Dopamine d3 / d2 receptor modulating compounds

Abstract

The present disclosure provides compounds of formula (I) and pharmaceutically acceptable salts thereof, where R1 has any of the values defined in the specification, which compounds and pharmaceutically acceptable salts thereof are useful as agents for the treatment of diseases and conditions, including schizophrenia. Also provided are pharmaceutical compositions comprising the compounds of Formula (I). # imgabs0 #

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 479,530, filed on January 11, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] 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 Art

[0004] Dysfunction of the dopaminergic 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, 16, 295). The effects of dopamine are mediated by at least five different dopamine receptors, which belong to the dopamine D1 (i.e., D1 and D5) or D2 (i.e., D2, D3, and D4) receptor families. 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 states. Dopamine D2 antagonists are used, for example, as antipsychotics. However, extensive antagonism of D2 receptors leads to undesirable side effects such as extrapyramidal motor symptoms, psychomotor sedation, or cognitive retardation. Antipsychotics that preferentially target the D3 receptor have provided successful therapeutic intervention in the treatment of schizophrenia.

[0005] There remains a need in the art for improved compounds that are D3 / D2 receptor selective. Summary of the Invention

[0006] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0007]

[0008] in

[0009] R 1 It is a C1-C3 alkyl group.

[0010] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is CH3.

[0011] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is CH2CH3.

[0012] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is CH2CH2CH3.

[0013] In some embodiments, the present disclosure provides a compound of formula (I), wherein the compound is selected from the group consisting of:

[0014] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea;

[0015] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(methoxymethyl)-N-methylurea; and

[0016] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-methyl-N-(propyloxymethyl)urea; or a pharmaceutically acceptable salt thereof.

[0017] In some embodiments, the present 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.

[0018] In some embodiments, the present 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.

[0019] In some embodiments, the present disclosure provides a compound of formula (I), wherein the compound is a pharmaceutically acceptable salt of N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea.

[0020] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier.

[0021] In some embodiments, the present disclosure provides a method for treating schizophrenia, comprising the step of administering to a subject a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0022] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use as a medicament.

[0023] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in treating schizophrenia.

[0024] In some embodiments, the present disclosure provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating schizophrenia. DETAILED DESCRIPTION

[0025] This disclosure describes compounds that modulate the activity of dopamine D3 and dopamine D2 receptors.

[0026] The compounds disclosed herein may contain one or more variables that appear more than once in any substituent or formula herein. The definition of a variable at each occurrence is independent of its definition at every other occurrence. Furthermore, combinations of substituents are permissible only if such combinations result in stable compounds. A stable compound is one that can be isolated from a reaction mixture.

[0027] definition

[0028] Certain terms used in this specification are intended to refer to the following definitions as described in detail below.

[0029] Note 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 identical or different compounds. Reference to "a pharmaceutically acceptable carrier" refers to a single pharmaceutically acceptable carrier as well as one or more pharmaceutically acceptable carriers, etc.

[0030] As used in the specification and appended claims, the following terms have the indicated meanings unless otherwise indicated:

[0031] Unless otherwise specified, as used herein, the term "C1-C3 alkyl" refers to a saturated hydrocarbon chain radical having one, two, or three carbon atoms. Representative examples of C1-C3 alkyl include methyl, ethyl, and n-propyl.

[0032] In some cases, the number of carbon atoms in a moiety is indicated by the prefix "C x -C y ” means, where x is the minimum number of carbon atoms in the substituent and y is the maximum number of carbon atoms. 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.

[0033] The phrase "pharmaceutical composition" refers to a composition suitable for administration in medicine.

[0034] The phrase "pharmaceutically acceptable salts" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio.

[0035] The term "stable" refers to compounds that possess sufficient stability to allow manufacture and maintain the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein.

[0036] The phrase "therapeutically effective amount" refers to that amount of a compound or a pharmaceutically acceptable salt thereof, which, when administered for treatment in a particular subject or population of subjects, is sufficient to prevent the manifestation of the condition or disorder being treated or to alleviate to some extent one or more of the symptoms of the condition or disorder being treated.

[0037] As used herein, the term "treating" refers to a method of alleviating or eliminating a disease and / or its attendant symptoms.

[0038] Compound

[0039] The compounds of the present disclosure have the general formula (I) as described herein.

[0040] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0041]

[0042] in

[0043] R 1 It is a C1-C3 alkyl group.

[0044] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is a C1 alkyl group.

[0045] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is a C2 alkyl group.

[0046] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is a C3 alkyl group.

[0047] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 Selected from the group consisting of CH3, CH2CH3 and CH2CH2CH3.

[0048] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is CH3.

[0049] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is CH2CH3.

[0050] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is CH2CH2CH3.

[0051] In some embodiments, the present 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.

[0052] In some embodiments, the present 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.

[0053] In some embodiments, the present disclosure provides a compound of formula (I), wherein the compound is a pharmaceutically acceptable salt of N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea.

[0054] In some embodiments, the present 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; or a pharmaceutically acceptable salt thereof.

[0055] In some embodiments, the present 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.

[0056] In some embodiments, the present disclosure provides a compound of formula (I), wherein the compound is a pharmaceutically acceptable salt of N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(methoxymethyl)-N-methylurea.

[0057] In some embodiments, the present 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-(propyloxymethyl)urea; or a pharmaceutically acceptable salt thereof.

[0058] In some embodiments, the present 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-(propyloxymethyl)urea.

[0059] In some embodiments, the present disclosure provides a compound of formula (I), wherein the compound is a pharmaceutically acceptable salt of N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-methyl-N-(propyloxymethyl)urea.

[0060] The compounds and intermediates of the present disclosure were prepared by using the ACD / Name 2021.1.3 (file version N15E41, build 123232, July 7, 2021) software program and / or by using Named using the Struct=Name naming algorithm that is part of Professional v.20.1.1.125.

[0061] Exemplary compounds of formula (I) include, but are not limited to, the compounds shown in Table 1 below and pharmaceutically acceptable salts thereof.

