Dopamine d3 / d2 receptor modulating compounds

Abstract

To provide pharmaceutical compositions, comprising compounds useful as agents in the treatment of diseases and conditions including schizophrenia.SOLUTION: The present disclosure provides compounds of Formula (I), where R1 is a linear C1-C3 alkyl, or pharmaceutically acceptable salts thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 479,530, filed January 11, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to compounds that modulate the activity of dopamine D2 and D3 receptors, methods of making such compounds, compositions containing the compounds, and methods of treatment using the compounds. [Background technology]

[0003] Dysfunction of the dopamine neurotransmitter system can be observed in the pathology of neuropsychiatric disorders, including schizophrenia and affective or cognitive dysfunction (Sokoloff, P. et al.: Nature, 1990, 347.146; Schwartz, J.-C. et al.: Clin. Neuropharmacology., 1993, vol. 16, p. 295). The action of dopamine is mediated by at least five different dopamine receptors, which belong to the dopamine D1 receptor family (i.e., D1 and D5) or the D2 receptor family (i.e., D2, D3 and D4). D3 receptors have a characteristic distribution in the central dopaminergic system. Dopamine D2 receptors are widely distributed in the brain and are involved in many physiological functions and pathological conditions. Dopamine D2 antagonists are used, for example, as drugs for treating schizophrenia. However, massive antagonism of D2 receptors results in unwanted side effects, such as extrapyramidal motor symptoms, psychomotor sedation, cognitive slowing, etc. Antipsychotics that preferentially target D3 receptors have been successful therapeutic interventions in the treatment of schizophrenia. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Sokoloff, P. et al.: Nature, 1990, 347.146 [Non-Patent Document 2] Schwartz, J.-C. et al.: Clin. Neuropharmacology., 1993, vol. 16, p. 295 Summary of the Invention [Problem to be solved by the invention]

[0005] There remains a need in the art for improved compounds that have D3 / D2 receptor selectivity. [Means for solving the problem]

[0006] (Summary) In some embodiments, the present invention provides a method for the preparation of a compound of formula (I):

[0007] [ka] (In the formula, R 1 is C1-C3 alkyl), or a pharma- ceutically acceptable salt thereof.

[0008] In some embodiments, the present disclosure provides 1 is CH3, or a pharma- ceutically acceptable salt thereof.

[0009] In some embodiments, the present disclosure provides 1 is CH2CH3, or a pharma- ceutically acceptable salt thereof.

[0010] In some embodiments, the present disclosure provides 1 is CH2CH2CH3, or a pharma- ceutically acceptable salt thereof.

[0011] In some embodiments, the disclosure provides a compound of formula (I), N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea; N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(methoxymethyl)-N-methylurea; and N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-methyl-N-(propoxymethyl)urea; or a pharma- ceutically acceptable salt thereof.

[0012] In some embodiments, the disclosure provides a compound of formula (I), wherein the compound is N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea; or a pharmaceutically acceptable salt thereof.

[0013] In some embodiments, the disclosure provides a compound of formula (I), wherein the compound is N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea.

[0014] In some embodiments, the disclosure provides a compound of formula (I), wherein the compound is a pharma- ceutically acceptable salt, N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea.

[0015] In some embodiments, the disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharma- ceutically acceptable salt thereof, in combination with a pharma- ceutically acceptable carrier.

[0016] In some embodiments, the disclosure provides a method for treating schizophrenia, the method comprising administering to a subject a therapeutically effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt thereof.

[0017] In some embodiments, the disclosure provides a compound of formula (I) or a pharma- ceutically acceptable salt thereof for use as a medicament.

[0018] In some embodiments, the disclosure provides a compound of formula (I), or a pharma- ceutically acceptable salt thereof, for use in the treatment of schizophrenia.

[0019] In some embodiments, the disclosure provides the use of a compound of formula (I) or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament for the treatment of schizophrenia. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure describes compounds that modulate the activity of dopamine D3 and dopamine D2 receptors.

[0021] The compounds disclosed herein may contain one or more variables, which may occur more than once in any substituent or formula herein.The definition of a variable at each occurrence is independent of its definition at other occurrences.Furthermore, combinations of substituents are permissible only if such combinations result in stable compounds.Stable compounds are compounds that can be isolated from reaction mixtures.

[0022] definition Certain terms used herein are intended to refer to the following definitions, as detailed below.

[0023] It should be noted that, as used in this specification and the intended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "compound" includes a single compound as well as one or more of the same or different compounds. Reference to a "pharmaceutically acceptable carrier" means a single pharmaceutically acceptable carrier as well as one or more pharmaceutically acceptable carriers, and the like.

[0024] As used in this specification and the appended claims, unless otherwise specified, the following terms have the meanings indicated.

[0025] As used herein, the term "C1-C3 alkyl" refers to a saturated hydrocarbon chain radical having 1, 2 or 3 carbon atoms, unless otherwise specified. 1~3 Representative examples of "alkyl" include methyl, ethyl, and n-propyl.

[0026] In some cases, the number of carbon atoms in a moiety is indicated by the prefix "C x ~C y」 where x is the minimum value and y is the maximum number of carbon atoms in the substituent. Thus, for example, "C1-C6 alkyl" means an alkyl substituent containing from 1 to 6 carbon atoms, and "C1-C3 alkyl" means an alkyl substituent containing from 1 to 3 carbon atoms.

[0027] The phrase "pharmaceutical composition" refers to a composition suitable for administration for medical use.

[0028] The phrase "pharmaceutically acceptable salt" refers to salts that are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, and the like, and that are commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0029] The term "stable" refers to a compound that has sufficient stability to permit manufacture and maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein.

[0030] The phrase "therapeutically effective amount" refers to the amount of a compound, or a pharma- ceutically acceptable salt thereof, that, when administered therapeutically in a particular subject or population of subjects, is sufficient to prevent the occurrence of, or alleviate to some extent, one or more of the symptoms of, the condition or disorder being treated.

[0031] As used herein, the terms "treat," "treating," and "treatment" refer to a method of alleviating or arresting diseases and / or their associated symptoms.

[0032] compound The compounds of the present disclosure have the general formula (I) described herein.

[0033] In some embodiments, the present disclosure provides a compound of formula (I):

[0034] [ka] (In the formula, R 1 is C1-C3 alkyl), or a pharma- ceutically acceptable salt thereof.

[0035] In some embodiments, the present disclosure provides 1 is C1 alkyl, or a pharma- ceutically acceptable salt thereof.

