SUBSTITUTED BENZOTRIAZINONE METABOLITES OF A GPR139 AGONIST

Abstract

Compounds that are mammalian metabolites of an agonist of G-protein-coupled receptor 1.39 (GPR139), intermediates used in the synthesis of such metabolites, pharmaceutical compositions comprising such metabolites, and the use of such metabolites as biomarkers and agents in the treatment of schizophrenia, for example, negative and/or cognitive symptoms of schizophrenia, disorders associated with social and cognitive dysfunction, as well as several other disorders related to modulated of GPR139.

Claims

1. A compound of formula I ##STR00081## or a pharmaceutically acceptable salt thereof, wherein: each R.sup.1 is independently chosen from —OH, —OSO.sub.3H, —O-glucuronide, —SH, a —S(O)C.sub.1-C.sub.6 alkyl group, a —S(O).sub.2C.sub.1-C.sub.6 alkyl group, a —SC.sub.1-C.sub.6 alkyl group, a —SC.sub.1-C.sub.6 alkyl-NHC(O)C.sub.1-C.sub.6 alkyl group, an amino acid, and a peptide, wherein each hydrogen atom in a C.sub.1-C.sub.6 alkyl group is optionally replaced by OH, oxo, —CO.sub.2H, —O-glucuronide, —NH.sub.2, a —NHC(O)C.sub.1-C.sub.6 alkyl group, or -a N(H)C.sub.1-C.sub.6 alkyl-CO.sub.2H group; R.sup.2 is H, —OH, or —O-glucuronide; R.sup.3 is a C.sub.1-C.sub.6 alkyl group optionally substituted with —OH, oxo, —O-glucuronide, —NH.sub.2, an —NHC(O)C.sub.1-C.sub.6 alkyl group, or —NHCH.sub.2COOH; each R.sup.4 is independently —OH or —O-glucuronide; n is an integer from 0 to 4; and m is an integer from 0 to 4; provided that: (a) n is at least 1, (b) m is at least 1, (c) R.sup.2 is not H, and/or (d) R.sup.3 is substituted.

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.2 is H.

3. The compound or pharmaceutically acceptable salt thereof according to claim 2, wherein R.sup.3 is methyl.

4. The compound or pharmaceutically acceptable salt thereof according to claim 3, wherein m is 0.

5. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein n is 1.

6. The compound or pharmaceutically acceptable salt thereof according to claim 5, wherein R.sup.1 is at C-1.

7. The compound or pharmaceutically acceptable salt thereof according to claim 5, wherein R.sup.1 is at C-2.

8. The compound or pharmaceutically acceptable salt thereof according to claim 5, wherein R.sup.1 is at C-3.

9. The compound or pharmaceutically acceptable salt thereof according to claim 5, wherein R.sup.1 is at C-4.

10. The compound or pharmaceutically acceptable salt thereof according to claim 3, wherein n is 2.

11. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R.sup.1 is an amino acid.

12. The compound or pharmaceutically acceptable salt thereof according to claim 11, wherein the amino acid is cysteine.

13. The compound or pharmaceutically acceptable salt thereof according to claim 12, wherein the cysteine is attached through the side chain thiol.

14. The compound or pharmaceutically acceptable salt thereof according to claim 13, wherein the amino acid is N-terminally acetylated.

15. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R.sup.1 is a peptide.

16. The compound or pharmaceutically acceptable salt thereof according to claim 15, wherein the peptide comprises a cysteine.

17. The compound or pharmaceutically acceptable salt thereof according to claim 15, wherein the peptide is attached through the side chain thiol of the cysteine.

18. The compound or pharmaceutically acceptable salt thereof according to claim 16, wherein the peptide is 2-6 residues in length.

19. The compound or pharmaceutically acceptable salt thereof according to claim 18, wherein the peptide is N-terminally acetylated.

20. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein n is 0.

21. The compound or pharmaceutically acceptable salt thereof according to claim 20, wherein m is an integer from 1 to 4.

22. The compound or pharmaceutically acceptable salt thereof according to claim 21, wherein R.sup.2 is H.

23. The compound or pharmaceutically acceptable salt thereof according to claim 22, wherein R.sup.3 is methyl.

24. The compound or pharmaceutically acceptable salt thereof according to claim 23, wherein m is 1.

25. The compound or pharmaceutically acceptable salt thereof according to claim 24, wherein R.sup.4 is located at C-5.

26. The compound or pharmaceutically acceptable salt thereof according to claim 24, wherein R.sup.4 is located at C-6.

27. The compound or pharmaceutically acceptable salt thereof according to claim 24, wherein R.sup.4 is located at C-7.

28. The compound or pharmaceutically acceptable salt thereof according to claim 24, wherein R.sup.4 is located at C-8.

29. The compound or pharmaceutically acceptable salt thereof according to claim 20, wherein m is 0.

30. The compound or pharmaceutically acceptable salt thereof according to claim 29, wherein R.sup.2 is H.

31. The compound or pharmaceutically acceptable salt thereof according to claim 30, wherein R.sup.3 is substituted with —OH, oxo, —O-glucuronide, —NH.sub.2, a —NHC(O)C.sub.1-C.sub.6 alkyl group, or —NHCH.sub.2COOH.

32. The compound or pharmaceutically acceptable salt thereof according to claim 29, wherein R.sup.3 is methyl.

33. The compound or pharmaceutically acceptable salt thereof according to claim 32, wherein R.sup.2 is —OH or —O-glucuronide.

34. A compound of formula II ##STR00082## or a pharmaceutically acceptable salt thereof, wherein: R.sup.5 and R.sup.6 are independently chosen from C.sub.1-C.sub.6 alkyl, —NH—C.sub.1-C.sub.6 alkyl, —NH-aryl, and —NH-heteroaryl groups, wherein each hydrogen atom in a C.sub.1-C.sub.6 alkyl group is optionally replaced by —OH, oxo, or —CO.sub.2H.

35. The compound or pharmaceutically acceptable salt thereof according to claim 34, wherein R.sup.5 is methyl.

36. The compound or pharmaceutically acceptable salt thereof according to claim 35, wherein R.sup.6 is a —NH—C.sub.1-C.sub.6 alkyl group.

