Patent Application
20240239738
This application is being filed electronically via EFS-Web and includes an electronically submitted Sequence Listing in .txt format. The .txt file contains a sequence listing entitled “702581_02111_ST25.txt” created on Apr. 4, 2022 and is 4,198 bytes in size. The Sequence Listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
Liver cancer is the sixth most common cancer and the second leading cause of cancer-related deaths. Among all primary liver cancers, hepatocellular carcinoma (HCC) is the most common neoplasm, accounting for approximately 85-90% of cases. To date, only four small molecules are available on the market as treatment options for HCC, while they lack target selectivity, thereby resulting in various off-target effects and drug resistance. Therefore, there is a significant unmet need for this area. The human ornithine d-aminotransferase (hOAT) gene was found to be overexpressed in liver cancer tissues, while pharmacological inhibition of hOAT leads to promising tumor growth reduction. Therefore, the discovery of hOAT inhibitors may provide a novel therapeutic approach for HCC. In this work, we identified (S)-3-amino-4-(difluoromethylene)cyclohex-1-ene-1-carboxylic acid (WZ-1-181, 14) as a potent and selective inactivator of hOAT. Moreover, the preliminary assessment suggests that WZ-1-181 exhibits favorable DMPK properties, and it is being investigated in the PDX mouse models of HCC.
Disclosed are compounds, compositions, and related methods of use for the selective inhibition of human ornithine aminotransferase. The disclosed compounds, compositions, and methods can be utilized to treat diseases and disorders associated with human ornithine aminotransferase activity such as cell proliferation diseases and disorders such as cancer.
The disclosed compounds may be described as substituted methylenecyclohexene and substituted methylenecyclohexane compounds. In particular, the disclosed compounds may be described as amino-substituted, halomethylene-substituted cyclohexene carboxylic acid compounds and amino-substituted, halomethylene-substituted cyclohexane carboxylic acid compounds. The disclosed compounds and compositions thereof may be utilized in methods for modulating human ornithine aminotransferase (hOAT) activity, including methods for treating diseases or disorders associated with hOAT activity or expression such as cell proliferative diseases and disorders including cancer.
The disclosed compounds may be directed to a compound of the following formula or a dissociated form, a non-protonated form, a zwitterion form, or a salt thereof:
wherein a double bond is optionally present between the α and ζ carbons or wherein a double bond is optionally present between the α and β carbons, and the compound has a formula:
wherein each of R1 and R2 is a halogen such as F, Cl, Br, and I (e.g., F).
In some embodiments, the disclosed compounds may be directed to a compound of the following formula or a dissociated form, a non-protonated form, a zwitterion form, or a salt thereof:
wherein a double bond is present between the α and ζ carbons or a double bond is present between the α and β carbons, wherein each of R1 and R2 is a halogen such as F, Cl, Br, and I (e.g., F).
In other embodiments, the compound is a compound of the following formula or a dissociated form, a non-protonated form, a zwitterion form, or a salt thereof:
wherein each of R1 and R2 is a halogen such as F, Cl, Br, and I (e.g., F).
In certain embodiments, the compound is a compound of the following formula or a dissociated form, a non-protonated form, a zwitterion form, or a salt thereof:
wherein each of R1 and R2 is a halogen such as F, Cl, Br, and I (e.g., F).
In certain embodiments, the compound is a compound of the following formula or a dissociated form, a non-protonated form, a zwitterion form, or a salt thereof:
wherein each of R1 and R2 is a halogen such as F, Cl, Br, and I (e.g., F).
As indicated, the disclosed compounds may be protonated, for example to form an ammonium moiety, optionally where the compound is present as a salt (e.g., an ammonium salt). The disclosed compounds also may be non-protonated and/or dissociated, for example, where the carboxylic acid moiety is dissociated to from a carboxylate moiety, optionally where the compound is present as a salt (e.g., a carboxylate salt). The disclosed compounds may be in zwitterionic form where the compound comprises a protonated ammonium moiety and a dissociated carboxylate moiety, optionally where the compound is present as a salt.
The disclosed compounds and compositions may be utilized in methods for modulating human ornithine aminotransferase (hOAT) activity. Such methods can comprise providing a compound as disclosed herein, such as a compound of the following formula or a dissociated form, a zwitterion form, or a salt thereof, and contacting hOAT with the compound:
wherein a double bond is optionally present between the α and ζ carbons or wherein a double bond is optionally present between the α and β carbons, wherein each of R1 and R2 is a halogen such as F, Cl, Br, and I (e.g., F). In certain embodiments, no double bond is present between the α and ζ carbons and no double bond is presented between the α and β carbons. In certain embodiments, the double bond is between the α and ζ carbons. In other embodiments, the double bond is between the α and β carbons. In certain embodiments, R1 and R2 are F.
In certain embodiments, the disclosed methods may be directed to reducing activity of an hOAT expressed by a cancer, which may include but is not limited to hepatocellular cancer (HCC), non-small cell lung cancer (NSCLC), and colorectal cancer, or other cancers that express or overexpress hOAT. Such a method can comprise providing a compound as disclosed herein, such as a compound of the following formula or a dissociated form, a non-protonated form, a zwitterion form, or a salt thereof, and contacting the cancer with the compound:
wherein a double bond is optionally present between the α and ζ carbons or wherein a double bond is optionally present between the α and β carbons, wherein each of R1 and R2 is a halogen such as F, Cl, Br, and I (e.g., F). In certain embodiments, no double bond is present between the α and ζ carbons and no bond is presented between the α and β carbons. In certain embodiments, the double bond is between the α and ζ carbons. In other embodiments, the double bond is between the α and β carbons. In certain embodiments, R1 and R2 are F.
In certain embodiments, the disclosed methods may be directed to treating a cell proliferative disease or disorder in a subject in need thereof, which methods comprise administering an effective amount of the compounds disclosed herein or pharmaceutical compositions comprising an effective amount of the compounds disclosed herein for treating the cell proliferative disease or disorder. Suitable cell proliferative diseases and disorders may include cancers that express or overexpress hOAT such as, but not limited to, hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), and colorectal cancer. Such a method can comprise administering to such a subject in need thereof an effective amount of a compound of the following formula or a dissociated form, a non-protonated form, a zwitterion form, or a salt thereof (or a pharmaceutical composition comprising an effective amount of the compound, a dissociated form, a non-protonated form, a zwitterion form, or a salt thereof:
wherein a double bond is optionally present between the α and ζ carbons or wherein the double bond is optionally present between the α and β carbons, wherein each of R1 and R2 is independently selected from a halogen such as F, Cl, Br, and I (e.g., F). In certain embodiments, no double bond is present between the α and ζ carbons and no double bond is present between the α and β carbons. In certain embodiments, the double bond is between the α and ζ carbons. In other embodiments, the double bond is between the α and β carbons. In certain embodiments, R1 and R2 are F.
The compounds disclosed herein are without stereochemical or configurational limitation and encompass all stereochemical or configurational isomers, unless stereochemical or configurational limitations are indicated. As illustrated and discussed below, such compounds and/or their intermediates are available as single enantiomers, racemic mixtures from which isomers can be resolved, or diastereomers from which the corresponding enantiomers can be separated. Accordingly, any stereocenter can be (S) or (R) with respect to any other stereocenter(s). As another separate consideration, various compounds can be present as an acid or base salt, either partially or fully protonated, for example at the amino group to form an ammonium moiety, and/or either partially or fully dissociated, for example at the carboxyl group to form a carboxylate substituent or moiety. In certain such embodiments, with respect to an ammonium substituent or moiety, the counter ion can be a conjugate base of a protic acid. In certain such or other embodiments, with respect to a carboxylate substituent or moiety, the counter ion can be an alkaline, alkaline-earth or ammonium cation. Further, it will be understood by those skilled in the art that any one or more the compounds disclosed herein can be provided as part of a pharmaceutical composition comprising a pharmaceutically-acceptable carrier component for use in conjunction with a treatment method or medicament.
In certain embodiments, the disclosed methods are directed to a disease or disorder associated with hOAT activity and/or expression or overexpression, including cell proliferative diseases and disorders such as cancers associated with hOAT activity and/or expression or overexpression. Suitable diseases and disorders may include, but are not limited to cell proliferative diseases and disorders, which may include but are not limited to hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), and colorectal cancer in a human subject in need of such a treatment. In certain embodiments, such a compound can be provided as part of a pharmaceutical composition.
In certain embodiments, the disclosed methods are directed to reducing or modulating activity of an ornithine aminotransferase expressed by a cancer (e.g., hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC)), and colorectal cancer. Such a method can comprise providing a compound of the sort discussed above or described elsewhere and contacting such a compound with a cellular medium comprising a cancer expressing a human ornithine aminotransferase with an amount of such a compound effective to reduce human ornithine aminotransferase activity. In certain embodiments, such a compound can be provided as part of a pharmaceutical composition. Regardless, such contact can be in vitro or in vivo.
More generally, the disclosed methods may be directed to inhibiting a human ornithine aminotransferase. Such a method can comprise providing a compound of the sort discussed above or described below, whether or not part of a pharmaceutical composition, and administering an effective amount of such a compound for contact with a human ornithine aminotransferase. Such contact can be, as would be understood in the art, for experimental and/or research purposes or as may be designed to simulate one or more in vivo or physiological conditions. Such compounds can include but are not limited to those illustrated by the following examples, referenced figures, incorporated references and/or accompanying synthetic schemes. In certain such embodiments, such a compound and/or combination thereof can be present in an amount at least partially sufficient to inhibit hOAT, cell proliferation and/or tumor growth.
The disclosed compounds preferably inhibit ornithine aminotransferase relative to other aminotransferases. In some embodiments, the disclosed compounds specifically inhibit ornithine aminotransferases (OATs) versus γ-aminobutyric acid aminotransferases (GABA-ATs). In some embodiments, a disclosed compound inhibits an OAT (e.g., hOAT) and has a KI which is at least 10, 20, 50, 100, 200, 500, or 1000× lower than the compound's KI for a GABA-AT.
