Substituted benzotriazinone metabolites of GPR139 agonists

Abstract

Compounds that are mammalian metabolites of agonists of G protein-coupled receptor 139 (GPR139), intermediates used in the synthesis of such metabolites, pharmaceutical compositions containing such metabolites, and the use of such metabolites as biomarkers and drugs in the treatment of schizophrenia, e.g., disorders associated with negative and / or cognitive symptoms of schizophrenia, social and cognitive dysfunction, and several other disorders associated with modulation of GPR139.

Description

Technical Field

[0001] This application claims the benefit of priority of U.S. Patent Application No. 63 / 021,895, filed May 8, 2020, the entire content of which is incorporated herein by reference.

[0002] This disclosure relates to pharmaceutical chemistry, pharmacology, and medicine.

Background Art

[0003] GPR139 is an orphan G protein-coupled receptor. GPR139 can be linked to Gs, Gq, and Gi signaling and appears to be constitutively active when recombinantly expressed in mammalian cells. GPR139 is abundantly expressed in the CNS (central nervous system), to a lesser extent in the pancreas and pituitary gland, and at low levels in other peripheral tissues.

[0004] GPR139 is highly conserved among different species. For example, the GPR139 protein sequences of human, mouse, and rat share more than 94% identity at the amino acid level. The predominant expression in the brain and the high degree of sequence homology across different species suggest that GPR139 has an important role in physiology.

[0005] GPR139 has its strongest expression in the nucleus raphe magnus of the mouse. The raphe receives input from the basal ganglia and limbic system and sends output to midbrain and forebrain structures, including dopaminergic and serotonergic neurons. The nucleus raphe magnus is involved in pain processing, reproductive behavior, nutrition, sleep-wake cycle, stress response, and learning.

[0006] In particular, several findings have suggested a role for the habenula in schizophrenia. Large calcifications in the pineal gland and habenula are more common in people with schizophrenia than in normal controls. Furthermore, fMRI studies have shown changes in habenula activation in schizophrenia patients. Also, after errors in a difficult matching-to-sample task, the habenula was activated in control subjects but not in schizophrenia patients. Chronic treatment with cocaine or amphetamine damages the output pathway of the habenula in rats, resulting in a schizophrenia-like state. Accordingly, modulators of GPR139 are expected to be useful for treating other CNS disorders such as schizophrenia and depression. There is a need for treating such conditions and other conditions described herein using compounds that are metabolites of GPR139 agonists and that may be useful for treating diseases, disorders, or symptoms associated with GPR139, including use as biomarkers. The present disclosure provides metabolites that may be agonists of GPR139, as well as methods of using GPR139 metabolite agonists for treating diseases, disorders, and symptoms associated with GPR139 and other embodiments described herein in the form of compounds of Formulas I, II, and III. Certain activators of GPR139 are described in International Publication No. WO 2014 / 152917. Certain agonists of GPR139 are described in J. Chem. Inf. Model. 2014, 54, 1553-1557 and Med. Chem. Lett. 2011, 2, 303-306.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Non-Patent Documents

[0008]

Non-Patent Document 1

Non - Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0009] The compounds of the present disclosure are metabolites of GPR139 agonists and may be useful for the treatment of diseases, disorders, or conditions related to GPR139, including use as biomarkers.

[0010] In one aspect, the present disclosure provides a compound of formula I

Chemical Formula

[0011] In another aspect, the disclosure relates to a compound of formula II

Chemical formula

[0012] In another aspect, the disclosure relates to a compound of formula III

Chemical formula

[0013] In another aspect, the disclosure relates to (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 relates to a compound selected therefrom or a pharmaceutically acceptable salt thereof.

[0014] Another aspect of the present disclosure provides a compound or pharmaceutically acceptable salt defined in the previous paragraph for use in the treatment of a disease, disorder, or symptom selected from schizophrenia, autism spectrum disorder, sleep disorder, depression, bipolar disorder, cognitive impairment, attention deficit hyperactivity disorder, post-traumatic stress disorder, substance abuse, drug addiction, eating disorder, obsessive-compulsive disorder, anxiety disorder, pain, and fibromyalgia.

[0015] A further aspect of the present disclosure provides a method for treating a disease, disorder, or symptom associated with GPR139 in a subject, the method comprising administering an effective amount of a compound or pharmaceutically acceptable salt defined in the previous paragraph.

[0016] A further aspect of the present disclosure provides a method for treating a disease, disorder, or symptom in a subject, the method comprising administering an effective amount of a compound or pharmaceutically acceptable salt defined in the previous paragraph, wherein the disease, disorder, or symptom is selected from schizophrenia, autism spectrum disorder, sleep disorder, depression, bipolar disorder, cognitive impairment, attention deficit hyperactivity disorder, post-traumatic stress disorder, substance abuse, drug addiction, eating disorder, obsessive-compulsive disorder, anxiety disorder, pain, and fibromyalgia.

[0017] Another aspect of the present disclosure provides the use of a compound or pharmaceutically acceptable salt defined in the previous paragraph for the manufacture of a medicament for treating a disease, disorder, or symptom associated with GPR139.

[0018] A further aspect of the present disclosure provides a combination comprising a compound or pharmaceutically acceptable salt defined in the previous paragraph and at least one additional pharmacologically active agent.

[0019] Another aspect of the present disclosure provides a metabolite of a GPR193 agonist for use as a biomarker.

[0020] A further aspect of the present disclosure provides a method for preparing metabolites and / or intermediates of a GPR139 agonist.

Mode for Carrying Out the Invention

[0021] Further embodiments, features, and advantages of the present disclosure will become apparent from the following detailed description. The compounds of the present disclosure can be described as embodiments of any of the following listed clauses. It will be understood that any of the embodiments described herein can be used in connection with any other embodiment described herein, provided the embodiments do not conflict with each other.

[0022] As used herein, the term "C" 1~6 "alkyl" refers to a straight-chain or branched alkyl chain of 1 to 6 carbon atoms.

[0023] As used herein, the terms "halogen" and "halo" refer to chloro, fluoro, bromo, or iodo.

[0024] As used herein, the term "oxo" represents a carbonyl oxygen. For example, cyclopentyl substituted with oxo is cyclopentanone.

[0025] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a pharmaceutically acceptable organic or inorganic acid or base, including those described in Journal of Pharmaceutical Science, 66, 2-19 (1977). A non-limiting example is hydrochloride.

[0026] As used herein, the term "amino" refers to -NH2.

[0027] As used herein, the terms "hydroxy" or "hydroxyl" refer to -OH.

[0028] As used herein, the term "-O-glucuronide" refers to the α and β isomers of, also sometimes called, glucuronic acid, [Chem.] .

[0029] As used herein, the term "aryl" refers to a monocyclic or polycyclic condensed ring group of 6 to 12 carbon atoms having a fully conjugated π electron system. In some embodiments, it will be understood that aryl can be of limited size, such as C6-C 10 aryl. Exemplary aryl groups include, but are not limited to, phenyl, naphthylenyl, and anthracenyl. An aryl group may be unsubstituted or substituted, as described for alkyl or as described in the various embodiments provided herein.

[0030] As used herein, the term "heteroaryl" refers to a monocyclic or fused ring group of 5 to 12 ring atoms containing 1, 2, 3, or 4 ring heteroatoms selected from nitrogen, oxygen, and sulfur, with the remaining ring atoms being carbon atoms and having a fully conjugated pi electron system. In some embodiments, it will be understood that heteroaryl can be of a limited size, such as 3- to 7-membered heteroaryl, 5- to 7-membered heteroaryl, etc. Heteroaryl may be unsubstituted or substituted as described for alkyl or as described in the various embodiments provided herein. Exemplary heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl, pyrimidinyl, quinolinyl, isoquinolinyl, purinyl, tetrazolyl, triazinyl, pyrazinyl, tetrazinyl, quinazolinyl, quinoxalinyl, thienyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzoisoxazolyl, benzoisothiazolyl, and carbazolyl. Examples of heteroaryl groups shown in graphical representation include the following entities in the form of appropriately linked moieties:

Chemical formula

[0031] As used herein, the term "optionally" or "optionally" means that the subsequently described event or situation may occur, but need not occur, and that the description includes both the case where the event or situation occurs and the case where it does not occur. For example, "wherein C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C 10Each hydrogen atom in an aryl, or monocyclic or bicyclic heteroaryl group, is independently optionally substituted by a C1-C6 alkyl", means that by replacement of each hydrogen atom by an alkyl group, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3-7 membered heterocycloalkyl, C6-C 10 Alkyl may or may not be present on either an aryl, or monocyclic or bicyclic heteroaryl group, and the description thereof includes C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3-7 membered heterocycloalkyl, C6-C 10 The situation where an aryl, or monocyclic or bicyclic heteroaryl group is substituted with an alkyl group, and C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3-7 membered heterocycloalkyl, C6-C 10 Means including the situation where an aryl, or monocyclic or bicyclic heteroaryl group is not substituted with an alkyl group.