37. The compound or pharmaceutically acceptable salt thereof according to claim 36, wherein R.sup.6 is —NH-ethyl, and wherein at least one hydrogen atom in ethyl is replaced by —OH, oxo, or —CO.sub.2H.

38. The compound or pharmaceutically acceptable salt thereof according to claim 35, wherein R.sup.6 is —NH(1,2,3-oxadiazole).

39. A compound of formula III ##STR00083## or a pharmaceutically acceptable salt thereof, wherein: R.sup.7 is a C.sub.1-C.sub.6 alkyl group, wherein each hydrogen atom in the C.sub.1-C.sub.6 alkyl group is optionally replaced by —OH, oxo, —CO.sub.2H, or —NH.sub.2; provided that the compound is not ##STR00084##

40. The compound or pharmaceutically acceptable salt thereof according to claim 39, wherein R.sup.7 is ethyl, wherein at least one hydrogen atom in ethyl is replaced by —OH, oxo, —CO.sub.2H, or —NH.sub.2.

41. A compound selected from: (S)-2-(6-hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(5-hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(7-hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(8-hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid; (2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid; (2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid; (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid; (S)-4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl hydrogen sulfate; (S)-4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl hydrogen sulfate; (S)-4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl hydrogen sulfate; (S)-4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl hydrogen sulfate; N5-(1-((carboxymethyl)amino)-1-oxo-3-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)thio)propan-2-yl)glutamine; N5-(1-((carboxymethyl)amino)-1-oxo-3-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)thio)propan-2-yl)glutamine; N5-(1-((carboxymethyl)amino)-1-oxo-3-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)thio)propan-2-yl)glutamine; N5-(1-((carboxymethyl)amino)-1-oxo-3-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl)thio)propan-2-yl)glutamine; S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)cysteinylglycine; S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)cysteinylglycine; S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)cysteinylglycine; S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl)cysteinylglycine; S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4 dihydrobenzo[d][1,2,3]triazin-6-yl)cysteine; S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)cysteine; S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)cysteine; S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl)cysteine; N-acetyl-S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)cysteine; N-acetyl-S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)cysteine; N-acetyl-S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)cysteine; N-acetyl-S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl)cysteine; (S)-2-(6-mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(5-mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(7-mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(8-mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(6-(methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(5-(methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(7-(methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(8-(methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; 2-(6-(methylsulfinyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N—((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; 2-(5-(methylsulfinyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N—((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; 2-(7-(methylsulfinyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N—((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; 2-(8-(methylsulfinyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N—((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(6-(methylsulfonyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(5-(methylsulfonyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(7-(methylsulfonyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-(8-(methylsulfonyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-oxo-3-((4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)thio)propanoic acid; (S)-2-oxo-3-((4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)thio)propanoic acid; (S)-2-oxo-3-((4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)thio)propanoic acid; (S)-2-oxo-3-((4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl)thio)propanoic acid; (S)—N-(1-(2-hydroxy-4-(trifluoromethoxy)phenyl)ethyl)-2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide; (S)—N-(1-(3-hydroxy-4-(trifluoromethoxy)phenyl)ethyl)-2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide; (R)—N-(2-hydroxy-1-(4-(trifluoromethoxy)phenyl)ethyl)-2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide; N-(1-hydroxy-1-(4-(trifluoromethoxy)phenyl)ethyl)-2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide; (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-(5-((S)-1-(2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamido)ethyl)-2-(trifluoromethoxy)phenoxy)tetrahydro-2H-pyran-2-carboxylic acid; (2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(2-((S)-1-(2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamido)ethyl)-5-(trifluoromethoxy)phenoxy)tetrahydro-2H-pyran-2-carboxylic acid; (2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-((S)-1-(2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamido)-1-(4-(trifluoromethoxy)phenyl)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid; (2R,3R,4R,5S,6S)-3,4,5-trihydroxy-6-((R)-2-(2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamido)-2-(4-(trifluoromethoxy)phenyl)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid; 2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide; 2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetic acid; (S)-2-hydroxy-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide; (S)-2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)acetic acid; and (S)—N-(1-(4-(trifluoromethoxy)phenyl)ethyl)-1,2,3-oxadiazol-5-amine, or a pharmaceutically acceptable salt thereof.

Description

EXAMPLES

[0161] The following examples are intended to be illustrative and non-limiting, and represent specific embodiments of the present disclosure.

[0162] Example chemical entities useful in methods of the description will now be described by reference to illustrative synthetic schemes for their general preparation below and the specific examples that follow. Artisans will recognize that, to obtain the various compounds herein, starting materials may be suitably selected so that the ultimately desired substituents will be carried through the reaction scheme with or without protection as appropriate to yield the desired product. Alternatively, it may be necessary or desirable to employ, in the place of the ultimately desired substituent, a suitable group that may be carried through the reaction scheme and replaced as appropriate with the desired substituent. Furthermore, one of skill in the art will recognize that the transformations shown in the schemes below may be performed in any order that is compatible with the functionality of the particular pendant groups.

[0163] Proton nuclear magnetic resonance (NMR) spectra were obtained for many of the compounds in the following examples. Characteristic chemical shifts (δ) are given in parts-per-million downfield from tetramethylsilane using conventional abbreviations for designation of major peaks, including s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad). The following abbreviations are used for common solvents: CDCl.sub.3 (deuterochloroform), DMSO-d.sub.6 (deuterodimethylsulfoxide), and CD.sub.3OD (deuteromethanol or methanol-d.sub.4). The mass spectra were recorded using either electrospray ionization (ESI) or atmospheric pressure chemical ionization.

[0164] As used herein, terms have their using conventional abbreviations, unless otherwise indicated, for example: room temperature (RT), methanol (MeOH), ethanol (EtOH), isopropanol (IPA), acetonitrile (MeCN or AcCN), tetrahydrofuran (THF), ethyl acetate (EtOAc), dichloromethane (DCM), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), hydrochloric acid (HCl), diisopropylethylamine (DIEA or DIPEA), hydroxybenzotriazole (HOBT), N-(3-dimethylaminopropyl)-N′-ethylcarbonate (EDC), and the like.

