WO2021197295A1 - Crystalline form of macrocyclic indole derivative and preparation method therefor - Google Patents

Abstract

The present disclosure relates to a crystalline form of a macrocyclic indole derivative and a preparation method therefor. In particular, the present disclosure relates to a crystalline form of a compound of formula (I) and a preparation method therefor. The new crystalline form of the present disclosure has good physicochemical properties and is more useful with regard to clinical treatment.

Description

Crystal form of indole macrocyclic derivative and preparation method thereof

This application claims the priority of the Chinese patent application CN202010236480.7 whose filing date is March 30, 2020. This application quotes the full text of the aforementioned Chinese patent application.

Technical field

The present disclosure relates to a crystalline form of an indole macrocyclic derivative and a preparation method thereof, in particular to a crystalline form of a compound of formula (I) and a preparation method thereof.

Background technique

An important feature that distinguishes tumor cells from normal cells is that apoptosis is inhibited, which gives them a greater survival advantage. Apoptosis is also called programmed death, which can be divided into exogenous apoptosis and endogenous apoptosis. Among them, endogenous apoptosis is an important barrier to the occurrence and development of cancer. BCL-2 family proteins are important regulators of endogenous apoptosis.

BCL-2 family proteins mainly exist on the mitochondrial membrane, and can be divided into two categories: anti-apoptotic proteins and pro-apoptotic proteins according to their functions. Anti-apoptotic proteins include BCL-2, BCL-XL, BCL-w and MCL-1. Pro-apoptotic proteins include Bax, Bak and BH3-only proteins. When Bax and Bak are activated, multimer cavities are formed, which increases the permeability of cell mitochondrial membranes and promotes the release of cytochrome C into the cytoplasm, leading to cell death. The BH3-only protein contains only the BH3 domain. When cells are alive, BH3-only proteins (such as Bim) bind to anti-apoptotic proteins. When the cell is under external pressure, the balance of binding is broken, BH3-only protein is released and binds to BAX on mitochondria, promotes BAX/BAK to form multimers, promotes the release of cytochrome C and SMAC into the cytoplasm, and activates downstream apoptosis path.

Existing clinical data indicate that MCL-1 is overexpressed in a variety of tumors. For example, overexpression of MCL-1 is detected in 55% of breast cancer and 84% of lung cancer samples. In multiple myeloma samples, as the degree of cancer progression increased, the expression of MCL-1 increased significantly, but the expression of BCL-2 did not change. In addition, the expression of MCL-1 is negatively correlated with the survival rate of patients. Both breast cancer and multiple myeloma patients have observed high expression of MCL-1 accompanied by lower survival rates. This shows that MCL-1 is an important tumor treatment target.

PCT/CN2019/108322 relates to a compound represented by formula (I), the chemical name is (Ra)-17-chloro-5,13,14,22-tetramethyl-28-oxa-2,9-disulfide heteroaryl -5,6,12,13,24- five azepan ^{[27.6.1.1 4,7 .0 11,15 .0 16,21 .0} 20,24 .0 30,34] thirty-seven -1 (36),4(37),6,11,14,16,18,20,22,29,34-undecene-23-carboxylic acid, this compound is a new type of MCL-1 inhibitor, in clinical efficacy or adaptation Symptoms and safety have been improved. Its structure is as follows:

The crystal structure of medicinal active ingredients often affects the chemical stability of the drug. The difference in crystallization conditions and storage conditions may lead to changes in the crystal structure of the compound, sometimes accompanied by the production of other crystalline forms. Generally speaking, amorphous drug products have no regular crystal structure and often have other defects, such as poor product stability, finer crystallization, difficult filtration, easy agglomeration, and poor fluidity. Therefore, it is necessary to improve the properties of the above-mentioned products. We need in-depth research to find new crystal forms with higher crystal purity and good chemical stability.

Summary of the invention

The purpose of the present disclosure is to provide a new crystal form of the compound represented by formula (I), which has good stability and can be better applied in clinics.