[0062] Table 1

[0063]

[0064]

[0065] The compounds of formula (I) may be used in the form of pharmaceutically acceptable salts.The compounds of formula (I) may contain basic or acidic functional groups or both and, if desired, may be converted into pharmaceutically acceptable salts by use of an appropriate acid or base.

[0066] Methods of Preparing Exemplary Compounds

[0067] The compounds of the present disclosure can be better understood in conjunction with the following synthetic schemes and methods that illustrate the means by which the compounds can be prepared. The compounds of the present disclosure can be prepared by various synthetic procedures. Representative synthetic procedures are shown in, but not limited to, Schemes 1-2. Variable R 1 The definition of is as detailed herein, for example in the Summary of the Invention.

[0068] plan

[0069] Solution 1

[0070]

[0071] As shown in Scheme 1, the compound of formula (I) (wherein R 1 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 A1. Thus, a compound of formula (1) can be treated with commercially available paraformaldehyde or formaldehyde (saturated aqueous solution with methanol as a stabilizer) and with a suitable alcohol of formula (2) at ambient temperature to reflux, in which alcohol R 1 As described herein, and the alcohol is commercially available or can be prepared according to methods known in the art. The reaction is typically carried out in a suitable solvent (such as, but not limited to, alcohol (2) or tetrahydrofuran) for 1 to 168 hours to provide a compound of formula (I).

[0072] Option 2

[0073]

[0074] 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 in a suitable solvent (such as a mixture of acetic acid and tetrahydrofuran) at ambient temperature to reflux for 1 hour to 24 hours to provide compounds of formula (3). Compounds of formula (2) can be prepared with a suitable alcohol (wherein R 1The compound of formula (3) is treated with a suitable base (such as potassium hydroxide) or a suitable acid (such as sulfuric acid or methanesulfonic acid) as described herein to provide a compound of formula (I). The reaction is typically carried out in a suitable solvent (such as tetrahydrofuran or toluene) at ambient temperature to reflux for 1 to 24 hours.

[0075] Specific procedures are provided in the Synthetic Examples section. Unless otherwise stated, starting materials and reagents are either commercially available or can be prepared from commercially available materials by one skilled in the art using methods described in the chemical literature.

[0076] Pharmaceutical composition

[0077] When used as a medicine, the compounds of the present disclosure can be administered in the form of a pharmaceutical composition. Such a composition can comprise a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0078] In some embodiments, a pharmaceutical composition is provided, comprising a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier.

[0079] How to use

[0080] The compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof can be administered to a subject suffering from schizophrenia. The term "administering" refers to a method of contacting a subject with a compound.

[0081] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, the compound of formula (I) or a pharmaceutically acceptable salt thereof, and the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof are used as medicaments.

[0082] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, the compound of formula (I) or a pharmaceutically acceptable salt thereof, and the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof are used to treat schizophrenia.

[0083] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating schizophrenia.

[0084] Example

[0085] The following examples are provided for illustrative purposes and should not be considered to narrow the scope of the invention.

[0086] Unless otherwise stated, all reagents were of commercial grade and used as received without further purification. Reactions were performed under an inert atmosphere using commercially available anhydrous solvents. Reagent grade solvents were used in all other cases unless otherwise stated. 1 Chemical shifts (δ) for H NMR spectra are reported in parts per million (ppm) relative to tetramethylsilane (δ 0.00) as an internal standard or the appropriate residual solvent peak, CHCl 3 (δ 7.27).

[0087] Unless otherwise stated, the following abbreviations have the designated meanings:

[0088] NMR Nuclear magnetic resonance imaging s Single peak br s Broad single peak d Doublet or doublet m Multiplets t Triple Peak q Quadruple Peak sxt Sextet LC / MS or LCMS Liquid chromatography-mass spectrometry min minute mL milliliters μL Slightly higher L Lift g gram mg mg mmol millimoles HPLC High-pressure liquid chromatography ppm parts per million ESI Electrospray ionization M Molar concentration (mol / L)

[0089] Synthesis Example

[0090] Example 1

[0091] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea

[0092] 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, eluting with acetone. The crude material was mixed with diethyl ether and filtered to provide the title compound. 1H NMR(400MHz,DMSO-d6)δppm 0.88-1.02(m,2H),1.08(t,J=7.0Hz,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=7.0Hz,2H),3.31-3.44(m,1H),4.63(s,2H),6.03(d,J=7.8Hz,1H),7.10-7.18(m,1H),7.25-7.34(m,2H); LC-MS(ESI)m / z 471.3(M+H) + .

[0093] Example 2

[0094] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(methoxymethyl)-N-methylurea

[0095] 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 (containing about 10% methanol in water) was stirred at 85°C for 20 hours. The reaction mixture was concentrated in vacuo. The residue was purified by preparative HPLC on a Phenomenex 5μm C18 On an AXIA packed LC column (150 mm x 21.2 mm); using isocratic 20 mmol / L NH4HCO3 in water + 0.1% diethylamine (A) (over 3 minutes); 40%-50% gradient of 20 mmol / L NH4HCO3 in water + 0.1% diethylamine (A) and methanol (B) (over 16 minutes), at a flow rate of 21.2 ml / min, purified at 40°C to provide the title compound. 1H NMR (400 MHz, dimethyl sulfoxide-d6) δ ppm 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) + .

[0096] Example 3

[0097] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-methyl-N-(propyl (oxymethyl)urea

[0098] Example 3A

[0099] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-[(1H-imidazole- 1-amino)methyl]-N-methylurea

[0100] 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 hours. Dichloromethane (100 mL) and 50 mL of saturated NaHCO3 aqueous solution 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 provide the title compound. LC-MS (ESI) m / z 493.3 (M+H) + .