Example 1

[0165] Extraction of Metabolites from Rat Plasma

[0166] Rat plasma samples were obtained from a study in which male and female Sprague Dawley rats were administered repeat 3-mg/kg oral doses of Compound A QD for 14 days. Whole blood was obtained from rats at selected time points on Day 14 (predose and 0.5, 1, 2, 4, 8, and 24 hours postdose) and processed into plasma for analysis. Plasma from 3 animals/sex/time point were pooled (30 μL each), combined with 360 μL of ice cold ACN and mixed to precipitate proteins. Samples were centrifuged, and the supernatants were taken and dried down under a stream of nitrogen. Each sample was reconstituted with 100 μL of 5% ACN, and 10 μL was analyzed by LC/MS/MS for profiling and metabolite identification.

Example 2

[0167] Extraction of Metabolites from Dog Plasma

[0168] Dog plasma samples were obtained from a study in which male and female beagle dogs were administered repeat 30-mg/kg oral doses of Compound A QD for 14 days. Whole blood was obtained from dogs at selected time points on Day 14 (predose and 0.5, 1, 2, 4, 8, and 24 hours postdose) and processed into plasma for analysis. Plasma from 2 animals/sex/time point were pooled (50 μL each), combined with 400 μL of ice cold ACN and mixed to precipitate proteins. Samples were centrifuged, and the supernatants were taken and dried down under a stream of nitrogen. Each sample was reconstituted with 100 μL of 5% ACN, and 10 μL was analyzed by LC/MS/MS for profiling and metabolite identification.

Example 3

[0169] Extraction of Metabolites from Dog Urine, Bile, Liver Tissue, and Kidney Tissue

[0170] Dog urine, bile, and liver and kidney tissue samples were obtained from a study in which male and female beagle dogs were administered repeat 60-mg/kg oral doses of Compound A QD for 13 weeks. Urine and bile were centrifuged and supernatant was analyzed by LC/MS/MS without further processing or dilution. Kidney and centrilobular, midzonal, and periportal liver tissue samples were diluted 3-fold with saline solution and homogenized. Homogenized samples were crashed with 4 volumes of ACN, vortexed, and centrifuged at 15,000×g for 10 minutes. The resulting supernatant was reconstituted with 100 μL of 5% ACN, and 10 μL was analyzed by LC/MS/MS for profiling and metabolite identification.

Example 4

[0171] Extraction of Metabolites from Monkey Plasma

[0172] Cynomolgus monkey plasma and urine samples were obtained from a study in which animals were administered a single 200 mg/kg oral dose of Compound A. Plasma samples from each monkey was pooled using Hamilton's method using the area under the plasma concentration-time curve from time 0 to 24 hours. Proteins were precipitated with 3 volumes of ACN and mixed thoroughly. The samples were vortexed and centrifuged. The supernatant was removed, dried under a stream of nitrogen, and reconstituted with 80/20 water/ACN (v/v) for analysis by LC/MS/MS for profiling and metabolite identification.

[0173] Urine was pooled across subjects (1000 μL each, 6 subjects) from 0 to 6, 6 to 12, 12 to 24, 24 to 48, and 48 to 72 hours postdose and centrifuged at 4000×g for 10 minutes to remove any pellet. Predose and placebo urine samples were also pooled by combining equivalent volumes (1,000 μL) and processed in the same manner. Samples were then analyzed by LC/MS/MS for profiling and metabolite and identification.

Example 5

[0174] Extraction of Metabolites from Human Plasma & Urine

[0175] Human plasma and urine samples were obtained from study in which healthy volunteers were administered either a single 40-mg oral dose of Compound A or placebo as part of a single rising dose (SRD) study.

[0176] Plasma samples from 6 subjects were pooled using Hamilton's method using the area under the plasma concentration-time curve from time 0 to 48 hours for each subject, and further pooled across the subjects (250 μL each) to generate a single plasma pool. As controls, equal volumes (250 μL) of predose plasma were pooled, as well as equal volumes (625 μL) of plasma were pooled from 2 subjects who received placebo.

[0177] Proteins were precipitated with 3 volumes of ACN and mixed thoroughly. The samples were centrifuged and supernatant was removed. The pellets were resuspended with 2 mL of 80/20 ACN/water (volume-over-volume [v/v]), mixed, and centrifuged. Supernatants were combined, dried under a stream of nitrogen, and reconstituted with 80/20 water/ACN (v/v) with 0.1% formic acid (FA) for analysis by LC/MS/MS for profiling and metabolite identification.

[0178] Urine was pooled across subjects (1000 μL each, 6 subjects) from 0 to 6, 6 to 12, 12 to 24, 24 to 48, and 48 to 72 hours postdose and centrifuged at 4000×g for 10 minutes to remove any pellet. Predose and placebo urine samples were also pooled by combining equivalent volumes (1000 μL) and processed in the same manner. Samples were then analyzed by LC/MS/MS for profiling and metabolite identification.

Example 6

Qualitative Assessment of the Metabolites of Compound A in Rat, Dog, Monkey, and Human Hepatocytes

[0179] A 2 μL aliquot of a 10 mM stock solution Compound A in dimethyl sulfoxide (DMSO) was added to 998 μL of Krebs-Henseleit buffer (KHB; pH 7.4) to prepare a 20-μM working solution for the hepatocyte incubation. The hepatocyte incubations consisted of (final concentrations): 10-μM Compound A, 1×10.sup.6 cells/mL hepatocytes (Life Technologies [Grand Island, N.Y., USA]; Sprague Dawley rat, lot RS688, 3 male donors; beagle dog, lot DB295, 1 male donor; cynomolgus monkey, lot CY359, 1 male donor; and human, lot HUE 115, 5 male and 5 female donors), 0.1% DMSO, and KHB at pH 7.4 made up to a final volume of 100 μL. Hepatocytes were thawed, processed, and prepared according to the protocol recommended by the vendor. A 96-well plate was used for this study. The reaction was initiated by adding Compound A to hepatocytes in the plate. Plates were incubated at 37° C. for 0 and 120 minutes. The reactions were terminated by adding equal volume of ice cold ACN. Precipitated protein was removed by centrifugation (5,000 rpm for 10 minutes at room temperature) and the supernatant was analyzed by LC/MS/MS.

