2-[(thiophene-2-yl)carboxamido]-n-(phenyl)-2-methylpropanamide derivatives and their use as pharmaceuticals

Abstract

The present invention relates to novel 2-[(thiophene-2-yl)formamido]-N-(phenyl)-2- methylpropanamides of general formula A, methods for their preparation, pharmaceutical compositions containing them, and their use in therapy, particularly in the treatment or prevention of conditions in which inhibition of the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme can be beneficial.

Description

[0001] This invention relates to a novel 2-[(thiophen-2-yl)carbamate]-N-(phenyl)-2-methylpropionamide of general formula A.

[0002]

[0003] Its preparation method, pharmaceutical compositions containing it, and its use in therapy, particularly for the treatment or prevention of symptoms in which inhibition of indoleamine 2,3-dioxygenase 1 (IDO1) enzyme may be beneficial.

[0004] The compound of the present invention according to general formula A is an IDO1 inhibitor.

[0005] Extensive research over the past two decades has shown that IDO1 enzyme activity can play a role in the symptoms of a variety of neurological and psychiatric disorders, including major depressive disorder, schizophrenia, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, chronic pain, and obesity.

[0006] To date, inhibition of IDO1 has been primarily used for oncology indications. Most compounds used in these studies are non-brain-penetrating, excluding them for neurological or psychiatric applications. In these conditions, IDO1 expression is increased in brain immune cells, primarily microglia and infiltrating macrophages. The resulting changes in tryptophan metabolites (e.g., quinolinic acid, kynurenic acid) in the kynurenine pathway affect brain function. For this reason, targeting the IDO1 enzyme located in the brain is crucial for treating these diseases.

[0007] The compounds disclosed herein have been developed for the treatment of central nervous system disorders and therefore exhibit good permeability, low / no efflux, low clearance, and pharmacokinetic properties suitable for once-daily treatment. Central nervous system disorders may include neuropsychiatric and neurobehavioral disorders, neurodegenerative diseases, and the adverse consequences of increased IDO1 activity following head trauma or cerebrovascular events. The compounds are suitable as adjunctive therapy for such neurological indications, demonstrating a metabolic profile that minimizes drug-drug interactions.

[0008] IDO1 activity is also known to play a role in the symptoms of many somatic diseases due to its upregulation during inflammatory states. Inhibition of IDO1 may have beneficial effects following bacterial and viral infections (such as tuberculosis and human immunodeficiency virus (HIV) and meningitis). These compounds, optimized for central nervous system effects, are suitable for treating both central and peripheral symptoms of these diseases, such as the cognitive and emotional effects of HIV or the long-term neurological consequences of meningitis. Retinal inflammation has been observed in retinal diseases (such as diabetic retinopathy and geographic atrophy), where the ensuing increase in IDO1 is thought to lead to tissue degeneration. Metabolic disorders (such as obesity) are associated with increased inflammation and IDO1, suggesting that inhibition may be beneficial.

[0009] WO2006 / 034822 discloses a compound of formula (I), which is a human factor Xa inhibitor and can be used as an antithrombotic agent.

[0010]

[0011] The compound of this invention differs structurally from formula (I) of WO2006 / 034822 in that it contains:

[0012] i) 5-chlorothiophene or 5-bromothiophene moiety instead of 5-ethynylthiophene moiety; and

[0013] ii) A benzene ring substituted with one or two halogens, rather than a benzene ring substituted with the heterocyclic A portion.

[0014] Formula (I) covers the specific embodiment 5-ethynyl-N-{1-trifluoromethyl-1-[3-methyl-4-(3-oxomorpholin-4-yl)phenylaminocarboxyloyl]ethyl}thiophen-2-carboxamide (WO2006 / 034822, page 91, lines 7-8, 5-ethynyl-thiophen-2-carboxamide-N-{1-trifluoromethyl-1-[3-methyl-4-(3-oxomorpholin-4-yl)phenylaminocarboxyloyl]ethyl}-amide), which also exhibits a trifluoromethyl moiety in Formula (I).

[0015] Example 5 - Ethynyl-N-{1-trifluoromethyl-1-[3-methyl-4-(3-oxomorpholin-4-yl)phenylaminoformyl]ethyl}thiophene-2-carboxamide (WO2006 / 034822, page 91, lines 7-8, 5-ethynyl-thiophene-2-carboxamide-N-{1-trifluoromethyl-1-[3-methyl-4-(3-oxomorpholin-4-yl)phenylaminoformyl]ethyl}-amide)

[0016]

[0017] Example from WO2006 / 034822: 5-ethynyl-N-{1-trifluoromethyl-1-[3-methyl-4-(3-oxomorpholin-4-yl)phenylaminoformyl]ethyl}thiophene-2-carboxamide (page 91, lines 7-8, 5-ethynyl-thiophene-2-carboxamide-N-{1-trifluoromethyl-1-[3-methyl-4-(3-oxomorpholin-4-yl)phenylaminoformyl]ethyl}-amide) inhibits human factor Xa enzyme and exhibits a high efflux ratio.

[0018] Inhibition of human factor Xa enzymes is associated with undesirable and serious side effects, such as major bleeding. Currently available factor Xa inhibitors, such as rivaroxaban and apixaban, are used as antithrombotic agents to prevent deep vein thrombosis, pulmonary embolism, and blood clots in atrial fibrillation. An antidote (andexanet alfa) has been developed for rapid reversal of the effects. Other uses for factor Xa inhibitors may include CNS indications, such as neurodegeneration.

[0019] The compound of this invention differs structurally from formula (I) of WO2006 / 034822 in that it contains:

[0020] i) 5-chlorothiophene or 5-bromothiophene moiety instead of 5-ethynylthiophene moiety; and

[0021] ii) A benzene ring substituted with one or two halogens, rather than a benzene ring substituted with the heterocyclic A portion.

[0022] The structural differences unexpectedly led to an increased selectivity in inhibiting IDO1 over human factor Xa enzymes and produced a low efflux ratio in MDCK-MDR1 cell analysis, while WO2006 / 034822’s 5-ethynyl-N-{1-trifluoromethyl-1-[3-methyl-4-(3-oxomorpholin-4-yl)phenylaminocarbamoyl]ethyl}thiophene-2-carboxamide (page 91, lines 7-8, 5-ethynyl-thiophene-2-carboxamide-N-{1-trifluoromethyl-1-[3-methyl-4-(3-oxomorpholin-4-yl)phenylaminocarbamoyl]ethyl}-amide) inhibited human factor Xa enzymes and showed a high efflux ratio (Table 3a).

[0023] Therefore, the target technical problem is to provide a potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitor.

[0024] According to the present invention, the compounds disclosed herein have been unexpectedly found to be potent, selective, metabolically stable, and brain-penetrating IDO1.

[0025] The compounds of the present invention are expected to exhibit favorable brain penetration due to their potent inhibition of IDO1, increased selectivity for human factor Xa enzymes, and low extracorporeal efflux, which is essential for effective brain-targeting drugs, and are expected to have an acceptable window between efficacy and undesirable serious side effects such as massive hemorrhage.

[0026] Therefore, the compounds of the present invention are certainly more feasible for human use.

[0027] WO2007 / 003536 discloses a compound of formula (I), which is a human factor Xa inhibitor used as an antithrombotic agent.

[0028]

[0029] Formula (I) encompasses specific embodiments 4, 6 and 159 that present the heterobicyclic portion D within Formula (I), in contrast to the distinctly different monocyclic aromatic portion within general Formula A.

[0030] Example 4 (page 66)

[0031]

[0032] Example 6 (page 68)

[0033]

[0034] Example 159 (page 137)

[0035]

[0036] Inhibition of human factor Xa enzymes is associated with undesirable and serious side effects, such as major bleeding. Currently available factor Xa inhibitors (such as rivaroxaban and apexaban) are used as antithrombotic agents to prevent deep vein thrombosis, pulmonary embolism, and blood clots in atrial fibrillation. An antidote (andresenel α) has been developed for rapid reversal of the effects. Other uses for factor Xa inhibitors may include CNS indications, such as neurodegeneration.

[0037] The compounds of the present invention differ structurally from formula (I) of WO2007 / 003536 in that they contain a benzene ring substituted with one or two halogens instead of a heterobicyclic moiety D.

[0038] The structural differences unexpectedly led to increased metabolic stability in human hepatocytes and did not cause in vitro efflux in MDCK-MDR1 cell analysis—and for Examples 3 and 4, no in vitro efflux was caused—while Examples 4, 6 and 159 of WO2007 / 003536 showed low metabolic stability and / or high in vitro efflux ratios in human hepatocytes (Table 3b), and also showed high in vivo efflux (Table 7).

[0039] Therefore, the target technical problem is to provide a potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitor.

[0040] According to the present invention, the compounds disclosed herein have been unexpectedly found to be potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitors.

[0041] The compounds of the present invention are expected to exhibit favorable brain penetration due to their potent inhibition of IDO1, increased selectivity for human factor Xa enzymes, and low extracorporeal efflux, which is essential for effective brain-targeting drugs, and have an acceptable window between efficacy and undesirable serious side effects such as massive hemorrhage.

[0042] Therefore, the compounds of the present invention are certainly more feasible for human use.

[0043] WO98 / 22432 discloses a compound of formula (I) with anti-androgenic activity, which can be used as a preventive or therapeutic agent for prostate cancer, benign prostatic hyperplasia, analgesia, hirsutism, baldness, acne and seborrhea.

[0044]

[0045] Formula (I) covers specific examples 53 and 86 (Table 1) that present the thiophene structure within group Z.

[0046] Table 1. Examples 53 and 86 of WO98 / 22432 present the thiophene structure within group Z of formula (I).

[0047]

[0048] Examples 53 and 86 of WO98 / 22432 exhibit anti-androgenic activity and show extremely low metabolic stability in human hepatocytes.

[0049] Inhibition of human androgen receptors is associated with potentially serious side effects such as loss of libido, erectile dysfunction, fatigue, osteoporosis and induced skeletal complications, hot flashes, changes in body composition, arteriosclerosis, new-onset diabetes, cognitive decline, and increased cardiovascular morbidity and mortality (doi:10.1016 / j.eururo.2008.10.008).

[0050] Formula (I) of WO98 / 22432 generally covers the compounds of this invention. The compounds of this invention differ structurally from Examples 53 and 86 of WO98 / 22432 (representing the closest analogues) in that they contain:

[0051] i) a benzene ring substituted with one or two halogens instead of a 4-cyano-3-trifluoromethyl-phenyl group; and

[0052] ii) 2-chloro-thiophene or 2-bromo-thiophene moiety instead of unsubstituted thiophene or 2-methyl-thiophene moiety.

