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

Abstract

This invention discloses an A-crystal form of the NK3R antagonist shown in formula (I), its preparation method, and its applications. The A-crystal form, when subjected to Cu-Kα radiation, exhibits characteristic peaks at 10.40±0.20°, 11.79±0.20°, 19.51±0.20°, and 20.81±0.20° in X-ray powder diffraction at 2θ angles. The A-crystal form provided by this invention is stable, has low hygroscopicity, is suitable for long-term storage, is suitable for pharmaceutical preparation, and can effectively inhibit NK3R.

Description

[0001] This application claims priority to the following two earlier applications: patent application No. 202211282020.3, filed with the China National Intellectual Property Administration on October 19, 2022, entitled "A Crystal Form of an NK3R Antagonist and Its Preparation Method and Application", and patent application No. 202311315851.0, filed with the China National Intellectual Property Administration on October 11, 2023, also entitled "A Crystal Form of an NK3R Antagonist and Its Preparation Method and Application". The entire contents of the aforementioned earlier applications are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of pharmaceutical compounds, specifically relating to the crystal form of an NK3R antagonist, its preparation method, and its application. Background Technology

[0003] Tachykinin receptors are targets of a family of structure-related peptides collectively named "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. Currently, three tachykinin receptors are known: neurokinin-1 (NK-1) receptor, neurokinin-2 (NK-2) receptor, and neurokinin-3 (NK-3) receptor. Tachykinin receptors belong to the rhodopsin-like seven-transmembrane G protein-coupled receptor family. SP has the highest affinity and is considered an endogenous ligand for the NK-1 receptor; NKA is an endogenous ligand for the NK-2 receptor; and NKB is an endogenous ligand for the NK-3 receptor. NK-1, NK-2, and NK-3 receptors have been identified in different species. NK-1 and NK-2 receptors are expressed in a variety of peripheral tissues, and NK-1 receptors are also expressed in the CNS, while NK-3 receptors are mainly expressed in the CNS.

[0004] Neurokinin receptors mediate a variety of biological effects stimulated by tachykinins, including transmitting signals to excitatory neurons in the CNS and periphery (e.g., pain), regulating smooth muscle contraction, modulating immune and inflammatory responses, inducing hypotensive effects by dilating the peripheral vascular system, and stimulating the secretion of endocrine and exocrine glands.

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

[0006] Kisspeptin / neurokine 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, thus maintaining hormone levels within a reasonable range. Simultaneously, KNDy neurons are also associated with the thermoregulatory signaling pathway. By releasing NKB ligands, which bind to NK-3 receptors on the preoptic nucleus, they regulate body temperature within a certain range by inhibiting shivering and vasoconstriction, and promoting sweating and vasodilation. In postmenopausal women, the decline in estrogen levels leads to a lack of negative feedback, resulting in overactivation of KNDy neurons. This leads to the release of large amounts of endogenous NKB ligands, which bind to NK-3 receptors on the preoptic nucleus, causing sweating, vasodilation, and hot flashes. Therefore, developing antagonists targeting KNDy neurons and NK-3 receptors on the preoptic nucleus holds promise for the treatment of hot flashes.

[0007] In the central nervous system (CNS), NK-3 receptors are expressed in regions including the medial prefrontal cortex, hippocampus, thalamus, and amygdala. Furthermore, NK-3 receptors are expressed on dopaminergic neurons. Activation of NK-3 receptors has been shown to regulate the release of dopamine, acetylcholine, and serotonin, suggesting the potential therapeutic efficacy of NK-3 receptor modulators for a variety of conditions, including psychotic disorders, anxiety disorders, depression, schizophrenia, as well as obesity, pain, or inflammation.

[0008] Developing novel NK-3R antagonists and drug solid forms of such compounds suitable for pharmaceutical preparation, such as solid forms that improve stability, hygroscopicity, and / or efficacy, thereby achieving good results in the pharmaceutical and administration stages, has always been a technical problem that those skilled in the art have been committed to solving. Summary of the Invention

[0009] To solve the above-mentioned technical problems, the present invention provides a crystal form A of the compound shown in formula (I).

[0010]

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

[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 A-type crystal 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°.

[0014] In some embodiments of the present invention, the above-mentioned crystal form A has essentially the following characteristics: Figure 1 The XRPD map shown.

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

[0016] Table 1

[0017]

[0018]

[0019] In some embodiments of the present invention, the XRPD spectral analysis data of the above-mentioned A crystal form are shown in Table 2:

[0020] Table 2

[0021]

[0022]

[0023] In some embodiments of the present invention, the above-mentioned crystal form A loses 0.244% of its weight at 150±3°C.

[0024] In some embodiments of the present invention, the above-mentioned crystal form A has essentially the following characteristics: Figure 2 The TGA spectrum shown.

[0025] In some embodiments of the present invention, the above-mentioned crystal form A has a peak value of the endothermic peak at 272.53±3℃.

[0026] In some embodiments of the present invention, the above-mentioned crystal form A has essentially the following characteristics: Figure 3 The DSC spectrum shown.

[0027] The present invention also provides a method for preparing the A crystal form of the compound represented by formula (I), comprising using the compound represented by formula (I) as raw material and preparing it by antisolvent method, solvent evaporation method, cooling precipitation method, suspension stirring method, temperature cycling method, gas-solid permeation method, polymer induction method or grinding method.

