3-amino-4,4-dihalocyclopenta-1-enecarboxylic acid as a selective inactivator of human ornithine aminotransferase

Abstract

Amino, fluoro-substituted cyclopentene carboxylic acid compounds are disclosed. The disclosed compounds and compositions thereof can be used in methods of modulating human ornithine delta-aminotransferase (hOAT) activity, including methods for treating diseases or disorders associated with hOAT activity or manifestations such as cell proliferative diseases and disorders.

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims priority to U.S. Patent Application No. 63 / 156,147, filed on 3 March 2021, which is incorporated by reference in its entirety.

[0002] Statement on federally funded research or development This invention was made with government support under DA030604, awarded by the Ministry of Health and Human Services, the National Institutes of Health, and the National Institute on Drug Abuse. The government has certain rights with respect to this invention. [Background technology]

[0003] Ornithine δ-aminotransferase (hOAT; EC2.6.1.13) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes two conjugated amino group transfer reactions. It converts L-ornithine (L-Orn) to produce L-glutamic acid-γ-semialdehyde (L-GSA), and in the second reaction, it produces L-glutamic acid (L-Glu) from α-ketoglutarate (α-KG). 1 The resulting intermediate L-GSA is a spontaneous equilibrium species of Δ1-pyrroline-5-carboxylic acid (P5C), and P5C can be catalyzed by P5C reductase (PYCR) to yield L-proline. 2 Meanwhile, the generated L-Glu is also converted to P5C by pyrroline-5-carboxylate synthase (P5CS) and is involved in proline metabolism. Proline biosynthesis has been identified as the most substantially altered amino acid metabolism in human tumor tissue of hepatocellular carcinoma (HCC), characterized by accelerated proline consumption, hydroxyproline accumulation, increased α-fetoprotein (AFP) levels, and correlation with poor prognosis of HCC. 3 Furthermore, glutamine synthetase (GS) catalyzes the conversion of L-Glu to L-glutamine (L-Gln). 4 L-Gln is highly required by cancer cells to support the anabolic process and promotes cell proliferation.

[0004] HCC is the main liver malignancy and one of the most common causes of cancer-related deaths worldwide. In previous studies, the OAT gene was identified as one of seven overexpressed genes in spontaneously occurring HCC-developed livers from Psammomys obesus (sand rat) identified by DNA microarray analysis 5 . Furthermore, treatment with the selective hOAT mechanism-based inactivator (MBI) BCF3 (1) (0.1 and 1.0 mg / kg; PO) significantly decreased serum AFP levels and inhibited tumor growth in an HCC mouse model 5 and emphasized the antitumor effect of pharmacological selective hOAT inhibition. MBI is a type of molecule that initially acts as an alternative substrate for the target enzyme and is then converted into a reactive species that can further inactivate the enzyme via specific covalent modification, strong binding electrostatic interaction, or other functionally irreversible inhibition 6-7 . MBI typically does not react prior to the initial binding to the active site of the target enzyme, thereby usually exhibiting significant target specificity and selectivity 8 . Overall, hOAT is considered a potential therapeutic target for HCC, and selectively inactivating hOAT may offer new opportunities for discovering effective HCC treatments.

[0005] However, the main challenge in discovering selective MBI for hOAT is to overcome irreversible inhibition against other aminotransferases 6 , especially γ-aminobutyric acid aminotransferase (GABA-AT), which has a high structural similarity to hOAT 1 . There are only two significant differences in the active site pocket of the homodimeric structure: Tyr85 and Tyr55 in hOAT are replaced by Ile72 and Phe351 in GABA-AT, respectively * . 1 . Furthermore, Ile72 and Phe351 *This is related to the fact that GABA-AT has a slightly narrower active site but is more hydrophobic compared to hOAT. In contrast, the hydroxyl group of Tyr55 acts as a hydrogen bond acceptor that interacts with the charged C-2 amino group of the substrate, while Tyr85 is a key determinant of substrate specificity and is conformally flexible to accommodate bulky substrates. 1 .

[0006] Due to the high similarity between these two aminotransferases, preliminary screening for hOAT was previously performed using stock GABA-AT inhibitors. 5 A cyclopentane analog called BCF3(1), which has a bis(trifluoromethyl) group as a warhead, was identified as a selective MBI of hOAT, but only showed reversible mmol inhibition of GABA-AT. Recent mechanistic studies have revealed that one of its trifluoromethyl groups causes fluoride ion elimination, and the ligand covalently modifies the catalyst Lys292 through conjugate addition. 9-10 The sterically bulky bis(trifluoromethyl) group does not easily access the relatively narrow pocket of GABA-AT, affecting the initial binding pose between the ligand and the enzyme, which may be the cause of the reversible inhibition of GABA-AT. BCF3 has been demonstrated to be effective in vivo as described above. 5 It has been investigated in extensive IND-labeled toxicity evaluations and efficacy studies in xenotransplant (PDX) models derived from HCC patients. Based on a similar strategy, the ring system was expanded and tested, leading to the discovery of a cyclohexene-based analog WZ-2-051(2) containing a difluoro group. 11 WZ-2-051 has a lower inactivation efficiency (k) for hOAT compared to BCF3. inact / K I The ratio (defined by the ratio) improved 23-fold, but it showed 13.3 times higher selectivity than GABA-AT. Subsequent mechanistic studies revealed that WZ-2-051 undergoes two-step fluoride ion elimination, ultimately inactivating hOAT via an addition-aromatization mechanism. 11A further example of a selective hOAT inactivator is 5-FMOrn(3), which is stimulated by the structure of the hOAT substrate L-Orn and the structure of the non-selective GABA-AT inactivator AFPA. 12 , by forming a ternary adduct, it inactivates hOAT via the enamine pathway. 13 .

[0007] It should be noted that the α-amino group of 5-FMOrn forms a strong hydrogen bond with the phenol group of Tyr55 in the hOAT crystal complex (PDB-invaded 2OAT). 14 Furthermore, hydrogen bonding between the carboxylate groups of BCF3 (PDB-entering 6OIA) and WZ-2-051 (PDB-entering 6V8C) and Tyr55 was observed in their hOAT crystal complexes. 9,11 This demonstrated that the interaction with Tyr55 plays an important role in ligand specificity and selectivity. However, among the published hOAT inactivators, there is a strong irreversible inhibition of hOAT and a weak reversible inhibition of GABA-AT (K i It does not show inhibition of aspartate aminotransferase (Asp-AT) and alanine aminotransferase (Ala-AT) up to 4 mM, resulting in promising hOAT selectivity. 5 Therefore, discovering novel selective MBIs for hOAT would facilitate research into hOAT inactivators as a potential therapeutic approach for HCC.

[0008] In 2000, (1R,4S)-4-amino-3,3-difluorocyclopentanecarboxylic acid (4) was found to be a reversible inhibitor of GABA-AT (K i (=0.19mM) 15 Fifteen years later, it was further proven to be an hOAT inactivator. However, compound 4 showed high binding affinity to hOAT (KI = 7.8 mM) and a low maximum inactivation rate (k) for hOAT. inact =0.02 minutes -1 ) shows a moderate inactivation efficiency (k inact / K I =0.003 minutes-1 mM -1 This leads to inactivation and there are limitations in elucidating the turnover mechanisms.

[0009] This technology provides novel cyclopentene analogs, including SS-1-148(6), by incorporating an additional double bond into a cyclopentane ring system of 4, which has been demonstrated to be a highly potent and selective hOAT inactivator. Mechanistic studies using crystallization, protein and molecular mass spectrometry, transient state measurements, and computational simulations revealed a novel non-covalent inactivation mechanism of SS-1-148. [Overview of the Initiative]

[0010] Disclosed are compounds, compositions, and related methods of use for the selective inhibition of human ornithine δ-aminotransferase (hOAT). The disclosed compounds, compositions, and methods can be used to treat diseases and disorders associated with human ornithine δ-aminotransferase (hOAT).

[0011] The disclosed compounds may be described as substituted cyclopentene compounds. In particular, the disclosed compounds may be described as amino, halosubstituted cyclopentenecarboxylic acid compounds. The disclosed compounds and their compositions may be used in methods to modulate human ornithine δ-aminotransferase (hOAT) activity, including methods for treating diseases or disorders related to the expression of hOAT activity or cell proliferation disorders and disorders.

[0012] The disclosed compounds can be directed to compounds of the following formula, or to dissociated forms, aprotonated forms, zwitterionic forms, or salts thereof.

[0013] [ka]

[0014] In the formula, a double bond exists between the α-carbon and the ε-carbon, or between the α-carbon and the β-carbon.

[0015] [ka]

[0016] In the formula, R 1 and R 2 Each of these is independently selected from halogens such as F, Cl, Br, and I.

[0017] As shown, the disclosed compounds can be protonated to form, for example, an ammonium moiety, and the compound may exist as a salt. The disclosed compounds may also be deprotonated and / or dissociated, for example, a carboxylic acid moiety dissociates from the carboxylic acid moiety, and the compound may exist as a salt. The disclosed compounds may also be in a zwitterionic form in which the compound comprises a protonated ammonium moiety and a dissociated carboxylate moiety, and the compound may exist as a salt.

[0018] The disclosed compounds and compositions can be used in methods for modulating human ornithine δ-aminotransferase (hOAT) activity. Such methods may include providing the compounds disclosed herein, such as compounds of the following formulas, or their dissociated forms, zwitterionic forms, or salts thereof, and contacting hOAT with the compounds.

[0019] [ka]

[0020] In the formula, a double bond exists between the α-carbon and the ε-carbon, or between the α-carbon and the β-carbon, R 1 and R 2 Each of these is independently selected from halogens such as F, Cl, Br, and I.

[0021] In some embodiments, the disclosed methods may be aimed at reducing the activity of hOAT expressed by cancer, including, but not limited to, hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC), or other cancers that express or overexpress hOAT. Such methods may include providing the compounds disclosed herein, such as compounds of the following formulas, or dissociated, aprotonated, zwitterionic, or salts thereof, and contacting cancer with the compound.

[0022] [ka]

[0023] In the formula, a double bond exists between the α-carbon and the ε-carbon, or between the α-carbon and the β-carbon, R 1 and R 2 Each of these is independently selected from halogens such as F, Cl, Br, and I.

[0024] In one embodiment, the disclosed method may be directed towards treating a cytoproliferative disorder or impairment in a subject requiring it. Appropriate cytoproliferative disorders and impairments may include, but are not limited to, cancers that express or overexpress hOAT, such as hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC). Such a method may include administering a compound of the following formula, or its dissociated form, unprotonated form, zwitterionic form, or salt thereof, to a subject requiring it.

[0025] [ka]

[0026] In the formula, a double bond exists between the α-carbon and the ε-carbon, or between the α-carbon and the β-carbon, R 1 and R 2 Each of these is independently selected from halogens such as F, Cl, Br, and I.

[0027] The compounds disclosed herein are without stereochemical or constitutive limitations and, unless otherwise indicated, encompass all stereochemical or constitutive isomers. As illustrated and described below, such compounds and / or their intermediates are available as single enantiomers, racemic mixtures in which isomers can be separated, or diastereomers in which the corresponding enantiomers can be separated. Thus, any stereocenter can be (S) or (R) with respect to any other stereocenter. As another consideration, various compounds can be partially or completely protonated, for example at an amino group, to form an ammonium moiety, and / or partially or completely dissociated, for example at a carboxyl group, to form a carboxylate substituent or moiety. In certain such embodiments, with respect to the ammonium substituent or moiety, the counterion may be a conjugate base of a protic acid. In certain such or other embodiments, with respect to the carboxylate substituent or moiety, the counterion may be an alkali, alkaline earth, or ammonium cation. Furthermore, it will be understood by those skilled in the art that any one or more of 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 therapeutic method or agent.

[0028] In one embodiment, the disclosed method relates to diseases or disorders associated with hOAT activity and / or overexpression (including proliferative disorders and disorders such as cancers associated with hOAT activity and / or expression or overexpression). Appropriate diseases and disorders include, but are not limited to, proliferative disorders and disorders, including, but not limited to, hepatocellular carcinoma and non-small cell lung cancer in human subjects requiring such treatment. In one embodiment, such compounds can be provided as part of a pharmaceutical composition.

[0029] In one embodiment, the disclosed method aims to reduce or modulate the activity of human ornithine δ-aminotransferase expressed by cancer (e.g., hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC)). Such a method may include the steps of providing a compound of the type described above or elsewhere in this specification, and contacting a cell medium containing a cancer expressing human ornithine δ-aminotransferase with such a compound in an amount effective to reduce human ornithine δ-aminotransferase activity. In one embodiment, such a compound may be provided as part of a pharmaceutical composition. In any case, such contact may be in vitro or in vivo.

[0030] More generally, the disclosed methods may be aimed at inhibiting or inactivating human ornithine δ-aminotransferase. Such methods may include the steps of providing a compound of the kinds described above or below, whether or not it is part of a pharmaceutical composition, and administering an effective amount of such a compound for contact with human ornithine δ-aminotransferase. Such contact may be designed for experimental and / or research purposes, or to simulate one or more in vivo or physiological conditions, as understood in the art. Such compounds include, but are not limited to, those illustrated by the following examples, reference figures, references, and / or accompanying synthesis schemes. In certain such embodiments, such compounds and / or combinations thereof may be present in amounts at least partially sufficient to inhibit hOAT, cell proliferation, and / or tumor growth. [Brief explanation of the drawing]

[0031] [Figure 1] hOAT and related metabolic pathways (A); Structures of inactivators BCF3 (1), WZ-2-051 (2), and 5-FMOrn (3) (B) based on the hOAT mechanism. [Figure 2]Comparison of the active sites of hOAT (A; PDB entry 1 OAT) and GABA-AT (B; PDB entry 1 OHV). C) Structure of hOAT inactivators 4-6. [Figure 3A] The main metabolite of SS-1-148 in hOAT(A) and GABA-AT(B). [Figure 3B] The main metabolite of SS-1-148 in hOAT(A) and GABA-AT(B). [Figure 4] Crystal structures of hOAT obtained from immersion experiments (A, PDB entry 7LK1) and co-crystallization (B, PDB entry 7LK0) using compound SS-1-148. The immersion structure of SS-1-148 is shown in two conformations. One is a structure in which the carboxylate group interacts with Tyr55 (Conformation A), and the other is a structure in which the carboxylate forms a salt bridge with Arg413 (Conformation B). For this particular chain, the conformational occupancy is 0.51 (Conformation A) and 0.49 (Conformation B). hOAT residues and SS-1-148 are shown as rods. The H-bond distances between hexagonal atoms are shown by dashed lines. [Figure 5A] Denaturation, intact (A) and natural (B) protein mass spectrometry of hOAT inactivated with SS-1-148. [Figure 5B-1] Denaturation, intact (A) and natural (B) protein mass spectrometry of hOAT inactivated with SS-1-148. [Figure 5B-2] Denaturation, intact (A) and natural (B) protein mass spectrometry of hOAT inactivated with SS-1-148. [Figure 6]Transient absorption changes observed at 275, 420, and 560 nm of hOAT reacting with SS-1-148. OAT (12.7 μM final) was mixed with SS-1-148 (126, 251, 502, 1004, 2008, 4016 μM), and CCD spectra were collected over a time frame of 0.009 to 49.2 seconds. (A) The data observed at 420 nm fits a linear combination of three exponential terms according to equation (3) described in the supporting information. The arrows indicate the trend of amplitude observed as the inhibitor concentration increases. (B) The observed rate constant dependence of the first phase observed at 420 nm fits equation (5) described in the supporting information. The values ​​of k2 and k3 are the average values ​​obtained from the fit in Figure 5A. The fit is indicated by a red dash. (C) Data observed at 560 nm. The curved arrows indicate the observed amplitude trend as the inhibitor concentration increases. These data were fitted to a linear combination of two (1004, 2008, and 4016 μM) or three (126, 251, and 502 μM) exponential terms according to equation (4) in the supporting information. The fit is indicated by a red dash. (D) Observations at 275 nm over 2000 seconds obtained in the presence of 8032 μM SS-1-148 were fitted to a linear combination of two exponential terms according to equation (4) in the supporting information. The fit is indicated by a red dash. [Figure 7A-B] Partial resolution by singular value decomposition (SVD) of transient absorption changes observed in hOAT reacting with SS-1-148. hOAT (6.94 μM; final concentration) was reacted at 10°C in a stopped-flow spectrophotometer containing SS-1-148 (1040 μM; final concentration). A composite CCD absorbance dataset spanning 250–800 nm and 0.0137–9843 seconds was prepared by splicing averaged short- and long-time frame datasets together to obtain sufficient temporal resolution to analyze velocities over a four-order-of-magnitude range. These data were fitted to a linear irreversible four-stage model constrained by rate constants determined from single-wavelength analysis (Figure 6). Deconvolution composite spectrum derived from SVD analysis (A). Seed concentration profile based on rate constants used to fit dataset (B). [Figure 7C]Three-dimensional view of a subset of spectra from the analyzed dataset (C). [Figure 8] Theoretical pKa calculation of hydrogen at the Cγ position using the DFT / B3LYP method (A), and electron density maps encoded with the electrostatic potential of the intermediate and the ESP charges at the Cδ and C4' positions (B). [Figure 9] Titration of hOAT using SS-1-148. The loss of enzyme activity was measured as a function of the ratio of inactivation to enzyme concentration. Linear regression was used on the linear portion of the curve to obtain the X-intercept, which is the number of turns (distribution ratio = number of turns - 1). [Figure 10] Time-dependent dialysis of hOAT was partially or completely inhibited by SS-1-148 at various concentrations. [Figure 11] Porter map of SS-1-148 with the intermediate immersed at the 4σ level. [Figure 12] Porter map of SS-1-148 cocrystal at the 4σ level. [Figure 13] Natural, unmodified hOAT was examined using native MS. Left: Untreated hOAT; Right: Untreated hOAT with applied collisional dissociation energy (NCE:15). [Figure 14-1] Gibb's free energy for M7 and related tautomers. [Figure 14-2] Gibb's free energy for M7 and related tautomers. [Figure 15-1] Major metabolites of 4-6 in hOAT. (A) Primary metabolite of 4 in hOAT. (B) Primary metabolite of 5 in hOAT. Primary metabolite of SS-1-148(6) in hOAT (C). [Figure 15-2] Major metabolites 4-6 in hOAT. [Figure 15-3] Major metabolites 4-6 in hOAT. [Figure 15-4] Major metabolites 4-6 in hOAT. [Modes for carrying out the invention]

[0032] The disclosed subject matter may be further described using the following definitions and terms. The definitions and terms used herein are for the purpose of describing only specific embodiments and are not intended to limit them.