[0053] The structural differences unexpectedly resulted in potent IDO1 inhibition and increased selectivity for human androgen receptors, while examples 53 and 86 of WO98 / 22432 showed weaker IDO1 inhibition and no or poor selectivity for human androgen receptors. Furthermore, the compounds of the present invention have been found to be metabolically stable in human hepatocytes (Table 3c).

[0054] Therefore, the target technical problem is to provide a potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitor.

[0055] According to the present invention, the compounds disclosed herein have been unexpectedly found to be potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitors.

[0056] The compounds of the present invention are expected to be effective in vivo for treating diseases and symptoms of CNS origin due to their potent inhibition of IDO1, increased selectivity for human androgen receptors, and metabolic stability in human hepatocytes, with an acceptable window between efficacy and undesirable effects, such as loss of libido, erectile dysfunction, fatigue, osteoporosis and induced skeletal complications, hot flashes, changes in body composition, arteriosclerosis, new-onset diabetes, cognitive decline, and increased cardiovascular morbidity and mortality.

[0057] Therefore, the compounds of the present invention are certainly more feasible for human use.

[0058] WO2005 / 111029 discloses a compound of formula (I), which is a human factor Xa inhibitor used as an antithrombotic agent.

[0059]

[0060] Formula (I) covers specific examples 8 and 28, which present heterocyclic amide moiety A (Table 2).

[0061] Table 2. Examples 8 and 28 of WO2005 / 111029 present the heterocyclic amide portion A in formula (I).

[0062]

[0063] Examples 8 and 28 of WO2005 / 111029 inhibit human factor Xa enzyme and show high efflux.

[0064] Inhibition of human factor Xa enzymes is associated with undesirable and serious side effects, such as major bleeding. Currently available factor Xa inhibitors (such as rivaroxaban and apexaban) are used as antithrombotic agents to prevent deep vein thrombosis, pulmonary embolism, and blood clots in atrial fibrillation. An antidote (andresenel α) has been developed for rapid reversal of the effects. Other uses for factor Xa inhibitors may include CNS indications, such as neurodegeneration.

[0065] The compounds of the present invention differ structurally from formula (I) of WO2005 / 111029 in that they contain benzene rings substituted with one or two halogens rather than benzene rings substituted with the heterocyclic amide portion A.

[0066] Structural differences unexpectedly led to increased selectivity for human factor Xa enzyme and resulted in a low efflux ratio in MDCK-MDR1 cell analysis, while Examples 8 and 28 of WO2005 / 111029 inhibited human factor Xa enzyme and showed a high efflux ratio (Table 4).

[0067] Therefore, the target technical problem is to provide a potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitor.

[0068] According to the present invention, the compounds disclosed herein have been unexpectedly found to be potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitors.

[0069] The compounds of the present invention are expected to exhibit favorable brain penetration due to their potent inhibition of IDO1, increased selectivity for human factor Xa enzymes, and low extracorporeal efflux, which is essential for effective brain-targeting drugs, and have an acceptable window between efficacy and undesirable serious side effects such as massive hemorrhage.

[0070] Therefore, the compounds of the present invention are certainly more feasible for human use.

[0071] WO2005 / 111013 discloses a compound of formula (I), which is a human factor Xa inhibitor used as an antithrombotic agent.

[0072]

[0073] Formula (I) covers specific embodiment 22, which presents the heterocyclic portion A within formula (I).

[0074]

[0075] Example 22 (page 154)

[0076] Example 22 of WO2005 / 111013 inhibits human factor Xa enzyme and exhibits moderate metabolic stability in human hepatocytes.

[0077] Inhibition of human factor Xa enzymes is associated with undesirable and serious side effects, such as major bleeding. Currently available factor Xa inhibitors (such as rivaroxaban and apexaban) are used as antithrombotic agents to prevent deep vein thrombosis, pulmonary embolism, and blood clots in atrial fibrillation. An antidote (andresenel α) has been developed for rapid reversal of the effects. Other uses for factor Xa inhibitors may include CNS indications, such as neurodegeneration.

[0078] The compounds of the present invention differ structurally from formula (I) of WO2005 / 111013 in that they contain benzene rings substituted with one or two halogens rather than benzene rings substituted with saturated heterocyclic portion A.

[0079] Structural differences unexpectedly resulted in potent IDO1 inhibition and increased selectivity for human factor Xa enzymes and increased metabolic stability in human hepatocytes, while example 22 of WO2005 / 111013 inhibited human factor Xa enzymes and showed moderate metabolic stability in human hepatocytes (Table 5).

[0080] Therefore, the target technical problem is to provide a potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitor.

[0081] According to the present invention, the compounds disclosed herein have been unexpectedly found to be potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitors.

[0082] The compounds of the present invention are expected to be effective in vivo for the treatment of diseases and conditions of CNS origin due to their potent inhibition of IDO1, increased selectivity for human factor Xa enzymes, and increased metabolic stability in human hepatocytes, with an acceptable window between efficacy and undesirable serious side effects (such as massive bleeding).

[0083] Therefore, the compounds of the present invention are certainly more feasible for human use.

[0084] WO2005 / 111014 discloses a compound of formula (I), which is a human factor Xa inhibitor used as an antithrombotic agent.

[0085]

[0086] Formula (I) covers specific Example 4, which presents the amide portion within Formula (I).

[0087]

[0088] Example 4 (page 137)

[0089] Example 4 of WO2005 / 111014 inhibits human factor Xa enzyme and shows high efflux.

[0090] Inhibition of human factor Xa enzymes is associated with undesirable and serious side effects, such as major bleeding. Currently available factor Xa inhibitors (such as rivaroxaban and apexaban) are used as antithrombotic agents to prevent deep vein thrombosis, pulmonary embolism, and blood clots in atrial fibrillation. An antidote (andresenel α) has been developed for rapid reversal of the effects. Other uses for factor Xa inhibitors may include CNS indications, such as neurodegeneration.

[0091] The compounds of the present invention differ structurally from formula (I) of WO2005 / 111014 in that they contain benzene rings substituted with one or two halogens rather than benzene rings partially para-substituted with amides.

[0092] The structural differences unexpectedly led to an increased selectivity in inhibiting IDO1 over human factor Xa enzymes and resulted in a low efflux ratio in MDCK-MDR1 cell analysis, while Example 4 of WO2005 / 111014 inhibited human factor Xa enzymes and showed high efflux (Table 6).

[0093] Therefore, the target technical problem is to provide a potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitor.

[0094] According to the present invention, the compounds disclosed herein have been unexpectedly found to be potent, selective, metabolically stable, and brain-penetrating IDO1.

[0095] The compounds of the present invention are expected to exhibit favorable brain penetration due to their potent inhibition of IDO1, increased selectivity for human factor Xa enzymes, and low extracorporeal efflux, which is essential for effective brain-targeting drugs, and have an acceptable window between efficacy and undesirable serious side effects such as massive hemorrhage.

[0096] Therefore, the compounds of the present invention are certainly more feasible for human use.

[0097] None of the factor Xa enzyme inhibitors described above teach or indicate the compounds of the present invention or their advantageous properties as IDO1 inhibitors as described herein.

[0098] Furthermore, the compounds of the present invention are metabolically stable in human hepatocytes (see Tables 3b, 3c, and 5 for details on metabolic stability). Therefore, the compounds of the present invention are expected to have favorable in vivo clearance and thus the desired duration of action in humans.

[0099] Stability in human hepatocytes refers to the ease with which a compound can undergo biotransformation when selected and / or designed to have favorable pharmacokinetic properties. The liver is the primary metabolic side for many drugs. Human hepatocytes contain cytochrome P450 (CYP) and other phase II metabolic enzymes (e.g., phosphatases and sulfatases), and therefore represent a model system for studying in vitro drug metabolism. Enhanced stability in hepatocytes is associated with several advantages, including increased bioavailability and sufficient half-life, which allows patients to reduce dosage and dosing frequency. Therefore, enhanced stability in hepatocytes is a favorable feature for compounds intended for use as drugs.

[0100] Furthermore, the compounds of the present invention exhibit good membrane permeability and low to moderate in vitro efflux (see Tables 4 and 6 for MDCK analysis of MDR1(P-gp)). Therefore, the compounds of the present invention are expected to exhibit the favorable brain penetration required for effective CNS drugs.

[0101] MDCK analysis provides information about the potential of compounds to cross the blood-brain barrier. Permeability measurements across polarized, confluent MDCK-MDR1 cell monolayers grown on permeable filterable carriers serve as an in vitro absorption model: the apparent permeability (PE) of compounds across the MDCK-MDR1 cell monolayer was measured in both apex-to-base (AB) and base-to-apex (BA) delivery directions (pH 7.4, 37°C). AB permeability (PEAB) indicates drug absorption from the blood into the brain, and BA permeability (PEBA) indicates drug return from the brain to the blood via both passive permeability and active delivery mechanisms mediated by efflux and absorption transporters expressed on MDCK-MDR1 cells, primarily mediated by overexpressed human MDR1. Identical or similar permeability in both delivery directions indicates passive permeability, while vector permeability points to other active delivery mechanisms. A PEBA higher than PEAB (PEBA / PEAB>3) indicates involvement of MDR1-mediated active efflux, which may impair the achievement of adequate brain exposure. Therefore, this analysis provides valuable support for the selection of compounds suitable for further in vivo testing. The high permeability of the blood-brain barrier, unrestricted by efflux, is an advantageous feature for the use of compounds as drugs that primarily function in the CNS. Therefore, to ensure high permeability of the blood-brain barrier, it is highly preferable to minimize the efflux of the MDR1 transporter (efflux < 3%).

[0102] The compounds of the present invention exhibit favorable in vivo pharmacokinetic properties when administered orally to rats or mice, as indicated by plasma and brain exposure levels.

[0103] To estimate the amount of drug administered into the brain, the brain-to-plasma ratio was used as a proxies. When efflux transporters (e.g., P-gp) are located at the blood-brain barrier, the compound acting as a substrate is actively effluxed out of the brain. Because these efflux transporters are not expressed in muscle tissue, the relative distribution ratio of the compound between muscle and brain tissue can be used to indicate in vivo efflux in rats or mice. In the absence of detectable in vivo efflux (in rats / mice) and in the absence of in vitro efflux (MDCK-MDR1(P-gp)), brain exposure in patients can be expected [see Table 7 for a comparison of in vitro efflux data (MDCK-MDR1 analysis) with in vivo efflux data (tissue distribution in rats / mice after oral administration)].