[0101] Example 3B

[0102] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-methyl-N-(propyl (oxymethyl)urea

[0103] To a solution of 87 mg of methanesulfonic acid (0.9 mmol) in 273 mg of 1-propanol (4.55 mmol) was added 20 mL of toluene and 443 mg (1 mmol) of N'-[(1r, 4r)-4-{2-[4-(2,3-dichlorophenyl)piperazine-1-yl]ethyl}cyclohexyl]-N-[(1H-imidazole-1-yl)methyl]-N-methylurea at room temperature, and the reaction mixture was stirred at 90 ° C for 2 hours. Ethyl acetate (30 mL) and 30 mL of saturated NaHCO3 aqueous solution 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, eluted with dichloromethane / methanol (97:3). The crude material was mixed with n-pentane and filtered to provide the title compound. 1 H NMR(400MHz, DMSO-d6)d ppm 0.84(t,J=7.2Hz,3H),0.88-1.03(m,2H),1.13-1.29(m,3H),1.30-1.40(m,2H),1.48(sxt,J=7.0Hz,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=6.5Hz,2H),3.31-3.46(m,1H),4.64(s,2H),6.02(d,J=7.8Hz,1H),7.11-7.17(m,1H),7.27-7.33(m,2H); LC-MS (ESI) m / z 485.3(M+H) + .

[0104]

[0105] Reference Example 1

[0106] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-methyl-N-propyl Urea

[0107] The title compound was prepared as described in WO2005012266.

[0108]

[0109] Reference Example 2

[0110] N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N,N-dimethylurea (CAR)

[0111] The title compound was prepared as described in WO2005012266.

[0112]

[0113] Reference Example 3

[0114] N-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N'-methylurea (DCAR)

[0115] The title compound was prepared as described in WO2005012266.

[0116] Kinetic solubility studies

[0117]

[0118] *In the presence of 1% DMSO co-solvent, RT, 2 hours

[0119] To determine kinetic solubility, 3 mL of a 10 mM (5 mM, 1 mM) DMSO stock solution of each compound was pipetted into a 96-well plate (Millipore HTS -PCF filter plates, MilliporeSigma, St. Louis, MO, USA) containing 295 mL of PBS pH 7.4. The suspension was shaken on an orbital shaker at 300 rpm for 2 h and then filtered by vacuum filtration ( The suspension was clarified using a Vacuum Manifold (Millipore Sigma). Following the filtration step, 160 mL of the filtrate was immediately transferred to 40 mL of acetonitrile to prevent precipitation of the compound from the saturated solution. The concentration of the study compound was determined by HPLC-UV-(MS) detection using an external standard prepared from the same batch of the study compound. The results are shown in Table 2.

[0120] Thermodynamic solubility study (37°C, 4 hours)

[0121] To measure thermodynamic solubility: typically, 200 μL to 400 μL solution is added to 1 mg to 2 mg of solid test material (n=5). The resulting mixture / supersaturated solution is shaken at 37°C for 4 hours (to solution equilibrium) and filtered on a Millipore MultiScreen filter plate (0.45 μm). The concentration of the filtrate is determined based on a 5-point calibration by LC / UV / MS. (When the solid material is completely dissolved in the aqueous test medium, the result should be considered as a minimum.) The results are shown in Table 2.

[0122] Thermodynamic solubility studies (RT, 24 h)

[0123] To measure thermodynamic solubility: typically, 200 μL to 400 μL of solution were added to 1 mg to 2 mg of solid test material (n=5). The resulting mixture was shaken at room temperature (RT) for 24 hours (to solution equilibrium) and filtered on a Millipore MultiScreen filter plate (0.45 μm). The concentration of the filtrate was determined based on a 5-point calibration by LC / UV / MS. (When the solid material was completely dissolved in the aqueous test medium, the result should be considered as a minimum.) The results are shown in Table 2.

[0124] Table 2

[0125]

[0126] SIF: Simulated intestinal fluid (0.68% (w / v) KH2PO4, 0.09% (w / v) NaOH) without pancreatic enzymes, pH 6.8

[0127] PBS: pH 7.4 (0.01 M phosphate buffered saline, 0.138 M NaCl, 0.0027 M KCl, without Ca 2+ Mg 2 + )**24 hours

[0128] RT = Room temperature

[0129] The kinetic and thermodynamic solubility data for the disclosed examples demonstrate unexpectedly enhanced solubility compared to that of the reference compounds.

[0130] Biological assays

[0131] In in vitro biological studies, the following abbreviations are commonly used: BSA stands for bovine serum albumin, cAMP stands for cyclic adenosine monophosphate, DMEM stands for Dulbecco's modified Eagle's medium, DMSO stands for dimethyl sulfoxide, EDTA stands for ethylenediaminetetraacetic acid tetrasodium salt, EGTA stands for ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, FBS stands for fetal bovine serum, G418 stands for geneticin, IBMX stands for 3-isobutyl-1-methylxanthine, and Tris stands for tris(hydroxymethyl)aminomethane.

[0132] User D 2L Receptor research

[0133] Used in competitive binding assays [ 3 H] Raclopride determines human D 2L Receptor affinity

[0134] Recombinant human D 2L CHO-K1 cells (DRD2L cAMP Hunter TM , Gi Cell lines (Eurofins DiscoverX, Fremont, CA, USA) were homogenized in a 4x (v / v) buffer solution (50 mM Tris, 5 mM MgCl2, 1 mM EGTA, pH 7.4, at 25°C) using a Dounce tissue grinder and centrifuged at 40,000 x g for 10 minutes at 4°C. The supernatant was removed, and the pellet was resuspended in 4x (v / v) buffer and centrifuged. The resulting pellet was resuspended in the above buffer solution at a volume of 12.5 mL / g of original weight. The membrane preparation was then aliquoted and stored at -70°C.

[0135] The test compound was 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). The 5-fold concentrated compound in binding buffer (50 μL) was transferred to a 96-well deep-well plate (BRAND, Wertheim, Germany). The membrane preparation was thawed and washed once in binding buffer. In the same buffer, 10 μg protein / well was mixed with 2 nM [ 3 H] Racloprid (PerkinElmer, Waltham, MA, USA) was incubated in a volume of 250 μL at 25°C for 120 minutes in the presence or absence of the test compound (to determine binding inhibition or total binding of the test compound, respectively). Nonspecific binding (NSB) was determined in the presence of 10 μM haloperidol (a D2 receptor antagonist). After incubation, the cells were lysed using Filtermate TM Harvester (PerkinElmer) The samples were filtered onto GF / B plates (PerkinElmer) and washed four times with 1 mL of ice-cold binding buffer. The plates were dried at 40°C for one hour and 40 μL of Microscint was added to each well. TM -20 scintillation cocktail (PerkinElmer). Radioactivity was determined using a microplate counter (PerkinElmer).

