]amino]-4-methylsulfanyl-butanoate (1.55 g, 3.89 mmol) in methanol (120 mL) was added NaOH 1N (58 mL) and the mixture was stirred at rt for 4 hr. The mixture was concentrated, diluted with HCl 1N and extracted with EtOAc. The organic extract was dried over Na2SO4, filtered and concentrated to give (2S)-2-[[5-[1,1- di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzoyl]amino]-4-methylsulfanyl-butanoic acid (1.49 g, 98% yield).1H NMR (400 MHz, DMSO-d6) δ 12.87 (s, 1H), 11.41 (s, 1H), 9.13 (s, 1H), 8.91 (d, J = 7.4 Hz, 1H), 7.93 (d, J = 2.7 Hz, 1H), 7.37 (d, J = 9.0 Hz, 1H), 6.85 (d, J = 8.8 Hz, 1H), 4.64 – 4.54 (m, 1H), 2.60 – 2.48 (m, 2H), 2.16-2.02 (m, 5H), 1.47 (s, 9H). Step c:

[0168] To a stirred suspension of (2S)-2-[[5-[1,1-di(methyl)ethoxycarbonylamino]-2- oxidanyl-benzoyl]amino]-4-methylsulfanyl-butanoic acid (1.5 g, 3.90 mmol) in DCM (700 mL) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.12 g, 5.85 mmol) , followed by hydroxybenzotriazole (790 mg, 5.85 mmol). The mixture was stirred at rt for 20 h and concentrated. The crude was purified by flash column chromatography (EtOAc- hexane) to give 1,1-di(methyl)ethyl N-[(3S)-3-(2-methylsulfanylethyl)-5-oxidanylidene-2-oxo- 3,4-dihydro-1,4-benzoxazepin-7-yl]carbamate (950 mg, 66% yield) as a white solid.1H NMR (400 MHz, Chloroform-d) δ 7.81 – 7.71 (m, 2H), 7.17 (d, J = 8.9 Hz, 1H), 6.89 (d, J = 5.3 Hz, 1H), 6.70 (s, 1H), 4.24 – 4.16 (m, 1H), 2.69 (t, J = 6.7 Hz, 2H), 2.41-2.32 (m, 1H), 2.17-2.09 (m, 1H), 2.08 (s, 3H), 1.53 (s, 9H). ESI-MS [M+H]+: 366.9. Step d:

[0169] A mixture of 1,1-di(methyl)ethyl N-[(3S)-3-(2-methylsulfanylethyl)-5- oxidanylidene-2-oxo-3,4-dihydro-1,4-benzoxazepin-7-yl]carbamate (300 mg, 0.82 mmol) , Lawesson's reagent (330 mg, 0.82 mmol) in anhydrous THF (5 mL) in a sealed vial was stirred at 65 °C for 1.5 h. The mixture was concentrated and purified by flash column chromatography (EtOAc-hexane) to give 1,1-di(methyl)ethyl N-[(3S)-3-(2- methylsulfanylethyl)-2-oxo-5-sulfanylidene-3,4-dihydro-1,4-benzoxazepin-7-yl]carbamate (270 mg, 86% yield) as a yellow solid.

[0170] 1H NMR (400 MHz, Chloroform-d) δ 8.37 (d, J = 5.8 Hz, 1H), 7.98 (d, J = 2.8 Hz, 1H), 7.77 (s, 1H), 7.12 (d, J = 8.9 Hz, 1H), 6.70 (s, 1H), 4.38 (q, J = 6.5 Hz, 1H), 2.75-2.64 (m, 2H), 2.44-2.35 (m, 1H), 2.21 – 2.12 (m, 1H), 2.08 (s, 3H), 1.52 (s, 9H). Step e: - 43 -

[0171] To a solution of 1,1-di(methyl)ethyl N-[(3S)-3-(2-methylsulfanylethyl)-2-oxo-5- sulfanylidene-3,4-dihydro-1,4-benzoxazepin-7-yl]carbamate (200 mg, 0.52 mmol) in DCM (3 mL) was added trifluoroacetic acid (1.0 mL, 12.98 mmol). After stirring at rt for 1.5 h, the mixture was concentrated and purified by flash column chromatography (EtOAc-hexane) to give product as a yellow solid. (3S)-7-azanyl-3-(2-methylsulfanylethyl)-5-sulfanylidene-3,4- dihydro-1,4-benzoxazepin-2-one: (S)-I-5 (59 mg, 40% yield).1H NMR (400 MHz, Chloroform- d) δ 8.16 (s, 1H), 7.41 (d, J = 2.9 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 6.84 (dd, J = 8.7, 2.9 Hz, 1H), 4.40 (q, J = 6.6 Hz, 1H), 2.77 – 2.63 (m, 2H), 2.43-2.35 (m, 1H), 2.19-2.12 (m, 1H), 2.09 (s, 3H). ESI-MS [M+H]+: 283.1. Example 1F: (3S,4S)-7-azanyl-4-methyl-3-(1-methylethyl)-5-sulfanylidene-3H-1,4- benzoxazepin-2-one: (S)-I-6

[0172] To a solution of 1,1-di(methyl)ethyl N-[(3S)-3-(1-methylethyl)-5-oxidanylidene- 2-oxo-3,4-dihydro-1,4-benzoxazepin-7-yl]carbamate (200 mg, 0.60 mmol) in DMF (3 mL) at 0 °C was added sodium hydride (60% dispersion in mineral oil) (24 mg, 0.60 mmol) in portions. After stirring for 15 min at 0 °C, iodomethane (56 μL, 0.90 mmol) was added dropwise and the mixture was stirred at 0 °C for 30 min. The mixture was diluted with EtOAc and washed with sat. NH4Cl. The organic extract was dried over Na2SO4, filtered and concentrated. The crude was purified by flash column chromatography (EtOAc-hexane) to give 1,1-di(methyl)ethyl N- [(3S,4S)-4-methyl-3-(1-methylethyl)-5-oxidanylidene-2-oxo-3H-1,4-benzoxazepin-7- yl]carbamate as a white solid (124 mg, 60% yield).1H NMR (400 MHz, Chloroform-d) δ 7.84 (s, 1H), 7.69 (s, 1H), 7.14 (d, J = 8.9 Hz, 1H), 6.73 (s, 1H), 3.80 (d, J = 11.1 Hz, 1H), 3.32 (br, 3H), 1.58-1.55 (m, 10H), 0.94 (br, 6H). ESI-MS [M+H]+: 349.3.

[0173] A mixture of 1,1-di(methyl)ethyl N-[(3S,4S)-4-methyl-3-(1-methylethyl)-5- oxidanylidene-2-oxo-3H-1,4-benzoxazepin-7-yl]carbamate (120 mg, 0.34 mmol) and Lawesson's Reagent (140 mg, 0.34 mmol) in THF (2 mL) was stirred at 65 °C for 2 h. Additional Lawesson's Reagent (140 mg, 0.34 mmol) was added and reaction was continued for another 16 h. The mixture was concentrated and purified by flash column chromatography (EtOAc- - 44 -hexane) to give 1,1-di(methyl)ethyl N-[(3S,4S)-4-methyl-3-(1-methylethyl)-2-oxo-5- sulfanylidene-3H-1,4-benzoxazepin-7-yl]carbamate as a yellow solid. ESI-MS [M+H]+: 365.2.

[0174] To a solution of 1,1-di(methyl)ethyl N-[(3S,4S)-4-methyl-3-(1-methylethyl)-2- oxo-5-sulfanylidene-3H-1,4-benzoxazepin-7-yl]carbamate (50 mg, 0.14 mmol) in DCM (2 mL) at rt was added trifluoroacetic acid (1 mL) . After stirring at for 1 h, the mixture was concentrated and diluted with aq. NaHCO3. The mixture was extracted with DCM, the organic extract dried over Na2SO4and concentrated. The crude was purified by flash column chromatography (DCM 100%) and to give (3S,4S)-7-azanyl-4-methyl-3-(1-methylethyl)-5- sulfanylidene-3H-1,4-benzoxazepin-2-one: (S)-I-6 as a yellow solid (20 mg, 55% yield).