[0032] As used herein, the term "independently" means that the subsequently described event or situation is read on its own with respect to other similar events or situations. For example, in a situation where several equivalent hydrogen groups are optionally substituted by another group described in the situation, the use of "independently optionally" means that each instance of a hydrogen atom on that group may be substituted by another group, and the groups substituting each of the hydrogen atoms may be the same or different. As an example, when multiple groups are present and all of them can be selected from a set of possibilities, the use of "independently" means that each of these groups can be selected from a set of possibilities separate from any other group, and the groups selected in the situation may be the same or different.

[0033] As used herein, the term "agonist" refers to both full agonists and partial agonists and other agonists.

[0034] As used herein, the term "pharmaceutically acceptable excipient" refers to those typically used in the preparation of pharmaceutical compositions and should be pharmaceutically pure and non-toxic in the amounts used. These are generally solid, semi-solid, or liquid materials that can function as a vehicle or medium for the active ingredient in an aggregate. Some examples of pharmaceutically acceptable excipients can be found in Remington’s Pharmaceutical Sciences and the Handbook of Pharmaceutical Excipients and include diluents, vehicles, carriers, ointment bases, binders, disintegrants, lubricants, glidants, sweeteners, flavorants, gel bases, sustained-release matrices, stabilizers, preservatives, solvents, suspending agents, buffers, emulsifiers, dyes, aerosol propellants, coating agents, and the like.

[0035] As used herein, the term "substantially enantiomerically pure" refers to an enantiomeric purity of greater than 90% for a given stereocenter. Thus, the term "substantially enantiomerically pure" refers to greater than 80% ee (enantiomeric excess). In the case of a compound that exists as stereoisomers, such stereoisomers may be substantially enantiomerically pure or may have, for example, an enantiomeric purity of greater than 97% or, for example, an enantiomeric purity of greater than 99% at the stereocenter.

[0036] One of ordinary skill in the art will understand that certain compounds of the present disclosure may exist as isomers. All stereoisomers of the compounds of the present disclosure, including geometric isomers, enantiomers, and diastereomers in any ratio, are considered to be within the scope of the present disclosure.

[0037] One of ordinary skill in the art will understand that certain compounds of the present disclosure may exist as tautomers. All tautomeric forms of the compounds of the present disclosure are considered to be within the scope of the present disclosure.

[0038] The compounds of the disclosure also include all isotopic variants in which at least one atom has an atomic mass different from the most commonly found atomic mass in nature, but has the same atomic number.

[0039] As used herein, terms such as "the compounds of the disclosure" and "a compound of the disclosure" include embodiments of Formula I, Formula II, Formula III, as well as other more specific embodiments encompassed by Formulas I, II, and III described herein, each of the exemplary compounds described herein, and pharmaceutically acceptable salts of each of these embodiments. For the sake of clarity, it is understood that certain features of the disclosure described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for the sake of brevity, the various features of the disclosure described in the context of a single embodiment may be provided separately or in any suitable partial combination. All combinations of embodiments related to chemical groups represented by variable elements are specifically encompassed by the disclosure, and are disclosed herein as if each and every such combination were individually and explicitly disclosed, to the extent such combinations are compounds that are stable compounds (i.e., compounds that can be isolated, characterized, and tested for biological activity). Further, all partial combinations of the chemical groups recited in the embodiments describing such variable elements are also specifically encompassed by the disclosure and are disclosed herein as if each and every such partial combination of chemical groups were individually and explicitly disclosed herein.

[0040] The compounds of Formulas I, II, and III can be identified as metabolites of Compound A.

Chemical formula

[0041] The synthesis of Compound A can be found in U.S. Patent No. 9,556,130, the entire disclosure of which is incorporated herein by reference.

[0042] Metabolites of Compound A can be generated by administering Compound A to a subject. Subsequently, a body fluid or tissue can be collected and analyzed for the metabolite. In some embodiments, the body fluid is whole blood, urine, bile, urine, or any other body fluid suitable for analyzing metabolites. In some embodiments, the tissue is liver tissue, kidney tissue, or any other suitable tissue for analyzing metabolites.

[0043] Exemplary subjects include humans and non-human animals, such as mammals like monkeys, mice, rats, guinea pigs, dogs, cats, rabbits, cows, horses, sheep, goats, and pigs. The term also includes birds, fish, reptiles, amphibians, etc. In some embodiments, the subject is human. In some embodiments, the subject is a non-human mammal such as a mouse, rat, dog, or any other animal suitable for testing.

[0044] In some embodiments, the metabolite is generated in vitro. In some embodiments, the metabolite is generated using hepatocytes.

[0045] Some embodiments relate to a compound of Formula I

Chemical Formula

[0046] In some embodiments, each R 1is independently selected from -OH, -OSO3H, -O-glucuronide, -SH, -S(O)C1-C6 alkyl group, -S(O)2C1-C6 alkyl group, -SC1-C6 alkyl group, -SC1-C6 alkyl-NHC(O)C1-C6 alkyl group, amino acids, and peptides. In some embodiments, each hydrogen atom in the C1-C6 alkyl group is optionally replaced by OH, oxo, -CO2H, -O-glucuronide, -NH2, -NHC(O)C1-C6 alkyl group, or -N(H)C1-C6 alkyl-CO2H group.

[0047] In some embodiments, R 1 is at C-1. In some embodiments, R 1 is at C-2. In some embodiments, R 1 is at C-3. In some embodiments, R 1 is at C-4. As an example, when n is 2 or 3, R 1 can be at any combination of C-1, C-2, C-3, and C-4.

[0048] In some embodiments, R 1 is an amino acid. Exemplary amino acids include natural amino acids, non-natural amino acids, and their derivatives. In some embodiments, when R 1 is an amino acid, the amino acid is attached through the side chain of the amino acid. As an example, when the amino acid is cysteine, the amino acid can be attached to the compound of formula I through the side chain thiol of cysteine. In some embodiments, the amino acid is N-terminally acetylated.

[0049] In some embodiments, R 1 is a peptide. In some embodiments, the peptide is 2 to 6 residues in length. In some embodiments, the peptide is 2 residues in length. In some embodiments, the peptide is 3 residues in length. In some embodiments, the peptide contains cysteine. In some embodiments, R 1When it is a peptide, the peptide can be linked through the side chains of the amino acids of the peptide. As an example, when the peptide contains cysteine, the peptide can be linked to the compound of formula I through the side chain thiol of cysteine. In some embodiments, the peptide is N-terminally acetylated.

[0050] In some embodiments, R 2 is H, -OH, or -O-glucuronide. In some embodiments, R 2 is H. In some embodiments, R 2 is -OH or -O-glucuronide.

[0051] In some embodiments, R 3 is a C1-C6 alkyl group optionally substituted by -OH, oxo, -O-glucuronide, -NH2, -NHC(O)C1-C6 alkyl group, or -NHCH2COOH. In some embodiments, R 3 is a methyl, ethyl, propyl, butyl, pentyl, or hexyl group optionally substituted. In some embodiments, R 3 is methyl. In some embodiments, R 3 is substituted by -OH, oxo, -O-glucuronide, -NH2, -NHC(O)C1-C6 alkyl group, or -NHCH2COOH.

[0052] In some embodiments, R 4 is -OH or -O-glucuronide.

[0053] In some embodiments, R 4 is at C-5. In some embodiments, R 4 is at C-6. In some embodiments, R 4 is at C-7. In some embodiments, R 4 is at C-8. As an example, when m is 2 or 3, R 4 can be at any combination of C-5, C-6, C-7, and C-8.

[0054] In some embodiments, n is an integer from 0 to 4. In some embodiments, n is 1, 2, 3, or 4. In some embodiments, n is 0. In some embodiments, n is 1.