Example 7

Qualitative Assessment of the Metabolite Profile of Compound A in HepatoPac Incubation

[0180] HepatoPac application and maintenance media were prepared per instructions from the vendor. HepatoPac cells pre-plated in 24-well plates were received from the vendor and a full medium change was performed with the species-specific maintenance medium. Rat wells and associated stromal and blank wells were filled with 300 μL of maintenance media and multi-species wells and associated stromal and blank wells were filled with 400 μL of maintenance media. Plates were incubated at 37° C. in a 10% CO.sub.2 atmosphere with >95% humidity for 48 hours.

[0181] After incubation, two application media were prepared and warmed to 37° C. Compound A (2.00 mg) was dispensed into a 4-mL glass vial and stored frozen at −20° C. until use. DMSO (509.8 μL) was added to the vial to generate a 10-mM stock solution. An 80-μL aliquot of the 10-mM stock solution was added to 40-mL each of the pre-warmed rat and multi-species application media inside a 50-mL conical tube to generate a 20-μM dosing solution. The fortified media was capped and stored at 37° C. in a water bath until use.

[0182] The maintenance media was withdrawn from all plates replaced with the species-specific application media. The application media was withdrawn a second time and replaced with species-specific application media (300 μL for rat HepatoPac, stromal, and blank wells and 400 μL for multi-species HepatoPac, stromal, and blank wells). Cells were stored in the incubator until dosing. When ready for dosing, media in all wells were removed and replaced with fresh application media at half the final dosing volume (150 μL for rat, stromal, and blank wells and 200 μL for multi-species, stromal, and blank wells). An equal volume of species-specific 20-μM dosing solution was applied to each well and gently swirled to initiate the incubation.

[0183] The plates were incubated and samples were collected at 0, 2, 4, 24, 48, 168, and 336 hours postdose from multi-species wells and associated stromal and blank wells; a single well was used for each time point. For the 0-, 2-, and 4-hour time point samples, the blank application media was already prewarmed; for the 24-, 48-, 168-, and 336 hour time point samples, the blank application media was prewarmed to 37° C. for 30 minutes before sampling. At each time point, 250 μL (rat wells) and 350 μL (multi-species wells) of media were removed from the HepatoPac, stromal and blank wells and quenched with 500 μL (rat wells) or 700 μL (multi-species wells) of ice-cold ACN in polypropylene tubes; these were referred to as the primary samples. A 250-μL (rat wells) or 350 μL (multi-species wells) aliquot of blank application media was added back to each well and all contents were removed and quenched with 600 μL (rat wells) or 800 μL (multi species wells) of ice-cold ACN in polypropylene tubes; these were referred to as the wash samples. Ice cold ACN (600 μL for rat wells and 800 μL for multi-species wells) was added back to the wells. Using the edge of a 1,000 μL pipette tip, the well was scraped thoroughly side to side starting at the top and moving to the bottom. After thorough scraping, all ACN was removed and transferred to polypropylene tubes; these were referred to as the cell lysate samples. After all samples were collected, an aliquot of 300 μL (rat wells) or 400 μL (multi-species wells) of application media was returned to the wells to maintain the local humidity for the remaining samples.

[0184] Cell lysate samples were stored frozen at −80° C. for future analysis. Primary and wash samples were gently rotated manually several times to ensure thorough mixing of sample and quench solutions, then centrifuged at 3,000 rpm for 30 minutes in an Allegra® X-14R centrifuge (Beckman Coulter [Brea, Calif., USA]). Supernatant (500 μL for rat samples and 800 μL for multi species samples) was collected and transferred to fresh polypropylene tubes and stored frozen at −80° C. until analysis. Tubes containing residual quench solution and protein pellet were stored frozen at −80° C. for possible future analysis. Immediately before liquid chromatography with mass spectrometry (LC/MS) analysis, the supernatants from the primary and wash samples were combined, and dried under a stream of nitrogen. Samples were reconstituted in water containing 5% ACN and analyzed by LC/MS.

Example 8

LC-MS/MS Analysis Conditions for Metabolite Identification

[0185] HPLC analysis was conducted using an Agilent 1290 binary pump (Agilent Technologies, Inc. [Santa Clara, Calif., USA]) with a PAL autosampler (Leap Technologies). Separation was achieved on a Kinetex 5-μm C18 column (2.1×150 mm; Phenomenex, Inc [Torrance, Calif., USA]) under ambient conditions. The HPLC eluent was introduced via electrospray positive ionization directly into an MDS SCIEX TripleTOF 5600 mass spectrometer with a source temperature of 500° C., IonSpray voltage floating set to 5,000, declustering potential of 80, and MS.sup.2 collision energy set to 20. The samples were analyzed in full scan mode with independent data acquisition triggered product ion scanning and mass defect filtering enabled.

[0186] Using the methods described above, the following compounds were identified in which ESI-MS m/z [M+H].sup.+ for each of the compounds matched the calculated value for the exact mass of the neutral species to about 2 decimal places.

(S)-2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0187] ##STR00010##

Exact mass calc'd for C.sub.18H.sub.15FN.sub.4O.sub.3, 392.11.

(S)-2-(6-hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0188] ##STR00011##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.4, 408.10.

(S)-2-(5-hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0189] ##STR00012##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.4, 408.10.

(S)-2-(7-hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0190] ##STR00013##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.4, 408.10.

(S)-2-(8-hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0191] ##STR00014##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.4, 408.10.

(2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid

[0192] ##STR00015##

Exact mass calc'd for C.sub.24H.sub.23F.sub.3N.sub.4O.sub.10, 584.14.

(2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid

[0193] ##STR00016##

Exact mass calc'd for C.sub.24H.sub.23F.sub.3N.sub.4O.sub.10, 584.14.

(2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid

[0194] ##STR00017##

Exact mass calc'd for C.sub.24H.sub.23F.sub.3N.sub.4O, 584.14.

(2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid

[0195] ##STR00018##

Exact mass calc'd for C.sub.24H.sub.23F.sub.3N.sub.4O, 584.14.

(S)-4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl hydrogen sulfate

[0196] ##STR00019##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.7S, 488.06.

(S)-4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl hydrogen sulfate

[0197] ##STR00020##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.7S, 488.06.

(S)-4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl hydrogen sulfate

[0198] ##STR00021##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.7S, 488.06.

