An oxazaspiro compound, its salt form and its crystal form

Abstract

This invention relates to an oxazaspirocyclic compound, its salt form, and its crystal form. Specifically, it relates to Compound III and its pharmaceutically acceptable salt, its crystal form, and its preparation method, as well as its use as an LSD1 inhibitor in the treatment of hematologic malignancies, small cell lung cancer, squamous non-small cell lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, glioma, or Ewing's sarcoma. #imgabs0#

Description

[0001] This application claims priority to:

[0002] CN202110179260.X, application date February 9, 2021. Technical Field

[0003] The present invention relates to an oxazaspiro compound, its salt form and crystal form and a preparation method, and particularly to compound III and its pharmaceutically acceptable salt, and the crystal form of its salt form. Background Art

[0004] The methylation status of histones is regulated by both histone methyltransferases and histone demethylases. Lysine specific demethylase 1 (LSD1, also known as KDM1A) was the first reported histone lysine demethylase. By regulating the methylation status of histone lysine, it is widely involved in transcriptional regulation, influencing numerous physiological processes, including cell proliferation and differentiation, and embryonic stem cell pluripotency. [Yujiang Shi, Fei Lan, Caitlin Matson et al., Cell, 2004, 941–953] [Daniel P. Mould, Alison E. McGonagle, Daniel H. Wiseman et al., Medicinal Research Reviews, 2015, 35, 586–618]. The LSD1 structure consists of three main components: an N-terminal SWIRM domain, a C-terminal amino oxidase domain (AOL), and a central Tower domain. [Ruchi Anand, Ronen Marmorstein, Journal of Biological Chemistry, 2007, 35425–35429]. The C-terminal amino oxidase domain contains two active pockets: one for FAD binding and the other for substrate recognition and binding [Pete Stavropoulos, Günter Blobel, André Hoelz, Nature Structural & Molecular Biology, 2006, 626–632]. The function of the SWIRM domain has not yet been clearly determined; it does not directly bind FAD or substrates. However, mutation or deletion of this region reduces LSD1 activity, leading to speculation that this region may affect the active domain by adjusting its conformation [Yong Chen, Yuting Yang, Feng Wang et al., Biochemistry, 2006, 13956–13961]. The Tower domain is the binding site for LSD1 and other protein factors. LSD1 binds to different protein factors and acts on different substrates, thereby exerting different regulatory effects on histones and gene expression. Furthermore, LSD1 also regulates the methylation status of some non-histone substrates, including the tumor suppressor gene p53 and DNA methyltransferase 1 (DNMT1) [Yi Chao Zheng, Jinlian Ma, Zhiru Wang, Medicinal Research Reviews, 2015, 1032–1071].

[0005] LSD1 is a FAD-dependent amino oxidase, with proton transfer being the most likely mechanism of its oxidation [Zheng YC, Yu B, Chen ZS, et al. Epigenomics, 2016, 8, 651-666]. First, through proton transfer, the N-CH3 bond of the substrate is converted into an imine bond. This imine ion intermediate undergoes hydrolysis, generating a demethylated amine on one side and formaldehyde on the other. During this catalytic cycle, FAD is reduced to FADH2, which is then oxidized back to FAD by a molecule of oxygen, simultaneously generating a molecule of H2O2 [Yujiang Shi, Fei Lan, Caitlin Matson, Cell, 2004, 941–953].

[0006] LSD1 is abnormally expressed in many different types of tumors. LSD1 is highly expressed in acute myeloid leukemia (AML) subtypes and is an important factor in maintaining the potential of leukemia stem cells (LSCs). LSD1 is highly expressed in a variety of solid tumors such as lung cancer, breast cancer, prostate cancer, liver cancer, and pancreatic cancer, and is closely related to poor tumor prognosis. LSD1 inhibits the expression of cadherin and is closely related to tumor invasion and epithelial-mesenchymal transition (EMT) [Hosseini A, Minucci S. Epigenomics, 2017, 9, 1123-1142.].

[0007] While no LSD1 inhibitors have been approved for marketing, eight are currently in clinical development, primarily for the treatment of hematologic malignancies, small cell lung cancer, and Ewing's sarcoma. However, facing a significant unmet medical need, this field still requires candidate compounds with improved activity and pharmacokinetic parameters to advance into clinical trials and address these therapeutic needs. Summary of the Invention

[0008] The present invention provides compound III:

[0009]

[0010] The present invention provides compound IV':

[0011]

[0012] Wherein, n is 0-2, preferably 1.5-2.

[0013] In some embodiments of the present invention, the compound IV' has the structure of compound IV:

[0014]

[0015] The present invention provides a crystalline form A of compound IV, whose X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at the following 2θ angles: 7.42±0.20°, 16.90±0.20° and 19.99±0.20°;

[0016]

[0017] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.42±0.20°, 8.46±0.20°, 15.88±0.20°, 16.90±0.20°, 17.80±0.20°, 18.77±0.20°, 19.99±0.20° and 22.65±0.20°.

[0018] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.42±0.20°, 8.46±0.20°, 15.88±0.20°, 16.90±0.20°, 17.80±0.20°, 18.27±0.20°, 18.77±0.20°, 19.99±0.20°, 21.98±0.20° and 22.65±0.20°.

[0019] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.42±0.20°, 8.46±0.20°, 10.79±0.20°, 11.69±0.20°, 13.66±0.20°, 14.78±0.20°, 15.88±0.20°, 16.40±0.20°, 16.90±0.20° , 17.80±0.20°, 18.27±0.20°, 18.77±0.20°, 19.57±0.20°, 19.99±0.20°, 21.23±0.20°, 21.64±0.20°, 21.98±0.20°, 22.30±0.20°, 22.65±0.20°, 23.15±0.20° and 23.44±0.20°.

[0020] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.42°, 8.46°, 10.79°, 11.69°, 13.66°, 14.78°, 15.88°, 16.40°, 16.90°, 17.80°, 18.27°, 18.77°, 19.57°, 19.99°, 21.23°, 21.64°, 21.98°, 22.30°, 22.65°, 23.15° and 23.44°.

[0021] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.42±0.20°, 19.99±0.20°, and / or 16.90±0.20°, and / or 8.46±0.20°, and / or 10.79±0.20°, and / or 11.69±0.20°, and / or 13.66±0.20°, and / or 14.78±0.20°, and / or 15.88±0.20°, and / or 16. 40±0.20°, and / or 17.80±0.20°, and / or 18.27±0.20°, and / or 18.77±0.20°, and / or 19.57±0.20°, and / or 21.23±0.20°, and / or 21.64±0.20°, and / or 21.98±0.20°, and / or 22.30±0.20°, and / or 22.65±0.20°, and / or 23.15±0.20°, and / or 23.44±0.20°.

[0022] In some embodiments of the present invention, the above-mentioned crystal form A has an XRPD pattern substantially as follows Figure 1 shown.

[0023] In some embodiments of the present invention, the XRPD of the above-mentioned crystal form A uses Cu-Kα radiation.

[0024] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form A is shown in Table 1.

[0025] Table 1 XRPD analysis data of Form A of Compound IV

[0026]

[0027] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned crystal form A has an endothermic peak starting point at 215.4±5°C.

[0028] In some embodiments of the present invention, the DSC analysis method of the above-mentioned crystal form A is as follows: the scanning rate is 10°C / min, and the temperature range is 25-350°C.

[0029] In some embodiments of the present invention, the above-mentioned crystal form A has a DSC spectrum as shown below: Figure 2 shown.

[0030] In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned crystal form A shows a weight loss of 0.89% at 180±5°C.

[0031] In some embodiments of the present invention, the TGA analysis method of the above-mentioned crystal form A is as follows: the scanning rate is 10°C / min, and the temperature range is room temperature-350°C.

[0032] In some embodiments of the present invention, the TGA spectrum of the above-mentioned A crystal form is as follows Figure 3 The present invention provides compound V:

[0033]

[0034] The present invention provides a crystalline form B of compound V, whose X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at the following 2θ angles: 16.33±0.20°, 18.98±0.20° and 22.27±0.20°;

[0035]

[0036] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 7.08±0.20°, 10.51±0.20°, 14.54±0.20°, 16.33±0.20°, 17.37±0.20°, 17.91±0.20°, 18.98±0.20° and 22.27±0.20°.

[0037] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 7.08±0.20°, 10.51±0.20°, 14.54±0.20°, 16.33±0.20°, 17.37±0.20°, 17.91±0.20°, 18.98±0.20°, 19.56±0.20°, 21.30±0.20°, 22.27±0.20°, 23.58±0.20° and 26.66±0.20°.