The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only, and are not intended to be limiting.
As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.
As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the claimed subject matter and does not pose a limitation on the scope of the claimed subject matter.
Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”
All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.
The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”
As used herein, a “subject in need thereof” may include a human and/or non-human animal. A “subject in need thereof” may include a subject having a disease or disorder associated with ornithine aminotransferase (OAT) activity. A “subject in need thereof” may include a subject having a cell proliferative disease or disorder, which may include, but is not limited to hepatocellular carcinoma (HCC), lung cancer (non-small cell lung cancer (NSCLC)), and colorectal cancer.
New chemical entities and uses for chemical entities are disclosed herein. The chemical entities may be described using terminology known in the art and further discussed below.
As used herein, a dash “-” an asterisk “*” or a plus sign “+” may be used to designate the point of attachment for any radical group or substituent group.
The term “alkyl” as contemplated herein includes a straight-chain or branched alkyl radical in all of its isomeric forms, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C1-C12 alkyl, C1-C10-alkyl, and C1-C6-alkyl, respectively.
The term “alkylene” refers to a diradical of straight-chain or branched alkyl group (i.e., a diradical of straight-chain or branched C1-C6 alkyl group). Exemplary alkylene groups include, but are not limited to —CH2—, —CH2CH2—, —CH2CH2CH2—, —CH(CH3)CH2—, —CH2CH(CH3)CH2—, —CH(CH2CH3)CH2—, and the like.
The term “halo” refers to a halogen substitution (e.g., —F, —Cl, —Br, or —I). The term “haloalkyl” refers to an alkyl group that is substituted with at least one halogen. For example, —CH2F, —CHF2, —CF3, —CH2CF3, —CF2CF3, and the like.
The term “heteroalkyl” as used herein refers to an “alkyl” group in which at least one carbon atom has been replaced with a heteroatom (e.g., an O, N, or S atom). One type of heteroalkyl group is an “alkoxy” group.
The term “alkenyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12-alkenyl, C2-C10-alkenyl, and C2-C6-alkenyl, respectively.
The term “alkynyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12-alkynyl, C2-C10-alkynyl, and C2-C6-alkynyl, respectively.
The term “cycloalkyl” refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as “C4-8-cycloalkyl,” derived from a cycloalkane. Unless specified otherwise, cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido or carboxyamido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. In certain embodiments, the cycloalkyl group is not substituted, i.e., it is unsubstituted.
The term “cycloheteroalkyl” refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons in which at least one carbon of the cycloalkane is replaced with a heteroatom such as, for example, N, O, and/or S.
The term “cycloalkylene” refers to a cycloalkyl group that is unsaturated at one or more ring bonds.
The term “partially unsaturated carbocyclyl” refers to a monovalent cyclic hydrocarbon that contains at least one double bond between ring atoms where at least one ring of the carbocyclyl is not aromatic. The partially unsaturated carbocyclyl may be characterized according to the number oring carbon atoms. For example, the partially unsaturated carbocyclyl may contain 5-14, 5-12, 5-8, or 5-6 ring carbon atoms, and accordingly be referred to as a 5-14, 5-12, 5-8, or 5-6 membered partially unsaturated carbocyclyl, respectively. The partially unsaturated carbocyclyl may be in the form of a monocyclic carbocycle, bicyclic carbocycle, tricyclic carbocycle, bridged carbocycle, spirocyclic carbocycle, or other carbocyclic ring system. Exemplary partially unsaturated carbocyclyl groups include cycloalkenyl groups and bicyclic carbocyclyl groups that are partially unsaturated. Unless specified otherwise, partially unsaturated carbocyclyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido or carboxyamido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. In certain embodiments, the partially unsaturated carbocyclyl is not substituted, i.e., it is unsubstituted.
The term “aryl” is art-recognized and refers to a carbocyclic and/or heterocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, pyridinyl, quinolinyl, furanyl, thionyl, and the like. The term “aryl” includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and/or aryls. Unless specified otherwise, the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido or carboxyamido, carboxylic acid, —C(O)alkyl, -CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, —CF3, —CN, or the like. In certain embodiments, the aromatic ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In certain other embodiments, the aromatic ring is not substituted, i.e., it is unsubstituted. In certain embodiments, the aryl group is a 6-10 membered ring structure.
The terms “heterocyclyl” and “heterocyclic group” are art-recognized and refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, alternatively 3- to 7-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The number of ring atoms in the heterocyclyl group can be specified using 5 Cx-Cx nomenclature where x is an integer specifying the number of ring atoms. For example, a C3-C7 heterocyclyl group refers to a saturated or partially unsaturated 3- to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The designation “C3-C7” indicates that the heterocyclic ring contains a total of from 3 to 7 ring atoms, inclusive of any heteroatoms that occupy a ring atom position.
The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines (e.g., mono-substituted amines or di-substituted amines), wherein substituents may include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.
The terms “alkoxy” or “alkoxyl” are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxy groups include methoxy, ethoxy, tert-butoxy and the like.
An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, and the like.
The term “carbonyl” as used herein refers to the radical —C(O)—.
The term “oxo” refers to a divalent oxygen atom —O—.
The term “carboxy” or “carboxyl” as used herein refers to the radical —COOH or its corresponding salts, e.g. —COONa, etc. A carboxy alkyl ester refers to a compound having a moiety —C(O)O—R, where R is alkyl.
The term “carboxamido” as used herein refers to the radical —C(O)NRR′, where R and R′ may be the same or different. R and R′, for example, may be independently hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, formyl, haloalkyl, heteroaryl, or heterocyclyl.
The term “amide” or “amido” or “amidyl” as used herein refers to a radical of the form —R1C(O)N(R2)—, —R1C(O)N(R2)R3—, —C(O)NR2R3, or —C(O)NH2, wherein R1, R2 and R3, for example, are each independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, or nitro.
The compounds of the disclosure may be isomeric. In some embodiments, the disclosed compounds may be isomerically pure, wherein the compounds represent greater than about 99% of all compounds within an isomeric mixture of compounds. Also contemplated herein are compositions comprising, consisting essentially of, or consisting of an isomerically pure compound and/or compositions that are isomerically enriched, which compositions may comprise, consist essential of, or consist of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single isomer of a given compound.
The compounds of the disclosure may contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term “stereoisomers” when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “S,” or “+” or “−” depending on the configuration of substituents around the chiral or stereogenic carbon atom and or the optical rotation observed. The disclosed compounds encompass various stereo isomers and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated (±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. It is understood that graphical depictions of chemical structures, e.g., generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise. Also contemplated herein are compositions comprising, consisting essentially of, or consisting of an enantiopure compound and/or compositions that are enantiomer enriched, which compositions may comprise, consist essential of, or consist of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of a given compound (e.g., at least about 95% of an R enantiomer of a given compound).
Various non-limiting embodiments of the disclosed compounds and methods of use can be considered with an understanding of a catalytic mechanism of hOAT and mechanism of inactivation of GABA-AT and hOAT. In a some embodiments, the disclosed subject matter relates to one or more hOAT inhibitors or inactivators, as set forth above, formulated into compositions together with one or more physiologically tolerable or acceptable diluents, carriers, adjuvants or vehicles that are collectively referred to herein as carriers. Compositions suitable for such contact or administration can comprise physiologically acceptable aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, whether or not sterile. The resulting compositions can be, in conjunction with the various methods described herein, for administration or contact with a human ornithine aminotransferase. Whether or not in conjunction with a pharmaceutical composition, “contacting” means that a human ornithine aminotransferase and one or more inhibitor or inactivator compounds are brought together for purpose of binding and/or complexing such an inhibitor or inactivator compound to the enzyme. Amounts of a compound effective to inhibit or inactivate a human ornithine aminotransferase may be determined empirically, and making such determinations is within the skill in the art. Inhibition or otherwise affecting a human ornithine aminotransferase activity includes reduction, mitigation and/or modulation, as well as elimination of hOAT activity (i.e., inactivation), glutamate production, glutamine synthesis, cell proliferation and/or tumor growth.
It is understood by those skilled in the art that dosage amount will vary with the activity of a particular inhibitor or inactivator compound, disease state, route of administration, duration of treatment, and like factors well-known in the medical and pharmaceutical arts. In general, a suitable dose will be an amount which is the lowest dose effective to produce a therapeutic or prophylactic effect. If desired, an effective dose of such a compound, pharmaceutically-acceptable salt thereof, or related composition may be administered in two or more sub-doses, administered separately over an appropriate period of time.
Methods of preparing pharmaceutical formulations or compositions include the step of bringing an inhibitor or inactivator compound into association with a carrier and, optionally, one or more additional adjuvants or ingredients. For example, standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
Regardless of composition or formulation, those skilled in the art will recognize various avenues for medicament administration, together with corresponding factors and parameters to be considered in rendering such a medicament suitable for administration. Accordingly, with respect to one or more non-limiting embodiments, the disclosed compounds may be utilized as inhibitor or inactivator compounds for the manufacture of a medicament for therapeutic use in the treatment or prevention of a disease or disorder associated with hOAT activity, expression, or overexpression. Suitable diseases or disorders may include cell proliferative diseases or disorders, which may include but are not limited to hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), and colorectal cancer.
Generally, with respect to various embodiments, the disclosed subject matter can be directed to method(s) for the treatment of a pathologic proliferative disorder. As used herein, the term “disorder” refers to a condition in which there is a disturbance of normal functioning. A “disease” is any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with the person. Sometimes the term is used broadly to include injuries, disabilities, syndromes, symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts these may be considered distinguishable categories. It should be noted that the terms “disease”, “disorder”, “condition” and “illness”, are equally used herein.
According to certain embodiments, the disclosed methods can be specifically applicable for the treatment of malignant proliferative disorders, including malignant proliferative disorders that express human ornithine aminotransferase (hOAT). As used herein, “cancer”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. Accordingly, the compounds, compositions, and methods disclosed herein may be used in the treatment of non-solid and solid tumors.