[0033] As used herein and in the claims, the singular forms “a,” “an,” and “the” include the plural form unless the context explicitly indicates otherwise. For example, the term “substituent” should be interpreted as “one or more substituents” unless the context explicitly indicates otherwise.

[0034] As used herein, the terms “about,” “about,” “substantial,” and “significant” will be understood by those skilled in the art and will vary to some extent depending on the context in which they are used. Given the context in which the terms are used, if there is any use of a term that is not clear to those skilled in the art, “about” and “about” mean up to ±10% of a particular term, and “substantial” and “significantly” mean more than ±10% of a particular term.

[0035] 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 “open” transitional terms that allow for the inclusion of additional components beyond those described in the claims. The terms “consist” and “consisting of” should be interpreted as “closed” transitional terms that do not allow for the inclusion of additional components other than those described in the claims. The term “consisting essentially of” should be interpreted as partially closed and may include only additional components that do not fundamentally alter the nature of the claimed subject matter.

[0036] The phrase “such things” should be interpreted as “for example, including,” and furthermore, any use of any illustrative language including but not limited to such things is intended merely to better illuminate the claimed subject matter and not to limit the scope of the claimed subject matter.

[0037] Furthermore, where conventions similar to “at least one of A, B, C, etc.” are used, such structures are generally intended to be understood by those skilled in the art (for example, “A system having at least one of A, B, C includes, but is not limited to, systems having only A, only B, only C, between A and B, between A and C, between B and C, and / or between A, B and C”). It will be further understood by those skilled in the art that substantially any separator and / or phrase presenting two or more alternative terms in either the specification or the drawings should be understood to take into account the possibility of including either one or both of the terms, or one of either of the terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B”.

[0038] All terms such as "maximum," "minimum," "more than," and "less than" refer to a range that includes the listed number and can then be broken down into ranges and subranges. A range includes its individual members. For example, a group with 1 to 3 members refers to a group with 1, 2, or 3 members. Similarly, a group with 6 members refers to a group with 1, 2, 3, 4, or 6 members, and so on.

[0039] A modal verb refers to a preferred use or selection of one or more options or choices of a particular embodiment or feature contained within the same. If no options or choices relating to a particular embodiment or feature contained within the same are disclosed, a modal verb may refer to an affirmative action relating to the method or use and aspects of a particular embodiment or feature contained within the same, or a definitive decision to use a particular skill relating to a particular embodiment or feature contained within the same. In the latter context, the modal auxiliary verb "may" has the same meaning and implications as the auxiliary verb "can".

[0040] As used herein, “subjects requiring it” may include humans and / or non-human animals. “Subjects requiring it” may include subjects having diseases or disorders related to human ornithine δ-aminotransferase (hOAT) activity. “Subjects requiring it” may include, but are not limited to, subjects having cell proliferation disorders or disorders, including hepatocellular carcinoma (HCC).

[0041] chemicals Novel chemical substances and their uses are disclosed herein. Chemical substances may be described using terms known in the art, which are further described below.

[0042] When used in this specification, a dash "-" or an asterisk " * You can use the '' or plus sign "+" to specify a bond point to any radical group or substituent.

[0043] As intended herein, the term "alkyl" includes all its isomers, for example, linear or branched alkyl radicals in linear or branched groups of 1 to 12, 1 to 10, or 1 to 6 carbon atoms, referred herein to as C1-C12 alkyl, C1-C10-alkyl, and C1-C6-alkyl, respectively.

[0044] The term "alkylene" refers to a diradical of a linear or branched alkyl group (i.e., a diradical of a linear or branched C1-C6 alkyl group). Exemplary alkylene groups include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)CH2-, and -CH(CH2CH3)CH2CH3.

[0045] The term "halo" refers to halogen substitution (e.g., -F, -Cl, -Br, or -I). The term "haloalkyl" refers to an alkyl group substituted with at least one halogen. Examples include -CH2F, -CHF2, -CF3, -CH2CF3, and -CF2CF3.

[0046] As used herein, the term “heteroalkyl” refers to an “alkyl” group in which at least one carbon atom is substituted with a heteroatom (e.g., an O, N, or S atom). One type of heteroalkyl group is an “alkoxy” group.

[0047] As used herein, the term "alkenyl" refers to an unsaturated linear or branched hydrocarbon having at least one carbon-carbon double bond, such as a linear or branched group of 2 to 12, 2 to 10, or 2 to 6 carbon atoms, which are referred to herein as C2-C12-alkenyl, C2-C10-alkenyl, and C2-C6-alkenyl, respectively.

[0048] The term "alkynyl" in this specification refers to unsaturated linear or branched hydrocarbons having at least one carbon-carbon triple bond, such as linear or branched groups of 2 to 12, 2 to 10, or 2 to 6 carbon atoms, respectively, referred to as C2-C12-alkynyl, C2-C10-alkynyl, and C2-C6-alkynyl.

[0049] The term "cycloalkyl" refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group derived from cycloalkanes, referred herein as "C4-8-cycloalkyl." Unless otherwise specified, cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amide or carboxyamide, amino, arylalkyl, arylalkyl, azide, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, formyl, ether, haloalkyl, heteroaryl, heteroaryl, hydroxyl, imino, ketone, nitro, phosphate, phosphonate, phosphine, sulfate, sulfonamide, sulfonyl, or thiocarbonyl. In some embodiments, the cycloalkyl group is unsubstituted, i.e., unsubstituted.

[0050] The term "0055" refers to a monovalent saturated cyclic, bicyclic, or bridging cyclic hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbon atoms in which at least one carbon of the cycloalkane is substituted with a heteroatom such as N, O, and / or S.

[0051] The term "cycloalkylene" refers to a cycloalkyl group that is unsaturated in one or more ring bonds.

[0052] The term “partially unsaturated carbocyclyl” refers to a monovalent cyclic hydrocarbon in which at least one carbocyclyl ring contains at least one double bond between non-aromatic ring atoms. Partially unsaturated carbocyclyls can be characterized according to the number of carbon atoms. For example, a partially unsaturated carbocyclyl may contain 5-14, 5-12, 5-8, or 5-6 ring carbon atoms and is therefore called a 5-14, 5-12, 5-8, or 5-6 membered partially unsaturated carbocyclyl, respectively. Partially unsaturated carbocyclyls may be in the form of monocyclic, bicyclic, tricyclic, bridged, spirocyclic, or other carbocyclic systems. Exemplary partially unsaturated carbocyclyl groups include cycloalkenyl groups and partially unsaturated bicyclic carbocyclyl groups. Unless otherwise specified, the partially unsaturated carbocyclyl group may be substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amide or carboxyamide, amino, arylalkyl, arylalkyl, azide, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, formyl, ether, halogen, heteroaryl, heteroaryl, hydroxyl, imino, ketone, nitro, phosphate, phosphonate, phosphine, sulfate, sulfonamide, sulfonyl, or thiocarbonyl. In some embodiments, the partially unsaturated carbocyclyl is unsubstituted, i.e., unsubstituted.

[0053] The term "aryl" is technically recognized and refers to carbocyclic and / or heterocyclic aromatic groups. Representative aryl groups include phenyl, naphthyl, anthracenyl, pyridinyl, quinolinyl, furanyl, and thionyl. The term "aryl" includes polycyclic ring systems having two or more carbocyclic rings, where two or more carbons are common to two adjacent rings (the rings are "fused rings"), where at least one of the rings is aromatic, and the other ring may be, for example, a cycloalkyl, cycloalkenyl, cycloalkynyl, and / or aryl. Unless otherwise specified, the aromatic ring may be substituted at one or more ring positions with, for example, halogens, azides, alkyls, aralkyls, alkenyls, alkynyls, cycloalkyls, hydroxyls, alkoxyls, aminos, nitros, sulfhydryls, iminos, amides or carboxyamides, carboxylic acids, -C(O) alkyls, -CO2 alkyls, carbonyls, carboxyls, alkylthios, sulfonyls, sulfonyls, sulfonamides, ketones, aldehydes, esters, heterocyclyls, aryl or heteroaryl moieties, -CF3, -CN, etc. In some embodiments, the aromatic ring is substituted at one or more ring positions with halogens, alkyls, hydroxyls, or alkoxyls. In certain other embodiments, the aromatic ring is unsubstituted, i.e., unsubstituted. In some embodiments, the aryl group has a 6- to 10-membered ring structure.

[0054] The terms "heterocyclyl" and "heterocyclic group" are technically recognized and refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, or 3- to 7-membered rings, which contain 1 to 4 heteroatoms such as nitrogen, oxygen, and sulfur. The number of ring atoms in a heterocyclyl group can be specified using the 5Cx~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 1 to 4 heteroatoms such as nitrogen, oxygen, and sulfur. The designation "C3~C7" indicates that the heterocycle contains 3 to 7 ring atoms, including the heteroatoms occupying the ring atom positions.

[0055] The terms "amine" and "amino" are technically recognized and refer to both unsubstituted and substituted amines (e.g., monosubstituted amines or disubstituted amines), where substituents may include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.

[0056] The terms "alkoxy" or "alkoxyl" are technically recognized and refer to alkyl groups as defined above, to which an oxygen radical is bonded. Typical alkoxy groups include methoxy, ethoxy, and tert-butoxy.

[0057] An ether is a hydrocarbon composed of two hydrocarbons covalently bonded by oxygen. Therefore, the alkyl substituents that alkylate an ether are alkoxyls or alkyl-like substituents, represented by -O-alkyl, -O-alkenyl, -O-alkynyl, etc.

[0058] As used herein, the term "carbonyl" refers to the radical -C(O)-.

[0059] The term "oxo" refers to a divalent oxygen atom, -O-.

[0060] As used herein, the terms "carboxy" or "carboxyl" refer to the radical -COOH or its corresponding salt, such as -COONa. "Carboxyalkyl ester" refers to a compound having a partial -C(O)OR, where R is alkyl.

[0061] As used herein, the term "carboxamide" refers to the radical-C(O)NRR', where R and R' may be the same or different. R and R' may independently be, for example, hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, formyl, haloalkyl, heteroaryl, or heterocyclyl.

[0062] The terms "amide," "amido," or "amidyl" are used herein with the -R symbol. 1 C(O)N(ROB)-, -R 1 C(O)N(ROD)R 3 -, -C(O)NR 2 R 3 , or refers to a base in the form of -C(O)NH2, where R 1 , R 2 and R 3 Each of these is independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, amide, amide, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, hydrogen, hydroxyl, ketone, or nitro.

[0063] The compounds of this disclosure may be isomers. In some embodiments, the disclosed compounds may be pure as isomers, where the compound is greater than about 99% of all compounds in a mixture of isomers of the compound. Also intended herein are compositions comprising, or comprising, essentially pure isomeric compounds and / or isomerically concentrated compositions, which may contain at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single isomer of a given compound.

[0064] The compounds disclosed herein may contain one or more chiral centers and / or double bonds, and thus exist as stereoisomers such as geometric isomers, enantiomers, or diastereomers. As used herein, the term “stereoisomer” comprises all geometric isomers, enantiomers, or diastereomers. These compounds can be represented by the symbols “R” or “S” or “+” or “-” depending on the configuration of substituents around the chiral carbon atom and the observed optical rotation. The disclosed compounds encompass a variety of stereoisomers and mixtures thereof. Stereoiomers include enantiomers and diastereomers. A mixture of enantiomers or diastereomers may be nominally referred to as “(±)”, but those skilled in the art will recognize that the structure may implicitly indicate a chiral center. It is understood that illustrations of chemical structures, such as general chemical structures, encompass all stereoisomeric forms of a particular compound unless otherwise specified. Furthermore, this specification also intends compositions that essentially consist of or are rich in enantiomers, comprising a single enantiomer of a given compound (e.g., at least about 95% of the R enantiomer of a given compound) and which may consist of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the enantiomer.

[0065] Various non-limiting embodiments and uses of the disclosed compounds can be considered by understanding the catalytic mechanism of OAT and the inactivation mechanisms of GABA-AT and OAT. In some embodiments, the disclosed subject matter relates to one or more of the above-mentioned OAT inhibitors formulated into compositions with one or more physiologically acceptable or tolerable diluents, carriers, adjuvants or vehicles, collectively referred herein as carriers. Such compositions suitable for contact or administration may include physiologically acceptable aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, whether sterile or not. The resulting compositions can be obtained, together with the various methods described herein, for administration or contact with human ornithine δ-aminotransferase. Whether or not to "contact" with the pharmaceutical composition means that human ornithine δ-aminotransferase and one or more inhibitor compounds are brought together for the purpose of conjugating and / or compounding such inhibitor compounds to the enzyme. The amount of compound effective in inhibiting human ornithine δ-aminotransferase can be determined empirically, and making such a determination is within the scope of the art. Inhibition or other effects of human ornithine δ-aminotransferase activity include reduction, mitigation, and / or regulation of OAT activity, glutamate production, glutamine synthesis, cell proliferation, and / or tumor growth, as well as removal.

[0066] Those skilled in the art will understand that the dosage varies depending on the activity of a particular inhibitory compound, the pathological condition, the route of administration, the duration of treatment, and similar factors well known in the fields of medicine and pharmacy. Generally, the appropriate dose will be the minimum dose effective in producing a therapeutic or prophylactic effect. If desired, effective doses of such compounds, pharmaceutically acceptable salts thereof, or related compositions may be administered separately in two or more subdoses over an appropriate period of time.

[0067] A method for preparing a pharmaceutical formulation or composition includes the step of conjugating an inhibitor compound to a carrier and, optionally, one or more additional adjuvants or components. For example, standard pharmaceutical formulation techniques such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA can be used.

[0068] Regardless of composition or formulation, those skilled in the art will recognize various routes for drug administration, along with corresponding factors and parameters to be considered in making such drugs suitable for administration. Accordingly, with respect to one or more non-limiting embodiments, the disclosed compounds can be used as inhibitory compounds for the manufacture of agents for therapeutic use in the treatment or prevention of diseases or disorders related to hOAT activity, expression, or overexpression. Appropriate diseases or disorders may include, but are not limited to, cell proliferation disorders or disorders, hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC).

[0069] In general, with respect to various embodiments, the disclosed subject matter may cover methods for the treatment of pathological proliferative disorders. As used herein, the term “disorder” refers to a condition in which there is an impairment of normal function. “Disease” refers to an abnormal physical or mental condition that causes discomfort, dysfunction, or distress to the patient or those who come into contact with it. Often, this term is used broadly to encompass injuries, disorders, syndromes, symptoms, deviant behaviors, and atypical changes in structure and function, and in other contexts these are considered distinct categories. Note that the terms “disease,” “disorder,” “condition,” and “illness” are used interchangeably herein.

[0070] According to one embodiment, the disclosed method may be particularly applicable to the treatment of malignant proliferative disorders, including malignant proliferative disorders expressing human ornithine δ-aminotransferase (hOAT). As used herein, “cancer,” “tumor,” and “malignant tumor” are all equivalent to hyperplasia of tissue or organ. If the tissue is part of the lymphatic system or immune system, malignant cells may include non-solid tumors of circulating cells. Malignant tumors of other tissues or organs may cause solid tumors. Accordingly, the compounds, compositions, and methods disclosed herein can be used to treat non-solid and solid tumors.

[0071] The malignancies intended herein may be selected from the group consisting of melanoma, carcinoma, leukemia, lymphoma, and sarcoma that express hOAT. Malignancies that express OAT (including leukemia, lymphoma, and myeloproliferative disorders), hypoplastic and aplastic anemia (both viral and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune-mediated and idiopathic), and solid tumors (including bladder, rectum, stomach, cervix, ovary, kidney, lung, liver, breast, colon, prostate, gastrointestinal tract, pancreas, and carposi) that can be treated by the methods disclosed herein. More specifically, according to certain embodiments, compounds and compositions can be used in combination for methods of treating or suppressing hematopoietic malignancies such as non-solid tumors, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), mast cell leukemia, hairy cell leukemia, Hodgkin's disease, non-Hodgkin lymphoma, Burkitt lymphoma, and multiple myeloma, as well as for treating or suppressing solid tumors such as tumors of the lips and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, and stomach. Small intestine, colon, colon, colon, rectum, colic canal, liver, anal canal, colon, anal canal, liver, ampulla of the anal canal, pancreas, pleural mesothelioma, bone, soft tissue sarcoma, cancer and skin, breast, vulva, vulva, vagina, cervix, uterine body, fallopian tube, ovary, fallopian tube, gestational choriocarcinoma, penis, prostate, testes, renal pelvis, kidney, ureter, ureter, urethra, eyelid cancer, conjunctival cancer, malignant melanoma, conjunctival malignant melanoma, retinoblastoma, lacrimal gland cancer, lacrimal gland cancer, cerebral sarcoma, orbit, brain, spinal cord, vascular system, angiosarcoma, Kaposi's sarcoma.

[0072] The compounds and compositions disclosed herein can be administered by therapeutic methods known in the art, and therefore various such compounds and compositions can be administered in any suitable manner, in conjunction with such methods. For example, administration may include oral administration, intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, parenteral administration, transdermal administration, vaginal administration, intranasal administration, mucosal administration, sublingual administration, topical administration, rectal administration or subcutaneous administration, or any combination thereof.

[0073] According to some embodiments, the treated subject may be a mammalian subject. The methods disclosed herein are intended in particular for the treatment of proliferative disorders in humans, but other mammals are also included. Non-limiting examples of mammalian subjects include monkeys, horses, cattle, dogs, cats, mice, rats, and pigs.

[0074] As used herein and in the claims, the terms “treatment,” “therapy,” and “therapy” mean improvement of one or more clinical indicators of disease activity in a subject having a pathological disorder. “Treatment” means a therapeutic procedure. A subject in need of treatment is a mammalian subject having some pathological disorder. “Patient” or “subject in need” means any mammal for which administration of a compound of the type described herein or any pharmaceutical composition is desirable to prevent, overcome, regulate or slow such a load. To provide “preventive treatment” or “prophylactic treatment” is to act in a defensive manner to defend against or prevent something, in particular a condition or disease.