[0038] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 7.08±0.20°, 7.40±0.20°, 10.51±0.20°, 12.27±0.20°, 12.47±0.20°, 14.15±0.20°, 14.54±0.20°, 16.33±0.20°, 17.37±0.20°, 17.91±0.20°, 18.60±0.20°, 18.98±0.20°, 19.20±0.20°, 19.56±0.20°, 20.13±0.20°, 21.06±0.20° , 21.30±0.20°, 22.27±0.20°, 22.74±0.20°, 23.58±0.20°, 24.68±0.20°, 25.16±0.20°, 25.84±0.20°, 26.66±0.20°, 27.77±0.20°, 29.03±0.20°, 30.18±0.20°, 30.70±0.20°, 31.54±0.20°, 32.18±0.20°, 32.96±0.20°, 34.03±0.20°, 34.53±0.20°, 36.26±0.20° and 37.41±0.20°.

[0039] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 7.08°, 7.40°, 10.51°, 12.27°, 12.47°, 14.15°, 14.54°, 16.33°, 17.37°, 17.91°, 18.60°, 18.98°, 19.20°, 19.56°, 20.13 °, 21.06°, 21.30°, 22.27°, 22.74°, 23.58°, 24.68°, 25.16°, 25.84°, 26.66°, 27.77°, 29.03°, 30.18°, 30.70°, 31.54°, 32.18°, 32.96°, 34.03°, 34.53°, 36.26° and 37.41°.

[0040] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 16.33±0.20°, 18.98±0.20°, and / or 7.08±0.20°, and / or 7.40±0.20°, and / or 10.51±0.20°, and / or 12.27±0.20°, and / or 12.47±0.20°, and / or or 14.15±0.20°, and / or 14.54±0.20°, and / or 17.37±0.20°, and / or 17.91±0.20°, and / or 18.60±0.20°, and / or 19.20±0.20°, and / or 19.56±0.20°, and / or 20.13±0.20°, and / or 21.06±0.20°, and / or 21. 30±0.20°, and / or 22.27±0.20°, and / or 22.74±0.20°, and / or 23.58±0.20°, and / or 24.68±0.20°, and / or 25.16±0.20°, and / or 25.84±0.20°, and / or 26.66±0.20°, and / or 27.77±0.20°, and / or 29.03±0 .20°, and / or 30.18±0.20°, and / or 30.70±0.20°, and / or 31.54±0.20°, and / or 32.18±0.20°, and / or 32.96±0.20°, and / or 34.03±0.20°, and / or 34.53±0.20°, and / or 36.26±0.20°, and / or 37.41±0.20°.

[0041] In some embodiments of the present invention, the XRPD pattern of the above-mentioned Form B is substantially as follows Figure 4 shown.

[0042] In some embodiments of the present invention, the XRPD of the above-mentioned Form B uses Cu-Kα radiation.

[0043] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form B is shown in Table 2.

[0044] Table 2 XRPD analysis data of Form B of Compound V

[0045]

[0046]

[0047] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned B crystal form has an endothermic peak at 127.4±5°C.

[0048] In some embodiments of the present invention, the DSC analysis method of the above-mentioned Form B is as follows: the scanning rate is 10°C / min, and the temperature range is 25-350°C.

[0049] In some embodiments of the present invention, the above-mentioned B crystal form has a DSC spectrum as shown in FIG. Figure 5 shown.

[0050] In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned Form B shows a weight loss of 8.84% at 150±5°C.

[0051] In some embodiments of the present invention, the TGA analysis method of the above-mentioned Form B is as follows: the scanning rate is 10°C / min, and the temperature range is room temperature-350°C.

[0052] In some embodiments of the present invention, the TGA spectrum of the above-mentioned B crystal form is as follows Figure 6 shown.

[0053] The present invention also provides a method for preparing compound II.

[0054]

[0055] It includes the following steps:

[0056]

[0057] in,

[0058] The organic solvent is methanol, ethanol, isopropanol, acetonitrile or isopropyl acetate;

[0059] Chiral acid is

[0060] In some embodiments of the present invention, the method for preparing the above-mentioned compound II comprises the following steps:

[0061]

[0062] in,

[0063] The organic solvent is methanol, ethanol, isopropanol, acetonitrile or isopropyl acetate;

[0064] Chiral acid is

[0065] Solvent 1 is saturated sodium bicarbonate solution;

[0066] The extractant is dichloromethane.

[0067] In some embodiments of the present invention, the organic solvent is acetonitrile.

[0068] In some embodiments of the present invention, the above The molar ratio to compound I is 0.5-1:1; preferably 1:1.

[0069] In some embodiments of the present invention, the volume mass ratio V:m of the above-mentioned organic solvent to compound I is 5-20 (mL / g).

[0070] In some embodiments of the present invention, the volume mass ratio V:m of the above-mentioned acetonitrile and compound I is 5-20 (mL / g).

[0071] In some embodiments of the present invention, the reaction temperature for preparing compound Ⅰ-A from compound Ⅰ is 25-85°C.

[0072] The present invention also provides use of compound III, compound IV', compound IV, compound V, crystal form A of compound IV, and crystal form B of compound V in the preparation of drugs for treating LSD1-related diseases.

[0073] The present invention also provides the use of Compound III, Compound IV', Compound IV, Compound V, Compound IV's Crystal Form A, and Compound V's Crystal Form B in the preparation of a medicament for treating LSD1-related diseases; the diseases being hematologic malignancies, small cell lung cancer, squamous non-small cell lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, glioma, or Ewing's sarcoma. The hematologic malignancy is preferably human acute myeloid leukemia.

[0074] Technical Effects

[0075] Compound III, compound IV', compound V, compound IV crystal form A and compound V crystal form B of the present invention have good pharmacokinetic properties in rodents, including good oral bioavailability, oral exposure, half-life and clearance rate; compound IV crystal form A has good in vitro activity and in vivo efficacy; compound IV crystal form A is stable, is less affected by light, heat and humidity, and has broad prospects for drug development; the benefits of intermediate resolution are: a single configuration with good optical purity can be obtained through chemical resolution condition screening.

[0076] Definition and Description

[0077] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0078] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0079] The chemical reactions of the present invention are carried out in suitable solvents that are compatible with the chemical transformations of the present invention and the reagents and materials required. To obtain the compounds of the present invention, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0080] The structures of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compounds, the absolute configuration can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data on the cultured single crystal using a Bruker D8 venture diffractometer, using CuKα radiation as the light source and scanning mode: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0081] The present invention will be described in detail below through examples, which are not intended to limit the present invention in any way.

[0082] All solvents used in the present invention were commercially available and used without further purification.

[0083] The present invention uses the following abbreviations: DCM stands for dichloromethane; DMF stands for N,N-dimethylformamide; DMSO stands for dimethyl sulfoxide; EtOH stands for ethanol; MeOH stands for methanol; 2-MeTHF stands for 2-methyltetrahydrofuran; Dioxane stands for dioxane; ACN stands for acetonitrile; Toluene stands for toluene; Acetone stands for acetone; EtOAc stands for ethyl acetate; THF stands for tetrahydrofuran; H2O stands for water; TosOH stands for p-toluenesulfonic acid; FaSSIF stands for simulated fasting artificial intestinal fluid; FeSSIF stands for simulated full artificial intestinal fluid; SGF stands for simulated artificial gastric fluid; LOQ stands for limit of quantification.

[0084] Compounds are named according to the conventional nomenclature in the art or using Software naming, commercially available compounds use supplier catalog names. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 : XRPD pattern of Form A of Compound IV;

[0086] Figure 2 : DSC spectrum of Form A of Compound IV;

[0087] Figure 3 : TGA spectrum of Form A of Compound IV;

[0088] Figure 4 : XRPD pattern of Form B of Compound V;

[0089] Figure 5 : DSC spectrum of Form B of Compound V;

[0090] Figure 6 : TGA spectrum of Form B of Compound V;

[0091] Figure 7 : Single crystal ellipsoid diagram of compound IV. DETAILED DESCRIPTION

[0092] The test parameters of the X-ray powder diffractometer (XRPD) method of the present invention are shown in Table 3.

[0093] Table 3 XRPD test parameters

[0094]

[0095] The test parameters of the differential scanning calorimeter (DSC) method of the present invention are shown in Table 4.

[0096] Table 4 DSC test parameters

[0097]

[0098] Thermogravimetric analysis (TGA) method of the present invention and test parameters are shown in Table 5.