Malignancy, as contemplated herein, may be selected from the group consisting of melanomas, carcinomas, leukemias, lymphomas and sarcomas, which express hOAT. Malignancies that can be treated by the methods disclosed herein, including malignancies that express hOAT can comprise but are not limited to hematological malignancies (including leukemia, lymphoma and myeloproliferative disorders), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic) and solid tumors (including bladder, rectum, stomach, cervix, ovarian, renal, lung, liver, breast, colon, prostate, GI tract, pancreas and Karposi). More particularly, according to certain embodiments, the compounds and compositions used in conjunction can be used in methods for the treatment or inhibition of non-solid cancers, e.g. hematopoietic malignancies such as all types of leukemia, e.g. acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndrome (MDS), mast cell leukemia, hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphomas, Burkitt's lymphoma and multiple myeloma, as well as for the treatment or inhibition of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extraliepatic bile ducts, ampulla of Vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignant melanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestational trophoblastic tumors, penis, prostate, testis, kidney, renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma of the conjunctiva, malignant melanoma of the conjunctiva, malignant melanoma, retinoblastoma, carcinoma of the lacrimal gland, sarcoma of the orbit, brain, spinal cord, vascular system, hemangiosarcoma and Kaposi's sarcoma.
The compounds and compositions disclosed herein may be administered in methods of treatment as known in the art. Accordingly, various such compounds and compositions can be administered in conjunction with such a method in any suitable way. For example, administration may comprise oral, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, parenteral, transdermal, intravaginal, intranasal, mucosal, sublingual, topical, rectal or subcutaneous administration, or any combination thereof.
According to some embodiments, the treated subject may be a mammalian subject. Although the methods disclosed herein are particularly intended for the treatment of proliferative disorders in humans, other mammals are included. By way of non-limiting examples, mammalian subjects include monkeys, equines, cattle, canines, felines, mice, rats and pigs.
The terms “treat, treating, treatment” as used herein and in the claims mean ameliorating one or more clinical indicia of disease activity in a subject having a pathologic disorder. “Treatment” refers to therapeutic treatment. Those in need of treatment are mammalian subjects suffering from any pathologic disorder. By “patient” or “subject in need” is meant any mammal for which administration of a compound or any pharmaceutical composition of the sort described herein is desired, in order to prevent, overcome, modulate or slow down such infliction. To provide a “preventive treatment” or “prophylactic treatment” is acting in a protective manner, to defend against or prevent something, especially a condition or disease.
More generally, the disclosed methods may be directed to affecting, modulate, reducing, inhibiting and/or preventing the initiation, progression and/or metastasis (e.g., from the liver elsewhere or to the liver from any other organ or tissue) of a malignant pathologic proliferative disorder associated with hOAT activity. (See, e.g., Lucero O M, Dawson D W, Moon R T, et al. A re-evaluation of the “oncogenic” nature of Wnt/beta-catenin signaling in melanoma and other cancers. Curr Oncol Rep 2010, 12, 314-318; Liu Wei; Le Anne; Hancock Chad; Lane Andrew N; Dang Chi V; Fan Teresa W-M; Phang James M. Reprogramming of proline and glutamine metabolism contributes to the proliferative and metabolic responses regulated by oncogenic transcription factor c-MYC. Proc. Natl. Acad. Sci. USA 2012, 109(23), 8983-8988; and Tong, Xuemei; Zhao, Fangping; Thompson, Craig B. The molecular determinants of de novo nucleotide biosynthesis in cancer cells. Curr. Opin. Genet. Devel. 2009, 19(1), 32-37.)
The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter. The following non-limiting Examples and data illustrate various aspects and features relating to the disclosed compounds, compositions, and methods including the treatment of diseases and disorders associated with hOAT activity, expression, or overexpression, and/or reduction of human ornithine aminotransferase activity, such as cell proliferative diseases and disorders including, but not limited to hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), and colorectal cancer. While the utility of this invention is illustrated through the use of several compounds and compositions which can be used therewith, it will be understood by those skilled in the art that comparable results are obtainable with various other compound(s), as are commensurate with the scope of this invention.
Human ornithine aminotransferase (hOAT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme, which was recently found to play an important role in the metabolic reprogramming of hepatocellular carcinoma (HCC) via proline and glutamine metabolic pathways. The selective inhibition of hOAT by compound 10 exhibited potent in vivo anti-tumor activity, along with dramatically reduced alpha-fetoprotein (AFP, a biomarker for HCC) levels. We rationally designed, synthesized, and evaluated a series of six-membered ring analogs. Among them, analog 14 was identified as a novel selective hOAT inhibitor and demonstrated 18.6 times more potent than 10. Three types of protein mass spectrometry and crystallography of hOAT-14 revealed the formation of the final adduct (28′) in the catalytic pocket, which surprisingly establishes two covalent bonds with nearby residues. Later, molecular dynamics (MD) simulations and soaking crystallography studies were carried out to disclose the potential binding pose of active intermediate 27′, leading to the initial reaction of the warhead with *Thr322 and the following nucleophilic attack of catalytic Lys292. Besides, the turnover mechanism of 14 by hOAT was also revealed via the mass spectrometry-based analysis of metabolites and fluoride ion release experiments.
Hepatocellular carcinoma (HCC) is the most common liver cancer subtype and the second leading cause of cancer death worldwide.1-4 The disease is highly malignant, radiotherapy-resistant, and refractory to the standard-of-care chemotherapy, sorafenib.5-8 Recently, human ornithine aminotransferase (hOAT) was identified as a metabolic regulator of HCC progression via proline and glutamine metabolic pathways.9-10 OAT (E.C. 2.6.1.13) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme,11 which is found in the mitochondrial matrix of most human and animal tissues.12 Mechanically, two coupled half-reactions are required to complete one transamination cycle of OAT (Scheme 1).13 In the first half-reaction, ornithine binds with cofactor PLP in the catalytic pocket of OAT to form Schiff base M1, followed by the tautomerization to intermediate M2; the subsequent hydrolysis of M2 generates pyridoxamine 5′-phosphate (PMP) and glutamate γ-semialdehyde (GyS) that spontaneously cyclizes to Δ1-pyrroline-5-carboxylate (PSC). In the second-half reaction, OAT catalyzes the conversion of α-ketoglutarate (α-KG) and PMP to L-glutamate (L-Glu) and PLP. Glutamate and glutamine metabolism have acknowledged roles in supporting the anabolic demands of tumor growth and it is increasingly clear that proline metabolism promotes liver cancer tumorigenesis.14-17 Not only L-Glu and proline have direct effects on metabolic rewiring, but they also induce a hypoxia-inducible factor-1α (HIF1α) transcriptional program in HCC leading to sorafenib resistance and retained proliferative capacity under oxygen starvation.18 hOAT is commonly overexpressed in HCC due to dysregulation and activation of the Wnt/β-catenin pathway.19-20 The selective inhibition of hOAT exhibited potent in vivo anti-tumor activity in the HCC mouse model, along with dramatically reduced alpha-fetoprotein (AFP, a biomarker for HCC) levels.10 Recently, upregulation of hOAT was found in non-small cell lung cancer (NSCLC) cells, which contributes to the promotion of proliferation, invasion, and migration, the inhibition of apoptosis, and the change of cell cycle.21 The specific knockdown of hOAT in NSCLC inhibited the cell proliferation in vitro and suppressed the tumor growth in vivo.21 Thus, hOAT can serve as a promising therapeutic target for the treatment of HCC and related cancers with similar underlying metabolic alterations.
OAT belongs to the same enzyme subgroup22 as γ-aminobutyric acid aminotransferase (GABA-AT, E.C. 2.6.1.19) that demonstrates a similar catalytic mechanism as OAT,23 in which GABA is converted to succinic semialdehyde (SSA) in the first-half reaction.24 In past decades, our laboratory has been devoting efforts to the rational design of mechanism-based inactivators (MBIs) of GABA-AT25 for the potential treatment of epilepsies and addictions. MBIs usually act as native substrates initially and inhibit the target enzymes after the conversion to active intermediates in their catalytic sites,25-28 which can demonstrate higher potency and reduced off-target effects when compared with traditional inhibitors.10, 25, 29-30 For example, vigabatrin (1)31, as an MBI of GABA-AT, is an FDA-approved drug for the treatment of infantile spasms and refractory complex partial seizures (Scheme 2).32 Mechanism study33 revealed that 1 initially forms Schiff base with PLP in the catalytic pocket of GABA-AT, followed by the tautomerization as the native substrates do, to afford intermediate 3. Catalytic residue Lys329 attacks at the Michael acceptor of 3 to form covalent adduct 4, which accounts for 70% of the inactivation pathway of 1 (Scheme 2).33 Based on this mechanism, difluoro-substituted conformationally-rigid analog 5 was synthesized and exhibited 186 times more efficient as an inactivator than 1, determined by their kinact/KI values (Scheme 2).34-35 Analogue 5 was also found to generate Michael acceptor intermediate 7 via similar steps as shown in the mechanism pathway of 1.36 Interestingly, highly electrophilic 7 reacts with water molecules existing in the pocket, instead of Lys329, leading to the formation of tight-binding adduct 8, in which the newly generated carboxylate forms the electrostatic interaction with Arg445 (Scheme 2).36 Currently, 5 is investigated in clinical trial for the potential treatment of infantile spasms and cocaine dependency.37-38 Later, cyclopentene analog 9 (
When comparing the catalytic pockets of hOAT and GABA-AT for differences, hOAT has a more flexible and larger catalytic pocket, resulting from the replacement of Phe351 and Ile72 in GABA-AT with Tyr55 and Tyr85 in hOAT, which can accommodate ornithine that is one carbon longer than GABA.39 In 2015, relative bulky trifluoromethyl analog 10 (
We aim to discover potent and selective hOAT inactivators for the treatment of HCC and reveal the underlying mechanism of inactivators for further improved rational design. Herein, we designed and synthesized six-membered ring analogs 12-15 based on GABA-AT inactivators 5 and 9 (
The synthetic route of 12-15 is shown in Scheme 3. Chirally-pure lactam 1641 was prepared via a reported procedure. Intermediate 16 was treated with silver trifluoroacetate, followed by Dess-Martin oxidation, to afford ketones 17a and 17b. Intermediate 18 was obtained from 17b with the protection of PMB, followed by the treatment34-35 of CHF2PO(OEt)2 and tBuLi, to provide intermediate 19. Deprotection of PMB with CAN and the ring-opening reaction under acid conditions afforded the desired product 12. Chirally-pure ester 21 was prepared as reported,40 which was treated42 with 2-PySO2CF2H and tBuOK in DMF to give difluoromethylene intermediate 22. Interestingly, the treatment of 22 with PhSeCl and KHMDS afforded bicyclic lactam 23 as the major product. The sequential Boc protection, ring-opening reaction, and oxidative elimination provided two isomers 24a and 24b. The desired products 13-15 were afforded from the corresponding esters under acid conditions, respectively.