[0055] In some embodiments, m is an integer from 0 to 4. In some embodiments, m is 1, 2, 3, or 4.

[0056] In some embodiments, when n is 0, m is at least 1, or R 2 is not H, or R 3 is substituted, or a combination thereof. In some embodiments, when m is 0, n is at least 1, or R 2 is not H, or R 3 is substituted, or a combination thereof. In some embodiments, when n is 0 and m is 0, R 2 is not H, or R 3 is substituted, or a combination thereof.

[0057] Some embodiments include a compound of formula II

Chemical formula

[0058] In some embodiments, R 5 and R 6 are independently selected from C1-C6 alkyl, -NH-C1-C6 alkyl, -NH-aryl, and -NH-heteroaryl groups, and each hydrogen atom in the C1-C6 alkyl group is optionally replaced by -OH, oxo, or -CO2H.

[0059] In some embodiments, R 5 is methyl.

[0060] In some embodiments, R 6is a -NH-C1-C6 alkyl group, and each hydrogen atom in the C1-C6 alkyl group is optionally replaced by -OH, oxo or -CO2H. In some embodiments, R 6 is -NH-ethyl. In some embodiments, at least one hydrogen atom in ethyl is replaced by -OH, oxo or -CO2H. In some embodiments, R 6 is -NH(1,2,3-oxadiazole).

[0061] Some embodiments are compounds of formula III

Chemical formula

[0062] In some embodiments, R 7 is a C1-C6 alkyl group, and each hydrogen atom in the C1-C6 alkyl group is optionally replaced by -OH, oxo, -CO2H, or -NH2. In some embodiments, R 7 is ethyl, and at least one hydrogen atom in ethyl is replaced by -OH, oxo, -CO2H, or -NH2.

[0063] The compounds of the present disclosure can be administered alone or in the form of a pharmaceutical composition. In some embodiments, the compounds of the present disclosure are administered in the form of a pharmaceutical composition, i.e., admixed with at least one pharmaceutically acceptable excipient. The proportion and nature of any pharmaceutically acceptable excipient are determined by the properties of the selected compound of the present disclosure, the selected route of administration, and standard pharmaceutical practice.

[0064] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt disclosed herein; and at least one pharmaceutically acceptable excipient.

[0065] When treating patients who require such treatment, the compounds of the present disclosure or pharmaceutically acceptable salts thereof can be administered in any form and via any route that makes the compound biologically available. By way of example, the compounds and pharmaceutically acceptable salts of the present disclosure can be administered by various routes, including orally, using, for example, tablets and capsules. The compounds of the present disclosure can be administered by parenteral routes, such as by inhalation, subcutaneously, intramuscularly, intravenously, intraarterially, transdermally, intranasally, rectally, intravaginally, intravitreally, topically, sublingually, and buccally, intraperitoneally, intralipally, intrathecally, and via local delivery, for example, by catheter or stent.

[0066] One of ordinary skill in the art can readily select the appropriate form and route of administration depending on the specific characteristics of the selected compound, the disorder or condition being treated, the stage of the disorder or condition, and other relevant circumstances. The pharmaceutical compositions of the present disclosure can be administered to a patient, for example, in the form of tablets, capsules, cachets, papers, lozenges, wafers, elixirs, ointments, transdermal patches, aerosols, inhalants, suppositories, solutions, and suspensions.

[0067] The pharmaceutical compositions of the present disclosure can be prepared by methods well known in the pharmaceutical art and can contain at least one of the compounds disclosed herein as an active ingredient. The amount of the compounds of the present disclosure can vary depending on the specific form, and in some embodiments, can be between 1% and about 50% by weight of the unit dosage form.

[0068] In some embodiments, the pharmaceutical composition is formulated in unit dosage form, and each dosage contains about 0.5 mg to about 100 mg of the compound of the present disclosure or a pharmaceutically acceptable salt thereof. The term "unit dosage form" refers to physically discrete units containing a predetermined amount of the active ingredient in association with the appropriate pharmaceutical excipient, and one or more are used over the course of the dosing regimen to provide the desired therapeutic effect. To affect the treatment dosage, one or more "unit dosage forms" can typically be taken on a daily schedule.

[0069] In some embodiments, the pharmaceutical composition is a pharmaceutical composition suitable for oral administration, such as, for example, a tablet or capsule suitable for oral administration, or a liquid formulation, such as a solution or suspension. In some embodiments, the pharmaceutical composition is a liquid formulation suitable for parenteral administration.

[0070] In some embodiments, the present disclosure provides a method of treating a disease, disorder, or condition associated with GPR139, the method comprising administering to a patient in need thereof an effective amount of a compound or pharmaceutically acceptable salt disclosed herein. In some embodiments, a compound or pharmaceutically acceptable salt of the present disclosure for use as a medicament is provided. The present disclosure also provides the use of a compound or pharmaceutically acceptable salt disclosed herein, including use for manufacturing a medicament for treating a disease, disorder, or condition associated with GPR139 described herein. In some embodiments, the compounds of the present disclosure are GPR139 agonists for treating various subjects (e.g., humans, non-human mammals, and non-mammals). In some embodiments, the subject is human.

[0071] As used herein, the terms "condition," "disorder," and "disease" relate to any unhealthy or abnormal condition. The compounds of the present disclosure are metabolites of GPR139 agonists and may be useful for treating various conditions. The term "disease, disorder or condition associated with GPR139" includes conditions, disorders, and diseases for which an agonist of GPR139 may provide a therapeutic benefit, such as CNS disorders, pancreatic disorders such as pancreatitis, phenylketonuria, and pituitary disorders.

[0072] The term "disease, disorder or condition associated with GPR139" includes, but is not limited to, among others, CNS disorders such as schizophrenia, autism spectrum disorder, sleep disorders, depression, bipolar disorder, cognitive disorders including mild cognitive impairment, Alzheimer's disease, disorders affecting short-term memory, disorders affecting long-term memory, attention deficit hyperactivity disorder, post-traumatic stress disorder, substance abuse, drug addiction, eating disorders, obsessive-compulsive disorder, anxiety disorders including generalized anxiety disorder and social anxiety disorder, pain, fibromyalgia, as well as other disorders mentioned herein.

[0073] Schizophrenia is a chronic and severe disabling disorder characterized in part by negative symptoms such as affective flattening, deficits in social functioning, anhedonia, avolition and poverty of speech, as well as cognitive impairments associated with schizophrenia (CIAS) such as impairments in attention, working memory, executive function and social cognition. Autism spectrum disorder is a group of developmental disorders that can cause significant social, communication and behavioral problems (repetitive and stereotyped behaviors). Due to the prosocial effects expected from GPR139 agonists, the compound may treat schizophrenia and autism spectrum disorder.

[0074] As used herein, the term "disease, disorder or condition associated with GPR139" includes schizophrenia.

[0075] As used herein, the term "disease, disorder or condition associated with GPR139" includes autism spectrum disorder.

[0076] As used herein, the term "disease, disorder or condition associated with GPR139" includes addiction. Non-limiting examples include addiction to nicotine, alcohol, and / or cocaine.

[0077] As used herein, the term "disease, disorder or condition associated with GPR139" includes attention deficit hyperactivity disorder.

[0078] As used herein, the term "disease, disorder or condition associated with GPR139" includes bipolar disorder.

[0079] As used herein, the term "disease, disorder or condition associated with GPR139" includes depression such as major depressive disorder.

[0080] As used herein, the terms "treating", "treatment", and "treat" include the improvement of the symptoms described herein. The terms "treating", "treatment" and "treat" include all processes that result in a delay, interruption, suppression, control, or halt in the state or progression of the symptoms described herein, but do not necessarily indicate the complete elimination of all symptoms or the cure of the symptoms. The terms "treating", "treatment" and "treat" are intended to include the therapeutic treatment of such disorders. The terms "treating", "treatment" and "treat" are intended to include the prophylactic treatment of such disorders.

[0081] As used herein, the terms "patient" and "subject" include humans and non-human animals, such as mammals, for example, mice, rats, guinea pigs, dogs, cats, rabbits, cows, horses, sheep, goats, and pigs. The term also includes birds, fish, reptiles, amphibians, etc. In some embodiments, the patient is human. In some embodiments, the patient is a non-human mammal, such as a mouse, rat, or dog.

