Dopamine D3 / D2 receptor modulating compound

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 on January 11, 2023, the content of which is incorporated herein by reference in its entirety.

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

Background Art

[0003] Dysfunction of the dopamine neurotransmitter system can be observed in the pathogenesis of neuropsychiatric disorders, including schizophrenia and mood or cognitive dysfunctions (Sokoloff, P. et al.: Nature, 1990, 347.146; Schwartz, J.-C. et al.: Clin. Neuropharmacology., 1993, Vol. 16, p. 295). The actions of dopamine are mediated by at least five different dopamine receptors belonging to the dopamine D1 receptor family (i.e., D1 and D5) or the D2 receptor family (i.e., D2, D3, and D4). The D3 receptor has a characteristic distribution in the central dopaminergic system. The dopamine D2 receptor is widely distributed in the brain and is involved in numerous physiological functions and pathological states. Dopamine D2 antagonists are used, for example, as therapeutic agents for schizophrenia. However, massive antagonism of the D2 receptor causes unwanted side effects such as extrapyramidal motor symptoms, psychomotor sedation, and cognitive function dulling. Therapeutic agents for schizophrenia that preferentially target the D3 receptor have achieved therapeutic intervention results in the treatment of schizophrenia.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] In the art, there is still a need for improved compounds having selectivity for the D3 / D2 receptor.

Means for Solving the Problems

[0006] (Abstract) In some embodiments, the present invention provides a compound of formula (I)

[0007]

Chemical Formula

[0008] In some embodiments, the present disclosure provides a compound of formula (I) wherein R 1 is CH 3 , or a pharmaceutically acceptable salt thereof.

[0009] In some embodiments, the present disclosure provides a compound of formula (I) wherein R 1 is CH 2 CH 3 , or a pharmaceutically acceptable salt thereof.

[0010] In some embodiments, the present disclosure provides a compound of formula (I) wherein R 1 is CH 2 CH 2 CH 3 , or a pharmaceutically acceptable salt thereof.

[0011] In some embodiments, the present disclosure provides a compound of formula (I), which compound is 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 pharmaceutically acceptable salt thereof, selected from the group consisting of.

[0012] In some embodiments, the present disclosure provides a compound of formula (I), which 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 present disclosure provides a compound of formula (I), which 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 present disclosure provides a compound of formula (I), which compound is the pharmaceutically 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 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.

[0016] In some embodiments, the present 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 pharmaceutically acceptable salt thereof.

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

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

[0019] 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 the treatment of schizophrenia. DETAILED DESCRIPTION OF THE INVENTION

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

[0021] The compounds disclosed herein can include one or more variables that are present in any substituent or two or more times in the formulas herein. The definition of a variable in each occurrence is independent of its definition in another occurrence. Further, combinations of substituents are permitted only if such combinations result in a stable compound. A stable compound is a compound that can be isolated from a reaction mixture.

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

[0023] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" are to be noted as including plural referents. Thus, for example, a reference to "a compound" includes a single compound as well as one or more of the same or different compounds. A 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 indicated meanings.

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

[0026] In certain 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, "C 1 ~C 6 alkyl" means an alkyl substituent containing 1 to 6 carbon atoms, and "C 1 ~C 3 alkyl" means an alkyl substituent containing 1 to 3 carbon atoms.

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

[0028] The term "pharmaceutically acceptable salt" refers to a salt that is free of excessive toxicity, irritation, allergic reaction, and the like, and is suitable for use in contact with the tissues of humans and lower animals within the scope of sound medical judgment, with a reasonable benefit / risk ratio.

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

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

[0031] As used herein, the terms "treat", "treating", and "treatment" refer to methods of alleviating or suppressing a disease and / or its attendant symptoms.

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

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

[0034]

Chemical formula

[0035] In some embodiments, the present disclosure provides compounds of formula (I) wherein R 1 is C 1 alkyl, or a pharmaceutically acceptable salt thereof.

[0036] In some embodiments, the present disclosure provides compounds of formula (I) wherein R 1 is C 2 alkyl, or a pharmaceutically acceptable salt thereof.

[0037] In some embodiments, the present disclosure provides a compound of formula (I) wherein R 1 is C 3 alkyl, or a pharmaceutically acceptable salt thereof.

[0038] In some embodiments, the present disclosure provides a compound of formula (I) wherein R 1 is CH 3 , CH 2 CH 3 and CH 2 CH 2 CH 3 selected from the group consisting of, or a pharmaceutically acceptable salt thereof.

[0039] In some embodiments, the present disclosure provides a compound of formula (I) wherein R 1 is CH 3 or a pharmaceutically acceptable salt thereof.

[0040] In some embodiments, the present disclosure provides a compound of formula (I) wherein R 1 is CH 2 CH 3 or a pharmaceutically acceptable salt thereof.

