Crystal form of NK3R antagonist as well as preparation method and application of crystal form

Abstract

The invention discloses an A crystal form of an NK3R antagonist as shown in a formula (I) as well as a preparation method and application of the A crystal form. Wherein the crystal form A is radiated by Cu-K alpha, and X-ray powder diffraction represented by a 2 theta angle has characteristic peaks at the positions of 10.40 + / -0.20 degrees, 11.79 + / -0.20 degrees, 19.51 + / -0.20 degrees and 20.81 + / -0.20 degrees. The crystal form A provided by the invention is stable in property, weak in hygroscopicity, suitable for long-term preservation and patent medicine, and capable of effectively inhibiting NK3R.

Description

A crystal form of NK3R antagonist and its preparation method and application

[0001] This application claims priority to two prior applications: Patent Application No. 202211282020.3, filed with the State Intellectual Property Office of China on October 19, 2022, entitled “A Crystalline Form of a NK3R Antagonist, Method for Preparing the Same, and Use thereof”; and Patent Application No. 202311315851.0, filed with the State Intellectual Property Office of China on October 11, 2023, entitled “A Crystalline Form of a NK3R Antagonist, Method for Preparing the Same, and Use thereof.” The entire contents of both prior applications are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of pharmaceutical compounds, and particularly relates to a crystal form of an NK3R antagonist, a preparation method thereof, and an application thereof. Background Art

[0003] Tachykinin receptors are targets of a family of structurally related peptides collectively designated "tachykinins," including substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). Tachykinins are synthesized in the central nervous system (CNS) and peripheral tissues, where they exert a variety of biological activities. Three tachykinin receptors are currently known: neurokinin-1 (NK-1), neurokinin-2 (NK-2), and neurokinin-3 (NK-3). Tachykinin receptors belong to the rhodopsin-like seven-transmembrane G protein-coupled receptor family. SP has the highest affinity and is considered the endogenous ligand for the NK-1 receptor, NKA for the NK-2 receptor, and NKB for the NK-3 receptor. NK-1, NK-2, and NK-3 receptors have been identified in different species. NK-1 receptor and NK-2 receptor are expressed in various peripheral tissues and NK-1 receptor is also expressed in the CNS, whereas NK-3 receptor is expressed predominantly in the CNS.

[0004] Neurokinin receptors mediate a variety of biological effects stimulated by tachykinins, including transmission of excitatory neuronal signals in the CNS and periphery (e.g., pain), modulation of smooth muscle contractile activity, regulation of immune and inflammatory responses, induction of antihypertensive effects via dilation of peripheral vasculature, and stimulation of endocrine and exocrine gland secretion.

[0005] The NK-3 receptor, encoded by the TACR3 gene, is involved in regulating the hypothalamic-pituitary-gonadal axis. TACR3 knockout or mutant mice exhibit abnormal reproductive organ development, low sex hormone levels, and severely reduced reproductive capacity. Carrying TACR3 gene mutations can lead to abnormal gonadotropin release, resulting in sexual infantilism and infertility. A significant proportion of familial hypogonadism is caused by TACR3 gene mutations.

[0006] Kisspeptin / neurokinin B / dynorphin (KNDy) neurons participate in the gonadotropin-releasing hormone (GnRH) signaling pathway, promoting estrogen production through the GnRH neuron-pituitary-sex organ pathway. This signaling pathway is regulated by a negative feedback mechanism, thereby maintaining hormone levels within a reasonable range. KNDy neurons are also involved in thermoregulatory signaling pathways, releasing NKB ligands that bind to NK-3 receptors in the median preoptic nucleus (MPN). This regulates body temperature within a certain range by inhibiting shivering and vasoconstriction and promoting sweating and vasodilation. In menopausal women, decreased estrogen levels lead to a loss of this negative feedback mechanism, leading to overactivation of KNDy neurons and the release of large amounts of endogenous NKB ligands, which bind to NK-3 receptors in the MPN. This leads to sweating, vasodilation, and the symptoms of hot flashes. Therefore, the development of antagonists targeting KNDy neurons and the NK-3 receptor in the MPN has the potential to be a promising therapeutic approach for hot flashes.

[0007] In the CNS, NK-3 receptors are expressed in regions including the medial prefrontal cortex, hippocampus, thalamus, and amygdala. In addition, NK-3 receptors are expressed on dopaminergic neurons. Activation of the NK-3 receptor has been shown to regulate the release of dopamine, acetylcholine, and serotonin, suggesting the therapeutic utility of NK-3 receptor modulators in treating a variety of conditions, including psychotic disorders, anxiety disorders, depression, schizophrenia, and obesity, pain, or inflammation.

[0008] The development of novel structural NK-3R antagonists and pharmaceutical solid forms of such compounds suitable for drug formulation, such as solid forms with improved stability, hygroscopicity and / or efficacy, so as to achieve good results in the pharmaceutical preparation and administration stages, has always been a technical problem that those skilled in the art have been committed to solving.

[0009] Summary of the Invention

[0010] In order to solve the above technical problems, the present invention provides a crystal form A of a compound represented by formula (I),

[0011] The A crystal form uses Cu-Kα radiation, and the X-ray powder diffraction expressed in 2θ angles has characteristic peaks at 10.40±0.20°, 11.79±0.20°, 19.51±0.20°, and 20.81±0.20°.

[0012] Preferably, the A crystal form uses Cu-Kα radiation, and the X-ray powder diffraction expressed in 2θ angles has characteristic peaks at 10.40±0.20°, 11.79±0.20°, 15.92±0.20°, 16.92±0.20°, 19.51±0.20°, 20.81±0.20°, 21.19±0.20°, and 22.91±0.20°.

[0013] In some embodiments, the Form A further has characteristic peaks at 3.17±0.20°, 8.50±0.20°, 13.24±0.20°, 15.15±0.20°, 19.03±0.20°, 25.03±0.20°, and / or 26.95±0.20° in X-ray powder diffraction expressed in 2θ angles using Cu-Kα radiation.

[0014] In some embodiments of the present invention, the above-mentioned Form A has an XRPD pattern substantially as shown in FIG1 .

[0015] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form A are shown in Table 1:

[0016] Table 1

[0017] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form A are shown in Table 2:

[0018] Table 2

[0019] In some embodiments of the present invention, the above-mentioned crystal form A loses weight by 0.244% at 150±3°C.

[0020] In some embodiments of the present invention, the above-mentioned crystal form A has a TGA pattern substantially as shown in Figure 2.