[0075] More generally, the disclosed methods may be intended to influence, modulate, reduce, inhibit, and / or prevent the initiation, progression, and / or metastasis (e.g., to the liver from other sites or to the liver from other organs or tissues) of malignant pathological proliferative disorders associated with OAT activity. (e.g., Lucero OM, Dawson DW, Moon RT, 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 WM; Phang James M. Reprogramming of proline and glutamine metabolism contributes to the proliferative and metabolic responses regulated by oncogenic transcription factor cMYC. 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. See 2009, 19(1), 32-3).

[0076] Exemplary Embodiments The following embodiments are illustrative and should not be construed as limiting the scope of the claimed subject matter.

[0077] Embodiment 1. A compound of the following formula, or its dissociated form, zwitterionic form, or salt thereof.

[0078] [ka]

[0079] In the formula, a double bond exists between the α-carbon and the ε-carbon, or between the α-carbon and the β-carbon, R 1 and R 2 Each of these is independently selected from halogens such as F, Cl, Br, and I.

[0080] Embodiment 2. The compound according to Embodiment 1 in a zwitterionic form, comprising an ammonium moiety and a carboxylate moiety.

[0081] Embodiment 3. The compound according to Embodiment 1, wherein the double bond is located between the α-carbon and the ε-carbon.

[0082] Embodiment 4. The compound according to Embodiment 1, wherein the double bond is located between the α-carbon and β-carbon.

[0083] R 1 and R 2 The compound according to Embodiment 1, wherein at least one of the elements is F.

[0084] Embodiment 6. The compound according to Embodiment 5, wherein the compound is a salt comprising a substituent selected from an ammonium substituent, a carboxylate substituent, and a combination thereof.

[0085] Embodiment 7. The compound according to Embodiment 6, wherein the ammonium salt has a counterion that is the conjugate base of the proton acid.

[0086] Embodiment 8. The compound according to Embodiment 1 in a pharmaceutical composition containing a pharmaceutically acceptable carrier component.

[0087] Embodiment 9. Compound of Embodiment 1 of Formula:

[0088] [ka]

[0089] In the formula, R 1 and R 2 At least one of them is F.

[0090] Embodiment 10.R 1 and R 2 The compound according to Embodiment 9, wherein each of the elements is F.

[0091] Embodiment 11. The compound of Embodiment 1 having the following formula.

[0092] [ka]

[0093] Embodiment 12. The compound of Embodiment 1 having the following formula.

[0094] [ka]

[0095] Embodiment 12. The compound according to Embodiment 9, wherein the compound is a salt comprising a substituent selected from an ammonium substituent, a carboxylate substituent, and a combination thereof.

[0096] Embodiment 14. The compound according to Embodiment 13, wherein the ammonium salt has a counterion that is a conjugate base of a protonic acid.

[0097] Embodiment 15. The compound according to Embodiment 9 in a pharmaceutical composition containing a pharmaceutically acceptable carrier component.

[0098] Embodiment 16. The compound according to Embodiment 11 or 12, wherein the compound is a salt comprising a substituent selected from an ammonium substituent, a carboxylate substituent, and a combination thereof.

[0099] Embodiment 17. The compound according to Embodiment 16, wherein the ammonium salt has a counterion that is the conjugate base of the proton acid.

[0100] Embodiment 18. The compound according to Embodiment 11 or 12 in a pharmaceutical composition containing a pharmaceutically acceptable carrier component.

[0101] Embodiment 19. A pharmaceutical composition comprising (i) the compound of Embodiment 1; and (ii) a pharmaceutically appropriate carrier, diluent, or excipient.

[0102] Embodiment 20. A pharmaceutical composition comprising (i) the compound of Embodiment 9; and (ii) a pharmaceutically appropriate carrier, diluent, or excipient.

[0103] Embodiment 21. A pharmaceutical composition comprising (i) the compound of Embodiment 11 or 12; and (ii) a pharmaceutically suitable carrier, diluent or excipient.

[0104] Embodiment 22. A method for regulating human ornithine δ-aminotransferase (hOAT) activity, comprising contacting the compound of Embodiment 1 with a culture medium containing hOAT, wherein the compound is present in an amount sufficient to regulate hOAT activity.

[0105] Embodiment 23. The method according to Embodiment 22, wherein the double bond is located between the α-carbon and ε-carbon.

[0106] Embodiment 24. The method according to Embodiment 22, wherein the double bond is located between the α-carbon and β-carbon.

[0107] Embodiment 25.R 1 and R 2 The method according to embodiment 22, wherein at least one of is F.

[0108] Embodiment 26. The method according to Embodiment 22, wherein the compound is a salt comprising a substituent selected from ammonium substituents, carboxylate substituents, and combinations thereof.

[0109] Embodiment 27. The method according to Embodiment 26, wherein the ammonium salt has a counterion that is a conjugate base of a protonic acid.

[0110] Embodiment 22. The method according to Embodiment 22, wherein the contact is in vivo.

[0111] Embodiment 29. A method for reducing the activity of hOAT expressed by human cancer, comprising contacting a cancer expressing hOAT with the compound of Embodiment 1, wherein the compound is present in an amount effective to reduce hOAT activity.

[0112] Embodiment 30. The method according to Embodiment 29, wherein the double bond is located between the α-carbon and the ε-carbon.

[0113] Embodiment 31. The method according to Embodiment 29, wherein the double bond is located between the α-carbon and β-carbon.

[0114] Embodiment 32.R 1 and R 2 The method according to embodiment 29, wherein at least one of is F.

[0115] Embodiment 33. The method according to Embodiment 32, wherein the compound is provided in a pharmaceutical composition.

[0116] Embodiment 34. The method according to Embodiment 33, wherein the contact is in vivo.

[0117] Embodiment 35. The method according to Embodiment 34, wherein the contact is contact with a human subject that requires it.

[0118] Embodiment 36. A method for treating cancer in a subject requiring the treatment thereof, comprising administering a therapeutically effective amount of the compound of Embodiment 1 to the subject.

[0119] Embodiment 37. The method according to Embodiment 36, wherein the double bond is located between the α-carbon and ε-carbon.

[0120] Embodiment 38. The method according to Embodiment 36, wherein the double bond is located between the α-carbon and β-carbon.

[0121] Embodiment 39.R 1 and R 2The method according to embodiment 36, wherein at least one F is present.

[0122] Embodiment 40. The method according to Embodiment 36, wherein the compound is a compound of the formula.

[0123] [ka]

[0124] Embodiment 41. The compound is of the following formula:

[0125] [ka]

[0126] [In the formula, R 1 and R 2 [At least one of them is F] The method according to Embodiment 36, wherein the compound is or a salt thereof.

[0127] Embodiment 42.R 1 and R 2 The method according to embodiment 36, wherein each of them is F.

[0128] Embodiment 43. The method according to Embodiment 36, wherein the cancer is characterized by the expression or overexpression of human ornithine δ-aminotransferase (hOAT).

[0129] Embodiment 44. The method according to Embodiment 36, wherein the cancer is hepatocellular carcinoma.

[0130] Embodiment 45. The method according to Embodiment 36, wherein the cancer is non-small cell lung cancer (NSCLC). [Examples]

[0131] The following examples are illustrative and should not be construed as limiting 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, but not limited to, the treatment of diseases and disorders associated with the hOAT activity, expression or overexpression, and / or reduction of human ornithine aminotransferase activity, such as proliferative disorders and disorders including hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC). While the usefulness of the present invention is illustrated through the use of several compounds and compositions that can be used together, it will be understood by those skilled in the art that equivalent results can be obtained using a variety of other compounds, in accordance with the scope of the present invention.

[0132] Example 1 Title - Discovery and Mechanistic Study of SS-1-148, an Inactivator Based on the Selective Mechanism of Human Ornithine Aminotransferase introduction Human ornithine δ-aminotransferase (hOAT; EC2.6.1.13) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes two conjugated amino group transfer reactions. It converts L-ornithine (L-Orn) to L-glutamic acid-γ-semialdehyde (L-GSA) in the first half of the reaction, while the second half involves the formation of L-glutamic acid (L-Glu) from α-ketoglutarate (α-KG) in the second half (Figure 1A). 1 The resulting intermediate L-GSA is a spontaneous equilibrium species of Δ1-pyrroline-5-carboxylic acid (P5C), and P5C can be catalyzed by P5C reductase (PYCR) to yield L-proline. 2 On the other hand, the generated L-Glu is also converted to P5C by pyrroline-5-carboxylate synthase (P5CS) and is involved in proline metabolism (Figure 1A). 2 Proline biosynthesis was identified as the most substantially altered amino acid metabolism in human tumor tissue of hepatocellular carcinoma (HCC), characterized by accelerated proline consumption, hydroxyproline accumulation, increased alpha-fetoprotein (AFP) levels, and correlation with poor prognosis of HCC. 3Furthermore, glutamine synthetase (GS) catalyzes the conversion of L-Glu to L-glutamine (L-Gln). 4 L-Gln is highly required by cancer cells to support the anabolic process and promotes cell proliferation.

[0133] HCC is the leading liver malignancy and one of the most common causes of cancer-related death worldwide. Our previous research identified the OAT gene as one of seven overexpressed genes in spontaneously occurring HCC-developing livers from Psammomys obesus (sand rat) identified by DNA microarray analysis. 5 Furthermore, treatment with the selective hOAT mechanism-based inactivator (MBI) BCF3(1) (0.1 and 1.0 mg / kg; PO) significantly reduced serum AFP levels and inhibited tumor growth in HCC mouse models, highlighting the antitumor effect of pharmacologically selective hOAT inhibition. 5 MBIs are a type of molecule that initially acts as an alternative substrate for a target enzyme, and are then converted into an active species that can further inactivate the enzyme through specific covalent modification, strong electrostatic interactions, or other functionally irreversible inhibition. 6-7 MBI typically does not react before initial binding to the active site of the target enzyme, thereby usually exhibiting remarkable target specificity and selectivity. 8 Overall, hOAT is considered a potential therapeutic target for HCC, and selectively inactivating hOAT may offer new opportunities for discovering effective HCC treatments.

[0134] However, a major challenge in discovering the selective MBI of hOAT is other aminotransferases. 6 In particular, it is necessary to overcome irreversible inhibition of γ-aminobutyric acid aminotransferase (GABA-AT), which has a high structural similarity to hOAT. 1 There are only two significant differences in the active site pockets of the homodimer structure. Tyr85 and Tyr55 of hOAT correspond to Ile72 and Phe351 of GABA-AT, respectively. * It has been replaced by (Figure 2) 1Furthermore, Ile72 and Phe351 * are involved in the slightly narrower but more hydrophobic active site of GABA-AT compared to hOAT. In contrast, the hydroxyl group of Tyr55 acts as a hydrogen bond acceptor interacting with the charged C-2 amino group of the substrate, while Tyr85 is an important determinant of substrate specificity and is conformationally flexible to accommodate bulky substrates 1 .

[0135] In accordance with the high similarity between these two aminotransferases, a preliminary screening against hOAT was previously performed using the authors' stock of GABA-AT inhibitors 5 . A cyclopentane-based analog called BCF3 (1, Figure 1B) with a bis(trifluoromethyl) group as the warhead was identified as a selective MBI of hOAT but showed only millimolar reversible inhibition against GABA-AT. Recent mechanistic studies have revealed that one of its trifluoromethyl groups undergoes fluoride ion elimination and the ligand covalently modifies the catalytic Lys292 via conjugate addition (Scheme 1A) 9-10 . The sterically bulky bis(trifluoromethyl) group may not easily access the relatively narrow pocket of GABA-AT and affect the initial binding pose between the ligand and the enzyme, which may account for the reversible inhibition against GABA-AT. BCF3 has been demonstrated to be effective in vivo as described above and is under investigation in extensive IND-labeled toxicity evaluations and efficacy tests in a patient-derived xenograft (PDX) model of HCC patients. A cyclohexene-based analog WZ-2-051 (2, Figure 1B) has an expanded ring system and a difluoride group 11 . WZ-2-051 showed a 23-fold improvement in the inactivation efficiency (defined by the k inact / K I ratio) against hOAT compared to BCF3 but was 13.3-fold more selective than GABA-AT. Subsequent mechanistic studies have revealed that WZ-2-051 undergoes two-step fluoride ion elimination and finally inactivates hOAT via an addition-aromatization mechanism (Scheme 1B) 11。A further example of a selective hOAT inactivator is 5-FMOrn(3), which was inspired by the structures of the hOAT substrate L-Orn and the non-selective GABA-AT inactivator AFPA 12 and inactivates hOAT via the enamine pathway by forming a ternary adduct (Scheme 1C) 13 .

[0136]

Chemical Structure

[0137] It should be noted that the α-amino group of 5-FMOrn forms a strong hydrogen bond with the phenolic group of Tyr55 in the hOAT crystal complex (PDB entry 2OAT) 14 . Furthermore, hydrogen bonds between their carboxylate groups and Tyr55 in the hOAT crystal complexes with BCF3 (PDB entry 6OIA) and WZ-2-051 (PDB entry 6V8C) were observed 9,11 , indicating that the interaction with Tyr55 plays an important role in ligand specificity and selectivity. However, among the published hOAT inactivators, strong irreversible inhibition against hOAT, weak reversible inhibition against GABA-AT (K i = 4.2 mM), and no inhibition up to 4 mM against aspartate aminotransferase (Asp-AT) and alanine aminotransferase (Ala-AT) were shown, resulting in promising hOAT selectivity 5 . Therefore, discovering novel selective MBI of hOAT would promote the research of hOAT inactivators as potential therapeutic approaches for HCC

[0138] In 2000, (1R,4S)-4-amino-3,3-difluorocyclopentanecarboxylic acid (4, Figure 2C) was found to be a reversible inhibitor against GABA-AT (K i = 0.19 mM) 15 . Fifteen years later, it was further proven to be an hOAT inactivator 5However, compound 4 has a high binding affinity (K) for hOAT. I It exhibits a low maximum deactivation rate (k = 7.8 mM) relative to hOAT. inact =0.02 minutes -1 ) shows a moderate inactivation efficiency (k inact / K I =0.003 minutes -1 mM -1 This leads to inactivation and there are limitations in elucidating the turnover mechanisms.

[0139] This technology provides novel cyclopentene analogs, including SS-1-148(6), by incorporating an additional double bond into a cyclopentane ring system of 4, which has been demonstrated to be a highly potent and selective hOAT inactivator. Mechanistic studies using crystallization, protein and molecular mass spectrometry, transient state measurements, and computational simulations revealed a novel non-covalent inactivation mechanism of SS-1-148.

[0140] Results and Discussion Synthesis of cyclopentene analogs 5 and 6 having a difluoro group Incorporating double bonds has been shown to be an effective strategy for improving deactivation efficiency, as this affects the stereochemistry of the initial external aldimine and the acidity of the coprotons provided by the α,β-unsaturated carboxylate. 16-17 Therefore, in order to apply this approach to the development of more potent hOAT inactivators, cyclopentene analogs 5 and 6 having a difluoro group were designed based on the structure of the parent compound 4 (Figure 2C).

[0141] Synthesis routes to schemes 2.4 and 5 aReagents and conditions: (a) i) p-anisyl alcohol, concentrated HCl, rt; ii) NaH, TBAI, THF / DMF (10:1), 0℃-rt; (b) DBDMH, Ac2O, rt; (c) K2CO3, MeOH / H2O, rt; (d) (COCl)2, DMSO, TEA, THF, -78℃-rt; (e) Bu3SnH, AIBN, benzene, reflux; (f) Deoxo-Fluor (2.7M in toluene), THF, 120℃ (MW); (g) Ceric ammonium nitrate, CH3CN / H2O, rt; (h) 4N HCl, AcOH, 70 °C; (i) HCl (1.2 M) in EtOH, 70 °C; ii) Boc2O, TEA, DCM, rt; (j) PhSeCl, KHMDS (3.0 eq., 0.5 M in toluene), -78 °C-rt; (k) 4N HCl, AcOH, 70 °C.

[0142] [ka]

[0143] The synthetic route for producing compound 5 is (1R)-(-)-2-azabicyclo[2.2.1 hept-5-en-3-one(7; vinlactam 18 Starting from (CAS#:79200-56-9), we obtain the key biring intermediate 9 following the previously developed procedure. 15 Next, the acetyl group of 9 was converted to a hydroxyl group under acidic conditions, followed by Swern oxidation to obtain ketone intermediate 11. Subsequently, following the procedure described in the literature, the bromo group at the bridgehead of 11 was replaced with hydrogen by a dehydrogenation reaction, producing 12 under Bu3SnH / AIBN conditions. 15Intermediate 12 was further reacted with Deoxo-Fluor reagent under microwave conditions to obtain difluoro intermediate 13. Next, PMB deprotection was performed on 13 using ammonium cerium nitrate (CAN), and the resulting lactam 14 was treated with HCl / EtOH to obtain ethyl ester cyclopentane under reflux conditions. Following the introduction of the Boc protecting group, intermediate 15 was obtained. Interestingly, when attempting to prepare an intermediate of 15 having a selenenyl group at the α-position of the carboxylate group of 15 under KHMDS (3.0 eq) conditions for a follow-up α-elimination reaction, intermediate 16 having an α,ε-conjugated carboxylate group was directly obtained as the sole product. 16 1D and 2D NMR were performed to verify the structure of 16. Finally, after deprotection under acidic conditions, new cyclopentene-based analog 5 was appropriately obtained. The parent cyclopentane-based analog 4 was also prepared from intermediate 14, easily subjected to one-step acid hydrolysis, and then evaluated together with the new analog and BCF3 in subsequent kinetic studies.

[0144] Synthetic route to Scheme 3.6a a Reagents and conditions: (a) m-CPBA, CHCl3, reflux; (b) BF3·OEt2, AcOH, DCM, rt; (c) MOMCl, DIPEA, DCM, rt; (d) K2CO3, MeOH / H2O, rt; (e) (COCl)2, DMSO, TEA, THF, -78 °C - rt; (f) Deoxo-Fluor (2.7 M in toluene), THF, 120 °C (MW); (g) ceric ammonium nitrate, CH3CN / H2O, rt; (h) i) HCl (1.2 M in MeOH), 85 °C, sealed; ii) Boc2O, MeOH, rt; (i) Burgess reagent, THF, 70 °C; (l) 4N HCl, AcOH, 70 °C.