[0041] In some embodiments, the present disclosure provides a compound of formula (I) wherein R 1 is CH 2 CH 2 CH 3 or a pharmaceutically acceptable salt thereof.

[0042] In some embodiments, the present 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 pharmaceutically acceptable salt thereof.

[0043] In some embodiments, the present 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.

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

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

[0047] In some embodiments, the present disclosure provides a compound of formula (I), which is a pharmaceutically 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 present disclosure provides a compound of formula (I); or a pharmaceutically acceptable salt thereof, which 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 present disclosure provides a compound of formula (I), which 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 present disclosure provides a compound of formula (I), which is a pharmaceutically 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 present disclosure were named by using the ACD / Name 2021.1.3 (file version N15E41, Build 123232, July 7, 2021) software program and / or by 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 shown in Table 1 below, and pharmaceutically 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 or both basic and acidic functional groups and can be converted to pharmaceutically acceptable salts by using appropriate acids or bases, if desired.

[0055] Methods for preparing exemplary compounds The compounds of the present disclosure can be better understood in connection with the following synthetic schemes and methods that illustrate 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 Schemes 1 - 2, among others. The variable R 1 is defined as detailed herein, for example, as described in the summary. Scheme

[0056]

Chemical Formula

[0057] As shown in Scheme 1, the compound of formula (I) in which R 1 is as defined herein can be prepared from the compound of formula (1) according to methods known in the art, such as International Publication No. WO 2010 / 070370. Thus, the compound of formula (1) can be treated with commercially available paraformaldehyde or formaldehyde (saturated aqueous solution containing a methanol stabilizer), and an appropriate alcohol of formula (2) in which R 1 is as described herein, which are commercially available or can be prepared by refluxing at ambient temperature according to methods known in the art. The reaction is usually carried out in a suitable solvent, such as alcohol (2) or tetrahydrofuran, for 1 - 168 hours to produce the compound of formula (I).

[0058]

Chemical Formula

[0059] As shown in Scheme 2, the compound of formula (I) can be prepared from the compound of formula (1). Thus, the compound of formula (1) can be treated with commercially available 1 - hydroxymethylimidazole at ambient temperature and refluxed for 1 - 24 hours in a suitable solvent, such as a mixture of acetic acid and tetrahydrofuran, to produce the compound of formula (3). The compound of formula (3) is then treated with R 1As described herein, using a suitable alcohol of formula (2), it can be treated 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 usually carried out at ambient temperature and refluxed for 1 to 24 hours in a suitable solvent such as tetrahydrofuran or toluene.

[0060] Detailed procedures are shown in the section of Synthesis Examples. Unless otherwise stated, starting materials and reagents can be commercially available or prepared by those skilled in the art from commercially available materials using methods described in the chemical literature.

[0061] Pharmaceutical composition 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 together with a pharmaceutically acceptable carrier.

[0062] In some embodiments, there is provided 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.

[0063] Method of use The compounds of formula (I) or pharmaceutically acceptable salts thereof, and pharmaceutical compositions comprising the compounds of formula (I) or pharmaceutically acceptable salts thereof can be administered to a subject suffering from schizophrenia. The term "administer" refers to a method of bringing the compound into contact with the subject.

[0064] In some embodiments, the present disclosure provides a 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 for use as a medicine.

[0065] In some embodiments, the present disclosure provides a 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, for use in the treatment of schizophrenia.

[0066] In some embodiments, the present disclosure provides the use of a 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, for the manufacture of a medicament for the treatment of schizophrenia.

Example

[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 used as received without further purification unless otherwise stated. Commercial 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 The chemical shifts (δ) of the 1H NMR spectra were reported in parts per million (ppm) relative to tetramethylsilane (δ 0.00) or an appropriate residual solvent peak as an internal reference, i.e., CHCl 3 (δ 7.27).

[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 subjected to chromatography on silica gel eluting with acetone. The crude material was mixed with diethyl ether and filtered to give the title compound. 1 H NMR (400 MHz, DMSO-d 6 ) 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 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) were stirred at 85 °C for 20 hours. 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 for 3 minutes, 20 mmol / L NH 4 HCO 3 ; 20 mmol / L NH 4 HCO 3 in water + 0.1% diethylamine (A) and methanol (B), gradient from 40 to 50% of (A) over 16 minutes at 40 °C with a flow rate of 21.2 mL / min to give the title compound. 1 H NMR (400 MHz, dimethyl sulfoxide-d 6 ) 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) + .