As shown in Table 1, six-membered ring analogs 12-15 exhibited a lower binding affinity to GABA-AT with much greater KI values than 5 or 9, which is well consistent with our initial design strategy. The enlarged ring could potentially obstruct the initial binding with GABA-AT,40 which has a relatively small and rigid catalytic pocket.39 Although cyclohexane analogs 12 and 13 showed enhanced selectivity for OAT, their potency was greatly decreased. The introduction of the double bond significantly increased their binding affinity with OAT, which may result from the reduced steric hindrance of cyclohexene.40 α, β-unsaturated analog 14 is more potent than the other cyclohexene analog 15, possibly due to the reduced acidity of the γ-position, which is critical for the tautomerization step and further inactivation.25
As the most potent analog, 14 is 18.6-fold more efficient as an inactivator of OAT than 7 (kinact/KI=0.87 min−1 mM−1) that significantly suppressed HCC tumor growth in vivo,10 along with good selectivity over GABA-AT with lower efficiency constants (kinact/KI=0.29 min−1 mM−1). Besides, it demonstrated little or no inhibitory activities against alanine aminotransferase (Ala-AT) and aspartate aminotransferase (Asp-AT) at high concentrates (up to 20 mM,
akinact and KI values were determined by the equation: kobs = kinact*[I]/(KI + [I]) and are presented as means and standard errors.
bratio of kinact/KI was determined by the slope of kobs = kinact*[I]/(KI + [I]).
Cyclopentene analog 9 was found to inactivate GABA-AT in a similar manner as cyclopentane analog 5 (Scheme 2), where a tight-binding adduct was formed via the nucleophilic attack of water molecules at Michael acceptor position.29 Not surprisingly, 9 could also inactivate hOAT due to the high similarity of catalytic pockets between these two aminotransferases, but its inactivation mechanism with hOAT has not to be disclosed yet. Thus, we obtained the cocrystal structure of hOAT inactivated by 9, which provided us some clues for the inactivation study of its cyclohexene analog (14).
Cocrystal Structure for hOAT Inactivated by 9.
The structure of hOAT inactivated by 9 was solved by molecular replacement using a monomer from a previously reported structure of hOAT43 (PDB code 1OAT), while water molecules and ligand atoms were deleted. As shown in
Although the formation of these two covalent bonds is quite clear as shown in this crystal structure, the electron density for the carboxylate group part is relatively weak and is completely missing in two out of six copies of the ligand in an asymmetric unit. This observation may result from the weak hydrogen bond interaction between the carboxylate group and nearby Tyr55. Aminotransferases inactivators usually are converted to active intermediates after binding with the cofactor PLP in the catalytic pocket, leading to the formation of various kinds of final adducts.25 Thus, it would be difficult to determine the final adduct of MBI merely based on X-ray cocrystal, compared with the cases for traditional covalent inhibitors. However, this preliminary result provided us some new ideas for mechanism study, in which Lys292 or/and *Thr322 could be involved in the inactivation of hOAT by 14.
MBIs of aminotransferases are reported to inactivate the enzymes through different mechanism pathways, along with the formation of covalent or tight-binding adducts occupying the catalytic pockets.25 Based on previous reports (Scheme 2) and preliminary cocrystal results for hOAT-9 (
Previously, five-membered ring analogs 5 and 9 were identified as partially irreversible inhibitors of GABA-AT, with the formation of the tight-binding adducts.29-30 Thus, time-dependent reactivation experiments of hOAT for cyclohexene analog 14 were carried out to determine if similar reversible components were also involved during the inactivation. After hOAT activity was partially or fully abolished by 0.6-2.0 equivalents of 14, aliquots at different time intervals were collected and assayed for the return of enzyme activity. No matter how many equivalents of 14 were used, no enzyme activity recovered, even after 91 h of dialysis (
Intact protein mass spectrometry has served as an efficient tool for the identification of inactivation mechanisms for aminotransferases.44, 50-51 The hydrolysis of unstable groups under acidic conditions, such as imine or enamine groups, has been observed with intact protein mass spectrometry to create masses that correspond to stable covalent adducts alone.42 Accordingly, unstable adducts for 14 may suffer from similar hydrolysis and release PMP or result in cleavage of newly formed covalent bonds with Lys292/*Thr322. As shown in
By intact protein mass spectrometry, unmodified, dimeric hOAT resolved almost entirely as monomeric protein due to acidic conditions and reverse-phase ultra-high performance liquid chromatography (
We next turned to top-down proteomics to localize the 14-induced adduct to the hOAT primary sequence. Fragmentation of hOAT-14 was first assessed using electron transfer dissociation (ETD) as this method preserves weaker covalent interactions and cross-linked peptides, and any further adduct loss was of primary concern to this study.52-53 ETD fragmentation of hOAT-14 created a complex MS2 spectrum distinct to unmodified control hOAT and only 6 z-ions (compared to 54 z-ions for the control sample) could be mapped the protein C-terminus, all occurring after Thr322 (
Intact mass and complementary fragmentation studies support *Thr322 covalent modification and the crosslinking adduct 28 in pathway a1 (Scheme 4), but they cannot clearly explain the observed hOAT-14 dimer adduct mass nor the loss of adduct cofactor for hOAT-14. Therefore, to identify the adduct mass on dimeric hOAT-14, we turned to native mass spectrometry (nMS), which preserves non-covalent substrate binding and protein tertiary structure.56 To evaluate the applicability of nMS for the study of MBIs, native untreated hOAT, hOAT-11b40, and hOAT-Gabaculine47 samples were first used as positive controls. In these cases, the catalytic pocket is occupied by an unstable covalent adduct, a stable covalent adduct, and a tight-binding adduct, respectively. Theoretical mass shifts for control samples were calculated based on known mechanisms; hOAT-14 mass shifts were calculated based on the proposed inactivation pathways a1-a4 and b (
Although mass spectrometry-based studies strongly indicated the formation of adduct 28 (pathway a1, Scheme 4) at the catalytic pocket of hOAT when inactivated by 14, top-down proteomics was unable to provide direct evidence for the covalent attachment to catalytic Lys292. Hence, protein crystallography of hOAT inactivated by 14 was conducted to interpret the adduct formed in the active site.
The hOAT cocrystal with compound 14 was obtained and solved via a similar approach as compound 9. Similarly, two covalent bonds were formed with the adduct from Lys292 and *Thr322, which was consistent with mass spectrometry studies. Analog 14 possesses a six-member ring in its structure instead of a five-member ring as in 9, and the extra carbon provides enough length for the carboxylate group of the adduct to form hydrogen bonds with Tyr55. Because of this relatively stable interaction with the nearby residue, the intensity of electron density for the carboxylate group of adduct generated from 14 is much stronger than the adduct generated from 9 (
Intermediate 27 was proposed to be the active Michael acceptor for the inactivation process, whose warhead is positioned near the internal H-bond between protonated imine and oxygen anion (Scheme 4). Interestingly, the cocrystal structure for 14 demonstrated the newly formed covalent linkage is moved to the phosphate side (
Although the structure of the final adduct for hOAT-14 is well asserted with mass spectrometry and crystallography studies, the inactivation process is not quite clear. Two covalent bonds were formed with catalytic Lys292 and *Thr322 from the other chain, which potentially resulted from two times nucleophilic attacks at the Michael acceptor intermediate (Scheme 4), but it is hard to conclude which covalent bond is formed first. No doubt, catalytic Lys292 is more nucleophilic than *Thr322, which was found to react with active intermediates previously. However, the highly electrophilic difluoro Michael acceptor was previously disclosed to react with water molecules in the active site of the aminotransferase (Scheme 2),29, 36 suggesting its possibility to react with *Thr322 initially. Fortunately, the cocrystal structure of hOAT inactivated by 14 (
Intermediate 25 showed similar docking poses in the catalytic pockets of hOAT with the salt bridge maintained (
When the salt bridge of hOAT is disrupted, strong interaction between Arg413 and the carboxylate of intermediate 27 was observed in the docking studies, which forced the warhead to move to the other side near Tyr55 and *Thr322 (
As indicated in the above molecular docking and MD simulation studies, the order for the nucleophilic addition of two residues could be potentially determined by different binding poses of 27 (
Intermediate 36 (Scheme 8) was trapped in the soaking study of hOAT and 14 (
MBIs act as native substrates to bind at the catalytic pockets of target enzymes initially, thus it is common that certain amounts of MBIs will be converted to corresponding metabolites during the inactivation process.25 Based on the reported turnover mechanism for 1 and 5 with GABA-AT,33, 36 two possible turnover pathways for 14 by hOAT are proposed with the release of PMP or PLP, respectively. The Lys292-assisted deprotonation of Schiff base 25 affords intermediate 26, followed by the protonation at different positions to give intermediate 27′ (pathway a) or 33 (pathway b). Intermediate 27 could be further hydrolyzed with the release of PMP and metabolite 37 (pathway a1) or 38 (pathway a2), which is similar to the degradation of ornithine by hOAT (Scheme 1). On the other hand, the reaction of intermediate 33 with Lys292 could afford intermediate 39, followed by the release of PLP and metabolite 40 (pathway b1) or 42 (pathway b2). Enamine 40 could be hydrolyzed to give ketone 41; imine 42 could undergo aromatization to generate metabolite 43 or 45. To identify which above-mentioned turnover pathway is involved, partition ratio and fluoride ion release experiments and the mass spectrometric analysis of metabolites were carried out.