[0021] In some embodiments of the present invention, the above-mentioned crystal form A has an endothermic peak at 272.53±3°C.

[0022] In some embodiments of the present invention, the above-mentioned Form A has a DSC spectrum substantially as shown in FIG3 .

[0023] The present invention also provides a method for preparing the crystal form A of the compound represented by formula (I), comprising using the compound represented by formula (I) as a raw material and preparing it by an antisolvent method, a solvent volatilization method, a cooling precipitation method, a suspension stirring method, a temperature cycling method, a gas-solid permeation method, a polymer induction method or a grinding method.

[0024] Preferably, the preparation method of the crystal form A comprises the following steps:

[0025] (a) adding the compound represented by formula (I) to a solvent to form a suspension; the solvent is selected from an organic solvent, water, or a mixed solvent of an organic solvent and water;

[0026] (b) stirring the suspension at 25-60° C. for 8-120 hours;

[0027] (c) After step (b), centrifugation and drying (e.g., drying for 8 to 16 hours) are performed to obtain the Form A.

[0028] In some embodiments of the present invention, the organic solvent can be selected from one or more of methanol, ethanol, isopropanol, acetone, methyl isobutyl ketone, heptane, toluene, m-xylene, dichloromethane, chloroform, anisole, methyl tert-butyl ether, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, 2-methyltetrahydrofuran, 1,4-dioxane, acetonitrile, tetrahydrofuran, ethyl acetate and isopropyl acetate.

[0029] The present invention also provides a pharmaceutical composition containing the above-mentioned crystal form A.

[0030] According to an embodiment of the present invention, the pharmaceutical composition further contains pharmaceutically acceptable excipients, such as, but not limited to, one or more of excipients, fillers, lubricants, binders, disintegrants, inorganic salts, solvents, dissolution aids, suspending agents, isotonic agents, buffers, preservatives, antioxidants, colorants, foaming agents and flavoring agents.

[0031] According to an embodiment of the present invention, the pharmaceutical composition may further comprise one or more other active ingredients in addition to the above-mentioned crystal form A.

[0032] In the pharmaceutical composition, the dosage of the crystal form A and one or more other active ingredients is a therapeutically effective amount.

[0033] The present invention also provides use of the above-mentioned crystal form A or the pharmaceutical composition in preparing a pharmaceutical preparation, wherein the pharmaceutical preparation is an NK-3 receptor antagonist.

[0034] According to an embodiment of the present invention, the pharmaceutical preparation is used for preventing and / or treating diseases mediated by NK-3 receptors; for example, for preventing and / or treating depression, anxiety, psychosis, schizophrenia, psychotic disorders, bipolar disorder, cognitive disorders, Parkinson's disease, Alzheimer's disease, attention deficit hyperactivity disorder (ADHD), pain, convulsions, obesity, inflammatory diseases, vomiting, preeclampsia, airway-related diseases, reproductive disorders, contraception and sex hormone-dependent diseases and / or gynecological diseases.

[0035] According to an embodiment of the present invention, the sex hormone-dependent diseases include but are not limited to benign prostatic hyperplasia (BPH), prostatic hyperplasia, metastatic prostate cancer, testicular cancer, breast cancer, ovarian cancer, androgen-dependent acne, male pattern baldness, endometriosis, puberty abnormalities, uterine fibrosis, uterine fibroids, hormone-dependent cancers, hyperandrogenism, hirsutism, virilization, polycystic ovary syndrome (PCOS), premenstrual dysphoric disorder (PMDD), HAIR-AN syndrome (hyperandrogenism, insulin resistance and acanthosis nigricans), ovarian theca cell hyperplasia (HAIR-AN with luteinizing theca cell proliferation in the ovarian stroma), other manifestations of high intraovarian androgen concentrations (such as follicular maturation arrest, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility), androgen-producing tumors (masculinizing ovarian tumors or adrenal tumors), menorrhagia and / or adenomyosis.

[0036] According to an embodiment of the present invention, the airway-related diseases include but are not limited to chronic obstructive pulmonary disease, asthma, airway hyperresponsiveness, bronchoconstriction and / or cough.

[0037] In some embodiments, the pharmaceutical preparation is used to treat and / or prevent diseases associated with menopausal syndrome, wherein the menopausal syndrome includes symptoms such as hot flashes, sweating, palpitations, dizziness and / or obesity.

[0038] According to an embodiment of the present invention, the pharmaceutical preparation can be in the form of powder, tablet (e.g., coated tablet, sustained-release or controlled-release tablet), lozenge, capsule (e.g., soft capsule or hard capsule), granule, pill, dispersible powder, suspension, solution, emulsion, elixir, syrup, aerosol, cream, ointment, gel, injection, lyophilized powder injection or suppository.

[0039] According to an embodiment of the present invention, the pharmaceutical preparation can be administered in any of the following ways: orally, buccal administration, sublingually, inhalation, topical application, intravenous, subcutaneous, acupuncture point or intramuscular injection via parenteral administration, and rectal administration.

[0040] The present invention also provides a method for preventing and / or treating a disease, comprising administering a therapeutically effective amount of the crystal form A or the pharmaceutical composition to a patient;

[0041] The disease is a disease mediated by NK-3 receptors, preferably depression, anxiety, psychosis, schizophrenia, psychotic disorders, bipolar disorder, cognitive disorders, Parkinson's disease, Alzheimer's disease, attention deficit hyperactivity disorder, pain, convulsions, obesity, inflammatory diseases, vomiting, preeclampsia, airway-related diseases, reproductive disorders, contraception and sex hormone-dependent diseases and / or gynecological diseases;

[0042] Alternatively, the disease is a disease related to menopausal syndrome, and the menopausal syndrome includes symptoms of hot flashes, sweating, palpitations, dizziness and / or obesity. Beneficial effects

[0043] The crystal form A provided by the present invention has stable properties, weak hygroscopicity, is suitable for long-term storage, is suitable for drug formulation, and can effectively inhibit NK3R.

[0044] Definitions and Explanations of Terms

[0045] Unless otherwise indicated, the definitions of terms in this specification and claims, including definitions used as examples, exemplary definitions, preferred definitions, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with each other. Such combinations and couplings shall fall within the scope of this specification.

[0046] The term "crystalline form" refers to forms of a crystal having the same chemical composition but a different spatial arrangement of the molecules and / or ions that form the crystal.

[0047] The compound represented by formula (I) is the "free base", and the "free base crystalline form A" is the "crystalline form A of the compound represented by formula (I)".