[0036] In some embodiments, the present disclosure provides 1 is C2 alkyl, or a pharma- ceutically acceptable salt thereof.

[0037] In some embodiments, the present disclosure provides 1 is C alkyl; or a pharma- ceutically acceptable salt thereof.

[0038] In some embodiments, the present disclosure provides1 is selected from the group consisting of CH3, CH2CH3, and CH2CH2CH3, or a pharma- ceutically acceptable salt thereof.

[0039] In some embodiments, the present disclosure provides 1 is CH3, or a pharma- ceutically acceptable salt thereof.

[0040] In some embodiments, the present disclosure provides 1 is CH2CH3, or a pharma- ceutically acceptable salt thereof.

[0041] In some embodiments, the present disclosure provides 1 is CH2CH2CH3, or a pharma- ceutically acceptable salt thereof.

[0042] In some embodiments, the disclosure provides a compound of formula (I) which is N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea; or a pharma- ceutically acceptable salt thereof.

[0043] In some embodiments, the disclosure provides a compound of formula (I), wherein the compound is N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea.

[0044] In some embodiments, the disclosure provides a compound of formula (I), which is a pharma- ceutically acceptable salt of N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea.

[0045] In some embodiments, the disclosure provides a compound of formula (I); or a pharma- ceutically acceptable salt thereof, wherein the compound is N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(methoxymethyl)-N-methylurea.

[0046] In some embodiments, the disclosure provides a compound of formula (I), wherein the compound is N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(methoxymethyl)-N-methylurea.

[0047] In some embodiments, the disclosure provides a compound of formula (I), which is a pharma- ceutically acceptable salt of N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(methoxymethyl)-N-methylurea.

[0048] In some embodiments, the disclosure provides a compound of formula (I); or a pharma- ceutically acceptable salt thereof, wherein the compound is N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-methyl-N-(propoxymethyl)urea.

[0049] In some embodiments, the disclosure provides a compound of formula (I), wherein the compound is N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-methyl-N-(propoxymethyl)urea.

[0050] In some embodiments, the disclosure provides a compound of formula (I), which is a pharma- ceutically acceptable salt of N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-methyl-N-(propoxymethyl)urea.

[0051] The compounds and intermediates of the disclosure were named using the ACD / Name 2021.1.3 (file version N15E41, Build 123232, July 7, 2021) software program and / or using the Struct=Name naming algorithm as part of CHEMDRAW® Professional v.20.1.1.125.

[0052] Exemplary compounds of Formula (I) include, but are not limited to, the compounds set forth in Table 1 below, and pharma- ceutically acceptable salts thereof.

[0053] [Table 1]

[0054] The compounds of formula (I) can be used in the form of pharmaceutically acceptable salts. The compounds of formula (I) can contain either basic or acidic functional groups, or both, and can be converted to pharmaceutically acceptable salts, if desired, by using an appropriate acid or base.

[0055] Methods for making exemplary compounds The compounds of the present disclosure may be better understood in connection with the following synthetic schemes and methods illustrating the means by which the compounds may be prepared. The compounds of the present disclosure may be prepared by a variety of synthetic procedures. Representative synthetic procedures are shown in Schemes 1-2, but are not limited thereto. The variable R 1 is defined as detailed herein, e.g., in the Abstract. Scheme

[0056] [ka]

[0057] As shown in Scheme 1, R 1Compounds of formula (I), where R is as defined herein, can be prepared from compounds of formula (1), which can be prepared according to methods known in the art, such as, for example, WO 2010 / 070370. Thus, compounds of formula (1) can be prepared by reacting commercially available paraformaldehyde or formaldehyde (saturated aqueous solution with methanol stabilizer) and R 1 The reaction can be treated with an appropriate alcohol of formula (2), where X is as described herein, which are either commercially available or can be prepared according to methods known in the art at reflux at ambient temperature. The reaction is typically carried out in a suitable solvent, such as alcohol (2) or tetrahydrofuran, for 1-168 hours to produce a compound of formula (I).

[0058] [ka]

[0059] As shown in Scheme 2, compounds of formula (I) can be prepared from compounds of formula (1). Thus, compounds of formula (1) can be treated with commercially available 1-hydroxymethylimidazole at ambient temperature and refluxed in a suitable solvent, such as a mixture of acetic acid and tetrahydrofuran, for 1-24 hours to produce compounds of formula (3). Compounds of formula (3) can be reacted with R 1 may be treated with an appropriate alcohol of formula (2), wherein X is as described herein, in the presence of a suitable base, such as potassium hydroxide, or a suitable acid, such as sulfuric acid or methanesulfonic acid, to produce a compound of formula (I). The reaction is typically carried out at ambient temperature and reflux in a suitable solvent, such as tetrahydrofuran or toluene, for 1-24 hours.

[0060] Detailed procedures are provided in the Synthetic Examples section. Unless otherwise noted, starting materials and reagents are either commercially available or can be prepared from commercially available materials by one of ordinary skill in the art using methods described in the chemical literature.

[0061] Pharmaceutical Compositions When used as pharmaceuticals, the compounds of the present disclosure can be administered in the form of a pharmaceutical composition. Such a composition can include a therapeutically effective amount of a compound of formula (I), or a pharma- ceutically acceptable salt thereof, together with a pharma- ceutically acceptable carrier.

[0062] In some embodiments, a pharmaceutical composition is provided that includes a therapeutically effective amount of a compound of formula (I), or a pharma- ceutically acceptable salt thereof, in combination with a pharma- ceutically acceptable carrier.

[0063] How to use The compound of formula (I), or a pharma- ceutically acceptable salt thereof, and pharmaceutical compositions comprising the compound of formula (I), or a pharma- ceutically acceptable salt thereof, can be administered to a subject suffering from schizophrenia. The term "administration" refers to the method of contacting a compound with a subject.

[0064] In some embodiments, the disclosure provides a compound of formula (I), or a pharma- ceutically acceptable salt thereof, and pharmaceutical compositions comprising a compound of formula (I), or a pharma- ceutically acceptable salt thereof, for use as a medicament.

[0065] In some embodiments, the disclosure provides a compound of formula (I), or a pharma- ceutically acceptable salt thereof, and a pharmaceutical composition comprising a compound of formula (I), or a pharma- ceutically acceptable salt thereof, for use in the treatment of schizophrenia.

[0066] In some embodiments, the disclosure provides the use of a compound of formula (I), or a pharma- ceutically acceptable salt thereof, and a pharmaceutical composition comprising a compound of formula (I), or a pharma- ceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of schizophrenia. EXAMPLES

[0067] The following examples can be used for illustrative purposes and should not be considered as limiting the scope of the invention.