[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 NaHCO 3 aqueous solution (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 give 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 were added 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 at room temperature, and the reaction mixture was stirred at 90 °C for 2 h. Ethyl acetate (30 mL) and saturated NaHCO 3 aqueous solution (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 subjected to silica gel chromatography eluting with dichloromethane / methanol (97:3). The crude material was mixed with n-pentane and filtered to give the title compound. 11H NMR (400 MHz, DMSO-d 6 ) δ 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, 2H), 6.02 (d, J = 7.8 Hz, 1H), 7.11 - 7.17 (m, 1H), 7.27 - 7.33 (m, 2H); LC-MS (ESI) m / z 485.3 (M+H) + .

[0076]

Chem.

[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 International Publication No. WO 2005 / 012266.

[0078]

Chem.

[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 International Publication No. WO 2005 / 012266.

[0080] [Chemical]

[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 International Publication No. 2005012266.

[0082] Kinetic Solubility Test

[0083] [Table 3]

[0084] For the determination of kinetic solubility, 3 mL of a 10 mM (5 mM, 1 mM) DMSO stock solution of each compound was pipetted into the wells of a 96-well plate (Millipore Multiscreen® HTS -PCF Filter Plate, 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 hours and then 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 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.

[0085] Thermodynamic Solubility Test (37 °C, 4 hours) When measuring the thermodynamic solubility: Usually, 200 - 400 μL of this solution was added to 1 - 2 mg of the solid test substance (n = 5). The resulting mixture / supersaturated solution was shaken at 37 °C for 4 hours (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 five - point calibration. (If the solid material is completely dissolved in the aqueous test medium, the result should be considered the minimum value.). The results are shown in Table 2.

[0086] Thermodynamic solubility test (RT, 24 hours) When measuring the thermodynamic solubility: Usually, 200 - 400 μL of this solution was added to 1 - 2 mg of the solid test substance (n = 5). The resulting mixture was shaken at room temperature (RT) for 24 hours (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 five - point calibration. (If the solid material is completely dissolved in the aqueous test medium, the result should be considered the minimum value.). The results are shown in Table 2.

[0087]

Table 4

[0088] The kinetic and thermodynamic solubility data of the examples of the present disclosure show that their solubilities are unexpectedly improved compared to the solubility of the reference compounds.

[0089] Biological assay In in vitro biological studies, the following abbreviations were generally used: BSA is bovine serum albumin, cAMP is cyclic adenosine monophosphate, DMEM is Dulbecco's modified Eagle's medium, DMSO is dimethyl sulfoxide, EDTA is tetrasodium ethylenediaminetetraacetate, 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 Test using the receptor In a competitive binding assay 3 Using [H] raclopride for human D 2L Determination of the affinity of the receptor Recombinant human D 2L Cultured CHO-K1 cells expressing the receptor (DRD2L cAMP Hunter (trademark), G i cell line, Eurofins DiscoverX, Fremont, CA, USA) was homogenized using a Dounce tissue grinder in 4× (v / v) buffer (50 mM Tris, 5 mM MgCl 2 , 1 mM EGTA, pH 7.4, 25 °C), centrifuged at 40,000×g for 10 minutes at 4 °C. The supernatant was removed, and the pellet was resuspended in 4× buffer (v / v) and centrifuged. The resulting pellet was resuspended in the above buffer at a volume of 12.5 mL / g of the original weight. Then, the membrane samples were aliquoted and stored at -70 °C.

[0091] The test compound was serially diluted with DMSO and then diluted to 5% DMSO (v / w) in binding buffer (50 mM Tris, 5 mM MgCl 2 , 5 mM KCl, 1 mM CaCl 2 , 120 mM NaCl, 1 mM EDTA). 5× concentrated compound in binding buffer (50 μL) was transferred to a 96-well deep well plate (BRAND, Wertheim, Germany). The aliquoted membrane samples were thawed and washed once in binding buffer. In the same buffer, 10 μg of protein per well was incubated at 25 °C for 120 minutes at a volume of 250 μL in the presence or absence of the test compound (to determine the binding inhibition or total binding of the test compound, respectively) with 2 nM 3 [H] raclopride (PerkinElmer, Waltham, MA, USA). Nonspecific binding (NSB) was determined with D 2Determined in the presence of 10 μM haloperidol, an antagonist of the receptor. After incubation, samples were filtered through 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 at 40 °C for 1 hour and 40 μL of Microscint™-20 scintillation cocktail (PerkinElmer) was added to each well. Radioactivity was determined using a MicroBeta 2 (registered trademark) microplate counter (PerkinElmer).

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

[0093]

Table 5

[0094] 3 Determination of the affinity of human D 2L receptor in a competitive binding assay using spiperone Human recombinant D 2L CHO-K1 cells expressing the receptor were washed with phosphate-buffered saline (PBS). The cells were scraped 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 MgCl 2 , 1 mM EDTA, and 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 samples were then aliquoted and stored at -70 °C.