[0048] The term "therapeutically effective amount" refers to an amount of the crystalline Form A of the present invention and one or more other active ingredients sufficient to achieve the intended application (including but not limited to the treatment of a disease as defined below). The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the subject and disease condition being treated, such as the weight and age of the subject, the severity of the disease condition, and the mode of administration, which can be readily determined by one of ordinary skill in the art. The specific dosage will vary depending on the specific active ingredient selected, the dosage regimen used, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system used.

[0049] The term "patient" refers to any animal including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses or primates, and most preferably humans.

[0050] The term "plurality" means two or more, for example, two or more than two.

[0051] The term "150±3°C" represents 147-153°C, that is, it can be 147°C, 148°C, 149°C, 150°C, 151°C, 152°C, 153°C or a value between any two of the foregoing points.

[0052] The term "272.53±3°C" represents 269.53-275.53°C, that is, it can be 269.53°C, 270.00°C, 271.00°C, 272.00°C, 275.53°C or a value between any two of the foregoing points. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1: XRPD pattern of the crystalline form of compound A represented by formula (I);

[0054] Figure 2: TGA spectrum of the crystalline form of compound A represented by formula (I);

[0055] Figure 3: DSC spectrum of the crystalline form of compound A represented by formula (I);

[0056] Figure 4: DVS image of the crystalline form of compound A represented by formula (I);

[0057] Figure 5: XRPD comparison of the crystalline form of compound A represented by formula (I) before and after DVS testing;

[0058] Figure 6: XRPD comparison of the crystalline form of compound A represented by formula (I) before and after stability evaluation. DETAILED DESCRIPTION

[0059] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0060] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0061] The following are the instruments, parameters, characterizations and test methods used in the examples:

[0062] (1) X-ray powder diffractometer (XRPD) method

[0063] Model: PANalytical X-ray powder diffraction analyzer X'Pert3;

[0064] X-ray: Cu, kα, 1.540598; 1.544426; Kα2 / Kα1 intensity ratio: 0.50;

[0065] X-ray tube setting: 45kV, 40mA;

[0066] Divergence slit: fixed 1 / 8°;

[0067] Scan mode: continuous;

[0068] Scanning range (°2Theta): 3-40;

[0069] Scan time per step (s): 46.7;

[0070] Scan step (°2Theta): 0.0263;

[0071] Test time (minutes): 5.

[0072] (2) Thermogravimetric analysis (TGA) method

[0073] Instrument model: Discovery 5500;

[0074] Sample pan: open or closed aluminum pan;

[0075] Starting temperature: room temperature (below 35°C);

[0076] Termination temperature: 300℃ or when weight <80% (w / w) the next stage is terminated

[0077] (weight loss of the compound does not exceed 20% (w / w));

[0078] Heating rate: 10℃ / min;

[0079] Nitrogen flow rate: stable at 10 mL / min; sample chamber 25 mL / min;

[0080] Sample amount: approximately 2-10 mg.

[0081] (3) Differential Scanning Calorimeter (DSC) method

[0082] Instrument model: TA Discovery 2500 or Q2000;

[0083] Sample pan: Tzero pan and Tzero sealing cover with a pin hole of 0.7 mm diameter;

[0084] Temperature range: 30 to 300℃ or before decomposition;

[0085] Heating rate: 10℃ / min;

[0086] Nitrogen flow rate: 50 mL / min;

[0087] Sample amount: about 0.5-2 mg;

[0088] (4) Dynamic moisture adsorption (DVS) method

[0089] DVS curves were acquired on a DVS Intrinsic instrument from SMS (Surface Measurement Systems). Relative humidity at 25°C was calibrated based on the deliquescent points of LiCl, Mg(NO3)2, and KCl.

[0090] Temperature: 25℃;

[0091] Sample amount: 10-20 mg;

[0092] Protective gas and flow rate: N2, 200 mL / min;

[0093] dm / dt: 0.002% / min;

[0094] Minimum dm / dt balance time: 10min;

[0095] Maximum equilibration time: 180 min;

[0096] RH range: 0%RH~95%RH~0%RH;

[0097] RH gradient: 10%RH (0%RH-90%RH&90%RH-0%RH); 5%RH (90%RH-95%RH&95%RH-90%RH)

[0098] (5) Ultra-high performance liquid chromatography (UPLC) method

[0099] A Waters H-Class ultra-high performance liquid chromatograph was used to collect solubility and purity data for the project. Specific instrument and test parameters are shown in Table 3:

[0100] Table 3

[0101] Patent application PCT / CN2022 / 087947 describes the compound represented by formula (I), and all contents involved in this patent application are added to the present invention by reference.

[0102] Comparison table of Chinese and English names of solvents used in the experiment

[0103] Preparation Example Compound represented by formula (I)

[0104] The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR chemical shifts (δ) are given in parts per million (ppm). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer, using deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), and deuterated chloroform (CDCl3) as the solvents, with tetramethylsilane (TMS) as the internal standard.

[0105] Liquid chromatography-mass spectrometry (LC-MS) was performed using an Agilent 1200 Infinity Series mass spectrometer. HPLC was performed using an Agilent 1200DAD high-pressure liquid chromatograph (Sunfire C18 150×4.6 mm column) and a Waters 2695-2996 high-pressure liquid chromatograph (Gimini C18 150×4.6 mm column).

[0106] Thin layer chromatography silica gel plates use Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The specifications used for TLC are 0.15mm-0.20mm, and the specifications used for thin layer chromatography separation and purification products are 0.4mm-0.5mm. Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

[0107] Unless otherwise specified, all reactions of the present invention are carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere, with dry solvents and reaction temperatures in degrees Celsius.

[0108] 1.1 Synthesis of Intermediate 001

[0109] Preparation of (4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)methanone (001)

[0110] Step 1: Preparation of 2-(chloromethyl)-3-methylpyrazine

[0111] 2,3-Dimethylpyrazine 001a (10 g, 92.47 mmol) was added to carbon tetrachloride (250 mL), followed by N-chlorosuccinimide (14.83 g, 110.96 mmol) and benzoyl peroxide (224 mg, 9.25 mmol). The reaction mixture was allowed to react at 80°C for 16 hours under nitrogen. After completion of the reaction, monitored by LCMS, the solvent was dried, and water (150 mL) and dichloromethane (3 × 100 mL) were added. The mixture was extracted and washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified on a silica gel column (petroleum ether / ethyl acetate = 10:1) to obtain the title product, 2-(chloromethyl)-3-methylpyrazine 001b (3.20 g, colorless oil) in a yield of 22%.