[0099] Table 5 TGA test parameters

[0100]

[0101] The test parameters of the dynamic vapor sorption analysis (DVS) method of the present invention are shown in Table 6.

[0102] Table 6 DVS test parameters

[0103] Instrument manufacturer and model SMS / DVS intrinsic Test conditions Weigh about 10 mg of sample and test temperature 25℃ balance dm / dt:0.01% / min dry Dry at 25°C, 0% RH for 2 hours RH(%) test steps 5% RH RH(%) test step range 0%-95%-0%RH

[0104] The classification of moisture absorption evaluation is shown in Table 7 below:

[0105] Table 7 Moisture absorption evaluation classification table

[0106] Hygroscopicity classification ΔW% deliquescence Absorb enough water to form a liquid Highly hygroscopic ΔW%≥15% Hygroscopic 15%>ΔW%≥2% Slightly hygroscopic 2%>ΔW%≥0.2%

[0107] No or almost no hygroscopicity ΔW%<0.2%

[0108] Note: ΔW% indicates the weight gain of the test sample at 25±1℃ and 80±2%RH.

[0109] Example 1: Preparation of Compound I

[0110]

[0111] Synthesis route:

[0112]

[0113] first step

[0114] Compound 1 (200 g, 706 mmol) was dissolved in dichloromethane (600 mL) to form a suspension. The reaction solution was cooled to 0°C, and sodium hydroxide solution (1 mol / L, 706 mL) was slowly added to the reaction solution. The reaction solution was stirred at 25°C for 1 hour. The reaction solution was diluted with water H2O (200 mL), extracted with dichloromethane (600 mL x 2), and the organic phase was washed with water (500 mL x 1) and saturated brine (1000 mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 2. 1 H NMR (400MHz, CDCl3) δ7.18-7.14(m,2H),7.08-7.06(m,1H),6.95-6.93(m,2H) ,2.48-2.44(m,1H),1.76-1.77(m,1H),0.97-0.94(m,1H),0.92-0.88(m,1H).

[0115] Step 2

[0116] Compound 2 (86.6 g, 650 mmol) was dissolved in dichloromethane (2000 mL), and compound 3 (182.6 g, 715 mmol) and sodium acetate borohydride (344.5 g, 1.63 mol) were added to the reaction solution. The reaction solution was stirred at 25 ° C. for 3 hours, quenched with saturated sodium bicarbonate (2500 mL), and extracted with dichloromethane (1500 mL x 2). The combined organic phases were washed with saturated brine (1000 mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain crude compound I. 1H NMR (400 MHz, CDCl3) δ 7.19-7.18 (m, 2H), 7.17-7.00 (m, 1H), 6.96-6.94 (m, 2H), 3.91-3.90 (m, 1H), 3.58-3.51 (m, 1H), 3.50-3.24 (m, 3H), 3.23-3.22 (m, 2H), 2.26-2.22 (m, 1H), 1.96 (m, 1H), 1.56-1.55 (m, 1H), 1.53-1.52 (m, 4H), 1.51-1.50 (m, 1H), 1.39-1.38 (m, 9H), 1.00-0.99 (m, 1H), 0.92 (m, 1H). MS-ESI calcd. [M+H] + 373 Measured value: 373.

[0117] Example 2: Preparation of Compound II

[0118]

[0119] Synthesis route:

[0120]

[0121] first step

[0122] Chemical separation condition screening - solvent and chiral acid type screening

[0123] At room temperature, raw material I (50 mg) and 1 mL of each solvent were added to separate reaction flasks, followed by the addition of different chiral acid reagents (1 eq). The reaction was stirred at 50-60°C for 2 h, then cooled to 25°C and stirred for 12 h. Precipitated solids were collected and freed with a 1 mol / L NaOH solution before SFC analysis (chiral column: Chiralpak AD-3 50×4.6 mm ID, 3 μm; mobile phase: [CO2-0.05% diethylamine in methanol]; gradient: 40% (0.05% diethylamine in methanol), 3 mL / min). The retention time of the first peak was 0.425 min, and the retention time of the second peak was 0.678 min. The experimental results are shown in Table 8 below:

[0124]

[0125] Table 8 Screening of solvents and chiral acids

[0126]

[0127] Conclusion: (-)-O,O-di-p-toluoyl-L-tartaric acid (chiral acid 5) and acetonitrile as solvent showed high selectivity. Based on this, further optimization of the separation conditions was conducted. "-" indicates no significant solid precipitation.

[0128] Chiral acid equivalent screening:

[0129] At room temperature, raw material I (500 mg) and acetonitrile (5 mL) were added to a reaction flask, followed by varying amounts of (-)-O,O-di-p-toluoyl-L-tartaric acid. The mixture was stirred at 50-60°C for 2 hours, then cooled to 25°C and stirred for 12 hours. Solid precipitated and was collected. The solid was then freed with a 1 mol / L NaOH solution and analyzed by SFC. The results are shown in Table 9 below:

[0130] Table 9 Chiral acid equivalent screening

[0131] Acid equivalent 0.5eq 0.6eq 0.8eq 1.0eq Yield 32.12％ 40.61％ 44.77％ 44.61％ The second peak ee value 98.674％ 97.582％ 95.840％ 96.672％

[0132] Conclusion: The yield and ee value are relatively good when the chiral acid is 1 eq, and further optimization is carried out based on this.

[0133] The volume of solvent acetonitrile and the mass ratio of raw material I are V mL / m filter

[0134] At room temperature, raw material I (500 mg), (-)-O,O-di-p-toluoyl-L-tartaric acid (1 eq), and varying volumes of acetonitrile were added to different reaction bottles. The reaction was stirred at 50-60°C for 2 h and allowed to stand at 0°C for 12 h. Solid precipitated and was collected. The solid was then freed with NaOH (1 mol / L) solution and analyzed by SFC. The experimental results are shown in Table 10 below:

[0135] Table 10 Screening of solvent acetonitrile volume and raw material I mass ratio

[0136]

[0137] When V / m = 20, the precipitated solid has good stirring uniformity and the ee value remains above 90%. Through the above condition screening, it was found that the crystallization temperature should not be too low. Therefore, the final splitting conditions are 1 eq of chiral acid, 20V of solvent acetonitrile, and a single configuration is obtained by splitting at a temperature of 60-25°C.

[0138] Preparation of Compound IA

[0139] Compound I (121.1 g x 2, 299.6 mmol x 2) was dissolved in acetonitrile (2422 mL x 2), and compound (-)-O,O-di-p-toluoyl-L-tartaric acid (115.8 g x 2, 299.6 mmol x 2) was added to the reaction solution. The reaction solution was stirred at 56 ° C for 2 hours, naturally cooled to 25 ° C and stirred for 12 hours. The reaction solution was filtered, and the solid was washed with acetonitrile (600 mL x 2) and vacuum dried at 40 ° C. Acetonitrile (3858 mL) was added to the solid, and the reaction solution was stirred at 85 ° C for 1.5 hours, at 60 ° C for 1 hour, at 50 ° C for 0.5 hour, at 40 ° C for 1 hour, at 30 ° C for 1 hour, at 26 ° C for 0.5 hour, and allowed to stand at 26 ° C for 12 hours. The reaction solution was filtered, and the solid was washed with acetonitrile (500 mL x 2). 2) After washing, the product was dried under vacuum to obtain compound IA. This product was analyzed by SFC (chiral column: Chiralpak AD-3 50×4.6 mm ID, 3 μm; mobile phase: [CO2-0.05% diethylamine in methanol]; gradient: 40% (0.05% diethylamine in methanol), 3 mL / minn). The second peak had a retention time of 0.676 min and an ee value of 99.208%.

[0140] Step 2

[0141] Compound IA (101.1 g, 133 mmol) was dissolved in saturated sodium bicarbonate solution (1250 mL), and the reaction solution was stirred at 25°C for 0.5 hour. The reaction solution was extracted with dichloromethane (900 mL x 2), and the organic phase was washed with saturated brine (1247 mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound II. 1 H NMR (400 MHz, CD3OD) 7.14-7.10 (m, 2H), 7.03-7.00 (m, 1H), 6.96-6.94 (m, 2H), 3.90-3.87 (m, 1H), 3.54-3.43 (m, 4H), 3.22-3.21 (m, 2H), 2.21-2.17 (m, 1H), 2.05-1.96 (m, 1H), 1.85-1.80 (m, 1H), 1.62-1.48 (m, 4H), 1.43-1.35 (m, 1H), 1.35 (s, 9H), 0.97-0.88 (m, 2H). MS-ESI calcd. [M+H] + 373 Measured value: 373.