[0082] As used herein, the term "effective amount" refers to the amount of a compound of the present disclosure that, when administered once or multiple times, treats a patient suffering from the above symptoms. The effective amount can be readily determined by an attending diagnostician, as a person skilled in the art, by using known techniques and by observing the results obtained in similar situations. When determining the effective amount and dosage, several factors are considered by the attending diagnostician, including but not limited to the species of the patient; its size, age, and general health; the specific symptoms, disorders, or diseases involved; the degree or involvement or severity of the symptoms, disorders, or diseases; the response of the individual patient; the specific compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dosing regimen selected; the use of concomitant medications; and other related circumstances. In some embodiments, the effective amount, treatment dosage of the present disclosure is in the range of 1 mg to 100 mg. The specific amount can be determined by a person skilled in the art. These dosages are based on an average human subject having a mass of about 60 kg to about 70 kg, but a physician would be able to determine an appropriate dosage for a patient having a mass outside this weight range.

[0083] The compounds of the present disclosure may be combined with one or more other pharmacologically active compounds or treatments for treating one or more disorders, diseases, or symptoms to which GPR139 applies, and may be administered simultaneously, sequentially, or separately in combination with one or more compounds or treatments for treating specific diseases, disorders, or symptoms related to GPR139.

[0084] In some embodiments, in the treatment of schizophrenia, the compounds or pharmaceutically acceptable salts of the present disclosure are combined with one or more sedatives, hypnotics, anxiolytics, antipsychotics, anxiolytics, cyclopyrrolones, imidazopyridines, pyrazolopyrimidines, minor tranquillizers, melatonin agonists and antagonists, melatonin agonists, benzodiazepines, barbiturates, mGlu2 / 3 agonists, 5HT-2 antagonists, PDE10 antagonists, GlyT1 inhibitors, etc., for example: azinazolam, allobarbital, allonimid, alprazolam, amisulpride, amitriptyline, amobarbital, amoxapine, aripiprazole, bentazepam, benzocutamide, brotizolam, bupropion, busprione, butabarbital, butalbital, caprid, carbocloral, chloral betaine, chloral hydrate, clomipramine, clonazepam, cloperidone, clorazepate, chlordiazepoxide, chloretate, chlorpromazine, clozapine, cyprazepam, desipramine, dexcramol, diazepam, dichloralphenazone, divalproex, diphenhydramine, doxepin, estazolam, ethchlorvynol, etomidate, phenobarb, flunitrazepam, flupentixol, fluphenazine, flurazepam, fluvoxamine, fluoxetine, flurazepam, fosazepam, glutethimide, halazepam, haloperidol, hydroxyzine, imipramine, lithium, lorazepam, lormetazepam, maprotiline, meclocarone, melatonin, mephobarbital, meprobamate, metacalone, midafuril, midazolam, nefazodone, nisobamate, nitrazepam, nortriptyline, olanzapine, oxazepam, paraldehyde, paroxetine, pentobarbital, perlapine, perphenazine, phenelzine, phenobarbital, prazepam, promethazine, propofol, protriptyline, quazepam, quetiapine, reclazepam, risperidone, loreclezole, secobarbital, sertraline, sproclon, temazepam, thioridazine, thiothixene, tracazolate, kanylcypromaine, trazodone, triazolam, trepipam, tricetamide, trichloroethane, trifluoperazine,It can be administered in combination with trimethadione, trimipramine, urazepam, venlafaxine, zaleplon, diprasidone, zolazepam, zolpidem, etc.

[0085] In some embodiments, in the treatment of depression, the compounds or pharmaceutically acceptable salts of the present disclosure are norepinephrine reuptake inhibitors (including tertiary amine tricyclics and secondary amine tricyclics), selective serotonin reuptake inhibitors (SSRI), monoamine oxidase inhibitors (MAOI), reversible inhibitors of monoamine oxidase (RIMA), serotonin and norepinephrine reuptake inhibitors (SNRI), corticotropin-releasing factor (CRF) antagonists, adrenergic receptor antagonists, neurokinin-1 receptor antagonists, antidepressants for atypical depression, benzodiazepines, 5-HTA agonists or antagonists, particularly 5-HTA partial agonists, and corticotropin-releasing factor (CRF) antagonists. It can be administered in combination with antidepressants or anxiolytics. Specific drugs include amitriptyline, clomipramine, doxepin, imipramine and trimipramine; amoxapine, desipramine, maprotiline, nortriptyline and protriptyline; fluoxetine, fluvoxamine, paroxetine and sertraline; isocarboxazid, phenelzine, tranylcypromine and selegiline; moclobemide, venlafaxine; duloxetine; aprepitant; bupropion, lithium, nefazodone, trazodone and viloxazine; alprazolam, chlordiazepoxide, clonazepam, chlazepate, diazepam, halazepam, lorazepam, oxazepam and prazepam; buspirone, flesinoxan, gepirone and ipsapirone, etc.

[0086] In some embodiments, in the treatment of Alzheimer's disease or mild cognitive impairment, the compounds or pharmaceutically acceptable salts of the present disclosure may be administered in combination with one or more anti-Alzheimer's agents, β-secretase inhibitors, γ-secretase inhibitors, HMG-CoA reductase inhibitors, NSAIDs including ibuprofen, vitamin E, anti-amyloid antibodies, sedatives, hypnotics, anxiolytics, antipsychotics, anti-anxiety agents, and tranquilizers, and other pharmaceuticals as used in the treatment of Alzheimer's disease or mild cognitive impairment.

[0087] The activity of the compounds as metabolites of GPR139 agonists and GPR103 agonists can be determined by various methods including in vitro and in vivo assays.

[0088] One of ordinary skill in the art will recognize that the species listed or exemplified herein are not exhaustive and that additional species within the scope of these defined terms may be selected.

Examples

[0089] Examples The following examples are intended to be illustrative and non-limiting and represent specific embodiments of the present disclosure.

[0090] Exemplary chemical entities useful in the methods herein are described herein by reference to the exemplary synthetic schemes for the following general preparations and the following specific examples. One of ordinary skill in the art will recognize that starting materials can be appropriately selected such that the ultimately desired substituents are carried through the reaction scheme, protected or unprotected as necessary to obtain the desired product. Alternatively, it may be necessary or desirable to use appropriate groups that are carried through the reaction scheme and replaced with the ultimately desired substituents as necessary. Further, one of ordinary skill in the art will recognize that the transformations shown in the following schemes can be carried out in any order compatible with the functionality of the particular pendant groups.

[0091] Proton nuclear magnetic resonance (NMR) spectra were obtained for many of the compounds of the following examples. Characteristic chemical shifts (δ) are given in parts per million on the low magnetic field side from tetramethylsilane using conventional abbreviations to designate major peaks, including s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad). The following abbreviations are used for common solvents: CDCl3 (deuterochloroform), DMSO-d6 (deuterodimethyl sulfoxide), and CD3OD (deuteromethanol or methanol-d4). Mass spectra were recorded using either electrospray ionization (ESI) or atmospheric pressure chemical ionization.

[0092] As used herein, unless otherwise indicated, terms are using conventional abbreviations: 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'-ethylcarbodiimide (EDC), and the like. Example 1 Extraction of Metabolites from Rat Plasma

[0093] Rat plasma samples were obtained from a study in which male and female Sprague Dawley rats were repeatedly dosed orally with 3 mg / kg of Compound A QD for 14 days. Whole blood was obtained from the rats at selected time points on Day 14 (before dosing and at 0.5, 1, 2, 4, 8, and 24 hours after dosing) and processed to plasma for analysis. Plasma from 3 animals / sex / time point was pooled (30 μL each), combined with 360 μL of ice-cold ACN, and mixed to precipitate proteins. The samples were centrifuged, the supernatant was removed, and dried under a nitrogen stream. Each sample was reconstituted in 100 μL of 5% ACN, and 10 μL was analyzed by LC / MS / MS for profiling and metabolite identification. Example 2 Extraction of Metabolites from Canine Plasma

[0094] Canine plasma samples were obtained from a study in which male and female beagle dogs were repeatedly dosed orally with 30 mg / kg of Compound A QD for 14 days. Whole blood was obtained from the dogs at selected time points on Day 14 (before dosing and at 0.5, 1, 2, 4, 8, and 24 hours after dosing) and processed to plasma for analysis. Plasma from 2 animals / sex / time point was pooled (50 μL each), combined with 400 μL of ice-cold ACN, and mixed to precipitate proteins. The samples were centrifuged, the supernatant was removed, and dried under a nitrogen stream. Each sample was reconstituted in 100 μL of 5% ACN, and 10 μL was analyzed by LC / MS / MS for profiling and metabolite identification. Example 3 Extraction of Metabolites from Canine Urine, Bile, Liver Tissue, and Kidney Tissue