[0112] MS m / z(ESI):143.2[M+1] + .

[0113] 1 H NMR (400MHz, CDCl3) δ8.45 (d, J = 2.0 Hz, 1H), 8.38 (d, J = 2.0 Hz, 1H), 4.71 (s, 2H), 2.69 (s, 3H).

[0114] Step 2: Preparation of 2-((3-methylpyrazin-2-yl)methyl)isoindoline-1,3-dione

[0115] 2-(Chloromethyl)-3-methylpyrazine 001b (3.20 g, 22.44 mmol) was added to N,N-dimethylformamide solvent, followed by potassium phthalimide (6.23 g, 33.66 mmol). The reaction solution was reacted at 110°C under nitrogen protection for 8 hours. After completion of the reaction monitored by LCMS, the solvent was dried, and water and ethyl acetate were added. The extract was then washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified on a silica gel column with petroleum ether / ethyl acetate = 1:1 to obtain the title product, 2-((3-methylpyrazin-2-yl)methyl)isoindoline-1,3-dione 001c, 3.10 g, as a light yellow solid in a yield of 95%.

[0116] MS m / z(ESI):254.2[M+1]+.

[0117] 1H NMR (400MHz, CDCl3) δ8.33(d,J=2.4Hz,1H),8.24(d,J=2.4Hz,1H),7.90(dd,J=5.6,3.2Hz,2H),7.75(dd,J=5.6,3.2Hz,2H),5.02(s,2H),2.70(s,3H).

[0118] Step 3: Preparation of 2-(aminomethyl)-3-methylpyrazine

[0119] 2-((3-Methylpyrazin-2-yl)methyl)isoindoline-1,3-dione 001c (2.00 g, 7.90 mmol) was added to ethanol (50 mL) and hydrazine hydrate (3.95 g, 79 mmol). The reaction mixture was reacted at 80°C under nitrogen for 6 hours. After completion of the reaction, as monitored by LCMS, the solvent was dried and water (150 mL) was added. The mixture was extracted with dichloromethane / methanol (1 / 1, v / v, 50 mL). The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to afford the crude product, 2-(aminomethyl)-3-methylpyrazine 001d (300 mg, yellow oil), in a yield of 28%.

[0120] MS m / z(ESI):124.3[M+1] + .

[0121] 1 H NMR (400MHz, CDCl3) δ8.36 (s, 1H), 8.33 (d, J = 2.4Hz, 1H), 4.02 (s, 2H), 2.54 (s, 3H).

[0122] Step 4: Preparation of 3-methyl-N-((3-methylpyrazin-2-yl)methyl)-1,2,4-thiadiazole-5-carboxamide

[0123] 3-Methyl-1,2,4-thiadiazole-5-carboxylic acid (280 mg, 1.94 mmol) was added to dichloromethane (10 mL), followed by 0.5 mL of oxalyl chloride (0.5 mL) and N,N-dimethylformamide (0.1 mL). The reaction mixture was allowed to react at 25°C for 0.5 hours. After completion of the reaction, as monitored by LCMS, the solvent was evaporated to dryness to obtain the crude product, 3-methyl-1,2,4-thiadiazole-5-carbonyl chloride.

[0124] 2-(Aminomethyl)-3-methylpyrazine 001d (200 mg, 1.62 mmol) and triethylamine (246 mg, 2.43 mmol) were added to dichloromethane (10 mL). Crude 3-methyl-1,2,4-thiadiazole-5-carbonyl chloride dissolved in dichloromethane (5 mL) was slowly added dropwise. The reaction mixture was allowed to react at 25°C for 0.5 h. After completion of the reaction, monitored by LCMS, water (30 mL) and dichloromethane (3 × 20 mL) were added. The mixture was extracted and washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified on a silica gel column (petroleum ether / ethyl acetate = 1:1) to afford the title product, 3-methyl-N-((3-methylpyrazin-2-yl)methyl)-1,2,4-thiadiazole-5-carboxamide 001e (170 mg, yellow solid) in a 40% yield.

[0125] MS m / z(ESI):250.2[M+1] + .

[0126] 1 H NMR (400MHz, CDCl3) δ8.59 (s, 1H), 8.46 (s, 2H), 4.78 (d, J = 4.8Hz, 2H), 2.76 (s, 3H), 2.65 (s, 3H).

[0127] Step 5: Preparation of 3-methyl-5-(8-methylimidazo[1,5-a]pyrazin-3-yl)-1,2,4-thiadiazole

[0128] 3-Methyl-N-((3-methylpyrazin-2-yl)methyl)-1,2,4-thiadiazole-5-carboxamide 001e (400 mg, 1.60 mmol) was added to acetonitrile (10 mL), followed by phosphorus oxychloride (0.74 g, 4.80 mmol) and N,N-dimethylformamide (0.2 mL). The reaction mixture was incubated at 85°C under nitrogen for 48 hours. After completion of the reaction, the solvent was removed by evaporation. Saturated sodium bicarbonate solution (50 mL) and ethyl acetate (3×20 mL) were added, and the mixture was extracted and separated. The mixture was washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The mixture was purified on a silica gel column (petroleum ether / ethyl acetate = 1:1) to give the title product, 3-methyl-5-(8-methylimidazo[1,5-a]pyrazin-3-yl)-1,2,4-thiadiazole 001f (250 mg, yellow solid), in a yield of 60%.

[0129] MS m / z(ESI):232.2[M+1] + .

[0130] 1H NMR (400MHz, CDCl3) δ9.58 (d, J = 3.2Hz, 1H), 8.51 (s, 1H), 7.81 (s, 1H), 3.22 (s, 3H), 2.83 (s, 3H).

[0131] Step 6: Preparation of 7-(4-methoxybenzyl)-8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)imidazo[1,5-a]pyrazin-7-ium

[0132] 3-Methyl-5-(8-methylimidazo[1,5-a]pyrazin-3-yl)-1,2,4-thiadiazole 001f (1.0 g, 4.3 mmol) was added to acetonitrile (6 mL), followed by potassium iodide (357 mg, 2.15 mmol) and 1-(chloromethyl)-4-methoxybenzene (1.30 g, 8.60 mmol). The reaction mixture was incubated at 8°C under nitrogen for 16 hours. After completion of the reaction, as monitored by LCMS, the solvent was evaporated to dryness to afford the title crude product, 7-(4-methoxybenzyl)-8-methyl-3-(3-methyl-1,2,4-thiadiazole-5-yl)imidazo[1,5-a]pyrazin-7-ium 001g (600 mg, yellow solid), in a yield of 34%.