[0142] Example 3: Preparation of Compound III

[0143]

[0144] Synthesis route:

[0145]

[0146] first step

[0147] Compound II (48.7 g, 128 mmol) was dissolved in dichloromethane (600 mL), the reaction solution was cooled to 0 ° C, triethylamine (19.4 g, 192 mmol) and trifluoroacetic anhydride (40.4 g, 192 mmol) were added to the reaction solution, and the reaction solution was reacted at 26 ° C for 10 hours. The reaction solution was washed with saturated sodium bicarbonate (800 mL), dilute hydrochloric acid (0.1 mol / L, 800 mL), water (800 mL) and saturated brine (800 mL) in sequence, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound III-A. 1 H NMR (400MHz, CDCl3) δ7.26-7.23(m,2H),7.19-7.15(m,1H),7.00-6.98(m,2H),4.58-4.54(m,1H),4.03-3.99(m,1H),3.90-3.86(m, 1H),3.50-3.47(m,2H),3.22-3.17(m,2H),3.02-2.65(m,1H),2.3-2.26(m,1H),2.01-1.99(s,2H),1.62-1.52(m,6H),1.38(m,9H). MS-ESI[M+Na] + 491, measured value 491.

[0148] Step 2

[0149] Compound III-A (57.2 g, 117 mmol) was dissolved in ethyl acetate (306 mL), the reaction solution was cooled to 0°C, and hydrogen chloride ethyl acetate solution (4 mol / L, 117 mL) was added to the reaction solution. The reaction solution was stirred at 25°C for 2 hours. The reaction solution was filtered, and the filter cake was washed with n-heptane (150 mL x 2) to obtain compound III-B. 1 H NMR (400MHz, CD3OD)7.33-7.29(m,2H),7.24-7.20(m,1H),7.15-7.13(m,2H),4.71-4.64(m,1H),4.15-4.04(m,2H) ,3.25-3.22(m,5H),2.49-2.44(m,1H),2.30-2.18(m,2H),2.03-1.77(m,4H),1.66-1.61(m,1H),1.49-1.43(m,1H). MS-ESI[M+H] +369, measured value 369.

[0150] Step 3

[0151] Compound III-C (10.92 g, 51.56 mmol), carbonyldiimidazole (9.16 g, 56.47 mmol), and diisopropylethylamine (19.04 g, 147.32 mmol) were dissolved in acetonitrile (100 mL) and stirred at 28°C for 3 hours. Compound III-B (20 g, 49.11 mmol) was added to the reaction solution, and the reaction solution was stirred at 28°C for 12 hours. The reaction solution was diluted with ethyl acetate (250 mL) and washed sequentially with hydrochloric acid (1%, 100 mL x 4), saturated sodium bicarbonate (100 mL x 1), and saturated brine (100 mL x 1). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1 to 1 / 1) to obtain III-D. 1 H NMR (400MHz, CD3OD) δ7.31-7.28(m,2H),7.22-7.18(m,1H),7.13-7.11(m,2H),4.69- 4.65(m,1H),4.13-4.09(m,1H),4.03-3.99(m,1H),3.86-3.78(m,2H),3.47-3.33(m, 2H), 3.23-3.12 (m, 1H), 2.46-2.41 (m, 1H), 2.20-2.08 (m, 2H), 1.77-1.62 (m, 4H), 1.56-1.50 (m, 1H), 1.46-1.44 (m, 1H), 1.41 (s, 9H), 1.25-1.22 (m, 2H), 0.89-0.91 (m, 2H). MS-ESI calculated value [M+H] + 552, measured value 552.

[0152] Step 4

[0153] Compound III-D (20.0 g, 36.09 mmol) was dissolved in ethyl acetate (50 mL). A solution of sulfuric acid (10.84 g, 108.27 mmol) in ethyl acetate (50 mL) was added at 0°C, and the mixture was allowed to react at 25°C for 2 hours. Saturated sodium carbonate (30 mL) and saturated sodium bicarbonate (20 mL) were added to the reaction solution at 0°C. After 5 minutes, the mixture was extracted with ethyl acetate (100 mL × 2). The organic phases were combined, washed with saturated brine (50 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1 to 20 / 1) to obtain compound III-E. 1H NMR(400MHz,CD3OD)δ7.31-7.27(m,2H),7.22-7.18(m,1H),7.13-7.11(m,2H),4.69-4 .65(m,1H),4.14-4.10(m,1H),4.04-4.00(m,1H),3.95-3.87(m,2H),3.47-3.38(m,2H) ,3.21-3.15 (m, 2H),2.46-2.41 (m, 1H),2.23-2.18 (m, 1H),2.14-2.09 (m, 1H),1.79-1.72 (m, 2H),1.62-1.54 (m, 2H),1.46-1.41 (m, 1H),0.94-0.91 (m, 2H),0.82-0.79 (m, 2H). MS-ESI calculated value [M+H] + 452, measured value 452.

[0154] Step 5

[0155] Compound III-E (15.1 g, 33.15 mmol) was dissolved in methanol (150 mL), potassium carbonate (9.16 g, 66.30 mmol) was added, and the mixture was reacted at 28°C for 2 hours. The reaction solution was filtered, the filtrate was diluted with water (150 mL), and extracted with dichloromethane (200 mL, 100 mL, 50 mL x 2). The organic phases were combined, washed with saturated brine (100 mL x 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound III. 1 H NMR (400MHz, CD3OD) δ7.24-7.20(m,2H),7.13-7.09(m,1H),7.06-7.04(m,2H),4. 02-3.99(m,1H),3.94-3.89(m,2H),3.67-3.58(m,2H),3.48-3.45(m,2H),2.31-2. 27 (m, 1H), 2.15-2.10 (m, 1H), 1.95-1.90 (m, 1H), 1.81-1.74 (m, 2H), 1.70-1.65 (m, 2H), 1.61-1.58 (m, 1H), 1.08-0.99 (m, 2H), 0.95-0.92 (m, 2H), 0.82-0.79 (m, 2H). MS-ESI calculated value [M+H] + 356, measured value 356.

[0156] Example 4: Preparation of Compound IV and its Crystal Form A

[0157]

[0158] Synthesis route:

[0159]

[0160] Compound III-D (2.8 g, 5.08 mmol) was dissolved in methanol (30 mL). Potassium carbonate (1.40 g, 10.15 mmol) was added to the solution in one step. The reaction solution was stirred at 28°C for 12 hours and filtered. Water (30 mL) was added to the filtrate and the mixture was extracted with ethyl acetate (130 mL x 2). The combined organic phases were washed with saturated brine (50 mL x 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound III-F. MS-ESI calculated value [M+H] + 456, measured value 456.

[0161] Step 2

[0162] Compound III-F (1 g, 2.19 mmol) was stirred and mixed with 2-MeTHF (10 mL). A solution of p-toluenesulfonic acid monohydrate (1.07 g, 5.49 mmol) in 2-MeTHF (5 mL) was added to the suspension. The reaction solution was stirred at 50 ° C for 12 hours. A large amount of solid precipitated in the reaction solution, which was filtered. The filter cake was washed with 2-MeTH (3 mL x 3) and dried in vacuo to obtain Form A of Compound IV. 1 H NMR(400MHz,CD3OD)δ7.70(d,J=8.0Hz,4H),7.33-7.26(m,2H),7.26-7.20(m, 5H),7.18-7.13(m,2H),4.21-4.09(m,2H),4.08-4.02(m,1H),4.01-3.88(m,2 H),3.49-3.35(m,2H),3.03-2.99(m,1H),2.56-2.50(m,1H),2.42-2.32(m,7H ),2.03-1.96(m,1H),1.91-1.76(m,3H),1.65-1.50(m,2H),1.42-1.28(m,5H). MS-ESI calculated value [M+H] + 356, measured value 356. XRPD, DSC, TGA test results are as follows Figure 1 、 Figure 2 and Figure 3 .

[0163] Take an appropriate amount of compound IV to prepare a saturated solution of methanol, and let it stand for about 4 days to grow a single crystal. Transparent crystals are generated and sent for SC-XRD test. The single crystal structure analysis results are as follows: Figure 7 shown.