[0095] Canine urine, bile, and liver and kidney tissue samples were obtained from a study in which male and female beagle dogs were repeatedly dosed with 60 mg / kg oral doses of Compound A QD for 13 weeks. Urine and bile were centrifuged, and the supernatants were analyzed by LC / MS / MS without further treatment or dilution. Kidney and liver tissue samples from the centrilobular, central, and periportal regions were diluted 3-fold with saline and homogenized. The homogenized samples were disrupted with 4 volumes of ACN, vortexed, and centrifuged at 15,000×g for 10 minutes. The resulting supernatants were reconstituted with 100 μL of 5% ACN, and 10 μL was analyzed by LC / MS / MS for profiling and metabolite identification. Example 4 Extraction of Metabolites from Monkey Plasma

[0096] Cynomolgus monkey plasma and urine samples were obtained from a study in which animals were dosed with a single oral dose of 200 mg / kg of Compound A. Plasma samples from each monkey were pooled using the Hamilton method using the area under the plasma concentration-time curve from time 0 to 24 hours. Protein was precipitated with 3 volumes of ACN and mixed thoroughly. The samples were vortexed and centrifuged. The supernatant was removed and dried under a nitrogen stream and reconstituted with 80 / 20 water / ACN (v / v) for analysis by LC / MS / MS for profiling and metabolite identification.

[0097] Urine collected at 0 - 6, 6 - 12, 12 - 24, 24 - 48, and 48 - 72 hours post-dose was pooled across subjects (1000 μL each, 6 subjects) and centrifuged at 4000×g for 10 minutes to remove the pellet. Pre-dose and placebo urine samples were also pooled by combining equal volumes (1000 μL) and treated in the same manner. Samples were then analyzed by LC / MS / MS for profiling and metabolite identification. Example 5 Extraction of Metabolites from Human Plasma and Urine

[0098] Human plasma and urine samples were obtained from a study in which healthy volunteers were dosed with either a single oral dose of 40 mg of Compound A or placebo as part of a single ascending dose (SRD) study.

[0099] Plasma samples from six subjects were pooled using the Hamilton method using the area under the plasma concentration-time curve from time 0 to 48 hours for each subject, and further pooled across subjects (250 μL each) to generate a single plasma pool. As a control, an equal volume (250 μL) of pre-dose plasma was pooled, and at the same time, an equal volume (625 μL) of plasma from two subjects administered placebo was pooled.

[0100] The protein was precipitated with three volumes of ACN and mixed thoroughly. The sample was centrifuged and the supernatant removed. The pellet was resuspended in 2 mL of 80 / 20 ACN / water (v / v), mixed, and centrifuged. The supernatants were combined and dried under a nitrogen stream and reconstituted in 80 / 20 water / ACN (v / v) containing 0.1% formic acid (FA) for analysis by LC / MS / MS for profiling and metabolite identification.

[0101] Urine at 0 - 6, 6 - 12, 12 - 24, 24 - 48, and 48 - 72 hours post-dose was pooled across subjects (1000 μL each, six subjects) and centrifuged at 4000 × g for 10 minutes to remove the pellet. Pre-dose and placebo urine samples were also pooled by combining equal volumes (1000 μL) and processed in the same manner. The samples were then analyzed by LC / MS / MS for profiling and metabolite identification. Example 6 Qualitative evaluation of metabolites of Compound A in rat, dog, monkey, and human hepatocytes

[0102] An aliquot of 2 μL of a 10 mM stock solution of 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 hepatocyte incubation. Hepatocyte incubation was carried out with (final concentration): Compound A at 10 μM in a final volume of 100 μL, 1 × 10 6Consists of hepatocytes (Life Technologies, Grand Island, NY, USA; Sprague Dawley rats, lot RS688, 3 male donors; beagle dogs, lot DB295, 1 male donor; cynomolgus monkeys, lot CY359, 1 male donor; and humans, lot HUE115, 5 male and 5 female donors) at cells / mL, 0.1% DMSO, and KHB at pH 7.4. The hepatocytes were thawed, processed, and prepared according to the protocol recommended by the vendor. A 96-well plate was used for this test. The reaction was initiated by adding Compound A to the hepatocytes in the plate. The plate was incubated at 37 °C for 0 and 120 minutes. The reaction was terminated by adding an equal volume of ice-cold ACN. The precipitated protein was removed by centrifugation (5000 rpm for 10 minutes at room temperature), and the supernatant was analyzed by LC / MS / MS. Example 7 Qualitative evaluation of the metabolite profile of Compound A in HepatoPac incubation

[0103] HepatoPac application medium and maintenance medium were prepared according to the vendor's instructions. HepatoPac cells seeded in a 24-well plate in advance were received from the vendor, and a complete medium exchange was performed with the species-specific maintenance medium. The rat well and the associated stromal well and blank well were filled with 300 μL of maintenance medium, and the multi-species well and the associated stromal well and blank well were filled with 400 μL of maintenance medium. The plate was incubated at 37 °C for 48 hours in a 10% CO2 atmosphere with humidity above 95%.

[0104] 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 each of 40 mL of pre-warmed rat and multi-species application media in 50 mL conical tubes to generate a 20 μM dosing solution. The fortified medium was capped and stored in a water bath at 37 °C until use.

[0105] Removed from all plates where the maintenance medium was replaced with the species-specific application medium. The application medium was removed again and replaced with the species-specific administration medium (300 μL for rat HepatoPac, stroma, and blank well; 400 μL for multiple species HepatoPac, stroma, and blank well). The cells were stored in the incubator until administration. When ready for administration, the medium in all wells was removed and replaced with fresh application medium at half of the final administration volume (150 μL for rat, stroma, and blank well; 200 μL for multiple species, stroma, and blank well). An equal volume of the species-specific 20 μM administration solution was applied to each well, and gentle swirling was performed to initiate incubation.

[0106] The plates were incubated and samples were collected at 0, 2, 4, 24, 48, 168, and 336 hours post - administration from multi - well as well as related stromal wells and blank wells; a single well was used for each time point. For samples at the 0 - hour, 2 - hour, and 4 - hour time points, the blank application medium was already pre - heated; for samples at the 24 - hour, 48 - hour, 168 - hour, and 336 - hour time points, the blank application medium was pre - heated at 37 °C for 30 minutes before sampling. At each time point, 250 μL (rat well) and 350 μL (multi - well) of the medium were removed from the HepatoPac, stromal, and blank wells and quenched with 500 μL (rat well) or 700 μL (multi - well) of ice - cold ACN in polypropylene tubes; these were called primary samples. An aliquot of 250 μL (rat well) or 350 μL (multi - well) of the blank application medium was returned to each well, all the contents were removed, and quenched with 600 μL (rat well) or 800 μL (multi - well) of ice - cold ACN in polypropylene tubes; these were called wash samples. The ice - cold ACN (600 μL for rat wells and 800 μL for multi - wells) was returned to the wells. Using the end of a 1000 μL pipette tip, starting from the top and moving down, the wells were thoroughly scraped left and right. After thorough scraping, all the ACN was removed and transferred to polypropylene tubes; these were called cell lysate samples. After all samples were collected, an aliquot of 300 μL (rat well) or 400 μL (multi - well) of the application medium was returned to the wells to maintain local humidity for the remaining samples.