Usually, the partition ratio is used to determine the ratio of degradation and inactivation, calculated by titrating the enzyme with varying equivalents of inactivator. Ideally, a linear relationship (between enzyme activity remaining and equivalents of compounds) can be extrapolated to yield the exact equivalents (the intercept with the x-axis, turnover number) required to inactivate the enzyme completely. Since this obtained number includes the one molecule of inactivator required to inactivate one enzyme monomer, the partition ratio is the turnover number minus one. Therefore, the partition ratios of 5 and 14 were determined by titrating hOAT with varying equivalents of inactivators (
Different equivalents of fluoride ions can be released in PLP and PMP turnover pathways. If PMP is formed in the turnover mechanism, it cannot be converted back to PLP in the absence of α-KG, which results in at most two equivalents of fluoride ion released (pathway a); more equivalents of fluoride ion would be released in the presence of α-KG if PMP is formed after the fluoride ion release step (pathway a1). If PLP is regenerated during the turnover mechanism, at least two equivalents of fluoride ions will be released in the absence of α-KG (pathway b), which should be close to the equivalents in the presence of α-KG. Based on the partition ratio and the confirmed inactivation mechanism for 14, the theoretical equivalents of fluoride ions, which are released per enzyme active site in different turnover pathways, can be calculated in the presence/absence of α-KG (Table 3). The concentration of released fluoride ions can be monitored by a fluoride ion-selective electrode. When OAT was inactivated with an excess of 14, 2.95 equivalents of fluoride ions were released in the presence of α-KG and 2.66 equivalents of fluoride ions were released in the absence of α-KG (Table 3). These results indicate the regeneration of PLP is involved in the turnover mechanism for 14, in which the experimental fluoride ions released are close to the theoretical calculation result in turnover pathway b2.
To identify the potential metabolites generated during the inactivation process of OAT by 14, inactivated OAT sample was filtered through a 10 kDa MWCO filter, followed by the untargeted metabolomics (+/−ESI HRMS) analysis of the obtained filtrate. Among the possible metabolites proposed in Scheme 5, only metabolite 43 (observed: 170.0610 m/z, [M+H]+; theoretical: 170.0612 m/z) in pathway b2 was detected, followed by the confirmation of its fragmentation spectrum (
Based on the above inactivation and turnover mechanism studies, a modified pathway for 14 with hOAT is proposed in Scheme 6. Initially, Schiff base 25 was generated from 14 and PLP, followed by the deprotonation at the y-position. According to the partition ratio (0.6) of 14 (
Recently, selective pharmacological inhibition of human ornithine aminotransferase (hOAT) has been shown as a potential therapeutic approach for hepatocellular carcinoma (HCC) and other related cancers. A novel series of six-membered ring analogs (12-15) was rationally designed and chirally synthesized based on the difference in the catalytic pockets of aminotransferases. Among them, analog 14 was identified as a selective hOAT inhibitor and 18.6 times more potent than analog 10 that has demonstrated good in vivo anti-cancer activities. A plausible mechanism for 14 with hOAT was proposed (Scheme 6) based on four distinct applications of mass spectrometry, two types of protein crystallography, and molecular dynamics (MD) simulations with the aid of a dialysis experiment, total turnover, and measurement of fluoride ion release. Notably, this is the first time to successfully apply native mass spectrometry and top-down proteomics to the mechanistic study of aminotransferase inactivators. Surprisingly, double-covalent adduct 28′ was formed in the catalytic pocket of hOAT via sequential nucleophilic attacks of *Thr322 and Lys292 at the warhead of active intermediate 27′. This is the first example of one single adduct forming two covalent bonds with two residues in the catalytic pocket of an enzyme, indicating a non-catalytic residue of the aminotransferase could be covalently modified by an MBI. Moreover, an interesting turnover mechanism is also demonstrated, in which aromatic metabolite 43 was generated along with the re-generation of cofactor PLP. Interestingly, no water molecule was involved in the inactivation mechanism of hOAT for 14 and its cyclopentene analog 9, while water molecules played an important role in the mechanism of 5/9 and GABA-AT (Scheme 2). We believe this mechanistic difference might derive from the enzymatic machinery of these two aminotransferases with GABA analogs, which could contribute to the further rational design of selective inactivators.
DMP, Dess-martin periodinane; PMB, p-methoxybenzyl; CAN, cerium (IV) ammonium nitrate; Boc, tert-butyloxycarbonyl; KHMDS, potassium bis(trimethylsilyl)amide; DIPEA, N,N-diisopropylethylamine; BoczO, di-tert-butyl decarbonate; m-CPBA, meta-chloroperoxybenzoic acid; tBuOK, potassium tert-butoxide; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; DCM, dichloromethane.
General Synthetic Methods. All chemicals were purchased from Sigma Aldrich, Acros Organics, or Combi-block and used without further purification. Anhydrous solvents (THF, CH3CN, DMF) were purified before use by passing through a column composed of activated alumina and a supported copper redox catalyst. Yields refer to chromatographically homogeneous materials. Analytical thin-layer chromatography (TLC) was performed using Merck Silica Gel 60 Å F-254 precoated plates (0.25 mm thickness), and components were visualized by ultraviolet light (254 nm) and/or ceric ammonium molybdate stain and/or ninhydrin stain. Flash column chromatography was performed on a Teledyne Combiflash Rf Plus automated flash purification system with various Taledyne cartridges (4-80 g, 40-63 μm, 60 Å). Purifications were performed with hexanes and ethyl acetate unless otherwise noted. 1H and 13C NMR spectra were recorded on a Bruker Avance-III NMR spectrometer at 500 MHz and 126 MHz, respectively, in CDC13, CD3OD or DMSO-d6. Chemical shifts were reported in ppm; multiplicities are indicated by s=singlet, brs=broad singlet, d=doublet, t=triplet, q=quartet, dd=doublet of doublet, dt=doublet of triplet, dq=doublet of quartet, m=multiplet resonance. Coupling constants ‘J’ were reported in Hz. High-resolution mass spectral data were obtained on an Agilent 6210 LC-TOF spectrometer in the positive ion mode using electrospray ionization with an Agilent G1312A HPLC pump and an Agilent G1367B autoinjector at the Integrated Molecular Structure Education and Research Center (IMSERC), Northwestern University. Analytical HPLC was performed using a reversed-phase Agilent Infinity 1260 HPLC with a Phenomenex Kintex C-18 column (50×2.1 mm, 2.6 μm), detecting with UV absorbance at 254 nm.
(1R,4S)-2-azabicyclo[2.2.2]octane-3,6-dione (17a) & (1S,5S)-6-azabicyclo[3.2.1 ]octane-4,7-dione (17b). To a stirred solution of solution of the lactam 161 (3.79 g, 15.1 mmol, 1.0 equiv) in nitromethane (40 mL) was added silver(I) trifluoroacetate (5.00 g, 22.6 mmol, 1.5 equiv) at 0° C. (the flask was wrapped with aluminum foil). The reaction was warmed to r.t. and stirred for 16 h. The reaction was diluted with Et2O and filtered through a pad of Celite. The filtrate was concentrated in vacuo, followed by the addition of ammonia solution (7 N in methanol, 29 mL). After the reaction was stirred at r.t. for 1 h, it was filtered through a cotton plug and the obtained filtrate was concentrated in vacuo. To a solution of the obtained crude in dry CH3CN (60 mL) was added NaHCO3 (6.34g, 75.5 mmol, 5.0 equiv) and Dess-Martin periodinane (7.68 g, 18.1 mmol, 1.2 equiv). After the suspension was stirred at r.t. for 1 h, it was diluted with EtOAc and then filtered. The filtrate was concentrated and purified by silica gel chromatography (15% to 45% EtOAc in hexane) to afford a white solid (17a, 420 mg, 20%) 1H NMR (500 MHz, CDCl3): δ 7.80-7.60 (brs, N—H, 1H), 3.77 (ddd, J=5.3, 4.2, 1.6 Hz, H1, 1H), 2.86-2.82 (m, H4, 1H), 2.58 (ddd, J=19.0, 2.8, 2.8 Hz, 1H), 2.33 (dd, J=19.0, 2.8 Hz, 1H), 2.16-1.78 (m, 4H); 13C NMR (126 MHz, CDCl3): δ=206.5, 176.5, 58.6, 39.7, 39.1, 25.3, 22.3; HRMS-ESI (m/z) [M+H]+ calc'd for C7H10NO2=140.0706, found=140.0711. and a white solid (17b, 420 mg, 20%). 1H NMR (500 MHz, CDCl3): δ 7.10-6.80 (brs, 1H), 3.78 (dd, J=6.0, 1.0 Hz, 1H), 2.80 (ddd, J=16.7, 11.05, 9.6 Hz, 1H), 2.67 (dddd, J=12.1, 6.1, 5.6, 3.1 Hz, 1H), 2.61-2.57 (m, H1, 1H), 2.43 (ddd, J=16.7, 7.2, 7.2 Hz, H3/H3′, 1H), 2.21 (dddd, J=13.5, 9.5, 3.1, 3.1 Hz, 1H), 2.02-1.93 (m, 1H), 1.89 (dddd, J=13.5, 7.4, 7.2, 3.1 Hz, 1H); 13C NMR (126 MHz, CDCl3): δ=207.8, 179.5, 61.8, 39.2, 37.5, 34.4, 25.4.