[0028] Preferably, the method for preparing the A crystal form includes the following steps:

[0029] (a) The compound represented by formula (I) is added to a solvent to form a suspension; the solvent is selected from organic solvents, water, or a mixture of organic solvents and water;

[0030] (b) The suspension is stirred at 25–60°C for 8–120 hours;

[0031] (c) After step (b) is completed, centrifuge and dry (e.g., dry for 8 to 16 hours) to obtain the A crystal form.

[0032] In some embodiments of the present invention, the organic solvent may 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.

[0033] The present invention also provides a pharmaceutical composition comprising the above-described crystal form A.

[0034] According to embodiments of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable pharmaceutical excipients, such as, but not limited to, one or more of the following: excipients, fillers, lubricants, binders, disintegrants, inorganic salts, solvents, solubilizers, suspending agents, isotonic agents, buffers, preservatives, antioxidants, colorants, foaming agents, and flavoring agents.

[0035] According to embodiments of the present invention, the pharmaceutical composition may further comprise one or more other active ingredients besides the above-described crystal form A.

[0036] In the pharmaceutical composition described herein, the dosage of crystal form A and one or more other active ingredients is a therapeutically effective amount.

[0037] The present invention also provides the use of the above-described crystal form A or the pharmaceutical composition thereof in the preparation of a pharmaceutical formulation, wherein the pharmaceutical formulation is an NK-3 receptor antagonist.

[0038] According to embodiments of the present invention, the pharmaceutical preparation is used for the prevention and / or treatment of diseases mediated by NK-3 receptors; for example, for the prevention and / or treatment of depression, anxiety disorders, psychosis, schizophrenia, psychotic disorders, bipolar disorder, cognitive impairment, Parkinson's disease, Alzheimer's disease, attention deficit hyperactivity disorder (ADHD), pain, seizures, obesity, inflammatory diseases, vomiting, preeclampsia, airway-related diseases, reproductive disorders, contraceptive and sex hormone-dependent diseases and / or gynecological diseases.

[0039] According to embodiments 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 fibroids, uterine fibroids, hormone-dependent cancers, hyperandrogenemia, hirsutism, virilization, polycystic ovary syndrome (PCOS), premenstrual dysmenorrhea (PMDD), HAIR-AN syndrome (hyperandrogenemia, insulin resistance, and acanthosis nigricans), ovarian theca cell hyperplasia (HAIR-AN with luteinized theca cells in the ovarian stroma), other manifestations of high intraovarian androgen concentrations (e.g., follicular arrest, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility), androgen-producing tumors (virilizing ovarian tumors or adrenal tumors), menorrhagia, and / or adenomyosis.

[0040] According to embodiments 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.

[0041] In some embodiments, the pharmaceutical preparation is used to treat and / or prevent menopausal syndrome-related conditions, including symptoms such as hot flashes, sweating, palpitations, dizziness, and / or obesity.

[0042] According to embodiments of the present invention, the pharmaceutical preparation may 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 for injection or suppository, etc.

[0043] According to embodiments of the present invention, the pharmaceutical preparation can be administered in any of the following ways: oral, oral, sublingual, inhalation, topical application, intravenous, subcutaneous, acupoint or intramuscular injection via parenteral administration, or rectal administration.

[0044] The present invention also provides a method for preventing and / or treating a disease, comprising administering a therapeutically effective amount of the A crystal form or the pharmaceutical composition to a patient;

[0045] The disease is mediated by NK-3 receptors, preferably depression, anxiety, psychosis, schizophrenia, psychotic disorders, bipolar disorder, cognitive impairment, Parkinson's disease, Alzheimer's disease, attention deficit hyperactivity disorder, pain, seizures, obesity, inflammatory diseases, vomiting, preeclampsia, airway-related diseases, reproductive disorders, contraceptive and sex hormone-dependent diseases and / or gynecological diseases.

[0046] Alternatively, the disease may be a menopausal syndrome-related disease, which includes symptoms such as hot flashes, sweating, palpitations, dizziness, and / or obesity.

[0047] Beneficial effects

[0048] The A-type crystal provided by this invention is stable, has low hygroscopicity, is suitable for long-term storage, is suitable for pharmaceutical preparation, and can effectively inhibit NK3R.

[0049] Terminology Definitions and Explanations

[0050] Unless otherwise stated, the definitions of terms recorded in this application specification and claims, including definitions as examples, exemplary definitions, preferred definitions, and definitions of specific compounds in the embodiments, can be arbitrarily combined and combined with each other. Such combinations and combinations shall fall within the scope of this application specification.

[0051] The term "crystal form" refers to a crystal form that has the same chemical composition but different spatial arrangements of molecules and / or ions that form crystals.

[0052] The compound shown in formula (I) is the "free base", and "free base crystal form A" is the "crystal form A of the compound shown in formula (I)".

[0053] The term "therapeutic effective amount" refers to the amount of the A crystal form of the present invention, or one or more other active ingredients, sufficient to achieve the intended application (including, but not limited to, the treatment of diseases as defined below). Therapeutic effective amount can vary depending on factors such as the intended application (in vitro or in vivo), the subject being treated, and the condition of the disease, such as the subject's weight and age, the severity of the disease, and the route of administration, which can be readily determined by those skilled in the art. The specific dosage will vary depending on factors such as the specific active ingredient selected, the administration regimen, whether it is administered in combination with other compounds, the timing of administration, the tissue to which the drug is administered, and the physical delivery system used.