[0164] Example 5: Stability test of Form A of Compound IV in different solvents

[0165] About 100 mg of Form A of Compound IV was weighed and prepared into suspensions at 50°C in different solvents. The suspensions were stirred at 50°C for 24 hours. The reaction solution was cooled to room temperature, and the resulting solid was collected by filtration and subjected to XRPD analysis. The experimental results are shown in Table 11 below:

[0166] Table 11 Stability test results of Form A of Compound IV in different solvents

[0167] Test number solvent appear Solid crystal form 1 Isopropyl acetate suspension A crystal form 2 1,4-Dioxane suspension A crystal form 3 2-Methyltetrahydrofuran suspension A crystal form 4 n-heptane suspension A crystal form 5 Acetonitrile / n-heptane, 1:1 suspension A crystal form 6 Acetonitrile / 2-methyltetrahydrofuran, 1:1 suspension A crystal form 7 Acetonitrile / 1,4-dioxane, 1:1 suspension A crystal form 8 Acetonitrile / isopropyl acetate, 1:1 suspension A crystal form 9 Water / 2-methyltetrahydrofuran, 1 / 35 Dissolve and precipitate A crystal form 10 Tetrahydrofuran suspension A crystal form 11 Ethyl acetate suspension A crystal form 12 Acetonitrile suspension A crystal form 13 Methyl tert-butyl ether suspension A crystal form 14 2-Methyltetrahydrofuran / n-heptane, 1:1 suspension A crystal form 15 2-Methyltetrahydrofuran / methyl tert-butyl ether, 1:1 suspension A crystal form

[0168] Conclusion: Crystal form A of compound Ⅳ has good stability in solvents and is a stable crystal form.

[0169] Example 6: Test of Salt Content Coefficient of Form A of Compound IV

[0170] Based on the structural difference between p-toluenesulfonic acid and the main component, the p-toluenesulfonic acid content in Form A of Compound IV was tested using HPLC. The specific chromatographic conditions of the HPLC analysis method are shown in Table 12, and the test results are shown in Table 13.

[0171] Table 12 Chromatographic conditions for testing p-toluenesulfonic acid content

[0172]

[0173] Table 13 Test results of p-toluenesulfonic acid content of Form A of Compound IV

[0174]

[0175] Conclusion: The measured values ​​of the p-toluenesulfonic acid content of each batch of Form A of Compound IV are consistent with the theoretical value, and the error between the measured value and the theoretical value is less than 0.03. This product contains two p-toluenesulfonic acids.

[0176] Example 7: Study on Hygroscopicity of Form A of Compound IV

[0177] With reference to the Guiding Principles for Hygroscopicity Testing of Pharmaceuticals (Chinese Pharmacopoeia 2020 Edition (Volume IV) General Chapter 9103), the hygroscopicity of Form A of Compound IV was investigated. The results of the hygroscopicity test are shown in Table 14.

[0178] Table 14 Moisture absorption test results

[0179]

[0180] Conclusion: Based on the weight gain after moisture absorption, the crystal form A of compound IV is slightly hygroscopic.

[0181] Example 8: Preliminary stability test study of Form A solid of Compound IV

[0182] With reference to Part IV 9001 "Guidelines for Stability Testing of Active Pharmaceutical Ingredients and Preparations" of the 2020 edition of the Chinese Pharmacopoeia, the "Technical Guidelines for Stability Studies of Chemical Drugs (Active Pharmaceutical Ingredients and Preparations) (Revised)" issued by the Drug Review Center of the State Food and Drug Administration, and the requirements of ICH Q1B, an influencing factor test was conducted on the A crystal form of compound IV.

[0183] 1. Photostability test:

[0184] Weigh two samples of Compound IV Form A, one for the illumination sample and one for the illumination control sample. Place the illumination sample in a clean weighing bottle, spread it into a single layer without covering it with anything, and expose it in a light box with the light box open. The illumination conditions are 5000±500 lux (visible light) and 90μw / cm 2 (UV). The control sample was packaged identically to the light-exposed sample, but the weighing bottle was covered with aluminum foil. Both samples were observed simultaneously for 10 days. The XRPD results are shown in Table 15 below.

[0185] 2. Temperature influence test:

[0186] A sample of Form A of Compound IV was weighed into an open weighing bottle and placed at 60° C. for 30 days. The XRPD test results are shown in Table 15 below.

[0187] 3. Humidity impact test:

[0188] Each sample was placed in an open weighing bottle and placed under conditions of 25°C / 92.5% RH for 30 days. The XRPD test results are shown in Table 15 below.

[0189] Table 15 Solid stability test results of Form A of Compound IV

[0190]

[0191] Conclusion: Crystal form A of compound Ⅳ has good stability under high temperature, high humidity and strong light conditions, and crystal form A is a stable crystal form.

[0192] Example 9: Solubility determination of Form A of Compound IV in buffer solutions at different pH values

[0193] Step 1: Weigh approximately 3 mg of Form A sample of Compound IV into a 1.5 mL liquid phase vial, add 1 mL of medium, and place on a thermomixer (37°C, 800 rpm). Observe the dissolution. If the compound dissolves under the medium conditions, proceed to Step 2; otherwise, proceed to Step 4.

[0194] Step 2: Weigh approximately 7 mg of Form A sample of Compound IV into a 1.5 mL liquid phase vial, add 1 mL of medium, and place on a thermomixer (37°C, 800 rpm). Observe the dissolution. If the compound dissolves under the medium conditions, proceed to Step 3; otherwise, proceed to Step 4.

[0195] Step 3: Weigh approximately 10 mg of Form A sample of Compound IV into a 1.5 mL liquid phase vial, add 1 mL of medium, and place on a thermomixer (37°C, 800 rpm);

[0196] Step 4: Take samples after 18 hours and record the dissolution status. When the sample solution is completely clear, measure the pH value and measure the concentration by HPLC after dilution. The data are shown in Table 16.

[0197] Table 16 Solubility test results of Form A of Compound IV (37°C)

[0198]

[0199] Conclusion: Compound IV's crystal form A exhibits high solubility in buffer solutions below pH 7.4 and purified water.

[0200] Biological activity evaluation

[0201] Experimental Example 1: Pharmacokinetic Evaluation of Form A of Compound IV

[0202] 1.1 Pharmacokinetic evaluation of compound IV crystal form A in rodents

[0203] 1.1.1 Pharmacokinetic evaluation of the compound in SD rats

[0204] Experimental purpose: To test the pharmacokinetics of the compound in SD rats

[0205] Experimental Materials:

[0206] SD rats (male, 150-180 g, Beijing Weitonglihua)

[0207] Experimental operation:

[0208] The pharmacokinetic characteristics of the compounds in rodents after intravenous and oral administration were tested using a standard protocol. In the experiment, the candidate compounds were prepared into clear solutions and administered to SD rats by single intravenous and oral administration. The intravenous and oral solvents were a mixed solvent of 10% dimethyl sulfoxide and 90% 10% hydroxypropyl β-cyclodextrin. This project used four male SD rats. Two SD rats were administered intravenously at a dose of 0.5 mg / kg, and plasma samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration. The other two SD rats were administered orally by gavage at a dose of 1 mg / kg, and plasma samples were collected at 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration. The blood drug concentration was quantitatively analyzed by LC-MS / MS analysis, and pharmacokinetic parameters such as peak concentration (C max ), clearance (CL), half-life (T 1 / 2 ), tissue distribution (Vdss), area under the drug-time curve (AUC 0-last ), bioavailability (F), etc. The experimental results are shown in Table 17:

[0209] Table 17 Pharmacokinetic test results

[0210]

[0211] 1.1.2 Pharmacokinetic evaluation of the compound in CD-1 mice

[0212] Experimental purpose: To test the pharmacokinetics of the compound in CD-1 mice

[0213] Experimental Materials:

[0214] CD-1 mice (male, 7-9 weeks old, Shanghai Institute of Family Planning Science)

[0215] Experimental operation:

[0216] The standard protocol was used to test the pharmacokinetic characteristics of rodents after intravenous and oral administration of the compound. In the experiment, the candidate compound was prepared into a clear solution and given a single intravenous injection and oral administration to mice. The intravenous and oral solvents were a mixed solvent of 10% dimethyl sulfoxide and 90% 10% hydroxypropyl beta-cyclodextrin. The project used four male CD-1 mice. Two mice were intravenously administered with a dose of 1 mg / kg. Plasma samples were collected at 0h (before administration) and 0.0833, 0.25, 0.5, 1, 2, 4, 8, and 24h after administration. The other two mice were orally gavaged with a dose of 2 mg / kg. Plasma samples were collected at 0h (before administration) and 0.25, 0.5, 1, 2, 4, 8, and 24h after administration. Whole blood samples were collected within 24 hours, and blood drug concentrations were quantitatively analyzed by LC-MS / MS analysis. Pharmacokinetic parameters such as peak concentration (C max ), clearance (CL), half-life (T 1 / 2 ), tissue distribution (Vdss), area under the drug-time curve (AUC 0-last ), bioavailability (F), etc. The experimental results are shown in Table 18:

[0217] Table 18 Pharmacokinetic test results

[0218]

[0219] Conclusion: Crystal form A of compound IV of the present invention has good pharmacokinetic properties in rodents, including good oral bioavailability, oral exposure, half-life and clearance.