(1S,5S)-6-(4-methoxybenzyl)-6-azabicyclo[3.2.1]octane-4,7-dione (18). To a stirred solution of 17 (19.1 mg, 0.137 mmol, 1.0 equiv) in DMF (0.14 mL) at 0° C. was added sodium hydride (60 wt. % dispersion in mineral oil, 6.6 mg, 0.165 mmol, 1.2 equiv). After 1 h, p-methoxybenzyl chloride (20.1 μL, 0.151 mmol, 1.1 equiv) was added via syringe. The reaction mixture was stirred for 16 h before quenching with a 1:1 saturated solution of aq. sodium bicarbonate and water. The solution was partitioned with a 1:1 mixture of ethyl acetate and hexanes. The organic layer was removed, and the aqueous layer was further extracted with the ethyl acetate hexanes mixture (7×). The combined organic layer was concentrated in vacuo and purified by silica gel chromatography (10% to 100% EtOAc in hexane) to afford a colorless oil (18, 23.5 mg, 66%). 1H NMR (500 MHz, CDCl3): δ 7.18 (d, J=8.6 Hz, 2H), 6.83 (d, J=8.6 Hz, 2H), 4.56 (d, J=14.6 Hz, 1H), 4.17 (d, J=14.6 Hz, 1H), 3.78 (s, 3H), 3.65 (d, J=5.9 Hz, 1H), 2.73-2.68 (m, 1H), 2.53 (dddd, J=11.5, 6.0, 5.4, 2.9 Hz, 1H), 2.40-2.20 (m, 3H), 1.91-1.82 (m, 2H). 13C NMR (126 MHz, CDCl3): δ 207.5, 176.2, 159.4, 130.2, 127.6, 114.2, 65.5, 55.3, 53.4, 45.2, 39.9, 35.6, 34.6, 26.4. HRMS-ESI (m/z) [M+Na]+ calc'd for C15H17NNaO3: 282.1101, found: 282.1100.
(1S,5S)-4-(difluoromethylene)-6-(4-methoxybenzyl)-6-azabicyclo[3.2.1]octan-7-one (19). To a stirred solution of diethyl (difluoromethyl)phosphonate (128 mg, 0.678 mmol, 1.2 equiv) in THF (12 mL) at −100° C. was added 1-Butyllithium (1.7 M in pentanes, 0.38 mL, 0.649 mmol, 1.1 equiv). After 10 min, a solution of 18 (153 mg, 0.590 mmol, 1.0 equiv) in THF (2 mL) was added rapidly via syringe. The reaction mixture was stirred 2 h before being warm to r.t. The reaction was heated and refluxed for 16 h. The reaction was cooled to r.t. and quenched with aqueous NH4Cl solution. The solution was partitioned with diethyl ether, and the aqueous layer was separated and further extracted with Et2O (4×). The combined organic layer was concentrated in vacuo and purified by silica gel chromatography (50% EtOAc in hexane) to afford a colorless oil (19, 95 mg, 55%). 1H NMR (500 MHz, CDCl3): δ 7.18 (d, J=8.5 Hz, 2H), 6.86 (d, J=8.5 Hz, 2H), 4.76 (d, J=14.6 Hz, 1H), 4.10 (dd, J=3.8, 3.4 Hz, 1H), 3.80 (d, J=14.6 Hz, 1H), 3.80 (s, 3H), 2.65-2.60 (m, 1H), 2.38 (d, J=15.6, 6.6 Hz, 1H), 2.26-2.20 (m, 1H), 2.05-1.97 (m, 1H), 1.97-1.85 (m, 1H), 1.66-1.58 (m, 1H), 1.61 (d, J=11.3 Hz, 1H). 13C NMR (126 MHz, CDCl3): δ=176.5, 159.2, 151.1 (dd, J=287.2, 284.4 Hz), 129.7, 128.3, 114.0, 85.2 (dd, J=17.8, 17.8 Hz), 55.3, 53.2 (dd, J=4.5, 1.6 Hz), 43.8, 40.4, 36.6 (d, J=2.3 Hz), 25.0, 17.7. HRMS-ESI (m/z) [M+H]+ calc'd for C16H18F2NO2: 294.1300, found: 294.1295.
(1S,5S)-4-(difluoromethylene)-6-azabicyclo[3.2.1]octan-7-one (20). To a stirred solution of lactam 19 (68.0 mg, 0.232 mmol, 1.0 equiv) in acetonitrile (2.32 mL) was added a solution of cerium (IV) ammonium nitrate (318.0 mg, 0.580 mmol, 2.5 equiv) in water (0.23 mL) at 0° C. (the flask was wrapped with aluminum foil). The reaction was warmed to r.t. and stirred for 10 h. The solution was partitioned with EtOAc and the aqueous layer was further extracted with EtOAc (4×). The combined organic solution was concentrated in vacuo and purified by silica gel chromatography (40% EtOAc in hexane to 100% EtOAc) to afford a white solid (20, 32 mg, 80%). 1H NMR (500 MHz, CDCl3): δ 6.40-6.00 (brs, 1H), 4.39-4.35 (m, 1H), 2.53-2.47 (m, 1H), 2.48-2.32 (m, 2H), 2.30-2.18 (m, 1H), 2.00-1.90 (m, 1H), 1.70 (d, J=11.0 Hz, 1H), 1.68-1.56 (m, 1H). 13C NMR (126 MHz, CDCl3): δ 180.1, 150.1 (dd, J=286.3, 283.8 Hz), 87.0 (dd, J=18.0, 18.0 Hz), 50.2 (dd, 4.6, 1.4 Hz), 39.9, 38.8, 24.6, 17.7. HRMS-ESI (m/z) [M+H]+ calc'd for C8H10F2NO: 174.0725, found: 174.0724.
(1S,3S)-3-amino-4-(difluoromethylene)cyclohexane-1-carboxylic acid (12). A suspension of lactam 20 (31.0 mg, 0.179 mmol, 1.0 equiv) in aqueous HCl (4N, 5 mL) was heated to 75° C. and stirred for 5 h. The reaction mixture was concentrated to dryness in vacuo. The white solid was redissolved in a small quantity of water and the mixture was once again concentrated to dryness. The crude residue was concentrated and purified by C-18 chromatography (0-10% CH3CN in H2O) to give a white solid (20, 33.0 mg, 96%). 1H NMR (500 MHz, D2O): δ 4.16 (dd, J=5.5, 1.4 Hz, 1H), 2.78-2.70 (m, 1H), 2.30-2.20 (m, 2H), 2.12-1.94 (m, 2H), 1.90-1.80 (m, 2H); 13C NMR (126 MHz, D2O): δ=182.4, 151.8 (dd, J=286.0, 286.0 Hz), 85.5 (dd, J=23.1, 13.6 Hz), 44.3 (d, J=4.0 Hz), 41.2, 30.2, 27.4, 18.9; HRMS-ESI (m/z) [M+H]+ calc'd for C8H12F2NO2: 192.0831, found: 192.0830.
Ethyl (1R,3S)-3-((tert-butoxycarbonyl)amino)-4-(difluoromethylene)cyclohexane-1-carboxylate (22). To a stirred solution of 212 (2.9 g, 10.16 mmol, 1.0 equiv) and 2-PySO2CF2H (2.36 g, 12.20 mmol, 1.2 equiv) in dry DMF (20 mL) at −60° C. under Ar was added a solution of tBuOK (2.05 g, 18.29 mmol, 1.8 equiv) in dry DMF (20 mL) dropwise over 1 h. The solution was slowly warmed to −40° C. after the addition. A solution of NH4Cl (sat., 10 mL) was then added slowly, followed by the addition of HCl (3 M, 10 mL). The reaction was slowly warmed to r.t. and stirred overnight. The solution was diluted with EtOAc (150 mL). The organic phase was separated, washed with water (50 mL) and brine (50 mL), and dried with anhydrous Na2SO4. The solution was concentrated and purified by silica gel chromatography (30% EtOAc in hexane) to afford a yellow oil (22, 2.5 g, 77%). 1H NMR (500 MHz, CDCl3) δ 4.76 (brs, 1H), 4.54 (brs, 1H), 4.09 (q, J=7.1 Hz, 2H), 2.60 (brs, 1H), 2.40 (d, J=14.3 Hz, 1H), 2.14 (d, J=12.9 Hz, 1H), 2.07-1.90 (m, 2H), 1.64 (td, J=13.4, 3.9 Hz, 1H), 1.41 (s, 10H), 1.21 (t, J=7.1 Hz, 3H). HRMS-ESI (m/z) [M+Na]+ calc'd for C15H23F2NNa: 3452.1487, found: 342.1473.
(1R,5S)-4-(Difluoromethylene)-1-(phenylselanyl)-6-azabicyclo[3.2.1]octan-7-one (23). To a stirred solution of KHMDS (IM in THF, 2.76 mL, 2.76 mmol, 2.2 equiv) was slowly added dry THF (5 mL), and a solution of 22 (400 mg, 1.25 mmol, 1.0 equiv) in dry THF (5 mL) at −78° C. under Ar over 30 minutes. The solution was stirred at −78 ° C. for an additional 3 h, followed by the addition of a solution of PhSeCl (264 mg, 1.38 mmol, 1.1 equiv) in THF (5 mL). The solution was then slowly warmed to r.t. and stirred overnight. The reaction was quenched with the addition of sat. NH4Cl (10 mL). The solution was then diluted with EtOAc (100 mL) and the organic phase was separated. The aqueous phase was extracted with EtOAc (50 mL) twice. The combined organic phase was washed with brine (ca. 10 mL) and dried with anhydrous Na2SO4. The solution was concentrated and purified by silica gel chromatography (50% EtOAc in hexane) to give a colorless oil (23, 236 mg, 57%). 1H NMR (400 MHz, Chloroform-d) δ 7.66 (dd, J=8.1, 1.3 Hz, 2H), 7.39-7.35 (m, 1H), 7.30 (t, J=7.3 Hz, 2H), 6.45 (s, 1H), 4.30 (brs, 1H), 2.56-2.45 (m, 2H), 2.37-2.24 (m, 1H), 2.20-2.12 (m, 1H), 1.80 (dd, J=12.3, 7.0 Hz, 1H), 1.75 (d, J=9.9 Hz, 1H). HRMS-ESI (m/z) [M+Na]+ calc'd for C9H12F3N4OSe: 352.0023, found: 352.0018.