[0136] Nonspecific binding was subtracted to obtain specific binding, which was normalized to vehicle-treated samples and converted to % shift values. IC was performed using GraFit 6.0 (Erithracus Software, Horley, UK). 50 Determination of IC values ​​and curve fitting. Determination of IC values ​​from concentration-displacement curves by sigmoidal fitting 50 The inhibition constant (K) was calculated using the Cheng-Prusoff equation below. i ):K i =IC 50 / [1+([L] / K D )], where [L] is the radioligand concentration and K D is the affinity of the labeled ligand for the receptor. D Determined by separate saturation experiments. pK i The value is expressed in mol / L by calculating K i The experiment was performed in triplicate and the results are shown in Table 3. The results show that the embodiments of the present disclosure are human recombinant D 2L High affinity ligand for the receptor.

[0137] Table 3

[0138] Example <![CDATA[D 2L pK i [ 3 H]Raclopride]]> 1 8.71 2 8.90 3 8.94 Reference Example 2 8.71

[0139] Used in competitive binding assays [ 3 H] spiropyridine determines human D 2L Receptor affinity

[0140] The cells expressing human recombinant D 2L Recipient CHO-K1 cells were scraped from the plate and centrifuged at 1000 x g. The cells were disrupted with a pestle homogenizer in a buffer containing 25 mM Tris-HCl (pH 7.4), 6 mM MgCl2, 1 mM EDTA, and 10 mM phenylmethylsulfonyl fluoride (PMSF). The resulting suspension was centrifuged at 1000 x g. The supernatant was collected and centrifuged at 41,000 x 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.

[0141] An aliquot of the membrane preparation was mixed with 0.16 nM [ 3H] spiropyrone (PerkinElmer) was incubated in the presence or absence of the test compound in a 96-well plate at 25° C. in an incubation buffer (pH 7.4) containing 50 mM Tris-HCl, 1.4 mM ascorbic acid, 0.001% BSA, 150 mM NaCl with 1% DMSO in a final reaction volume of 222 μL. Nonspecific binding (NSB) was determined in the presence of 10 μM haloperidol (Sigma-Aldrich). After incubation, the samples were plated at 4° C. for 120 minutes. Filter onto GF / C plates (PerkinElmer), wash, and add Microscint TM -20 scintillation cocktail. Radioactivity was determined using a microplate counter (PerkinElmer).

[0142] NSB was subtracted from the scintillation counts to obtain specific binding, which was normalized to vehicle-treated samples and converted to % shift values. 50 The value is obtained using MathIQ TM (ID Business Solutions Ltd., Surrey, UK) was determined by nonlinear least squares regression analysis. K i The values ​​are the observed IC values ​​of the test compounds using the Cheng-Prusoff formula. 50 , the concentration of radioligand used in the assay and the K of the ligand D pK is calculated based on the historical value of i The value is expressed in mol / L by calculating K i The negative logarithm of the value is obtained and is shown in Table 4. The results show that when [ 3 H] spirocycline as a radioligand, the embodiment of the present disclosure is human recombinant D 2L High affinity ligand for the receptor.

[0143] Table 4

[0144] Example <![CDATA[D 2L pK i [ 3 H]Spirocyclidine]]> 1 9.06 2 8.93 3 8.93 Reference Example 2 9.20

[0145] Characterization of human D using cyclic AMP detection 2L Receptor agonism

[0146] Using cAMP G i The kit (Cisbio / PerkinElmer) was used to detect the presence of Measurement Expression D 2LcAMP levels in human D 2L Agonist activity at the receptor. 2L Recipient CHO-K1 cells were cultured in HamF12 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 measurement, cryopreserved cells were thawed and seeded at 10,000 cells / well in white-walled half-area 96-well plates. AssayComplete TM Cells were plated in 2 (CP2) reagent (Eurofins DiscoverX) and incubated overnight at 37° C. in a humidified atmosphere with 5.0% CO 2 . Prior to cAMP measurement, the CP2 reagent was removed from the cells and replaced with 20 μL of assay buffer (140 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 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 containing the compound or vehicle and incubated at ambient temperature for 20 minutes. After an additional 30-minute incubation step with 0.5 μM forskolin (Eurofins DiscoverX) at ambient temperature, cell stimulation was terminated by adding detection reagents (20 μL cAMP-d2 and 20 μL anti-cAMP cryptate, Cisbio / PerkinElmer) diluted in lysis buffer. After a 60-minute incubation at ambient temperature, the cells were stained using standard The time-resolved fluorescence signal was quantified using a PHERAstar FS multimode reader (BMG Labtech, Ortenberg, Germany) with a 40 nm laser excitation setting and a 337 nm laser. The results were calculated as the ratio of the acceptor fluorescence signal (A665 nm) to the donor fluorescence signal (A620 nm) × 10 4The ΔF% values ​​were calculated using the following formula: 100 × (sample ratio - negative control ratio) / negative control ratio. In the experiment, all treatments were measured in parallel in multiple wells, and the average ΔF% value was used for further analysis. The agonist activity value was calculated as the percentage inhibition of forskolin-stimulated cAMP accumulation, which was normalized to the response induced by the maximally effective concentration of dopamine tested in the same experiment. All calculations used (Microsoft, Redmond, WA, USA). The pEC values ​​shown in Table 5 50 The values ​​(negative logarithm of the concentration of agonist in mol / L that produces 50% inhibition of forskolin-stimulated cAMP accumulation) were obtained by fitting a 4-parameter sigmoid curve to the concentration-effect data (with the lower asymptote constrained to zero) using GraphPad Prism (GraphPad, San Diego, CA, USA). The results show that the embodiments of the present disclosure are human recombinant D 2L A potent agonist of the G protein-coupled signaling pathway of the receptor.