[0054] The term "patient" refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates, with humans being the most preferred.

[0055] The term "multiple" means two or more, such as two or more.

[0056] The term "150±3℃" represents 147 to 153℃, which can be 147℃, 148℃, 149℃, 150℃, 151℃, 152℃, 153℃ or any two of the aforementioned points.

[0057] The term "272.53±3℃" represents 269.53 to 275.53℃, which can be 269.53℃, 270.00℃, 271.00℃, 272.00℃, 275.53℃ or any two of the aforementioned points. Attached Figure Description

[0058] Figure 1 XRPD pattern of compound A crystal form shown in formula (I);

[0059] Figure 2 TGA spectrum of compound A crystal form as shown in formula (I);

[0060] Figure 3 DSC spectrum of compound A crystal form shown in formula (I);

[0061] Figure 4 DVS diagram of crystal form A of compound shown in formula (I);

[0062] Figure 5 XRPD comparison diagram of compound A crystal form shown in formula (I) before and after DVS test;

[0063] Figure 6 XRPD comparison diagram of compound A before and after the stability assessment of its crystal form, as shown in formula (I). Detailed Implementation

[0064] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory 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 covered within the scope of protection intended by the present invention.

[0065] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0066] The following are the instruments, parameters, characterization, and testing methods used in the examples:

[0067] (1) X-ray powder diffractometer (XRPD) method

[0068] Model: PANalytical X-ray Powder Diffraction Analyzer X'Pert3;

[0069] X-rays: Cu, kα 1.540598; 1.544426; Kα2 / Kα1 intensity ratio: 0.50;

[0070] X-ray tube settings: 45kV, 40mA;

[0071] Diverging slit: Fixed at 1 / 8°;

[0072] Scan mode: Continuous;

[0073] Scan range (°2Theta): 3~40;

[0074] Scan time per step (s): 46.7;

[0075] Scan step size (°2Theta): 0.0263;

[0076] Test time (minutes): 5.

[0077] (2) Thermogravimetric analysis (TGA) method

[0078] Instrument model: Discovery 5500;

[0079] Sample tray: Open or closed aluminum tray;

[0080] Starting temperature: Room temperature (below 35℃);

[0081] Termination temperature: 300℃ or stop the next stage when weight < 80% (w / w).

[0082] (The weight loss of the compound shall not exceed 20% (w / w));

[0083] Heating rate: 10℃ / min;

[0084] Nitrogen flow rate: stabilized at 10 mL / min; sample chamber flow rate: 25 mL / min;

[0085] Sample size: Approximately 2-10 mg.

[0086] (3) Differential Scanning Calorimeter (DSC) method

[0087] Instrument model: TA Discovery 2500 or Q2000;

[0088] Sample tray: Tzero tray and Tzero sealing cap with pin hole of 0.7mm diameter;

[0089] Temperature range: 30 to 300℃ or before decomposition;

[0090] Heating rate: 10℃ / min;

[0091] Nitrogen flow rate: 50 mL / min;

[0092] Sample volume: Approximately 0.5-2 mg;

[0093] (4) Dynamic Water Adsorption (DVS) Method

[0094] DVS curves were acquired via the DVS Intrinsic using SMS (Surface Measurement Systems). Relative humidity at 25°C was calibrated based on the deliquescence points of LiCl, Mg(NO3)2, and KCl.

[0095] Temperature: 25℃;

[0096] Sample size: 10–20 mg;

[0097] Protective gas and flow rate: N2, 200 mL / min;

[0098] dm / dt: 0.002% / min;

[0099] Minimum dm / dt equilibration time: 10 min;

[0100] Maximum equilibration time: 180 min;

[0101] RH range: 0%RH~95%RH~0%RH;

[0102] RH gradient: 10%RH (0%RH-90%RH&90%RH-0%RH); 5%RH (90%RH-95%RH&95%RH-90%RH)

[0103] (5) Ultra-high performance liquid chromatography (UPLC) method

[0104] The Waters H-Class ultra-high performance liquid chromatograph was used to collect solubility and purity data in the project. Specific instrument and test parameters are shown in Table 3.

[0105] Table 3

[0106]

[0107]

[0108] Patent application PCT / CN2022 / 087947 describes the compound represented by formula (I), and all contents involved in that patent application are incorporated herein by reference.

[0109] A table of Chinese and English names of solvents used in the experiment

[0110]

[0111] Preparation Example The compound shown in formula (I)

[0112] The structures of the compounds of this invention were 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 with deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), and deuterated chloroform (CDCl3) as solvents, and tetramethylsilane (TMS) as the internal standard.

[0113] Liquid chromatography-mass spectrometry (LC-MS) was performed using an Agilent 1200 Infinity Series mass spectrometer. HPLC was performed using an Agilent 1200DAD high-performance liquid chromatograph (Sunfire C18 150×4.6 mm column) and a Waters 2695-2996 high-performance liquid chromatograph (Gimini C18 150×4.6 mm column).

[0114] Thin-layer chromatography (TLC) uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The standard size for TLC is 0.15mm to 0.20mm, while the standard size for separating and purifying products using TLC is 0.4mm to 0.5mm. Column chromatography generally uses Yantai Huanghai 200-300 mesh silica gel as the carrier.