One aspect of the present disclosure provides a crystal form of the compound represented by formula (I),

Another aspect of the present disclosure provides a crystal form A of the compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 5.999, 7.002, 9.760, 11.061, 11.645, 12.118, 13.700, 16.517, 17.717, There are characteristic peaks at 19.406, 19.852, 20.554, 21.523, 22.279, 23.007, 23.510, 24.018, 24.890, 25.614, 26.618, 27.637, 28.283, 28.932, 31.283.

In certain embodiments, the present disclosure provides a crystal form A of the compound represented by formula (I), and its X-ray powder diffraction pattern is shown in FIG. 2.

The present disclosure further provides a method for preparing crystal form A of the compound represented by formula (I), the method comprising:

The compound represented by formula (I) is mixed with an appropriate amount of solvent to be beaten to separate the solids. The solvent may be one or more of n-heptane, n-hexane, and cyclohexane.

Another aspect of the present disclosure provides a crystal form B of the compound represented by formula (I), the X-ray powder diffraction pattern of which is at 2θ angles of 5.281, 8.322, 10.667, 12.483, 14.297, 17.295, 18.241, 19.934, 27.042 There are characteristic peaks.

In certain embodiments, the present disclosure provides a crystal form B of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 5.281, 6.901, 8.322, 10.667, 12.483, 14.297, 15.175, 17.295 , 18.241, 18.744, 19.188, 19.934, 20.679, 20.931, 22.075, 22.517, 23.137, 23.839, 25.249, 26.496, 27.042, 27.828, 29.134, 30.526, 31.518, 32.762, 33.417, 34.713, 37.933, 38.135, 48.442 have characteristic peaks .

In certain embodiments, the present disclosure provides a crystal form B of the compound represented by formula (I), and its X-ray powder diffraction pattern is shown in FIG. 3.

The present disclosure further provides a method for preparing crystal form B of the compound represented by formula (I), the method comprising:

Mix the compound represented by formula (I) with an appropriate amount of solvent to volatilize and crystallize. The solvent may be one or more of isopropyl acetate, methyl isobutyl ketone, isoamyl alcohol, and butyl acetate; or ,

The compound represented by the formula (I) is mixed with an appropriate amount of solvent to be slurried, and the solid is separated. The solvent can be one or more of methanol/water, ethanol/water, and ethyl acetate/n-heptane.

In some embodiments, the beating is performed under heating conditions, for example, the beating temperature is 40-80°C, preferably 50-65°C.

Another aspect of the present disclosure provides a crystal form C of the compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 7.000, 9.202, 10.827, 12.192, 13.421, 14.204, 14.751, 14.938, 16.097, There are characteristic peaks at 18.268, 19.438, 21.026, 21.374, 22.303, 23.161, 23.748, 24.682, 25.853, 29.444.

In certain embodiments, the present disclosure provides a crystal form C of the compound represented by formula (I), and its X-ray powder diffraction pattern is shown in FIG. 4.

The present disclosure further provides a method for preparing crystal form C of the compound represented by formula (I), the method comprising:

The compound represented by formula (I) is mixed with an appropriate amount of solvent to be slurried, and the solid is separated. The solvent can be one or more of ethanol, isopropanol, ethanol/water, and ethyl acetate/ethanol; or

The compound represented by formula (I) is dissolved in solvent A, and then mixed with solvent B to precipitate crystals and separate. The solvent A can be isopropyl acetate and the solvent B is isopropanol.

Another aspect of the present disclosure provides a crystal form D of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 6.737, 8.298, 9.742, 11.242, 11.887, 12.954, 13.585, 14.115, 15.047, 16.243, 16.766, 17.925, 18.183, 18.767, 19.693, 20.366, 21.067, 22.181, 22.640, 22.962, 23.833, 24.469, 24.890, 25.254, 25.938, 26.555, 27.711, 28.258, 28.507, 29.235, 30.094, 30.363, 30.727, 31.363 There are characteristic peaks at 33.015, 33.288, 34.508, 35.993, 39.325, 40.039, 41.734, 42.711.

In certain embodiments, the present disclosure provides a crystal form D of the compound represented by formula (I), and its X-ray powder diffraction pattern is shown in FIG. 5.