[0133] MS m / z(ESI):352.2[M+1] + .

[0134] Step 7: Preparation of 5-(7-(4-methoxybenzyl)-8-methyl-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazin-3-yl)-3-methyl-1,2,4-thiadiazole

[0135] 7-(4-methoxybenzyl)-8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)imidazo[1,5-a]pyrazin-7-ium 0.01 g (600 mg, 1.7 mmol) was added to ethanol (10 mL) solvent, followed by acetic acid (0.1 mL) and sodium cyanoborohydride (320 mg, 5.1 mmol). The reaction solution was heated at 0 ℃ The reaction was continued for 0.5 hours. After the reaction was completed as monitored by LCMS, the solvent was dried. Water (50 mL) and dichloromethane (3×20 mL) were added, and the extract was separated. The extract was then washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified on a silica gel column (petroleum ether / ethyl acetate = 1:1) to obtain the title product 5-(7-(4-methoxybenzyl)-8-methyl-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazin-3-yl)-3-methyl-1,2,4-thiadiazole 001h (300 mg, yellow solid) in a yield of 24%.

[0136] MS m / z(ESI):356.2[M+1] + .

[0137] Step 8: Preparation of 3-methyl-5-(8-methyl-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazin-3-yl)-1,2,4-thiadiazole

[0138] 5-(7-(4-methoxybenzyl)-8-methyl-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazin-3-yl)-3-methyl-1,2,4-thiadiazole 001h (200 mg, 0.56 mmol) was added to trifluoroacetic acid (3 mL). The reaction mixture was incubated at 100°C for 16 hours under nitrogen. After completion of the reaction, the mixture was cooled to room temperature, as monitored by LCMS. The solvent was evaporated to dryness to obtain the crude product, which was then purified using a reverse-phase column chromatography (acetonitrile / water = 1:10) to afford the title product, 3-methyl-5-(8-methyl-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazin-3-yl)-1,2,4-thiadiazole 001i (120 mg, white solid) in a yield of 72%.

[0139] MS m / z(ESI):236.2[M+1] + .

[0140] HNMR: 1 H NMR (400 MHz, DMSO- d6 )δ9.47(s,1H),7.32(s,1H),4.95-4.88(m,1H),4.70(q,J=6.4Hz,1H),4.45-4.35 (m,1H),3.86-3.79(m,1H),3.60-3.51(m,1H),2.66(s,3H),1.63(d,J=6.8Hz,3H).

[0141] Step 9: Preparation of (4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)methanone

[0142] 3-Methyl-5-(8-methyl-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazin-3-yl)-1,2,4-thiadiazole 001i (100 mg, 0.42 mmol) was dissolved in dichloromethane (4 mL). Triethylamine (64 mg, 0.63 mmol) and p-fluorobenzoyl chloride (80 mg, 0.50 mmol) were added sequentially. The reaction mixture was allowed to react at 25°C for 2 hours. After the reaction, water (20 mL) and dichloromethane (2 x 20 mL) were added. The mixture was extracted and washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The resulting residue was purified by reverse-phase chromatography (acetonitrile / water = 1:1) to afford 001 (10.20 mg, white solid) in a yield of 25%.

[0143] MS m / z(ESI):358.0[M+1] + .

[0144] 1 H NMR (400MHz, CDCl3) δ7.47 (dd, J=8.6, 5.3Hz, 2H), 7.16 (t, J=8.6Hz, 2H), 7.07 (s, 1H), 5.71 (br s,1H),5.06(dd,J=13.8,2.4Hz,1H),4.43–4.35(m,1H),4.24–4.17(m,1H),3.54(t,J=12.7Hz,1H),2.68(s,3H),1.61(d,J=6.8Hz,3H).

[0145] 1.2 Separation of Intermediate 001

[0146] Preparation of (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)methanone (002) and (S)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)methanone (003)

[0147] (4-Fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)methanone 001 (100 mg) was resolved by perp-SFC (CO2 / MeOH(0.2NH4.OH)) to give (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)methanone (002) (39.20 mg, white solid) and (S)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)methanone. (methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)methanone (003) (39.20 mg, white solid).

[0148] The data corresponding to intermediate 002 are as follows:

[0149] t R =3.62min

[0150] HNMR: 1 H NMR (400MHz, CDCl3) δ7.47 (dd, J=8.6, 5.6Hz, 2H), 7.16 (t, J=8.6Hz, 2H), 7.06 (s, 1H), 5.93-5.52 (m, 1H), 5 .06(dd,J=13.8,2.4Hz,1H),4.54-4.11(m,2H),3.54(t,J=12.4Hz,1H),2.68(s,3H),1.61(d,J=6.8Hz,3H).

[0151] The data corresponding to intermediate 003 are as follows:

[0152] t R =1.82min

[0153] MS m / z(ESI):358.0[M+1] + .

[0154] HNMR: 1 H NMR (400MHz, CDCl3) δ7.51-7.43(m,2H),7.16(t,J=8.6Hz,2H),7.06(s,1H),5.91-5.47(m,1H),5.05( dd,J=13.8,2.4Hz,1H),4.52-4.06(m,2H),3.54(t,J=12.6Hz,1H),2.67(s,3H),1.61(d,J=6.8Hz,3H).

[0155] The SFC splitting conditions are:

[0156] Chromatographic column: Daicel CHIRALPAK OZ-H 250mm*20mm ID, 5μm

[0157] Mobile phase: CO2 / MeOH (0.2% NH4·OH) = 70 / 30

[0158] Flow rate: 50g / min.

[0159] 1.3 Synthesis of the compound represented by formula (I)

[0160] Step 1: Preparation of (R)-(1-bromo-8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)(4-fluorophenyl)methanone (004)

[0161] Dissolve (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)methanone 002 (50 mg, 0.013 mmol) in dichloromethane (10 mL), then add N-bromosuccinimide (25 mg, 0.013 mmol). Stir at room temperature for 1 hour. The reaction solution was extracted with dichloromethane (10 mL), washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by reverse phase column (40% acetonitrile / water) to give (R)-(1-bromo-8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)(4-fluorophenyl)methanone 004 (16 mg, white solid), yield: 26%.