[0220] 1.2 Pharmacokinetic evaluation of Compound IV crystal form A in large animals

[0221] Experimental purpose: To test the pharmacokinetics of the compound in beagle dogs

[0222] Experimental Materials:

[0223] Beagle (male, 5-15kg, Beijing Mas Biotechnology Co., Ltd.)

[0224] Experimental operation:

[0225] The pharmacokinetic characteristics of the compound after oral administration in beagle dogs were tested using a standard protocol. In the experiment, the candidate compound was prepared into a clear solution and administered to the beagle dogs by single intravenous injection and oral administration. The intravenous solvent was a mixed solvent of 10% dimethyl sulfoxide and 90% 10% hydroxypropyl beta-cyclodextrin. The oral solvent was 0.5% methylcellulose (4000cp). This project used four male beagle dogs. Two beagle dogs were administered intravenously at a dose of 0.5 mg / kg, and plasma samples were collected at 0.033, 0.0833, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after administration. Two beagle dogs were administered orally by gavage at a dose of 1 mg / kg, and plasma samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after administration. The blood drug concentration was quantitatively analyzed by LC-MS / MS analysis method, and pharmacokinetic parameters such as peak concentration (C max ), clearance (CL), half-life (T 1 / 2 ), tissue distribution (Vdss), area under the drug-time curve (AUC 0-last ), bioavailability (F), etc. The experimental results are shown in Table 19:

[0226] Table 19 Pharmacokinetic test results

[0227]

[0228] Conclusion: Crystal Form A of Compound IV of the present invention has good pharmacokinetic properties in large animals, including good oral bioavailability, oral exposure, half-life and clearance.

[0229] Experimental Example 2: Evaluation of IV enzyme activity of compounds

[0230] Experimental purpose: To analyze the inhibitory effect of test compound Ⅳ on LSD1 enzyme activity using enzyme fluorescence coupling method.

[0231] Principle: LSD1 binds to a histone H3K4 monomethylated peptide substrate, generating demethylation activity and H2O2. The inhibitory effect of compounds on LSD1 activity is determined by detecting the H2O2 generated by the enzymatic reaction using peroxidase and the fluorescent reagent Amplex Red.

[0232] Experimental method: This experiment was set up with double replicates and repeated twice.

[0233] Preparation of compound working solution concentration:

[0234] 1) Dilute 10 mM test compound to 2 mM with 100% DMSO. Place 5 μL of compound stock solution into column 1 of a 386-well plate, then add 20 μL of 100% DMSO and mix thoroughly.

[0235] 2) Add 20 μL of 100% DMSO to columns 2 to 10 of a 384-well compound plate;

[0236] 3) Take 10 μL of compound from column 1 and transfer it to column 2 and mix thoroughly. Then take 10 μL of compound from column 2 and transfer it to column 3 and mix thoroughly. Repeat the above steps to column 10 to complete the serial dilution of the compound.

[0237] 4) Preparation of compound working solution: According to the compound arrangement diagram, transfer 1 μL of compound from columns 1 to 10 of the compound plate to the corresponding wells of a new compound plate, and add 39 μL of 1X LSD1 buffer per well. For the positive control wells, transfer 1 μL of 100% DMSO per well and add 39 μL of 1X LSD1 buffer per well for later use.

[0238] Experimental steps:

[0239] 1) 5 μL of compound working solution was added to each well of a 384-well assay plate according to the experimental layout. For the positive control, 5 μL of 1X LSD1 buffer containing 2.5% DMSO was added to each well. For the blank control, 5 μL of 1X LSD1 buffer was added to each well.

[0240] 2) Thaw the LSD1 enzyme stock solution on ice and keep it on ice during the experiment.

[0241] 3) After the enzyme is completely dissolved, dilute the enzyme stock solution to 12.5 ng / μL with 1X LSD1 buffer. That is, take 2.6 μL of enzyme stock solution (4090 ng / μL) and dilute it with 848 μL of 1X LSD1 buffer.

[0242] 4) Add 10 μL of enzyme solution per well to a 384-well plate. Add 10 μL of 1X LSD1 buffer per well to the blank control wells. The enzyme amount is 125 ng per well.

[0243] 5) Incubate the enzyme and compound at 25°C for 30 minutes;

[0244] 6) Dissolve the dry powder of histone H3K4 monomethylated peptide substrate in 500 μL of water and place on ice until ready for use.

[0245] 7) After the incubation of the compound and enzyme is completed, 10 μL of substrate mixture solution is added to each well of the test plate. The 10 μL substrate mixture solution includes 7.5 μL of 2X LSD1 buffer and 2.5 μL of histone H3K4 monomethylated peptide substrate solution. The test plate is sealed and incubated at 25°C for 60 minutes.

[0246] 8) Prepare the assay mixture by adding 20 μL of 10 mM Amplex Red and 40 μL of 10 U / mL Peroxidase to 1940 μL of 1X LSD1 buffer. After incubation, add 25 μL of the assay mixture to each well of the assay plate. Incubate the plate at 25°C for 5 minutes.

[0247] 9) After incubation, immediately use Nivo to detect fluorescence (detection wavelength: excitation 530nm, emission 580nm).

[0248] Data analysis: The inhibition percentage was calculated from the original readings according to the following formula, and then Prism was used to generate the graphs and calculate the IC of the compound. 50 value.

[0249] % inhibition rate = 100-(FI 化合物 -FI 空白对照 ) / (FI 阳性对照 -FI 空白对照 )×100%

[0250] The blank control was a control well without enzyme, and the positive control was a control well containing enzyme, substrate, and 0.5% DMSO. The results are shown in Table 20.

[0251] Table 20 Inhibitory effect of compounds on LSD1 enzyme activity

[0252]

[0253] Conclusion: Compound IV of the present invention has significant inhibitory activity against LSD1.

[0254] Experimental Example 3: Evaluation of Cellular Activity of Compound IV

[0255] 1.1 Evaluation of Kasumi-1 cell viability

[0256] Experimental purpose: To analyze the inhibitory effect of compound Ⅳ on the proliferation of human acute myeloid leukemia Kasumi-1 cells.

[0257] Experimental Materials: RPMI 1640 medium was purchased from Ecosine Biotechnology Co., Ltd., penicillin / streptomycin antibiotics were purchased from HyClone, CellTiter-Glo Luminescent Cell Viability Assay (Cell Viability Chemiluminescent Detection Reagent) was purchased from Promega, fetal bovine serum (FBS) and Kasumi-1 cells were purchased from the American Type Culture Collection (ATCC). Nivo Multilabel Analyzer (PerkinElmer) was used.

[0258] Experimental methods:

[0259] 1) Kasumi-1 cells were seeded in a 96-well plate, with 80 μL of cell suspension per well, including 1×10 4 cells / mL. The cell plates were cultured in a carbon dioxide incubator.

[0260] 2) Dilute the compound to 50 μM in cell culture medium and place it in column 1 of the compound plate (1.5 μL of 10 mM stock solution + 300 μL of cell culture medium). Add 80 μL of cell culture medium to the wells in columns 2 through 9. Add 20 μL of compound from column 1 to column 2 and mix thoroughly. Then, add 20 μL of compound from column 2 to column 3 and mix thoroughly. Repeat this step until column 9.

[0261] 3) Place 20 μL of the serially diluted compound from the compound plate into the corresponding position of the cell culture plate. The final concentration of the compound is now 10 μM to 0.128 nM. Return the cell plate to the incubator containing 5% CO2 and continue incubation for 6 days.

[0262] 4) After 6 days of cell culture, remove the 96-well cell culture plate and add 50 μL / well of CTG reagent. Mix well and centrifuge. Incubate at room temperature for 15 minutes. Read the plate using the Envision Multi-label Analyzer.

[0263] Data Analysis:

[0264] 1) Calculation of inhibition rate:

[0265] % inhibition rate = (RFU sample - RFU negative control) / (RFU positive control - RFU negative control) × 100%

[0266] 2)IC 50 Calculation: The inhibition rate was calculated using Prism 8 software. 50 The calculation results are shown in Table 21.