[0068] All reagents were of commercial grade and were used as is without further purification unless otherwise stated. Commercially available anhydrous solvents were used for reactions carried out under an inert atmosphere. Reagent grade solvents were used in all other cases unless otherwise specified. 1 Chemical shifts (δ) for H NMR spectra are reported in parts per million (ppm) relative to tetramethylsilane (δ 0.00) or the appropriate residual solvent peak, i.e., CHCl3 (δ 7.27), as internal reference.

[0069] The following abbreviations have the indicated meanings unless otherwise specified:

[0070] [Table 2]

[0071] Synthesis Example [Example 1] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea A mixture of 5.0 g (12.5 mmol) of N-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N'-methylurea, 250 mL of ethanol, 2.5 mL of acetic acid, and 3.75 g (125 mmol) of paraformaldehyde was stirred at 70°C for 40 hours. The reaction mixture was concentrated in vacuo. The residue was chromatographed on silica gel eluted with acetone. The crude material was mixed with diethyl ether and filtered to yield the title compound. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.88-1.02 (m, 2H), 1.08 (t, J = 7.0 Hz, 3H), 1.14-1.28 (m, 3H), 1.31-1.40 (m, 2H), 1.69-1.81 (m, 4H), 2.31-2.39 (m, 2H), 2.42-2.61 (br m, 4H), 2.79 (s, 3H), 2.90-3.04 (br m, 4H), 3.35 (q, J = 7.0 Hz, 2H), 3.31-3.44 (m, 1H), 4.63 (s, 2H), 6.03 (d, J = 7.8 Hz, 1H), 7.10-7.18 (m, 1H), 7.25-7.34 (m, 2H); LC-MS (ESI) m / z 471.3 (M+H) + .

[0072] [Example 2] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(methoxymethyl)-N-methylurea A mixture of 1 g (2.4 mmol) of N-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N'-methylurea, 20 mL of methanol, 0.5 mL of acetic acid, and 2 mL (26.8 mmol) of 37% formaldehyde solution (aqueous solution containing approximately 10% methanol) was stirred for 20 hours at 85° C. The reaction mixture was concentrated in vacuo. The residue was purified by preparative HPLC on a Phenomenex Kinetex® 5 μm C18 100 Å AXIA Packed LC Column (150 mm×21.2 mm); isocratic over 3 min, 20 mmol / L NH4HCO3 in water + 0.1% diethylamine (A); 40-50% gradient over 16 min of 20 mmol / L NH4HCO3 in water + 0.1% diethylamine (A) and methanol (B) (A) at 40° C. with a flow rate of 21.2 mL / min to yield the title compound. 1H NMR (400 MHz, dimethyl sulfoxide-d6) δppm 0.88-1.06 (m, 2H), 1.15-1.28 (m, 3H), 1.31-1.40 (m, 2H), 1.68-1.82 (m, 4H), 2.31-2.39 (m, 2H), 2.46-2.59 (br m, 4H), 2.79 (s, 3H), 2.90 -3.05 (br m, 4H), 3.12 (s, 3H), 3.25-3.45 (m, 1H), 4.59 (s, 2H), 6.05 (d, J = 7.9 Hz, 1H), 7.11-7.17 (m, 1H), 7.27-7.34 (m, 2H); LC-MS (ESI) m / z 457.3 (M+H) + .

[0073] [Example 3] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-methyl-N-(propoxymethyl)urea

[0074] [Example 3A] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(1H-imidazol-1-yl)methyl]-N-methylurea A mixture of 3.0 g (7.25 mmol) of N-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N'-methylurea, 1.070 g (10.9 mmol) of (1H-imidazol-1-yl)methanol, 9 mL (200 mmol) of acetic acid and 70 mL of tetrahydrofuran was stirred at 80°C for 18 h. Dichloromethane (100 mL) and saturated aqueous NaHCO3 (50 mL) were added to the mixture. The organic layer was separated, dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo. The residue was mixed with a mixture of diethyl ether (200 mL) and methanol (5 mL) and filtered to produce the title compound. LC-MS (ESI): m / z 493.3 (M+H) + .

[0075] [Example 3B] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-methyl-N-(propoxymethyl)urea To a solution of 87 mg (0.9 mmol) of methanesulfonic acid in 273 mg (4.55 mmol) of 1-propanol, 20 mL of toluene and 443 mg (1 mmol) of N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-[(1H-imidazol-1-yl)methyl]-N-methylurea were added at room temperature and the reaction mixture was stirred at 90° C. for 2 h. Ethyl acetate (30 mL) and saturated aqueous NaHCO3 (30 mL) were added to the mixture. The organic layer was separated, dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo. The residue was chromatographed on silica gel eluting with dichloromethane / methanol (97:3). The crude material was mixed with n-pentane and filtered to yield the title compound. 1 H NMR (400 MHz, DMSO-d6) d ppm 0.84 (t, J =7.2 Hz, 3H), 0.88-1.03 (m, 2H), 1.13-1.29 (m, 3H), 1.30-1.40 (m, 2H), 1.48 (sxt, J = 7.0 Hz, 2H), 1.69-1.81 (br m, 4H), 2.31-2.40 (m, 2H), 2.43-2.60 (br m, 4H), 2.79 (s, 3H), 2.90-3.05 (br m, 4H), 3.26 (t, J = 6.5 Hz, 2H), 3.31-3.46 (m, 1H), 4.64 (s, LC-MS (ESI) m / z 485.3 (M+H) + .

[0076] [ka]

[0077] Reference example 1 N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-methyl-N-propylurea The title compound was prepared as described in WO2005012266.

[0078] [ka]

[0079] Reference example 2 N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N,N-dimethylurea (CAR) The title compound was prepared as described in WO2005012266.

[0080] [ka]

[0081] Reference example 3 N-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N'-methylurea (DCAR) The title compound was prepared as described in WO2005012266.

[0082] Kinetic Solubility test

[0083] [Table 3]

[0084] For kinetic solubility determination, 3 mL of a 10 mM (5 mM, 1 mM) DMSO stock solution of each compound was placed in a 96-well plate (Millipore Multiscreen®) containing 295 mL of PBS (pH 7.4). HTS The suspension was pipetted into the wells of a 100-PCF Filter Plate (MilliporeSigma, St. Louis, MO, USA). After shaking the suspension on an orbital shaker at 300 rpm for 2 h, the suspension was clarified by vacuum filtration (Multiscreen® Vacuum Manifold; MilliporeSigma). Immediately after the filtration step, 160 mL of the filtrate was transferred to 40 mL of acetonitrile to avoid precipitation of the compound from the saturated solution. The concentration of the studied compound was determined by HPLC-UV-(MS) detection by using an external standard made from the same batch of the studied compound. The results are shown in Table 2.