[0162] MS.m / z.(ESI):436.20[M+1] + .

[0163] 1 H NMR (400MHz, CDCl3) δ7.49-7.45(m,2H),7.19-7.17(m,2H),6.02-5.81(m,1H),5.13-4 .92(m,2H),4.25-4.13(m,1H),3.68-3.49(m,1H),2.68(s,3H),1.64(d,J=6.4Hz,3H).

[0164] HPLC: 254nm (99.76%), 214nm (99.53%)

[0165] Step 2: Preparation of (R)-1-(7-(4-fluorobenzoyl)-8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazin-1-yl)-pyrrolidin-2-one

[0166] (R)-(1-Bromo-8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)(4-fluorophenyl)methanone 004 (40 mg, 0.09 mmol) was dissolved in 1,4-dioxane (2 mL), and then pyrrolidin-2-one (100 mg, 0.19 mmol), potassium carbonate (38 mg, 0.28 mmol), cuprous iodide (1 mg, 0.005 mmol), 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl (18 mg, 0.04 mmol), and (1R,2R)-N1,N2-dimethylcyclohexane-1,2-diamine (3 mg, 0.02 mmol) were added in sequence. The reaction mixture was heated and stirred at 120°C under nitrogen for 16 hours. After completion, the reaction was quenched with water and extracted with ethyl acetate (3 × 10 mL). The organic phases were combined and washed with saturated brine (20 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50 / 50) to obtain a crude product. The crude product was then purified by reverse phase column separation (mobile phase: acetonitrile / water = 52 / 48) to obtain (R)-1-(7-(4-fluorobenzoyl)-8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazin-1-yl)-pyrrolidin-2-one (6.53 mg), the compound of formula (I), in a yield of 15%.

[0167] MS m / z(ESI):441.1[M+1] + .

[0168] HPLC: 90.94% (214nm), 97.04% (254nm).

[0169] 1 H NMR (400MHz, CDCl3) δ7.56–7.45(m,2H),7.21–7.12(m,2H),5.99(s,1H),5.11(d,J=12.8Hz,1H),5.01–4.72(m ,1H),4.28–4.12(m,2H),3.64(s,1H),3.43(s,1H),2.69(s,3H),2.52(s,2H),2.30–2.12(m,2H),1.36(s,3H).

[0170] Example 1: Preparation of the crystalline form of compound A represented by formula (I)

[0171] Antisolvent addition test

[0172] A total of 13 anti-solvent addition tests were set up using different solvents. About 15 mg of the starting sample of the compound represented by formula (I) was weighed into a 20 mL vial. After dissolving it with a certain amount of solvent, the corresponding anti-solvent was gradually added to the clear solution, and the solution was stirred dropwise (500 rpm) until solid precipitated. If no solid precipitated after adding 5 mL of anti-solvent, the test was stopped. The clear solution was transferred to 5°C / -20°C and stirred to induce solid precipitation. If it was still clear, it was transferred to room temperature for volatilization. The solid was collected and subjected to XRPD testing. The results are shown in Table 4. Free base form A was obtained in the anti-solvent addition test, and no other forms were found.

[0173] Table 4 Summary of Antisolvent Addition Test

[0174] *: No solid was obtained after adding the antisolvent. The solution remained clear after stirring at 5°C / -20°C. The solution was further evaporated at room temperature.

[0175] #: No solid was obtained after adding the anti-solvent. The solid precipitated after stirring at 5°C.

[0176] Slow evaporation test

[0177] Five slow evaporation experiments were conducted using different solvents. Approximately 15 mg of a starting sample of the compound of formula (I) was weighed into a 3 mL vial. An appropriate amount of the corresponding solvent was added to dissolve the sample. The solution was filtered through a 0.45 μm PTFE filter to obtain a clear solution. The solution was sealed with parafilm at room temperature, punctured four to five times, and allowed to evaporate naturally. The resulting solid was collected and analyzed by XRPD. The test results are shown in Table 5. The slow evaporation experiment yielded the free base crystalline form A; no other crystalline forms were observed.

[0178] Table 5 Summary of slow volatilization test

[0179] Slow cooling test

[0180] A total of 8 slow cooling tests were set up using different solvents. About 15 mg of the starting sample of the compound represented by formula (I) was weighed into a 3 mL vial, the corresponding solvent was added, and the supernatant was filtered after stirring at 50 ° C for 2 hours. The resulting filtrate was cooled from 50 ° C to 5 ° C at a rate of 0.1 ° C / min and kept at 5 ° C. If the sample is a clear solution, it was transferred to -20 ° C to induce solid precipitation. If it is still clear, it was transferred to room temperature for volatilization. The precipitated solid was collected and subjected to XRPD testing. The test results are shown in Table 6. The slow cooling test obtained free base crystal form A, and no other crystal forms were found.

[0181] Table 6 Summary of slow cooling test

[0182] *: No solid was obtained by slow cooling. After transferring to -20°C, the solution remained clear and was further transferred to room temperature for evaporation.

[0183] #: No solid was obtained by slow cooling, but solid precipitated after transfer to -20℃.

[0184] Suspension stirring (room temperature / 50℃) test

[0185] A total of 38 suspension stirring tests were set up using different temperatures and solvents. About 15 mg of the starting sample of the compound represented by formula (I) was weighed into an HPLC vial, and 1.0 mL of the solvent listed in Table 7 was added respectively. The resulting suspension was stirred at the corresponding temperature for a period of time, and then the solid was collected by centrifugation and subjected to XRPD testing. If the sample is a clear solution, transfer it to -20 ° C and stir to induce solid precipitation. If it is still clear, transfer it to room temperature for volatilization. Collect the precipitated solid and perform XRPD testing. The test results are shown in Table 7. The suspension stirring test obtained free base crystal form A, and no other crystal forms were found.

[0186] Table 7 Summary of suspension stirring test

[0187] *: Dissolve after suspension and stirring at room temperature or 50℃. Transfer to -20℃ and stir to obtain a clear solution. Further transfer to room temperature to evaporate.

[0188] Temperature cycle test

[0189] A total of 12 temperature cycle tests were set up using different solvents. About 15 mg of the starting sample of the compound represented by formula (I) was weighed into an HPLC bottle, an appropriate amount of solvent was added, and the suspension was placed in a 50°C biochemical incubator for a cyclic temperature rise and fall experiment (procedure: constant temperature at 50°C for 2 hours, then reduced to 5°C at a rate of 0.1°C / min, constant temperature at 5°C for 2 hours, and then heated to 50°C at a rate of 0.1°C / min. A total of 2 cycles were performed. Finally, all samples were kept at 5°C). The solid obtained after the cyclic temperature rise and fall program was characterized by XRPD. If the sample is a clear solution, transfer it to -20°C and stir to induce solid precipitation. If it is still clear, transfer it to room temperature for volatilization. The test results are shown in Table 8. The temperature cycle test obtained free base crystal form A, and no other crystal forms were found.