Ethyl (S)-3-((tert-butoxycarbonyl)amino)-4-(difluoromethylene)cyclohex-1-ene-1-carboxylate (24a) & Ethyl (S)-5-((tert-butoxycarbonyl)amino)-4-(difluoro methylene)cyclohex-1-ene-1-carboxylate (24b). To a stirred solution of 23 (150 mg, 0.866 mmol, 1.0 equiv) in DCM (10 mL) was added BoczO (283 mg, 1.3 mmol, 1.5 equiv), DIPEA (0.22 mL, 1.3 mmol, 1.5 equiv) and DMAP (11 mg, 0.086 mmol, 0.1 equiv) sequentially. The solution was stirred at r.t. overnight. The completion of reaction was determined by TLC (hexane: EtOAc=2:1). The solution was concentrated and diluted with EtOH (5 mL), followed by the addition of K2CO3 (360 mg, 2.6 mmol, 3.0 equiv). The suspension was stirred at r.t. for 3 h. The mixture was concentrated and diluted with DCM (20 mL) and HCl (0.5 M, 10 mL). The organic phase was separated and then washed with water (10 mL), NaHCO3 (aq. 10 mL), and brine (10 mL). The organic phase was dried over anhydrous Na2SO4. To this stirred solution was added m-CPBA (291 mg, 1.3 mmol, 1.5 equiv). The solution was stirred at r.t. for 3 h. The completion of reaction was determined by LC-MS. The reaction was quenched with sat. Na2S2O3 (aq. 10 mL). The organic phase was separated, washed with saturated NaHCO3 (aq. 10 mL) and brine (10 mL), and dried with anhydrous Na2SO4. The solution was concentrated and purified by silica gel chromatography (5-10% EtOAc in hexane) to afford a colorless oil (24a, 35 mg, 24%) 1H NMR (500 MHz, CDCl3) δ 6.86-6.82 (m, 1H), 4.98 (brs, 1H), 4.66 (brs, 1H), 4.21 (qd, J=7.1, 2.1 Hz, 2H), 2.61-2.49 (m, 2H), 2.18 (dddd, J=19.1, 10.3, 5.1, 2.3 Hz, 1H), 1.99 (ddtd, J=14.4, 11.8, 5.6, 3.3 Hz, 1H), 1.45 (s, 9H), 1.29 (t, J=7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 166.6, 154.6, 152.7 (t, J=286.9 Hz), 135.5, 133.4, 86.4, 80.3, 61.0, 42.9, 28.5, 24.4, 18.2, 14.3. HRMS-APCI (m/z) [M-H]− calc'd for C15H21F2NO4: 316.1366, found: 316.1361. and a colorless oil (24b, 32 mg, 22%). 1H NMR (500 MHz, CDCl3) δ 6.97 (brs, 1H), 4.70 (brs, 1H), 4.62 (brs, 1H), 4.20 (q, J=7.1 Hz, 2H), 3.02 (d, J=21.7 Hz, 1H), 2.90 (d, J=21.9 Hz, 1H), 2.65 (d, J=17.8 Hz, 1H), 2.49 (d, J=17.6 Hz, 1H), 1.43 (s, 9H), 1.29 (t, J=7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 166.5, 155.1, 151.6 (t, J=286.2 Hz), 136.2, 127.8, 84.2, 80.0, 61.0, 42.4, 30.8, 28.5, 21.9, 14.4. HRMS-APCI (m/z) [M−H]− calc'd for C15H20F2NO4: 316.1366, found: 316.1361.
General procedure A. To a solution of aq. HCl (4 M, 1.5 mL) and AcOH (1.5 mL) was added the ester intermediate under Ar. The solution was sealed and heated to 80° C. and stirred overnight. The completion of reaction was determined by LC-MS. The solution was concentrated and purified by C-18 chromatography (0-10% CH3CN in H2O) to give product.
(1R,3S)-3-Amino-4-(difluoromethylene)cyclohexane-1-carboxylic acid (13). 22 (200 mg, 0.626 mmol) was deprotected by procedure A to give a white solid (13, 87 mg, 61%). 1H NMR (500 MHz, CD3OD) δ 4.37 (brs, 1H), 2.75 (tt, J=12.3, 3.5 Hz, 1H), 2.59-2.51 (m, 1H), 2.27 (d, J=14.8 Hz, 1H), 2.24-2.14 (m, 2H), 1.91 (ddd, J=14.9, 13.1, 4.6 Hz, 1H), 1.50 (qd, J=13.3, 12.7, 4.4 Hz, 1H). 13C NMR (126 MHz, CD3OD) δ 175.6, 152.8 (t, J=287.9 Hz), 84.3 (dd, J=23.2, 15.4 Hz), 44.3 (dd, J=5.3, 1.2 Hz), 36.2, 31.1, 27.3, 18.6. HRMS-ESI (m/z) [M−H]− calc'd for C8H10F2NO2: 190.0685, found: 190.0684.
(S)-3-Amino-4-(difluoromethylene)cyclohex-1-ene-1-carboxylic acid (14). 24a (30 mg, 0.095 mmol) was deprotected by procedure A to give a white solid (14, 11 mg, 52%). 1H NMR (500 MHz, CD3OD) δ 6.89 (brs, 1H), 4.72 (brs, 1H), 2.71 (d, J=14.4 Hz, 1H), 2.65 (d, J=8.9 Hz, 1H), 2.32-2.17 (m, 2H). 13C NMR (126 MHz, CD3OD) δ 168.7, δ 155.0 (t, J=287.6 Hz), 139.2, 130.5, 84.7 (dd, J=24.6, 15.5 Hz), 44.2 (d, J=5.8 Hz), 25.0, 17.8. HRMS-ESI (m/z) [M−H]− calc'd for C8H8F2NO2: 188.0529, found: 188.0534.
(S)-5-Amino-4-(difluoromethylene)cyclohex-1-ene-1-carboxylic acid (15). 24b (25 mg, 0.095 mmol) was deprotected by procedure A to give a white solid (15, 15 mg, 67%). 1H NMR (500 MHz, CD3OD) δ 7.1 (brs, 1H), 4.5 (brs, 1H), 3.2 (d, J=22.3 Hz, 1H), 3.1-3.0 (m, 1H), 2.9 (d, J=18.7 Hz, 1H), 2.6 (ddq, J=18.4, 6.0, 3.1 Hz, 1H). 13C NMR (126 MHz, CD3OD) δ 168.8, 154.2 (t, J=288.4 Hz), 137.0 (d, J=1.8 Hz), 126.9, 82.7 (dd, J=23.1, 17.6 Hz), 44.1 (d, J=6.2 Hz), 28.9, 21.8. HRMS-ESI (m/z) [M−H]− calc'd for C8H8F2NO2: 188.0529, found: 188.0534.
hOAT and PYCR1 were expressed, grown, and purified according to literature procedures.3-4 GABA-AT was isolated from pig brains and purified according to a literature procedure.5 Coupled enzyme assays for GABA-AT, hOAT, Ala-AT, and Asp-AT were carried out according to previous procedures.6-7
The dialysis experiment was conducted using previous protocols.6, 8-9
The partition ratio was calculated using previous protocols.6, 8-9
The fluoride ion release assay was conducted using previous protocols.2 The final concentration of hOAT in the sample was determined to be 61.7±4.1 ug/mL (monomer, 1.34±0.12 μM) via BSA assay and calculation of dilution. A calibration curve of voltage (V, mV) was generated from varying concentrations of NaF (F, μM) to get the equation: F−=10{circumflex over ( )}((V−181.4)/−54.21). For accurate detection of fluoride ion concentration, 2.0 μM of fluoride ion was added to each control and sample. The number of fluoride ions released per active site was calculated by the ratio of the fluoride ion release concentration and hOAT concentration.
Co-crystallization of hOAT with 14 and 9: The freshly prepared enzyme was buffer exchanged into 50 mM Tricine pH 7.8 and concentrated to a protein concentration of 6 mg/mL. Previously reported crystallization condition8 was optimized using the hanging drop vapor diffusion method by varying PEG 6000 (8-12%), NaCl (100-250 mM), glycerol (0%-10%) with 100 mM Tricine pH 7.8 was kept constant as the buffer. For each hanging drop, 2 μl of protein solution was mixed with equal volume of well solution and 0.5 μl of 10 mM compound 181 or OV215. The crystals with the best morphology and size grew in a final condition containing 12% PEG 8000, 200 mM NaCl, 10% glycerol. Crystals were transferred to a cryo-protectant solution (well solution supplemented with 30% glycerol) before being flash-frozen in liquid nitrogen.
Crystal Soaking of hOAT with 14: The holoenzyme crystals were first grown via a hanging drop vapor diffusion method. Each drop contained 2 μL of protein and 2 μL of well solution. The best crystallization condition contained 8% PEG 6000, 100 mM NaCl, 5% glycerol, and 50 mM Tricine pH 7.8. Once the holoenzyme crystals reached their maximum size within five days, 1 μL of 10 mM 14 was added to the drop with crystals. Within the first three minutes of 14 addition, the hOAT crystals turned their color from yellow to transparent. The crystals were soaked for varying time periods from 17 to 44 minutes, transferred into cryoprotective solution (well solution supplemented with 30% glycerol), and then flash-frozen in liquid nitrogen.