[0104] This invention provides a novel 2-[(thien-2-yl)carbamate]-N-(phenyl)-2-methylpropionamide of formula A.

[0105]

[0106] in

[0107] R 1 Indicates chlorine and bromine;

[0108] R 2 Indicates hydrogen and fluorine;

[0109] R 3 Indicates chlorine and bromine;

[0110] Or its salts, especially its pharmaceutically acceptable salts.

[0111] The present invention provides a novel 2-[(thiophen-2-yl)carbamoyl]-N-(phenyl)-2-methylpropionamide of formula A, which is unexpectedly a potent IDO1 inhibitor.

[0112] Another aspect of the present invention relates to a compound of formula A as a potent and selective IDO1 inhibitor.

[0113] Another aspect of the invention relates to a compound of formula A as a potent IDO1 inhibitor, which has suitable selectivity for human androgen receptors.

[0114] Another aspect of the invention relates to a compound of formula A as a potent IDO1 inhibitor, which has suitable selectivity for human factor Xa enzyme.

[0115] Another aspect of the invention relates to a compound of formula A as a potent and selective IDO1 inhibitor, having suitable membrane permeability and low in vitro efflux.

[0116] Another aspect of the present invention relates to a compound of formula A as a potent and selective IDO1 inhibitor, which has high metabolic stability in human hepatocytes.

[0117] Another aspect of the present invention relates to a compound of formula A as a potent and selective IDO1 inhibitor, which has appropriate membrane permeability, low extracorporeal efflux and high metabolic stability in human hepatocytes.

[0118] Another aspect of the present invention relates to a compound of formula A as a potent, selective, and brain-penetrating IDO1 inhibitor.

[0119] Another aspect of the present invention relates to a compound of formula A as a potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitor.

[0120] Another aspect of the invention relates to a pharmaceutical composition comprising at least one compound of formula A, optionally together with one or more inert carriers and / or diluents.

[0121] Another aspect of the present invention relates to a compound of formula A, which is used for the prevention and / or treatment of conditions associated with IDO1 inhibition.

[0122] Another aspect of the present invention relates to a method for manufacturing the compounds of the present invention.

[0123] preparation

[0124] The following procedures should generally illustrate how to prepare compounds of general formula A and their corresponding intermediates using the examples. Unless otherwise defined in the context of the procedures, abbreviations for substituents are as defined above.

[0125] Process 1: Method A

[0126]

[0127] Starting with a commercially available compound (I), wherein PGA is a suitable protecting group for forming alkyl or aryl esters (PGA is selected from the group consisting of methyl, ethyl, n-butyl, phenyl, p-nitrobenzene), compound (III) can be obtained by reacting it with an activated compound (II) (G represents a halogenated or ureon moiety, such as chlorine, bromine, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea or O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea) in a solvent (such as tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane or toluene) in the presence of an organic base (such as triethylamine, N-ethyl-diisopropylamine or N-methylmorpholine) at a temperature ranging from -50°C to 120°C.

[0128] Compound (III) is hydrolyzed in a solvent or solvent mixture such as methanol, ethanol, tetrahydrofuran, 2-methyltetrahydrofuran, or water at a temperature ranging from -20°C to 100°C, using an inorganic base such as lithium hydroxide, sodium hydroxide, or potassium hydroxide. Before reacting with compound (V) to obtain compound (VI), compound (IV) needs to be activated with a reagent such as O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroborate or O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate at a temperature ranging from -50°C to 120°C.

[0129] Process 2: Method B

[0130]

[0131] To obtain compound (VIII), prior to the addition of compound (V), a commercially available compound (VII) needs to be activated with a reagent (such as O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroboronic acid or O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate) in a solvent (such as tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane or toluene) in the presence of an organic base (such as triethylamine, N-ethyl-diisopropylamine or N-methylmorpholine) at a temperature ranging from -50°C to 120°C, wherein PGB is a suitable protecting group for forming a carbamate (PGB is selected from the group consisting of tert-butoxycarbonyl, benzoxycarbonyl, allyloxycarbonyl or 9-fluorenylmethoxycarbonyl). Compound (IX) is formed by removing the protecting group in a solvent (such as dichloromethane, tetrahydrofuran, 1,4-dioxane, or water) with an acid (such as hydrogen chloride, hydrogen bromide, acetic acid, or trifluoroacetic acid) or an organic base (such as triethylamine, N-ethyl-diisopropylamine, or N-methylmorpholine) at a temperature ranging from 0°C to 100°C.

[0132] To obtain compound (VI), compound (IX) needs to be reacted with activated compound (II) (G represents a halogen or ureon moiety (such as chlorine, bromine, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea or O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea) in a solvent (such as tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane or toluene) in the presence of an organic base (such as triethylamine, N-ethyl-diisopropylamine or N-methylmorpholine) at a temperature ranging from -50°C to 120°C.

[0133] In processes 1 and 2, all substituents R 1 R 2 and R 3 Having the meaning as defined for general formula A, the abbreviations PGA and PGB have the meaning of a protecting group, and the abbreviation G has the meaning of an activating group. All embodiments of the invention directly referred to herein have, in particular, the meaning as defined in the claims.

[0134] General definition

[0135] Terms not specifically defined herein shall be given the meanings by those skilled in the art in accordance with the invention and the text.

[0136] In the case of any inconsistency between the chemical names and chemical formulas of the compounds described in this invention, the chemical formula shall prevail.

[0137] An asterisk in a subformula can be used to indicate a bond that is attached to the core molecule or to a substituent as defined thereto.

[0138] As used herein, the term “substitution” means the replacement of one or more hydrogen atoms on a specified atom with a selected specified group, subject to the condition that the substitution does not exceed the feasible valence number of the specified atom, and that the substitution produces a stable compound.

[0139] Stereochemistry:

[0140] Unless otherwise specified, throughout the specification and the appended claims, the given chemical formulas or names shall cover rotational isomers and tautomers, as well as mixtures in different proportions or mixtures of any of the foregoing forms (if such isomers are present), and salts, including pharmaceutically acceptable salts thereof.

[0141] Salt:

[0142] The phrase “pharmaceutically acceptable” is used herein to refer to compounds, materials, compositions and / or dosage forms that are suitable for use within the bounds of reasonable medical judgment without excessive toxicity, irritation, allergic reactions or other problems or complications and in proportion to a reasonable benefit / risk ratio.

[0143] As used herein, "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound in which the parent compound forms a salt or complex with an acid or base.

[0144] Examples of acids that form pharmaceutically acceptable salts with parent compounds containing a basic moiety include inorganic or organic acids such as benzenesulfonic acid, benzoic acid, citric acid, ethanesulfonic acid, fumaric acid, gentian acid, hydrobromic acid, hydrochloric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, 4-methylbenzenesulfonic acid, phosphoric acid, salicylic acid, succinic acid, sulfuric acid, or tartaric acid.

[0145] Examples of cations and bases that form pharmaceutically acceptable salts with parent compounds containing acidic moieties include Na. + K + Ca 2+ Mg 2+ NH4 + L-arginine, 2,2'-imine diethanol, L-lysine, N-methyl-D-glucosamine, or tris(hydroxymethyl)-aminomethane.

[0146] Pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing basic or acidic moieties using conventional chemical methods. Generally, such salts can be prepared by reacting the free acidic or basic form of these compounds with a sufficient amount of a suitable base or acid in water or an organic diluent (such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, or mixtures thereof). In addition to the salts mentioned above, salts of other acids suitable for purifying or isolating the compounds of the present invention (e.g., trifluoroacetates) also constitute a part of the present invention.

[0147] Bioanalysis and Data

[0148] Abbreviation list

[0149] BSA bovine serum albumin

[0150] cDNA complementary DNA

[0151] CSF cerebrospinal fluid

[0152] DMSO (dimethyl sulfoxide)

[0153] HEPES 2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid

[0154] IFN interferon

[0155] K3EDTA 1,2-Diaminoethane-N,N,N',N'-Tetraacetic acid tripotassium salt

[0156] LPS lipopolysaccharide

[0157] MDCK Maddard II canine kidney cells

[0158] MDR1 multidrug resistance protein 1

[0159] P-gp p-glycoprotein

[0160] %QH hepatic blood flow

[0161] rpm revolutions per minute

[0162] rt room temperature

[0163] U unit

[0164] WBA Whole Blood Analysis

[0165] The data provided in analyses A through F are the arithmetic mean (IC50) of the individual analysis results. 50 The values ​​(%QH and outflow ratio) and standard deviations are given, where N is the number of available data points for the corresponding analysis.

[0166] The bioactivity of the compound was determined by the following methods:

[0167] Analysis of in vitro tests for A.IDO1 efficacy:

[0168] In vitro pharmacological activity assay

[0169] This analysis assessed the reduction of kynurenine (an enzymatic catabolite of L-tryptophan) in human whole blood stimulated with LPS and IFN-γ in response to inhibitor doses. Kynurenine was quantified by LC-MS / MS for D4-kynurenine (internal standard). This analysis was used to determine specific IC50 and IC90 values ​​of the compound in a human model.

[0170] Materials and reagents:

[0171] Human whole blood from healthy donors was obtained using a blood sampling syringe equipped with lithium heparin anticoagulant (SARSTEDT Monovette, LH9mL, orange code).

[0172] 96-well tray (THERMO FISHER Abgene 96-well storage tray, 0.8 mL, catalog number AB0765)

[0173] Stomatal zone (QIAGEN)

[0174] RPMI 1640 (GIBCO, catalog number 61870010) with GlutaMAX supplement.

[0175] Lipopolysaccharide from E. coli dissolved in RPMI, aseptically filtered (SIGMA ALDRICH, catalog number L3129-25MG, E. coli 0127:B8, batch number 016M4187V).

[0176] Recombinant human interferon-γ (PEPROTECH|TEBU-BIO, catalog number 167300-02-B, batch number 121527) was dissolved in DPBS containing 0.1% recombinant human serum albumin (SIGMA ALDRICH, catalog number A6608-100MG, batch number SLBQ1282V).

[0177] CHROMASOLV acetonitrile (SIGMA ALDRICH, catalog number 34851-2.5L) for HPLC

[0178] L-kynurenine D4 (BUCHEM BV), 5 mM in DMSO

[0179] DMSO

[0180] The analytical grade was 100% anhydrous acetic acid (MERCK, catalog number 34885-2.5L).