The present disclosure further provides a method for preparing crystal form D of the compound represented by formula (I), the method comprising:

The compound represented by the formula (I) is mixed with an appropriate amount of solvent, heated to dissolve, and cooled to precipitate crystals and separated. The solvent may be methanol, n-propanol, methanol/water, or isopropanol/water.

Another aspect of the present disclosure provides a crystal form E of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 5.967, 6.999, 9.626, 10.105, 11.043, 11.617, 12.753, 13.239, 13.641, There are characteristic peaks at 14.988, 15.486, 16.437, 16.796, 17.659, 18.046, 19.326, 19.820, 21.314, 21.775, 22.162, 22.896, 23.333, 23.999, 24.305, 25.907, 26.563, 26.976, 27.526, 28.113, 28.988, 29.984, 31.165.

In certain embodiments, the present disclosure provides a crystal form E of the compound represented by formula (I), and its X-ray powder diffraction pattern is shown in FIG. 6.

The present disclosure further provides a method for preparing the E crystal form of the compound represented by formula (I), the method comprising:

Mix the compound represented by formula (I) with an appropriate amount of solvent to volatilize and crystallize, and the solvent may be acetone/nitromethane; or,

Mix the compound represented by formula (I) with an appropriate amount of solvent to make a slurry to separate the solids, and the solvent may be nitromethane; or

The compound represented by formula (I) is dissolved in solvent A, and then mixed with solvent B to precipitate crystals and separate. The solvent A can be isopropyl acetate and the solvent B is nitromethane.

Another aspect of the present disclosure provides a crystal form F of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 10.118, 10.726, 11.073, 11.875, 12.361, 12.944, 14.098, 14.697, 15.197, There are characteristic peaks at 15.585, 16.597, 17.494, 18.577, 18.880, 19.684, 19.837, 20.481, 21.682, 22.575, 23.073, 23.653, 24.166, 25.480, 26.315, 27.880, 28.511, 30.314, 30.868, 34.194, 40.719.

In certain embodiments, the present disclosure provides a crystal form F of the compound represented by formula (I), and its X-ray powder diffraction pattern is shown in FIG. 7.

The present disclosure further provides a method for preparing the F crystal form of the compound represented by formula (I), the method comprising:

The compound represented by formula (I) is mixed with an appropriate amount of solvent to be beaten to separate the solid, and the solvent may be p-xylene.

Another aspect of the present disclosure provides a crystal form G of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 5.934, 6.937, 9.695, 10.983, 12.020, 13.604, 16.396, 16.547, 17.623, There are characteristic peaks at 19.309, 19.797, 21.438, 22.107, 22.954, 23.501, 23.880, 25.648, 26.542, 27.559, 28.142, and 31.182.

In certain embodiments, the present disclosure provides a crystal form G of the compound represented by formula (I), the X-ray powder diffraction pattern of which is shown in FIG. 8.

The present disclosure further provides a method for preparing the G crystal form of the compound represented by formula (I), the method comprising:

The compound represented by formula (I) is mixed with an appropriate amount of solvent to make a slurry, and the solid is separated. The solvent can be acetonitrile, acetonitrile/methanol.

Another aspect of the present disclosure provides a crystal form H of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 7.718, 8.594, 13.426, 14.282, 14.788, 15.886, 16.517, 17.384, 18.271, There are characteristic peaks at 19.160, 20.587, 21.144, 22.083, 22.743, 23.314, 24.215, 25.834, 28.090, 30.658.

In certain embodiments, the present disclosure provides a crystal form H of the compound represented by formula (I), the X-ray powder diffraction pattern of which is shown in FIG. 9.

The present disclosure further provides a method for preparing the H crystal form of the compound represented by formula (I), the method comprising:

The compound represented by formula (I) is mixed with an appropriate amount of solvent to be beaten to separate the solid. The solvent may be tetrahydrofuran.

Another aspect of the present disclosure provides a crystal form I of the compound represented by formula (I), and its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 8.508, 10.914, 12.724, 14.787, and 16.260.

In certain embodiments, the present disclosure provides a crystalline form I of the compound represented by formula (I), the X-ray powder diffraction pattern of which is shown in FIG. 10.