(S)-4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl hydrogen sulfate

[0199] ##STR00022##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.7Si, 488.06.

N5-(1-((carboxymethyl)amino)-1-oxo-3-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)thio)propan-2-yl)glutamine

[0200] ##STR00023##

Exact mass calc'd for C.sub.28H.sub.30F.sub.3N.sub.7O.sub.9S, 697.18.

N5-(1-((carboxymethyl)amino)-1-oxo-3-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)thio)propan-2-yl)glutamine

[0201] ##STR00024##

Exact mass calc'd for C.sub.28H.sub.30F.sub.3N.sub.7O.sub.9S, 697.18.

N5-(1-((carboxymethyl)amino)-1-oxo-3-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)thio)propan-2-yl)glutamine

[0202] ##STR00025##

Exact mass calc'd for C.sub.28H.sub.30F.sub.3N.sub.7O.sub.9S, 697.18.

N5-(1-((carboxymethyl)amino)-1-oxo-3-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl)thio)propan-2-yl)glutamine

[0203] ##STR00026##

Exact mass calc'd for C.sub.28H.sub.30F.sub.3N.sub.7O.sub.9S, 697.18.

S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)cysteinylglycine

[0204] ##STR00027##

Exact mass calc'd for C.sub.23H.sub.23F.sub.3N.sub.6O.sub.6S, 568.14.

S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)cysteinylglycine

[0205] ##STR00028##

S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)cysteinylglycine

[0206] ##STR00029##

Exact mass calc'd for C.sub.23H.sub.23F.sub.3N.sub.6O.sub.6S, 568.14.

S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl)cysteinylglycine

[0207] ##STR00030##

Exact mass calc'd for C.sub.23H.sub.23F.sub.3N.sub.6O.sub.6S, 568.14.

S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4 dihydrobenzo[d][1,2,3]triazin-6-yl)cysteine

[0208] ##STR00031##

Exact mass calc'd for C.sub.21H.sub.23F.sub.3N.sub.5O.sub.5S, 511.11.

S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)cysteine

[0209] ##STR00032##

Exact mass calc'd for C.sub.21H.sub.20F.sub.3N.sub.5O.sub.5S, 511.11.

S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)cysteine

[0210] ##STR00033##

Exact mass calc'd for C.sub.21H.sub.20F.sub.3N.sub.5O.sub.5S, 511.11.

S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl)cysteine

[0211] ##STR00034##

Exact mass calc'd for C.sub.21H.sub.20F.sub.3N.sub.5O.sub.5S, 511.11.

N-acetyl-S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)cysteine

[0212] ##STR00035##

Exact mass calc'd for C.sub.23H.sub.22F.sub.3N.sub.5O.sub.6S, 553.12.

N-acetyl-S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)cysteine

[0213] ##STR00036##

Exact mass calc'd for C.sub.23H.sub.22F.sub.3N.sub.5O.sub.6S, 553.12.

N-acetyl-S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)cysteine

[0214] ##STR00037##

Exact mass calc'd for C.sub.23H.sub.22F.sub.3N.sub.5O.sub.6S, 553.12.

N-acetyl-S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl)cysteine

[0215] ##STR00038##

Exact mass calc'd for C.sub.23H.sub.22F.sub.3N.sub.5O.sub.6S, 553.12.

(S)-2-(6-mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0216] ##STR00039##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.3S, 424.08.

(S)-2-(5-mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0217] ##STR00040##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.3S, 424.08.

(S)-2-(7-mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0218] ##STR00041##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.3S, 424.08.

(S)-2-(8-mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0219] ##STR00042##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.3S, 424.08.

(S)-2-(6-(methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0220] ##STR00043##

Exact mass calc'd for C.sub.19H.sub.17F.sub.3N.sub.4O.sub.3S, 438.10.

(S)-2-(5-(methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0221] ##STR00044##

(S)-2-(7-(methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0222] ##STR00045##

Exact mass calc'd for C.sub.19H.sub.17F.sub.3N.sub.4O.sub.3S, 438.10.

(S)-2-(8-(methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0223] ##STR00046##

Exact mass calc'd for C.sub.19H.sub.17F.sub.3N.sub.4O.sub.3S, 438.10.

2-(6-(methylsulfinyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N—((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0224] ##STR00047##

Exact mass calc'd for C.sub.19H.sub.17F.sub.3N.sub.4O.sub.4S, 454.09.

2-(5-(methylsulfinyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N—((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0225] ##STR00048##

Exact mass calc'd for C.sub.19H.sub.17F.sub.3N.sub.4O.sub.4S, 454.09.

2-(7-(methylsulfinyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N—((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0226] ##STR00049##

Exact mass calc'd for C.sub.19H.sub.17F.sub.3N.sub.4O.sub.4S, 454.09.

2-(8-(methylsulfinyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N—((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0227] ##STR00050##

Exact mass calc'd for C.sub.19H.sub.17F.sub.3N.sub.4O.sub.4S, 454.09.

(S)-2-(6-(methylsulfonyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0228] ##STR00051##

Exact mass calc'd for C.sub.19H.sub.17F.sub.3N.sub.4O.sub.5S, 470.09.

(S)-2-(5-(methylsulfonyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0229] ##STR00052##

Exact mass calc'd for C.sub.19H.sub.17F.sub.3N.sub.4O.sub.5S, 470.09.

(S)-2-(7-(methylsulfonyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0230] ##STR00053##

Exact mass calc'd for C.sub.19H.sub.17F.sub.3N.sub.4O.sub.5S, 470.09.

(S)-2-(8-(methylsulfonyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0231] ##STR00054##

Exact mass calc'd for C.sub.19H.sub.17F.sub.3N.sub.4O.sub.5S, 470.09.

(S)-2-oxo-3-((4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)thio)propanoic acid

[0232] ##STR00055##

Exact mass calc'd for C.sub.21H.sub.17F.sub.3N.sub.4O.sub.6S, 510.08.

(S)-2-oxo-3-((4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)thio)propanoic acid

[0233] ##STR00056##

Exact mass calc'd for C.sub.21H.sub.17F.sub.3N.sub.4O.sub.6S, 510.08.