[0147] Table 5

[0148] Example <![CDATA[D 2L cAMP pEC 50 ]]> 1 8.43 2 8.88 Reference Example 2 8.43

[0149] β-arrestin to human dopamine D 2L Measurement of receptor recruitment

[0150] The person who will express the marker D 2L Receptor and labeled β-arrestin-2 CHO-K1 cells (Eurofins DiscoverX, Fremont, CA, USA) were seeded at a density of 20,000 cells / well in 96-well white-walled, clear-bottom tissue culture plates using 90 μl / cell AssayComplete. TM Cell plating 2 (CP2) reagent (Eurofins DiscoverX). The plate was incubated overnight at 37°C in a humidified atmosphere with 5% CO2. Twenty to twenty-four hours later, 20 μL of the test compound or vehicle in CP2 reagent containing 2.2% DMSO was added to the cells and incubated at 37°C for 90 minutes. Then, 55 μL of Detection reagent (Eurofins DiscoverX), and the plate was incubated at 25°C for 60 minutes, followed by Luminescence detection was performed using a FS multimode plate reader (BMG Laboratories, Ortenberg, Germany). Raw data were converted to percentage stimulation above basal values. Values ​​were further converted to percentage of the maximal stimulation of β-arrestin recruitment by 30 μM dopamine. EC 50 The value is to use The pEC was calculated by sigmoidal fitting from concentration-response curves of at least six concentrations run in duplicate using ELISA 7.5 software (OriginLab Corporation, Northampton, MA, USA) and was defined as the concentration of agonist with half-maximal stimulation. 50 Values ​​were calculated as EC in mol / L 50 The negative logarithm of the value is shown in Table 6. The results show that the embodiments of the present disclosure are human recombinant D 2L A potent agonist of the G protein-independent signaling pathway of the receptor.

[0151] Table 6

[0152] Example <![CDATA[D 2L inhibitory protein pEC 50 ]]> 1 8.96 2 9.14 Reference Example 2 8.71

[0153] Studies using human D3 receptors

[0154] Used in competitive binding assays [ 3 Determination of the affinity of raclopride at the human D3 receptor

[0155] Cell cultures (CHO-K1) expressing recombinant human D3 receptor (DRD3, GenBank ID DU32499, purchased from Euroscreen Fast, Brussels, BE) were homogenized in 4x (v / v) buffer (15 mM Tris, 2 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, pH 7.4 at 25°C) using a Dounce tissue grinder and centrifuged at 40,000 x g for 25 minutes 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, at 25°C) at a volume of 12.5 mL / g of original cell weight. The membrane preparation was then aliquoted and stored at -70°C.

[0156] The 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 5 times the final concentration in 5% DMSO-buffered solution. Aliquots of the membrane preparation were thawed and washed once in binding buffer. In the same buffer, 3.3 μg of protein / assay was mixed with approximately 2.7 nM [ 3 H] raclopride (PerkinElmer) were incubated in a volume of 250 μL in a 96-well deep-well plate (BRAND) at 25°C for 120 minutes in the presence or absence of the test compound. Nonspecific binding (NSB) was determined in the presence of 10 μM haloperidol. In all reactions, the final DMSO concentration was 1% (v / v). After incubation, the cells were lysed using Filtermate TM Harvester (PerkinElmer) The samples were filtered onto GF / B plates (PerkinElmer) and washed with 4 x 1 mL of ice-cold binding buffer. The plates were dried at 40°C for one hour and 40 μL of Microscint was added to each well. TM -20 scintillation cocktail (PerkinElmer). Radioactivity was determined using a microplate counter (PerkinElmer).

[0157] Using raw scintillation counts, NSB was subtracted to obtain specific binding, which was normalized to vehicle-treated samples and converted to percent displacement values. Curve fitting and calculations were performed using GraFit 6.0 (Erithracus Software, Horley, UK). IC was determined from the concentration-displacement curves by sigmoidal fitting. 50 The inhibition constant (K) was calculated using the Cheng-Prusoff equation below. i ):K i =IC 50 / [1+([L] / K D )], where [L] is the radioligand concentration and K D is the affinity of the labeled ligand for the receptor. D Determined from separate saturation experiments. The pK values ​​shown in Table 7 i The value is expressed in mol / L by calculating K i The negative logarithm of the value was obtained. In all experiments, samples were run in triplicate. The results show that the embodiments of the present disclosure are high affinity ligands for human recombinant D3 receptor.

[0158] Table 7

[0159] Example <![CDATA[D3pK i [ 3 H]Raclopride]]> 1 8.99 2 9.15 Reference Example 2 9.51

[0160] Used in competitive binding assays [ 3 Determination of the affinity of [H]-spiroperidone at the human D3 receptor

[0161] Wash CHO-K1 cells expressing human recombinant D3 receptor with PBS. Scrape the cells from the plate and centrifuge at 1000 x g. Disrupt the cells with a pestle homogenizer in a buffer containing 25 mM Tris-HCl (pH 7.4), 6 mM MgCl2, 1 mM EDTA, and 10 mM PMSF. Centrifuge the suspension at 1000 x g. Collect the supernatant and centrifuge at 41,000 x g. Discard the supernatant and resuspend the pellet in the above buffer. Aliquot the membrane preparation and store at -70°C.

[0162] An aliquot of the membrane preparation was mixed with 0.7 nM [ 3 H] spiropyrone (PerkinElmer) was incubated in the presence or absence of the test compound in a 96-well plate at 37° C. in an incubation buffer (pH 7.4) containing 50 mM Tris-HCl, 1.4 mM ascorbic acid, 0.001% bovine serum albumin, and 150 mM NaCl with 1% DMSO in a final reaction volume of 222 μL. Nonspecific binding (NSB) was determined in the presence of 25 μM (S)-(-)-sulpiride (Sigma-Aldrich). After incubation, samples were filtered on GF / C filter plates (PerkinElmer), washed, and Microscint was added. TM -20 scintillation cocktail. Radioactivity was determined in a microplate counter (PerkinElmer).