[0115] Unless otherwise specified, all reactions in this invention are carried out under continuous magnetic stirring, in a dry nitrogen or argon atmosphere, using a dry solvent, and the reaction temperature is expressed in degrees Celsius.

[0116] 1.1 Synthesis of Intermediate 001

[0117] 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) methyl ketone (001)

[0118]

[0119] Step 1: Preparation of 2-(chloromethyl)-3-methylpyrazine

[0120] 2,3-Dimethylpyrazine 001a (10 g, 92.47 mmol) was added to carbon tetrachloride (250 mL) solvent, followed by N-chlorosuccinimide (14.83 g, 110.96 mmol) and benzoyl peroxide (224 mg, 9.25 mmol). The reaction mixture was reacted at 80 °C for 16 hours under nitrogen protection. After the reaction was completed by LCMS monitoring, the solvent was evaporated to dryness and water (150 mL) and dichloromethane (3 × 100 mL) were added. The mixture was extracted and separated, washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1) to obtain the title product 2-(chloromethyl)-3-methylpyrazine 001b (3.20 g, colorless oil), yield: 22%.

[0121] MS m / z(ESI): 143.2 [M+1] + .

[0122] 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).

[0123] The second step involves preparing 2-((3-methylpyrazin-2-yl)methyl)isoindololin-1,3-dione.

[0124] 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 mixture was reacted at 110 °C for 8 hours under nitrogen protection. After the reaction was completed by LCMS monitoring, the solvent was evaporated to dryness, and water and ethyl acetate were added. The mixture was extracted and separated, washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was purified by silica gel column chromatography with petroleum ether / ethyl acetate ratio of 1:1 to obtain the title product 2-((3-methylpyrazine-2-yl)methyl)isoindoline-1,3-dione 001c 3.10 g, a pale yellow solid, yield: 95%.

[0125] MS m / z(ESI): 254.2[M+1]+.

[0126] 1 H 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).

[0127] Step 3: Preparation of 2-(aminomethyl)-3-methylpyrazine

[0128] 2-((3-methylpyrazine-2-yl)methyl)isoindoline-1,3-dione 001c (2.00 g, 7.90 mmol) was added to ethanol (50 mL) solvent, followed by hydrazine hydrate (3.95 g, 79 mmol). The reaction mixture was reacted at 80 °C for 6 hours under nitrogen protection. After the reaction was completed as monitored by LCMS, the solvent was evaporated to dryness and water (150 mL) was added. The mixture was then extracted with dichloromethane / methanol (1 / 1, V / V, 50 mL), and 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 give crude product 2-(aminomethyl)-3-methylpyrazine 001d (300 mg, yellow oil), yield: 28%.

[0129] MS m / z (ESI): 124.3 [M+1] + .

[0130] 1 H NMR (400MHz, CDCl3) δ8.36 (s, 1H), 8.33 (d, J = 2.4Hz, 1H), 4.02 (s, 2H), 2.54 (s, 3H).

[0131] Step 4: Preparation of 3-methyl-N-((3-methylpyrazin-2-yl)methyl)-1,2,4-thiadiazole-5-carboxamide

[0132] 280 mg (1.94 mmol) of 3-methyl-1,2,4-thiadiazole-5-carboxylic acid was added to 10 mL of dichloromethane solvent, followed by the addition of 0.5 mL of oxaloyl chloride and 0.1 mL of N,N-dimethylformamide. The reaction mixture was reacted at 25 °C for 0.5 h. After the reaction was completed as monitored by LCMS, the solvent was evaporated to dryness to obtain the crude product 3-methyl-1,2,4-thiadiazole-5-carbonyl chloride.

[0133] 2-(aminomethyl)-3-methylpyrazine 001d (200 mg, 1.62 mmol) and triethylamine (246 mg, 2.43 mmol) were added to dichloromethane (10 mL) solvent. The crude product 3-methyl-1,2,4-thiadiazole-5-carbonyl chloride, dissolved in dichloromethane (5 mL), was then slowly added dropwise. The reaction mixture was reacted at 25 °C for 0.5 h. After the reaction was completed as monitored by LCMS, water (30 mL) and dichloromethane (3 × 20 mL) were added, followed by extraction and separation. The extract was washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purification was performed using a silica gel column (petroleum ether / ethyl acetate = 1:1) to obtain the title product 3-methyl-N-((3-methylpyrazine-2-yl)methyl)-1,2,4-thiadiazole-5-carboxamide 001e (170 mg, yellow solid), yield: 40%.

[0134] MS m / z(ESI): 250.2 [M+1] + .

[0135] 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).

[0136] Step 5: Preparation of 3-methyl-5-(8-methylimidazo[1,5-a]pyrazin-3-yl)-1,2,4-thiadiazole

[0137] 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 reacted at 85 °C for 48 hours under nitrogen protection. After the reaction was completed, the solvent was evaporated to dryness using LCMS. Add saturated sodium bicarbonate solution (50 mL) and ethyl acetate (3 × 20 mL), extract and separate the contents, wash with saturated sodium chloride solution (50 mL), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify by silica gel column chromatography (petroleum ether / ethyl acetate = 1:1) to obtain the title product 3-methyl-5-(8-methylimidazo[1,5-a]pyrazin-3-yl)-1,2,4-thiadiazole 001f (250 mg, yellow solid), yield: 60%.