[0107] The cell lysate samples were stored frozen at -80 °C for future analysis. The primary and wash samples were gently rotated manually several times to ensure complete mixing of the sample and the quench solution, and then centrifuged at 3000 rpm for 30 minutes in an Allegra® X-14R centrifuge (Beckman Coulter, Brea, California, USA). The supernatant (500 μL for rat samples and 800 μL for multi-species samples) was collected, transferred to a new polypropylene tube, and stored frozen at -80 °C until analysis. The tubes containing the residual quench solution and protein pellet were stored frozen at -80 °C for possible future analysis. Immediately prior to liquid chromatography / mass spectrometry (LC / MS) analysis combined with mass spectrometry, the supernatants from the primary and wash samples were combined and dried under a nitrogen stream. The samples were reconstituted in water containing 5% ACN and analyzed by LC / MS. Example 8 LC-MS / MS Analysis Conditions for Metabolite Identification

[0108] HPLC analysis was performed using an Agilent 1290 binary pump (Agilent Technologies, Inc., Santa Clara, California, USA) equipped with a PAL autosampler (Leap Technologies). Separation was achieved on a Kinetex 5 μm C18 column (2.1 × 150 mm; Phenomenex, Inc., Torrance, California, USA) under ambient conditions. The HPLC eluent was introduced directly into an MDS SCIEX TripleTOF 5600 mass spectrometer via electrospray positive ionization with a source temperature of 500 °C, an IonSpray voltage floating set at 5,000, a declustering potential of 80, and an MS 2 collision energy of 20. Samples were analyzed in full scan mode, enabling product ion scanning and mass defect filtering by independent data acquisition.

[0109] Using the above method, the ESI-MS m / z [M+H] for each of the compounds +The following compounds were identified that matched the calculated value for the exact mass of the neutral species to approximately two decimal places.

[0110] (S)-2-(4-Oxobenzotriazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chemical formula

[0111] (S)-2-(6-Hydroxy-4-oxobenzotriazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chemical formula

[0112] (S)-2-(5-Hydroxy-4-oxobenzotriazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chemical formula

[0113] (S)-2-(7-Hydroxy-4-oxobenzotriazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chemical formula

[0114] (S)-2-(8-Hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chem.

[0115] (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

Chem.

[0116] (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

Chem.

[0117] (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

Chem.

[0118] (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

Chem.

[0119] (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 sulfide salt

Chem.

[0120] (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 sulfide salt [Chemistry] C 18 H 15 The exact mass calculated for F3N4O7S is 488.06.

[0121] (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 sulfide [Chemistry] C 18 H 15 The exact mass calculated for F3N4O7S is 488.06.

[0122] (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 sulfide [Chemistry] C 18 H 15 The exact mass calculated for F3N4O7S1 is 488.06.

[0123] N5-(1-((Carboxymethyl)amino)-1-oxo-3-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydroxybenzo[d][1,2,3]triazin-6-yl)thio)propan-2-yl)glutamine [Chemistry] C 28 H 30 The exact mass calculated for F3N7O9S is 697.18.

[0124] N5-(1-((Carboxymethyl)amino)-1-oxo-3-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydroxybenzo[d][1,2,3]triazin-5-yl)thio)propan-2-yl)glutamine

Chem.

[0125] N5-(1-((Carboxymethyl)amino)-1-oxo-3-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydroxybenzo[d][1,2,3]triazin-7-yl)thio)propan-2-yl)glutamine

Chem.

[0126] N5-(1-((Carboxymethyl)amino)-1-oxo-3-((4-oxo-3-(2-oxo-2-(((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydroxybenzo[d][1,2,3]triazin-8-yl)thio)propan-2-yl)glutamine

Chem.

[0127] 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

Chem.

[0128] 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

Chem.

[0129] 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

Chem.

[0130] 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

Chem.

[0131] 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

Chem.

[0132] 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

Chem.

[0133] 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

Chem.

[0134] 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

Chem.

[0135] 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

Chem.

[0136] 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

Chem.

[0137] 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

Chem.

[0138] 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 [Chemistry] C 23 H 22 The exact mass calculated for F3N5O6S is 553.12.

[0139] (S)-2-(6-Mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide [Chemistry] C 18 H 15 The exact mass calculated for F3N4O3S is 424.08.

[0140] (S)-2-(5-Mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide [Chemistry] C 18 H 15 The exact mass calculated for F3N4O3S is 424.08.

[0141] (S)-2-(7-Mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide [Chemistry] C 18 H 15 The exact mass calculated for F3N4O3S is 424.08.

[0142] (S)-2-(8-Mercapto-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide [Chemistry] C 18 H 15 The exact mass calculated for F3N4O3S, 424.08.

[0143] (S)-2-(6-(Methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chem.

[0144] (S)-2-(5-(Methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chem.

[0145] (S)-2-(7-(Methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chem.

[0146] (S)-2-(8-(Methylthio)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chem.

[0147] 2-(6-(Methylsulfinyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chem.

[0148] 2-(5-(Methylsulfinyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chem.

[0149] 2-(7-(Methylsulfinyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chem.

[0150] 2-(8-(Methylsulfinyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-((S)-1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chem.

[0151] (S)-2-(6-(Methylsulfonyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chem.

[0152] (S)-2-(5-(Methylsulfonyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chem.

[0153] (S)-2-(7-(Methylsulfonyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chem.

[0154] (S)-2-(8-(Methylsulfonyl)-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide

Chem.

[0155] (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

Chem.

[0156] (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

Chem.

[0157] (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

Chem.

[0158] (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

Chemical formula

[0159] (S)-N-(1-(2-Hydroxy-4-(trifluoromethoxy)phenyl)ethyl)-2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide

Chemical formula

[0160] (S)-N-(1-(3-Hydroxy-4-(trifluoromethoxy)phenyl)ethyl)-2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide

Chemical formula

[0161] (R)-N-(2-Hydroxy-1-(4-(trifluoromethoxy)phenyl)ethyl)-2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide

Chemical formula

[0162] N-(1-Hydroxy-1-(4-(trifluoromethoxy)phenyl)ethyl)-2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide

Chemical formula

[0163] (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

Chemical formula

[0164] (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

Chemical formula

[0165] (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; [Chemistry] corresponding to C 24 H 23 F3N4O 10 The exact mass calculated for 584.137 C 24 H 23 F3N4O 10 The exact mass calculated for, 584.14.

[0166] (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 [Chemistry] C 24 H 23 F3N4O 10 The exact mass calculated for, 584.14.

[0167] 2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetamide [Chemistry] The exact mass calculated for C9H8N4O2, 204.06.

[0168] 2-(4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetic acid [Chemistry] The exact mass calculated for C9H7N3O3, 205.05.

[0169] (S)-2-hydroxy-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide [Chemistry] C 11 H 12 The exact mass calculated for F3NO3, 263.08.

[0170] (S)-2-Oxo-2-((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)acetic acid

Chem.

[0171] (S)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)-1,2,3-oxadiazol-5-amine

Chem.

[0172] The following preparations were carried out in a suitable container and typically stirred. Where indicated, the products of certain preparations and examples were purified by mass-triggered HPLC. Where indicated, the 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 a gradient of ACN (containing 0.035% TFA) and water (containing 0.005% TFA). A 10% - 100% ACN gradient was used unless otherwise indicated. SFC purification: Multigram II Berger SFC; A ChiralPak AD-H (5 μm, 21×150 mm) column was used and eluted with a gradient of liquid CO2 and isopropanol. After isolation by chromatography, the solvent was removed and the product was obtained by evaporation of the product-containing fractions (e.g., GeneVac®), rotary evaporator, vacuum flask, freeze-drying. Synthesis 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

Chemical Structure

[0173] 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 for 5 minutes at RT, (S)-1-(4-(trifluoromethoxy)phenyl)ethan-1-amine, HCl (536 mg, 2.218 mmol) and iPr2EtN (1290 μL, 7.39 mmol) were added. The reaction mixture was stirred for 1 hour at RT, then water was added and the white solid was filtered. The obtained crude material was dissolved in CH2Cl2 and purified via an ISCO automated purification system eluting with a gradient of 0 - 20% MeOH in DCM. The collected fractions were combined and the solvent was removed via rotary evaporation at 35 °C. Drying the resulting mixture in vacuo gave 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

[0174] To a vial, (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) were added, and then ethan-1,2-dithiol (71 μL, 0.849 mmol) was added. The vial was heated at 90 °C for 1 hour. Since the thiol formation was completed by UPLC, a solution of iodomethane (80 μl, 1.273 mmol) in 1 mL of DMF was added. The solution was stirred at RT for 16 hours. The resulting crude material was diluted with 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 the solvent was removed via rotary evaporation at 45 °C. Drying the resulting mixture in vacuo gave the title compound as a white solid (100 mg, 53%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.41 (d, J=7.0 Hz, 3 H), 2.66 (s, 3 H), 4.99 (t, J=7.3 Hz, 1 H), 5.07 (s, 2 H), 7.31 - 7.36 (m, 2 H), 7.45 - 7.50 (m, 2 H), 7.89 - 7.99 (m, 2 H), 8.12 (d, J=8.5 Hz, 1 H), 8.81 (d, J=8.0 Hz, 1 H); ESI-MS m / z [M+H] + 439.1. Synthesis 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