[0163] NSB was subtracted from the raw scintillation counts to obtain specific binding, which was normalized to vehicle-treated samples and converted to % shift values. 50 The value is obtained using MathIQ TM (ID Business Solutions Ltd, Surrey, UK) was determined by nonlinear least squares regression analysis. i The values ​​are the observed IC values ​​of the test compounds using the Cheng-Prusoff formula. 50 , the concentration of radioligand used in the assay and the K of the ligand D The pK values ​​shown in Table 8 are calculated based on the historical values. i The value is expressed in mol / L by calculating K i The results show that when using [ 3When [H] spiropiperidone is used as the radioligand, the disclosed embodiments are high affinity ligands for the human recombinant D3 receptor.

[0164] Table 8

[0165] Example <![CDATA[D3 pK i [ 3 H]Spirocyclidine]]> 1 10.00 2 9.77 3 9.74 Reference Example 2 9.80

[0166] Characterizing Agonism at the Human D3 Receptor Using Cyclic AMP Assays

[0167] Using cAMP G iAgonist activity at the human D3 receptor was determined by homogeneous time-resolved fluorescence (HTRF) using a kit (Cisbio / PerkinElmer) using cAMP levels in HEK293 cells (a cell line developed by Gedeon Richter) expressing recombinant human D3 receptor stably co-expressing adenylate cyclase V (ACV) (BioXtal, Saint-Félix, France). 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 resuspended in assay buffer (140 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES), 10 mM glucose, pH 7.4) in a white-walled half-area 96-well microplate at a density of 15,000 cells / well in a volume of 20 μL. The assay buffer was supplemented with 100 μM IBMX (Sigma-Aldrich, St. Louis, MO, USA). After adding the test compound (4x concentrated) or vehicle (DMSO) at 10 μL / well, the cells were incubated with assay buffer or various concentrations of the test compound for 20 minutes. After an additional 30-minute incubation with 1.5 μM forskolin (0.3% final DMSO concentration) at ambient temperature, cell stimulation was terminated by the addition of detection reagents (20 μL cAMP-d2 and 20 μL anti-cAMP cryptate) diluted in lysis buffer (PerkinElmer). After 60 minutes of incubation at ambient temperature, time-resolved fluorescence signals (TRF) were quantified using a PHERAstar FS multimode reader (BMG Labtech, Ortenberg, Germany) using standard HTRF settings and laser excitation at 337 nm.

[0168] The results were calculated by the ratio of the acceptor fluorescence signal (A665nm) to the donor fluorescence signal (A620nm) × 10 4Calculate and express ΔF% values ​​using the following formula: 100 x (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 inhibition of forskolin-stimulated cAMP accumulation, normalized to the response induced by the maximally effective concentration of dopamine tested in the same experiment. All calculations were performed using Excel (Microsoft Corporation, Redmond, WA, USA). pEC 50 The values ​​(negative logarithm of the concentration of agonist in moles / liter that produced 50% inhibition of forskolin-stimulated cAMP accumulation) were obtained by fitting the concentration-effect data with a 4-parameter sigmoidal curve, with the lower asymptote constrained to zero, using GraphPad Prism (GraphPad Software, San Diego, CA, USA) and are shown in Table 9. The results indicate that the disclosed embodiments are highly potent agonists of the G protein-dependent signaling pathway of the human recombinant D3 receptor.

[0169] Table 9

[0170] Example <![CDATA[D3 cAMP pEC 50 ]]> 1 8.45 2 8.71 Reference Example 2 8.71

[0171] Measurement of β-arrestin recruitment to human dopamine D3 receptors

[0172] expressing labeled human D3 receptor and labeled β-arrestin-2 CHO-K1 cells (Eurofins DiscoverX, Fremont, CA, USA) were seeded at a density of 25,000 cells / well in 96-well white-walled, clear-bottom tissue culture plates using 90 μL of AssayComplete. TM Cells were plated in CP2 reagent (Eurofins DiscoverX) and incubated overnight at 37°C in a humidified atmosphere containing 5% CO2. Twenty to twenty-four hours later, 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 Detection reagent (Eurofins DiscoverX), and the plate was incubated at 25°C for 60 minutes, followed by Luminescence detection was performed using a FS multimode plate reader (BMG Laboratories, Ortenberg, Germany).

[0173] Raw data were first converted to % stimulation above basal values. This % stimulation above basal value was further converted to % maximal stimulation of β-arrestin recruitment by 1 μM dopamine. 50 The value is to use 7.5 software (Northampton, Massachusetts, USA pEC) was calculated by sigmoidal fitting from the concentration-response curves of at least six concentrations in duplicate and was defined as the concentration of agonist with half-maximal stimulation. 50 Values ​​were calculated as EC in mol / L 50 The results indicate that the disclosed embodiments are highly potent agonists of the G protein-independent signaling pathway of the human recombinant D3 receptor.

[0174] Table 10

[0175]

[0176] 5-HT2 A Receptor assay

[0177] Determined in competitive binding assays for human 5-HT 2A Receptor affinity

[0178] From stably expressing human 5-HT 2A CHO-K1 recombinant receptor Cell line (Perkin Elmer, Waltham, Massachusetts, USA) prepares receptor membrane.Cell is suspended in buffer A (15mM Tris-HCl (pH 7.5), 2mM MgCl , 0.3mM EDTA, 1mM EGTA) (1g cell-4mL buffer) with 4 times of volumes, and homogenized in Dounce homogenizer.After two continuous centrifugal steps of centrifugal 25 minutes with 40,000xg, collect thick membrane fraction, these two continuous centrifugal steps are separated by the washing step of washing in buffer A.Final precipitate is resuspended in buffer B (75mM Tris-HCl (pH 7.5), 12.5mM MgCl , 0.3mM EDTA, 1mM EGTA, 250mM sucrose) with the concentration of 80mg wet cell weight in 0.5mL buffer, divided equally and quick freezing 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 a standard.