[0138] MS m / z(ESI): 232.2 [M+1] + .

[0139] 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).

[0140] Step 6: Preparation of 7-(4-methoxybenzyl)-8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)imidazo[1,5-a]pyrazine-7-onium

[0141] 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-methoxybenzyl (1.30 g, 8.60 mmol). The reaction mixture was reacted at 8 °C for 16 hours under nitrogen protection. After the reaction was completed as monitored by LCMS, the solvent was evaporated to dryness to give the crude product 7-(4-methoxybenzyl)-8-methyl-3-(3-methyl-1,2,4-thiadiazole-5-yl)imidazo[1,5-a]pyrazin-7-onium 001g (600 mg, yellow solid), yield: 34%.

[0142] MS m / z(ESI): 352.2 [M+1] + .

[0143] 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

[0144] 0.01 g (600 mg, 1.7 mmol) of 7-(4-methoxybenzyl)-8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)imidazo[1,5-a]pyrazine-7-onium was added to 10 mL of ethanol, followed by the addition of 0.1 mL of acetic acid and 320 mg (5.1 mmol) of sodium cyanoborohydride. The reaction mixture was incubated under nitrogen protection at 0°C. ℃ The reaction was carried out for 0.5 hours. After the reaction was completed by LCMS monitoring, the solvent was evaporated to dryness. Water (50 mL) and dichloromethane (3 × 20 mL) were added, the mixture was extracted and separated, washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:1) to give 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), yield: 24%.

[0145] MS m / z(ESI): 356.2 [M+1] + .

[0146] Step 8: Preparation of 3-methyl-5-(8-methyl-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazin-3-yl)-1,2,4-thiadiazole

[0147] 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) solvent. The reaction mixture was reacted at 100 °C for 16 h under nitrogen protection. After the reaction was completed as monitored by LCMS, the reaction mixture was cooled to room temperature. The solvent was evaporated to dryness to obtain the crude product, which was purified by reverse column chromatography (acetonitrile / water = 1:10) to give 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), yield: 72%.

[0148] MS m / z(ESI): 236.2 [M+1] + .

[0149] HNMR: 1 H NMR (400MHz, 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).

[0150] 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) methyl ketone

[0151] 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), followed by the addition of triethylamine (64 mg, 0.63 mmol) and p-fluorobenzoyl chloride (80 mg, 0.50 mmol). The reaction mixture was reacted at 25 °C for 2 hours. After the reaction was complete, water (20 mL) and dichloromethane (2 × 20 mL) were added, and the mixture was extracted and separated. The extract was washed with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by reversed-phase chromatography (acetonitrile / water = 1:1) to obtain 001 (10.20 mg, white solid), yield: 25%.

[0152] MS m / z(ESI): 358.0 [M+1] + .

[0153] 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).

[0154] 1.2 Splitting of Intermediate 001

[0155] 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) methyl ketone (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) methyl ketone (003)

[0156] (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl) methyl ketone 001 (100 mg) was resolved by perp-SFC (CO2 / MeOH(0.2NH4.OH)) to obtain (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl) methyl ketone 001 (100 mg). (S)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl) ketone (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) ketone (003) (39.20 mg, white solid).

[0157] The data corresponding to intermediate 002 is as follows:

[0158] t R =3.62min

[0159] 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).

[0160] The data corresponding to intermediate 003 is as follows:

[0161] t R =1.82min

[0162] MS m / z(ESI): 358.0 [M+1] + .

[0163] 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).

[0164] The conditions for splitting SFC are:

[0165] Column: Daicel CHIRALPAK OZ-H 250mm*20mm ID, 5μm

[0166] Mobile phase: CO2 / MeOH (0.2% NH4·OH) = 70 / 30

[0167] Flow rate: 50 g / min.

[0168] 1.3 Synthesis of the compound shown in formula (I)

[0169]

[0170] 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) methyl ketone (004)

[0171] (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl) methyl ketone 002 (50 mg, 0.013 mmol) was dissolved in dichloromethane (10 mL), and then N-bromosuccinimide (25 mg, 0.013 mmol) was added. The mixture was stirred 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 residue was purified by reverse-phase column chromatography (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) methyl ketone 004 (16 mg, white solid), yield: 26%.

[0172] MS.m / z.(ESI):436.20[M+1] + .

[0173] 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).

[0174] HPLC: 254nm (99.76%), 214nm (99.53%)

[0175] 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)-pyrrolidine-2-one

[0176] (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) methyl ketone 004 (40 mg, 0.09 mmol) was dissolved in 1,4-dioxane (2 mL), followed by the addition of pyrrolidone-2-one (100 mg, 0.19 mmol), potassium carbonate (38 mg, 0.28 mmol), cuprous iodide (1 mg, 0.005 mmol), 2-dicyclohexylphospho-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). The reaction was carried out under nitrogen protection and heated at 120°C with stirring for 16 hours. After the reaction was completed, water was added to quench the reaction, and the mixture was 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 residue was subjected to silica gel column chromatography (petroleum ether / ethyl acetate = 50 / 50) to obtain a crude product. The crude product was purified by reverse column chromatography (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)-pyrrolidine-2-one (6.53 mg), i.e., the compound shown in formula (I), yield: 15%.

[0177] MS m / z(ESI): 441.1 [M+1] + .

[0178] HPLC: 90.94% (214nm), 97.04% (254nm).