[0095] In a 96-well plate, in an incubation buffer containing 50 mM Tris-HCl, 1.4 mM ascorbic acid, 0.001% BSA, 150 mM NaCl (pH 7.4) with 1% DMSO in a final reaction volume of 222 μL, aliquotted membrane samples were incubated at 25 °C for 120 min in the presence or absence of test compounds with 0.16 nM 3 [H] spiperone (PerkinElmer). Nonspecific binding (NSB) was determined in the presence of 10 μM haloperidol (Sigma-Aldrich). After incubation, the samples were filtered through a UniFilter® GF / C plate (PerkinElmer), washed, and Microscint™-20 scintillation cocktail was added. Radioactivity was determined using a MicroBeta 2 ® microplate counter (PerkinElmer).

[0096] Specific binding was obtained by subtracting NSB from the scintillation counts, normalized to vehicle-treated samples, and converted to percent displacement values. IC 50 ​The values were determined by non-linear least squares regression analysis using MathIQ (trademark) (ID Business Solutions Ltd., Surrey, UK). K i The values were calculated using the Cheng-Prusoff equation with the observed IC 50 of the test compound, the concentration of the radioligand used in the assay, and the historical value of the K D of the ligand. pK i values were derived by calculating the negative logarithm of the K i values expressed in mol / liter and are shown in Table 4. From these results, the examples of the present disclosure 3 When [H] spiperone was used as the radioligand, it was shown to be a high-affinity ligand for the human recombinant D 2L receptor.

[0097]

Table 6

[0098] Characterization of agonism of the human D 2L receptor using cyclic adenosine monophosphate detection The agonistic activity at the human D 2L receptor was assayed by measuring cAMP levels in CHO-K1 cells expressing the human D i receptor (Eurofins DiscoverX Corp., Fremont, CA, USA) using a cAMP G 2L kit (Cisbio / PerkinElmer) by homogeneous time-resolved fluorescence (HTRF®: homogenous time-resolved fluorescence). CHO-K1 cells expressing the human D 2L receptor were cultured in Ham's F12 medium supplemented with 10% FBS, 1% penicillin-streptomycin antimycotic solution, and 800 μg / mL of G418 (Thermo Fisher Scientific, Waltham, MA, USA), and CO 2It was maintained at 37°C in a humidified atmosphere containing 5.0%. For cAMP measurement, the cryopreserved cells were thawed and seeded at 10,000 cells per well in a 96-well plate with a white wall of half the area in PathHunter® AssayComplete™ Cell Plating 2 (CP2) reagent (Eurofins DiscoverX), and CO 2 It was incubated overnight at 37°C in a humidified atmosphere containing 5.0%. Before 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) containing 100 μM IBMX and incubated at ambient temperature for 20 minutes. After further incubation at ambient temperature for 30 minutes with 0.5 μM forskolin (Eurofins DiscoverX), cell stimulation was stopped by adding detection reagent (20 μL of cAMP-d2 and 20 μL of anti-cAMP cryptate, Cisbio / PerkinElmer) diluted with lysis buffer. After incubation at ambient temperature for 60 minutes, the time-resolved fluorescence signal was quantified by a PHERAstar FS multimode reader (BMG Labtech, Ortenberg, Germany) using the standard HTRF® setting with a laser excitation of 337 nm. The results were the ratio of the acceptor fluorescence signal (A665 nm) to the donor fluorescence signal (A620 nm) × 10 4Calculated from and expressed as ΔF% values 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 and normalized to the response induced by the maximum effective concentration of dopamine tested in the same experiment. All calculations were performed using Microsoft Excel® (Microsoft Corporation, Redmond, WA, USA). The pEC 50 value (negative logarithm of the agonist concentration that inhibits forskolin-stimulated cAMP accumulation by 50%, expressed in mol / liter) was obtained using GraphPad Prism (GraphPad Software, San Diego, CA, USA) by fitting a four-parameter sigmoid curve to the concentration-effect data with the lower asymptote restricted to zero. From these results, the examples of the present disclosure are shown to be potent agonists of the G-protein coupled signaling pathway of the human recombinant D 2L receptor.

[0099]