(S)-2-oxo-3-((4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)thio)propanoic acid

[0234] ##STR00057##

Exact mass calc'd for C.sub.21H.sub.17F.sub.3N.sub.4O.sub.6S, 510.08.

(S)-2-oxo-3-((4-oxo-3-(2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-8-yl)thio)propanoic acid

[0235] ##STR00058##

Exact mass calc'd for C.sub.21H.sub.17F.sub.3N.sub.4O.sub.6S, 510.08.

(S)—N-(1-(2-hydroxy-4-(trifluoromethoxy)phenyl)ethyl)-2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide

[0236] ##STR00059##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.4, 408.10.

(S)—N-(1-(3-hydroxy-4-(trifluoromethoxy)phenyl)ethyl)-2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide

[0237] ##STR00060##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.4, 408.10.

(R)—N-(2-hydroxy-1-(4-(trifluoromethoxy)phenyl)ethyl)-2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide

[0238] ##STR00061##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.4, 408.10.

N-(1-hydroxy-1-(4-(trifluoromethoxy)phenyl)ethyl)-2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide

[0239] ##STR00062##

Exact mass calc'd for C.sub.18H.sub.15F.sub.3N.sub.4O.sub.4, 408.10.

(2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-(5-((S)-1-(2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamido)ethyl)-2-(trifluoromethoxy)phenoxy)tetrahydro-2H-pyran-2-carboxylic acid

[0240] ##STR00063##

Exact mass calc'd for C.sub.24H.sub.23F.sub.3N.sub.4O.sub.10, 584.14.

(2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(2-((S)-1-(2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamido)ethyl)-5-(trifluoromethoxy)phenoxy)tetrahydro-2H-pyran-2-carboxylic acid

[0241] ##STR00064##

Exact mass calc'd for C.sub.24H.sub.23F.sub.3N.sub.4O.sub.10, 584.14.

[0242] (2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-((S)-1-(2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamido)-1-(4-(trifluoromethoxy)phenyl)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid; exact mass 584.137 calc'd for C.sub.24H.sub.23F.sub.3N.sub.4O.sub.10, corresponding to:

##STR00065##

Exact mass calc'd for C.sub.24H.sub.23F.sub.3N.sub.4O.sub.10, 584.14.

(2R,3R,4R,5S,6S)-3,4,5-trihydroxy-6-((R)-2-(2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamido)-2-(4-(trifluoromethoxy)phenyl)ethoxy)tetrahydro-2H-pyran-2-carboxylic acid

[0243] ##STR00066##

Exact mass calc'd for C.sub.24H.sub.23F.sub.3N.sub.4O.sub.10, 584.14.

2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide

[0244] ##STR00067##

Exact mass calc'd for C.sub.9H.sub.8N.sub.4O.sub.2, 204.06.

2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetic acid

[0245] ##STR00068##

Exact mass calc'd for C.sub.9H.sub.7N.sub.3O.sub.3, 205.05.

(S)-2-hydroxy-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0246] ##STR00069##

Exact mass calc'd for C.sub.11H.sub.12F.sub.3NO.sub.3, 263.08.

(S)-2-oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)acetic acid

[0247] ##STR00070##

Exact mass calc'd for C.sub.11H.sub.10F.sub.3NO.sub.4, 277.06.

(S)—N-(1-(4-(trifluoromethoxy)phenyl)ethyl)-1,2,3-oxadiazol-5-amine

[0248] ##STR00071##

Exact mass calc'd for C.sub.11H.sub.10F.sub.3N.sub.3O.sub.2, 273.07.

[0249] The preparations below were carried out in appropriate vessels and were typically stirred. Where indicated, products of certain preparations and examples are purified by mass-triggered HPLC. Where indicated products of the preparations and examples were purified by the following methods: HPLC Method A: Pump: Shimadzu LC-8A; UV/Vis: SPD-20A; Software: LCsolution. A Phenomenex Gemini® C18, 5 μm, ID 30×100 mm column was used and eluted with gradients of ACN (containing 0.035% TFA) and water (containing 0.005% TFA). A 10% to 100% ACN gradient was used unless otherwise indicated. SFC purification: Multigram II Berger SFC; ChiralPak AD-H (5 μm, 21×150 mm) column was used and eluted with gradients of liquid CO.sub.2 and isopropanol. After isolation by chromatography, the solvent was removed and the product was obtained by evaporating product containing fractions (e.g., GeneVac™), rotary evaporator, evacuated flask, lyophilization, etc.

Synthetic Example 1

Preparation of (S)-2-(6-(methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0250] ##STR00072##

Step 1: (S)-2-(6-bromo-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0251] To a vial containing 2-(6-bromo-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetic acid (420 mg, 1.479 mmol), HOBt (316 mg, 1.774 mmol), and EDC (368 mg, 1.922 mmol) was added DMF (2957 μL). After stirring at RT for 5 minutes, (S)-1-(4-(trifluoromethoxy)phenyl)ethan-1-amine, HCl (536 mg, 2.218 mmol) and iPr.sub.2EtN (1290 μL, 7.39 mmol) were added. The reaction mixture was stirred at RT for 1 hour, then water was added and the white solid was filtered. The resulting crude material was dissolved in CH.sub.2Cl.sub.2 and purified via ISCO automated purification system, eluting with a gradient of 0-20% MeOH in DCM. The collected fractions were combined and solvent was removed via rotary evaporation at 35° C. The resulting mixture was dried in vacuo to give the title compound as a white solid (454 mg, 65%).

Step 2: (S)-2-(6-(methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0252] To a vial was added (S)-2-(6-bromo-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide (200 mg, 0.424 mmol), copper(II) sulfate (3 mg, 0.021 mmol), cesium carbonate (691 mg, 2.122 mmol) and DMSO (2.1 mL) and then ethane-1,2-dithiol (71 μL, 0.849 mmol). The vial was heated at 90° C. for 1 hour. Thiol formation was complete by UPLC so added solution of iodomethane (80 μl, 1.273 mmol) in 1 mL DMF. The solution was stirred at RT for 16 hours. The resulting crude material was diluted in DMF, filtered through a hydrophilic PTFE 0.45 μm filter (Millipore Millex-LCR) and purified via HPLC Method A. The collected fractions were combined and solvent was removed via rotary evaporation at 45° C. The resulting mixture was dried in vacuo to afford the title compound as a white solid (100 mg, 53%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.41 (d, J=7.0 Hz, 3H), 2.66 (s, 3H), 4.99 (t, J=7.3 Hz, 1H), 5.07 (s, 2H), 7.31-7.36 (m, 2H), 7.45-7.50 (m, 2H), 7.89-7.99 (m, 2H), 8.12 (d, J=8.5 Hz, 1H), 8.81 (d, J=8.0 Hz, 1H); ESI-MS m/z [M+H].sup.+ 439.1.