[0085] Thermodynamic solubility test (37℃, 4 hours) For thermodynamic solubility measurements: Typically, 200-400 μL of this solution was added to 1-2 mg of solid test substance (n=5). The resulting mixture / supersaturated solution was shaken for 4 h (until solution equilibrium) at 37°C and filtered through a Millipore multiscreen filter plate (0.45 μm). The concentration of the filtrate was determined by LC / UV / MS based on a 5-point calibration. (If the solid material was completely dissolved in the aqueous test medium, the result should be considered as the minimum value.) The results are shown in Table 2.

[0086] Thermodynamic solubility test (RT, 24 hours) For thermodynamic solubility measurements: Typically, 200-400 μL of this solution was added to 1-2 mg of solid test substance (n=5). The resulting mixture was shaken at room temperature (RT) for 24 h (until solution equilibrium) and filtered through a Millipore multiscreen filter plate (0.45 μm). The concentration of the filtrate was determined by LC / UV / MS based on a 5-point calibration. (If the solid material was completely dissolved in the aqueous test medium, the result should be considered as the minimum value.) The results are shown in Table 2.

[0087] [Table 4]

[0088] Kinetic and thermodynamic solubility data for the examples of the present disclosure demonstrate unexpected improvements in their solubilities compared to those of the reference compounds.

[0089] Biological assays The following abbreviations were commonly used in in vitro biological studies: BSA is bovine serum albumin, cAMP is cyclic adenosine monophosphate, DMEM is Dulbecco's modified Eagle's medium, DMSO is dimethyl sulfoxide, EDTA is ethylenediaminetetraacetic acid tetrasodium salt, EGTA is ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, FBS is fetal bovine serum, G418 is geneticin, IBMX is 3-isobutyl-1-methylxanthine, and Tris is tris(hydroxymethyl)aminomethane.

[0090] Human D 2L Receptor-based testing In competitive binding assays 3 H]raclopride in human D 2L Determining receptor affinity Recombinant human D 2L Cultured CHO-K1 cells expressing the receptor (DRD2L cAMP Hunter™, G i Cell lines (Eurofins DiscoverX, Fremont, CA, USA) were homogenized in 4x (v / v) buffer (50 mM Tris, 5 mM MgCl2, 1 mM EGTA, pH 7.4, 25°C) using a Dounce tissue grinder and centrifuged at 40,000 x g for 10 min at 4°C. The supernatant was removed and the pellet was resuspended in 4x buffer (v / v) and centrifuged. The resulting pellet was resuspended in the above buffer at a volume of 12.5 mL / g original weight. Membrane preparations were then aliquoted and stored at -70°C.

[0091] Test compounds were serially diluted in DMSO and subsequently diluted to 5% DMSO (v / w) in binding buffer (50 mM Tris, 5 mM MgCl2, 5 mM KCl, 1 mM CaCl2, 120 mM NaCl, 1 mM EDTA). 5x concentrated compounds in binding buffer (50 μL) were transferred to a 96-well deep-well plate (BRAND, Wertheim, Germany). Aliquot membrane preparations were thawed and washed once in binding buffer. In the same buffer, 10 μg protein per well was incubated at 2 nM [ 3 H]raclopride (PerkinElmer, Waltham, MA, USA). Non-specific binding (NSB) was determined in the presence of 10 μM haloperidol, an antagonist of the D2 receptor. After incubation, samples were filtered onto UniFilter® GF / B plates (PerkinElmer) using a Filtermate™ harvester (PerkinElmer) and washed four times with 1 mL of ice-cold binding buffer. Plates were dried for 1 h at 40°C and 40 μL of Microscint™-20 scintillation cocktail (PerkinElmer) was added to each well. Radioactivity was determined using MicroBeta 2 The concentrations were determined using a Microplate Counter (PerkinElmer).

[0092] Non-specific binding was subtracted to obtain specific binding, which was normalized to vehicle-treated samples and converted to % displacement values. GraFit 6.0 (Erithracus Software, Horley, UK) was used to calculate IC 50 The IC values ​​were determined and curve fitting was performed. 50 The inhibition constant (K) (i.e., the concentration of compound that inhibits 50% of specific binding) was determined from the concentration-displacement curves by sigmoidal fitting. i ) was calculated using the Cheng-Prusoff equation: K i =IC 50 / [1+([L] / K D )] (where [L] is the radioligand concentration, K D is the affinity of the labeled ligand for the receptor). K D was determined from a separate saturation experiment. i Values ​​are expressed in mol / liter, K i The results were derived by calculating the negative logarithm of the value. The experiment was performed three times, and the results are shown in Table 3. From these results, the examples of the present disclosure show that the human recombinant D 2L It is shown to be a high affinity ligand for the receptor.

[0093] [Table 5]

[0094] [ 3 Human D in a competitive binding assay with [H]spiperone 2L Determining receptor affinity Human recombinant D 2L CHO-K1 cells expressing the receptor were washed with phosphate-buffered saline (PBS). The cells were scraped off from the plate and centrifuged at 1000×g. The cells were disrupted using a Teflon® pestle homogenizer in a buffer containing 25 mM Tris-HCl, pH=7.4, 6 mM MgCl2, 1 mM EDTA, 10 mM phenylmethylsulfonyl fluoride (PMSF). The resulting suspension was centrifuged at 1000×g. The supernatant was collected and centrifuged at 41,000×g. The supernatant was discarded and the pellet was resuspended in the above buffer. The membrane preparation was then aliquoted and stored at −70° C.

[0095] In a 96-well plate, 0.16 nM [ 3Aliquots of membrane preparations were incubated with [H]spiperone (PerkinElmer) for 120 min at 25°C. Non-specific binding (NSB) was determined in the presence of 10 μM haloperidol (Sigma-Aldrich). After incubation, samples were filtered onto UniFilter® GF / C plates (PerkinElmer), washed, and Microscint™-20 scintillation cocktail was added. Radioactivity was measured using a MicroBeta 2 The concentrations were determined using a Microplate Counter (PerkinElmer).