[0175] 1H NMR (400 MHz, Chloroform-d) δ 7.42 (dd, J = 8.3, 2.9 Hz, 1H), 6.96 – 6.87 (m, 1H), 6.83-6.76 (m, 1H), 4.17-4.13 (m, 1H), 3.83 (br, 2H), 3.55 (s, 3H), 2.62-2.53 (m, 1H), 1.05-1.03 (m, 6H). Minor isomer signals at 3.80 (s), 1.73-1.63 (m), 0.90-0.88 (m). ESI-MS [M+H]+: 265.2. Example 1G: N-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7- yl]acetamide: (S)-I-9

[0176] To a mixture of (3S)-7-amino-3-isopropyl-5-thioxo-3,4-dihydro-1,4- benzoxazepin-2-one;2,2,2-trifluoroacetic acid salt (70 mg, 0.19 mmol) in DCM (2 mL) at 0°C was added triethylamine (53 μL, 0.38 mmol), followed by the dropwise addition of acetic anhydride (16 μL, 0.172 mmol). The resulting mixture was then stirred at rt for 5 h. Additional triethylamine (75 μL, 0.576 mmol) and acetic anhydride (30 μL, 0.318 mmol) were added and the reaction was stirred for another hour. The mixture was diluted with EtOAc and washed with aq. NaHCO3. The organic extract was dried over Na2SO4, concentrated and purified by flash column chromatography (EtOAc-hexane) to give N-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4- dihydro-1,4-benzoxazepin-7-yl]acetamide: (S)-I-9 (10 mg, 18% yield).1H NMR (400 MHz, Chloroform-d) δ 8.06 (d, J = 8.8 Hz, 1H), 8.00 (d, J = 2.8 Hz, 2H), 7.37 (s, 1H), 7.17 (d, J = 8.9 Hz, 1H), 3.87 – 3.80 (m, 1H), 2.38 (s, 1H), 2.23 (s, 3H), 1.16 (dd, J = 17.0, 6.7 Hz, 6H). ESI- MS [M+H]+293.2. Example 1H: N-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7-yl]-2,2- dimethyl-propanamide: (S)-I-10 - 45 -

[0177] xo-3,4-dihydro-1,4- benzoxazepin-2-one (80 mg, 0.32 mmol), triethylamine (135 μL, 0.96 mmol) in DCM (2 mL) at 0°C was added trimethylacetyl chloride (39 μL, 0.32 mmol) dropwise. The mixture was stirred at rt for 3 h, diluted with aq. NaHCO3and extracted with EtOAc. The organic extract was dried over Na2SO4and concentrated under reduced pressure. The crude was purified by flash column chromatography (EtOAc-hexane), followed by C18 reverse phase chromatography (CH3CN-H2O). Fractions containing product were partitioned between aq. NaHCO3and EtOAc. The organic layer was dried over Na2SO4, concentrated and lyophilized to give N- [(3S)-3-isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7-yl]-2,2-dimethyl- propanamide: (S)-I-10 (12 mg, 11% yield) as a yellow solid.1H NMR (400 MHz, Chloroform- d) δ 8.10 (dd, J = 8.9, 2.7 Hz, 1H), 8.01 (d, J = 2.7 Hz, 2H), 7.49 (s, 1H), 7.17 (s, 1H), 3.82 (dd, J = 8.2, 6.3 Hz, 1H), 2.38 (s, 1H), 1.35 (s, 9H), 1.15 (dd, J = 17.1, 6.7 Hz, 6H). ESI-MS [M+H]+: 335.2. Example 1I: Methyl N-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7- yl]carbamate: (S)-I-11

[0178] To a mixture of (3S)-7-amino-3-isopropyl-5-thioxo-3,4-dihydro-1,4- benzoxazepin-2-one (80 mg, 0.32 mmol), triethylamine (135 μL, 0.96 mmol) in DCM (2 mL) was added dropwise methyl chloroformate (22 μL, 0.29 mmol). The mixture was stirred at rt for 3 h, diluted with aq. NaHCO3and extracted with EtOAc. The organic extract was dried over Na2SO4and concentrated under reduced pressure. The crude was purified by flash column chromatography (EtOAc-hexane, followed by C18 reverse phase chromatography (CH3CN-H2O). Fractions containing product were partitioned between aq. NaHCO3and EtOAc. The organic extract was dried over Na2SO4, concentrated and lyophilized to give methyl N-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7-yl]carbamate: (S)- I-11 (3.5 mg, 4% yield) as a yellow solid.1H NMR (400 MHz, Chloroform-d) δ 7.99 (d, J = 2.8 - 46 -Hz, 2H), 7.86 (d, J = 9.1 Hz, 1H), 7.17 (d, J = 8.9 Hz, 1H), 6.77 (s, 1H), 3.83 (s, 4H), 2.42 – 2.32 (m, 1H), 1.19 – 1.10 (m, 6H). ESI-MS [M+H]+: 309.2. Example 1J: N-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7- yl]formamide: (S)-1-12

[0179] 3,4-dihydro-1,4- benzoxazepin-2-one (70 mg, 0.28 mmol) in ethyl formate (2 mL) was stirred at 60°C for 3 h. The mixture was diluted with aq. NaHCO3and extracted with EtOAc. The organic extract was dried over Na2SO4, concentrated under reduced pressure and purified by flash column chromatography (EtOAc-hexane) to give N-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4- benzoxazepin-7-yl]formamide: (S)-I-12 (9 mg, 12% yield) as a yellow solid.1H NMR (400 MHz, Chloroform-d) δ 8.74 – 8.45 (d, J = 1.6 Hz, d, J = 11.3 Hz, 1H), 8.11– 7.75 (m, 3H), 7.43 – 7.30 (m, 1H), 7.22 (d, J = 8.8 Hz, 1H), 3.87 – 3.81 (m, 1H), 2.40 (s, 1H), 1.20 – 1.13 (m, 6H). ESI-MS [M+H]+: 279.2. Example 1K: (3S)-3-isopropyl-7-(methylamino)-5-thioxo-3,4-dihydro-1,4-benzoxazepin-2- one: (S)-I-13

[0180] To a mixture of (3S)-7-amino-3-isopropyl-5-thioxo-3,4-dihydro-1,4- benzoxazepin-2-one (50 mg, 0.20 mmol), formaldehyde solution (37%, 15.0 μL, 0.20 mmol) in 1,2-dichloroethane (2 mL) at rt was added sodium triacetoxy borohydride (170 mg, 0.80 mmol). After stirring for 2 h, the mixture was quenched with aq. NaHCO3and extracted with EtOAc. The organic extract was dried over Na2SO4, concentrated under reduced pressure and purified by flash column chromatography (EtOAc-hexane) to give (3S)-3-isopropyl-7- (methylamino)-5-thioxo-3,4-dihydro-1,4-benzoxazepin-2-one: (S)-I-13 (16 mg, 30% yield).1H NMR (400 MHz, Chloroform-d) δ 7.92 (s, 1H), 7.32 (d, J = 3.0 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 6.79 (dd, J = 8.8, 3.0 Hz, 1H), 3.96 (s, 1H), 3.85 (dd, J = 8.2, 6.4 Hz, 1H), 2.91 (s, 3H), 2.37 (s, 1H), 1.16 (dd, J = 17.4, 6.7 Hz, 6H). ESI-MS [M+H]+: 265.20 - 47 -Example 1L: 1-ethyl-3-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7- yl]urea: (S)-I-14

[0181] , . (3S)-7-amino-3- isopropyl-5-thioxo-3,4-dihydro-1,4-benzoxazepin-2-one (50 mg, 0.20 mmol) was dissolved in DCM (1 mL). The mixture was stirred at rt for 8 h, then at 45°C for another 14 h. Solvent was removed under reduced pressure and the crude was purified via flash column chromatography (EtOAc-Hexanes) to give 1-ethyl-3-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4- dihydro-1,4-benzoxazepin-7-yl]urea: (S)-I-14 (21 mg, 32% yield).1H NMR (400 MHz, DMSO- d6) δ 11.01 (s, 1H), 8.82 (s, 1H), 8.01 (s, 1H), 7.68 (dd, J = 8.7, 2.7 Hz, 1H), 7.12 (d, J = 8.9 Hz, 1H), 6.16 (s, 1H), 3.91 (s, 1H), 3.16 – 3.08 (m, 3H), 1.06 (t, J = 7.1 Hz, 3H), 0.97 (dd, J = 20.2, 6.4 Hz, 6H). ESI-MS [M+H]+: 322.2. Example 1M :1-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7-yl]-3- phenyl-urea: (S)-I-15