Synthetic Example 2

Preparation of 2-(6-(methylsulfinyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N—((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0253] ##STR00073##

[0254] To a RT solution of (S)-2-(6-(methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide (15 mg, 0.034 mmol) in CH.sub.2Cl.sub.2 (170 μL) was added mCPBA (5.9 mg, 0.034 mmol). The reaction was stirred at RT for 30 minutes. The resulting crude material was diluted in DMF, filtered through a hydrophilic PTFE 0.45 μm filter (Millipore Millex-LCR) and purified via HPLC Method A. The collected fractions were combined and solvent was removed via rotary evaporation at 45° C. The resulting mixture was dried in vacuo to provide the title compound as a white solid (6.1 mg, 39%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.42 (d, J=7.0 Hz, 3H), 2.89 (s, 3H), 4.99 (t, J=7.2 Hz, 1H), 5.13 (s, 2H), 7.34 (d, J=7.8 Hz, 2H), 7.45-7.50 (m, 2H), 8.35-8.39 (m, 1H), 8.40-8.45 (m, 1H), 8.54 (dd, J=1.9, 1.1 Hz, 1H), 8.84 (d, J=8.0 Hz, 1H); ESI-MS m/z [M+H].sup.+ 455.1.

Synthetic Example 3

Preparation of (S)-2-(6-(methylsulfonyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0255] ##STR00074##

[0256] To a RT solution of (S)-2-(6-(methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide (15 mg, 0.034 mmol) in CH.sub.2Cl.sub.2 (170 μL) was added mCPBA (12 mg, 0.068 mmol). The reaction was stirred at RT for 1 hour. The resulting crude material was diluted in DMF, filtered through a hydrophilic PTFE 0.45 μm filter (Millipore Millex-LCR) and purified via HPLC Method A. The collected fractions were combined and solvent was removed via rotary evaporation at 45° C. The resulting mixture was dried in vacuo to provide the title compound as a white solid (9.9 mg, 62%). .sup.1HNMR (400 MHz, DMSO-d.sub.6) δ ppm 1.42 (d, J=7.0 Hz, 3H), 3.41 (s, 3H), 4.95-5.05 (m, 1H), 5.15 (s, 2H), 7.31-7.37 (m, 2H), 7.45-7.50 (m, 2H), 8.47-8.52 (m, 1H), 8.55-8.60 (m, 1H), 8.70 (d, J=1.5 Hz, 1H), 8.85 (d, J=7.3 Hz, 1H); ESI-MS m/z [M+H].sup.+ 471.1.

Synthetic Example 4

Preparation of N-acetyl-S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)cysteine

[0257] ##STR00075##

Step 1: (S)-2-(6-mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0258] To a vial was added (S)-2-(6-bromo-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide (100 mg, 0.212 mmol), copper(II) sulfate (2 mg, 10.61 μmol), cesium carbonate (346 mg, 1.061 mmol) and DMSO (1.1 mL) and then ethane-1,2-dithiol (36 μl, 0.424 mmol). The vial was heated at 90° C. for 1 hour, then poured into 1 M HCl, extracted with EtOAc (2×10 mL), washed with water (2×20 mL), dried over MgSO.sub.4, filtered and concentrated to yield the title compound as a yellow solid, which was used without further purification.

Step 2: N-acetyl-S-(4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)cysteine

[0259] To a solution of (S)-2-(6-mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide (20 mg, 0.047 mmol) and 2-acetamidoacrylic acid (6 mg, 0.043 mmol) in dioxane (36 μL) was added 2 drops of piperidine. The reaction mixture was heated at reflux for 2 hours. The resulting crude material was diluted in DMF, filtered through a hydrophilic PTFE 0.45 μm filter (Millipore Millex-LCR) and purified via HPLC Method A. The collected fractions were combined and solvent was removed via rotary evaporation at 45° C. The resulting mixture was dried in vacuo to provide the title compound as a white solid (8.5 mg, 36%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.41 (d, J=7.2 Hz, 3H), 1.82 (d, J=1.7 Hz, 3H), 3.34-3.43 (m, 2H), 3.59-3.69 (m, 1H), 4.99 (t, J=7.4 Hz, 1H), 5.07 (s, 2H), 7.34 (d, J=8.4 Hz, 2H), 7.47 (d, J=8.8 Hz, 2H), 7.96-8.04 (m, 2H), 8.08-8.15 (m, 1H), 8.82 (d, J=7.9 Hz, 1H); ESI-MS m/z [M+H].sup.+ 554.1.

Synthetic Example 5

Preparation of (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid (Compound B)

[0260] ##STR00076##

[0261] To a solution of (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (155 mg, 0.214 mmol) in methanol (3 mL) was added 1.0 M aq. lithium hydroxide solution (1.070 ml, 1.070 mmol). The solution stirred at 20° C. for 1.5 hours and then the reaction was quenched with acetic acid (0.037 mL, 0.642 mmol). The reaction mixture (white slurry) was diluted with acetonitrile (1.5 mL) and water (1.5 mL) to dissolve all solids. Acetic acid (0.037 ml, 0.642 mmol) was added to give a solution with pH 6, which was subsequently filtered thought a syringe filter, rinsed with ACN/water, and purified by preparative HPLC (ACN/water, basic mode) to give the title compound as a white solid (57 mg, 46%). .sup.1H NMR (400 MHz, 1:1 deuterium oxide/CD.sub.3CN) δ ppm 1.94 (d, J=7.06 Hz, 3H) 3.99-4.16 (m, 3H) 4.37 (d, J=9.35 Hz, 1H) 5.46-5.52 (m, 1H) 5.52-5.59 (m, 1H) 5.61-5.68 (m, 1H) 5.73 (d, J=7.24 Hz, 1H) 7.77 (d, J=8.25 Hz, 2H) 7.93 (d, J=8.62 Hz, 2H) 8.24-8.33 (m, 2H) 8.67 (d, J=8.89 Hz, 1H); ESI-MS m/z [M+H].sup.+ 585.