Table 7

[0100] Human dopamine D 2L Measurement of β-arrestin recruitment to the receptor Tagged human D 2L receptor and tagged β-arrestin-2 (Eurofins DiscoverX, Fremont, CA, USA)-expressing PathHunter® CHO-K1 cells were seeded into a 96-well tissue culture plate with a white wall and a transparent bottom at a density of 20,000 cells per well in 90 μL of AssayComplete™ Cell Plating 2 (CP2) reagent (Eurofins DiscoverX) per cell. The plate was incubated at 37°C in CO 2Incubated overnight in a humidified atmosphere containing 5%. After 20 - 24 hours, 20 μL of the test compound or vehicle containing and 2.2% DMSO in CP2 reagent was added to the cells and they were incubated at 37 °C for 90 minutes. Then, 55 μL of PathHunter® detection reagent (Eurofins DiscoverX) was added per well and the plate was incubated at 25 °C for 60 minutes, after which luminescence was detected using a PHERAstar® FS multimode plate reader (BMG Labtech, Ortenberg, Germany). The raw data was converted to percent stimulation above the basal value. The values were further converted to percent of the maximum 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 defined as the concentration of the agonist at which the stimulation is half - maximal. pEC 50 Values were expressed in mol / liter, the EC 50 values were calculated as the negative logarithm and are shown in Table 6. From these results, the examples of the present disclosure are shown to be potent agonists of the G - protein - independent signaling pathway of the human recombinant D 2L receptor.

[0101]

Table 8

[0102] Human D 3 Receptor - based assay 3 Determination of the affinity of the human D 3 receptor in a competitive binding assay using [H] raclopride Recombinant human D 3 ​Cell culture (CHO-K1) expressing the receptor (DRD3, purchased from Euroscreen Fast, Brussels, BE, GenBank ID U32499) was homogenized using a Dounce tissue grinder in 4× buffer (v / v) solution (15 mM Tris, 2 mM MgCl 2 2, 0.3 mM EDTA, 1 mM EGTA, pH 7.4, 25 °C) and centrifuged at 40,000×g for 25 minutes at 4 °C. The supernatant was removed, and the pellet was resuspended in 4× (v / v) buffer and centrifuged. This process was repeated twice, and the pellet was resuspended in storage buffer (75 mM Tris, 12.5 mM MgCl 2 2, 0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose, pH 7.4, 25 °C) at 12.5 mL / g of the original cell weight. Then, the membrane samples were aliquoted and stored at -70 °C.

[0103] The compound was diluted in DMSO and binding buffer (containing 50 mM Tris, 5 mM MgCl 2 2, 5 mM KCl, 1 mM CaCl 2 2, 120 mM NaCl, 1 mM EDTA), and 50 μL of each solution was transferred to a deep well plate (BRAND) at 5-fold the final concentration in 5% DMSO-buffer. The aliquoted membrane samples were thawed and washed once in binding buffer. In the same buffer, 3.3 μg of protein per assay was added in the presence or absence of the test compound into a 96-well deep well plate (BRAND) at approximately 2.7 nM 3Using raclopride (PerkinElmer), incubation was carried out at 25 °C for 120 minutes in a volume of 250 μL. Nonspecific 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, the samples were filtered through UniFilter® GF / B plates (PerkinElmer) using a Filtermate™ harvester (PerkinElmer) and washed with 4 × 1 mL of ice-cold binding buffer. The plates were dried at 40 °C for 1 hour, and 40 μL of Microscint™-20 scintillation cocktail (PerkinElmer) was added to each well. Radioactivity was determined using a MicroBeta 2 (registered trademark) microplate counter (PerkinElmer).

[0104] Using the raw scintillation counts, NSB was subtracted to obtain specific binding, which was normalized to vehicle-treated samples and converted to percent displacement values. GraFit 6.0 (Erithracus Software, Horley, UK) was used for curve fitting and calculations. IC 50 values (i.e., the concentration of the compound that inhibits specific binding by 50%) were determined from concentration-displacement curves by sigmoidal fitting. The inhibition constant (K i ) was calculated using the Cheng-Prusoff equation: K i = IC 50 / [1 + ([L] / K D )] (where [L] is the radioactive ligand concentration and K D is the affinity of the labeled ligand for the receptor). K D was determined from a separate saturation experiment. The pK i values shown in Table 7 were derived by calculating the negative logarithm of the K i values expressed in mol / liter. In all experiments, the samples were run in triplicate. From these results, the examples of the present disclosure are shown to be high-affinity ligands for the human recombinant D 3 receptor.

[0105]

Table 9

[0106] 3 Determination of the Affinity of Human D Receptor in a Competitive Binding Assay Using Spiperone 3 Human Recombinant D 3 CHO-K1 cells expressing the human D receptor were washed with PBS. The cells were scraped from the plate and centrifuged at 1000 × g. The cells were disrupted in a buffer containing 25 mM Tris-HCl (pH = 7.4), 6 mM MgCl 2 , 1 mM EDTA, and 10 mM PMSF using a Teflon (registered trademark) 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. The membrane preparation was aliquoted and stored at -70 °C.