[0182] A mixture of phenyl isocyanate (22 μL, 0.20 mmol) and (3S)-7-amino-3- isopropyl-5-thioxo-3,4-dihydro-1,4-benzoxazepin-2-one (50 mg, 0.200 mmol) was dissolved in DCM (1 mL) and stirred at rt for 4 h. The resulting reaction mixture was filtered and washed with diethyl ether before being evaporated under reduced pressure to give 1-[(3S)-3-isopropyl- 2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7-yl]-3-phenyl-urea: (S)-I-15 (18 mg, 24% yield).1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 9.08 (s, 1H), 8.73 (s, 1H), 8.12 (d, J = 2.8 Hz, 1H), 7.72 (dd, J = 8.9, 2.7 Hz, 1H), 7.47 (d, J = 8.0 Hz, 2H), 7.30 (t, J = 7.8 Hz, 2H), 7.20 (d, J = 8.9 Hz, 1H), 6.99 (t, J = 7.3 Hz, 1H), 3.97 (d, J = 10.2 Hz, 1H), 2.34 (s, 1H), 0.98 (dd, J = 19.5, 6.5 Hz, 6H). ESI-MS [M+H]+: 370.2. Example 1N: (3S)-7-(dimethylamino)-3-isopropyl-5-thioxo-3,4-dihydro-1,4-benzoxazepin-2- one: (S)-I-16 - 48 -pine-2,5- dione (200 mg, 0.854 mmol) in DCM (1 mL) at 0°C was added triethylamine (0.95 mL, 6.83 mmol), followed by dropwise addition of iodomethane (0.26 mL, 4.27 mmol). The reaction mixture was stirred at rt for 24 h, diluted with water and extracted with DCM. The organic layer was dried over Na2SO4 and the solvent was evaporated at reduced pressure. The resulting crude was purified using flash column chromatography (EtOAc-Hexane) to afford (3S)-7- (dimethylamino)-3-isopropyl-3,4-dihydro-1,4-benzoxazepine-2,5-dione (30 mg, 13% yield) as a white solid.1H NMR (400 MHz, Chloroform-d) δ 7.09 (m, 2H), 6.90 (m, 1H), 6.51 (d, 1H), 3.67 (m, 1H), 3.02 (s, 6H), 2.29 (m, 1H), 1.11 (dd, J = 19.5, 6.5 Hz, 6H).

[0184] A mixture of (3S)-7-(dimethylamino)-3-isopropyl-3,4-dihydro-1,4- benzoxazepine-2,5-dione (40 mg, 0.14 mmol) and Lawesson’s reagent (60 mg, 0.14 mmol) in THF (1 mL) was stirred at 65 °C for 2 h. The mixture was concentrated under reduced pressure to give a crude which was purified by flash column chromatography (EtOAc-hexane) to give (3S)-7-(dimethylamino)-3-isopropyl-5-thioxo-3,4-dihydro-1,4-benzoxazepin-2-one: (S)- I-16 (20 mg, 48% yield) as a yellow solid.1H NMR (400 MHz, Chloroform-d) δ 7.95 (s, 1H), 7.40 (m, 1H), 6.92 (m, 1H), 6.89 (m, 1H), 3.85 (m, 1H), 3.03 (s, 6H), 2.37 (m, 1H), 1.14 (dd, J = 19.5, 6.5 Hz, 6H). ESI-MS [M+H]+: 279.2. Example 1O: 1-ethyl-3-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7- yl]thiourea: (S)-I-17

[0185] To a solution of (3S)-7-amino-3-isopropyl-5-thioxo-3,4-dihydro-1,4- benzoxazepin-2-one (30 mg, 0.20 mmol) in ethanol (2 mL) at rt was added ethyl isothiocyanate (16 μL, 0.18 mmol). The resulting mixture was stirred for 24 h and purified by flash column chromatography (EtOAc-hexane), followed by C18 reverse phase chromatography (CH3CN-H2O). Fractions containing product were combined and partitioned between EtOAc and aq. NaHCO3. The organic extract was dried over Na2SO4, concentrated under reduced pressure and lyophilized to give 1-ethyl-3-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4- - 49 -dihydro-1,4-benzoxazepin-7-yl]thiourea: (S)-I-17 (6 mg, 15% yield) as a yellow solid.1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 9.73 (s, 1H), 8.01 (d, J = 2.7 Hz, 1H), 7.86 (dd, J = 8.8, 2.8 Hz, 2H), 7.20 (d, J = 8.8 Hz, 1H), 3.94 (dd, J = 10.3, 6.0 Hz, 1H), 3.49 (s, 3H), 1.14 (t, J = 7.1 Hz, 3H), 0.98 (dd, J = 16.2, 6.5 Hz, 6H). ESI-MS [M+H]+: 338.1. Example 1P: N-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7- yl]methanesulfonamide: (S)-I-18

[0186] o a sou on o ( )- -amno- -sopropy- - ioxo-3,4-dihydro-1,4- benzoxazepin-2-one (50 mg, 0.20 mmol) in acetonitrile (2 mL) at rt was added NaHCO3(50 mg, 0.60 mmol), followed by methanesulfonic anhydride (50 μL, 0.30 mmol). The reaction mixture was stirred for 18 h and the solvent was evaporated under reduced pressure. The crude was purified on C18 reverse phase chromatography (CH3CN-H2O) to give N-[(3S)-3- isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4-benzoxazepin-7-yl]methanesulfonamide: (S)-I-18 (5 mg, 6% yield).1H NMR (400 MHz, Chloroform-d) δ 7.96 (s, 1H), 7.41 (d, J = 3.2 Hz, 1H), 7.06 (d, J = 9.0 Hz, 1H), 6.90 (dd, J = 9.0, 3.2 Hz, 1H), 3.85 (dd, J = 8.2, 6.3 Hz, 1H), 3.04 (s, 3H), 2.37 (d, J = 5.7 Hz, 1H), 1.16 (dd, J = 18.0, 6.7 Hz, 6H). ESI-MS [M+H]+: 329.1. Example 1Q: 1,1,1-trifluoro-N-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4- benzoxazepin-7-yl]methanesulfonamide: (S)-I-19

[0187] To a solution of (3S)-7-amino-3-isopropyl-5-thioxo-3,4-dihydro-1,4- benzoxazepin-2-one (80 mg, 0.319 mmol) in acetonitrile (2 mL) at rt was added NaHCO3(80 mg, 0.96 mmol), followed by trifluoromethanesulfonic anhydride (160 μL, 0.96 mmol). The reaction mixture was stirred for 18 h and concentrated. The crude was purified using C18 reverse phase chromatography (CH3CN-H2O). Fractions containing product were combined and lyophilized to give 1,1,1-trifluoro-N-[(3S)-3-isopropyl-2-oxo-5-thioxo-3,4-dihydro-1,4- benzoxazepin-7-yl]methanesulfonamide: (S)-I-19 (10 mg, 8 % yield).1H NMR (400 MHz, - 50 -DMSO-d6) δ 11.12 (d, J = 6.5 Hz, 1H), 7.86 (d, J = 2.8 Hz, 1H), 7.47 (dd, J = 8.8, 2.8 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 3.99 (dd, J = 10.3, 6.4 Hz, 1H), 2.30 (s, 1H), 0.98 (dd, J = 17.1, 6.5 Hz, 6H).19F NMR (376 MHz, DMSO-d6) δ -75.76. ESI-MS [M+H]+: 383.1. Example 1R: methyl (2S)-2-[(5-amino-2-hydroxy-benzenecarbothioyl)amino]propanoate (S)- I-20ep a:

[0188] A mixture of methyl (2S)-2-[[5-(tert-butoxycarbonylamino)-2-hydroxy- benzoyl]amino]propanoate (350 mg, 1.03 mmol), Lawesson’s reagent (418 mg, 1.03 mmol) in THF (6 mL) was stirred at 66 °C in a sealed vial under nitrogen for 1 h. The mixture was concentrated and purified by flash column chromatography (EtOAc-hexane). Fractions containing product were combined and concentrated to give a crude of methyl (2S)-2-[[5-(tert- butoxycarbonylamino)-2-hydroxy-benzenecarbothioyl]amino]propanoate (200 mg, 55% yield), which was used for the next step. ESI-MS [M+H]+: 354.9. Step b:

[0189] A mixture of methyl (2S)-2-[[5-(tert-butoxycarbonylamino)-2-hydroxy- benzenecarbothioyl]amino]propanoate (200 mg, 0.59 mmol) in trifluoroacetic acid (1.14 mL) and DCM (6.86 mL) was stirred at rt for 2 h. Solvent was evaporated and the crude was purified by flash column chromatography (EtOAc-hexane) to give methyl (2S)-2-[(5-amino-2-hydroxy- benzenecarbothioyl)amino]propanoate (22 mg, 13% yield).1H NMR (400 MHz, DMSO-d6) δ 10.74 (d, J = 6.7 Hz, 1H), 9.59 (br, 3H), 7.80 (d, J = 2.8 Hz, 1H), 7.17 (dd, J = 8.6, 2.8 Hz, 1H), 6.97 (d, J = 8.6 Hz, 1H), 5.09-5.02 (m, 1H), 3.69 (s, 3H), 1.51 (d, J = 7.2 Hz, 3H). ESI-MS [M+H]+: 254.9. Example 1S: (2S)-2-[(5-azanyl-2-oxidanyl-benzenecarbothioyl)amino]-3-methyl-butanoate hydrochloride ((S)-I-21); (2S)-2-[(5-azanyl-2-oxidanyl-benzenecarbothioyl)amino]-3-methyl- butanoic acid hydrochloride ((S)-I-22) - 51 -Step a:

[0190] To a stirred solution of^2-acetyloxy-5-[1,1- di(methyl)ethoxycarbonylamino]benzoic acid (1.5 g, 5.08 mmol) and L-valine methyl ester hydrochloride (1.11 g, 6.60 mmol)^in^DMF (10 mL)^at 0 °C was added^HATU (2.12 g, 5.59 mmol),^followed by^DIPEA (2.2 mL, 12.70 mmol). The resulting mixture was warmed to room temperature and stirred overnight.^The mixture was diluted with water and saturated sodium bicarbonate and then extracted with ethyl acetate.^The combined organic extracts were washed with brine / water / 1 M HCl (1:1:1, 2 times), brine / water (1:1), and brine.^The organic extract was dried, filtered and concentrated in vacuo and purified flash column chromatography (EtOAc-hexanes). The product fractions were concentrated and dried to give a foamy solid of^methyl (2S)-2-[[2-acetyloxy-5-[1,1- di(methyl)ethoxycarbonylamino]benzoyl]amino]-3-methyl-butanoate (1.2 g, 2.94 mmol, 58% yield).1H NMR (400 MHz, Chloroform-d) δ 7.76 (d, J = 8.9 Hz, 1H), 7.69 (d, J = 2.7 Hz, 1H), 7.14 (d, J = 8.5 Hz, 1H), 7.10 (d, J = 8.8 Hz, 1H), 6.60 (s, 1H), 4.78 (dd, J = 8.5, 4.6 Hz, 1H), 3.79 (s, 3H), 2.42 (d, J = 1.0 Hz, 3H), 2.33 – 2.20 (m, 1H), 1.54 (d, J = 2.4 Hz, 9H), 0.99 (dd, J = 8.4, 6.8 Hz, 6H); ESI-MS [M+H]+: 409. Step b:

[0191] To a stirred solution of^methyl (2S)-2-[[2-acetyloxy-5-[1,1- di(methyl)ethoxycarbonylamino]benzoyl]amino]-3-methyl-butanoate (1.2 g, 2.94 mmol)^in^THF (15 mL)^under argon at room temperature was added^Lawesson's reagent (594.57 mg, 1.47 - 52 -mmol). The resulting suspension was heated to 60 °C and stirred for 1 h.^ After this time, additional Lawesson's reagent (100 mg)^was added and stirring was continued for another hour. The mixture was diluted with ethyl acetate and washed with brine / aq. NaHCO3(1:1, 2 times), followed by brine.^ The organic extract was dried, filtered and concentrated in vacuo and purified by flash column chromatography (EtOAc - hexanes).^The product containing fractions were concentrated in vacuo and dried to give a pale yellow^foamy solid of^methyl (2S)-2-[[2-acetyloxy-5-[1,1-di(methyl)ethoxycarbonylamino]benzenecarbothioyl]amino]-3- methyl-butanoate (1.18 g, 2.78 mmol, 95% yield).1H NMR (400 MHz, Chloroform-d) δ 8.14 (d, J = 8.1 Hz, 1H), 7.62 (d, J = 2.6 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H), 6.53 (s, 1H), 5.22 (dd, J = 8.0, 4.8 Hz, 1H), 3.82 (s, 3H), 2.46 – 2.36 (m, 1H), 2.34 (s, 3H), 1.53 (s, 9H), 1.07 (d, J = 6.9 Hz, 3H), 1.03 (d, J = 6.9 Hz, 3H); ESI-MS [M+H]+: 425. Step c: ((S)-I-21)

[0192] To a solution of^acetyl chloride (0.093 mL, 1.53 mmol)^in^methanol (3 mL)^was added solution of methyl (2S)-2-[[2-acetyloxy-5-[1,1- di(methyl)ethoxycarbonylamino]benzenecarbothioyl] amino]-3-methyl-butanoate (130 mg, 0.306 mmol)^in^methanol (3 mL). The mixture was stirred at rt for^2 h and then concentrated in vacuo. A pale-yellow oil was obtained, to which diethyl ether was added and the mixture was stirred for 20 min at rt. The formation of a yellow solid was observed and the mixture was filtered, the solid was further dried under vacuum to obtain the desired product^methyl (2S)-2- [(5-azanyl-2-oxidanyl-benzenecarbothioyl)amino]-3-methyl-butanoate hydrochloride (55 mg, 0.17 mmol, 56 % yield)^HCl salt as a yellow solid.^1H NMR (400 MHz, Chloroform-d): d 10.75 (d, J = 8 Hz, 1H), 8.01 (m, 1H), 7.31 (dd, J = 4.0, 8.0 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 4.98 (t, J = 4.0 Hz, 1H), 3.69 (s, 3H), 2.33 (m, 1H), 1.04 (d, J = 8.0 Hz, 3H), 1.00 (d, J = 8.0 Hz, 3H); ESI-MS [M+H]+: 283. Step d:

[0193] To a stirred solution of^methyl (2S)-2-[[2-acetyloxy-5-[1,1- di(methyl)ethoxycarbonylamino]benzenecarbothioyl]amino]-3-methyl-butanoate (1.18 g, 2.78 mmol)^in^THF (5 mL)^was added^a solution of lithium hydroxide (333 mg, 13.90 mmol) in water (5 mL),^methanol (2 mL)^was added to aid solubility.^The resulting mixture was stirred at room temperature for 20 min then at 50 °C for^2 h. The mixture was diluted with water and^acidified with 1 M HCl to pH 5.^The mixture was then diluted with brine and extracted with ethyl acetate; the organic phase was washed with brine / aq. HCl (9:1, 2 times). The organic phase was dried, filtered and concentrated in vacuo giving a yellow foamy solid of (2S)-2-[[5-[1,1- di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzenecarbothioyl]amino]-3-methyl-butanoic acid (1.0 g, 2.71 mmol, 97% yield).1H NMR (400 MHz, Methanol-d4) δ 8.18 (d, J = 2.8 Hz, - 53 -1H), 7.42 (d, J = 8.8 Hz, 1H), 6.85 (d, J = 8.8 Hz, 1H), 5.24 (d, J = 5.1 Hz, 1H), 2.47 (dd, J = 6.9, 1.9 Hz, 1H), 1.53 (s, 9H), 1.12 (dd, J = 16.4, 6.9 Hz, 6H); ESI-MS [M-H]+: 367. Step e: ((S)-I-22)

[0194] To (2S)-2-[[5-[1,1-di(methyl)ethoxycarbonylamino]-2-oxidanyl- benzenecarbothioyl]amino]-3-methyl-butanoic acid (1 g, 2.71 mmol)^was added HCl solution in dioxane (4 M, 6.78 mL)^and the mixture was stirred at room temperature for 1 h.^The mixture was concentrated in vacuo giving a sticky oil.^The oil was diluted with diethyl ether and concentrated again; the procedure was repeated 5 times.^At last iteration, the solid was stirred in diethyl ether overnight, filtered and dried to get yellow powder of^(2S)-2-[(5-azanyl-2- oxidanyl-benzenecarbothioyl)amino]-3-methyl-butanoic acid hydrochloride (802 mg, 2.63 mmol, 97.10% yield).1H NMR (400 MHz, DMSO-d6) δ 10.74 (d, J = 7.7 Hz, 1H), 9.98 (br, 3H), 8.11 (d, J = 2.8 Hz, 1H), 7.31 (dd, J = 8.7, 2.8 Hz, 1H), 7.07 (d, J = 8.7 Hz, 1H), 5.03 (dd, J = 7.7, 5.0 Hz, 1H), 2.42 – 2.29 (m, 1H), 1.04 (d, J = 6.9 Hz, 3H), 0.99 (d, J = 6.9 Hz, 3H); ESI- MS [M+H]+: 269. Example 1T: (2S)-2-[(5-azanyl-2-oxidanyl-benzenecarbothioyl)amino]-3,3-dimethyl- butanoicacid hydrochloride ((S)-I-23)Step a:

[0195] To a stirred suspension of 2-acetyloxy-5-[1,1- di(methyl)ethoxycarbonylamino]benzoic acid (1.5 g, 5.08 mmol) in CH3CN (20 mL) under argon at room temperature was added HATU (2.32 g, 6.10 mmol). The mixture was stirred for 1 min then treated with methyl (2S)-2-azanyl-3,3-dimethyl-butanoate hydrochloride (1.11 g, 6.10 mmol), followed by DIPEA (2.65 mL, 15.24 mmol). The mixture was stirred at room temperature for 3 h, diluted with water and saturated NaHCO3then extracted with ethyl acetate. The combined organics were washed with a mixture of brine / water / 1 M HCl (1:1:1), - 54 -followed by brine. The organic phase was dried, filtered and concentrated in vacuo then purified by flash column chromatography (EtOAc-hexane). Fractions containing products were concentrated in vacuo then stirred in hexanes, the suspension was filtered to give the desired methyl (2S)-2-[[2-acetyloxy-5-[1,1-di(methyl)ethoxycarbonylamino]benzoyl]amino]-3,3- dimethyl-butanoate (1.36 g, 3.22 mmol, 63 % yield) as a white solid.1H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.23 (d, J = 8.3 Hz, 1H), 7.64 (d, J = 2.7 Hz, 1H), 7.54 (dd, J = 8.8, 2.7 Hz, 1H), 7.09 (d, J = 8.8 Hz, 1H), 4.32 (d, J = 8.4 Hz, 1H), 3.67 (s, 3H), 2.20 (s, 3H), 1.49 (s, 9H), 1.00 (s, 9H). ESI-MS [M+H]+: 423.0. Step b:

[0196] A mixture of methyl (2S)-2-[[2-acetyloxy-5-[1,1- di(methyl)ethoxycarbonylamino]benzoyl] amino]-3,3-dimethyl-butanoate (820 mg, 1.94 mmol) , Lawesson’s reagent (785 mg, 1.94 mmol) in THF (10 mL) was heated at 66oC in a sealed vial for 2 h. The crude was concentrated and purified by flash column chromatography (EtOAc- hexane), followed by C18 reverse phase chromatography (CH3CN-H2O) to give methyl (2S)- 2-[[2-acetyloxy-5-[1,1-di(methyl)ethoxycarbonylamino]benzenecarbothioyl]amino]-3,3- dimethyl-butanoate (680 mg, 80% yield) as a yellow solid.1H NMR (400 MHz, Chloroform-d) δ 8.05 (d, J = 8.6 Hz, 1H), 7.61 – 7.51 (m, 2H), 7.00 (d, J = 8.7 Hz, 1H), 6.54 (s, 1H), 5.14 (d, J = 8.6 Hz, 1H), 3.80 (s, 3H), 2.35 (s, 3H), 1.53 (s, 9H), 1.09 (s, 9H). ESI-MS [M+H]+: 438.9. Step c:

[0197] To a solution of methyl (2S)-2-[[2-acetyloxy-5-[1,1- di(methyl)ethoxycarbonylamino]benzenecarbothioyl]amino]-3,3-dimethyl-butanoate (330 mg, 0.75 mmol) in methanol (5 mL) was added NaOH 2 M (5.64 mL). After 5 h, additional of NaOH 2 M (5.64 mL) was added and the reaction was stirred overnight. The mixture was concentrated, acidified with HCl 1N and extracted with EtOAc. The organic extract was dried over Na2SO4, filtered, and concentrated to give (2S)-2-[[5-[1,1- di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzenecarbothioyl]amino]-3,3-dimethyl- butanoic acid (260 mg, 90% yield) as a yellow solid. ESI-MS [M+H]+: 382.9. Step d:

[0198] A mixture of 1,1-di(methyl)ethyl N-[3-[4-[1,1-di(methyl)ethyl]-5-oxidanylidene- 4H-thiazol-2-yl]-4-oxidanyl-phenyl]carbamate (860 mg, 2.36 mmol) in HCl solution in dioxane (4 M, 20.51 mL) was stirred at rt for 1.5 h. The mixture was concentrated and stirred in diethyl ether for 2 h. The decanted solid was dried in vacuo to give 2-(5-azanyl-2-oxidanyl-phenyl)-4- [1,1-di(methyl)ethyl]thiazol-5-ol;chlorane (560 mg, 79% yield) as a yellow solid.1H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 3H), 7.77 (d, J = 2.6 Hz, 1H), 7.20 (dd, J = 8.7, 2.6 Hz, 1H), 7.05 (d, J = 8.6 Hz, 1H), 1.36 (s, 9H). ESI-MS [M+H]+: 283.2. - 55 -Example 1U: (S)-5-amino-2-(5-amino-2-hydroxyphenylthioamido)-5-thioxopentanoic acid hydrochloride ((S)-I-24) Step a:

[0199] A 100 ml round-bottom flask was charged with 2-acetyloxy-5-[1,1- di(methyl)ethoxycarbonylamino]benzoic acid (1.5 g, 5.08 mmol) and L-Glutamine methyl ester hydrochloride (1.20 g, 6.10 mmol) in DMF (10 mL) at 0 °C. To this mixture was added HATU (2.12 g, 5.59 mmol), followed by N,N-diisopropylethylamine (1.97 g, 15.24 mmol, 2.65 mL) . The mixture was warmed up to rt and stirred at this temperature for 12 h. Saturated solution of NaHCO3was added and the mixture was extracted with EtOAc. The combined organic extracts were washed with H2O (3 times), followed by brine. The organic extract was then dried over Na2SO4, filtered and concentrated. The crude was purified by flash column chromatography using MeOH-DCM to give the desired product methyl (2S)-2-[[2-acetyloxy- 5-[1,1-di(methyl)ethoxycarbonylamino]benzoyl]amino]-5-amino-5-oxo-pentanoate (1.9 g, 4.34 mmol, 86% yield) as a white solid. ESI-MS [M+H]+: 438.2. Step b:

[0200] To a stirred solution of methyl 2-[[2-acetyloxy-5-[1,1- di(methyl)ethoxycarbonylamino]benzoyl]amino]-5-azanyl-5-oxidanylidene-pentanoate (850 mg, 1.94 mmol) in THF (22 mL) under argon at room temperature was added Lawesson's reagent (1.57 g, 3.89 mmol). The resulting suspension was heated at 60 °C for 2 h. The mixture was diluted with EtOAc and washed with aq. NaHCO3, followed by brine. The organic phase was dried over Na2SO4, filtered, concentrated and purified by flash column chromatography (EtOAc – hexanes) to give the desired product methyl 2-[[2-acetyloxy-5-[1,1- - 56 -di(methyl) ethoxycarbonylamino]benzenecarbothioyl]amino]-5-azanyl-5-sulfanylidene- pentanoate (805 mg, 1.71 mmol, 88 % yield). ESI-MS [M+H]+: 470.2. Step c:

[0201] To a mixture of methyl 2-[[2-acetyloxy-5-[1,1-di(methyl) ethoxycarbonylamino]benzenecarbothioyl]amino]-5-azanyl-5-sulfanylidene-pentanoate (420 mg, 0.89 mmol) in methanol (15 mL) was added NaOH 2M (6.71 mL) at rt. The mixture was stirred at rt for 1.5 h. All the volatiles were removed under reduced pressure. The mixture was acidified with HCl 1M and extracted with EtOAc. The organic extract was washed with brine, dried over Na2SO4, filtered and concentrated. The oil product was triturated in diethyl ether and further purified by flash column to give the desired product 5-azanyl-2-[[5-[1,1- di(methyl)ethoxycarbonylamino]-2-oxidanyl-benzenecarbothioyl]amino]-5-sulfanylidene- pentanoic acid (48 mg, 12% yield). ESI-MS [M+H]+: 414.10. Step d:

[0202] 5-azanyl-2-[[5-[1,1-di(methyl)ethoxycarbonylamino]-2-oxidanyl- benzenecarbothioyl]amino]-5-sulfanylidene-pentanoic acid (60 mg, 0.15 mmol) was stirred in hydrogen chloride solution in dioxane (4 M, 0.36 mL) at room temperature for 1 h. The mixture was concentrated and triturated in diethyl ether. The obtained solid was further purified by prep-HPLC using a C18 reverse phase column (CH3CN-H2O) to give a yellow solid (6 mg, 0.017 mmol, 12% yield).1H NMR (400 MHz, DMSO-d6): ^ 10.50-10.90 (1H, br), 9.42 (1H, s), 9.25 (1H, s), 7.31 (1H, s), 6.59-6.66 (2H, m), 5.04 (1H, br), 2.56-2.58 (2H, m), 2.33-2.50 (2H, m), 2.23 (1H, m). ESI-MS [M+H]+: 314.29. Example 1V: (2S)-2-[(5-amino-2-hydroxy-benzenecarbothioyl)amino]-3-methyl-butanoic acid ((S)-I-25)Step a:

[0203] To a stirred solution of methyl (2S)-2-[[2-acetoxy-5-(tert- butoxycarbonylamino)benzenecarbothioyl]amino]-3-methyl-butanoate (7.53 g, 17.74 mmol) in MeOH (100 mL) was added 6N HCl (30 mL) using syringe at rt and the resulting solution was refluxed at 60oC for 1 h. The reaction mixture was cooled to rt and then cooled in an ice bath. pH of the mixture was adjusted between 4-5 using cold 5N NaOH. MeOH was evaporated by - 57 -rotavapor and the mixture was extracted with DCM. The organic extract dried over Na2SO4, concentrated and dried under high vacuum to give methyl (2S)-2-[(5-amino-2-hydroxy- benzenecarbothioyl)amino]-3-methyl-butanoate (4.32 g, 86% yield) as a bright yellow solid.1H NMR (400 MHz, Chloroform-d) δ 8.97 (s, 1H), 7.37 (br, 1H), 7.02-7.00 (m, 1H), 6.84 (d, J = 8.8 Hz, 1H), 5.13 (br, 1H), 3.81 (s, 3H), 2.44-2.35 (m, 1H), 1.05 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H). ESI-MS [M+H]+: 283.3. Step b:

[0204] To a stirred yellow solution of methyl (2S)-2-[(5-amino-2-hydroxy- benzenecarbothioyl)amino]-3-methyl-butanoate (4.30 g, 15.23 mmol) in MeOH (25 mL) was added a solution of LiOH (1.82 g, 76.14 mmol) in H2O (15 mL) at rt and the resulting solution was refluxed at 60 °C for 1 h. The reaction mixture was cooled to rt and MeOH was concentrated by rotavapor at 35 °C. The obtained brown mixture was cooled to 0-5 °C and pH was adjusted between 3-4 using cold 6N HCl. Precipitation observed, the slurry was stirred, and pH was kept between 3-4. The mixture was filtered, the solid washed with cold water (20 mL) (product is soluble in water), followed by hexane. The obtained solid was dried under high vacuum at 40 °C to give (2S)-2-[(5-amino-2-hydroxy-benzenecarbothioyl)amino]-3-methyl- butanoic acid (2.95 g, 72% yield) as a greyish-beige solid.1H NMR (400 MHz, DMSO-d6) δ 10.82 (br, 2H), 7.57 (d, J = 2.8 Hz), 6.69-6.61 (m, 2H), 5.06 (d, J = 4.8 Hz, 1H), 2.40-2.32 (m, 1H), 1.03 (d, J = 7.2 Hz, 3H), 0.98 (d, J = 7.2 Hz, 3H). ESI-MS [M+H]+: 269.4. Example 2: H2S quantification after incubation with FaSSIF (Fasted State Simulated Intestinal Fluid) and phosphate-buffered saline (PBS) using Methylene Blue method

[0205] Assay buffer was prepared according to the manufacturer’s instructions by mixing FaSSIF buffer concentrate (BioRelevant, UK; FASBUF), purified water and FaSSIF powder (BioRelevant). Pancreatin (Sigma-Aldrich, MO, USA) was added to obtain 1x stock according to provider’s indications.

[0206] Stock solutions of the test compounds in dimethyl sulfoxide (DMSO) were diluted, in a 96-well plate, in the buffer with and without pancreatin at a final concentration of 50 µM. After sealing the plate, it was incubated at 37 °C and 500 rpm for 2 hours. Samples were also incubated in PBS, following an equivalent procedure.

[0207] In order to prepare a standard curve, a stock solution containing 100 µM Na2S and 1 mM ZnSO4in PBS was prepared. This solution was diluted with FaSSIF assay buffer without pancreatin to obtain 7 additional standard points. - 58 -

[0208] H2S was quantified from incubated samples and standard curve by a modification of the methylene blue method (Giustarini D. et al. Biochem. Pharmacol.2014, 89, 246-254). Briefly, in a separate plate, 40 µL of 20 mM N-N-dimethyl-p-phenylenediamine sulfate (TCI America, OR, USA; D0782) were added in 60% HCl and 40 µL of FeCl3 (Combi- Blocks, CA, USA; QE-7694) in 10% HCl to each well, and 120 µL of the incubated samples or standards were transferred. The plates were sealed and incubated for additional 45 minutes. Then, the plate were centrifuged at 2500 rpm for 5 minutes and, after appropriate dilution with H2O:ACN 1:1 mixture, the supernatant was analyzed by HPLC using a Kinetix PS C18 (2.6 µM, 4.6x100 mm) column (Phenomenex, CA, USA) and coupled to a VWD detector setup at 660 nm (Agilent Technologies, CA, US).

[0209] Concentration of H2S was determined by comparison of the Area Under the ROC Curve (AUC) values from the incubated samples to the standard curve values, adjusted by lineal regression. Results are presented in Table 1. Table 1: H2S release in FaSSIF and PBS of selected exemplary compounds H2S releaseFassIF* PBSExample 3: Evaluation of efficacy on the TNBS induced colitis model in mice Materials and Reagents - 59 -

[0210] Dulbecco's phosphate-buffered saline (DPBS) was obtained from Corning, Trinitrobenzene sulfonic acid (TNBS) was obtained from Beijing OUHE Technology, Fecal occult blood test strip (improved pyramidon method) was obtained from Zhuhai Baso Diagnostics Inc., Avertin was obtained from Nanjing Aibei Biotechnology and mesalamine was obtained from Selleck. Equipment Equipment Vendor Model Electronical balance Tianzhiping balance Inc., YH-2000 Metods Experimental Animals

[0211] Female Balb / c mice (90) aged 8 weeks, around 18-20 g body weight purchased from Charles River Laboratories were bred in specific pathogen free individually ventilated (IV) cages (4 mice in each cage) in a temperature controlled room (20±2˚C) with a 12 hour light- dark cycle. Chow pellets and tap water were available ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Wuxi AppTec. The mice were acclimated at the animal facility at Wuxi for at least of three days before the experiments. TNBS induced acute colitis model

[0212] Mice in sham group received an intracolonic injection of 50% ethanol (0.1mL) at day 0, whereas mice in other TNBS colitis model groups received an intracolonic injection of 2% TNBS solution (0.1mL) at day 0. These mice were euthanized on day 7 (9thday of the experiment), and samples were immediately harvested at the endpoint. Mice grouping and dosing regimen

[0213] Two studies were conducted and for each study the mice were divided into relevant number of groups with 10 mice in each group. The dosing regimen for mice in the two studies are shown in Tables 2-3. Table 2. Grouping and Dosing Regimen for TNBS induced IBD Model for in-vivo study No.1 Group N Test articles Treatment Systematic dosing- 60 -Route Frequency 1 10 Sham NA NA NA. p g g g y o.2 Grou N Test articles Treatment Systematic dosing p (mg / kg) Route Frequency

[0214] Body weight and disease activity index (DAI) scoring were recorded daily to assess the severity of colitis. DAI score was the summation of the weight loss score, stool score and bleeding score. A blinded scoring system was employed to assess the colitis. The DAI scorer blinded to the group information and animal ID was responsible for the stool consistency and bleeding evaluations. DAI scoring standards to be followed are described in Table 3. Table 4. DAI scoring system Scoring system eFecal occult blood (FOB) test - 61 -

[0215] If there was no blood visible with naked eyes, the FOB test was performed with a fecal occult blood test strip (the improved pyramidon method). The FOB score (0-2) was interpreted as follows: 0, no color appeared after 2 min; 1, a dimmed color during 1-2 min; 2, a deeper color during 1-2 min. Colon collection and colon density measurement

[0216] The colons were harvested, mesentery and adipose tissue were carefully removed. After that, the length of colon was measured, and then colon weight was measured by removing and rinsing out the internal content of colon with cold PBS. PK colon tissue collection

[0217] On Day 7 (endpoint), the mice were subjected to PK dosing of the exemplary compounds. 20 hours post day 6, 2ndBID (twice a day) dosing and 4 hours post day 7 PK dosing, the colon tissues were collected, and the colon weight was recorded. The colon samples were frozen and reserved for assessment of the level of exemplary compounds of the application. Statistical analysis