[0179] 1H 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).

[0180] Example 1: Preparation of the crystal form of compound A shown in formula (I)

[0181] Antisolvent addition test

[0182] Thirteen antisolvent addition experiments were conducted using different solvents. Approximately 15 mg of the starting sample of the compound shown in formula (I) was weighed into a 20 mL vial, dissolved in a certain amount of solvent, and then the corresponding antisolvent was gradually added to the clear solution while stirring (500 rpm) until solid precipitated. If no solid precipitated after adding 5 mL of antisolvent, the experiment was stopped. The clear solution was then heated to 5℃ / -20℃ and stirred to induce solid precipitation. If the solution remained clear, it was allowed to evaporate at room temperature. The solid was collected and XRPD was performed. The results are shown in Table 4. Free basal crystal form A was obtained in the antisolvent addition experiments; no other crystal forms were found.

[0183] Table 4 Summary of Antisolvent Addition Experiments

[0184]

[0185]

[0186] *: The addition of antisolvent did not yield a solid. After stirring at 5℃ / -20℃, the mixture remained clear and was further transferred to room temperature for evaporation.

[0187] #: No solid was obtained after adding antisolvent. After being transferred to 5°C and stirred, solid precipitated.

[0188] Slow evaporation test

[0189] Five slow evaporation tests were conducted using different solvents. Approximately 15 mg of the starting sample of the compound shown in formula (I) was weighed into a 3 mL vial, and an appropriate amount of the corresponding solvent was added to dissolve the sample. The solution was filtered (using a 0.45 μm PTFE membrane) to obtain a clear solution, which was then sealed at room temperature with a sealing film. After puncturing 4–5 small holes, the solution was allowed to evaporate naturally. The resulting solid was collected and subjected to XRPD testing. The results are shown in Table 5. The slow evaporation test yielded a free alkali crystalline form A; no other crystalline forms were observed.

[0190] Table 5 Summary of Slow Evaporation Test

[0191]

[0192] Slow cooling test

[0193] Eight slow cooling experiments were conducted using different solvents. Approximately 15 mg of the starting sample of the compound shown in formula (I) was weighed into a 3 mL vial, the corresponding solvent was added, and the mixture was stirred at 50 °C for 2 hours. The supernatant was then filtered, and the resulting filtrate was cooled from 50 °C to 5 °C at a rate of 0.1 °C / min and held at 5 °C. If the sample was a clear solution, it was transferred to -20 °C to induce solid precipitation; if it remained clear, it was transferred to room temperature for evaporation. The precipitated solids were collected and subjected to XRPD testing. The experimental results are shown in Table 6. The slow cooling experiment yielded a free alkali crystalline form A; no other crystalline forms were observed.

[0194] Table 6 Summary of the Slow Cooling Experiment

[0195]

[0196]

[0197] *: Slow cooling did not yield a solid. After being transferred to -20°C, it remained a clear solution. Further transfer to room temperature resulted in evaporation.

[0198] #: Slow cooling did not yield a solid, but a solid precipitated after being transferred to -20℃.

[0199] Suspension stirring (room temperature / 50℃) test

[0200] Thirty-eight suspension-stirring experiments were conducted using different temperatures and solvents. Approximately 15 mg of the starting sample of the compound shown in formula (I) was weighed into an HPLC vial, and 1.0 mL of each of the solvents listed in Table 7 was added. The resulting suspensions were stirred at the corresponding temperatures for a period of time, then centrifuged to collect the solids and perform XRPD analysis. If the sample was a clear solution, it was transferred to -20°C and stirred to induce solid precipitation; if it remained clear, it was transferred to room temperature for evaporation. The precipitated solids were collected and XRPD analyzed. The experimental results are shown in Table 7. The suspension-stirring experiments yielded a free basal crystal form A; no other crystal forms were observed.

[0201] Table 7 Summary of Suspension and Stirring Tests

[0202]

[0203]

[0204] *: After suspension and stirring at room temperature or 50°C, the solution is clear. After stirring at -20°C, it remains a clear solution. Further transfer to room temperature for evaporation.

[0205] Temperature cycling test

[0206] Twelve temperature cycling experiments were conducted using different solvents. Approximately 15 mg of the starting sample of the compound shown in formula (I) was weighed into an HPLC vial, and an appropriate amount of solvent was added. The suspension was placed in a 50°C biochemical incubator for cyclic heating and cooling experiments (program: 50°C for 2 hours, decreasing to 5°C at a rate of 0.1°C / min, remaining at 5°C for 2 hours, then increasing to 50°C at a rate of 0.1°C / min. This was repeated twice. Finally, all samples were maintained at 5°C). The solid obtained after the cyclic heating and cooling program was characterized by XRPD. If the sample was a clear solution, it was transferred to -20°C and stirred to induce solid precipitation; if it remained clear, it was transferred to room temperature for evaporation. The experimental results are shown in Table 8. The temperature cycling experiment yielded a free alkali crystal form A; no other crystal forms were found.

[0207] Table 8 Summary of Temperature Cycling Tests

[0208]

[0209] *: After circulating heating and cooling with stirring, the solution is clear. After being transferred to -20℃ and stirred, it remains a clear solution. It is then transferred to room temperature to evaporate.