[0181] CHROMASOLV methanol (SIGMA-ALDRICH, catalog number 1.00063.2500) for HPLC

[0182] program:

[0183] The required analytical concentrations for 8-fold concentration of IFN-γ and LPS were diluted in RPMI medium:

[0184] IFN-γ 25ng / ml × 8 200ng / ml

[0185] LPS 500ng / ml × 8 4μg / mL

[0186] The LPS should be ultrasonically treated for at least 10 minutes and then directly vortexed before use.

[0187] IFN-γ is gently mixed by aspirating upwards and downwards.

[0188] Stimulant mixtures were prepared by mixing the two diluents in a 1:1 ratio, resulting in 4 times the desired final concentration of each stimulant.

[0189] Semi-log serial dilutions of the compound were prepared in 100% DMSO within the range of 4 mM to 4 μM. These dilutions were further diluted 1:100 in RPMI medium.

[0190] Prepare analytical trays and titration profiles for stimulants and inhibitors (3 technical replicates, 4 biological replicates). Inoculate all reagents at a 4-fold concentration using 60 μL per well. Each tray includes a positive control (no stimulation, no inhibitor treatment, basal activity) and a negative control (stimulation but no inhibitor treatment, maximum signal) (6 technical replicates). Before adding whole blood samples, top all wells to a final volume of 120 μL using RPMI medium.

[0191] Whole blood treated with lithium heparin was received directly from four donors within 2 hours of blood collection. Each sample was diluted 1:2 with RPMI medium. 120 μL of diluted blood sample was added to the designated wells to produce a final volume of 240 μL in each well, with 1x reagent concentration, a compound concentration range between 10 μM and 0.01 μM, and 0.25% DMSO.

[0192] The culture trays were sealed with vent tape and incubated at 37°C and 5% CO2 for 24 hours.

[0193] After the incubation period, the analysis dish was briefly shaken and then centrifuged to obtain plasma: 15 minutes, 2000×g, room temperature.

[0194] Pipe 200 μL of acetonitrile into each well of the new 96-well plate, and transfer 100 μL of plasma from each analytical sample into the plate. Incubate the plate on a shaker at 1000 rpm for 5 minutes, then incubate at -80°C for 20 to 60 minutes for further precipitation.

[0195] Preparation of internal standard solution: Dilute 5 mM D4-kynurenine / DMSO in MilliQ water at a ratio of 1:3333 to a 1.5 μM solution. Add 100 μL of the internal standard solution to each well to produce a final concentration of 0.375 μM D4-kynurenine in each well. Centrifuge the culture dish to allow the precipitate to settle: 20 min, 2000 × g, room temperature.

[0196] Transfer the supernatant to a new 96-well plate for LC / MS analysis.

[0197] LC parameters

[0198] Filter cartridge (type and column bed volume): C18 (12μL)

[0199] Flow rate pumps 1 / 2 / 3 [mL / min]: 1.5 / 1.25 / 1.25

[0200] LC solvent pump 1 / 2 / 3: Double-distilled H2O containing 0.5% acetic acid / Methanol containing 0.5% acetic acid / Methanol washing solvent containing 0.5% acetic acid (washing 1 / 2): H2O / MeOH

[0201] LabVIEW configuration file: ido.cfg

[0202] Aspiration time: 250 milliseconds

[0203] Washing time: 3000 milliseconds

[0204] Washout time: 3000 milliseconds

[0205] Equilibrium time: 500 milliseconds

[0206] Sip height: 2mm

[0207] MS parameters

[0208] Mass spectrometer: TSQ Vantage

[0209] Ion source: HESI II

[0210] Spray voltage: 2500V

[0211] Capillary temperature: 325℃

[0212] Sheath pressure: 30 psi

[0213] vaporizer temperature: 500℃

[0214] Auxiliary pressure: 45 psi

[0215] Scan width: 0.1 amu

[0216] Resolution: SRM (0.7 / 0.7)

[0217] MRM conversion of analyte / internal standard (Dalton)

[0218] L-Kynourishine 209.11 (parent structure) -94.10

[0219] D4-Kynourishine 213.05 (parent structure) -150.14

[0220] Results are given as the ratio of L-kynurenine to D4-kynurenine. Dose-response curves were calculated and fitted to the control inhibition percentage between 100% (negative control) and 0% (positive control, unstimulated basal activity).

[0221] Analysis of B. In vitro test of androgen receptor efficacy:

[0222] In vitro pharmacological activity assay

[0223] Human androgen receptor (AR) analysis was performed using Indigo Biosciences product #IB03001. The cell line provided in this kit carries a reporter gene induced upon AR stimulation. This reporter gene is cDNA encoding beetle luciferase (a 62kD protein derived from the North American firefly (Photinus pyralis)). This luciferase catalyzes the single oxidation of D-luciferin, producing oxidized luciferin, adenosine monophosphate, pyrophosphate, CO2, and photon emission. The luminescence intensity of the reaction was quantified using a luminometer.

[0224] For the analysis, cells were thawed by adding pre-warmed cell recovery medium to each tube and then placed in a 37°C water bath. 30,000 viable cells (in collagen-coated culture dishes) were seeded in 200 μL and incubated for 24 hours at 37°C, ≥85% humidity, and 5% CO2. The medium was replaced with compound screening medium. Cells were then challenged with 125 pM (EC80) 6a-Fl testosterone and administered a 10 μM to 10 nM semi-logarithmic dilution of the compound. The analysis dishes were then incubated for 22 to 24 hours at 37°C, ≥85% humidity, and 5% CO2. The medium was replaced with luciferase assay reagent and equilibrated for 5 minutes before luminescent quantification.

[0225] Data analysis consisted of calculating the percentage of controls between 100% (negative control: 6α-FL testosterone) and 0% (positive control: no 6α-FL testosterone). Standardized values ​​were fitted to a 4-parameter sigmoid binding curve (Log[concentration]) relative to the signal.

[0226] Analysis of in vitro tests for the efficacy of human factor Xa:

[0227] In vitro pharmacological activity assay

[0228] Active human factor Xa is measured using a specific chromogenic substrate labeled with a p-nitrophenylamino group (pNA). A color change occurs after the peptide substrate cleaves at the binding site with the pNA group, and this color change is detectable at 405 nm. The amount of cleaved substrate is directly proportional to the amount of active Xa.

[0229] 0.8 mg (100 U) of human factor Xa (Enzyme Research Laboratories; HFXa1011) was dissolved in 0.444 mL of H2O (Millipore), consistent with the stock solution of 19.28 μM or 225 U / mL. Other dilutions were prepared in analytical buffer (Tris 100 mM, NaCl 150 mM, adjusted to pH 7.8 with HCl containing 0.1% BSA and 0.05% Tween 20).

[0230] Dissolve 25 mg (1 vial) of the chromogenic substrate S2765 (Chromogenix; S2765) in 3.5 ml of H2O (Millipore) to obtain a 10 mM stock solution.

[0231] The compound was dissolved and diluted in DMSO, with the final dilution in the analysis being 1:10, resulting in a final concentration of 1% DMSO.

[0232] or

[0233] Dilute the compound dissolved in H2O.

[0234] - The final dilution step is performed 1:10 in analytical buffer containing 11% DMSO to produce 10% DMSO.

[0235] - The final dilution in the analysis was 1:10, yielding a final concentration of 1% DMSO.

[0236] The compounds were tested at concentrations ranging from 10 μM to 0.003 nM or lower.

[0237] Pipe 5 μl of human factor Xa (final concentration 0.86 nM), 2 μl of compound dilution buffer or analysis buffer (containing 10% DMSO), and 8 μl of analysis buffer into a 384-well plate three times and incubate in Thermomix at 24 °C for 10 minutes.

[0238] After adding 5 μl of chromogenic substrate (final concentration 0.5 mM), the enzyme-substrate reaction started and kinetic measurements were performed using Spectramax within 1 to 2 minutes.

[0239] Measurement parameters: (Spectramax, monochromator)

[0240] Absorbance brightness; 1 LM 405 nm

[0241] Kinetics: 16 minutes, repeated every 2 minutes (9 times)

[0242] Negative control (NC): 5 μl human factor Xa + 10 μl analysis buffer + 5 μl substrate

[0243] Positive control (PC): 5 μl human factor Xa + 2 μl nafamostat (commercially available, final concentration: 1 μM) + 8 μl assay buffer + 5 μl substrate

[0244] Blank control (optional): 15 μl analysis buffer + 5 μl substrate

[0245] Calculate IC50, KI, and the percentage (ratio) of action at the highest concentration.

[0246] For calculation purposes, the average value of Vmax is taken between 120 seconds and 720 seconds.

[0247] Create an x / y plot: x = logarithm(M, compound); y = vmax(drfu / min)

[0248] Analyzing data using a curve fitting model: Logarithm (inhibitor) relative to response – variable slope (four parameters)

[0249] The results of this fit are the highest, lowest, logarithmic (IC50, M), Hill slope, and IC50(M).

[0250] Analysis D. Assessing metabolic stability in human hepatocytes

[0251] The metabolic degradation of the test compounds in hepatocyte suspension was analyzed. After recovery from cryopreservation, human hepatocytes were cultured in Durbecco's modified eagle medium supplemented with 3.5 μg glucagon, 2.5 mg insulin, and 3.75 mg DHEA per 500 mL, containing 5% or 50% human serum, or serum-free medium.

[0252] After pre-culturing in a cell culture incubator (37°C, 10% CO2) for 30 minutes, 5 μl of the test compound solution (80 μM; obtained by diluting 2 mM DMSO stock solution 1:25 with culture medium) was added to 395 μl of hepatocyte suspension to produce a final cell density of 1 Mio cells / mL, a final test compound concentration of 1 μM, and a final DMSO concentration of 0.05%.

[0253] Cells were cultured for six hours (incubator, orbital shaker), and samples (25 μl) were removed from the culture at 0, 0.5, 1, 2, 4, and 6 hours. The samples were transferred to acetonitrile and collected by centrifugation (5 min). The supernatant was transferred to a new 96-well plate, evaporated and resuspended under nitrogen, and then analyzed for the attenuation of the parent compound by HPLC-MS / MS.

[0254] CLint is calculated as follows:

[0255] CL_intrinsic = k / CD × 1000 / 60

[0256] k: Slope of the decay regression line of the parent compound [h] -1 CD: Cell density of live cells [10e6 cells / mL]. The calculated in vitro intrinsic liver clearance was scaled up to the in vivo intrinsic liver clearance using a liver model (a well-mixed model). This in vivo intrinsic liver clearance was used to predict the in vivo hepatic blood clearance (CL).