[0218] The data was compared by ANOVA with post-hoc Dunnett's multiple comparisons test using Graph Pad Prism 6.0 software (San Diego, CA, USA). P-value < 0.05 was considered as statistically significant difference. Data was expressed as mean ± S.E.M. Results Exemplary compounds ameliorate TNBS colitis disease severity

[0219] Colitis was induced by intracolonic injection with 2% TNBS at Day 0. It was assessed whether a series of compounds of the application can ameliorate TNBS induced acute colitis model in mice. As expected, the reference compound mesalamine remarkably reduced the body weight loss of TNBS colitis mice when compared with that of vehicle group (Figures 1A and 3A). Exemplary compounds of the application showed a good effect on alleviating the body weight loss of colitis mice with (S)-I-3, (S)-I-6 and (S)-I-22 exhibiting an effect comparable to mesalamine. Meanwhile, it was also found that mesalamine and exemplary compounds showed the effect on improving DAI score, showing less diarrhea and rectal bleeding (Figures 1B and 3B). The data suggest exemplary compounds of the application should trigger the effect on alleviating colitis severity by reducing the body weight loss and improving the DAI score. Therefore, exemplary compounds of the application showed a good effect in the TNBS colitis model in mice. - 62 -Exemplary compounds maintain colon length and density

[0220] TNBS administration in mice usually leads to shortening of the colon and increased colon density, which reflects the severity of TNBS induced colitis. At the endpoint, colon density of the exemplary compounds of the application was significantly lower than that of vehicle group (Figures 2A and 4A). Consistently, colon length of exemplary compounds of the application was longer than that of vehicle group (Figures 2B and 4B). In addition, colon weight of exemplary compounds of the application was lighter than that of vehicle group (Figures 2C and 4C). All the data suggest exemplary compounds of the application improved the TNBS induced colon shortening and swelling. Conclusions

[0221] In conclusion, the in-vivo data suggests that exemplary compounds of the application showed an effect on reducing the severity of TNBS colitis. Promisingly, (S)-I-3, (S)- I-6 and (S)-I-22 showed good efficacy, showing a comparable effect as the reference mesalamine on alleviating the body weight loss and improving DAI score. Example 4: Evaluation of efficacy on the TNBS induced colitis model in rats Materials and Reagents

[0222] Dulbecco's phosphate-buffered saline (DPBS) was obtained from Corning, Trinitrobenzene sulfonic acid (TNBS) was obtained from Beijing OUHE Technology, Fecal occult blood test strip (improved pyramidon method) was obtained from Zhuhai Baso Diagnostics Inc., Avertin was obtained from Nanjing Aibei Biotechnology, mesalamine was obtained from Selleck and myeloperoxidase (MPO) Activity Assay Kit was obtained from Elabscience. Equipment Equipment Vendor Model MetExperimental Animals - 63 -

[0223] Male Sprague Dawley (SD) rats (100), aged 6 weeks, around 250 grams for body weight purchased from Charles River Laboratories, were grouped into 10 cohorts and were bred in specific pathogen free IV cages (4 rats in each cage) in a temperature controlled room (20±2˚C) with a 12 hour light-dark cycle. Chow pellets and tap water were available ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Wuxi AppTec. The rats were acclimated at the animal facility at Wuxi for at least three days before the experiments. TNBS induced acute colitis model

[0224] Rats in the sham group were treated with an intracolonic injection / rectal infusion of 50% ethanol (0.6 mL) at day 0, whereas rats in the active colitis model groups were treated with intracolonic injection / rectal infusion of 2% TNBS solution (0.6 mL) in 50% ethanol at day 0. The reference compound mesalamine and test compounds were administered from day-1 to day 6, with day 7 as the endpoint. Rats grouping and treatment regimen

[0225] The rats were divided into 7 groups with 10 rats in each treatment cohort. The dose regimen and group assignment are shown in Table 5. Table 5. Grouping and Treatment Regimen for TNBS induced IBD Model for in-vivo study Group N Test articles Treatment Systematic dosing (mg / kg) Route FrequencyEvaluation of colitis severity

[0226] Body weight and DAI scoring were recorded daily to assess the severity of colitis. DAI score was the summation of the weight loss score, stool score and bleeding score. A blinded scoring system was employed to assess the colitis. The DAI scorer, blinded to the group information and animal ID, was responsible for the stool consistency and bleeding evaluations. DAI scoring standards used are described in Table 6. - 64 -Table 6. DAI scoring system Scoring system Score Weight loss Stool consistency Bleeding e Fecal occ

[0227] If there were no obvious visible blood visible, a FOB test was performed with a fecal occult blood test strip (the improved pyramidon method). The FOB score (0-2) was interpreted as follows: 0, no color appeared after 2 min; 1, a dimmed color during 1-2 min; 2, a deeper color during 1-2 min. Colon collection and colon density measurement

[0228] The colons were harvested, mesentery and adipose tissue were carefully removed. Subsequently, the length of colon was measured, and then colon weight was measured by removing and rinsing out the internal content of colon with cold PBS. Myeloperoxidase activity measurement

[0229] Myeloperoxidase (MPO) activity was determined as indices of inflammation. MPO activity is associated with the severity of colonic mucosal injury. The colon tissues were removed from storage and allowed to thaw on ice. Once thawed, 1 mL buffer solution was added per 100 mg tissue for homogenization. Homogenates underwent a series of four freeze / thaw cycles before finally being centrifuged at 12,000 g for 10 min at 4 ℃. The supernatant was collected for measurement of MPO activity. At the time of assay 90 μL of standard and sample were added to appropriately labeled tubes. Inhibitor was added at a volume of 10 μL to initiate the reaction after 10 min to terminate the reaction. The absorbance changes were read at 535 nm and recorded. Statistical analysis

[0230] The data was compared by ANOVA with post-hoc Dunnett's multiple comparisons test using Graph Pad Prism 6.0 software (San Diego, CA, USA). P-value < 0.05 was considered as statistically significant difference. Data was expressed as mean ± S.E.M. Results - 65 -Exemplary compounds ameliorate TNBS colitis disease severity

[0231] Colitis was induced by intracolonic injected with 2% TNBS at Day 0. It was assessed whether a series of exemplary compounds of the application had beneficial effects on TNBS induced acute colitis in rats. As expected, the reference compound mesalamine remarkably reduced the body weight loss of TNBS colitis rats when compared with that of vehicle group (Figure 5A). Exemplary compounds, (S)-I-7, (S)-I-6, and (S)-I-25, showed a positive effect on alleviating the body weight loss and improved DAI score comparable to mesalamine (Figure 5A and 5B). The data suggest oral administration of exemplary compounds largely ameliorated the body weight loss and improved DAI scores. Therefore, exemplary compounds showed a good effect on the TNBS colitis model in rats. Exemplary compounds maintain colon length and density

[0232] TNBS administration in rats usually leads to shortening of the colon and increased colon density, which reflects the severity of TNBS induced colitis. At the endpoint, the colon density of the exemplary compound groups was significantly lower than that of vehicle group (Figure 6A). Consistently, colon length of the exemplary compound groups was significantly longer when compared with that of vehicle group (Figure 6B). In addition, when compared with vehicle rats, colon weights of the exemplary compound groups were significantly lower (Figure 6C). The data suggest exemplary compounds of the application improved the TNBS induced colon shortening and swelling. MPO activity

[0233] MPO activity in vehicle group was elevated significantly compared to the samples from other colitis model groups (Figure 7). The MPO activity of the exemplary compound groups was significantly lower than that of vehicle group. Exemplary compounds, (S)-I-7, (S)-I-6, and (S)-I-25 showed a positive effect on MPO activity, comparable to mesalamine. The data suggest exemplary compounds caused a significant inhibition of myeloperoxidase activity in the colon of TNBS-treated rats. Conclusions

[0234] In conclusion, the in-vivo rat data show that exemplary compounds of the application have an effect on reducing the severity of TNBS colitis. Further, exemplary compounds exhibited beneficial effects on TNBS induced colitis model in rats comparable to mesalamine. Example 5: Cytokine Profiling