[0210] Gas-solid permeation test

[0211] Thirteen gas-solid permeation tests were conducted using different solvents. Approximately 15 mg of the starting sample of the compound shown in formula (I) was weighed into a 3 mL vial. A separate 20 mL vial was taken and the corresponding solvent was added. The 3 mL vial was placed open inside the 20 mL vial, sealed, and allowed to stand at room temperature for one week. The solid was then collected and subjected to XRPD testing. If the solid completely dissolved, it was allowed to evaporate at room temperature. The results are shown in Table 9. Free alkali crystal form A was obtained in the gas-solid permeation tests; no other crystal forms were observed.

[0212] Table 9 Summary of Gas-Solid Permeation Test

[0213]

[0214]

[0215] *: After one week of gas-solid permeation, the solution is cleared and transferred to room temperature for volatilization.

[0216] Polymer-induced test

[0217] Six polymer-induced experiments were conducted using different solvents. Approximately 15 mg of the starting sample of the compound shown in formula (I) was weighed into a 3 mL vial and dissolved in a certain amount of solvent to obtain a clear solution. If the solution remained unclear after adding 1.5 mL of solvent, it was filtered (using a 0.45 μm PTFE membrane) to obtain a clear solution. Approximately 2 mg of the corresponding polymer was weighed and added to the corresponding 3 mL vial. The vials were sealed at room temperature with sealing film, four small holes were punctured, and the vials were placed in a fume hood for natural evaporation. The solids were collected and XRPD tests were performed. The results are shown in Table 10. Free alkali crystal form A was obtained in the polymer-induced experiments; no other crystal forms were found.

[0218] Table 10 Summary of Polymer Induction Tests

[0219]

[0220] Mixed polymer A: polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl chloride, polyvinyl acetate, hydroxypropyl methylcellulose and methylcellulose (mixed in equal masses);

[0221] Mixed polymer B: polycaprolactone, polyethylene glycol, polymethyl methacrylate, sodium alginate, and hydroxyethyl cellulose (mixed in equal masses).

[0222] Grinding test

[0223] Five grinding experiments were conducted using different solvents. Approximately 15 mg of the starting sample of the compound represented by formula (I) was placed in a mortar, and the solid was manually ground (for approximately 5 minutes) with or without the appropriate amount of the corresponding solvent added. After grinding, the obtained solid samples were subjected to XRPD testing. The experimental results are shown in Table 11. All grinding experiments yielded free alkali crystal form A.

[0224] Table 11 Summary of Grinding Test

[0225]

[0226]

[0227] Example 2 Characterization of the crystal form of compound A shown in formula (I)

[0228] The XRPD pattern of the free alkali crystal form A obtained in Example 1 is as follows: Figure 1 As shown, the analytical data is shown in Table 12.

[0229] Table 12 shows the XRPD spectrum analysis data of the free alkali crystal form A of the compound shown in formula (I).

[0230]

[0231]

[0232] The TGA spectrum of free alkali crystal form A is as follows: Figure 2 As shown, the weight loss reached 0.244% at 150℃.

[0233] The DSC spectrum of free alkali crystal form A is as follows: Figure 3 As shown, the peak temperature of the endothermic peak is 272.53℃.

[0234] The hygroscopic properties of free alkali crystal form A were evaluated by DVS testing under a constant temperature of 25°C. The DVS results are as follows: Figure 4 As shown, the water adsorption of free alkali crystalline form A at 25℃ / 80% RH is approximately 0.15%, indicating that free alkali crystalline form A has almost no hygroscopicity. The XRPD results before and after the DVS test are shown below. Figure 5 As shown, this indicates that the free alkali crystal form A did not undergo any crystal form change before and after the DVS test.

[0235] The solid-state stability of the free alkali crystal form A was evaluated. Appropriate amounts of sample were weighed and left exposed at 60°C for 1 day, and at 25°C / 60%RH and 40°C / 75%RH for 1 week and 4 weeks, respectively. The samples were characterized by XRPD and HPLC after these exposures to detect changes in crystal form and chemical purity. The test results are summarized in Table 13, and the XRPD comparison chart is shown below. Figure 6 The results showed that the purity of free alkali crystal form A did not change significantly (<0.2%, area%) after being left exposed at 60℃ for 1 day, and after being left exposed at 25℃ / 60%RH for 1 week and 4 weeks at 40℃ / 75%RH, respectively. The crystal form also remained unchanged, indicating that free alkali crystal form A has good physicochemical stability under the current evaluation conditions.

[0236] Table 13 shows the stability test results of the free alkali crystal form A of the compound represented by formula (I).

[0237]

[0238] Example 3 Biological evaluation of the compound shown in formula (I)

[0239] Determination of the activity of the compound represented by formula (I) against human NK-3 receptor

[0240] This method was used to determine the agonistic effect of the compound shown in formula (I) on the activity of human NK-3 receptor protein expressed in human NK-3R / HEK293 stable cell line.

[0241] 1. Experimental materials and instruments

[0242] 1.1 Culture medium

[0243] F12 (Gibco, Cat#11765-047);

[0244] FBS (Corning, Cat#35-076-CV);

[0245] Geneticin (Invitrogen, Cat#10131);

[0246] Penicillin / Streptomycin (Invitrogen, Cat#15140).

[0247] 1.2 Reagents

[0248] Fluo-4Direct(Invitrogen,Cat#F10471);

[0249] HBSS(Gibco,Cat#14025076);

[0250] HEPES (Gibco, Cat#15630080);

[0251] Bonine Serum Albumin (Sgima, Cat#B2064-100G).