[0145]

Chem.

[0146] The synthetic route for preparing 6 started from the synthesis of an important bicyclic intermediate 20 from PMB-protected vinclozolin lactam 8 and followed an efficient procedure recently reported. 1Next, the 20 hydroxyl groups were converted to the ketone intermediate 21 by Swern oxidation. The difluoro intermediate 22 was obtained by the same fluorination conditions as above. Further, the PMB group of 22 was removed under CAM conditions, followed by lactam hydrolysis and Boc protection to obtain the cyclopentane intermediate 24. The hydroxyl group at the β-position of the methyl carboxylate group of 24 was dehydrated using the Burgess reagent 19-20 under reflux conditions to form the cyclopentene intermediate 25 containing an α,β-conjugated carboxylate group. The final product 6 was thus obtained according to the above deprotection conditions.

[0147] Kinetic studies of analogs 4 - 6. The kinetic results in Table 1 showed that all three difluorobase compounds 4 - 6 exhibited irreversible inhibition against hOAT, but reversible inhibition against GABA-AT, indicating that they are selective hOAT inactivators. Furthermore, compared with 4 and 5, which showed high mmol binding affinity (K I ) for hOAT, 6 showed significant improvement (KI = 0.06 mM). The results of partition ratio determination and fluoride ion release (Table 1) revealed that most of 4 and 5 released a large amount of fluoride ions, resulting in their high binding affinity, while being involved in an alternative turnover pathway rather than the inactivation pathway. On the other hand, 6 showed the highest rate constant (k inact ) compared to 4 and 5, thereby resulting in an excellent inactivation efficiency (k inact / K I ) of 6 against the parent compound 4 (1.333 vs 0.003 min -1 mM -1(444-fold improvement). Furthermore, it was noted that the inhibitory activity of cyclopentene-based compounds 5 and 6 against GABA-AT was approximately 10 times weaker than that of cyclopentane-based compound 4, suggesting higher selectivity than GABA-AT. Compound 6, named SS-1-148, showed comparable inactivation efficiency against hOAT while retaining reversible inhibition against GABA-AT compared to preclinical-stage BCF3. SS-1-148 also did not show significant inhibition against Asp-AT and Ala-AT up to 10 mM. Therefore, elucidating the potential inactivation and turnover mechanism of SS-1-148 in hOAT is of interest.

[0148] [Table 1]

[0149] Proposed inactivation pathways for SS-1-148 (6). Based on previous mechanistic studies of other related GABA-AT / hOAT inactivators, three possible pathways were initially proposed. 7,11 This is summarized in Scheme 4. At the start of inactivation, SS-1-148 captures the PLP moiety from the internal aldimine Lys292-PLP, forming the external aldimine M1. Subsequently, M1 undergoes deprotonation to form the quinonoid species M2, followed by a fluoride ion elimination step to obtain the common intermediate M3 in the following three pathways. Pathway a was proposed based on recent findings related to the cyclohexene-based analog WZ-2-051. 11 Electrophilicity of intermediate M3 C δ The site is attacked by Lys292, forming a covalent bond (M4) via conjugate addition. The quinonoid species M4 undergoes a second fluoride ion elimination, giving the final adduct M5 (Scheme 4). Pathway b is stimulated by the inactivation mechanisms of CPP-115 and OV329 in GABA-AT. This mechanism is achieved by a water-mediated mechanism resulting in a strong electrostatic interaction between the carboxylate and arginine residues of the active site. 16,21 The water molecule catalyzed by Lys292 is electrophilic C of intermediate M3. δThe site is attacked, and then another fluoride ion elimination step is performed, giving an enol / carbonyl group (M7) rather than establishing a covalent bond with the residue in hOAT. Pathway c is proposed following a typical enamine mechanism. 22 Lys292 is C δ Instead of position, use the C of algimin. 4’ It attacks the site and releases the enamine intermediate M9, which recombines the imine bond of the internal aldimine (PLP-Lys292) to produce the covalent adduct M10.

[0150] [ka]

[0151] A reasonable metabolic turnover mechanism of SS-1-148 using hOAT and GABA-AT. SS-1-148 exhibited a relatively high distribution ratio (33.9 times, Table 1 and Figure 9), indicating that 34.9 equivalents of SS-1-148 were inverted per active site for each equivalent of the compound, leading to inactivation. Furthermore, since 33.9 ± 0.8 equivalents of fluoride ions (Table 1) were released with each inactivation event, it is suggested that the primary turnover pathway occurs in only one fluoride ion removal step. Previously, electrostatic potential (ESP) charge calculations showed that the fluorine atom clearly reduced the nucleophilicity of the enamine intermediate (similar in structure to M8). 11 This demonstrated that it inhibits the occurrence of enamine addition. Metabolomics studies of SS-1-148 in hOAT (Figure 3A) showed that the molecular weight and fragmentation of the primary metabolite were consistent with the structure of M10 in Scheme 5, which is the corresponding hydrolysis product from the enamine intermediate M8. Therefore, pathway c in Scheme 4 was considered to be the major turnover pathway of SS-1-148 in hOAT. On the other hand, the major metabolite of SS-1-148 in GABA-AT was identified. The molecular weight and fragmentation shown in Figure 3B suggest that SS-1-148 acts as a substrate to produce ketone M11, which has a difluoro group (Scheme 5).

[0152] To capture the major intermediate of the deinactivation pathway, immersion experiments were performed using hOAT holoenzyme crystals with SS-1-148 solution for 1 hour. The hOAT structure was determined by molecular substitution from a previously reported structure (PDB entry 1OAT). The space group of the SS-1-148 immersion structure is P 322 1, and the structure was found to contain three copies of the protein monomer in a single asymmetric unit. The crystal structures shown in Figures 4A and 11 (PDB entry 7LK1) show that PLP is covalently bonded to SS-1-148, while the immersion crystal revealed a covalent bond between Lys292 and SS-1-148. The covalent bond between Lys292 and SS-1-148 represents a stable gem-diamine species not observed in previous hOAT / ligand crystals. 9-11,23 This observation further validated the gemdiamine precursor (M12 or M13, scheme 5) of the enamine intermediate M8. Furthermore, two structural conformations of this intermediate were observed in immersed crystals with different carboxylate group positions of SS-1-148. The first conformation forms a hydrogen bond between Tyr55 and the carboxylate of SS-1-148, while the second conformation forms a salt bridge with Arg413. The conclusion regarding the two selective conformations of the intermediate structure was based on the positive density of the vicinity of Arg413 and Tyr55, and the relatively high B factor relative to its own single conformation. Another explanation is that there are two different, but structurally similar, intermediate species that may interact with the protein's active site in different ways.

[0153] [ka]

[0154] Overall, evidence suggests that the major turnover mechanisms of SS-1-148 in hOAT and GABA-AT are as proposed in Scheme 5, respectively. After capturing the PLP ligand from Lys292, M1 undergoes deprotonation, and the resulting quinonoid M2 undergoes fluoride ion elimination in hOAT (pathway a; Scheme 5) to give a monofluorinated aldimine intermediate M3. The majority of M3 is attacked by Lys292 at the C4' position to form the first gem-diamine M12, followed by proton transfer to obtain the second gem-diamine M13. 24-25 It is further converted to the enamine metabolite M8 and internal aldimine. Finally, M8 is hydrolyzed to the ketone M10, which is the major metabolite of SS-1-148 in hOAT. On the other hand, the quinonoid intermediate M2 undergoes electron transfer to produce the ketimine M14, releasing PMP and M11 as major metabolites of GABA-AT. The behavior of SS-1-148 in GABA-AT is similar to an aminotransfer reaction without the removal of fluoride ions, which is consistent with its reversible inhibition of GABA-AT. It should be noted that due to structural differences between hOAT and GABA-AT, the known selective hOAT inactivators (1-3) described above contain bulky moieties or α-amino groups to improve ligand specificity and selectivity compared to GABA-AT. However, the selectivity of SS-1-148 is related to its competition with the substrate GABA.

[0155] Possible inactivation mechanism of SS-1-148 by hOAT. The irreversibility of hOAT inhibition by SS-1-148 was evaluated by time-dependent dialysis experiments up to 48 hours with a buffer consisting of excess PLP and α-KG (Figure 10). The remaining activity of hOAT remained unchanged, indicating that SS-1-148 is an irreversible inhibitor of hOAT. To elucidate the structure of the final product in the inactivation reaction, the authors cocrystallized excess SS-1-148 with hOAT. The crystal structure (PDB entry 7LK0) was solved using the same method as above, and it was found that the space group of the SS-1-148 cocrystal (Figures 4B and 12) is P 311 2. Also, one asymmetric unit contains three copies of the protein monomer. Similar to the immersion crystal structure shown in Figure 4A, PLP is covalently bonded to the SS-1-148 portion in the cocrystal structure. However, no covalent bond was observed between Lys292 and the final product. Furthermore, in the published crystal structures of the natural enzyme (holo-hOAT), Arg413 generally forms a salt bridge with Glu235, and this was found to be intact for several cocrystals of hOAT with different inactivators. 14,23,26 In the hOAT / SS-1-148 immersion results (Figure 4A), the Arg413-Glu235 salt bridge was broken due to the presence of an alternative salt bridge between one intermediate and the carboxylate of Arg413. Interestingly, the Arg413-Glu235 salt bridge was also found to be cleaved in the hOAT / SS-1-148 cocrystal, but no direct interaction between Arg413 and the ligand was observed. Instead, Arg413, Gln266, and the final product are hydrogen-bonded to the same water molecule. Furthermore, the oxygen group of the ligand shows hydrogen bonding with Glu235 at a distance of 2.9 Å (Figure 4B), which may contribute to the stabilization of the ligand within the hOAT pocket. The ligand structure in the hOAT / SS-1-148 cocrystal indicates that inactivation is attributed to pathway B in scheme 4, producing a final product (theoretical mass: 370.06 Da) close to the structure of M7.

[0156] Intact protein mass spectrometry (intact MS) has been previously applied to elucidate the inactivation mechanism of hOAT inactivators. 9,11Here, through denaturation and intact LC-MS (Figure 5A, right) experiments using hOAT samples completely inactivated with SS-1-148, it was found that only 16% of the hOAT was covalently modified, leading to a mass shift of 370.01 Da. The majority of the hOAT remained unmodified (Figure 5A, top) and was compared to natural hOAT as a control (Figure 5A, bottom). The amount of added substance (370.01 Da) was found to be identical to the theoretical mass of gemdiamine M15 (370.06 Da; Scheme 6) that may be in equilibrium with the non-covalent form M7. However, under these conditions where the enzyme is completely inactivated, a greater abundance of covalently added enzyme is expected. Thus, this finding supports the main non-covalent, inactivation mechanism between hOAT and SS-1-148.

[0157] Further use of innate protein mass spectrometry (innate MS) was employed to identify non-covalent protein interactions in the solution state that are conserved in the gas phase, and to characterize the binding of SS-1-148 products in hOAT. The results shown in Figure 5B indicate that unmodified hOAT appears as a dimer 459 Da larger (92,737 ± 2 Da) than the theoretical apo-hOAT dimer. This mass shift is consistent with the two PLP-bound internal aldimines in the two active sites of the dimer (a 459 Da shift was observed; 460 Da theoretically). Proteolytic degradation morphology of unmodified hOAT (Figure 5B, unmodified hOAT( * )) was also observed, but only under control conditions. In contrast, natural dimeric hOAT samples completely inactivated with SS-1-148 were observed at two high masses of 93,020±4Da and 93,056±3Da, corresponding to mass shifts of 742Da and 778Da (Figure 5B, hOAT + SS-1-1-148 (left)). A larger number of species, possibly due to salt adducts, was observed (Figure 5B, hOAT + SS-1-1-148 (left)) *)). No unmodified, apo-hOAT, or PLP-bound hOAT was observed. The observed mass of 93,020 ± 4 Da was consistent with the theoretical mass of M7 (370.06 Da) in both active sites (theoretically 93,018 Da). Furthermore, the observed mass of 93,056 ± 3 Da was consistent with M7 and the water in the active sites of both protein chains (theoretically 93,056 Da), verifying the major water molecules observed in the final product and co-crystal structure (Figure 4B).

[0158] To further investigate the SS-1-148-hOAT interaction by mass spectrometry, high-energy collision dissociation (HCD) was applied to unmodified hOAT to dissociate the protein-ligand interaction using separately processed hOAT and release the ligand from the enzyme complex. Under subproteolytic dissociation conditions (HCD NCE:15), no mass defect was observed in unmodified hOAT (Figure 13). However, under these same dissociation conditions, two additional masses were generated for SS-1148-inactivated hOAT (Figure 5B, hOAT + SS-1-1-148 (right)). A mass consistent with apo-hOAT was observed at 45% relative abundance (observed: 92,275±4Da; theoretical: 92,278Da). A second mass of 92,597±5Da was observed at 100% relative abundance. Compared to previously observed dimer masses consistent with each active site bound to M7 and one water molecule, this species exhibits a mass defect of 457 Da. Strictly speaking, the large mass change in abundance cannot be explained by the loss of a single M7±H2O adduct from the protein dimer (theoretical mass of M7-hOAT: 92,648 Da; theoretical mass of 1M7-hOAT+H2O: 92,666 Da). However, the mass defect observed via HCD activation can be explained by the disappearance of PLP from both active sites, while the active site water and SS-1-148 moieties are retained (observed: 457 Da; theoretical: 460 Da). This finding is surprising, considering that the same collision energy does not release PLP from an unmodified protein dimer. However, based on the co-crystal structure that identified the binding of SS-1-148 to Tyr55, Glu235, Gln266, and Arg413 of water, protein stability increases upon SS-1-148 binding, and in the gas phase, blocking these interactions is undesirable compared to cleavage of PLP.

[0159] Since no apoenzymes were observed in natural MS, and approximately 84% of hOAT was in the apoenzyme state under denaturation conditions, the results indicated that non-covalent M7 is the first form after inactivation, while covalent M15 is a smaller form in equilibrium with M7. Therefore, M7 generated from water-mediated pathway b (Scheme 4) is thought to be the final product of SS-1-148 after inactivation. However, given its highly coupled structure, M7 is prone to tautomerization. Therefore, Gibbs free energy calculations were performed on MOPAC to evaluate the stability of M7 and related tautomers in the active site of hOAT. The results shown in Figure 14 are for enol form M7 (ΔG o = -26.36 kcal mol -1 ) compared to its corresponding ketone (tautomer 5, ΔG o = -48.07 kcal mol -1 ) and ketimine (tautomer 8, ΔG o The absorbance of -49.89 kcal (1) suggests that the active site is relatively stable. Furthermore, it should be noted that the species exhibiting absorbance at approximately 275 nm was determined to be the final product in subsequent transient state measurements (Figure 7C), thereby excluding all external aldimines (e.g., M7 and tautomer 5) as the absorbance at 420 nm is appropriate for the final form of SS-1-148. On the other hand, the above results indicate that the final state is something like a gemdiamine or ketimine, while their typical absorbance appears at approximately 330-340 nm. According to the literature, enolimines with neutral hydroxyl and aldimine groups exhibit a significantly lower absorption maximum (410 nm vs. 330 nm) compared to ketoenamine moieties containing protonated aldimines and deprotonated hydroxyls. 30 In summary, both gemdiamine M15, which is in equilibrium with M7, and ketimine M18, which is tautomerized from M7, may undergo an extra step of electron transfer to form M16 and M19, respectively. Due to the neutral state of gemdiamine M16 and ketimine M19, their absorption maxima can drop from 330 nm to 275 nm, thereby matching the absorbance of the final species observed in transient state measurements.

[0160] Overall, the inactivation pathway of SS-1-148 in hOAT is summarized in Scheme 6. The first external aldimine M1 undergoes deprotonation catalyzed by Lys292 to generate the first quinonoid M2. Following fluoride ion elimination, monofluoroaldimine M3 is obtained from M2. The C4’ position of most of M3 (about 97%; determined by the partition ratio) is attacked by Lys292, releasing an enamine metabolite that hydrolyzes to the ketone M10 as the primary metabolite (pathway a; Schemes 5 and 6). A small amount of M3 (about 3%) undergoes nucleophilic attack via water (pathway b; Scheme 6) to generate the second quinonoid M6. Intermediate M6 further undergoes another fluoride ion elimination to generate M7. Subsequently, a small amount of M7 (about 16%) covalently binds to Lys292 at the C4’ position via the gem-diamine form (M15) (pathway c; Scheme 6). The resulting gem-diamine M15 can be in equilibrium with M7, while further electron transfer can occur to generate the more stable neutral gem-diamine form 16. On the other hand, most of M7 (~84%) tautomerizes to the more favorable ketimine M18, followed by electron transfer to yield the ketimine M19 as the primary end product (pathway d; Scheme 6). δ undergoes nucleophilic attack via water (pathway b; Scheme 6) to generate the second quinonoid M6. Intermediate M6 further undergoes another fluoride ion elimination to generate M7. Subsequently, a small amount of M7 (about 16%) covalently binds to Lys292 at the C4’ position via the gem-diamine form (M15) (pathway c; Scheme 6). The resulting gem-diamine M15 can be in equilibrium with M7, while further electron transfer can occur to generate the more stable neutral gem-diamine form 16. On the other hand, most of M7 (~84%) tautomerizes to the more favorable ketimine M18, followed by electron transfer to yield the ketimine M19 as the primary end product (pathway d; Scheme 6).