[0190] Table 8 Summary of temperature cycle test

[0191] *: Dissolve in a clear solution by stirring at a temperature of -20°C. Stir to obtain a clear solution. Then transfer to room temperature for evaporation.

[0192] Gas-solid permeability test

[0193] A total of 13 gas-solid permeation tests were performed in different solvents. Approximately 15 mg of a starting sample of the compound represented by formula (I) was weighed into a 3 mL vial. Another 20 mL vial was taken and the corresponding solvent was added thereto. The 3 mL vial was left open and placed in a 20 mL vial. After sealing, the vial was left standing at room temperature for one week. The solid was then collected and subjected to XRPD analysis. If the solid completely dissolved, it was brought to room temperature for volatilization. The test results are shown in Table 9. The free base crystalline form A was obtained in the gas-solid permeation test, and no other crystalline forms were found.

[0194] Table 9 Summary of gas-solid permeability test

[0195] *: After one week of gas-solid penetration, the product will dissolve and evaporate at room temperature.

[0196] Polymer induction test

[0197] A total of 6 polymer induction tests were set up using different solvents. About 15 mg of the starting sample of the compound represented by formula (I) was weighed into a 3 mL vial and dissolved with a certain amount of solvent to obtain a clear solution. If the solution is still not clear after adding 1.5 mL of solvent, filter the solution (0.45 μm PTFE filter membrane) to obtain a clear solution. About 2 mg of the corresponding polymer was weighed and added to the corresponding 3 mL vial. Seal with sealing film at room temperature, pierce 4 small holes and place in a fume hood for natural volatilization. Collect the solid and perform XRPD testing. The results are shown in Table 10. Free base crystal form A was obtained in the polymer induction test, and no other crystal forms were found.

[0198] Table 10 Summary of polymer induction test

[0199] Mixed polymer A: polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl chloride, polyvinyl acetate, hydroxypropyl methylcellulose and methylcellulose (mixed in equal weight);

[0200] Mixed polymer B: polycaprolactone, polyethylene glycol, polymethyl methacrylate, sodium alginate and hydroxyethyl cellulose (mixed in equal amounts).

[0201] Grinding test

[0202] A total of five grinding tests were conducted using different solvents. Approximately 15 mg of a starting sample of the compound of formula (I) was placed in a mortar. No or an appropriate amount of the corresponding solvent was added to the mortar and the solid was manually ground for approximately 5 minutes. After grinding, the resulting solid sample was subjected to XRPD analysis. The test results are shown in Table 11. All grinding tests yielded free base Form A.

[0203] Table 11 Grinding test summary

[0204] Example 2 Characterization of the Crystalline Form of Compound A Represented by Formula (I)

[0205] The XRPD pattern of the free base crystalline form A prepared in Example 1 is shown in FIG1 , and its analytical data is shown in Table 12.

[0206] Table 12 XRPD pattern analysis data of the free base crystal form A of the compound represented by formula (I)

[0207] The TGA spectrum of the free base crystal form A is shown in FIG2 , and the weight loss at 150° C. is 0.244%.

[0208] The DSC spectrum of the free base crystal form A is shown in FIG3 , where the peak temperature of the endothermic peak is 272.53° C.

[0209] Freebase Form A was subjected to a DVS test at 25°C to evaluate its hygroscopicity. The DVS results, shown in Figure 4, show that Form A exhibited approximately 0.15% water adsorption at 25°C / 80% RH, indicating that Form A exhibits virtually no hygroscopicity. XRPD results before and after the DVS test are shown in Figure 5, demonstrating that Form A exhibited no crystalline change before and after the DVS test.

[0210] The solid-state stability of the free base crystal form A was evaluated. Appropriate amounts of samples were weighed and exposed at 60°C for 1 day, and at 25°C / 60% RH and 40°C / 75% RH for 1 week and 4 weeks. The samples after exposure were characterized by XRPD and HPLC to detect changes in crystal form and chemical purity. The test results are summarized in Table 13, and the XRPD comparison diagram is shown in Figure 6. The results showed that the purity of the free base crystal form A did not change significantly (<0.2%, area %) after being exposed at 60°C for 1 day, at 25°C / 60% RH and at 40°C / 75% RH for 1 week and 4 weeks, respectively, and the crystal form did not change, indicating that the free base crystal form A has good physical and chemical stability under the current evaluation conditions.

[0211] Table 13 Stability test results of the free base crystal form A of the compound represented by formula (I)

[0212] Example 3 Biological evaluation of the compound represented by formula (I)

[0213] Determination of the activity of the compound represented by formula (I) on human NK-3 receptor

[0214] This method is used to determine the agonistic effect of the compound represented by formula (I) on the activity of human NK-3 receptor protein expressed in a human NK-3R / HEK293 stably transfected cell line.

[0215] 1. Test materials and instruments

[0216] 1.1 Culture medium

[0217] F12 (Gibco, Cat#11765-047);

[0218] FBS (Corning, Cat#35-076-CV);

[0219] Geneticin (Invitrogen, Cat#10131);

[0220] Penicillin / Streptomycin (Invitrogen, Cat#15140).

[0221] 1.2 Reagents

[0222] Fluo-4Direct(Invitrogen,Cat#F10471);

[0223] HBSS (Gibco, Cat#14025076);

[0224] HEPES (Gibco, Cat#15630080);

[0225] Bonine Serum Albumin (Sgima, Cat#B2064-100G).

[0226] 1.3 Instrument consumables

[0227] 384 well Poly-D-Lysine protein coating plate (Greiner, Cat#781946);

[0228] FLIPR (Molecular Devices);

[0229] Vi-cell XR Cell Viability Analyzer (Beckman Coulter);

[0230] Incubator (Thermo).

[0231] 2. Experimental steps

[0232] 2.1 Stably transfected human NK-3R / HEK293 cells were seeded into 384-well cell culture plates at a density of 12,000 cells / well / 25 μL and cultured overnight at 37°C in 5% CO2.