[0235] To define the role of exemplary compounds of the application in enhancing the healing / recovery process from inflammation, mRNA levels of cytokines / chemokines were - 66 -evaluated in TNBS-induced colitis models in Sprague Dawley (SD) rats. The total RNA was extracted from seven groups of animals (Sham, vehicle, mesalamine 100mg / kg (daily), (S)-I- 7, (S)-I-6, (S)-I-25, mesalamine 39 mg / kg (twice daily)) containing ten animals each group using RNeasy Mini Kit (Qiagen # 74106) according to the manufacturer’s protocol. Briefly, 20- 30 mg of intestinal tissue was homogenized using highly specialized buffer system and centrifuged to obtain a clear lysate. The lysate was precipitated by ethanol and transferred to the RNA spin column where residual amount of genomic DNA was removed and RNA was extracted by washing with buffers provided in the kit. The quality and concentration of RNA was determined by NanoDrop One / OneCMicrovolume UV-Vis spectrophotometer (Thermo Scientific). The complementary DNA (cDNA) was prepared from 1 μg of total RNA by using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems # 4368814) according to the manufacturer’s instructions. To evaluate the marker gene expression of cytokines / chemokines rat-specific primers were designed for targets (INFγ, IL-2, IL-5, IL-12β, TNFα, IL-1α, IL-1β, CCL-5, IL-4, IL-10) and housekeeping genes (GAPDH, HPRT-1, ACTβ) using the National Centre for Biotechnology Information (NCBI) website. All primers were tested for specificity by analyzing the melt curve of a gene and the amplified product was run on the agarose gel. Real-time quantitative polymerase chain reaction (PCR) (RT-qPCR) was carried out with gene-specific primers using 1:3 dilution of cDNA. The RT-qPCR reactions of all genes were carried out according to the manufacturer’s instruction (480 SYBR Green I Master mix (Roche# 04887352001) with an annealing temperature at 60oC for 15 sec. The expression levels of mRNA were normalized with the geometric mean of three housekeeping genes (GAPDH, ACTβ and HPRT1). The cycle threshold (Ct) values were obtained by using the second derivative maximum method and the relative expression of mRNA levels was determined by using the standard delta-delta Ct (^^^^Ct) method. Table 7: Relative levels of Cytokines in TNBS-induced Colitis Model in Rats Mesalamine Mesalamine 100 mpk 39 mpk ce )- 67 -IL-1α * ** * * * * indicates h kines and *** indicates loConclusions:

[0236] As can be seen from Table 7, the expression of mRNA for INFΥ, IL-5 and IL- 12β were reduced after treatment with compounds (S)-I-7 and (S)-I-25, while levels of IL-2 remained the same. With compound (S)-I-6 treatment, the levels of INFΥ, IL-5 and IL-12β remained the same and the levels of IL-2 increased. It is hypothesized that the exemplary compounds of the application may induce cytokine effects which are associated with improved healing and recovery processes from disease insult, injury and inflammation. - 68 -

Claims

CLAIMS:

1. A compound of Formula I, or a pharmaceutically acceptable salt, solvate and / or prodrug thereof:wherein: R1is selected from H, C1-6alkyl, C(X1)R8, C(X1)X2R8, S(O)R8and SO2R8; X1and X2are independently selected from O, NH, NC1-4alkyl and S; R2is selected from H and C1-6alkyl; R3is selected from H, halo and C1-4alkyl; R4is selected from H and C1-4alkyl; R5is selected from H, C1-6alkyl, OC1-6alkyl and OH; R6is selected from C1-10alkyl, C1-6alkyleneC3-8cycloalkyl, C1-6alkyleneC3-8heterocycloalkyl, C1-6alkyleneC6-10aryl and C1-6alkyleneC6-10heteroaryl, wherein the alkyl is optionally interrupted by one to three heteromoieties independently selected from O, NH, NHC1-4alkyl and S and further optionally substituted by one to three of NH2, =O and =S; R7is selected from H and C1-6alkyl; or R5and R7are joined to form a 7 membered ring; R8is selected from H, C1-10alkyl, C3-8cycloalkyl, C3-8heterocycloalkyl, C6-10aryl and C5-10heteroaryl, wherein the alkyl is optionally substituted with one or more halo; and all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom.

2. The compound of claim 1, wherein R1is selected from H, C1-4alkyl, C(O)R8, C(O)OR8, C(O)NHR8, C(S)OR8, C(S)NHR8and SO2R8, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. - 69 -3. The compound of claim 1, wherein R1is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, C(CH3)3, CH(CH3)CH2CH3and CH2CH(CH3)2, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom.

4. The compound claim 1 or 2, wherein R8is selected from H, C1-6alkyl, C3-6cycloalkyl, C3-6heterocycloalkyl, phenyl and C6heteroaryl, wherein the alkyl is optionally substituted with one or more halo and all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom.

5. The compound of claim 4, wherein R8is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, C(CH3)3, CH(CH3)CH2CH3and CH2CH(CH3)2and phenyl, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom.

6. The compound of any one of claims 1 to 5, wherein R2is selected from H, CH3and CH2CH3.

7. The compound of claim 1, wherein R1and R2are both H.

8. The compound of any one of claims 1 to 7, wherein R3is H.

9. The compound of any one of claims 1 to 8, wherein R4is selected from H, CH3and CH2CH3.

10. The compound of any one of claims 1 to 9, wherein R5is selected from H, C1-4alkyl, OC1-4alkyl and OH, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom.

11. The compound of claim 10, wherein R5is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, C(CH3)3, CH(CH3)CH2CH3, CH2CH(CH3)2, OCH3, OCH2CH3, OCH2CH2CH3, OCH(CH3)2, OCH2CH2CH2CH3, OC(CH3)3, OCH(CH3)CH2CH3, OCH2CH(CH3)2and OH, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom.

12. The compound of any one of claims 1 to 12, R6is selected from C1-6alkyl, C1-4alkyleneC5-6cycloalkyl, C1-4alkyleneC5-6heterocycloalkyl, C1-4alkylenephenyl and C1-4alkyleneC6heteroaryl, wherein the alkyl is optionally interrupted by one heteromoiety selected from O and S and further optionally substituted by one of NH2, =O and / or =S, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom. - 70 -13. The compound of claim 12, wherein R6is selected from CH3, CF3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, C(CH3)3, CH(CH3)CH2CH3, CH2CH(CH3)2, CH2CH2SCH3, CH2CH2OCH3, CH2CH2SCF3, CH2CH2OCF3and CH2phenyl.

14. The compound of any one of claims 1 to 13, wherein R7is selected from H, CF3and CH3. In some embodiments, R7is H.

15. The compound of any one of claims 1 to 9, wherein R5and R7are joined to form a 7 membered ring and the compound of Formula I has the following structure: or a pharmaceutically accep, r prodrug thereof. wherein R1, R2, R3, R4, and R6are as defined for Formula I.

16. The compound of claim 15, wherein R6is selected from C1-6alkyl, C1-4alkyleneC5-6cycloalkyl, C1-4alkyleneC5-6heterocycloalkyl, C1-4alkylenephenyl and C1-4alkyleneC6heteroaryl, wherein the alkyl is optionally interrupted by one heteromoiety selected from O and S and further optionally substituted by one of NH2, =O and / or =S, wherein all available hydrogen atoms are optionally and independently replaced with a fluorine or deuterium atom.

17. The compound of claim 16, wherein R6is selected from CH3, CF3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, C(CH3)3, CH(CH3)CH2CH3, CH2CH(CH3)2, CH2CH2SCH3, CH2CH2OCH3, CH2CH2SCF3, CH2CH2OCF3and CH2phenyl.

18. The compound of any one of claims 1 to 17 wherein the stereochemistry at the carbon to which R6is bonded is S.

19. The compound of any one of claims 1 to 17 wherein the stereochemistry at the carbon to which R6is bonded is R.

20. The compound of claim 1, selected from the compounds listed below, or a pharmaceutically acceptable salt, solvate and / or prodrug thereof: Compound ID Structure- 71 -(S)-I-1- 72 -(S)-I-9- 73 -(S)-I-17- 74 -(S)-I-25 21.1 to 20, or a pharmaceutically acceptable salt, solvate and / or prodrug thereof, and a pharmaceutically acceptable carrier.

22. A method for treating diseases, disorders or conditions that benefit from treatment with mesalamine, the method comprising administering an effective amount of a compound of any one of claims 1 to 20 to a subject in need thereof.

23. The method of claim 22, wherein the disease, disorder or condition that benefits from treatment with mesalamine is an inflammatory condition.

24. The method of claim 23, wherein the inflammatory condition is an inflammatory condition of the gastrointestinal (GI) tract.

25. The method of claim 24, wherein the inflammatory condition of the GI tract is selected from inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS).

26. The method of claim 25, wherein the IBD is ulcerative colitis or Crohn's Disease.

27. The method of any one of claims 22 to 26, wherein, the subject is a mammal.

28. The method of any one of claims 22 to 27, wherein the administration of the compound reduces gastric damage compared to administration of an equivalent dose of mesalamine and / or increases anti-inflammatory effects compared to administration of an equivalent dose of mesalamine and / or enhances the healing and recovery processes compared to administration of an equivalent dose of mesalamine. - 75 -

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