[0096] From the scintillation counts, NSB was subtracted to obtain specific binding, which was normalized to vehicle-treated samples and converted to % displacement values. IC 50 Values ​​were determined by nonlinear least-squares regression analysis using MathIQ™ (ID Business Solutions Ltd., Surrey, UK). i The values ​​are the observed IC 50 , the concentration of radioligand used in the assay, and the K of the ligand D pK was calculated using the Cheng-Prusoff equation using historical values. i Values ​​are expressed in mol / liter, K i The results are shown in Table 4. From these results, the examples of the present disclosure 3 When [H]spiperone was used as the radioligand, human recombinant D 2L It is shown to be a high affinity ligand for the receptor.

[0097] [Table 6]

[0098] Human D using cyclic adenosine monophosphate detection 2L Characterization of receptor agonism Human D 2L Agonist activity at the cAMP G receptori Using a kit (Cisbio / PerkinElmer), human D 2L Human D was assayed by measuring cAMP levels in CHO-K1 cells expressing the receptor (Eurofins DiscoverX, Fremont, CA, USA). 2LCHO-K1 cells expressing the receptor were cultured in Ham's F12 medium supplemented with 10% FBS, 1% penicillin-streptomycin antimycotic solution, and 800 μg / mL G418 (Thermo Fisher Scientific, Waltham, MA, USA) and maintained at 37° C. in a humidified atmosphere containing 5.0% CO2. For cAMP measurements, cryopreserved cells were thawed and plated at 10,000 cells per well in half-area white-walled 96-well plates in PathHunter® AssayComplete™ Cell Plating 2 (CP2) reagent (Eurofins DiscoverX) and incubated overnight at 37° C. in a humidified atmosphere containing 5.0% CO2. Prior to cAMP measurements, CP2 reagent was removed from cells and replaced with 20 μL of compound or vehicle-containing assay buffer (140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES), 10 mM glucose, pH 7.4) supplemented with 100 μM IBMX and incubated for 20 min at ambient temperature. After a further 30 min incubation at ambient temperature with 0.5 μM forskolin (Eurofins DiscoverX), cell stimulation was stopped by adding detection reagents (20 μL cAMP-d2 and 20 μL anti-cAMP cryptate, Cisbio / PerkinElmer) diluted in lysis buffer. After 60 min of incubation at ambient temperature, the time-resolved fluorescent signal was quantified by a PHERAstar FS multimode reader (BMG Labtech, Ortenberg, Germany) using standard HTRF settings with laser excitation at 337 nm. Results were expressed as the ratio of the acceptor fluorescent signal (A665 nm) and the donor fluorescent signal (A620 nm) × 10 4The pEC values ​​were calculated from the ΔF ratio and expressed as ΔF% values ​​using the following formula: 100 × (sample ratio-negative control ratio) / negative control ratio. In an experiment, all treatments were measured in parallel in multiple wells, and the average ΔF% value was used for further analysis. Agonist activity values ​​were calculated as the percentage of inhibition of forskolin-stimulated cAMP accumulation and normalized to the response evoked by the maximal effective concentration of dopamine tested in the same experiment. All calculations were performed using Microsoft Excel® (Microsoft Corporation, Redmond, WA, USA). The pEC values ​​shown in Table 5 50 Values ​​(expressed as the negative logarithm of the agonist concentration that inhibits forskolin-stimulated cAMP accumulation by 50%, in moles per liter) were obtained by constraining the lower asymptote to zero and fitting a four-parameter sigmoidal curve to the concentration-effect data using GraphPad Prism (GraphPad, San Diego, CA, USA). These results demonstrate that the examples of the present disclosure are consistent with the results of the study of human recombinant D 2L It is shown to be a potent agonist of the receptor's G protein-coupled signaling pathway.

[0099] [Table 7]

[0100] Human Dopamine D 2L Measuring β-arrestin recruitment to receptors Tagged human D 2LPathHunter® CHO-K1 cells expressing receptor and tagged β-arrestin-2 (Eurofins DiscoverX, Fremont, CA, USA) were seeded into white-walled, clear-bottom 96-well tissue culture plates at a density of 20,000 cells per well in 90 μL of AssayComplete™ Cell Plating 2 (CP2) reagent (Eurofins DiscoverX) per cell. Plates were incubated overnight at 37°C in a humidified atmosphere containing 5% CO2. After 20-24 hours, 20 μL of test compound or vehicle in CP2 reagent and 2.2% DMSO was added to the cells, which were then incubated at 37°C for 90 minutes. PathHunter® detection reagent (Eurofins DiscoverX) was then added at 55 μL per well and the plate was incubated at 25° C. for 60 min, after which luminescence was detected using a PHERAstar® FS multimode plate reader (BMG Labtech, Ortenberg, Germany). Raw data were converted to percent stimulation above basal values. Values ​​were further converted to percent maximal stimulation of β-arrestin recruitment by 30 μM dopamine. EC 50 Values ​​were calculated from concentration-response curves of at least six concentrations performed in duplicate by sigmoidal fitting using Origin® 7.5 software (OriginLab Corporation, Northampton, MA, USA) and were defined as the concentration of agonist at which stimulation was half-maximal. 50 Values ​​are expressed in mol / liter, EC 50 The results were calculated as the negative logarithm of the values ​​and are shown in Table 6. From these results, it can be seen that the examples of the present disclosure have 2L It is shown to be a potent agonist of the receptor's G protein-independent signaling pathway.

[0101] [Table 8]

[0102] Testing with human D3 receptors [ 3 Determination of affinity of human D3 receptor in competitive binding assay with [H]raclopride A cell culture (CHO-K1) expressing recombinant human D3 receptor (DRD3, GenBank ID U32499, purchased from Euroscreen Fast, Brussels, BE) was homogenized in 4x buffer (v / v) solution (15 mM Tris, 2 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, pH 7.4, 25°C) using a Dounce tissue grinder and centrifuged at 40,000 x g for 25 min at 4°C. The supernatant was removed and the pellet was resuspended in 4x (v / v) buffer and centrifuged. This process was repeated twice and the pellet was resuspended in storage buffer (75 mM Tris, 12.5 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose, pH 7.4, 25°C) at 12.5 mL / g original cell weight. Membrane preparations were then aliquoted and stored at -70°C.