[0257] CL_inherent_body [ml / min / kg] = (CL_inherent [μL / min / 10e6 cells] × hepatocyte mass [10e6 cells / g liver] × liver factor [g / kg body weight]) / 1000

[0258] CL [ml / min / kg] = CL_intrinsic_in vivo [ml / min / kg] × hepatic blood flow [ml / min / kg] / (CL_intrinsic_in vivo [ml / min / kg] + hepatic blood flow [ml / min / kg])

[0259] Results are expressed as a percentage of hepatic blood flow:

[0260] QH[%]=CL[ml / min / kg] / liver blood flow[ml / min / kg])

[0261] Human hepatocyte count: 120 × 10⁶ cells / g liver

[0262] Human liver factor: 25.7 g / kg body weight

[0263] Human blood flow: 20.7 ml / (min×kg)

[0264] Analysis of E. MDCK and MDR-1 (P-gp)

[0265] The apparent permeability (P0) of the compound across the MDCK-MDR1 monolayer (MDCKII cells transfected with the human MDR1 gene) was measured in the apex-to-base (AB) and base-to-apex (BA) directions. app ).

[0266] MDCK-MDR1 cells (6×10) 5 cells / cm 2 The compounds were inoculated onto filter inserts (Corning, Transwell, polycarbonate, 0.4 μm pore size) and incubated for 9 to 10 days. Compounds dissolved in DMSO stock solutions (1–20 mM) were diluted with HTP-4 buffer supplemented with 0.25% BSA (128.13 mM NaCl, 5.36 mM KCl, 1 mM MgSO4, 1.8 mM CaCl2, 4.17 mM NaHCO3, 1.19 mM Na2HPO4, 0.41 mM NaH2PO4, 15 mM HEPES, 20 mM glucose, pH 7.4) to prepare transport solutions, resulting in a final test concentration of 1 μM or 10 μM and a final DMSO content of 0.5%. The transport solutions were applied to the donor side at the tip or outside the substrate to measure AB or BA permeability, respectively. The receiver side contained HTP-4 buffer supplemented with 0.25% BSA. Samples were collected from the donor side at the beginning and end of the experiment, and from the receiver side at various time intervals of up to 2 hours for concentration measurement by HPLC-MS / MS. The receiving volume of the sample was replaced with freshly prepared receiving solution.

[0267] All data reported by E were measured and analyzed at a final test concentration of 10 μM.

[0268] Analysis F. Determining tissue distribution in rats / mice after oral administration of the compound.

[0269] Male Han Wistar rats or C57BL / 6NRj mice were orally administered 10 μmol / kg of the study compound suspended in 0.5% Natrosol / 0.015% Tween 80. Blood was collected from the sublingual vein of rats under short-term isoflurane anesthesia or from the saphenous vein of conscious mice at 0.83, 0.25, 0.5, 1, 2, 3, 4, 8, and 24 hours after administration and placed in vials containing K3EDTA. Plasma was then prepared by centrifugation at 5000 rpm for 5 minutes and stored at -20°C.

[0270] Tissue and CSF samples were taken from the anesthetized animals. CSF was collected from the cisterna magna. After bloodletting, samples of the femoral muscle were collected. The cranial and dura mater material was removed, and the entire brain was collected. The muscle and brain samples were homogenized before being transferred to a weighing apparatus. Previously, the tissue was rinsed with saline solution and then dried.

[0271] To homogenize, add four (v / w) portions of homogenization buffer (37.5% acetonitrile, 37.5% methanol, 25% water). Homogenize the tissue using a rotary homogenizer. Add 5 μL of homogenate to a 70 μL mixture of acetonitrile and methanol in a 1:1 (v / v) ratio and freeze at -18°C for at least 10 minutes.

[0272] To prepare the final samples, the samples were centrifuged at 4000 rpm for 1 minute, and aliquots (30 μL) of the resulting supernatant were mixed with 170 μL of formic acid and then injected into LC-MS. Calibration curves were prepared for rat and mouse plasma samples accordingly. All animal care and experimental procedures at Boehringer Ingelheim were conducted in accordance with German and European animal welfare laws (EU Directive 2010 / 63 / EU) and were approved by the local German authority, the Tübingen City Government (reference number 35 / 9185.81-8 / 14-009-G).

[0273] It has been unexpected that the compounds of the present invention are potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitors (selective for human factor Xa and without extracorporeal efflux), while the example 5-ethynyl-N-{1-trifluoromethyl-1-[3-methyl-4-(3-oxomorpholin-4-yl)phenylaminocarbamoyl]ethyl}thiophene-2-carboxamide (page 91, lines 7-8, 5-ethynyl-thiophene-2-carboxamide-N-{1-trifluoromethyl-1-[3-methyl-4-(3-oxomorpholin-4-yl)phenylaminocarbamoyl]ethyl}-amide) is a potent human factor Xa inhibitor that exhibits a high efflux ratio, which may impair the achievement of adequate brain exposure (see Table 3a).

[0274] The compounds of the present invention need to have high selectivity for human factor Xa enzyme (i.e., potent IDO1 inhibition, showing a low IC50 value in analysis A, and low inhibition of human factor Xa enzyme (showing a high IC50 value in analysis C) and low efflux ratio (efflux <3 in analysis E), that is, the compounds of the present invention are potent, selective, metabolically stable and brain-penetrating IDO1 inhibitors.

[0275] Table 3a. Comparison of biological data of the compounds of the present invention with those of prior art compounds in WO2006 / 034822

[0276]

[0277] It has been unexpected that the compounds of the present invention are potent, metabolically stable, and brain-penetrating IDO1 inhibitors (without extracorporeal efflux), while Examples 4, 6, and 159 of WO2007 / 003536 exhibit low metabolic stability or high extracorporeal efflux ratios, which may impair the achievement of adequate brain exposure (see Table 3b). Specific Examples 4, 6, and 159 of WO2007 / 003536 show substantial in vivo efflux of these compounds after oral administration to rats (Analysis F, see Table 7 for data).

[0278] The compounds of the present invention need to have high stability in human hepatocytes (low QH% value in analysis D) and / or low efflux ratio (efflux <3 in analyses E and F), that is, the compounds of the present invention are potent, selective, metabolically stable and brain-penetrating IDO1 inhibitors.

[0279] Table 3b. Comparison of biological data of the compounds of the present invention with those of prior art compounds in WO2007 / 003536

[0280]

[0281]

[0282] It has been unexpected that the compounds of the present invention are potent IDO1 inhibitors and selective for human androgen receptors, while Examples 53 and 86 of WO98 / 22432 inhibit human androgen receptors and show weaker IDO1 inhibition and therefore show no or poor selectivity for human androgen receptors (see Table 3c). Furthermore, the compounds of the present invention show enhanced metabolic stability (low QH% values) in human hepatocyte analysis, while Examples 53 and 86 of WO98 / 22432 show extremely low metabolic stability in human hepatocytes (see Table 3c). Therefore, it has been unexpected that the compounds of the present invention are potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitors.

[0283] The compounds of the present invention need to have high selectivity for human androgen receptors (i.e., potent IDO1 inhibition, showing a low IC50 value in analysis A, and low inhibition of human androgen receptors, showing a high IC50 value in analysis B) and high stability in human hepatocytes (low QH% value in analysis D). In other words, the compounds of the present invention are potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitors.

[0284] Table 3c. Comparison of biological data of the compounds of the present invention with those of prior art compounds in WO98 / 22432

[0285]

[0286] It has been unexpected that the compounds of the present invention are potent, selective, metabolically stable and brain-penetrating IDO1 inhibitors (selective for human factor Xa and without extracorporeal efflux), while Examples 8 and 28 of WO2005 / 111029 are potent human factor Xa inhibitors exhibiting high efflux ratios, which may impair the achievement of adequate brain exposure (see Table 4).

[0287] The compounds of the present invention need to have high selectivity for human factor Xa enzyme (i.e., potent IDO1 inhibition, showing a low IC50 value in analysis A, and low inhibition of human factor Xa enzyme, showing a high IC50 value in analysis C) and low efflux ratio (efflux <3 in analysis E), that is, the compounds of the present invention are potent, selective, metabolically stable and brain-penetrating IDO1 inhibitors.

[0288] Table 4. Comparison of biological data of the compounds of the present invention with those of prior art compounds in WO2005 / 111029

[0289]

[0290]

[0291] It has been unexpected that the compounds of the present invention are potent, selective, metabolically stable and brain-penetrating IDO1 inhibitors (selective to human factor Xa and metabolically stable in human hepatocytes), while Example 22 of WO2005 / 111013 inhibits human factor Xa enzymes and shows moderate metabolic stability in human hepatocytes (see Table 5).

[0292] The compounds of the present invention require high selectivity for human factor Xa enzyme (i.e., potent IDO1 inhibition, showing a low IC50 value in analysis A, and low inhibition of human factor Xa enzyme, showing a high IC50 value in analysis C) and high stability in human hepatocytes (low QH% value in analysis D). In other words, the compounds of the present invention are potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitors.

[0293] Table 5. Comparison of biological data of the compounds of the present invention with those of prior art compounds in WO2005 / 111013

[0294]

[0295] It has been unexpected that the compounds of the present invention are potent, selective, metabolically stable and brain-penetrating IDO1 inhibitors (selective to human factor Xa and without extracorporeal efflux), while Example 4 of WO2005 / 111014 is a potent human factor Xa inhibitor exhibiting a high efflux ratio, which may impair the achievement of the goal of adequate brain exposure (see Table 6).

[0296] The compounds of the present invention need to have high selectivity for human factor Xa enzyme (i.e., potent IDO1 inhibition, showing a low IC50 value in analysis A; and low inhibition of human factor Xa enzyme, showing a high IC50 value in analysis C) and low efflux ratio (efflux <3 in analysis E), that is, the compounds of the present invention are potent, selective, metabolically stable and brain-penetrating IDO1 inhibitors.

[0297] Table 6. Comparison of biological data of the compounds of the present invention with those of prior art compounds in WO2005 / 111014

[0298]

[0299] It has been unexpectedly found that the compounds of the present invention, upon oral administration to rats or mice, exhibit favorable in vivo pharmacokinetic properties as indicated by plasma and brain exposure levels. Examples 3 and 4 have been unexpectedly found to be potent and selective IDO1 inhibitors (selective against human androgen receptors and human factor Xa), which are metabolically stable and show neither in vitro nor in vivo efflux (Tables 3a-7). Therefore, brain exposure in patients is expected, which is necessary for substantial inhibition of IDO1 in the brain over 24 hours.