[0107] In a 96-well plate, in an incubation buffer containing 50 mM Tris-HCl, 1.4 mM ascorbic acid, 0.001% bovine serum albumin, and 150 mM NaCl (pH 7.4) with 1% DMSO in a final reaction volume of 222 μL, aliquots of the membrane preparation were incubated at 37 °C for 120 min in the presence or absence of the test compound with 0.7 nM 3 H] spiperone (PerkinElmer). Nonspecific binding (NSB) was determined in the presence of 25 μM (S)-(-)-sulpiride (Sigma Aldrich). After incubation, the samples were filtered through a GF / C filter plate (PerkinElmer), washed, and Microscint (trademark)-20 scintillation cocktail was added. Radioactivity was determined in a MicroBeta 2 (registered trademark) microplate counter (PerkinElmer).

[0108] ​​The number of counts of the raw scintillation was subtracted by NSB to obtain the specific binding, which was normalized to the vehicle-treated samples and converted to the substitution % value. The IC 50 value was determined by non-linear least squares regression analysis using MathIQ (trademark) (ID Business Solutions Ltd., Surrey, UK). The K i value was calculated using the Cheng-Prusoff equation with the observed IC 50 of the test compound, the concentration of the radioactive ligand used in the assay, and the historical value of the K D of the ligand. The pK i value shown in Table 8 was derived by calculating the negative logarithm of the K i value expressed in mol / liter. From these results, the examples of the present disclosure show that 3 when [3H] spiperone is used as the radioactive ligand, it is shown to be a high-affinity ligand for the human recombinant D 3 receptor.

[0109]

Table 10

[0110] Characterization of the agonism of the human D 3 receptor using cyclic adenosine monophosphate detection The agonist activity at the human D 3 receptor was assayed by the cAMP level using a homogeneous time-resolved fluorescence method (HTRF) with a cAMP G i kit (Cisbio / PerkinElmer) in HEK293 cells (cell line developed by Gedeon Richter) stably co-expressing adenylate cyclase V (ACV) and expressing the recombinant human D 3 receptor (BioXtal, Saint-Felix, France). The recombinant human D 3HEK293 cells expressing the 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% CO 2 2. Before measuring cAMP, the cells were detached with Versene (Thermo-Fisher Scientific) and suspended 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) at a density of 15,000 cells per well in a volume of 20 μL. The assay buffer was supplemented with 100 μM IBMX (Sigma Aldrich, St Louis, MO, USA). After adding 10 μL per well of the test compound (4× concentrated) or vehicle (DMSO), the cells were incubated for 20 min with the assay buffer or various concentrations of the test compound. After further incubation for 30 min at ambient temperature with 1.5 μM forskolin (final DMSO concentration was 0.3%), cell stimulation was stopped by adding the detection reagents (20 μL of cAMP-d2 and 20 μL of anti-cAMP cryptate) diluted in lysis buffer (PerkinElmer). After incubation for 60 min at ambient temperature, the time-resolved fluorescence signal (TRF) was quantified by a PHERAstar FS multimode reader (BMG Labtech, Ortenberg, Germany) using a standard HTRF setting with a laser excitation of 337 nm.

[0111] The results were expressed as the acceptor fluorescence signal (A665 nm) and the donor fluorescence signal (A620 nm) × 10 4Calculated from the ratio and expressed as a ΔF% value 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. The agonist activity value was calculated as the percentage inhibition of forskolin-stimulated cAMP accumulation and normalized to the response induced by the maximum effective concentration of dopamine tested in the same experiment. All calculations were performed using Excel (Microsoft, Redmond, WA, USA). pEC 50 value (negative logarithm of the agonist concentration that inhibits 50% of forskolin-stimulated cAMP accumulation, expressed in mol / liter) was obtained using GraphPad Prism (GraphPad Software, San Diego, CA, USA) by fitting a four-parameter sigmoid curve to the concentration-effect data with the lower asymptote restricted to zero and is shown in Table 9. From these results, the examples of the present disclosure are shown to be potent agonists of the G-protein-dependent signaling pathway of the human recombinant D 3 receptor.

[0112]

Table 11

[0113] Human dopamine D 3 Measurement of β-arrestin recruitment to the receptor Tagged human D 3 receptor and tagged β-arrestin-2 (Eurofins DiscoverX, Fremont, CA, USA)-expressing PathHunter® CHO-K1 cells were seeded at a density of 25,000 cells per well in 90 μL of AssayComplete™ Cell Plating 2 (CP2) reagent (Eurofins DiscoverX) in a 96-well tissue culture plate with a white wall and a transparent bottom and incubated at 37°C in CO 2Incubated overnight in a humidified atmosphere at 5%. After 20 - 24 hours, 20 μL of the 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 minutes. Then, luminescence was detected using a PHERAstar® FS multimode plate reader (BMG Labtech, Ortenberg, Germany).