Synthetic Example 6

Preparation of (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-((4-oxo-3-(2S-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0262] ##STR00077##

[0263] A mixture of (S)-2-(6-hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide (54 mg, 0.132 mmol), (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (63.0 mg, 0.159 mmol) and silver (I) oxide (83 mg, 0.357 mmol) in anhydrous ACN (1 mL) was stirred in the dark at 20° C. for 5 hours. LCMS showed about 50% conversion. The mixture was filtered through a large syringe filter, rinsed with acetonitrile and methanol, and purified by preparative HPLC (10-100% acetonitrile/water under basic mode) to give the title compound as a white solid (35 mg, 37%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.40 (d, J=6.97 Hz, 3H) 1.98-2.08 (m, 9H) 3.62 (s, 3H) 4.83 (d, J=9.81 Hz, 1H) 4.98 (quin, J=7.13 Hz, 1H) 5.06 (s, 2H) 5.12 (t, J=9.67 Hz, 1H) 5.19 (dd, J=9.63, 7.70 Hz, 1H) 5.45-5.55 (m, 1H) 6.04 (d, J=7.70 Hz, 1H) 7.33 (d, J=8.34 Hz, 2H) 7.46 (d, J=8.71 Hz, 2H) 7.70 (dd, J=8.90, 2.75 Hz, 1H) 7.74 (d, J=2.75 Hz, 1H) 8.24 (d, J=8.89 Hz, 1H) 8.81 (d, J=7.70 Hz, 1H); ESI-MS m/z [M+H].sup.+ 725.

Synthetic Example 7

Preparation of (S)-2-(6-hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

[0264] ##STR00078##

[0265] To a solution of 2-(6-hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetic acid (4.0 g, 18.09 mmol), (S)-1-(4-(trifluoromethoxy)phenyl)ethan-1-amine hydrochloride (5.24 g, 21.70 mmol), HOBt (1.385 g, 9.04 mmol) and EDC (5.20 g, 27.1 mmol) in DMF (32 ml) was added Hunig's base (6.32 mL, 36.2 mmol). The solution was stirred at 20° C. for 18 hours, then diluted with isopropyl acetate (400 mL) and washed with saturated ammonium chloride (400 mL), water (400 mL), and brine (400 mL). A precipitate was collected via filtration before separating the final layers. The solid phase was dried under vacuum for 1 hour to give a first batch of the title compound as an off-white solid (2.768 g).

[0266] The filtrate was separated and the organic layer was dried over magnesium sulfate, concentrated onto Celite® and purified on a 120 g silica gel column, eluting with a gradient of 0 to 100% ethyl acetate in heptane. The solids were transferred from the flask by slurrying in heptane and diethyl ether, then collected by vacuum filtration and dried under vacuum to give a second batch of the title compound (3.45 g) as a white solid (total yield 6.22 g, 84%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.40 (d, J=6.97 Hz, 3H) 4.97 (t, J=7.24 Hz, 1H) 5.01 (s, 2H) 7.33 (d, J=8.34 Hz, 2H) 7.41-7.54 (m, 4H) 8.08 (d, J=8.71 Hz, 1H) 8.79 (d, J=7.70 Hz, 1H) 11.10 (br s, 1H); ESI-MS m/z [M+H].sup.+ 409.

Synthetic Example 8

Preparation of 2-(6-hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetic acid

[0267] ##STR00079##

[0268] To a solution of 6-hydroxy-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (9.0 g, 50.2 mmol) and glycine (4.15 g, 55.3 mmol) in water (100 mL) was added triethylamine (7.70 mL, 55.3 mmol). The reaction mixture was stirred at 40° C. for 4 hours. The slurry was cooled to 20° C. Concentrated HCl (aq) (25.1 mL, 301 mmol) was added dropwise (violent bubbling occurred with first drops) and sodium nitrite (4.33 g, 62.8 mmol) was added portion-wise (bubbling occurred upon addition) and the mixture was stirred at 20° C. for 3 days. The solids were collected by vacuum filtration, rinsed with water and dried under high vacuum to give the title compound as a tan solid (9.512 g, 86%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 5.07 (s, 2H) 7.43-7.55 (m, 2H) 8.11 (d, J=8.71 Hz, 1H) 11.17 (s, 1H) 12.90-13.62 (m, 1H); ESI-MS m/z [M+H].sup.+ 222.

Synthetic Example 9

Preparation of 6-hydroxy-2H-benzo[d][1,3]oxazine-2,4(1H)-dione

[0269] ##STR00080##

[0270] A solution of 2-amino-5-hydroxybenzoic acid (9.5 g, 62.0 mmol) and triphosgene (19.60 g, 66.1 mmol) in THF (270 mL) was heated at 70° C. for 5 hours and then cooled to 20° C. The solid phase was collected by vacuum filtration and rinsed with heptane to give the title compound as a gray solid (9.072 g, 82%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 7.03 (d, J=8.25 Hz, 1H) 7.15-7.27 (m, 2H) 9.83 (s, 1H) 11.47 (s, 1H); ESI-MS m/z [M+H].sup.+ 180.

Biological Example 1

Bile Salt Export Pump (BSEP) and Multidrug Resistance-Associated Protein (MRP) Inhibition by (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy))phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid (Compound B)

[0271] Inhibition of BSEP (dog and human) and MRP2 (dog and human) by Compound A and Compound B were tested in membrane vesicles. Compound B inhibited dog MRP2 with an IC.sub.50 value of 1.80 μM. Human MRP2 was also inhibited (IC.sub.50=83.0 μM). BSEP inhibition was not identified in this assay.

TABLE-US-00001 TABLE 1 IC.sub.50 (μM) BSEP MRP2 MRP3 MRP4 Compound Rat Dog Human Rat Dog Human Human Human A >200 72.3 >200 >200 >200 >200 >200 >200 B N/A >200 >200 N/A 1.8 83 N/A N/A