[0179] In the binding experiments, 15 μg of protein / pore membrane preparation was mixed with 1 nM ketanserin hydrochloride, [ethylene- 3H] (PerkinElmer) was incubated with compound or vehicle (DMSO, 1% (v / v) final concentration) in incubation buffer (50 mM Tris, 0.3% BSA, pH 7.4). Nonspecific binding (NSB) was determined in the presence of 1 μM mianserin hydrochloride (Tocris, Bristol, UK). Samples were incubated at 25°C for 15 minutes in a final volume of 250 μL. The cells were then incubated with 0.5% (v / v) polyethyleneimine (PEI) (dissolved in distilled water) for at least 1 hour using a 5% PBS solution. GF / C board, through Filtermate TM The binding reaction was terminated by rapid filtration using a harvester (PerkinElmer). The filter plate was washed three times with 0.5 mL of ice-cold wash buffer (50 mM Tris, pH 7.4). The washed filter plate was dried at 40°C for 60 minutes and 40 μL of Microscint was added to each well. TM -20 scintillation cocktail (PerkinElmer). Radioactivity was determined using a microplate counter (PerkinElmer).

[0180] Nonspecific binding was subtracted from the raw scintillation counts to obtain specific binding, which was normalized to vehicle-treated samples and converted to % shift values. 50 The value (ie, the concentration of compound that displaces 50% of the specifically bound radioligand) was calculated using 7.5 software (Northampton, Massachusetts, USA The K values ​​shown in Table 11 were determined from the concentration-displacement curves by sigmoidal fitting. i The inhibition constant (K) was calculated using the Cheng-Prusoff formula: i =IC 50 / [1+([L] / K D )], where [L] is the concentration of radioligand used and determined by scintillation counting, K D 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 in two independent experiments, each replicated three times. The pK values ​​shown in Table 11 are: i The value is expressed in mol / L by calculating K i The results show that the embodiment of the present disclosure is human recombinant 5-HT 2A Receptor ligand.

[0181] Table 11

[0182] Example <![CDATA[5-HT 2A pK i ]]> 1 7.64 2 7.81 Reference Example 2 7.53

[0183] Using fluorescent Ca 2+ Detection and characterization of human 5-HT 2A Antagonism at the receptor level

[0184] According to established protocols, 90% FBS / 10% DMSO was used as the culture medium and cells expressing human recombinant 5-HT were cultured. 2A Receptors and Gα 16 CHO-K1 cells (purchased from Euroscreen Fast, Brussels, Belgium) were cryopreserved. Before the experiment, the cells were thawed and resuspended in PowerCHO supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin antimycotic solution and 1% pyruvate. TM 2 culture medium (Lonza, Basel, Switzerland). Cells were seeded in 96-well microplates at a density of 40,000 cells / well and incubated overnight at 37°C in a humidified atmosphere containing 5.0% CO. On the day of the experiment, the plates were washed with assay buffer (140 mM NaCl, 5 mM KCl, 2 mM MgCl, 2 mM CaCl, 10 mM 2-[4-(2-hydroxyethyl)piperazine-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), followed by the addition of 50 μL / well of 4 μM Fluo-4 AM (Thermo Fisher Scientific) in assay buffer. After dye loading (60 min, 37°C, in the dark), the plates were washed with assay buffer using a plate washer, leaving a residual volume of 50 μL / well, and then 50 μL / well of assay buffer containing vehicle (3% DMSO in assay buffer) or test compound (3 times the final concentration) was added, and the cells were incubated for another 10 min at 37°C.

[0185] The final DMSO concentration for all treatments was 1% (v / v). To achieve this, a series of DMSO stock solutions were prepared from all test compounds. The stock solutions were stored at -20°C and further diluted in assay buffer to obtain the desired final concentration just before measurement. A stock solution of 5-HT (10 mM) was prepared by dissolving 5-HT in deionized ultrafiltered water.

[0186] use Baseline and agonist-induced [Ca] were monitored using a 96-well plate reader (Molecular Devices, San Jose, CA, USA). 2+] i (Intracellular Ca 2+ ) changes. For fluorescence imaging, The II settings were as follows: excitation at 485 nm; emission at 525 nm, using a cutoff filter at 515 nm. Fluorescence measurements were performed at 37°C with a 40-second run time and a 1.36-second sampling interval. Excitation and detection were performed through the bottom of the plate. A baseline was recorded for 20 seconds, followed by agonist stimulation, where 50 μL of a 3x concentrated agonist (at its EC) was pipetted at 75 μL / s using an onboard pipette. 80 5-HT) solution or assay buffer / vehicle at a level corresponding to 155 μL was added to all wells. Fluorescence was monitored for an additional 20 seconds.

[0187] According to the measurement software ( The EC values ​​determined on each plate were obtained using the built-in curve fitting module of Pro 5.2 (Molecular Devices, San Jose, CA, USA). 80 Compounds were evaluated by measuring 5-HT concentration.

[0188] Results are expressed as ΔF / F values, where F is the baseline fluorescence (defined as the mean fluorescence before and / or immediately after agonist administration) and ΔF is the increase in fluorescence after agonist administration; calculated as ΔF = F max -F, where F max is the maximum fluorescence between readings 17 and 29, and F is the average of fluorescence readings 2 to 13. 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 value was converted to an inhibition % (I%) value using the following formula: I% = 100 × [1-((ΔF / F)] 化合物 -ΔF / F 媒介物 ) / (ΔF / F 对照 -ΔF / F 媒介物 ))].use Pro software (Molecular Devices) was used to determine the IC of test compounds from 4-parameter sigmoidal concentration-effect curves fitted to the % inhibition data. 50 The pIC values ​​shown in Table 12 50 The further calculation results of the values ​​were obtained by calculating the IC in mol / L using the above software. 50 The results show that the embodiment of the present disclosure is human recombinant 5-HT 2A Receptor antagonists.