[0114] Raw data was first converted to % stimulation above the basal value. The % stimulation above the basal value was further converted to the % maximum stimulation of β - arrestin recruitment by 1 μM dopamine. EC 50 values were calculated from concentration - response curves of at least six concentrations in duplicate by sigmoidal fitting using Origin® 7.5 software (OriginLab Corporation, Northampton, MA, USA), and defined as the concentration of the agonist at which the stimulation is half - maximal. The pEC 50 values shown in Table 10 were expressed in mol / liter and calculated as the negative logarithm of the EC 50 value. From this result, it is shown that the examples of the present disclosure are very potent agonists of the G - protein - independent signaling pathway of the human recombinant D 3 receptor.

[0115]

Table 12

[0116] 5 - HT2 A receptor assay Determination of affinity for the human 5 - HT 2A receptor in a competitive binding assay Receptor membranes were prepared from human 5 - HT 2APrepared from a CHO-K1 recombinant AequoScreen® cell line (PerkinElmer, Waltham, MA, USA) that stably expresses the receptor. The cells were suspended in Buffer A (15 mM Tris-HCl, pH 7.5, 2 mM MgCl 2 2, 0.3 mM EDTA, 1 mM EGTA) at 4× volume (1 g of cells - 4 mL of buffer), and homogenized with a Dounce homogenizer. The crude membrane fraction was separated by two consecutive centrifugations at 40,000×g for 25 minutes by a washing step with Buffer A, and then recovered. The final pellet was resuspended in Buffer B (75 mM Tris-HCl, pH 7.5, 12.5 mM MgCl 2 2, 0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose) at a concentration of 80 mg of wet cell weight in 0.5 mL of buffer, aliquoted, and snap-frozen on dry ice. The protein content was determined using the bicinchoninic acid assay in the presence of a sulfhydryl reagent with bovine serum albumin (BSA) as a standard.

[0117] In the binding experiment, 15 μg of protein of the membrane specimen per well and 1 nM ketanserin hydrochloride as a radioactive ligand, [ethylene- 3H](PerkinElmer) was incubated with the compound or vehicle (DMSO, final concentration 1% (v / v)) in incubation buffer (50 mM Tris, 0.3% BSA, pH 7.4). Nonspecific binding (NSB) was determined in the presence of 1 μM mianserine hydrochloride (Tocris, Bristol, UK). Samples were incubated at 25 °C for 15 min in a final volume of 250 μL. The binding reaction was terminated by rapid filtration through a Filtermate™ harvester (PerkinElmer) using UniFilter® GF / C plates that had been pre-soaked in 0.5% (v / v) polyethyleneimine (PEI, dissolved in distilled water) for at least 1 h. The 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 determined using a MicroBeta 2 (registered trademark) 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 values (i.e., the concentration of compound that displaces 50% of the specifically bound radioligand) were determined from the concentration-displacement curve by sigmoidal fitting using Origin® 7.5 software (OriginLab® Corporation, Northampton, MA, USA). The K i values (i.e., the inhibition constant) shown in Table 11 were calculated using the Cheng-Prusoff equation: K i = IC 50 / [1 + ([L] / K D )] (where [L] is the radioligand concentration used, determined by scintillation counting, and K D(which is the affinity of the labeled ligand for the receptor determined in another experiment). The competitive binding assay was performed at a minimum of six concentrations in two independent experiments, with each concentration repeated three times. The pK i values shown in Table 11 were derived by calculating the negative logarithm of the K i values expressed in mol / liter. From these results, the examples of the present disclosure are shown to be ligands for the human recombinant 5-HT 2A receptor.

[0119] [Table 13]

[0120] Characterization of Antagonism in Human 5-HT 2+ Receptors Using Fluorescent Ca 2A Detection CHO-K1 cells expressing human recombinant 5-HT 2A receptor and Gα 16 (purchased from Euroscreen Fast, Brussels, BE) were cryopreserved according to an established protocol using 90% FBS / 10% DMSO as the medium. Before the experiment, the 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. The cells were seeded at a density of 40,000 cells per well in a 96-well microplate and incubated overnight at 37°C in a humidified atmosphere containing 5.0% CO 2 On the day of the experiment, the plate was washed using a plate washer (Elx405UCWS, Biotek, Winooski, VT, USA) with assay buffer (140 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2, washed with 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES), 10 mM glucose, 2 mM probenecid, pH 7.4, and then 50 μL per well of 4 μM Fluo-4 AM (Thermo Fisher Scientific) in assay buffer was added. After loading the dye (60 minutes, 37 °C, in the dark), the plate was washed with assay buffer using a plate washer, leaving a volume of 50 μL per well, and then 50 μL per well of assay buffer containing vehicle (3% DMSO in assay buffer) or test compound (3× final concentration) was added, and the cells were incubated at 37 °C for an additional 10 minutes.

[0121] The final DMSO concentration was 1% (v / v) for all treatments. 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 immediately before measurement. A stock solution (10 mM) of 5-HT was prepared by dissolving 5-HT in deionized ultrafilted water.