[0161]

Chemical Structure

[0162] Measurement of the transient state of hOAT inhibited by SS-1-148. Since various transient states are involved in the proposed mechanism, subsequent stopped-flow spectrophotometric measurements were performed to capture the kinetics of hOAT inhibition by SS-1-148. This approach utilizes the coupled species that accumulate continuously in the PLP-dependent transaminase reaction 10The inhibitory reaction occurring with SS-1-148 was interpreted in combination with the aforementioned X-ray structures obtained at different stages of the reaction (see below). The experimental data shown in Figures 6 and 7 indicate that the reaction between SS-1-148 and hOAT is complex. Four identifiable phases were observed in evidence of at least two parallel reaction pathways (Figure 6), indicating that the mechanistic conclusions drawn are necessarily derived from an undetermined model. Within the dead time of the stopped flow apparatus, the reaction between SS-1-148 and hOAT formed a spectrum (approximately 420 nm) consistent with the external aldimine (Figure 7A-B(A)). Titration of hOAT with SS-1-148 regulated the rate and extent of accumulation of a second external aldimine contribution, which is likely additive to the aldimine formed during the dead time (Figure 7A-B(B)). The second external aldimine showed rate dependence and a bimolecular reaction (6.2 × 10⁻⁶). 3 M -1 s -1 This indicates that SS-1-148 binds to hOAT via at least two pathways, resulting in parallel reaction pathways (Figure 6A-B). The intensity of the combined external aldimine spectrum partially decays with the accumulation of spectral transition properties of the quinononoid species (approximately 560 nm) at 0.4 s-1 (Figures 6A-B and 7A-B). The apparent quinonoid species is formed with the simultaneous and partial decay of the external aldimine transition and has an amplitude almost equal to that of the second external aldimine accumulation. This suggests that these chemical species are in the same reaction pathway.

[0163] The deprotonation of the initial external aldimine M1 and stepwise fluoride elimination process proposed in Scheme 6 is typically reported to be associated with the E1CB mechanism. 31-32 The electron-withdrawing effect of fluorine and protonated nitrogen of aldimine can stabilize the carbanion state generated during the elimination reaction. 32-33On the other hand, the quinononoid transient state M2 is thought to form between the first and second external aldimines (M1 and M3). However, stopped-flow experimental results show that antiperiplanar hydrogen and fluorine in M1 undergo an abnormal Lys292-assisted E2 mechanism simultaneously with proton loss, fluoride ion release, and alkene formation as a more favorable fluoride removal pathway. 31 This suggests that a second external aldimine M3 is given as a single transient state (pathway e; scheme 6). This result has not been experimentally observed in previous mechanistic studies of other related PLP-dependent aminotransferase inactivators. 1,7 .

[0164] Quinonoid extinction coefficient is approximately 30 mM -1 cm -1 Assuming this, the observed fractional accumulation of quinonoids at 1 mM SS-1-148 represents approximately 20% of all reaction species. Subsequently, the quinonoids evaporate in 0.09 s. -1 It decays at a rate of , while the residual transitions attributed to the external aldimine species spread and persist (Figure 7A-B(D)). Quinonoid M6 appears to be the first quinonoid species (approximately 560 nm) that can be observed based on the turnover (pathway a; scheme 5) and inactivation (pathway b; scheme 6) mechanisms. The observed final phase was 0.007 s. -1 This occurs with a rate constant of . At this stage, the external aldimine decay characteristic with a significant increase in absorption intensity at 275 nm indicates loss of conjugation, consistent with gemdiamine M16 and ketimine M19, which were proposed as the final products in Figure 6 (Figure 7A-B(E)).

[0165] In summary, these data support a dominant pathway consisting of multiple distinct extrinsic aldimine species, which ultimately degrade into less conjugated products (metabolic turnover mechanism; pathway e; and scheme 6). A second secondary pathway, which forms the initial extrinsic aldimine, degrades more slowly, then via quinonoid intermediates, forming unconjugated products (inactivation mechanism; pathway be; scheme 6). The data shown in Figure 7C demonstrate that the proportion of each pathway depends on the concentration of SS-1-148. High concentrations of SS-1-148 reduced the accumulation of quinonoid species but did not alter the observed rates of accumulation and decay, suggesting that a faster, dominant pathway sequesters a larger fraction of the enzyme at higher SS-1-148 concentrations.

[0166] Significance of the conjugated alkene of SS-1-148. After gaining a better understanding of the inactivation and turnover mechanisms of SS-1-148(6), calculations were performed to compare analogs 4-6. Our previous research revealed that incorporating an extra double bond into the cyclopentane ring system establishes an α,β-conjugated carboxylate, accelerating the deprotonation step by increasing the acidity of the auxiliary proton and increasing the rate constant. 16-17 DFT / B3LYP method in 298K 34 Theoretical pKa calculations were performed using this method to predict the acidity of the proton at the Cγ position in difluoro analogs 4-6 (Figure 2C). The results shown in Figure 8A show that the Cγ hydrogen of PLP-bonded SS-1-148(M1) with an α,β-conjugated carboxylate system showed the lowest pKa value among the three analogs, while the pKa value of the corresponding proton in PLP-bonded 5(M1) was C relative to the parent cyclopentane 4(M1'). α and C ε This suggests that the positional introduction of the double bond does not significantly affect the result. These characteristic k inact Taken together from the values ​​(Table 1), these findings suggest that the more acidic hydrogen at the Cγ position promotes the initial external aldimine of SS-1-148(M1), initiating the E2 fluoride ion elimination step rather than the typical E1CB elimination reaction, thereby contributing to its improved rate constant.

[0167] The results of the distribution ratio measurements and fluoride ion release (Table 1) revealed that cells 4 and 5 release large amounts of fluoride ions, while primarily being involved in the second turnover pathway rather than the inactivation pathway. Electron density map and ESP charge calculations were also performed. 11 (Figure 8B) shows C in transient state M3 of SS-1-148(6). δ The electrophilicity of M3 is much higher than that of the corresponding transient states 4 and 5 (M3' is 4, M3' is 5), and the C of M3 δ This indicates that the position is more reactive than that in the other two intermediates. On the other hand, the C4' position in the aldimine bond of M3' and M3'' shows comparable electrophilicity, much greater than that of M3, indicating that M3' and M3'' are attacked by the catalyst Lys292, triggering their turnover pathway and ultimately resulting in a significantly higher distribution ratio of 4 and 5. Furthermore, further metabolomics studies in hOAT revealed that cyclopentene analog 5, like SS-1-148 in hOAT, produces a monofluorinated ketone as its major metabolite (M5-1, Figure 15B). In contrast, cyclopentane 4 not only forms a monofluorinated ketone (M4-1, Figure 15A) but also produces a hydroxyl-containing ketone metabolite (M4-2, Figure 15A), which was not detected in the metabolomics results of the cyclopentene analog. According to the mechanism proposed by SS-1-148 in Scheme 6, the incorporated double bond is also thought to play an important role in stabilizing the conjugated system.

[0168] conclusion Previously, (S)-3-amino-4,4-difluorocyclohex-1-enecarboxylic acid (WZ-2-051,2) inactivated hOAT via a covalent addition-aromatization mechanism (Scheme 1B). However, it still exhibited apparent irresponsible inhibition of GABA-AT. (S)-3-amino-4,4-difluorocyclopenta-1-enecarboxylic acid (SS-1-148,6) was identified to exhibit inactivation efficiency comparable to the preclinical-stage selective hOAT inactivator BCF3, demonstrating its reversible inhibitory effect on GABA-AT. Kinetic studies and calculations provided evidence supporting the high hOAT inactivation efficiency of the conjugated alkene of SS-1-148 in the cyclopentene ring. Two crystallographic approaches were employed to capture the transient gem-diamine intermediate covalent bond with Lys292 in immersed crystals and the stable non-covalent final product in the cocrystal complex. The critical salt bridge of Arg413-Glu235 in hOAT was found to be destroyed in both crystalline complexes. Furthermore, natural / intact MS experiments further support a non-covalent pathway as the primary inactivation mechanism of SS-1-148, and covalent modifications observed as minor forms that may be gem-diamine structures in equilibrium with the non-covalent form. Finally, stop flow experiments suggested that the initial external aldimine of SS-1-148 undergoes an unusual E2 fluoride ion elimination reaction instead of a typical E1CB elimination reaction, forming a second external aldimine as a single transient state. Comprehensive mechanistic studies have shown that SS-1-148 inactivates hOAT non-covalently, primarily via a water-mediated mechanism. In addition, preliminary evaluations suggest that SS-1-148 exhibits good DMPK properties (data not shown) and is under investigation in a PDX mouse model of HCC.

[0169] Abbreviation AFPA, (S)-4-amino-5-fluoropentanoic acid; Boc2O, di-tert-butyl dicarbonate; Deoxo-Fluor, bis(2-methoxyethyl)aminosulfur trifluoride; DMPK, drug metabolism and pharmacokinetics; DIPEA, N,N-diisopropylethylamine; DBDMH, 1,3-dibromo-5,5-dimethylhydantoin; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DCM, dichloromethane; IND, new drug application in clinical trials; PO, oral administration; THF, tetrahydrofuran; TEA, triethylamine; TBAI, tetra-n-butylammonium iodide.

[0170] References 1. Lee, H.; Juncosa, JI; Silverman, RB, Ornithine aminotransferase versus GABA aminotransferase: implications for the design of new anticancer drugs. Med Res Rev 2015, 35 (2), 286-305. 2. Tanner, JJ; Fendt, SM; Becker, DF, The Proline Cycle As a Potential Cancer Therapy Target. Biochemistry 2018, 57 (25), 3433-3444. 3. Tang, L.; Zeng, J.; Geng, P.; Fang, C.; Wang, Y.; Sun, M.; Wang, C.; Wang, J.; Yin, P.; Hu, C.; Guo, L.; Yu, J.; Gao, P.; Li, E.; Zhuang, Z.; Xu, G.; Liu, Y., Global Metabolic Profiling Identifies a Pivotal Role of Proline and Hydroxyproline Metabolism in Supporting Hypoxic Response in Hepatocellular Carcinoma. Clin Cancer Res 2018, 24 (2), 474-485. 4. Cadoret, A.; Ovejero, J.; Terris, B.; Soil, E.; Levy, L.; Lamers, WH; Kitajewski, J.; Kahn, A.; Perret, C., New targets of beta-catenin signaling in the liver are involved in the glutamine metabolism. Oncogene 2002, 21 (54), 8293-301. 5. Zigmond, E.; Ben Ya'acov, A.; Lee, H.; Lichtenstein, Y.; Shalev, Z.; Smith, Y.; Zolotarov, L.; Ziv, E.; Kalman, R.; Le, HV; Lu, H.; Silverman, RB; Ilan, Y., Suppression of Hepatocellular Carcinoma by Inhibition of Overexpressed Ornithine Aminotransferase. ACS Med Chem Lett 2015, 6 (8), 840-4. 6. Eliot, A. C.; Kirsch, J. F., Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annu Rev Biochem 2004, 73, 383-415. 7. Silverman, R. B., Design and Mechanism of GABA Aminotransferase Inactivators. Treatments for Epilepsies and Addictions. Chem Rev 2018, 118 (7), 4037-4070. 8. Pan, Y.; Gerasimov, M. R.; Kvist, T.; Wellendorph, P.; Madsen, K. K.; Pera, E.; Lee, H.; Schousboe, A.; Chebib, M.; Brauner-Osborne, H.; Craft, C. M.; Brodie, J. D.; Schiffer, W. K.; Dewey, S. L.; Miller, S. R.; Silverman, R. B., (1S, 3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115), a potent gamma-aminobutyric acid aminotransferase inactivator for the treatment of cocaine addiction. J Med Chem 2012, 55 (1), 357-66. 9. Moschitto, M. J.; Doubleday, P. F.; Catlin, D. S.; Kelleher, N. L.; Liu, D.; Silverman, R. B., Mechanism of Inactivation of Ornithine Aminotransferase by (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)cyclopentane-1-carboxylic Acid. J Am Chem Soc 2019, 141 (27), 10711-10721. 10. Butrin, A.; Beaupre, B. A.; Kadamandla, N.; Zhao, P.; Shen, S.; Silverman, R. B.; Moran, G. R.; Liu, D., Structural and Kinetic Analyses Reveal the Dual Inhibition Modes of Ornithine Aminotransferase by (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)-cyclopentane-1-carboxylic Acid (BCF3). ACS Chemical Biology 2020. 11. Zhu, W.; Doubleday, P. F.; Catlin, D. S.; Weerawarna, P. M.; Butrin, A.; Shen, S.; Wawrzak, Z.; Kelleher, N. L.; Liu, D.; Silverman, R. B., A Remarkable Difference That One Fluorine Atom Confers on the Mechanisms of Inactivation of Human Ornithine Aminotransferase by Two Cyclohexene Analogues of gamma-Aminobutyric Acid. J Am Chem Soc 2020, 142 (10), 4892-4903. 12. Daune, G.; Gerhart, F.; Seiler, N., 5-Fluoromethylornithine, an irreversible and specific inhibitor of L-ornithine:2-oxo-acid aminotransferase. Biochem J 1988, 253 (2), 481-8. 13. Bolkenius, F. N.; Knodgen, B.; Seiler, N., DL-canaline and 5-fluoromethylornithine. Comparison of two inactivators of ornithine aminotransferase. Biochem J 1990, 268 (2), 409-14. 14. Storici, P.; Capitani, G.; Muller, R.; Schirmer, T.; Jansonius, J. N., Crystal structure of human ornithine aminotransferase complexed with the highly specific and potent inhibitor 5-fluoromethylornithine. J Mol Biol 1999, 285 (1), 297-309. 15. Qiu, J.; Silverman, R. B., A new class of conformationally rigid analogues of 4-amino-5-halopentanoic acids, potent inactivators of gamma-aminobutyric acid aminotransferase. J Med Chem 2000, 43 (4), 706-20. 16. Juncosa, J. I.; Takaya, K.; Le, H. V.; Moschitto, M. J.; Weerawarna, P. M.; Mascarenhas, R.; Liu, D.; Dewey, S. L.; Silverman, R. B., Design and Mechanism of (S)-3-Amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic Acid, a Highly Potent gamma-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Addiction. J Am Chem Soc 2018, 140 (6), 2151-2164. 17. Shen, S.; Doubleday, P. F.; Weerawarna, P. M.; Zhu, W.; Kelleher, N. L.; Silverman, R. B., Mechanism-Based Design of 3-Amino-4-Halocyclopentenecarboxylic Acids as Inactivators of GABA Aminotransferase. ACS Med Chem Lett 2020, 11 (10), 1949-1955. 18. Singh, R.; Vince, R., 2-Azabicyclo[2.2.1]hept-5-en-3-one: chemical profile of a versatile synthetic building block and its impact on the development of therapeutics. Chem Rev 2012, 112 (8), 4642-86. 19. Wang, B. L.; Gao, H. T.; Li, W. D., Total synthesis of (+)-iresin. J Org Chem 2015, 80 (10), 5296-301. 20. Gross, L. J.; Stark, C. B. W., Regioselective dehydration of alpha-hydroxymethyl tetrahydrofurans using Burgess' reagent under microwave irradiation. Org Biomol Chem 2017, 15 (20), 4282-4285. 21. Lee, H.; Doud, E. H.; Wu, R.; Sanishvili, R.; Juncosa, J. I.; Liu, D.; Kelleher, N. L.; Silverman, R. B., Mechanism of inactivation of gamma-aminobutyric acid aminotransferase by (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115). J Am Chem Soc 2015, 137 (7), 2628-40. 22. Shen, S.; Doubleday, P. F.; Weerawarna, P. M.; Zhu, W.; Kelleher, N. L.; Silverman, R. B., Mechanism-Based Design of 3-Amino-4-Halocyclopentenecarboxylic Acids as Inactivators of GABA Aminotransferase. ACS Medicinal Chemistry Letters 2020. 23. Mascarenhas, R.; Le, H. V.; Clevenger, K. D.; Lehrer, H. J.; Ringe, D.; Kelleher, N. L.; Silverman, R. B.; Liu, D., Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover. Biochemistry 2017, 56 (37), 4951-4961. 24. Di Salvo, M. L.; Scarsdale, J. N.; Kazanina, G.; Contestabile, R.; Schirch, V.; Wright, H. T., Structure-based mechanism for early PLP-mediated steps of rabbit cytosolic serine hydroxymethyltransferase reaction. Biomed Res Int 2013, 2013, 458571. 25. Soniya, K.; Awasthi, S.; Nair, N. N.; Chandra, A., Transimination Reaction at the Active Site of Aspartate Aminotransferase: A Proton Hopping Mechanism through Pyridoxal 5′-Phosphate. ACS Catalysis 2019, 9 (7), 6276-6283. 26. Shah, S. A.; Shen, B. W.; Brunger, A. T., Human ornithine aminotransferase complexed with L-canaline and gabaculine: structural basis for substrate recognition. Structure 1997, 5 (8), 1067-75. 27. Leney, A. C.; Heck, A. J., Native Mass Spectrometry: What is in the Name? J Am Soc Mass Spectrom 2017, 28 (1), 5-13. 28. Stewart, J. J., MOPAC: a semiempirical molecular orbital program. J Comput Aided Mol Des 1990, 4 (1), 1-105. 29. Karsten, W. E.; Ohshiro, T.; Izumi, Y.; Cook, P. F., Reaction of serine-glyoxylate aminotransferase with the alternative substrate ketomalonate indicates rate-limiting protonation of a quinonoid intermediate. Biochemistry 2005, 44 (48), 15930-6. 30. Thibodeaux, C. J.; Liu, H. W., Mechanistic studies of 1-aminocyclopropane-1-carboxylate deaminase: characterization of an unusual pyridoxal 5'-phosphate-dependent reaction. Biochemistry 2011, 50 (11), 1950-62. 31. Clift, M. D.; Ji, H.; Deniau, G. P.; O'Hagan, D.; Silverman, R. B., Enantiomers of 4-amino-3-fluorobutanoic acid as substrates for gamma-aminobutyric acid aminotransferase. Conformational probes for GABA binding. Biochemistry 2007, 46 (48), 13819-28. 32. Gokcan, H.; Konuklar, F. A., Theoretical study on HF elimination and aromatization mechanisms: a case of pyridoxal 5' phosphate-dependent enzyme. J Org Chem 2012, 77 (13), 5533-43. 33. Alunni, S.; De Angelis, F.; Ottavi, L.; Papavasileiou, M.; Tarantelli, F., Evidence of a borderline region between E1cb and E2 elimination reaction mechanisms: a combined experimental and theoretical study of systems activated by the pyridine ring. J Am Chem Soc 2005, 127 (43), 15151-60. 34. Ghalami-Choobar, B.; Dezhampanah, H.; Nikparsa, P.; Ghiami-Shomami, A., Theoretical calculation of the pKa values of some drugs in aqueous solution. International Journal of Quantum Chemistry 2012, 112 (10), 2275-2280.