[0267] Table 21 Inhibitory effect of compounds on kasumi-1 proliferation

[0268]

[0269]

[0270] Conclusion: Compound IV of the present invention showed significant anti-proliferative activity on Kasumi-1 cells.

[0271] 1.2 Evaluation of KG-1 cell viability

[0272] Experimental purpose: To analyze the inhibitory effect of compound Ⅳ on the proliferation of human acute myeloid leukemia KG-1 cells.

[0273] Experimental Materials: Fetal bovine serum (FBS) was purchased from Ecosine Biotechnology Co., Ltd., IMDM medium was purchased from the American Type Culture Collection (ATCC), and penicillin / streptomycin antibodies were purchased from HyClone. CellTiter-Glo Luminescent Cell Viability Assay (chemiluminescent cell viability detection reagent) was purchased from Promega. KG-1 cells were purchased from the European Collection of Authenticated Cell Cultures (ECACC). Nivo multilabel analyzer was purchased from PerkinElmer.

[0274] Experimental methods:

[0275] 1) KG-1 cells were seeded in a 96-well plate, with 100 μL of cell suspension per well, including 1×10 4 cells / mL. The cell plates were cultured in a carbon dioxide incubator.

[0276] 2) Transfer the prepared compound to the corresponding wells of the cell plate (the final concentration of the cell plate starts at 10 μM and decreases by 5X, with 9 concentrations). Place the cell plate in a cell culture incubator containing 5% CO2 at 37°C and culture for 6 days.

[0277] 3) On day 6, remove the 96-well cell culture plate and add 50 μL / well of CellTiter-Glo Luminescent Cell Viability Assay reagent. Mix and shake the plate for 10 minutes. Incubate at room temperature for 5 minutes. Read the fluorescence of the cell plate using an Envision Multilabel Plate Reader.

[0278] Data Analysis:

[0279] 1) Calculation of inhibition rate:

[0280] % inhibition rate = (RFU sample - RFU negative control) / (RFU positive control - RFU negative control) × 100%

[0281] 2)IC 50 Calculation: The inhibition rate was calculated using GraphPad Prism 9 software. 50 The calculation results are shown in Table 22.

[0282] Table 22 Inhibitory effect of compounds on cell proliferation

[0283]

[0284] Conclusion: Compound IV of the present invention showed significant anti-proliferative activity on KG-1 cells.

[0285] Experimental Example 4: In vivo pharmacodynamic study of compound IV on human acute myeloid leukemia Kasumi-1 cells in a subcutaneous transplant tumor model of CB-17 SCID mice

[0286] 4.1 Experimental Purpose

[0287] The purpose of this experiment was to evaluate the tumor inhibitory effect of compound IV on human acute myeloid leukemia Kasumi-1 cells in the CB-17 SCID mouse subcutaneous transplant tumor model.

[0288] 4.2 Experimental Animals

[0289] Species: Mouse; Strain: CB-17 SCID mice; Age and weight: 6-8 weeks old, weight 16-21 g; Gender: female; Supplier: Shanghai Jihui Experimental Animal Breeding Co., Ltd.

[0290] 4.3 Experimental methods and steps

[0291] 4.3.1 Cell culture

[0292] Human acute myeloid leukemia Kasumi-1 cells were cultured in suspension in RPMI-1640 medium supplemented with 20% fetal bovine serum, 1% penicillin and streptomycin at 37°C and 5% CO2. When the cell saturation reached 80%-90%, the cells were harvested, counted, and adjusted to 10×10 6 Resuspend cells / mL in phosphate buffered saline (PBS).

[0293] 4.3.2 Tumor cell inoculation

[0294] 0.2mL(10×10 6Kasumi-1 cells (with Matrigel, volume 1:1) were subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached approximately 135 mm 3 The patients were randomly divided into groups and the medication was started.

[0295] 4.3.3 Preparation of test substances

[0296] The experimental solvent was 0.5% methylcellulose solution: 2.5 g of methylcellulose was weighed and dissolved in 400 mL of ultrapure water. After stirring evenly, the volume was made up to 500 mL with ultrapure water and stored at 4°C.

[0297] Preparation of azacitidine (5-Azacytidine, manufacturer MedChemExpress, batch number 28452): Weigh 1 mg of 5-azacytidine, add 14.2 mL of PBS, dissolve to obtain a clear solution, and store at 4°C.

[0298] Preparation of compound IV: Weigh 14.2 mg of compound IV, add 16.026 mL of 0.5% MC, dissolve to obtain a clear solution with a concentration of 0.45 mg / mL, and store at 4°C.

[0299] 4.3.4 Tumor Measurement and Experimental Indicators

[0300] The experimental indicator is to examine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured twice a week with a vernier caliper. The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0301] The tumor inhibition efficacy of a compound was evaluated using the TGI (%). TGI (%) reflects the rate of tumor growth inhibition. TGI (%) is calculated as follows: TGI (%) = [1 - (mean tumor volume at the end of treatment in a given treatment group - mean tumor volume at the start of treatment in that treatment group) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] × 100%.

[0302] Statistical analysis: One-tailed T test was used to analyze the differences between each experimental group and the solvent control, and p < 0.05 was considered to be significantly different.

[0303] 4.4 Experimental results: The experimental results calculated based on the tumor volume on day 21 after administration are shown in Table 23.

[0304] Table 23 Evaluation of the antitumor efficacy of compound IV on human acute myeloid leukemia Kasumi-1 cells in a CB-17 SCID mouse subcutaneous transplant tumor model

[0305]

[0306] Conclusion: Compound IV alone and in combination with azacitidine significantly inhibited the growth of Kasumi-1 xenograft tumors. The combination of Compound IV and azacitidine demonstrated a more pronounced inhibitory effect than either Compound IV or azacitidine alone. Tumor-bearing mice showed good tolerance to Compound IV at all doses.

[0307] Experimental Example 5: In vivo pharmacodynamic study of compound IV on human acute myeloid leukemia KG-1 cells in a subcutaneous transplanted tumor model of BALB / c nude mice

[0308] 5.1 Experimental Purpose

[0309] The purpose of this experiment was to evaluate the tumor inhibitory effect of compound IV on human leukemia KG-1 cells in a subcutaneous transplanted tumor model of BALB / c nude mice.

[0310] 5.2 Experimental Animals

[0311] Species: Mouse; Strain: BALB / c nude mice; Age and weight: 6-8 weeks old, weight 18-24 g; Gender: female; Supplier: Shanghai Lingchang Biotechnology Co., Ltd.

[0312] 5.3 Experimental methods and steps

[0313] 5.3.1 Cell culture

[0314] Human acute myeloid leukemia KG-1 cells were cultured in monolayers in IMDM medium supplemented with 20% fetal bovine serum, 1% penicillin and streptomycin at 37°C and 5% CO2. When the cell saturation reached 80%-90%, the cells were harvested, counted, and the cell suspension was adjusted to 5×10 7 pieces / mL.

[0315] 5.3.2 Tumor Cell Inoculation

[0316] 0.2mL(10×10 6 KG-1 cells (with Matrigel, volume 1:1) were subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached approximately 106 mm 3 The patients were randomly divided into groups and the medication was started.

[0317] 5.3.3 Preparation of test substances

[0318] The experimental solvent was 0.5% methylcellulose solution: 2.5 g of methylcellulose was weighed and dissolved in 400 mL of ultrapure water. After stirring evenly, the volume was made up to 500 mL with ultrapure water and stored at 4°C.

[0319] Preparation of azacitidine (5-Azacytidine, manufacturer MedChemExpress, batch number 103024): Weigh 1.6 mg of 5-azacytidine, add 16 mL of PBS, dissolve to obtain a clear solution, and store at 4°C.

[0320] Preparation of compound IV: Weigh 5 mg of compound IV, add 4.224 mL of 0.5% MC, dissolve to obtain a clear solution with a concentration of 0.6 mg / mL, and store at 4°C.

[0321] 5.3.4 Tumor Measurement and Experimental Indicators

[0322] The experimental indicator is to examine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured twice a week with a vernier caliper. The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0323] The tumor inhibition efficacy of a compound was evaluated using the TGI (%). TGI (%) reflects the rate of tumor growth inhibition. TGI (%) is calculated as follows: TGI (%) = [1 - (mean tumor volume at the end of treatment in a given treatment group - mean tumor volume at the start of treatment in that treatment group) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] × 100%.