[0103] Compounds were diluted in DMSO and binding buffer (containing 50 mM Tris, 5 mM MgCl2, 5 mM KCl, 1 mM CaCl2, 120 mM NaCl, 1 mM EDTA) and 50 μL of each solution was transferred to a deep-well plate (BRAND) at 5x the final concentration in 5% DMSO-buffer. Aliquots of membrane preparations were thawed and washed once in binding buffer. In the same buffer, 3.3 μg of protein per assay was diluted in 96-well deep-well plates (BRAND) at approximately 2.7 nM [ 3H]raclopride (PerkinElmer) in a volume of 250 μL for 120 min at 25° C. Non-specific binding (NSB) was determined in the presence of 10 μM haloperidol. The final concentration of DMSO was 1% (v / v) in all reactions. After incubation, samples were filtered onto UniFilter® GF / B plates (PerkinElmer) using a Filtermate™ harvester (PerkinElmer) and washed with 4×1 mL of ice-cold binding buffer. Plates were dried for 1 h at 40° C. and 40 μL of Microscint™-20 scintillation cocktail (PerkinElmer) was added to each well. Radioactivity was determined using MicroBeta 2 The concentrations were determined using a Microplate Counter (PerkinElmer).

[0104] Raw scintillation counts were used to subtract NSB to obtain specific binding, which was normalized to vehicle-treated samples and converted to percent displacement. GraFit 6.0 (Erithracus Software, Horley, UK) was used for curve fitting and calculation. IC 50 The inhibition constant (K) (i.e., the concentration of compound that inhibits 50% of specific binding) was determined from the concentration-displacement curve by sigmoidal fitting. i ) was calculated using the Cheng-Prusoff equation: K i =IC 50 / [1+([L] / K D )] (where [L] is the radioligand concentration, K D is the affinity of the labeled ligand for the receptor). K D was determined from a separate saturation experiment. i Values ​​are expressed in mol / liter, K i The affinity of the IgG4 antibody was determined by calculating the negative logarithm of the IgG4 antibody value. In all experiments, samples were run in triplicate. These results show that the examples of the present disclosure are high affinity ligands of the human recombinant D3 receptor.

[0105] [Table 9]

[0106] [ 3 Determination of affinity of human D3 receptor in competitive binding assay with [H]spiperone CHO-K1 cells expressing human recombinant D3 receptor were washed with PBS. Cells were scraped off from the plate and centrifuged at 1000×g. Cells were disrupted in a buffer containing 25 mM Tris-HCl (pH=7.4), 6 mM MgCl2, 1 mM EDTA, 10 mM PMSF using a Teflon® pestle homogenizer. The suspension was centrifuged at 1000×g. The supernatant was collected and centrifuged at 41,000×g. The supernatant was discarded and the pellet was resuspended in the above buffer. Membrane preparations were aliquoted and stored at −70° C.

[0107] In a 96-well plate, 0.7 nM [ 3 Aliquots of membrane preparations were incubated with [H]spiperone (PerkinElmer) for 120 min at 37°C. Non-specific binding (NSB) was determined in the presence of 25 μM (S)-(-)-sulpiride (Sigma Aldrich). After incubation, samples were filtered onto GF / C filter plates (PerkinElmer), washed, and Microscint™-20 scintillation cocktail was added. Radioactivity was measured using a MicroBeta 2 The concentrations were determined in a Microplate Counter (PerkinElmer).

[0108] From the raw scintillation counts, NSB was subtracted to obtain specific binding, which was normalized to vehicle-treated samples and converted to % displacement values. IC 50Values ​​were determined by nonlinear least-squares regression analysis using MathIQ™ (ID Business Solutions Ltd., Surrey, UK). i The values ​​are the observed IC 50 , the concentration of radioligand used in the assay, and the K of the ligand D The pK values ​​were calculated using the Cheng-Prusoff equation using historical values ​​of i Values ​​are expressed in mol / liter, K i From these results, the examples of the present disclosure are 3 When [H]spiperone is used as a radioligand, it is shown to be a high affinity ligand for the human recombinant D3 receptor.

[0109] [Table 10]

[0110] Characterization of human D3 receptor agonism using cyclic adenosine monophosphate detection Agonist activity at the human D3 receptor was determined by cAMP G icAMP levels were assayed in HEK293 cells (a cell line developed by Gedeon Richter) expressing recombinant human D3 receptor (BioXtal, Saint-Felix, France) stably co-expressing adenyl cyclase V (ACV) by homogeneous time-resolved fluorescence (HTRF) using a kit (Cisbio / PerkinElmer). HEK293 cells expressing recombinant human D3 receptor and ACV were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin antimycotic solution, 1% pyruvate, 100 μg / mL G418 (Thermo Fisher Scientific), and 60 μg / mL hygromycin B, and maintained at 37°C in a humidified atmosphere containing 5% CO2. Prior to measuring cAMP, cells were detached with Versene (Thermo-Fisher Scientific) and suspended in assay buffer (140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES), 10 mM glucose, pH 7.4) in a volume of 20 μL at a density of 15,000 cells per well in white-walled half-area 96-well microplates. Assay buffer was supplemented with 100 μM IBMX (Sigma Aldrich, St Louis, MO, USA). After adding test compounds (4× concentrated) or vehicle (DMSO) at 10 μL per well, cells were incubated with assay buffer or various concentrations of test compounds for 20 min. After a further 30 min incubation at ambient temperature with 1.5 μM forskolin (final DMSO concentration was 0.3%), cell stimulation was stopped by adding detection reagents (20 μL cAMP-d2 and 20 μL anti-cAMP cryptate) diluted in lysis buffer (PerkinElmer). After 60 min incubation at ambient temperature, the time-resolved fluorescence signal (TRF) was quantified by a PHERAstar FS multimode reader (BMG Labtech, Ortenberg, Germany) using standard HTRF settings with laser excitation at 337 nm.

[0111] The results were calculated by multiplying the acceptor fluorescence signal (A665 nm) and the donor fluorescence signal (A620 nm) by 10 4 and expressed as ΔF% values ​​using the following formula: 100 × (sample ratio-negative control ratio) / negative control ratio. In all experiments, multiple wells were measured in parallel and the average ΔF% value was used for further analysis. Agonist activity values ​​were calculated as the percentage of inhibition of forskolin-stimulated cAMP accumulation and normalized to the response evoked by the maximally effective concentration of dopamine tested in the same experiment. All calculations were performed using Excel (Microsoft, Redmond, WA, USA). pEC 50 Values ​​(negative logarithm of the agonist concentration that inhibits forskolin-stimulated cAMP accumulation by 50%, expressed in mol / liter) were obtained by constraining the lower asymptote to zero and fitting a four-parameter sigmoidal curve to the concentration-effect data using GraphPad Prism (GraphPad Software, San Diego, CA, USA) and are shown in Table 9. The results indicate that examples of the present disclosure are potent agonists of the G protein-dependent signaling pathway of the human recombinant D3 receptor.