Chemical Structure

[0175] (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 CH2Cl2 (170 μL) at RT was added mCPBA (5.9 mg, 0.034 mmol). The reaction mixture was stirred at RT for 30 minutes. The resulting crude material was diluted with 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 the solvent was removed via rotary evaporation at 45 °C. Drying the resulting mixture in vacuo gave the title compound as a white solid (6.1 mg, 39%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.42 (d, J = 7.0 Hz, 3 H), 2.89 (s, 3 H), 4.99 (t, J = 7.2 Hz, 1 H), 5.13 (s, 2 H), 7.34 (d, J = 7.8 Hz, 2 H), 7.45 - 7.50 (m, 2 H), 8.35 - 8.39 (m, 1 H), 8.40 - 8.45 (m, 1 H), 8.54 (dd, J = 1.9, 1.1 Hz, 1 H), 8.84 (d, J = 8.0 Hz, 1 H); ESI-MS m / z [M+H] + 455.1. Synthesis 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

Chemical Structure

[0176] (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 CH2Cl2 (170 μL) was added to a solution at RT, and mCPBA (12 mg, 0.068 mmol) was added. The reaction mixture was stirred at RT for 1 hour. The resulting crude material was diluted with 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 the solvent was removed via rotary evaporation at 45 °C. Drying the resulting mixture in vacuo gave the title compound as a white solid (9.9 mg, 62%). 1 HNMR (400 MHz, DMSO-d6) δ ppm 1.42 (d, J=7.0 Hz, 3 H), 3.41 (s, 3 H), 4.95 - 5.05 (m, 1 H), 5.15 (s, 2 H), 7.31 - 7.37 (m, 2 H), 7.45 - 7.50 (m, 2 H), 8.47 - 8.52 (m, 1 H), 8.55 - 8.60 (m, 1 H), 8.70 (d, J=1.5 Hz, 1 H), 8.85 (d, J=7.3 Hz, 1 H); ESI-MS m / z [M+H] + 471.1. Synthesis 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

Chemical Structure

[0177] To a vial, (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) were added, and then ethan-1,2-dithiol (36 μl, 0.424 mmol) was added. 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 MgSO4, filtered and concentrated to give 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

[0178] 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-aminoacryloyl acid (6 mg, 0.043 mmol) in dioxane (36 μL) were added 2 drops of piperidine. The reaction mixture was heated at reflux for 2 hours. The crude obtained was diluted with 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 the solvent was removed via rotary evaporation at 45 °C. The resulting mixture was dried in vacuo to give the title compound as a white solid (8.5 mg, 36%). 11H NMR (400 MHz, DMSO-d6) δ ppm 1.41 (d, J=7.2 Hz, 3 H), 1.82 (d, J=1.7 Hz, 3 H), 3.34 - 3.43 (m, 2 H), 3.59 - 3.69 (m, 1 H), 4.99 (t, J=7.4 Hz, 1 H), 5.07 (s, 2 H), 7.34 (d, J=8.4 Hz, 2 H), 7.47 (d, J=8.8 Hz, 2 H), 7.96 - 8.04 (m, 2 H), 8.08 - 8.15 (m, 1 H), 8.82 (d, J=7.9 Hz, 1 H); ESI-MS m / z [M+H] + 554.1. Synthesis Example 5 (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) Preparation [Chemical formula]

[0179] 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 treated with 1.0 M lithium hydroxide solution (1.070 mL, 1.070 mmol). The solution was stirred at 20 °C for 1.5 h, 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 obtain a solution of pH 6, which was then filtered through 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%). 1 H NMR (400 MHz, 1:1 deuterium oxide / CD3CN) δ ppm 1.94 (d, J=7.06 Hz, 3 H) 3.99 - 4.16 (m, 3 H) 4.37 (d, J=9.35 Hz, 1 H) 5.46 - 5.52 (m, 1 H) 5.52 - 5.59 (m, 1 H) 5.61 - 5.68 (m, 1 H) 5.73 (d, J=7.24 Hz, 1 H) 7.77 (d, J=8.25 Hz, 2 H) 7.93 (d, J=8.62 Hz, 2 H) 8.24 - 8.33 (m, 2 H) 8.67 (d, J=8.89 Hz, 1 H); ESI-MS m / z [M+H] + 585. Synthesis Example 6 (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-triyltriacetate Preparation

Chem.

[0180] 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-triyltriacetate (63.0 mg, 0.159 mmol) and silver(I) oxide (83 mg, 0.357 mmol) in anhydrous ACN (1 mL) was stirred at 20 °C for 5 h in the dark. LCMS showed about 50% conversion. The mixture was filtered through a large syringe filter, washed with acetonitrile and methanol, and purified by preparative HPLC (10 - 100% acetonitrile / water in basic mode) to give the title compound as a white solid (35 mg, 37%). 11H NMR (400 MHz, DMSO-d6) δ ppm 1.40 (d, J=6.97 Hz, 3 H) 1.98 - 2.08 (m, 9 H) 3.62 (s, 3 H) 4.83 (d, J=9.81 Hz, 1 H) 4.98 (quin, J=7.13 Hz, 1 H) 5.06 (s, 2 H) 5.12 (t, J=9.67 Hz, 1 H) 5.19 (dd, J=9.63, 7.70 Hz, 1 H) 5.45 - 5.55 (m, 1 H) 6.04 (d, J=7.70 Hz, 1 H) 7.33 (d, J=8.34 Hz, 2 H) 7.46 (d, J=8.71 Hz, 2 H) 7.70 (dd, J=8.90, 2.75 Hz, 1 H) 7.74 (d, J=2.75 Hz, 1 H) 8.24 (d, J=8.89 Hz, 1 H) 8.81 (d, J=7.70 Hz, 1 H); ESI-MS m / z [M+H] + 725. Synthesis Example 7 (S)-2-(6-Hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)-N-(1-(4-(trifluoromethoxy)phenyl)ethyl)acetamide Preparation

Chemical Structure

[0181] 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)ethane-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 h, then diluted with isopropyl acetate (400 mL) and washed with saturated ammonium chloride (400 mL), water (400 mL), and brine (400 mL). The precipitate was recovered via filtration before separating the final layer. Drying the solid phase under vacuum for 1 h gave the first batch of the title compound as an off-white solid (2.768 g).

[0182] The filtrate was separated, the organic layer was dried over magnesium sulfate, concentrated over Celite®, and purified on a 120 g silica gel column eluting with a gradient of 0 - 100% ethyl acetate in heptane. The solid was transferred from the flask by slurrying in heptane and diethyl ether and then recovered by vacuum filtration and dried under vacuum to give the second batch of the title compound (3.45 g) as a white solid (total yield 6.22 g, 84%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.40 (d, J=6.97 Hz, 3 H) 4.97 (t, J=7.24 Hz, 1 H) 5.01 (s, 2 H) 7.33 (d, J=8.34 Hz, 2 H) 7.41 - 7.54 (m, 4 H) 8.08 (d, J=8.71 Hz, 1 H) 8.79 (d, J=7.70 Hz, 1 H) 11.10 (br s, 1 H); ESI-MS m / z [M+H] + 409. Synthesis Example 8 Preparation of 2-(6-hydroxy-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)acetic acid

Chemical Structure

[0183] 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 h. The slurry was cooled to 20 °C. Concentrated HCl (aqueous solution) (25.1 mL, 301 mmol) was added dropwise (vigorous foaming occurred with the first drop), sodium nitrite (4.33 g, 62.8 mmol) was added portionwise (foaming occurred upon addition), and the mixture was stirred at 20 °C for 3 days. The solid was collected by vacuum filtration, washed with water, and dried under high vacuum to give the title compound as a yellowish brown solid (9.512 g, 86%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 5.07 (s, 2 H) 7.43 - 7.55 (m, 2 H) 8.11 (d, J=8.71 Hz, 1 H) 11.17 (s, 1 H) 12.90 - 13.62 (m, 1 H); ESI-MS m / z [M+H] + 222. Synthesis Example 9 Preparation of 6-hydroxy-2H-benzo[d][1,3]oxazine-2,4(1H)-dione