The present disclosure further provides a method for preparing the I crystal form of the compound represented by formula (I), the method comprising:

The compound represented by formula (I) is mixed with an appropriate amount of solvent to volatilize and crystallize. The solvent may be chloroform.

Another aspect of the present disclosure provides a crystal form J of the compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 5.743, 10.683, 11.526, 13.219, 14.351, 15.821, 16.164, 17.091, 17.333, There are characteristic peaks at 18.566, 21.356, 23.311, 25.193, 26.034, 26.702, 27.038, 29.344, 29.977, 33.657, 35.309.

In certain embodiments, the present disclosure provides a crystal form J of the compound represented by formula (I), the X-ray powder diffraction pattern of which is shown in FIG. 11.

The present disclosure further provides a method for preparing the J crystal form of the compound represented by formula (I), the method comprising:

The compound represented by formula (I) is dissolved in solvent A, and then mixed with solvent B to precipitate crystals and separate. The solvent A can be one or more of acetone, isopropyl acetate, tetrahydrofuran, and dichloromethane, Solvent B is acetonitrile.

Another aspect of the present disclosure provides a crystal form K of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 5.838, 11.996, 13.592, 19.262, 19.745, 22.060, 22.912, 24.807, 25.774, There are characteristic peaks at 27.235, 27.627, 28.188, 30.500, and 31.247.

In certain embodiments, the present disclosure provides a crystal form K of the compound represented by formula (I), and its X-ray powder diffraction pattern is shown in FIG. 12.

The present disclosure further provides a method for preparing the K crystal form of the compound represented by formula (I), the method comprising:

The compound represented by formula (I) is dissolved in solvent A, and then mixed with solvent B to precipitate crystals and separate. The solvent A can be ethyl acetate or 2-butanone, and the solvent B is acetonitrile.

Another aspect of the present disclosure provides a crystal form L of the compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 6.878, 9.075, 10.695, 12.040, 13.241, 13.960, 15.964, 19.283, 20.308, There are characteristic peaks at 21.028, 21.888, 23.326, 24.236, 24.656, 25.568, 25.922, 28.155, 29.374, 31.777, 36.458, 40.122, 43.686, 47.006.

In certain embodiments, the present disclosure provides a crystal form L of the compound represented by formula (I), the X-ray powder diffraction pattern of which is shown in FIG. 13.

The present disclosure further provides a method for preparing the L crystal form of the compound represented by formula (I), the method comprising:

The compound represented by formula (I) is dissolved in solvent A, and then mixed with solvent B to precipitate crystals and separate. The solvent A can be 2-butanone and the solvent B is isopropanol.

Another aspect of the present disclosure provides a crystal form M of the compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 6.362, 8.845, 9.799, 10.421, 10.824, 12.128, 12.920, 14.178, 15.321, There are characteristic peaks at 16.941, 17.376, 17.940, 18.693, 19.853, 21.125, 22.604, 23.628, 24.456, 25.143, 25.748, 26.517, 27.482, 28.435, 29.464, 30.068, 32.062.

In certain embodiments, the present disclosure provides a crystal form M of the compound represented by formula (I), and its X-ray powder diffraction pattern is shown in FIG. 14.

The present disclosure further provides a method for preparing the M crystal form of the compound represented by formula (I), the method comprising:

The compound represented by formula (I) is dissolved in solvent A, and then mixed with solvent B to precipitate and crystallize. The solvent A can be tetrahydrofuran, and the solvent B can be one or more of n-heptane, n-hexane, and petroleum ether. kind.

Another aspect of the present disclosure provides a crystal form N of the compound represented by formula (I), the X-ray powder diffraction pattern of which is at the 2θ angles of 5.215, 8.261, 10.555, 12.382, 13.903, 16.676, 17.359, 18.083, 20.288. There are characteristic peaks.

In certain embodiments, the present disclosure provides a crystal form N of the compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 5.215, 8.261, 10.555, 12.382, 13.903, 14.528, 16.676, 17.359 There are characteristic peaks at, 18.083, 18.284, 20.288, 22.343, 23.296, 23.777, 26.812, 28.196, 29.067, 30.910, 32.931.