[0112] [Table 11]

[0113] Measurement of β-arrestin recruitment to human dopamine D3 receptors PathHunter® CHO-K1 cells expressing tagged human D3 receptor and tagged β-arrestin-2 (Eurofins DiscoverX, Fremont, CA, USA) were seeded at a density of 25,000 cells per well in 90 μL of AssayComplete™ Cell Plating 2 (CP2) reagent (Eurofins DiscoverX) in white-walled, clear-bottom 96-well tissue culture plates and incubated overnight at 37°C in a humidified atmosphere with 5% CO2. After 20-24 hours, 20 μL of test compound or vehicle in CP2 reagent containing 2.2% DMSO was added to the cells and the cells were incubated at 37°C for 90 minutes. After incubation, 55 μL of PathHunter® detection reagent (Eurofins DiscoverX) was added per well and the plate was incubated at 25° C. for 60 min, after which luminescence was detected using a PHERAstar® FS multimode plate reader (BMG Labtech, Ortenberg, Germany).

[0114] Raw data were first converted to % stimulation above basal. The % stimulation above basal was further converted to % maximal stimulation of β-arrestin recruitment by 1 μM dopamine. EC 50 Values ​​were calculated from duplicate concentration-response curves of at least six concentrations by sigmoidal fitting using Origin® 7.5 software (OriginLab Corporation, Northampton, MA, USA) and were defined as the concentration of agonist at which stimulation was half-maximal. pEC 50 Values ​​are expressed in mol / liter, EC 50 The results were calculated as the negative logarithm of the G-protein-independent signaling pathway of the human recombinant D3 receptor.

[0115] [Table 12]

[0116] 5-HT2 A Receptor assay Human 5-HT in competitive binding assays 2A Determining affinity to receptors The receptor membrane was then treated with human 5-HT 2A The receptor was stably expressed from the CHO-K1 recombinant AequoScreen® cell line (PerkinElmer, Waltham, MA, USA). Cells were suspended in Buffer A (15 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA) at 4× volume (1 g cells − 4 mL buffer) and homogenized with a Dounce homogenizer. Crude membrane fractions were collected after separation by two successive centrifugations at 40,000×g for 25 min, with washing steps in Buffer A. The final pellet was resuspended in buffer B (75 mM Tris-HCl, pH 7.5, 12.5 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose) at a concentration of 80 mg wet cell weight in 0.5 mL buffer, aliquoted, and flash frozen on dry ice. Protein content was determined using the bicinchoninic acid assay in the presence of a sulfhydryl reagent, with bovine serum albumin (BSA) as the standard.

[0117] In the binding experiments, 15 μg of membrane protein per well and 1 nM ketanserin hydrochloride, [ethylene- 3[H] (PerkinElmer) was incubated with compound or vehicle (DMSO, final concentration 1% (v / v)) in incubation buffer (50 mM Tris, 0.3% BSA, pH 7.4). Non-specific binding (NSB) was determined in the presence of 1 μM mianserin hydrochloride (Tocris, Bristol, UK). Samples were incubated for 15 min at 25° C. in a final volume of 250 μL. Binding reactions were terminated by rapid filtration through a Filtermate™ harvester (PerkinElmer) using UniFilter® GF / C plates presoaked for at least 1 h in 0.5% (v / v) polyethyleneimine (PEI, dissolved in distilled water). Filter plates were washed three times with 0.5 mL of ice-cold wash buffer (50 mM Tris, pH 7.4). The washed filter plates were dried at 40° C. for 60 min and 40 μL of Microscint™-20 scintillation cocktail (PerkinElmer) was added to each well. Radioactivity was assayed using a MicroBeta 2 The concentrations were determined using a Microplate Counter (PerkinElmer).

[0118] Nonspecific binding was subtracted from the raw scintillation counts to generate specific binding, which was normalized to vehicle-treated samples and converted to % displacement values. IC 50 K values ​​(i.e., the concentration of compound that displaces 50% of the specifically bound radioligand) were determined from the concentration-displacement curves by sigmoidal fitting using Origin® 7.5. software (OriginLab® Corporation, Northampton, MA, USA). i The value (i.e., the inhibition constant) was calculated using the Cheng-Prusoff equation: K i =IC 50 / [1+([L] / K D )] (where [L] is the concentration of radioligand used, determined by scintillation counting, and K D(pK is the affinity of the labeled ligand for the receptor, determined in a separate experiment). Competitive binding assays were performed at a minimum of six concentrations, with each concentration repeated three times, in two independent experiments. The pK i Values ​​are expressed in mol / liter, K i These results were derived by calculating the negative logarithm of the values. 2A It is shown to be a ligand for the receptor.

[0119] [Table 13]

[0120] Fluorescent Ca 2+ Detection of human 5-HT using 2A Characterization of receptor antagonism Human recombinant 5-HT in culture 2A Receptors and Gα 16CHO-K1 cells expressing IL-16 (purchased from Euroscreen Fast, Brussels, BE) were cryopreserved according to established protocols using 90% FBS / 10% DMSO as medium. Before the experiment, cells were thawed and resuspended in PowerCHO™2 medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin antimycotic solution, and 1% pyruvate. Cells were seeded at a density of 40,000 cells per well in 96-well microplates and incubated overnight at 37° C. in a humidified atmosphere containing 5.0% CO2. On the day of the experiment, plates were washed with assay buffer (140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES), 10 mM glucose, 2 mM probenecid, pH 7.4) using a plate washer (Elx405UCWS, Biotek, Winooski, VT, USA), and then 50 μL of 4 μM Fluo-4 AM (Thermo Fisher Scientific) in assay buffer was added per well. After dye loading (60 min, 37° C., in the dark), the plates were washed with assay buffer using a plate washer, leaving a volume of 50 μL per well, then 50 μL of assay buffer per well containing vehicle (3% DMSO in assay buffer) or test compound (3× final concentration) was added and the cells were incubated for an additional 10 min at 37° C.

[0121] The final DMSO concentration was 1% (v / v) for all treatments. To achieve this, a series of DMSO stock solutions were prepared for all test compounds. Stock solutions were stored at -20°C and further diluted in assay buffer to obtain the desired final concentration immediately prior to measurement. A stock solution of 5-HT (10 mM) was prepared by dissolving 5-HT in deionized ultrafiltered water.