[0171] Example 2 - DMPK results from Example 1

[0172] [Table 2]

[0173] Example 3 - Supplementary materials for Example 1

[0174] Supplementary table

[0175] [Table 3]

[0176] 4-6 synthesis General Procedure Commercially available reagents and solvents were used without further purification. All reactions were monitored by thin-layer chromatography (TLC) using a 0.25 mm SiliCycle superhardened 250 μM TLC plate (60F254), and spots were visualized under UV (254 nm) and cerium ammonium molybdate or ninhydrin staining. Flash chromatography was performed on a Combi-Flash® Rf system (Teledyne ISCO) using silica columns and reversed-phase C-18 columns. The purity of the final product was measured using an Agilent 1260 series instrument under the following conditions using analytical HPLC: column, Phenomenex Kintex C-18 column (50 × 2.1 mm, 2.6 μm); mobile phase, 5-100% acetonitrile / water containing 0.05% TFA at a flow rate of 0.9 mL / min for 6 minutes; UV detection at 254 nm. The purity of all compounds tested for in vitro biological testing was >95% by HPLC analysis. Using the Bruker AVANCE III 500MHz system and the Bruker NEO console w / QCI-F cryoprobe 600MHz system, 1 H, 13¹¹C and 2D NMR spectra were obtained. The chemical shift was CDCl3( 1 δ = 7.26 in 1H NMR. 13 ¹³C NMR spectrum (δ=77.16), CD3OD( 1 δ=3.31 in 1H NMR. 13 ¹³C NMR spectrum (δ=49.15), DMSO-d6( 1 δ = 2.50 in 1H NMR. 13 The ¹³C NMR spectrum was reported for δ=39.52). The following abbreviations were used for multiplicity: s=singlet, d=doublet, t=triplet, q=quadruplet, m=multilet, dd=doublet, dd=doublet, dt=triplet, dq=triplet-doublet, dd=quadruplet-doublet, ddt=triplet-doublet, ddd=doublet-doublet, dddd=doublet-doublet, ddt=triplet-doublet-doublet, br=broad singlet. Low-resolution mass spectra (LRMS) were obtained using a positive ion mode Thermo TSQ quantum system with atmospheric pressure chemical ionization (APCI) using the aforementioned Agilent Infinity 1260 HPLC system. High-resolution mass spectra (HRMS) were obtained using an Agilent 6210 LC-TOF spectrometer in positive ion mode, following electrospray ionization (ESI) with an Agilent G1312A HPLC pump and an Agilent G1367B automated injector at the Northwestern University Center for Integrated Molecular Structure Education and Research (IMSERC).

[0177] [ka]

[0178] (1R,4S)-2-(4-methoxybenzyl)-2-azabicyclo[2.2.1]hept-5-en-3-one(8) 2(i)p-Anisyl alcohol (10 mL) and concentrated hydrochloric acid (15 mL) were dissolved in a 100 mL round-bottom, three-necked flask at room temperature. The resulting mixture was stirred at room temperature for a further 1 hour. After the reaction was complete, the solution was poured onto ice and extracted with dimethyl phosphate (20 mL x 3). The combined organic layers were separated and washed to saturation. The Na2CO3 solution and brine were dried over Na2SO4 and concentrated under reduced pressure. Fresh 4-methoxybenzyl chloride was obtained as a colorless oil (9.2 g, 52%) and used directly in the next step without further purification. (ii) To a stirred solution of (1R)-(-)-2-azabicyclo[2.2.1]hept-5-en-3-one (1, CAS#79200-56-9, 5.8g, 53 mmol) in dry THF (300 mL), NaH (60%, 3.18 g, 80 mmol) suspended in DMF (30 mL) in an ice bath was added. The resulting mixture was stirred at the same temperature for 30 minutes, then 4-methoxybenzyl chloride (9.2 g, 63.6 mmol) and TBAI (1.96 g, 5.3 mmol) were added at 0°C, and the resulting mixture was slowly warmed to room temperature and stirred for a further 3 hours. After the reaction was complete, the solution was quenched with water (200 mL) and extracted with siRNA (100 mL x 3). The combined organic layers were separated and washed to saturation. The Na2CO3 solution and brine were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by Combi-Flash® chromatography (siRNA / hexane: 0-50%) to obtain a colorless oily substance (8, 7.8 g, 65%). 1 H NMR (500 MHz, CD3OD) δ 7.12 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 6.62 - 6.50 (m, 2H), 4.24 (d, J = 14.6 Hz, 1 H), 4.16 (q, J = 1.9 Hz, 1 H), 4.10 (d, J = 14.6 Hz, 1 H), 3.77 (s, 3H), 3.32 - 3.30 (m, 1 H), 2.25 (dt, J = 7.7, 1.8 Hz, 1 H), 2.07 (dt, J = 7.7, 1.6 Hz,1 H). 13 C NMR (126 MHz, CD3OD) δ 182.5, 160.8, 141.6, 137.6, 130.9 (2C), 129.2, 115.0 (2C), 64.6, 60.0, 55.9, 55.1, 47.8. C 14 H 16 NO2 [M+H] + LRMS (APCI) calculated value: 230.12; measured value: 230.21; TR = 2.28 mins.

[0179] [ka]

[0180] (1R,4R,7R)-7-bromo-2-(4-methoxybenzyl)-2-azabicyclo[2.2.1]heptane-3,6-dione (11). To a stirred solution of oxalyl chloride (3.69 mL, 42.9 mmol) in DCM (200 mL), DMSO (5.0 mL, 70.6 mmol) was slowly added under an argon atmosphere at -78°C. After stirring at -78°C for 10 minutes, 10 (10.0 g, 30.7 mmol) dissolved in DCM (200 mL) was added to the resulting mixture at the same temperature, and the mixture was then stirred at -78°C for another 10 minutes. Next, TEA (30 mL, 215 mmol) was added dropwise. After the addition was complete, the reaction mixture was stirred at -78°C for 10 minutes, warmed to room temperature, and quenched with 1N NH4Cl. The organic layer was separated, washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product was washed with EtOH to obtain 11 (7.8 g, 73%) as a brown solid, which was used in the next step without further purification. 1 H NMR (500 MHz, CDCl3) δ 7.22 - 6.99 (m, 2H), 6.92 - 6.75 (m, 2H), 4.72 (d, J = 14.7 Hz, 1 H), 4.35 (d, J = 2.3 Hz, 1 H), 3.94 (d, J = 14.7 Hz, 1H), 3.80 (s, 3H), 3.66 (d, J = 2.0 Hz, 1 H), 3.16 (dt, J = 4.1, 2.0 Hz, 1 H), 2.71 (dd, J = 17.8, 4.2 Hz, 1 H), 2.22 (dd, J = 17.9, 2.5 Hz, 1 H). 13 C NMR (126 MHz, CDCl3) δ 202.9, 172.6, 159.6, 129.8 (2C), 127.0, 114.4 (2C), 68.4, 55.3, 49.0, 48.2, 45.3, 32.1. C 14 H 15 BrNO3[M+H] + LRMS (APCI) calculated value: 324.02; measured value: 324.33; TR = 2.40 mins.

[0181] [ka]

[0182] (1S,4R)-2-(4-methoxybenzyl)-2-azabicyclo[2.2.1]heptane-3,6-dione (12). To a stirred solution of 11 (6.0 g, 18.5 mmol) in benzene (100 mL), Bn3SnH (7 mL, 26.0 mmol) and AIBN (151 mg, 0.925 mmol) were added at room temperature. The resulting mixture was then heated and refluxed overnight. After the reaction was complete, the solution was quenched with water and extracted with siRNA (100 mL x 3). The combined organic layers were separated, washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by Combi-Flash® chromatography (siRNA / hexane: 0-50%) to obtain 12 (3.1 g, 68%) as an off-white solid. 1 H NMR (500 MHz, CDCl3) δ 7.22 - 7.13 (m, 2H), 6.89 - 6.80 (m, 2H), 4.70 (d, J = 14.8 Hz, 1H), 3.88 (d, J = 14.8 Hz, 1 H), 3.80 (d, J = 1.1 Hz, 3H), 3.56 (q, J = 1.7 Hz, 1 H), 3.03 (qd, J = 2.3, 1.4 Hz, 1 H), 2.30 - 2.22 (m, 1 H), 2.18 (td, J = 4.0, 3.4, 2.1 Hz, 2H), 1.86 (dt, J = 10.7, 1.5 Hz, 1 H). 13 C NMR (126 MHz, CDCl3) δ 206.2, 176.6, 159.4, 129.7 (2C), 127.9, 114.2 (2C), 64.6, 55.3, 45.1, 42.5, 39.1, 35.2. C 14 H 16 NO3 [M+H] + LRMS (APCI) calculated value: 246.11; measured value: 246.10; TR = 1.81 mins.

[0183] [ka]

[0184] (1S,4R)-6,6-difluoro-2-(4-methoxybenzyl)-2-azabicyclo[2.2.1]heptan-3-one (13). 12 (245 mg, 1.0 mmol) was dissolved in THF (3 mL) in a microwave tube, and then Deoxo-Fluor (1.11 mL, 3.0 mmol, 2.7 M in toluene) was added at room temperature. The resulting mixture was heated in a microwave reactor at 120 °C for 40 minutes. After the reaction was complete, the solution was quenched to saturation. The NaHCO3 solution was extracted with ELISA (10 mL × 3). The combined organic layers were separated, washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by Combi-Flash® chromatography (ELISA / hexane: 0-100%) to obtain 13 (190 mg, 71%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 7.26 - 7.12 (m, 2H), 6.99 - 6.73 (m, 2H), 4.99 (d, J = 14.9 Hz, 1 H), 3.81 (s, 3H), 3.78 (s, 1 H), 3.61 (d, J = 2.0 Hz, 1 H), 2.85 (dq, J = 3.9, 1.8 Hz, 1 H), 2.32 - 2.10 (m, 2H), 2.01 (dtd, J = 10.4, 3.6, 1.8 Hz, 1 H), 1.85 (ddd, J = 10.6, 3.8, 1.9 Hz, 1 H). 13 C NMR (126 MHz, CDCl3) δ 175.9, 159.2, 129.5 (2C), 130.05 (t, J = 260.4 Hz), 128.2, 114.2 (2C), 61.47 (dd, J = 32.7, 21.0 Hz), 45.35 (d, J = 2.7 Hz), 43.69 (t, J = 4.0 Hz), 38.86 (d, J = 3.9 Hz), 36.7 (t, J = 23.9 Hz). C 14 H 16 F2NO2[M+H] + The calculated value of LRMS (APCI) is 268.11; the measured value is 268.07; TR=2.36 points.

[0185]

change

[0186] (1S,4R)-6,6-difluoro-2-azabicyclo[2.2.1]heptan-3-one (14). To a stirred solution of 13 (190 mg, 0.71 mmol) in CH3CN (8 mL), an aqueous solution of serine ammonium nitrate (1.16 g, 2.10 mmol in 3 mL of water) was added at room temperature. The resulting mixture was stirred for 2 hours until the starting material was completely gone. The residue was extracted with siRNA (10 mL x 3). The combined organic layers were separated and washed under saturated conditions. The Na2CO3 solution and brine were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by Combi-Flash® chromatography (siRNA / hexane: 0-100%) to obtain 14 (60 mg, 57%) as a white powder. 1 H NMR (500 MHz, CDCl3) δ 5.69 (br s, 1 H), 3.82 (t, J = 1.9 Hz, 1 H), 2.77 (dd, J = 4.5, 2.3 Hz, 1 H), 2.35 - 2.21 (m, 1 H), 2.23 - 2.16 (m, 1 H), 2.17 - 2.03 (m, 1 H), 1.98 (ddq, J = 10.5, 3.6, 1.7 Hz, 1 H). 13 C NMR (126 MHz, CDCl3) δ 179.0, 128.8 (t, J = 258.6 Hz), 59.4 (dd, J = 33.2, 22.6 Hz), 43.6 (t, J = 4.0 Hz), 39.1 (d, J = 3.1 Hz), 36.97 (dd, J = 25.1, 23.4 Hz). C6H8F2NO[M+H] + LRMS (APCI) calculated value: 148.06; measured value: 148.02; TR = 0.33 mins.

[0187] [ka]

[0188] (1R,4S)-ethyl 4-((tert-butoxycarbonyl)amino)-3,3-difluorocyclopentanecarboxylate (15). (i) 14 (150 mg, 1.02 mmol) was dissolved in HCl ethanol solution (2 mL, 1.2 M) at room temperature. The resulting mixture was heated overnight at 85°C in a pressure tube. After the reaction was complete, excess solvent was removed under vacuum. The crude product was obtained as a pale yellow solid and used directly in the next step without further purification. (ii) To a stirred solution of the intermediate in MeOH (5 mL), Boc2O (327 mg, 1.50 mmol) and TEA (0.21 mL, 1.50 mmol) were added at room temperature. The resulting mixture was stirred at room temperature for 2 hours. After the reaction was complete, excess solvent was removed under vacuum, the residue was quenched with water, and extracted with SiO2 (25 mL × 3). The combined organic layers were separated, washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by Combi-Flash® chromatography (Â / hexane: 0-100%) to obtain compound 15 as a white solid (240 mg, 82% in two steps). 1 H NMR (500 MHz, CDCl3) δ 5.14 - 4.64 (m, 1 H), 4.27 - 4.19 (m, 1 H), 4.16 (q, J = 7.2 Hz, 2H), 3.01 - 2.85 (m, 1 H), 2.56 - 2.32 (m, 3H), 1.77 (dtd, J = 12.1, 10.0, 1.8 Hz, 1 H), 1.45 (s, 9H), 1.26 (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CDCl3) δ 173.5, 155.1, 127.2 (dd, J = 255.6, 250.9 Hz), 80.2, 61.7, 55.2 (dd, J = 26.0, 20.6 Hz), 36.3, 35.8 (t, J = 25.8 Hz), 32.9 (d, J = 4.5 Hz), 28.3 (3C), 14.1. C8H 14 F2NO2[M-Boc+2H] +LRMS (APCI) calculated value: 194.10; measured value: 193.99; TR = 2.67 mins.

[0189] [ka]

[0190] (S)-ethyl 4-((tert-butoxycarbonyl)amino)-3,3-difluorocyclopenta-1-enecarboxylate (16). To a stirred solution of (100 mg, 0.34 mmol) of (16) in THF (8 mL), KHMDS (2.0 mL, 0.5 M in toluene) was added at -78°C. The resulting mixture was stirred at -78°C for 2 hours, and then phenyl chloride (78 mg, 0.41 mmol) was added in a solution of THF (2 mL) at temperature. The reaction mixture was slowly warmed to room temperature and stirred for a further 6 hours. The reaction mixture was quenched with saturated aqueous solution NH4Cl and extracted with siRNA (10 mL x 3). The combined organic layers were separated, washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by Combi-Flash® chromatography (siRNA / hexane: 0-100%) to obtain (20 mg, 20%) 16 as a white solid. 1 H NMR (500 MHz, CDCl3) δ 6.60 (s, 1 H), 4.91 (d, J = 9.0 Hz, 1 H), 4.57 - 4.53 (m, 1 H), 4.26 (q, J = 7.1 Hz, 2H), 3.17 (dt, J = 16.1, 7.4 Hz, 1 H), 2.42 (ddt, J = 14.8, 6.1, 3.0 Hz, 1 H), 1.47 (s, 9H), 1.32 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 163.1, 155.1, 143.1 (t, J = 10.1 Hz), 132.44 (dd, J = 30.7, 24.6 Hz), 127.2 (t, J = 251.0 Hz), 80.4, 61.5, 54.5 (t, J = 20.9 Hz), 35.8, 28.3 (3C), 14.1. C8H 12 F2NO2[M-Boc+2H] + LRMS (APCI) calculated value: 192.08; measured value: 191.96; TR = 2.71 mins.

[0191] [ka]

[0192] (S)-4-amino-3,3-difluorocyclopenta-1-enecarboxylic acid (5) To a stirred solution of 16 (20 mg, 0.07 mmol) in AcOH (1 mL), 4N HCl (1 mL) was added at room temperature. The resulting mixture was heated overnight at 70°C. After the reaction was complete, excess solvent was removed under vacuum. The crude product was purified by Combi-Flash® chromatography (C18 reverse column, CH3CN / H2O: 0-5%) to obtain 5 as a white powder hydrochloride (10 mg, 71%). 1 H NMR (600 MHz, CD3OD) δ 6.65 (s, 1 H), 4.22 (dq, J = 13.2, 7.1, 6.7 Hz, 1 H), 3.28 - 3.21 (m, 1 H), 2.80 - 2.64 (m, 1 H). 13C NMR (126 MHz, CD3OD) δ 165.23, 145.84 (dd, J = 11.3, 9.5 Hz), 131.65 (dd, J = 29.4, 25.5 Hz), 129.21 (t, J = 248.6 Hz), 54.50 (dd, J = 31.3, 20.0 Hz), 34.64 (d, J = 2.2 Hz). C6H8FNO2[M+H] + HRMS(ESI) calculated value: 164.0518; measured value: 164.0516.

[0193] [ka]

[0194] (1R,4S)-4-amino-3,3-difluorocyclopentanecarboxylate (4). 14 (60 mg, 0.41 mmol) was dissolved in 4N HCl (4 mL) at room temperature. The resulting mixture was heated at 70°C for 1 hour. After the reaction was complete, excess solvent was removed under vacuum. The crude product was purified by Combi-Flash® chromatography (C18 reverse column, CH3CN / H2O: 0-5%) to obtain 4 as a white powder hydrochloride (60 mg, 89%). 1 H NMR (500 MHz, CD3OD) δ 3.93 (qd, J = 11.4, 7.8 Hz, 1 H), 3.16 (tt, J = 10.0, 8.2 Hz, 1 H), 2.69 - 2.47 (m, 3H), 2.04 (dtd, J = 12.3, 10.7, 1.4 Hz, 1 H). 13 C NMR (126 MHz, CD3OD) δ 175.5, 128.3 (dd, J = 255.0, 252.6 Hz), 55.8 (dd, J = 29.6, 20.7 Hz), 37.9 (t, J = 4.5 Hz), 37.2 (t, J = 24.8 Hz), 31.9 (d, J = 4.8 Hz). C6H 10 FNO2[M+H] +HRMS(ESI) calculated value: 166.0674; measured value: 166.0671.