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

[0123] The compound was evaluated against the 5-HT concentration as determined on each plate with the integrated curve fitting module of the measurement software (SoftMax® Pro 5.2, Molecular Devices, San Jose, CA, USA), EC 80 5-HT concentration.

[0124] The results were expressed as ΔF / F values, where F is the baseline fluorescence defined as the average fluorescence before and / or immediately after agonist administration, and ΔF is the increase in fluorescence after agonist administration; ΔF = F max - F was calculated as, and F max was the maximum fluorescence between 17 - 29 between leads, and F was the average of 2 - 13 of the fluorescence leads. 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 ))]. The IC 50 value of the test compound was determined from a 4-parameter sigmoidal concentration effect curve that was fit to the inhibition% data using SoftMax® Pro software (Molecular Devices). The further calculation of the pIC 50 value shown in Table 12 was derived by calculating the negative logarithm of the IC 50 value expressed as mol / liter by the above software. From these results, the examples of the present disclosure are shown to be antagonists of the human recombinant 5-HT 2A receptor.

[0125]

Table 14

[0126] Phencyclidine-induced hyperactivity 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 surface, twice, near the bottom of the cage, to detect horizontal walking behavior. To detect rearing responses, additional photocell arrays (30 pairs) were installed at different heights (6.5 cm, 12 cm, 18 cm, and 23 cm) along the side surface on the opposite side of the cage. Signals caused by the interruption of the photocell beam were processed by motion analysis software (Experimetria), the spatial position of the animal was determined at a sampling frequency of 1 Hz, and the time spent by the rat walking was calculated by computer.

[0127] The test compound was administered orally or subcutaneously to the test animals at 10 animals per treatment group. In these studies, male Harlan-Wistar rats (Toxi-Coop, Budapest, HU) weighing 190 - 210 grams were used. After oral administration of the test compound or vehicle, the animals were individually acclimated to the activity monitor for 30 minutes. In the case of subcutaneous administration of the test compound, the rats were first acclimated to the activity monitor for 15 minutes, then treated with the test compound or vehicle, and then returned to the activity monitor for an additional 15 minutes of acclimation. After acclimation, the rats were treated subcutaneously with phencyclidine hydrochloride (PCP) and immediately returned to the experimental apparatus during the measurement period (1 hour).

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

[0129]

Table 15

[0130] The above data (Table 13) showed that the compounds of the examples of the present disclosure exhibited good and potent pharmacodynamic effects / activities in phencyclidine-induced hyperactivity in rats, indicating that they may have the efficacy of a substantial schizophrenia therapeutic agent.

[0131] Climbing and sniffing behaviors induced by apomorphine 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 a cylindrical cage. The cage was 15 cm high and 12 cm in diameter, and had vertical metal bar walls with diameters of 2 mm and 1 cm spaced apart, inserted into a smooth plastic surface.

[0132] Immediately after subcutaneous administration of the vehicle or test compound, the animals were placed in cages and acclimated for 10 minutes. At the end of the 10-minute acclimation period, 1.5 mg / kg of apomorphine hydrochloride was administered subcutaneously. After treatment, the animals were returned to the cylindrical cages. Measurement of climbing and sniffing behaviors began 10 minutes (11 minutes after that treatment) after apomorphine treatment and continued for 16 minutes. Throughout the entire process, climbing behavior was scored as follows: all four legs on the floor crawling (0 points), front legs touching the bar (1 point), all four legs grasping the bar (2 points). The animals also evaluated repetitive sniffing behavior as a measure of stereotypy according to the following actions: no sniffing behavior (0 points), moderate sniffing behavior with little nose contact with the cage wall or floor (1 point), constant sniffing behavior with continuous nose contact (2 points).

[0133] The data were analyzed using GraphPad Prism 9 software (GraphPad). The scores of both behaviors were tabulated for each individual (the maximum possible score was 32 points), and the group means were calculated. The drug efficacy was calculated as the percentage of suppression of the behaviors induced by apomorphine. From the data of the suppression percentage, a dose-response curve was plotted, and the ED 50 value was calculated by simple linear regression for the ED 50 value using a computer.

[0134]

Table 16

[0135] The above data (Table 14) show that the compounds of the examples of the present disclosure exhibit good and potent pharmacodynamic effects / activities in climbing and sniffing behaviors induced by apomorphine in mice, indicating that they may have the efficacy of a substantial schizophrenia therapeutic agent.

[0136] It is understood that the foregoing detailed description and the accompanying examples are merely illustrative and are not to be construed as limiting the scope of the disclosure, which is defined solely 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 related to the chemical structures, substituents, derivatives, intermediates, syntheses, formulations, and / or methods of use of the disclosure, may be made without departing from its spirit and scope. 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 structure: 【Chemistry 2】

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

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