[0324] Statistical analysis: Prism 6.02 was used to analyze the differences between the experimental groups and the solvent control, and p < 0.05 was considered to be significantly different.

[0325] 5.4 Experimental results: The experimental results calculated based on the tumor volume on day 21 after administration are shown in Table 24.

[0326] Table 24 Evaluation of the anti-tumor efficacy of compound IV on human acute myeloid leukemia KG-1 cells in a subcutaneous transplanted tumor model in BALB / c nude mice

[0327]

[0328]

[0329] Conclusion: Compound IV, alone, inhibited the growth of subcutaneous human leukemia KG-1 xenografts in BALB / c nude mice. Combination administration of Compound IV and azacitidine significantly inhibited tumor growth, demonstrating significantly superior efficacy compared to either Compound IV or azacitidine alone. All doses of Compound IV were well tolerated by tumor-bearing mice.

[0330] Experimental Example 6: Pharmacodynamic evaluation of compound IV against systemic human acute myeloid leukemia MV-4-11-Luc cells in an NCG mouse xenograft tumor model

[0331] 6.1 Experimental Purpose

[0332] The purpose of this experiment is to evaluate the anti-tumor effect of compound IV on human leukemia MV-4-11-Luc cells in the NCG mouse xenograft tumor model.

[0333] 6.2 Experimental Animals

[0334] Species: Mouse; Strain: NCG mouse; Age and weight: 6-7 weeks old, weight 19.1-24.7 g; Gender: female; Supplier: Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0335] 6.3 Experimental methods and steps

[0336] 6.3.1 Cell culture

[0337] MV-4-11-Luc cells were cultured in IMDM medium supplemented with 10% fetal bovine serum. MV-4-11-Luc cells were harvested during the exponential growth phase, resuspended in PBS to an appropriate concentration, and then inoculated into the tail vein of mice.

[0338] 6.3.2 Tumor Cell Inoculation

[0339] Female NCG mice were inoculated with 1×10 7 MV-4-11-Luc cells. All mice were imaged on day 7 after inoculation and grouped according to fluorescence values, with the day of group administration defined as day 0.

[0340] 6.3.3 Preparation of test substances

[0341] The experimental solvent was 0.5% methylcellulose solution: 2.5 g of methylcellulose was weighed and dissolved in 400 mL of ultrapure water. After stirring evenly, the volume was made up to 500 mL with ultrapure water and stored at 4°C.

[0342] Azacitidine (5-Azacytidine, manufacturer MedChemExpress, batch number 103024) preparation: Weigh 0.675 mg of 5-Azacytidine, add 9 mL of normal saline, vortex, and ultrasonically oscillate to obtain a clear solution, which is stored at 4°C.

[0343] Preparation of Compound IV: Weigh 4.8 mg of Compound IV, add 24.219 mL of 0.5% methylcellulose (4000 cps), vortex, and ultrasonicate to obtain a clear solution, which is stored at 4°C.

[0344] 6.3.4 Tumor Measurement and Experimental Indicators

[0345] The experimental target was to examine whether tumor growth was inhibited, delayed, or cured. In this experiment, tumor growth was monitored by in vivo bioluminescence imaging of mice. Imaging was performed twice weekly after group dosing. The imaging system used was the IVIS Lumina III Mouse Intravital Imager (PerkinElmer, USA). The specific experimental steps are as follows:

[0346] 1) Imaging mice were injected subcutaneously with D-Luciferin imaging substrate (PerkinElmer, XenoLight D-Luciferin (K + Salt), Catalog No. 122799) in the neck at a dose of 150 mg / kg and an injection volume of 5 μl / g;

[0347] 2) 10 minutes after substrate injection, the imaging mice were placed in an isoflurane anesthesia box for anesthesia;

[0348] 3) After the imaging mouse enters anesthesia, transfer the mouse to the imaging device and maintain anesthesia during imaging by placing the mouse's mouth and nose in the anesthesia system cannula. The mouse is placed from small to large ear size, from left to right, with the abdomen facing up, and the mouse's tail is placed in a black light-blocking cannula;

[0349] 4) Select bioluminescence imaging in the IVIS Lumina III imaging software, set the exposure time to automatic, and image the mouse.

[0350] 5) After imaging is complete, transfer the mice to a feeding cage and return them to the mouse feeding rack after all animals are confirmed to be awake.

[0351] The anti-tumor efficacy of the compound was evaluated by TGI (%), which reflects the tumor growth inhibition rate. T / C% = T RTV / C RTV × 100%; TGI% = (1-T / C) × 100%. In this experiment, TV is replaced by the fluorescence signal value.

[0352] Statistical analysis: p < 0.05 was considered to be significantly different.

[0353] 6.4 Experimental results: The experimental results calculated based on the tumor fluorescence intensity on day 21 after administration are shown in Table 25.

[0354] Table 25 Evaluation of the antitumor efficacy of compound IV on human acute myeloid leukemia MV-4-11-Luc cells in the NCG mouse system transplant tumor model

[0355]

[0356] Conclusion: Azacitidine at a dose of 0.75 mg / kg had no significant inhibitory effect on the human acute myeloid leukemia MV-4-11-Luc cell line in the NCG mouse systemic xenograft tumor model. Compound IV at doses of 0.5 mg / kg and 1.0 mg / kg had significant inhibitory effects on the human acute myeloid leukemia MV-4-11-Luc cell line in the NCG mouse systemic xenograft tumor model. The combination of Compound IV (0.50 mg / kg) and azacitidine (0.75 mg / kg) and Compound IV (1.0 mg / kg) with azacitidine (0.75 mg / kg) also showed significant inhibitory effects.

Claims

1. Compound IV:

2. Form A of Compound IV, having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2θ angles: 7.42±0.20°, 16.90±0.20°, and 19.99±0.20°; 3. The crystal form A according to claim 2, having an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 7.42±0.20°, 8.46±0.20°, 15.88±0.20°, 16.90±0.20°, 17.80±0.20°, 18.77±0.20°, 19.99±0.20°, and 22.65±0.20°; Alternatively, the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.42±0.20°, 8.46±0.20°, 15.88±0.20°, 16.90±0.20°, 17.80±0.20°, 18.27±0.20°, 18.77±0.20°, 19.99±0.20°, 21.98±0.20°, and 22.65±0.20°; Alternatively, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.42±0.20°, 8.46±0.20°, 10.79±0.20°, 11.69±0.20°, 13.66±0.20°, 14.78±0.20°, 15.88±0.20°, 16.40±0.20°, 16.90±0.20°, 17.80 ±0.20°, 18.27±0.20°, 18.77±0.20°, 19.57±0.20°, 19.99±0.20°, 21.23±0.20°, 21.64±0.20°, 21.98±0.20°, 22.30±0.20°, 22.65±0.20°, 23.15±0.20°, and 23.44±0.20°; Alternatively, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.42°, 8.46°, 10.79°, 11.69°, 13.66°, 14.78°, 15.88°, 16.40°, 16.90°, 17.80°, 18.27°, 18.77°, 19.57°, 19.99°, 21.23°, 21.64°, 21.98°, 22.30°, 22.65°, 23.15° and 23.44°.

4. The crystal form A according to claim 2, wherein the differential scanning calorimetry curve has an endothermic peak starting point at 215.4±5°C; Alternatively, a thermogravimetric analysis curve thereof shows a weight loss of 0.89% at 180±5°C.

5. The crystal form A according to claim 2, whose XRPD pattern is substantially as shown in Figure 1; Alternatively, its DSC spectrum is shown in Figure 2; Alternatively, its TGA spectrum is shown in Figure 3.

6. Preparation method of compound II, It includes the following steps: in, The organic solvent was acetonitrile; Chiral acid is 7. The preparation method according to claim 6, comprising the steps of: in, The organic solvent was acetonitrile; Chiral acid is Solvent 1 is saturated sodium bicarbonate solution; The extractant is dichloromethane.

8. The preparation method according to claim 6 or 7, The molar ratio with compound I is 0.5-1:1; Alternatively, the volume mass ratio V:m of the organic solvent to compound I is 5-20 mL / g; Alternatively, the reaction temperature for preparing compound I-A from compound I is 25-85°C.

9. The preparation method according to claim 8, The molar ratio with compound I is 1:

1.

10. Use of compound IV according to claim 1 and the crystal form A according to any one of claims 2 to 5 in the preparation of a medicament for treating an LSD1-related disease, wherein the disease is hematological malignancy, small cell lung cancer, squamous non-small cell lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, glioma or Ewing's sarcoma. The use according to claim 10 , wherein the disease is human acute myeloid leukemia.

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