In certain embodiments, the present disclosure provides a crystal form N of the compound represented by formula (I), the X-ray powder diffraction pattern of which is shown in FIG. 15.

The present disclosure further provides a method for preparing the N crystal form of the compound represented by formula (I), the method comprising:

The crystal form B of the compound represented by formula (I) is heated to greater than 120° C., and then the temperature is lowered to crystallize.

Another aspect of the present disclosure provides a crystal form O of the compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 7.026, 9.353, 11.154, 12.308, 15.113, 15.543, 16.396, 18.634, 18.980, There is a characteristic peak at 21.526.

In certain embodiments, the present disclosure provides a crystal form O of the compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 7.026, 9.353, 11.154, 12.308, 15.113, 15.543, 16.396, 16.717 There are characteristic peaks at, 17.734, 18.634, 18.980, 19.881, 20.897, 21.526, 21.750, 22.397, 23.206, 24.024, 25.173, 25.880, 26.643, 27.878, 27.962, 28.681, 29.495, 30.026, 30.891, 31.476, 32.653.

In certain embodiments, the present disclosure provides a crystal form O of the compound represented by formula (I), and its X-ray powder diffraction pattern is shown in FIG. 16.

The present disclosure further provides a method for preparing the O crystal form of the compound represented by formula (I), the method comprising:

The crystal form C of the compound represented by formula (I) is heated to greater than 90° C., and then the temperature is lowered to crystallize.

Another aspect of the present disclosure provides a crystal form P of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 7.085, 8.452, 9.395, 10.398, 10.701, 11.198, 12.375, 12.598, 13.134, 14.225, 15.163, 15.519, 16.400, 16.804, 17.488, 18.113, 18.685, 19.055, 20.905, 21.530, 21.845, 22.403, 23.520, 24.047, 25.241, 25.853, 26.153, 26.638, 27.164, 27.458, 27.795, 28.679, 29.437, 30.068 There are characteristic peaks at 31.466, 32.005, and 32.678.

In certain embodiments, the present disclosure provides a crystal form P of the compound represented by formula (I), the X-ray powder diffraction pattern of which is shown in FIG. 17.

The present disclosure further provides a method for preparing the P crystal form of the compound represented by formula (I), the method comprising:

The crystal form C of the compound represented by formula (I) is placed at 60°C for at least 1 day, preferably 3 days, more preferably 4 days.

Another aspect of the present disclosure provides a crystal form Q of the compound represented by formula (I), the X-ray powder diffraction pattern of which is at 2θ angles of 7.441, 11.821, 13.781, 14.683, 16.886, 17.614, 18.673, 19.838, 20.495 There are characteristic peaks.

In certain embodiments, the present disclosure provides a crystal form Q of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 7.441, 8.381, 10.030, 11.125, 11.821, 13.075, 13.781, 14.683 , 16.277, 16.886, 17.614, 18.673, 19.838, 20.495, 22.155, 22.724, 23.464, 23.938, 24.366, 24.975, 26.051, 26.406, 27.121, 27.963, 28.435, 31.415, 32.384, 33.183, 34.657 have characteristic peaks.

In certain embodiments, the present disclosure provides a crystal form Q of the compound represented by formula (I), the X-ray powder diffraction pattern of which is shown in FIG. 18.

The present disclosure further provides a method for preparing the Q crystal form of the compound represented by formula (I), the method comprising:

The compound represented by formula (I) is mixed with an appropriate amount of solvent to be beaten to separate the solid, and the solvent can be one or more of dichloromethane and n-heptane; or

The compound represented by the formula (I) is mixed with a solvent, dissolved at elevated temperature, and crystals are precipitated and separated after cooling. The solvent may be acetone.

Another aspect of the present disclosure provides a crystal form R of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 6.747, 7.763, 9.143, 11.137, 11.942, 13.115, 13.584, 14.113, 15.042, There are characteristic peaks at 16.240, 17.467, 17.983, 18.741, 19.343, 19.643, 21.045, 22.343, 23.785, 24.101, 24.646, 25.543, 26.822, 27.398, 27.926, 28.570, 29.164, 30.912, 31.584, 32.426, 33.352.