[0300] In contrast, specific examples 4, 6 and 159 of WO2007 / 003536 showed that these compounds, after oral administration to rats, exhibited substantial in vivo efflux in the same analysis (analysis F, see Table 7 for data).

[0301] Table 7. Comparison of in vitro elution data (MDCK-MDR1 analysis) and in vivo elution data (tissue distribution in rats / mice after oral administration) of the compounds of the present invention with those of the prior art compounds in WO2007 / 003536

[0302] Example MDCK-MDR1 outflow ratio (Analysis E) Flowing out of the body (Analysis F) Example 4 of WO2007 / 003536 1.0 3.3 (Rat) Example 6 of WO2007 / 003536 7.6 12.6 (Rat) Example 159 of WO2007 / 003536 1.8 6.2 (Rat) 3 0.50 0.60 (mice) 4 0.50 0.50 (rat)

[0303] This invention provides a compound of formula A, which is unexpectedly a potent, selective, metabolically stable, and brain-penetrating IDO1 inhibitor. That is, the compound exhibits high efficacy against human IDO1 enzymes, high selectivity against human androgen receptors and human factor Xa enzymes, high metabolic stability in human hepatocytes, and shows neither in vitro (MDCK-MDR1 analysis) efflux nor in vivo efflux (as indicated by muscle / brain tissue distribution in rats / mice).

[0304] The full efficacy in patients (i.e., IDO1 enzyme activity inhibition >90%) and the in vitro IC50 corresponding to IDO1 inhibition measured in human whole blood analysis 50 The value is related to the plasma trough concentration (DOI:10.1002 / jcph.855,DOI:10.1158 / 1078-0432.CCR-16-2272). In clinical indications involving the central nervous system (CNS), a brain concentration equal to the plasma trough concentration must be maintained in the brain for more than 24 hours to be suitable for treating diseases and conditions where IDO1 inhibition has a therapeutic benefit. However, effective plasma exposure to the compound must not produce unwanted side effects. To account for variability among patients, higher plasma compound exposures must be without side effects. The ratio between the effective plasma exposure and the plasma exposure at the first occurrence of a side effect is defined as the safety window.

[0305] The safety window is significantly reduced due to the following:

[0306] (i) Low efficacy, because achieving the desired effect would require high plasma levels, which could also lead to a higher risk of unwanted side effects.

[0307] (ii) Low selectivity for targets such as human androgen receptors or human factor Xa, because side effects may be caused by off-target affinity.

[0308] (iii) Low metabolic stability, because a high initial plasma exposure to the compound is required to ensure that CNS levels provide substantial inhibition within 24 hours.

[0309] (iv) The high efflux ratio at the blood-brain barrier (BBB) ​​is due to the higher concentration of the compound in plasma than in the CNS, implying higher initial plasma exposure and drug-drug interactions via transporter proteins, thus limiting the use of the compound (FDA-Clinical Drug Interaction Studies-Cytochrome P450 Enzyme-and Transporter-Mediated Drug Interactions, Guidance for Industry, January 2020).

[0310] Therefore, compounds that meet the following conditions are expected.

[0311] (i) is a potent IDO1 inhibitor.

[0312] (ii) It is selective for human androgen receptors and human factor Xa.

[0313] (iii) It has high metabolic stability, and

[0314] (iv) Has low / no outflow,

[0315] Drugs that are effective in vivo and have an acceptable safety window for use in treating diseases and symptoms of CNS origin, in which IDO1 inhibition has therapeutic benefits.

[0316] This invention provides compounds targeting the CNS. To achieve substantial inhibition of the IDO1 enzyme in the CNS at a reasonable human dose (once daily, <250 mg) and an acceptable safety window, the compounds of this invention should be potent (IC50 (IDO1) <300 nM in human whole blood analysis), selective (preferably selectivity >10, more preferably selectivity >30, with optimal selectivity for human androgen receptor and / or human factor Xa >30), metabolically stable (<20% QH in human hepatocytes), and exhibit low efflux (efflux <3) or no efflux both in vitro and in vivo.

[0317] The compounds of the present invention have been found to be potent (IC50 (IDO1) < 300 nM in human whole blood analysis), selective (preferably selectivity > 10, more preferably selectivity > 30, with optimal selectivity for human androgen receptor and / or human factor Xa > 30), metabolically stable (< 20% QH in human hepatocytes), and exhibit low efflux (efflux < 3) or no efflux in vitro and in vivo.

[0318] The basic principle for selecting compounds with no or weak P-gp substrate properties:

[0319] According to the free drug hypothesis, only unbound (free) drug molecules exert their pharmacological effects by binding to a target. In cases where the drug for a therapeutic indication needs to be delivered to a CNS site of action, the free brain concentration is the drug concentration most correlated with CNS activity in vivo (doi:10.1124 / jpet.107.119560).

[0320] Drug distribution in the central nervous system (CNS) is typically restricted by the repulsive effect of the blood-brain barrier (BBB). The BBB is composed of a tightly bound endothelial cell monolayer and is characterized by low paracellular permeability, fenestration of various membrane efflux transport proteins, and lack of expression, thereby providing highly effective xenobiotic protection for the CNS against circulation.

[0321] Therefore, the required pharmacological response of drugs targeting the CNS depends on their passive diffusion, which is related to the physicochemical properties of the compound, and the relative balance between active absorption and efflux transporters to overcome neuroprotection at the BBB.

[0322] Many transporters have been identified at the BBB, among which P-gp (P-glycoprotein) is considered the most important efflux transporter due to its gatekeeper function in preventing drugs from entering the brain and its expulsion mechanism that pumps compounds that have already entered the cytoplasm of brain endothelial cells out (doi:10.1007 / s11095-007-9502-2,doi:10.1124 / jpet.107.130294, / doi.org / 10.1517 / 17460441.3.6.677).

[0323] Because P-gp plays such a crucial role in limiting drug absorption into the brain due to its broad substrate specificity and inherent transport activity, it is essential to design compounds without significant P-gp efflux transport activity for CNS targeting. The substrate characteristics of P-gp imply a narrowed therapeutic window for CNS drugs. This narrowing window arises from the need for higher free plasma concentrations to compensate for efflux transport and drive free brain concentrations to desired levels. However, higher plasma concentrations increase the risk of peripheral toxicity. This supports the strategy that CNS drugs should not have substrate affinity for P-gp (doi:10.1124 / dmd.104.001230,10.1124 / jpet.107.130294).

[0324] Furthermore, interference with the BBB can lead to drug-drug interactions (DDIs), as observed with, for example, loperamide (a μ-opioid receptor agonist and potent P-gp substrate): loperamide reduces intestinal motility by activating peripheral μ-receptors in the gut. Due to its high P-gp efflux, brain penetration is limited at clinical doses and even higher, and its administration is not associated with central opioid effects such as respiratory depression. However, co-administration of quinidine, a potent P-gp inhibitor, causes significant respiratory depression that does not occur after loperamide alone (doi:10.1067 / mcp.2000.109156). Other examples of DDI involving P-gp at the BBB include the interaction of verapamil and cyclosporine A (doi:10.1016 / j.clpt.2005.01.022), verapamil and tariquidar (doi:10.1038 / jcbfm.2015.19), verapamil and tamoxifen (doi:10.1124 / jpet.111.180398), or etoposide and cyclosporine A (doi:10.1002 / pbc.20382), suggesting that P-gp interactions at the BBB may cause DDI in humans (doi:10.1124 / dmd.112.049577).

[0325] Given the physiological complexity of the BBB, P-gp-transfected Maddox canine kidney (MDCK) cell lines have proven to be a valuable alternative for predicting P-gp efflux in in vitro preclinical screening, representing the current state of technology for in vitro systems (doi:10.1016 / j.ejps.2017.04.016, doi:10.1124 / dmd.116.074245). The P-gp transport characteristics of compounds were tested using a two-way permeability assay and expressed as an efflux ratio. It has been confirmed that the in vitro efflux observed in the MDCK-P-gp permeability assay is sufficiently correlated with in vivo drug efflux at the rat BBB (doi:10.3390 / pharmaceutics11110595).

[0326] Approximately 95% of CNS drugs exhibited a P-gp efflux ratio <3 (doi:10.1124 / jpet.102.039255,10.1080 / 00498250701570285), confirming that most CNS drugs do not exhibit or exhibit weak P-gp-mediated efflux, and demonstrating that compounds should not be good P-gp substrates for successful CNS delivery.

[0327] Pharmaceutical Composition

[0328] Formulations suitable for administering the compounds of the present invention will be apparent to those skilled in the art, and include, for example, tablets, pills, capsules, suppositories, lozenges, sugar-coated tablets, solutions, syrups, elixirs, capsules, injectables, inhalants, powders, etc. The content of the pharmaceutically active compound may vary from 0.1 to 95% by weight, preferably from 5.0 to 90% by weight, of the total composition.

[0329] Suitable tablets can be obtained, for example, by mixing and extruding the compound of the invention with a known excipient (e.g., an inert diluent, a carrier, a disintegrant, an adjuvant, a surfactant, a binder, and / or a lubricant) to form a tablet.

[0330] Therapeutic uses / methods of use

[0331] The therapeutic applications of IDO1 inhibition in humans have been summarized in the review (doi:10.1007 / s11011-018-0290-7., doi:10.7150 / jca.31727., doi:10.1177 / 1178646917691938., doi:10.1016 / j.pnpbp.2017.04.035., doi:10.2741 / 4363., doi:10.3390 / molecules 23010191. and doi:10.1007 / s00018-017-2504-2.).

[0332] This invention relates to compounds suitable for treating mental illnesses, diseases, and symptoms, wherein IDO1 inhibition has therapeutic benefits, said mental illnesses, diseases, and symptoms include: (1) mood disorders and affective disorders; (2) schizophrenia spectrum disorders; (3) neurotic, stress-related, and body symptom disorders, including anxiety disorders; (4) psychosocial developmental disorders; (5) behavioral syndromes related to physiological disorders and physical factors; (6) substance-related and addiction disorders; (7) diseases related to negative and positive symptoms; (8) neurodegenerative diseases; (9) neuroimmunological diseases; (10) neurovascular diseases; (11) head trauma; (12) metabolic diseases; (13) viral infections; (14) bacterial infections; (15) retinal diseases; and (16) tumor indications.