[0189] Table 12

[0190] Example <![CDATA[5-HT 2A That 2+ bit 50 ]]> 1 6.80 2 6.88 Reference Example 2 6.75

[0191] Phencyclidine-induced hyperlocomotion in rats

[0192] Spontaneous locomotor activity was measured using a six-channel activity monitor manufactured by Experimetria Ltd. (Budapest, HU, Hungary). The apparatus consisted of an acrylic cage (48.5 cm × 48.5 cm × 40 cm) equipped with 2 × 30 pairs of photocells on the wall near the bottom of the cage to detect horizontal ambulatory behavior. To detect rearing responses, additional photocell arrays (30 pairs) were placed at different heights (6.5 cm, 12 cm, 18 cm, and 23 cm) along the opposite sides of the cage. The signals caused by the interruption of the photocell beam were processed by motion analysis software (Experimetria), which determined the spatial position of the animal at a sampling frequency of 1 Hz and calculated the time the rat spent ambulatory.

[0193] Test compound is administered orally or subcutaneously to research animal, ten animals of each treatment group.In these studies, the male Harlan-Wistar rats (Toxi-Coop company (Toxi-Coop, Budapest, Hungary, HU) of 190 grams -210 grams) of weight are used.After oral administration of test compound or vehicle, animal is adapted to activity monitor separately for 30 minutes.In the case of subcutaneous administration of test compound, rat is first adapted to activity monitor 15 minutes, then with test compound or vehicle process, subsequently they are put back in activity monitor and adapt to 15 minutes time period again.After adaptation, with phencyclidine hydrochloride (PCP) subcutaneous treatment rat, and immediately they are put back in experimental apparatus to enter measurement period (one hour).

[0194] use Data were analyzed using GraphPad 9 software (GraphPad, Inc., San Diego, CA, USA). Statistical evaluation was performed after one hour of activity (time spent ambulating). Drug effects were evaluated using analysis of variance (ANOVA, or, where appropriate, Welch's ANOVA) followed by Dunnett's multiple comparison test. ED was analyzed as appropriate. 50 Values ​​were determined from percent inhibition data from linear regression analysis. 50 The values ​​are shown in Table 13.

[0195] Table 13

[0196] Example PCP (MED, mg / kg) <![CDATA[PCP(ED 50mg / kg)]]> 1 0.4 0.07 Reference Example 2 0.1 0.09

[0197] MED = minimum effective dose

[0198] (lowest dose showing a statistically significant effect)

[0199] The above data (Table 13) show that the compounds of the Examples of the present disclosure exhibit good and potent pharmacodynamic effects / activity in phencyclidine-induced hyperlocomotion in rats, indicating that they may have significant antipsychotic efficacy.

[0200] Apomorphine-induced climbing and sniffing in mice

[0201] The experiment used males weighing 24g-29g Mice (Envigo, Horst, NL) (n = 12 per treatment group) were placed in cylindrical cages. Climbing and sniffing behaviors were measured by visual observation in the cages. The cages were 15 cm high and 12 cm in diameter, with walls consisting of vertical metal bars 2 mm in diameter and spaced 1 cm apart mounted on a smooth plastic surface.

[0202] After subcutaneous administration of vehicle or test compound, the animal is immediately placed in a cage for 10 minutes to adapt. At the end of the 10-minute adaptation period, 1.5mg / kg of apomorphine hydrochloride is administered subcutaneously. After treatment, the animal is returned to a cylindrical cage. After apomorphine treatment, climbing and sniffing behavior are measured 10 minutes (starting during the 11th minute) and continue for 16 minutes. Climbing behavior is scored as follows per minute: four paws are placed on the floor (0 point); forefoot touches the stick (1 point); and four paws grab the stick (2 points). The repeated sniffing of animals is also rated as a stereotyped measure according to the following scale: no sniffing (0 point); medium sniffing, nose almost not in contact with cage wall or floor (1 point); and continuous sniffing, continuous nose contact (2 points).

[0203] Data were analyzed using GraphPad Prism 9 software (GraphPad). Scores for both behaviors were calculated for each individual (maximum possible score was 32), and group means were calculated. Drug effects were calculated as the percentage inhibition of apomorphine-induced behaviors. Dose-response curves were drawn from the percentage inhibition data, and ED was calculated by simple linear regression. 50 value.

[0204] Table 14

[0205] Example <![CDATA[APO(ED 50 cl mg / kg)]]> <![CDATA[APO(ED 50 sn mg / kg)]]> 1 0.1 0.4 Reference Example 2 0.27 0.38

[0206] APO cl = apomorphine-induced climbing in mice;

[0207] APO sn = apomorphine-induced sniffing in mice

[0208] The above data (Table 14) show that the compounds of the Examples of the present disclosure exhibit good and potent pharmacodynamic effects / activities in apomorphine-induced climbing and sniffing in mice, indicating that they may have significant antipsychotic efficacy.

[0209] It should be understood that the foregoing detailed description and the accompanying examples are illustrative only and should not be regarded as limiting the scope of the present disclosure, which is limited only by the appended claims and their equivalents. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Without departing from the spirit or scope of the present disclosure, such changes and modifications may be made, including but not limited to those changes and modifications associated with the chemical structures, substituents, derivatives, intermediates, syntheses, formulations and / or methods of use of the present disclosure. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. A compound of general formula (I) or a pharmaceutically acceptable salt thereof, in R 1 It is a straight chain C1-C3 alkyl group.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 It is CH3.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 It is CH2CH3.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 It is CH2CH2CH3.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: 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-(propyloxymethyl)urea. 6 . The compound according to claim 1 , 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.

7. The compound according to claim 1, wherein the compound is N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea.

8. The compound according to claim 1, wherein the compound is a pharmaceutically acceptable salt of N'-[(1r,4r)-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}cyclohexyl]-N-(ethoxymethyl)-N-methylurea.

9. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier.

10. A method for treating schizophrenia in a subject suffering from schizophrenia, the method comprising the step of administering to the subject a therapeutically effective amount of the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

11. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, for use as a medicament.

12. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, for use in treating schizophrenia.

13. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating schizophrenia.

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