[0233] 2.2 Freeze-thaw 20X Component A to room temperature, dilute it to 2X working concentration with Assay Buffer, and store at room temperature for use;

[0234] 2.3 Equilibrate the cell culture plate at room temperature for 10 minutes, remove the culture medium, add 20 μL Assay Buffer and 20 μL 2X Component A, centrifuge at 200g for 3-5 seconds at room temperature, and incubate at 37°C for 2 hours.

[0235] 2.4 Dilute the compound three-fold in DMSO in a 384PP-DMSO plate. Then, transfer 240 nl / well of the compound to a working plate using an Echo 550 and incubate at 200g for 1 min at room temperature. Add 40 μl of Assay Buffer to the working plate and incubate at 200g for 1 min at room temperature. Then, incubate on an oscillator at 2500 rpm for 20 min, mix thoroughly, and incubate at 200g for 1 min at room temperature.

[0236] 2.5 Prepare 2.5 nM Neurokinin B TFA (6X) in Assay Buffer, transfer 50 μL to a 3657 plate and set aside;

[0237] 2.6 Remove the cell culture plate and let it stand at room temperature for 10 minutes. Add 10 μL of the compound diluted in step 2.4 to the corresponding wells and let it stand at 25°C for 30 minutes.

[0238] 2.7 Use FLIPR Tetra to add 10 μL of the compound diluted in step 2.5 to the corresponding experimental wells and collect data.

[0239] The antagonistic activity of the compound represented by formula (I) on human NK-3 receptor was determined by the above experiment, and the inhibition curve of the compound represented by formula (I) was obtained and the concentration of the compound that inhibited 50% of the reference agonist (IC 50 ), specific IC 50 See Table 14 for values.

[0240] Table 14 IC values ​​of compounds represented by formula (I) for antagonistic activity against human NK-3 receptor 50 value

[0241] Experimental conclusion:

[0242] The above data show that the compound represented by formula (I) is a potent NK-3 receptor antagonist.

[0243] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Crystal form A of the compound represented by formula (I), in, The A crystal form uses Cu-Kα radiation, and the X-ray powder diffraction expressed in 2θ angles has characteristic peaks at 10.40±0.20°, 11.79±0.20°, 19.51±0.20°, and 20.81±0.20°.

2. The crystal form A according to claim 1, wherein The A crystal form uses Cu-Kα radiation, and the X-ray powder diffraction expressed in 2θ angles has characteristic peaks at 10.40±0.20°, 11.79±0.20°, 15.92±0.20°, 16.92±0.20°, 19.51±0.20°, 20.81±0.20°, 21.19±0.20°, and 22.91±0.20°; Preferably, the A crystal form uses Cu-Kα radiation, and the X-ray powder diffraction expressed in 2θ angles also has characteristic peaks at 3.17±0.20°, 8.50±0.20°, 13.24±0.20°, 15.15±0.20°, 19.03±0.20°, 25.03±0.20°, and / or 26.95±0.20°.

3. The crystal form A according to claim 1, wherein The Form A has an XRPD pattern substantially as shown in FIG1 ; Preferably, the XRPD pattern analysis data of the A crystal form are shown in Table 1: Table 1 4. A method for preparing Form A of the compound represented by formula (I) according to any one of claims 1 to 3, wherein the preparation method comprises using the compound represented by formula (I) as a raw material and preparing it by an antisolvent method, a solvent volatilization method, a cooling precipitation method, a suspension stirring method, a temperature cycling method, a gas-solid permeation method, a polymer induction method or a grinding method.

5. The preparation method according to claim 4, wherein The preparation method of the A crystal form comprises the following steps: (a) adding the compound represented by formula (I) to a solvent to form a suspension; the solvent is selected from an organic solvent, water, or a mixed solvent of an organic solvent and water; (b) stirring the suspension at 25-60° C. for 8-120 hours; (c) After step (b), centrifugation and drying are performed to obtain the crystal form A; Preferably, the organic solvent is selected from one or more of methanol, ethanol, isopropanol, acetone, methyl isobutyl ketone, heptane, toluene, m-xylene, dichloromethane, chloroform, anisole, methyl tert-butyl ether, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, 2-methyltetrahydrofuran, 1,4-dioxane, acetonitrile, tetrahydrofuran, ethyl acetate and isopropyl acetate.

6. A pharmaceutical composition, wherein the pharmaceutical composition contains the crystal form A according to any one of claims 1 to 3.

7. The pharmaceutical composition according to claim 6, wherein The pharmaceutical composition further contains pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition further comprises one or more other active ingredients in addition to the above-mentioned crystal form A; Preferably, the dosage of the crystal form A and one or more other active ingredients is a therapeutically effective amount.

8. Use of the crystal form A according to any one of claims 1 to 3 or the pharmaceutical composition according to any one of claims 6 to 7 in the preparation of a pharmaceutical preparation.

9. The use according to claim 8, wherein The pharmaceutical preparation is used for preventing and / or treating diseases mediated by NK-3 receptors; Preferably, the drug is used for preventing and / or treating depression, anxiety, psychosis, schizophrenia, psychotic disorders, bipolar disorder, cognitive disorders, Parkinson's disease, Alzheimer's disease, attention deficit hyperactivity disorder, pain, convulsions, obesity, inflammatory diseases, vomiting, preeclampsia, airway-related diseases, reproductive disorders, contraception and sex hormone-dependent diseases and / or gynecological diseases; Alternatively, the pharmaceutical preparation is used to treat and / or prevent diseases related to menopausal syndrome, wherein the menopausal syndrome includes symptoms of hot flashes, sweating, palpitations, dizziness and / or obesity; Preferably, the pharmaceutical preparation is a NK-3 receptor antagonist.

10. A method for preventing and / or treating a disease, wherein: The method comprises administering a therapeutically effective amount of the crystal form A according to any one of claims 1 to 3 or the pharmaceutical composition according to any one of claims 6 to 7 to a patient; The disease is a disease mediated by NK-3 receptors, preferably depression, anxiety, psychosis, schizophrenia, psychotic disorders, bipolar disorder, cognitive disorders, Parkinson's disease, Alzheimer's disease, attention deficit hyperactivity disorder, pain, convulsions, obesity, inflammatory diseases, vomiting, preeclampsia, airway-related diseases, reproductive disorders, contraception and sex hormone-dependent diseases and / or gynecological diseases; Alternatively, the disease is a disease related to menopausal syndrome, and the menopausal syndrome includes symptoms of hot flashes, sweating, palpitations, dizziness and / or obesity.

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