[0252] 1.3 Instrument Consumables

[0253] 384 well Poly-D-Lysine protein coating plate (Greiner, Cat#781946);

[0254] FLIPR (Molecular Devices);

[0255] Vi-cell XR Cell Viability Analyzer (Beckman Coulter);

[0256] Incubator (Thermo).

[0257] 2. Experimental Procedure

[0258] 2.1 Human NK-3R / HEK293 stable cell line was seeded in 384-well cell culture plates at a seeding density of 12,000 cells / well / 25 μL and cultured overnight at 37°C with 5% CO2.

[0259] 2.2 Freeze-thaw 20X Component A to room temperature, dilute it to 2X working concentration with Assay Buffer, and let it stand at room temperature for later use;

[0260] 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;

[0261] 2.4 The compound was 3-fold diluted in a 384PP_DMSO plate using DMSO. Then, 240 nl / well of the compound was transferred to the working plate using an Echo 550, 200 g, room temperature, 1 min. 40 μl of Assay Buffer was added to the working plate, 200 g, room temperature, 1 min. After mixing, the mixture was oscillated at 2500 rpm for 20 min. After mixing, it was 200 g, room temperature, 1 min and ready for use.

[0262] 2.5 Prepare 2.5 nM Neurokinin B TFA (6X) using Assay Buffer, and transfer 50 μL to a 3657 plate for later use;

[0263] 2.6 Remove the cell culture plate and let it stand at room temperature for 10 min. Add 10 μL of the diluted compound from step 2.4 to the corresponding well and let it stand at 25°C for 30 min.

[0264] 2.7 Using FLIPR Tetra, add 10 μL of the diluted compound from step 2.5 to the corresponding well and collect data.

[0265] The antagonistic activity of the compound represented by formula (I) against the human NK-3 receptor was determined through the above experiments. The inhibition curve of the compound represented by formula (I) was obtained, and the concentration (IC50) of the corresponding compound that inhibited the reference agonist by 50% was determined. 50 ), specific IC 50 The values ​​are shown in Table 14.

[0266] Table 14 shows the IC50 values ​​of the compounds represented by formula (I) for their antagonistic activity against human NK-3 receptors. 50 value

[0267]

[0268] Experimental conclusion:

[0269] The data above show that the compound represented by formula (I) is a potent NK-3 receptor antagonist.

[0270] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The A-crystal form of the compound shown in formula (I), in, The A crystal form, when subjected to Cu-Kα radiation, exhibits characteristic peaks in X-ray powder diffraction at 10.40±0.20°, 11.79±0.20°, 19.51±0.20°, and 20.81±0.20° when expressed in 2θ angles.

2. The A-type crystal according to claim 1, wherein, The A crystal form, when subjected to Cu-Kα radiation, exhibits characteristic peaks in X-ray powder diffraction 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°, as expressed in 2θ angles.

3. The A-type crystal according to claim 2, wherein, The A crystal form, when subjected to Cu-Kα radiation, exhibits characteristic peaks in X-ray powder diffraction 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°, as indicated by the 2θ angle.

4. The A-type crystal according to claim 1, wherein, The A-type crystal has a basic XRPD pattern as shown in Figure 1.

5. The A-type crystal according to claim 1, wherein, The XRPD spectra analysis data of the A crystal form are shown in Table 1: Table 1 6. A method for preparing the A crystal form of the compound represented by formula (I) according to any one of claims 1-5, wherein, The method for preparing the A crystal form includes the following steps: (a) The compound represented by formula (I) is added to a solvent to form a suspension; the solvent is selected from organic solvents, water, or a mixture of organic solvents and water; (b) The suspension is stirred at 25–60°C for 8–120 hours; (c) After step (b) is completed, 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 to evaporate, then centrifuge and dry to obtain the A crystal form. 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.

7. A pharmaceutical composition, wherein the pharmaceutical composition comprises the A crystal form as described in any one of claims 1-5.

8. The pharmaceutical composition according to claim 7, wherein, The pharmaceutical composition also contains pharmaceutically acceptable pharmaceutical excipients.

9. The pharmaceutical composition according to claim 7 or 8, wherein, The pharmaceutical composition also contains one or more other active ingredients besides the A crystal form described above.

10. The pharmaceutical composition according to claim 9, wherein, The dosage of crystal form A and one or more other active ingredients is a therapeutically effective amount.

11. Use of the A crystal form according to any one of claims 1-5 or the pharmaceutical composition according to any one of claims 7-10 in the preparation of pharmaceutical formulations for the prevention and / or treatment of diseases mediated by NK-3 receptors.

12. The use according to claim 11, wherein, The diseases mentioned are selected from anxiety disorders, psychosis, cognitive impairment, attention deficit hyperactivity disorder, pain, seizures, obesity, inflammatory diseases, vomiting, preeclampsia, airway-related diseases, reproductive disorders, contraceptive and sex hormone-dependent diseases and / or gynecological diseases.

13. The use according to claim 11, wherein, The disease is a menopausal syndrome-related disease, which includes symptoms such as hot flashes, sweating, palpitations, dizziness, and / or obesity.

14. The use according to claim 11, wherein, The disease is selected from depression, schizophrenia, psychotic disorders, bipolar disorder, Parkinson's disease, or Alzheimer's disease.

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