Chemical formula

[0184] 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 h and then cooled to 20 °C. The solid phase was collected by vacuum filtration and washed with heptane to give the title compound as a gray solid (9.072 g, 82%). 11H NMR (400 MHz, DMSO-d6) δ ppm 7.03 (d, J=8.25 Hz, 1 H) 7.15 - 7.27 (m, 2 H) 9.83 (s, 1 H) 11.47 (s, 1 H); ESI-MS m / z [M+H] + 180. Biological Example 1 (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) inhibition of bile salt export pump (BSEP) and multidrug resistance-associated protein (MRP)

[0185] The inhibition of BSEP (dog and human) and MRP2 (dog and human) by Compound A and Compound B was tested in membrane vesicles. Compound B inhibited dog MRP2 at an IC 50 value of 1.80 μM. Human MRP2 was also inhibited (IC 50 = 83.0 μM). BSEP inhibition was not identified in this assay. [Table 1] In one embodiment, for example, the following items are provided. (Item 1) A compound of formula I [Chemical Structure] (wherein each R 1 is independently selected from -OH, -OSO 3 H, -O-glucuronide, -SH, -S(O)C 1 ~C 6 alkyl group, -S(O) 2 C 1 ~C 6 alkyl group, -SC 1 ~C 6 alkyl group, -SC 1 ~C 6 alkyl-NHC(O)C 1 ~C6 alkyl group, amino acid, and peptide, and each hydrogen atom in the C 1 ~C 6 alkyl group is optionally replaced by OH, oxo, -CO 2 H, -O-glucuronide, -NH 2 , -NHC(O)C 1 ~C 6 alkyl group, or -N(H)C 1 ~C 6 alkyl-CO 2 H group; R 2 is H, -OH, or -O-glucuronide; R 3 is -OH, oxo, -O-glucuronide, -NH 2 , -NHC(O)C 1 ~C 6 alkyl group, or a C 2 alkyl group optionally substituted with -NHCH 1 ~C 6 COOH; each R 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 2 is not H and / or (d) R 3 is substituted) or a pharmaceutically acceptable salt thereof. (Item 2) R 2 The compound according to Item 1 or a pharmaceutically acceptable salt thereof, wherein is H. R 3 (Item 3) The compound according to Item 2 or a pharmaceutically acceptable salt thereof, wherein is methyl. (Item 4) The compound according to Item 3 or a pharmaceutically acceptable salt thereof, wherein m is 0. R 1 (Item 5) The compound according to Item 4 or a pharmaceutically acceptable salt thereof, wherein R 1 n is 1. (Item 6) R 1 The compound according to Item 5 or a pharmaceutically acceptable salt thereof, wherein is at C-1. R 1 (Item 7) The compound according to Item 5 or a pharmaceutically acceptable salt thereof, wherein is at C-2. (Item 8) R 1 The compound according to Item 5 or a pharmaceutically acceptable salt thereof, wherein is at C-3. (Item 9) The compound according to Item 5 or a pharmaceutically acceptable salt thereof, wherein is at C-4. (Item 10) The compound according to Item 3 or a pharmaceutically acceptable salt thereof, wherein n is 2. (Item 11) The compound according to Item 4 or a pharmaceutically acceptable salt thereof, wherein is an amino acid. (Item 12) The compound according to Item 11 or a pharmaceutically acceptable salt thereof, wherein the amino acid is cysteine. (Item 13) The compound according to Item 12 or a pharmaceutically acceptable salt thereof, wherein the cysteine is bonded through a side chain thiol. (Item 14) The compound according to item 13, or a pharmaceutically acceptable salt thereof, wherein the amino acid is N-terminally acetylated. (Item 15) R 1 The compound according to item 4, or a pharmaceutically acceptable salt thereof, which is a peptide. (Item 16) The compound according to item 15, or a pharmaceutically acceptable salt thereof, wherein the peptide contains cysteine. (Item 17) The compound according to item 15, or a pharmaceutically acceptable salt thereof, wherein the peptide is linked through the side-chain thiol of cysteine. (Item 18) The compound according to item 16, or a pharmaceutically acceptable salt thereof, wherein the peptide is 2 to 6 residues in length. (Item 19) The compound according to item 18, or a pharmaceutically acceptable salt thereof, wherein the peptide is N-terminally acetylated. (Item 20) The compound according to item 1, or a pharmaceutically acceptable salt thereof, wherein n is 0. (Item 21) The compound according to item 20, or a pharmaceutically acceptable salt thereof, wherein m is an integer from 1 to 4. (Item 22) R 2 The compound according to item 21, or a pharmaceutically acceptable salt thereof, wherein is H. (Item 23) R 3 The compound according to item 22, or a pharmaceutically acceptable salt thereof, wherein is methyl. (Item 24) The compound according to item 23, or a pharmaceutically acceptable salt thereof, wherein m is 1. (Item 25) R 4 The compound according to item 24, or a pharmaceutically acceptable salt thereof, wherein is located at C-5. (Item 26) R 4 The compound according to item 24, or a pharmaceutically acceptable salt thereof, wherein is located at C-6. (Item 27) R 4 The compound according to item 24, or a pharmaceutically acceptable salt thereof, wherein is located at C-7. (Item 28) R 4 The compound according to item 24, or a pharmaceutically acceptable salt thereof, wherein is located at C-8. (Item 29) The compound according to item 20, or a pharmaceutically acceptable salt thereof, wherein m is 0. (Item 30) R 2 The compound according to item 29, or a pharmaceutically acceptable salt thereof, wherein is H. (Item 31) R 3 is substituted with -OH, oxo, -O-glucuronide, -NH 2 , -NHC(O)C 1 ~C 6 alkyl group, or -NHCH 2 COOH, the compound according to item 30, or a pharmaceutically acceptable salt thereof. (Item 32) R 3 The compound according to item 29, or a pharmaceutically acceptable salt thereof, wherein is methyl. (Item 33) R 2 The compound according to item 32, or a pharmaceutically acceptable salt thereof, wherein is -OH or -O-glucuronide. (Item 34) The compound of formula II

change

Chem.

Chem.

Claims

Claim 1 (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; (S)-4-Oxo-3-(2-oxo-2-(((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)thio)propan-2-yl)glutamine; (S)-4-Oxo-3-(2-oxo-2-(((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)thio)propan-2-yl)glutamine; (S)-4-Oxo-3-(2-oxo-2-(((1-(4-(trifluoromethoxy)phenyl)ethyl)amino)ethyl)-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)thio)propan-2-yl)glutamine; (S)-4-Oxo-3-(2-oxo-2-(((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)cysteiny-glycine; 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)cysteiny-glycine; 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)cysteiny-glycine; 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)cysteiny-glycine; 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; and (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; a compound selected therefrom or a pharmaceutically acceptable salt thereof. **Claim 2** A pharmaceutical composition comprising the compound or pharmaceutically acceptable salt or a pharmaceutically acceptable salt thereof according to claim 1, admixed with at least one pharmaceutically acceptable excipient. **Claim 3** Use of the compound or pharmaceutically acceptable salt according to claim 1 or the pharmaceutical composition according to claim 2 in the manufacture of a medicament for preventing or treating a disease, disorder, or condition in a mammal in need thereof. **Claim 4** Use according to claim 3, wherein the disease, disorder, or condition is related to GPR139. **Claim 5** Use according to claim 3, wherein the disease, disorder, or condition is selected from schizophrenia, autism spectrum disorder, sleep disorder, depression, bipolar disorder, cognitive disorder, attention deficit hyperactivity disorder, post-traumatic stress disorder, substance abuse, drug addiction, eating disorder, obsessive-compulsive disorder, anxiety disorder, pain, fibromyalgia. **Claim 6** Use of a composition comprising the compound or pharmaceutically acceptable salt according to claim 1 or the pharmaceutical composition according to claim 2 for use in a method of preventing or treating a disease, disorder, or condition in a mammal in need thereof. **Claim 7** The composition according to claim 6, wherein the disease, disorder, or condition is related to GPR139. **Claim 8** The composition according to claim 6, wherein the disease, disorder, or condition is selected from schizophrenia, autism spectrum disorder, sleep disorder, depression, bipolar disorder, cognitive disorder, attention deficit hyperactivity disorder, post-traumatic stress disorder, substance abuse, drug addiction, eating disorder, obsessive-compulsive disorder, anxiety disorder, pain, fibromyalgia.

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