[0195] [ka]

[0196] (1S,4S,7R)-2-(4-methoxybenzyl)-7-(methoxymethoxy)-2-azabicyclo[2.2.1]heptane-3,6-dione (21) was prepared from 20 (12.0 g, 39 mmol) according to the synthetic procedure in 12, and obtained as a colorless oil (10.8 g, 91%). 1 H NMR (500 MHz, CDCl3) δ 7.20 - 7.04 (m, 2H), 6.97 - 6.77 (m, 2H), 4.64 (d, J = 6.9 Hz, 1 H), 4.62 (d, J = 14.8 Hz, 1 H), 4.59 (d, J = 6.9 Hz, 1 H), 4.17 (q, J = 2.2 Hz, 1 H), 4.02 (d, J = 14.8 Hz, 1 H), 3.80 (s, 3H), 3.59 (t, J = 1.9 Hz, 1 H), 3.30 (d, J = 1.7 Hz, 3H), 3.11 - 3.05 (m, 1 H), 2.51 (dd, J = 17.5, 4.3 Hz, 1 H), 2.14 (dd, J = 17.4, 1.9 Hz, 1 H). 13 C NMR (126 MHz, CDCl3) δ 206.0, 173.4, 159.4, 129.7 (2C), 127.7, 114.3 (2C), 96.2, 80.5, 66.6, 56.3, 55.3, 46.9, 45.0, 31.5. C 16 H 20 NO5 [M+H] + LRMS (APCI) calculated value: 306.13; measured value: 306.39; TR = 2.36 mins.

[0197] [ka]

[0198] (1S,4S,7R)-6,6-difluoro-2-(4-methoxybenzyl)-7-(methoxymethoxy)-2-azabicyclo[2.2.1]-heptan-3-one (18) was synthesized from 17 (670 mg, 2.20 mmol) according to the synthetic procedure in 13, and obtained as a brown oily substance (490 mg, 72%). 1 H NMR (500 MHz, CDCl3) δ 7.15 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 5.02 (d, J = 14.9 Hz, 1 H), 4.59 (s, 3H), 4.03 (dt, J = 4.7, 2.0 Hz, 1 H), 3.80 (s, 3H), 3.79 (d, J = 14.9 Hz, 1 H), 3.58 (q, J = 1.8 Hz, 1 H), 3.48 - 3.34 (m, 2H), 3.33 (s, 3H), 2.96 - 2.84 (m, 1 H), 2.57 (dddd, J = 18.0, 13.8, 9.4, 4.3 Hz, 1 H), 2.16 (dddd, J = 15.9, 13.7, 6.2, 1.7 Hz, 1 H). 13 C NMR (126 MHz, CDCl3) δ 172.5, 159.3, 129.72 (dd, J = 274, 254 Hz), 129.6 (2C), 127.8, 114.3 (2C), 95.9, 81.2 (d, J = 4.0 Hz), 62.4 (dd, J = 29.9, 20.3 Hz), 56.2 (d, J = 1.7 Hz), 55.3, 48.5 (t, J = 3.8 Hz), 45.3 (d, J = 2.6 Hz), 34.8 (t, J = 24.7 Hz). C 16 H 20F2NO4[M+H] + LRMS (APCI) calculated value: 328.14; measured value: 328.46; TR = 2.58 mins.

[0199] [ka]

[0200] (1S,4S,7R)-6,6-difluoro-7-(methoxymethoxy)-2-azabicyclo[2.2.1]heptan-3-one (23). To a stirred solution of 22 (900 mg, 2.75 mmol) in CH3CN (40 mL), an aqueous solution of ammonium serine nitrate (4.52 g, 8.26 mmol) was added at room temperature. The resulting mixture was stirred for 2 hours until the starting materials were completely gone. The residue was extracted with siRNA (25 mL x 3). The combined organic layers were separated and washed under saturated conditions. The Na2CO3 solution and brine were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by Combi-Flash® chromatography (siRNA-hexane: 0-100%) to obtain 23 (340 mg, 60%) as a pale yellow solid. 1 H NMR (500 MHz, CDCl3) δ 5.91 (s, 1 H), 4.70 (s, 2H), 4.29 - 4.16 (m, 1 H), 3.83 (q, J = 2.0 Hz, 1 H), 3.41 (s, 3H), 2.84 (dt, J = 3.9, 1.9 Hz, 1 H), 2.62 (dddd, J = 18.3, 13.6, 8.9, 4.4 Hz, 1 H), 2.13 (dddd, J = 15.8, 13.8, 6.2, 1.8 Hz, 1 H). 13C NMR (126 MHz, CDCl3) δ 175.0, 128.2 (dd, J = 263, 253 Hz), 96.2, 81.7 (d, J = 3.3 Hz), 60.5 (dd, J = 30.5, 21.6 Hz), 56.24 (d, J = 1.8 Hz), 48.66 (dt, J = 4.1, 1.5 Hz), 34.11 (dd, J = 25.7, 24.2 Hz). C8H 12 F2NO3[M+H] + LRMS (APCI) calculated value: 208.08; measured value: 2008.07; TR = 0.79 mins.

[0201] [ka]

[0202] (1S,2R,3S)-methyl 3-((tert-butoxycarbonyl)amino)-4,4-difluoro-2-hydroxycyclopentanecarboxylate (24). (i) 23 was dissolved in HCl methanol solution (1.2 M) at room temperature. The resulting mixture was then heated overnight at 85°C in a pressure tube. After the reaction was complete, the excess solvent was removed under vacuum, and the residue was neutralized to saturation. The NaHCO3 solution was extracted with ELISA (25 mL × 3). The combined organic layers were separated, washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product was obtained as a pale yellow solid (160 mg, 70%) and used directly in the next step without further purification. (ii) Boc2O (268 mg, 1.23 mmol) was added at room temperature to a stirred solution of the intermediate (160 mg, 0.82 mmol) in MeOH (10 mL). The resulting mixture was then stirred overnight at room temperature. After the reaction was complete, excess solvent was removed under vacuum, and the residue was then quenched with water and extracted with RINKAN (25 mL x 3). The combined organic layers were separated, washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure to obtain 24 as an off-white solid (230 mg, 95%), which was used directly in the next step without further purification. 1 H NMR (500 MHz, CDCl3) δ 5.07 (s, 1H), 4.19 (t, J = 8.4 Hz, 1 H), 4.13 (s, 1 H), 4.09 - 3.98 (m, 1 H), 3.76 (s, 3H), 2.92 (q, J = 9.4 Hz, 1 H), 2.67 - 2.39 (m, 2H), 1.46 (s, 9H). 13 C NMR (126 MHz, CDCl3) δ 172.5, 156.7, 124.4 (t, J = 254 Hz), 81.2, 77.8 (d, J = 6.9 Hz), 62.9 (dd, J = 25.2, 17.9 Hz), 52.6, 44.6, 35.0 (t, J = 26.0 Hz), 28.2 (3C). C7H 12 F2NO3[M-Boc+2H] + LRMS (APCI) calculated value: 196.08; measured value: 196.03; TR = 2.20 mins.

[0203] [ka]

[0204] (S)-methyl 3-(((tert-butoxycarbonyl)amino)-4,4-difluorocyclopenta-1-enecarboxylate (25). To a stirred solution of 24 (230 mg, 0.78 mmol) in THF (5 mL), Burgess reagent (580 mg, 2.44 mmol) was added at room temperature under an argon atmosphere. The resulting mixture was then heated under reflux for 4 hours. After the reaction was complete, the solution was quenched with water and extracted with RINKAN (25 mL x 3). The combined organic layers were separated, washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by Combi-Flash® chromatography (RINKAN / hexane: 0-100%) to obtain 25 (110 mg, 49%) as an off-white solid. C7H 10 F2NO2[M-Boc+2H] + The calculated LRMS (APCI) value was 178.07; the measured value was 177.86; TR = 2.65 minutes.

[0205] [ka]

[0206] (S)-3-amino-4,4-difluorocyclopenta-1-enecarboxylate (SS-1-148,6). 200 mg (0.72 mmol) of 25 in acetic acid (10 mL) was stirred, and 10 mL of 4N HCl was added at room temperature. The resulting mixture was heated overnight at 70°C. After the reaction was complete, excess solvent was removed under vacuum. The crude product was purified by Combi-Flash® chromatography (C18 reverse column, CH3CN / H2O: 0-5%) to obtain 6 as a white powder HCl salt (72 mg, 50%). 1 H NMR (500 MHz, CD3OD) δ 6.58 (s, 1 H), 4.77 - 4.67 (m, 1 H), 3.29 - 3.16 (m, 2H). 13 C NMR (126 MHz, CD3OD) δ 165.2, 140.0, 133.4, 128.0 (t, J = 254.8 Hz), 61.0 (dd, J = 35.5, 20.7 Hz), 41.64 (t, J = 27.1 Hz). C6H8FNO2[M+H] + HRMS(ESI) calculated value: 164.0518; measured value: 164.0517.

[0207] hOAT expression and purification hOAT was expressed and purified using a previously published protocol. Briefly, E. coli BL21(DE3) cells containing the pMAL-t-hOAT plasmid were incubated at 37°C with shaking in Lysogeny Broth (LB) medium supplemented with 100 μg / mL ampicillin. When the culture OD600 reached 0.7, the expression of the MBP-t-hOAT fusion protein was induced by adding 0.3 mM isopropyl β-D-1-thiogalactopyranoside. The cells were then incubated at 25°C for a further 16-18 hours, harvested by centrifugation, washed with buffer A consisting of 20 mM Tris-HCl, 200 mM NaCl, and 100 μM PLP, pH 7.4, flash-frozen in liquid nitrogen, stored at -80°C, and then thawed. The frozen cell pellet was sonicated in buffer A and centrifuged at 40,000 × g for 20 minutes. The obtained supernatant was loaded onto an amylose affinity column pre-equilibrated with buffer A, the column was thoroughly washed, and the MBP-t-hOAT fusion protein was eluted from the column with 10 mM maltose. The fractions containing the fusion protein were combined and treated with TEV protease to remove the MBP tag. The cleaved hOAT protein was collected and concentrated using a centrifugal filter. Next, the protein was further purified by size exclusion chromatography using a HiLoad Superdex-200PG column. The column was pre-equilibrated, and the protein was eluted in a buffer containing 50 mM HEPES, 100 μM PLP, and 300 mM NaCl, pH 7.5.

[0208] Aminotransferases and coenzymes for kinetic studies All reagents for enzyme purification and assay were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Human OAT (0.672 mg / mL) was purified from E. coli BL21 (DE3) cells according to the procedure described above, and the coenzyme human recombinant pyrroline 5-carboxylate reductase (PYCR) was used. 1The enzymes were expressed, grown, and purified according to literature procedures. γ-aminobutyric acid aminotransferase (GABA-AT, 0.4 mg / mL) was purified from pig brain according to the procedure described above, and the coenzyme succinate semialdehyde dehydrogenase (SSDH) was purified from GABase (catalog number G7509-25UN, Sigma-Aldrich). A commercially available mixture of SSDH and GABA-AT was used according to known procedures. Commercially available L-aspartate aminotransferase (Asp-AT) (catalog no. G2751-2KU, Sigma-Aldrich) derived from porcine heart and malate dehydrogenase (catalog no. M2634-5KU, Sigma-Aldrich), a desired assay coenzyme derived from porcine, and L-alanine aminotransferase (Ala-AT) (catalog no. G8255-200UN, Sigma-Aldrich) derived from porcine heart and lactate dehydrogenase (catalog no. 427211-50KU, Sigma-Aldrich), a desired assay coenzyme derived from porcine, were used directly without further purification. All enzyme assays for inactivation, partition ratio, and dialysis experiments were recorded using a Synergy H1 hybrid multimode microplate reader (Biotek, USA) with clear 96-well plates.

[0209] Evaluation of compounds that act as time-dependent inhibitors of hOAT Time-dependent inactivation of hOAT was performed using a previously published optimized procedure. 10 μL of hOAT (0.025 mg / mL) was pre-incubated in a 96-well plate at 25°C in 100 mM potassium pyrophosphate buffer (pH 8.0, 5 mM α-ketoglutaric acid, and 5 mM β-mercaptoethanol) with 10 μL of various concentrations of test compounds. At time intervals, the PYCR in the potassium pyrophosphate buffer was incubated. 180 μL of an assay solution containing (0.5 μg), 1 mM NADH, and 25 mM L-ornithine was added to the incubation mixture, and OAT activity was assayed at 37°C for 30 minutes. The change in UV absorbance at 340 nm at 37°C based on the conversion of NADH to NADOA was monitored using a microplate reader. All assays were performed in duplicate, and the remaining OAT activity at each pre-incubation time for each inhibitor concentration was averaged. The natural logarithm of the percentage of remaining OAT activity was plotted against the pre-incubation time for each inhibitor concentration to obtain the k for each concentration. obs The (slope) value was obtained. obs The value is the rate constant representing inactivation at each inhibitor concentration. obs This is then re-implanted against the inhibitor concentration using nonlinear regression analysis with GraphPad Prism 8.0. I and k inact The value is given by equation (1)

[0210]

number

[0211] It is estimated from this, where k inact This is the maximum rate of inactivation, and K I [I] is the inhibitory concentration required for up to half of the inactivation, and [I] is the pre-incubation concentration of the test compound.

[0212] Evaluation of compounds as reversible inhibitors of GABA-AT Inhibition constant (K iThe activity of GABA-AT was determined by monitoring GABA-AT activity in the presence of 0–20 mM concentrations of the test compound using an assay combined with SSDH. An 80 μL assay mixture containing excess SSDH, 1 mM NADP+, 11.1 mM GABA, and 10 μL of various concentrations of the test compound was loaded into a 96-well plate. After incubation of the mixture at 25°C for 1 minute, 10 μL of GABA-AT (0.03 mg / mL) in the potassium pyrophosphate buffer was added to the 96-well plate. The microplate was shaken at 25°C for 1 minute, and the remaining enzyme activity was determined by observing the change in absorbance at 340 nm at 25°C based on the conversion of NADPOA to NADPH. Nonlinear regression analysis using GraphPad Prism 8.0 was used to determine the semi-maximal inhibitory concentration (IC1). 50 The value was obtained. The subsequent Ki value was determined using equation (2). All assays were performed twice.

[0213]

number

[0214] Here, [S] is the final concentration of the substrate GABA (8.88 mM), and Km is the Michaelis-Menten constant, which was determined to be 2.60 mM for GABA-AT in this batch isolated from pig brain.

[0215] Inhibition of Asp-AT and Ala-AT by SS-1-148 A 90 μL assay mixture containing 100 mM potassium phosphate, 5.55 mM α-ketoglutaric acid, 1.11 mM NADH, 5.55 mM L-aspartic acid, 5.55 mM malate dehydrogenase, and various concentrations of SS-1-148 was loaded into a 96-well plate at pH 7.4. After incubating the mixture at room temperature for several minutes, 10 μL of Asp-AT (2.0 units / mL in 100 mM potassium phosphate at pH 7.4) was added. The plate was shaken at room temperature for 1 minute, and absorbance was measured at 340 nm every 10 seconds for 90 minutes based on the conversion of NADH to NADOA. The remaining Asp-AT activity percentage was calculated according to the corresponding concentration. All assays were performed twice. The assay conditions for determining the inhibition of Ala-AT by SS-1-148 were the same as those for L-aspartic acid, except that L-alanine was used as the substrate and lactate dehydrogenase as the coenzyme.

[0216] Dialysis assay The dialysis experiment was performed using the previous protocol. 8-10 .

[0217] Distribution ratio experiment The distribution ratio was calculated using the previous protocol. 8-10 .

[0218] Fluoride ion release assay The fluoride ion release assay was performed using a previous protocol. 10 The final enzyme concentration in the samples was measured via the BCA protein assay kit (Pierce, cat:23225). Calibration curves for voltage (V, mV) were created from various concentrations of NaF (F, μM). For accurate detection of fluoride ion concentration, 10 μM fluoride ions were added to each control and sample. The number of fluoride ions released per active site was calculated by the ratio of fluoride ion release concentration to enzyme concentration.

[0219] Natural Protein Mass Spectrometry Processed and unmodified hOAT samples in a D-Tube® Dialyzer Mini (MWCO12-14KDa; Lot#:345246; Millipore) were separately desalted at 4°C for 96 hours by dialysis with 100 mM NH4OAc. The desalted samples were separately introduced into a Q-Exactive Ultra High Mass Range (UHMR) (Thermo Fisher Scientific) mass spectrometer equipped with a Nanospray Flex Ion Source (Thermo Fisher Scientific) with a spray voltage of +1500 to +2000V. The ion transfer tube was set to 310°C. The mass spectrometer was operated in +ESI mode. Full scan data collected from 4000 to 10,000 m / z with 200 microscans were used, along with target AGC with Orbitrap resolution of 35,000 or 17,500 (at 200 m / z) and a 1e6 charge. The S-lens RF level was set to 200%, and the extended trapping was set to 100. HCD collision activation was applied at normalized collision energies (NCE) set to 5–15 to release adducts from protein dimers. Raw spectra from each experiment were summed across multiple scans. Mass deconvolution was performed using mMass and / or UniDec to generate zero-charge mass and associated mass standard deviation.

[0220] Intact Protein Mass Spectrometry and Metabolism Purified hOAT samples, both treated and unmodified, were desalted 10 times with optimal grade water (Fisher) on an Amicon Ultra 10kDa molecular weight spin filter (Millipore). To separate the protein chromatographically, 0.5 μg of protein was loaded onto a 3 cm PLRP-S (Agilent) trap column using a Dionex Ultimate3000 liquid chromatography system (Thermo Fisher). The protein analytes were washed with an isocratic gradient of 10% solvent B (95% acetonitrile / 5% H2O / 0.2% formic acid) and 90% solvent A (5% acetonitrile / 95% H2O / 0.2% formic acid) for 10 minutes. The proteins were separated using a homemade nanopore capillary column packed with PLRP-S resin (Agilent), with a length of 75 μm and an ID of 15 cm. The LC system was operated at a flow rate of 0.3 μL / min with the following gradient: 0-10 min, 10% solvent B; 10-12 min, ~40% solvent B; 12-22 min, ~90% solvent B; 22-24 min, 90% solvent B; 24-26 min, ~10% solvent B; 26-30 min, isocratic with 10% solvent B for 26-30 minutes. The hOAT sample was introduced into a Thermo Fischer Orbitrap Fusion Lumos or Eclipse mass spectrometer, and all MS data were acquired as described above. As described above, the masses of small molecules and metabolites were identified on the Q-Exactive Orbitrap mass spectrometer (Thermo) and characterized by high-resolution LC-MS / MS in positive and negative modes.