[0333] The compounds of this invention, by virtue of their pharmacological effects, are suitable for treating diseases, illnesses, or symptoms selected from the list of the following:

[0334] (1) Treatment of mood disorders and affective disorders, including: depression, hypomania, mania and mixed bipolar I; bipolar II; depression, such as single depressive episodes or recurrent severe depression, mild depression, postpartum depression, depression with psychotic symptoms; severe depression with or without accompanying anxiety, mixed features, melancholic features, atypical features, mood-consistent psychosis features, mood-inconsistent psychosis features and catatonic symptoms.

[0335] (2) Treatment of mood disorders and other psychotic disorders that fall within the schizophrenia spectrum, including schizophrenia and schizoaffective disorder with related negative and cognitive symptoms.

[0336] (3) Treatment of the following conditions: neurotic, stress-related and body shape disorder, including anxiety disorder, general anxiety disorder, panic disorder with or without agoraphobia, specific phobia, social phobia, chronic anxiety disorder; obsessive-compulsive disorder; reactions and disorders to severe stress, such as post-traumatic stress disorder; other neurotic conditions, such as loss of self and loss of reality syndrome.

[0337] (4) Treatment of developmental disorders, including pervasive developmental disorders, including Asperger's syndrome and Rett's syndrome, autism, childhood autism and ADHD associated with intellectual disability and stereotyped movements, motor function-specific developmental disorders, organizational skills-specific developmental disorders, and attention deficit / hyperactivity disorder.

[0338] (5) Treatment of behavioral syndromes related to physiological disorders and physical factors, including postpartum mental and behavioral disorders, including postpartum depression; eating disorders, including anorexia nervosa and bulimia nervosa and other impulse control disorders.

[0339] (6) Treatment of substance-related and addiction disorders, including substance use disorders induced by alcohol, marijuana, hallucinogens, stimulants, hypnotics, and tobacco.

[0340] (7) Treat illnesses associated with negative and positive valence symptoms, including anhedonia, persistent threat and loss, and suicidal ideation.

[0341] (8) Treat diseases involving neuronal and / or glial cell degeneration, such as Huntington's disease or amyotrophic lateral sclerosis.

[0342] (9) Treatment of metabolic disorders, including obesity. This includes treatment of metabolic disorders with persistent chronic inflammation, as well as those that may be attributable to microbial activity.

[0343] (10) Treatment of viral infections. This includes treatments aimed at normalizing T cell function and reducing immune tolerance, or treatment of the consequences of neurological infections.

[0344] (11) Treatment of bacterial infections. This includes treatments aimed at normalizing T cell function and reducing immune tolerance, treatment of the consequences of neurological infections, or treatment of tuberculosis.

[0345] (12) Treatment of retinal diseases. This includes treatments for the prevention of retinal degeneration caused by high IDO1 expression, such as diabetic retinopathy or geographic atrophy associated with age-related macular degeneration.

[0346] As used herein, unless otherwise stated, the term “treating / treatment” shall include the management and care of a human individual or human patient for the purpose of combating a disease, symptom or condition, and includes the administration of the compounds of the present invention to prevent the onset of symptoms or complications, relieve symptoms or complications or eliminate the disease, symptom or condition.

[0347] As used herein, unless otherwise stated, the term “prevention” shall include (a) a reduction in the frequency of one or more symptoms; (b) a reduction in the severity of one or more symptoms; (c) delaying or avoiding the development of other symptoms; and / or (d) delaying or avoiding the development of symptoms or ailments.

[0348] According to another aspect, the present invention provides a compound of formula A or a pharmaceutically acceptable salt thereof for treating and / or preventing the symptoms mentioned above.

[0349] According to another aspect, the present invention provides a compound of formula A according to any of the foregoing aspects, characterized in that the compound of formula is also used in addition to behavioral therapy, TMS (transcranial magnetic stimulation), ECT (electroconvulsive therapy for convulsive shock) and other therapies.

[0350] Combination therapy

[0351] The focus of this invention is that the compounds according to the invention can be combined with other therapeutic options known in the art for use in treating any indication.

[0352] According to another aspect, the present invention provides a compound of formula A according to any of the foregoing aspects, characterized in that, in addition to treatment with one or more antidepressants, the compound of formula A is also administered, the one or more antidepressants being selected from the list of the following: duloxetine, escitalopram, bupropion, venlafaxine, desvenlafaxine, sertraline, paroxetine, fluoxetine, vortioxetine, mirtazapine, citalopram, vilazodone, trazodone, amitriptyline, clomipramine, agomelatine, levomilnacipran, lithium, doxepin, and nortriptyline. The term "antidepressant" should mean any medicine or drug that can be used to treat depression or a disorder associated with depressive symptoms.

[0353] According to another aspect, the present invention provides a compound of formula A according to any of the foregoing aspects, characterized in that, in addition to treatment with one or more antipsychotic drugs, the compound of formula A is also administered, the one or more antipsychotic drugs being selected from the list of the following: aripiprazole, paliperidone palmitate, lurasidone, quetiapine, risperidone, olanzapine, paliperidone, brexpiprazole, clozapine, asenapine, chlorpromazine, haloperidol, cariprazine, ziprasidone, amisulpride, iloperidone, fluphenazine, blonanserin, and aripiprazole lauroxil. The term "antipsychotic" should mean any medicine or drug that can be used to treat illnesses associated with psychosis or depressive symptoms.

[0354] According to another aspect, the present invention provides a compound of formula A in any of the foregoing aspects, characterized in that, in addition to treatment with one or more psychotropic drugs, the compound of formula A is also administered, wherein the one or more psychotropic drugs are selected from the list consisting of: lisdexamphetamine, methylphenidate, amphetamine, dexamfetamine, dexmethylphenidate, armodafinil, and modafinil. The term "psychotropic drug" should mean any agent or medicine that can be used to treat conditions such as mood disorders or impulse control disorders.

[0355] According to another aspect, the present invention provides a compound of formula A in any of the foregoing aspects, characterized in that, in addition to treatment with a nootropic agent, the compound of formula A is also administered, the nootropic agent being selected from the list of the following: oxiracetam, piracetam, or the natural product St. John's Wort.

[0356] According to another aspect, the present invention provides a compound of formula A, wherein, according to any of the foregoing aspects, in addition to treatment with one or more antidepressants, antipsychotics, psychostimulants, nootropics or natural products, the compound of formula A is also applied, characterized in that, in addition to behavioral therapy, TMS (transcranial magnetic stimulation), ECT (electroconvulsive therapy for convulsive shock) and other therapies, the compound of formula A is also used in combination with one or more antidepressants, antipsychotics, psychostimulants, nootropics or natural products.

[0357] According to another aspect, the present invention provides a compound of formula A in any of the foregoing aspects, characterized in that, in addition to treatment with any chorea suppressant drug (such as tetrabenazine, deutetrabenazine, and baclofen), compound of formula A is also administered.

[0358] According to another aspect, the present invention provides a compound of formula A in any of the foregoing aspects, characterized in that the compound of formula A is also applied in addition to treatments for slowing neurodegeneration (such as riluzole, rasagiline, and edavarone).

[0359] According to another aspect, the present invention provides a compound of formula A in any of the foregoing aspects, characterized in that the compound of formula A is also applied in addition to treatment with any antiviral or antibacterial drug.

[0360] Experimental Chapter

[0361] abbreviation

[0362] aq. aqueous solution

[0363] eq. equivalent

[0364] EtOAc (ethyl acetate)

[0365] h hours

[0366] HATU O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea-hexafluorophosphate

[0367] HCl hydrochloric acid

[0368] Min minutes

[0369] NaOH (sodium hydroxide)

[0370] MeOH (methanol)

[0371] rt room temperature

[0372] Rt retention time

[0373] TBTU O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroborate

[0374] THF Tetrahydrofuran

[0375] Abbreviations in spectrum data

[0376] 1H-NMR proton nuclear magnetic resonance

[0377] δ chemical shift

[0378] d double peak

[0379] dd Double peak

[0380] DMSO-d6 Hexadeuterium-Dimethyl Sulfoxide

[0381] H proton

[0382] Hz (Hz = 1 / second)

[0383] J Coupling constant

[0384] m multiplet

[0385] ppm (parts per million)

[0386] s Single peak

[0387] General Analysis

[0388] All reactions were performed using commercially available reagents and solvents. NMR spectra were recorded using Top-Spin 3.2pl6 software on a Bruker AVANCE IIIHD 400MHz instrument.

[0389] Chemical shifts are given in parts per million (ppm) based on the low field of the internal standard trimethylsilane, expressed in δ.

[0390] Unless otherwise specified, all 1H-NMR spectra are measured in DMSO-d6.

[0391] The selected data are reported as follows: chemical shift, multiplicity, coupling constant (J), integral.

[0392] Low-resolution mass spectra were obtained using a liquid chromatography-mass spectrometry (LCMS) system consisting of an Agilent 1100 series LC system coupled to an Agilent 6130 quadrupole mass spectrometer (electrospray positive ionization).

[0393] method:

[0394] For solvent mixtures used in HPLC-MS methods, solvent % is given as a volume percentage of the corresponding solvent.

[0395] HPLC-MS method:

[0396] Method Name: Z011_S03 Device description: Agilent 1200 with DA and MS detectors column: XBridge C18_3.0×30mm_2.5μm Column Manufacturer: Waters describe:

[0397]

[0398] Preparation of intermediates:

[0399] Intermediate 1a: 2-[(5-bromothiophen-2-yl)formamido]-2-methylpropionic acid

[0400]

[0401] 2-[(5-bromothiophen-2-yl)formamido]-2-methylpropionate methyl ester

[0402] Under a nitrogen atmosphere at room temperature, 3.40 mL (19.5 mmol, 3.0 equivalent) of diisopropylethylamine and 2.30 g (7.16 mmol, 1.1 equivalent) of TBTU were added to 40 mL of THF containing 1.35 g (6.51 mmol) of 5-bromothiophene-2-carboxylic acid. After 10 minutes, 1.00 g (6.51 mmol, 1.0 equivalent) of methyl 2-aminoisobutyrate hydrochloride was added, and the mixture was stirred continuously at room temperature for 3.5 hours. The solvent was evaporated, and water and EtOAc were added to the residue. The organic layer was separated, washed with 5% NaHCO3 aqueous solution, washed twice with water, and dried over Na2SO4. After filtration and solvent evaporation, the product was obtained and used in the next step without further purification.