X-ray Diffraction and Data Processing: Monochromatic X-ray diffraction data were collected at the LS-CAT beamline 21-ID-D at the Advanced Photon Source at Argonne National Laboratory. Data were collected at a wavelength of 1.127 Å and a temperature of 100 K using a Dectris Eiger 9M detector. Data sets were processed and analyzed with with Xia2 software.10-12
Model Building and Refinement: The hOAT structure was solved by molecular replacement using PHASER13 in Phenix. The starting search model was the previously published structure of hOAT14 (PDB code: 1OAT). The model building and refinement were accomplished in Coot15 and Phenix16, respectively, as an iterative process until the lowest possible Rfree/R factor values were attained. Structural depiction figures were prepared using UCSF Chimera17.
a Rmerge = Σ|Iobs − Iavg|/ΣIavg
bThe values for the highest-resolution bin are in parentheses
c Precision-indicating merging R
d Pearson correlation coefficient of two “half” data sets
eRwork = Σ|Fobs − Fcalc|/ΣFobs
fFive percent of the reflection data were selected at random as a test set, and only these data were used to calculate Rfree
g Root-mean square deviation
hOAT was analyzed into a Orbitrap Fusion Lumos or Orbitrap Eclipse (Thermo Fischer Scientific) mass spectrometer and full MS data was acquired as previously described.8 The liquid chromatography (LC) column heater compartment was set to 55° C. or was left at room temperature (25° C.) as indicated in the results section. hOAT was fragmented using front-end electron transfer dissociation (f-ETD)18 on an Orbitrap Fusion Lumos (Thermo Fischer Scientific) mass spectrometer. The same LC conditions described8 were used. The charge state +50 of modified or unmodified hOAT were quadrupole isolated using a 10 m/z window with a target AGC value of 1e6 charges and subjected to a fixed ETD reaction time of 10 ms with at targeted t-SIM-MS2 method. Given the limits instrument control software, the precursor charge was set to +24. ETD reagent target was set to 7e5 charges. Fragmentation data was collected from 350-2000 m/z in the Orbitrap at a resolution of 120,000 (at 200 m/z) with 4 microscans. Fragmentation spectra were summed across the elution peak of hOAT to increase the signal-to-noise of fragment ions. hOAT was also fragmented using higher energy collisional activation (HCD) on an Orbitrap Eclipse (Thermo Fischer Scientific) mass spectrometer. Protein was introduced to the mass spectrometer using LC and the same conditions previously described8 applying a source voltage of 1,700 V and an ion transfer tube temperature of 320° C. HCD data was collected in a data-dependent Top-N modality with cycle time of 2 s, full scan acquired from 600-1800 m/z with 10 microscans and an Orbitrap resolution of 7,500 (at 200 m/z) and a target AGC of 4e6 charges, and a default charge state of +15 using the advanced peak determination algorithm (APD). MS2 data was acquired with a target 5e5 charges and a 3 m/z isolation window with an HCD NCE of 28 and 1 microscan from 300-2000 m/z. Fragmentation data was deconvoluted to neutral mass values using the Xtract algorithm in the Freestyle 1.6 software package (Thermo Fisher) and analyzed using Prosight Lite software19 with a 10 ppm fragment mass tolerance and HCD or ETD as the fragmentation method depending on the method used to generate underlying data.
Treated and unmodified control OAT samples were desalted by dialysis against an Amicon 15 kDa molecular weight cutoff filter (Millipore) for 10 times at 4° C. Desalted samples were individually analyzed into a Q-Exactive Ultra High Mass Range (UHMR) (Thermo Fisher Scientific) mass spectrometer using a Nanospray Flex™ static source (Thermo Fisher Scientific) and medium-length borosilicate-coated emitters (Thermo Fisher Scientific). Spray voltages ranged between +1,500V to +2,000V and the ion transfer tube was set to 310° C. The mass spectrometer was acquired in positive ESI mode, with full scan data collected from 4,000-10,000 m/z with 5-20 microscans and an Orbitrap resolution of 35,000 or 17,500 (at 200 m/z). Target AGC of 1e6 charges and maximum injection time ranging from 50-400 ms. The S-lens RF level was set to 200% and extended trapping set to 100. Raw spectra from each experiment were summed across scans and m/z deconvolution was performed using mMass20 and UniDec21 to generate zero-charge masses and associated mass standard deviations.
Metabolomics assays were preformed and data was acquired as previously described.8 MS1 and MS2 spectra was manually interpreted to identify hOAT metabolite turnover products.
Docking models of ligands bound to GABA-AT or OAT were developed using the Molecular Operating Environment (MOE) computational suite's Builder utility.22-24 The energy minimization of ligands was conducted in the gas phase using the force field MMFF94X. The X-ray crystal structures of inactivated hOAT (salt bridge maintained, PDB: 1GBN) and inactivated hOAT (salt bridge disrupted, PDB: 7JX9) were uploaded to MOE respectively, followed by the receptor preparation. The tight-binding products in the active pockets were deleted, and catalytic Lys292 was neutralized. The docking sites were specified at the catalytic Lys292 atoms. Ligand dockings were carried out in the prepared aminotransferase enzyme models with unrelated substrates and the solvent atoms inactivated. Ligand placement employed the Alpha Triangle method with Affinity dG scoring generating 300 data points that were further refined using the induced fit method with GBVI/WSA dG scoring to obtain the top 50 docking results. The docking results of each ligand were analyzed for selection of the best docking pose, based on the score and reported X-ray structures.
A docking protocol was employed to generate the initial OAT complex corresponding to 14a and 14b for the classical MD simulation. The protein structure chosen for this study was the X-ray crystal structure of xxx (PDB ID: 1OHW) with a resolution of yyy Å. The protonationstates of the amino acid residues were determined using the H++ server. The structures of 14a and 14b were optimized using Gausian09 software at a HF/6-31+(G) level of theory.15 The inhibitor and protein structures were further refined using AutoDockTools-1.5.6 available with the MGLTools software package.25 Refined inhibitor structures were docked into the OAT active site using Autodock 4.2 software. A gridbox centered on the active site was generated using Autogrid 4.2 software with a grid spacing of 0.375 Å and dimensions of 55×55×55 points along the x, y, and z axes. Lamarckian genetic algorithm (GA) was used for the conformational search with a GA populationsize of 150 and a maximum number of evaluations of 2,500,000. The 100 generated poses were clustered according to their rmsd values (1.5 Å cutoff), and the lowest energy conformation of the best cluster was selected for the MD simulation. The electrostaticpotential energies (ESP) of the geometrically optimized inhibitors (HF/6-31+G) were calculated at the HF/6-31+G level of theory. Then these energies were used to derive the partial atomic charges of the inhibitors using the electrostatic potential square fit (ESP) method employed in the antechamber module available in the Amber12 program.26 This is the method used to derive the partialatomic charges of the original Amber forcefields. The inhibitor atoms were treated with general Amber force field (GAFF), and the automatically assigned GAFF atom types were manually adjusted to accurately represent their chemical environment before deriving the parameters. The parameters and topology files were generated using tLEaP and antechamber modules of the Amber12 program. Then these parameters and topology files were converted to GROMACS compatible format using ACPYPE.27 The forcefield parameters and the topology for covalently bound PLP were derived similarly. The modified Amberff99SB-ILDN forcefield with parameters for covalently bound PLP were used to simulate the protein. Parameters for the OAT dimer were derived using the PDB2gmx module available in the GROMACS 5.1.2 software package. All of the MD simulations were performed using GROMACS 5.1.2 software.28 The TIP3P model was used as the water model. The protein-inhibitor complex was immersed in a dodecahedron box filled with TIP3P water where the boundaries are extending at least 1.8 nm in all directions from the edges of the protein. Then, NaCl was added to the system up to a concentration of 0.15 M (physiological NaCl concentration) to neutralize the charge of the system. The solvated system was energy minimized with the steepest descent followed by the conjugate gradient method until it converged with a maximum force no greater than 500 KJ mol−1 nm−1.
The resulting energy minimized periodic system was the starting configuration for the MD simulation, which was carred out with the aid of the Extreme Science and Engineering Discovery Environment (XSEDE).29 Prior to the production MD simulation, the system was subjected to equilibration in two steps. First, it was equilibrated at constant NVT (number of particles, volume, and temperature) ensemble for 1 ns. Then the resulting system was equilibrated at constant NPT(number of particles, pressure, and temperature) ensemble for 3 ns. Temperature and pressure were controlled at 310 K and 1 bar by the V-rescale thermostat (time constant of 0.4 ps) and the Parrinello-Rahmanbarostat (time constant of 2 ps), respectively. In both of the equilibration steps positions of the heavy atoms were restrained by applying a force constant of 1000 KJ mol−1 nm−2. After that, the position restraints were gradually reduced from 500 to 100 KJ mol−1 nm−2 over two runs (1 ns each). Finally, a production MD simulation of the equilibrated system was carried out under the NPT condition for 15 ns (time step of 2 fs) without positional restraints. During the NPTequilibration, the V-rescale thermostat was replaced by the most accurate Nose-Hoover thermostat and used for the rest of the simulation protocol. The long-range electrostatic interactions were treated with the particle mesh Ewald (PME) method, while Coulomb and van der Waals interactions were cut off at 1.2 nm. Bond lengths of the atoms were restrained using the Linear Constraint Solver (LINCS) algorithm.
Important distance and dihedral angle measurements were taken using distance, mindist and angle tools available in the GROMACS software package.
aC0/Cmax
In accordance with this disclosure, various other compounds, varied structurally, stereochemically and/or configurationally, are available through such incorporated synthetic procedures and techniques or straight-forward modifications thereof, such modifications as would also be known and understood by those skilled in the art and made aware of this invention, such procedures, techniques and modifications limited only by the commercial or synthetic availability of any corresponding reagent or starting material.
This application claims benefit of priority to U.S. Patent Application No. 63/201,008, filed Apr. 8, 2021, the contents of which are incorporated by reference in its entirety.
This invention was made with government support under DA030604 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/024041 | 4/8/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63201008 | Apr 2021 | US |