[0221] Crystallization and crystal immersion of hOAT using SS-1-148

[0263] hOAT crystal immersion. Holoenzyme crystals were first grown by the suspended droplet vapor diffusion method. Each drop contained 2 μL of protein and 2 μL of well solution. The best crystallization conditions were 10% PEG8000, 200 mM NaCl, and 10% glycerol. When the holoenzyme crystals reached their maximum size, 2 μL of 16 mM SS-1-148 was added to the crystals in the droplet. Within the first 5 minutes of adding SS-1-148, the hOAT crystals changed color from yellow to blue. The crystals were immersed for 1 hour, transferred to a cryoprotection solution (well solution with 30% glycerol added), and then flash-frozen in liquid nitrogen.

[0222] hOAT cocrystallization. After purification, hOAT was buffer-exchanged in crystallization buffer (50 mM tricine, pH 7.8) supplied with 5 mM α-ketoglutaric acid. Next, the protein was concentrated to 6 mg / mL. Previously reported crystallization conditions. 8 The protein was optimized using the suspended droplet vapor diffusion method by maintaining a constant buffer solution of PEG6000 (8-12%), NaCl (100-250 mM), glycerol (0-10%), and 100 mM trichine at pH 7.8. For each suspended drop, 2 μL of the protein solution was mixed with an equal volume of well solution and 0.5 μL of 10 mM SS-1-148. Crystals with the best morphology and size grew under the final conditions containing 10% PEG8000, 200 mM NaCl, and 10% glycerol. The crystals were transferred to a cryoprotection solution (well solution with 30% glycerol added) and then flash-frozen in liquid nitrogen.

[0223] X-ray diffraction and data processing. Monochromatic X-ray diffraction data was collected at the LS-CAT beamline 21-ID-D of the Advanced Photon Source at Argonne National Laboratory. Data was collected using a Dectris Eiger 9M detector at a wavelength of 1.127 Å and a temperature of 100 K. The dataset was processed and analyzed using autoPROC software.

[0224] Model construction and refinement. The hOAT structure was solved by molecular substitution using Phaser in Phenix. The initial search model was the previously published hOAT structure (PDB code: 1OAT). Model construction and refinement were achieved in Coot and Phenix, respectively, as an iterative process until the lowest Rfree / R factor values ​​were achieved. A structural diagram was created using UCSF chimeras. 19 .

[0225] Transient state method. The reaction between SS-1-148 and hOAT was observed in transient conditions using a combination of a Hitech Scientific (TgK) stopped-flow spectrophotometer and charge-coupled device detection (260-800 nm). hOAT (12.68 μM) was reacted with SS-1-148 at various concentrations (125, 251, 502, 1004, 2008, 4016, 8032 μM) in a buffer solution (pH 7.5) containing 50 mM HEPES and 200 mM NaCl at 10°C. For each inhibitor concentration, CCD spectral data sets were collected overlapping for 50 seconds, and the overlaps were averaged. For the highest inhibitor concentration (8 mM), overlapping CCD data sets were obtained for two time frame periods (0.0025-12.4 seconds and 0.0025-2480 seconds). The replicated datasets for any given time frame were averaged and then spliced ​​together at 12 seconds to form a single dataset with sufficient temporal resolution to adequately describe rapid and slow processes. The hybrid dataset was deconvoluted based on a linear quaternary model using the Spectrafit singular value decomposition module of KinTek Explorer software. In this process, the rate constant was fixed to a value measured using analytical fitting to the data extracted for a single wavelength assuming a continuous first-order process according to equations (3), (4), and (5). Antibody is absorbance, Ax is the amplitude associated with each observed phase, kx is the corresponding rate constant, and C is the absorbance endpoint.

[0226]

number

[0227] Gibbs free energy calculation MOPAC 2016 is computational chemistry software based on quantum theory and thermodynamic concepts, employing several advanced mathematical concepts. It is a semi-empirical molecular orbital package used for studying solid states, nanostructures, molecular structures, and their reactions. 20-21In this context, MOPAC 2016 was used to establish the molecular geometry of each tautomer form of M7 (Figure 14) and subsequently to optimize it through the PM7 semi-empirical method. Solvation by water molecules was not considered in the calculations. For the bond free energy of each tautomer shown in Figure 14, the combination of molecular mechanical energy, polar and nonpolar energy, and entropy was considered. Enthalpy (ΔH o ) and entropy (ΔS o ) represents the ΔG of each tautomer. o The value contributes to the Gibbs free energy equation, ΔG o =ΔH o -TΔS o It is determined by [the following].

[0228] Theoretical pK a calculation The geometry of the neutral and deprotonated species M1, M1', and M1'' is based on the theoretical DFT B3LYP / 6-31G. ** It was fully optimized using levels. For all compounds examined, the change in the gas phase Gibbs free energy of the compound (ΔG) was calculated. o g) was calculated using the Gaussian09 software. 22 The solvation free energy is calculated using the same level theory and basis set (B3LYP / 6-31G). ** The calculations were performed by applying the polarizable continuum model (PCM) using ). The PCM calculations were used along with the UAHF atomic radius when calculating the Gibbs free energy of solvation when constructing the solvent cavity. The pKa values ​​were obtained by applying the following equations (6), (7), and (8) and thermodynamic cycle A presented by Ghalami-Choobar et al.

[0229]

number

[0230] Electrostatic potential (ESP) charge calculation Three-dimensional (3D) molecular models of M1, M1', and M1'' were constructed using Spartan'14 software (Wavefuction, Inc., 2014). The constructed structures were refined using molecular mechanics with the Merck molecular force field (MMFF94). Next, the lowest energy conformational isomer was selected, and the equilibrium geometry and molecular orbitals were set to 6-31G. * The calculations were performed using the theoretical Hartree-Fock (HF) model. Spartan'14 was also used to generate electron density and electrostatic potential maps.

[0231] Abbreviation Boc2O, di-tert-butyl dicarbonate; Deoxo-Fluor, bis(2-methoxyethyl)aminosulfur trifluoride; DMPK, drug metabolism and pharmacokinetics; DIPEA, N,N-diisopropylethylamine; DBDMH, 1,3-dibromo-5,5-dimethylhydantoin; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DCM, dichloromethane; THF, tetrahydrofuran; TEA, triethylamine; TBAI, tetra-n-butylammonium iodide.

[0232] References 1. Lu, H.; Silverman, RB, Fluorinated conformationally restricted gamma-aminobutyric acid aminotransferase inhibitors. J Med Chem 2006, 49 (25), 7404-12. 2. Shen, S.; Doubleday, P. F.; Weerawarna, P. M.; Zhu, W.; Kelleher, N. L.; Silverman, R. B., Mechanism-Based Design of 3-Amino-4-Halocyclopentenecarboxylic Acids as Inactivators of GABA Aminotransferase. ACS Med Chem Lett 2020, 11 (10), 1949-1955. 3. Mascarenhas, R.; Le, H. V.; Clevenger, K. D.; Lehrer, H. J.; Ringe, D.; Kelleher, N. L.; Silverman, R. B.; Liu, D., Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover. Biochemistry 2017, 56 (37), 4951-4961. 4. Christensen, E. M.; Patel, S. M.; Korasick, D. A.; Campbell, A. C.; Krause, K. L.; Becker, D. F.; Tanner, J. J., Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1. J Biol Chem 2017, 292 (17), 7233-7243. 5. Koo, Y. K.; Nandi, D.; Silverman, R. B., The multiple active enzyme species of gamma-aminobutyric acid aminotransferase are not isozymes. Arch Biochem Biophys 2000, 374 (2), 248-54. 6. Silverman, R. B.; Bichler, K. A.; Leon, A. J. J. Am. Chem. Soc. 1996, 118 (6), 1241-52. 7. Juncosa, J. I.; Lee, H.; Silverman, R. B., Two continuous coupled assays for ornithine-delta-aminotransferase. Anal Biochem 2013, 440 (2), 145-9. 8. Moschitto, M. J.; Doubleday, P. F.; Catlin, D. S.; Kelleher, N. L.; Liu, D.; Silverman, R. B., Mechanism of Inactivation of Ornithine Aminotransferase by (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)cyclopentane-1-carboxylic Acid. J Am Chem Soc 2019, 141 (27), 10711-10721. 9. Juncosa, J. I.; Takaya, K.; Le, H. V.; Moschitto, M. J.; Weerawarna, P. M.; Mascarenhas, R.; Liu, D. L.; Dewey, S. L.; Silverman, R. B., Design and Mechanism of (S)-3-Amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic Acid, a Highly Potent gamma-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Addiction. J Am Chem Soc 2018, 140 (6), 2151-2164. 10. Lee, H.; Doud, E. H.; Wu, R.; Sanishvili, R.; Juncosa, J. I.; Liu, D. L.; Kelleher, N. L.; Silverman, R. B., Mechanism of Inactivation of gamma-Aminobutyric Acid Aminotransferase by (1S,3S)-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115). J Am Chem Soc 2015, 137 (7), 2628-2640. 11. Niedermeyer, T. H.; Strohalm, M., mMass as a software tool for the annotation of cyclic peptide tandem mass spectra. PLoS One 2012, 7 (9), e44913. 12. Marty, M. T.; Baldwin, A. J.; Marklund, E. G.; Hochberg, G. K.; Benesch, J. L.; Robinson, C. V., Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal Chem 2015, 87 (8), 4370-6. 13. Zhu, W.; Doubleday, P. F.; Catlin, D. S.; Weerawarna, P. M.; Butrin, A.; Shen, S.; Wawrzak, Z.; Kelleher, N. L.; Liu, D.; Silverman, R. B., A Remarkable Difference That One Fluorine Atom Confers on the Mechanisms of Inactivation of Human Ornithine Aminotransferase by Two Cyclohexene Analogues of gamma-Aminobutyric Acid. J Am Chem Soc 2020, 142 (10), 4892-4903. 14. Vonrhein, C.; Flensburg, C.; Keller, P.; Sharff, A.; Smart, O.; Paciorek, W.; Womack, T.; Bricogne, G., Data processing and analysis with the autoPROC toolbox. Acta Crystallogr D Biol Crystallogr 2011, 67 (Pt 4), 293-302. 15. McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J., Phaser crystallographic software. J Appl Crystallogr 2007, 40 (Pt 4), 658-674. 16. Shen, B. W.; Hennig, M.; Hohenester, E.; Jansonius, J. N.; Schirmer, T., Crystal structure of human recombinant ornithine aminotransferase. J Mol Biol 1998, 277 (1), 81-102. 17. Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K., Features and development of Coot. Acta Crystallogr D Biol Crystallogr 2010, 66 (Pt 4), 486-501. 18. Liebschner, D.; Afonine, P. V.; Baker, M. L.; Bunkoczi, G.; Chen, V. B.; Croll, T. I.; Hintze, B.; Hung, L. W.; Jain, S.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R. D.; Poon, B. K.; Prisant, M. G.; Read, R. J.; Richardson, J. S.; Richardson, D. C.; Sammito, M. D.; Sobolev, O. V.; Stockwell, D. H.; Terwilliger, T. C.; Urzhumtsev, A. G.; Videau, L. L.; Williams, C. J.; Adams, P. D., Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol 2019, 75 (Pt 10), 861-877. 19. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E., UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 2004, 25 (13), 1605-12. 20. Dewar, M. J. S.; Thiel, W., Ground states of molecules. 39. MNDO results for molecules containing hydrogen, carbon, nitrogen, and oxygen. Journal of the American Chemical Society 1977, 99 (15), 4907-4917. 21. Stewart, J. J. P., Optimization of parameters for semiempirical methods VI: more modifications to the NDDO approximations and re-optimization of parameters. Journal of Molecular Modeling 2013, 19 (1), 1-32. 22. Gaussian 09, Revision A.02, MJ Frisch, GW Trucks, HB Schlegel, GE Scuseria, MA Robb, JR Cheeseman, G. Scalmani, V. Barone, GA Petersson, H. Nakatsuji, X. Li, M. Caricato, A. Marenich, J. Bloino, BG Jannucci, R. Gomper, B. Menucci, HP. Hratchian, JV Ortiz, AF Izmaylov, JL Sonnenberg, D. Williams-Young, F. Ding, F. Lipparini, F. Egidi, J. Goings, B. Peng, A. Petrone, T. Henderson, D. Ranasinghe, VG Zakrzewski, J. Gao, N. Rega, G. Zheng, W. Liang, M. Hada, M. Hada, E. K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, K. Throssell, JA Montgomery, Jr., JE Peralta, F. Ogliaro, M. Bearpark, JJ Heyd, E. Brothers, K. N. Keverdin, K. N. Keverdin, Keverdin, T. N. R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, JC Burant, SS Iyengar, J. Tomasi, M. Cossi, JM Millam, M. Klene, J. Adamo, R. Cammi, JW Ochterski, RL Martin, K. Morokuma, O. Farkas, JB Foresman, and DJFox, Gaussian, Inc., Wallingford CT, 2016. 23. Ghalami-Choobar, B.; Dezhampanah, H.; Nikparsa, P.; Ghiami-Shomami, A., Theoretical calculation of the pKa values ​​of some drugs in aqueous solution. International Journal of Quantum Chemistry 2012, 112 (10), 2275-2280.

[0233] According to this disclosure, various other compounds that change structurally, stereochemically, and / or conformally are available by modifications known and understood to those skilled in the art, such procedures, techniques, and modifications, such as incorporated synthetic methods and techniques, or direct modifications thereof, limited only by the commercial or synthetic availability of any corresponding reagents or starting materials.

Claims

1. The following formula: 【Chemistry 1】 [During the ceremony, The double bond is located between the α-carbon and ε-carbon, or between the α-carbon and β-carbon. R １ and R ２ Each of these is independently selected from halogens such as F, Cl, Br, and I. The compound, or its dissociated form, aprotonated form, zwitterionic form, or salt.

2. The compound according to claim 1, which is a zwitterionic form containing an ammonium moiety and a carboxylate moiety.

3. The compound according to claim 1, wherein the double bond is located between the α-carbon and the ε-carbon.

4. The compound according to claim 1, wherein the double bond is located between the α-carbon and β-carbon.

5. R １ and R ２ The compound according to claim 1, wherein at least one of them is F.

6. The compound according to claim 5, wherein the compound is a salt comprising a substituent selected from an ammonium substituent, a carboxylate substituent, and a combination thereof.

7. The compound according to claim 6, wherein the ammonium salt has a counterion that is a conjugate base of a protonic acid.

8. The compound according to claim 1 in a pharmaceutical composition containing a pharmaceutically acceptable carrier component.

9. formula: 【Chemistry 2】 [During the ceremony, R １ and R ２ At least one of them is F. The compound according to claim 1.

10. The compound according to claim 9, wherein the compound is a salt comprising a substituent selected from an ammonium substituent, a carboxylate substituent, and a combination thereof.

11. formula: 【Transformation 3】 The compound according to claim 1.

12. The compound according to claim 11, wherein the compound is a salt comprising a substituent selected from an ammonium substituent, a carboxylate substituent, and a combination thereof.

13. formula: 【Chemistry 4】 The compound according to claim 1.

14. The compound according to claim 13, wherein the compound is a salt comprising a substituent selected from an ammonium substituent, a carboxylate substituent, and a combination thereof.

15. (i) the compound described in claim 1; and (ii) a pharmaceutical composition comprising a pharmaceutically suitable carrier, diluent or excipient.

16. A method for regulating human ornithine δ-aminotransferase (hOAT) activity, the method comprising contacting the compound described in claim 1 with a culture medium containing hOAT, wherein the compound is present in an amount sufficient to regulate hOAT activity.

17. A pharmaceutical composition comprising the compound according to claim 1 for reducing the activity of hoAT expressed by human cancer.

18. A pharmaceutical composition comprising the compound described in Claim 1 for treating cancer in a subject requiring cancer treatment.

19. The pharmaceutical composition according to claim 18, wherein the cancer is hepatocellular carcinoma (HCC).

20. The pharmaceutical composition according to claim 18, wherein the cancer is non-small cell lung cancer (NSCLC).

21. The pharmaceutical composition according to claim 18, wherein the cancer is characterized by the expression or overexpression of human ornithine δ-aminotransferase (hOAT).

22. The pharmaceutical composition according to any one of claims 17 to 21, wherein the compound is in a zwitterionic form comprising an ammonium moiety and a carboxylate moiety.

23. A pharmaceutical composition according to any one of claims 17 to 21, wherein the double bond is located between the α-carbon and the ε-carbon.

24. A pharmaceutical composition according to any one of claims 17 to 21, wherein the double bond is located between the α-carbon and the β-carbon.

25. R １ and R ２ A pharmaceutical composition according to any one of claims 17 to 21, wherein at least one of the is F.

26. The pharmaceutical composition according to claim 25, wherein the compound is a salt comprising a substituent selected from ammonium substituents, carboxylate substituents, and combinations thereof.

27. The pharmaceutical composition according to claim 26, wherein the ammonium salt has a counterion that is a conjugate base of a protonic acid.

28. The pharmaceutical composition according to any one of claims 17 to 21, wherein the compound is present in a pharmaceutical composition containing a pharmaceutically acceptable carrier component.

29. The compound has the formula: 【Transformation 5】 [During the ceremony, R １ and R ２ where at least one of them is F) A pharmaceutical composition according to any one of claims 17 to 21, as represented by the following:

30. The pharmaceutical composition according to claim 27, wherein the compound is a salt comprising a substituent selected from an ammonium substituent, a carboxylate substituent, and a combination thereof.

31. The compound has the formula: 【Transformation 6】 A pharmaceutical composition according to any one of claims 17 to 21, as represented by the following:

32. The pharmaceutical composition according to claim 31, wherein the compound is a salt comprising a substituent selected from an ammonium substituent, a carboxylate substituent, and a combination thereof.

33. The compound has the formula: 【Transformation 7】 A pharmaceutical composition according to any one of claims 17 to 21, as represented by the following:

34. The pharmaceutical composition according to claim 33, wherein the compound is a salt comprising a substituent selected from an ammonium substituent, a carboxylate substituent, and a combination thereof.

Images