[0403] Yield: 1.94g (97%)

[0404] MS: 306 / 308(M+H)+(Br)

[0405] 1H-NMR (400mHz): δ = 1.44 (s, 6H), 7.29 (d, 1H, J = 4.06Hz), 7.70 (d, 1H, J = 4.06Hz), 8.66 (s, 1H)

[0406] 2-[(5-bromothiophene-2-yl)formamido]-2-methylpropionic acid

[0407] Add 11.8 mL of 1 M LiOH aqueous solution to 50 mL of THF and 60 mL of water containing 1.80 g (5.88 mmol) of methyl 2-[(5-bromothiophene-2-yl)formamido]-2-methylpropionate. Stir the reaction mixture overnight at room temperature, concentrate to about 30 mL, dilute with water, and acidify to pH 3-4 with 2 M HCl aqueous solution. Filter the precipitate, wash with water, and dry at 50 °C to obtain the desired product.

[0408] Yield: 1.61g (94%)

[0409] MS: 292 / 294(M+H)+(Br)

[0410] 1H-NMR (400mHz): δ = 1.43 (s, 6H), 7.28 (d, 1H, J = 3.93Hz), 7.69 (d, 1H, J = 3.93Hz), 8.50 (s, 1H), 12.25 (s, 1H)

[0411] Intermediate 1b: 2-[(5-chlorothiophen-2-yl)formamido]-2-methylpropionic acid

[0412]

[0413] 2-[(5-chlorothiophen-2-yl)formamido]-2-methylpropionate methyl ester

[0414] Under ice cooling, 13.6 mL (78.1 mmol, 3.0 equivalence) of diisopropylethylamine was added to a mixture of 4.00 g (26.0 mmol) of methyl 2-aminoisobutyrate hydrochloride and 4.23 g (26.0 mmol) of 5-chlorothiophene-2-carboxylic acid in 15 mL of N,N-dimethylformamide, followed by a small addition of 10.0 g (26.3 mmol, 1.0 equivalence) of HATU. The reaction mixture was stirred overnight at room temperature, water (100 mL) was added, the precipitate was filtered, washed with water, and dried in a desiccator.

[0415] Yield: 5.64g (83%)

[0416] MS: 262 / 264(M+H)+(Cl)

[0417] 1H-NMR (400mHz): δ = 1.44 (s, 6H), 3.58 (s, 3H), 7.19 (d, 1H, J = 4.18Hz), 7.75 (d, 1H, J = 4.06Hz), 8.67 (s, 1H)

[0418] 2-[(5-chlorothiophen-2-yl)formamido]-2-methylpropionic acid

[0419] Add 6.00 mL of 4M NaOH aqueous solution (24.0 mmol, 1.1 equivalents) to a mixture of 5.63 g (21.5 mmol) of methyl 2-[(5-chlorothiophen-2-yl)formamido]-2-methylpropionate in 30 mL of THF and 10 mL of MeOH. Stir continuously overnight at room temperature. Evaporate the solvent under reduced pressure, dissolve the residue in water, and acidify with 6.0 mL of 4M HCl aqueous solution. Filter the precipitate, wash with water, and dry in a desiccator.

[0420] Yield: 5.24g (98%)

[0421] MS: 248 / 250(M+H)+(Cl)

[0422] 1H-NMR (400mHz): δ = 1.44 (s, 6H), 7.18 (d, 1H, J = 4.06Hz), 7.74 (d, 1H, J = 4.18Hz), 8.51 (s, 1H), 12.25 (s, 1H)

[0423] Exemplary implementation plan

[0424] Example 1: 2-[(5-bromothiophene-2-yl)formamido]-N-(4-chloro-3-fluorophenyl)-2-methylpropionamide

[0425]

[0426] A mixture of 100 mg (0.34 mmol) 2-[(5-bromothiophene-2-yl)formamido]-2-methylpropionic acid, 50 mg (0.34 mmol) 4-chloro-3-fluoroaniline, and 132 μL (1.20 mmol, 3.5 equivalences) 4-methylmorpholine in 3 mL of THF was prepared at room temperature. Subsequently, 232 μL (0.39 mmol, 1.2 equivalences, 50% in ethyl acetate) of propylphosphonic anhydride was added, and the reaction mixture was heated at 60 °C overnight. The crude reaction mixture was purified by HPLC, and the eluates containing the product were combined and lyophilized.

[0427] Yield: 49 mg (34%)

[0428] HPLC-MS; Method: Z011_S03; Rt[min]: 1.06

[0429] MS: 419 / 421 / 423(M+H)+(Br / Cl)

[0430] 1H-NMR (400mHz): δ = 1.50 (s, 6H), 7.31 (d, 1H, J = 4.06Hz), 7.41-7.49 (m, 2H), 7.75-7.79 (m, 2H),, 8.50 (s, 1H), 9.74 (s, 1H)

[0431] Example 2: 2-[(5-bromothiophene-2-yl)formamido]-N-(4-bromophenyl)-2-methylpropionamide

[0432]

[0433] A mixture of 100 mg (0.34 mmol) 2-[(5-bromothiophene-2-yl)formamido]-2-methylpropionic acid, 59 mg (0.34 mmol) 4-bromoaniline, and 132 μL (1.20 mmol, 3.5 equivalences) 4-methylmorpholine was prepared in 3 mL of THF at room temperature. Subsequently, 232 μL (0.39 mmol, 1.2 equivalences, 50% in ethyl acetate) of propylphosphonic anhydride was added, and the reaction mixture was heated at 60 °C overnight. The crude reaction mixture was purified by HPLC, and the eluates containing the product were combined and lyophilized.

[0434] Yield: 81 mg (53%)

[0435] HPLC-MS; Method: Z011_S03; Rt[min]: 1.05

[0436] MS: 445 / 447 / 449(M+H)+(2Br)

[0437] 1H-NMR (400mHz): δ = 1.50 (s, 6H), 7.31 (d, 1H, J = 4.06Hz), 7.43-7.46 (m, 2H), 7.54-7.57 (m, 2H), 7.77 (d, 1H, J = 4.06Hz), 8.45 (s, 1H), 9.55 (s, 1H)

[0438] Example 3: 2-[(5-chlorothiophen-2-yl)formamido]-N-(4-chloro-3-fluorophenyl)-2-methylpropionamide

[0439]

[0440] A mixture of 100 mg (0.40 mmol) 2-[(5-chlorothiophene-2-yl)formamido]-2-methylpropionic acid, 59 mg (0.40 mmol) 4-chloro-3-fluoroaniline, and 155 μL (1.41 mmol, 3.5 equivalents) 4-methylmorpholine in 3 mL of THF was prepared at room temperature. Subsequently, 274 μL (0.46 mmol, 1.2 equivalents, 50% in ethyl acetate) of propylphosphonic anhydride was added, and the reaction mixture was heated at 60 °C overnight. The crude reaction mixture was purified by HPLC, and the eluates containing the product were combined and lyophilized.

[0441] Yield: 62 mg (41%)

[0442] HPLC-MS; Method: Z011_S03; Rt[min]: 1.05

[0443] MS: 375 / 377 / 379(M+H)+(2Cl)

[0444] 1H-NMR (400mHz): δ = 1.50 (s, 6H), 7.21 (d, 1H, J = 4.06Hz), 7.40-7.49 (m, 2H), 7. 77(dd,1H,J=2.15 / 12.29Hz),7.83(d,1H,J=4.06Hz),8.51(s,1H),9.74(s,1H)

[0445] Example 4: 2-[(5-chlorothiophen-2-yl)formamido]-N-(4-bromophenyl)-2-methylpropionamide

[0446]

[0447] A mixture of 100 mg (0.40 mmol) 2-[(5-chlorothiophene-2-yl)formamido]-2-methylpropionic acid, 69 mg (0.40 mmol) 4-bromoaniline, and 155 μL (1.41 mmol, 3.5 equivalents) 4-methylmorpholine in 3 mL of THF was prepared at room temperature. Subsequently, 274 μL (0.46 mmol, 1.2 equivalents, 50% in ethyl acetate) of propylphosphonic anhydride was added, and the reaction mixture was heated at 60 °C overnight. The crude reaction mixture was purified by HPLC, and the eluates containing the product were combined and lyophilized.

[0448] Yield: 70 mg (43%)

[0449] HPLC-MS; Method: Z011_S03; Rt[min]: 1.04

[0450] MS: 401 / 403 / 405(M+H)+(Br / Cl)

[0451] 1H-NMR (400mHz): δ = 1.50 (s, 6H), 7.21 (d, 1H, J = 4.06Hz), 7.42-7.47 (m, 2H), 7.54-7.58 (m, 2H), 7.82 (d, 1H, J = 4.06Hz), 8.46 (s, 1H), 9.55 (s, 1H)

[0452] Example 5: 2-[(5-chlorothiophen-2-yl)formamido]-N-(4-chlorophenyl)-2-methylpropionamide

[0453]

[0454] A mixture of 100 mg (0.40 mmol) 2-[(5-chlorothiophene-2-yl)formamido]-2-methylpropionic acid, 52 mg (0.40 mmol) 4-chloroaniline, and 155 μL (1.41 mmol, 3.5 equivalents) 4-methylmorpholine was prepared in 3 mL of THF at room temperature. Subsequently, 274 μL (0.46 mmol, 1.2 equivalents, 50% in ethyl acetate) of propylphosphonic anhydride was added, and the reaction mixture was heated at 60 °C overnight. The crude reaction mixture was purified by HPLC, and the eluates containing the product were combined and lyophilized.

[0455] Yield: 93 mg (64%)

[0456] HPLC-MS; Method: Z011_S03; Rt[min]: 1.03

[0457] MS: 357 / 359 / 361(M+H)+(2Cl)

[0458] 1H-NMR(400mHz):δ=1.50(s,6H),7.21(d,1H,J=4.06Hz),7.30-7.34(m,2H),7.59-7.63(m,2H),7.82(d,1H,J=4.06Hz),8.46(s,1H),9.55(s,1H)

Claims

1. A compound of formula A in R 1 Indicates chlorine and bromine; R 2 Indicates hydrogen and fluorine; R 3 It represents chlorine and bromine.

2. The compound according to claim 1, i.e., a compound selected from the group consisting of:

3. The compound according to any one of claims 1 or 2, having the following structure:

4. The compound according to any one of claims 1 or 2, having the following structure:

5. The compound according to any one of claims 1 or 2, having the following structure:

6. The compound according to any one of claims 1 or 2, having the following structure:

7. The compound according to any one of claims 1 or 2, having the following structure:

8. A salt of a compound according to any one of claims 1 to 7.

9. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 mixed with a pharmaceutically acceptable carrier.

10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 mixed with a pharmaceutically acceptable adjuvant.

11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 mixed with a pharmaceutically acceptable diluent.

12. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 mixed with a pharmaceutically acceptable adjuvant and / or diluent.

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