[0122] Baseline and agonist-evoked [Ca 2 +] i (Intracellular Ca2+ The changes in EC were monitored using a FlexStation® II 96-well plate reader (Molecular Devices, San Jose, CA, USA). The settings on the FlexStation® II were as follows for fluorescence imaging: excitation 485 nm, emission 525 nm, cut-off filter 515 nm. Fluorescence measurements were performed at 37° C. with a runtime of 40 s and a sampling interval of 1.36 s. Excitation and detection were performed through the bottom of the plate. A baseline was recorded for 20 s, followed by addition of 50 μL of 3× concentrated agonist (EC 80 Agonist stimulation was performed when 100 µL of 5-HT (5-HT) solution or assay buffer / vehicle was added to all wells at 75 µL / sec with the attached pipettor at a height corresponding to 155 µL. Fluorescence was monitored for an additional 20 sec.

[0123] Compounds were analyzed using EC200 values ​​as determined for each plate with the integrated curve fitting module of the measurement software (SoftMax® Pro 5.2, Molecular Devices, San Jose, CA, USA). 80 5-HT concentrations were evaluated.

[0124] Results are expressed as ΔF / F values, where F is baseline fluorescence defined as the mean fluorescence before and / or immediately after agonist administration, and ΔF is the increase in fluorescence after agonist administration; ΔF=F max -F, calculated as F max F ranged from 17 to 29 for maximum fluorescence between reads, and F ranged from 2 to 13 for the average of fluorescent reads. In all experiments, all treatments were measured in parallel in multiple wells, and the average ΔF / F was used for further analysis. The average ΔF / F values ​​were converted to % inhibition (I%) values ​​using the following formula: I% = 100 × [1-((ΔF / F compound -ΔF / F vehicle ) / (ΔF / F control -ΔF / F vehicle )). IC of test compound 50pIC values ​​were determined from 4-parameter sigmoidal concentration-effect curves fitted to the % inhibition data using SoftMax® Pro software (Molecular Devices). 50 Further calculation of the value was performed using the above software as IC 50 These results were derived by calculating the negative logarithm of the values. 2A It has been shown to be an antagonist of the receptor.

[0125] [Table 14]

[0126] Phencyclidine-induced hyperlocomotion in rats. Spontaneous locomotor activity was measured using a 6-channel activity monitor manufactured by Experimetria Ltd. (Budapest, HU). The apparatus consisted of an acrylic cage (48.5 cm × 48.5 cm × 40 cm) equipped with 30 pairs of photocells on the wall near the bottom of the cage, twice to detect horizontal locomotor behavior. Additional photocell arrays (30 pairs) were placed at different heights (6.5 cm, 12 cm, 18 cm, and 23 cm) along the opposite side of the cage to detect rearing responses. Signals elicited by the breaking of the photocell beams were processed by movement analysis software (Experimetria) to determine the animal's spatial position with a sampling frequency of 1 Hz, and the time the rat spent walking was calculated by computer.

[0127] Test compounds were administered orally or subcutaneously to test animals, 10 animals per treatment group. Male Harlan-Wistar rats (Toxi-Coop, Budapest, HU) weighing 190-210 grams were used in these studies. After oral administration of the test compound or vehicle, the animals were individually habituated to the activity monitor for 30 minutes. When the test compound was administered subcutaneously, the rats were first habituated to the activity monitor for 15 minutes, then treated with the test compound or vehicle, and then returned to the activity monitor for another 15 minutes of habituation. After habituation, the rats were treated with phencyclidine hydrochloride (PCP) subcutaneously and immediately returned to the experimental apparatus for the measurement period (1 hour).

[0128] Data were analyzed using GraphPad® Prism® 9 software (GraphPad, San Diego, CA, USA). Statistical evaluation was performed 1 h after activity (time spent walking). Drug effects were evaluated using analysis of variance (ANOVA or Welch's ANOVA, where appropriate) followed by Dunnett's multiple comparison test. ED 50 Values ​​were determined from percent inhibition data obtained from linear regression analysis. ED 50 The values ​​are shown in Table 13.

[0129] [Table 15]

[0130] The above data (Table 13) show that the compounds of the examples of the present disclosure exhibited good and potent pharmacodynamic effects / activity in phencyclidine-induced hyperlocomotion in rats, indicating that they may have substantial antipsychotic efficacy.

[0131] Apomorphine-induced climbing and sniffing behavior in mice. Male CD1 mice (Envigo, Horst, NL) weighing 24-29 g were used in the experiment (n=12 per treatment group). Climbing and sniffing behaviors were measured by visual observation in cylindrical cages. The cages were 15 cm high and 12 cm in diameter, with walls of vertical metal bars 2 mm in diameter and spaced 1 cm apart embedded in a smooth plastic surface.

[0132] Immediately after subcutaneous administration of vehicle or test compound, animals were placed in a cage and habituated for 10 min. At the end of the 10 min habituation period, apomorphine hydrochloride 1.5 mg / kg was administered subcutaneously. After treatment, animals were returned to the cylindrical cage. Measurements of climbing and sniffing behaviors began 10 min after apomorphine treatment (11 min after treatment) and continued for 16 min. Total climbing behaviors were scored as follows: four paws on the floor (0 points), front paws touching the horizontal bar (1 point), four paws gripping the horizontal bar (2 points). Animals were also scored for repetitive sniffing behaviors as a measure of stereotypy according to the following behaviors: sniffing (0 points), moderate sniffing with little nose contact to the wall or floor of the cage (1 point), constant sniffing with constant nose contact (2 points).

[0133] Data were analyzed using GraphPad Prism 9 software (GraphPad, Inc.). Scores for both behaviors were tallied for each individual (maximum possible score 32 points) and group means were calculated. Drug efficacy was calculated as the percentage inhibition of apomorphine-induced behaviors. From the percentage inhibition data, dose-response curves were plotted and ED 50 ED by simple linear regression 50 Values ​​were calculated by computer.

[0134] [Table 16]

[0135] The above data (Table 14) show that the example compounds of the present disclosure exhibit good and potent pharmacodynamic effects / activity in apomorphine-induced climbing and sniffing behavior in mice, indicating that they may have substantial antipsychotic efficacy.

[0136] It is understood that the foregoing detailed description and the accompanying examples are merely illustrative and should not be construed as limiting in the scope of the present disclosure, which is solely defined by the appended claims and their equivalents.Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art.Such changes and modifications, including but not limited to those relating to the chemical structures, substituents, derivatives, intermediates, synthesis, formulation and / or methods of use of the present disclosure, can be made without departing from the spirit and scope thereof.All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A compound having the structure: or a pharma- ceutically acceptable salt thereof. 【Chemistry 1】

2. A compound having the following structure: 【Chemistry 2】

3. A pharma- ceutically acceptable salt of a compound having the structure: 【Chemistry 3】

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