In certain embodiments, the present disclosure provides a crystal form R of the compound represented by formula (I), the X-ray powder diffraction pattern of which is shown in FIG. 19.

The present disclosure further provides a method for preparing the R crystal form of the compound represented by formula (I), the method comprising:

The compound represented by formula (I) is mixed with an appropriate amount of solvent to volatilize and crystallize. The solvent may be N-N dimethylacetamide.

Another aspect of the present disclosure provides a crystal form of the compound represented by formula (I), the X-ray powder diffraction pattern at 2θ angles is 5.884, 6.904, 9.686, 10.133, 10.886, 11.918, 13.186, 13.438, 13.883, There are characteristic peaks at 14.642, 16.189, 17.029, 18.329, 19.091, 19.642, 20.476, 21.513, 21.971, 23.245, 24.091, 24.616, 25.460, 26.198, 27.240, 27.945, 28.917, 29.560, 30.087, 30.806.

In certain embodiments, the present disclosure provides a crystal form of the compound represented by formula (I), the X-ray powder diffraction pattern of which is shown in FIG. 34.

Another aspect of the present disclosure provides a crystal form T of the compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 6.955, 9.287, 11.139, 12.276, 13.441, 14.039, 15.009, 16.255, 18.627, There are characteristic peaks at 21.362, 22.380, 23.973, 25.834, 31.627, and 45.406.

In certain embodiments, the present disclosure provides a crystal form T of the compound represented by formula (I), and its X-ray powder diffraction pattern is shown in FIG. 35.

Through X-ray powder diffraction pattern (XRPD) and differential scanning calorimetry (DSC), the crystal form obtained in the present disclosure was subjected to structure determination and crystal form study.

The crystallization method of the crystal form in the present disclosure is conventional, such as volatilization crystallization, cooling crystallization or crystallization at room temperature.

The starting material used in the method for preparing the crystal form of the present disclosure can be any form of the compound represented by formula (I), and the specific form includes, but is not limited to: amorphous, any crystal form, hydrate, solvate, and the like.

The present disclosure further provides a pharmaceutical composition comprising the crystal form of the compound represented by formula (I) and one or more pharmaceutically acceptable carriers or excipients.

The present disclosure further provides a pharmaceutical composition, which is prepared by the crystal form of the compound represented by formula (I) and one or more pharmaceutically acceptable carriers or excipients.

The present disclosure further provides a method for preparing a pharmaceutical composition, comprising the step of mixing the crystal form of the compound represented by formula (I) with a pharmaceutically acceptable carrier, preferably the crystal form is selected from the crystal form described in the present disclosure One or more of.

The present disclosure further provides the use of the crystal form or pharmaceutical composition of the compound represented by formula (I) described in the present disclosure in the preparation of a drug for inhibiting MCL-1.

The present disclosure further provides the use of the crystal form or pharmaceutical composition of the compound represented by formula (I) described in the present disclosure in the preparation of a medicament for the treatment of diseases mediated by MCL-1.

The present disclosure further provides the use of the crystal form or pharmaceutical composition of the compound represented by the formula (I) described in the present disclosure in the preparation of a medicament for the treatment or prevention of tumors, autoimmune diseases and immune system diseases. The tumor is preferably selected from bladder cancer, brain tumor, breast cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, leukemia (such as chronic myeloid leukemia, chronic lymphocytic leukemia, lymphoblastic leukemia or acute Myeloid leukemia), cancer, colon cancer, rectal cancer, colorectal cancer, esophageal cancer, liver cancer, stomach cancer, head and neck cancer, skin cancer, lymphoma, pancreatic cancer, melanoma, myeloma (such as multiple myeloma), Bone cancer, neuroblastoma, glioma, sarcoma, lung cancer (such as non-small cell lung cancer or small cell lung cancer), thyroid cancer, and prostate cancer.

In some embodiments, the crystal form described in the present disclosure is preferably selected from crystal form A, crystal form B, crystal form C, crystal form D, crystal form E, crystal form F, crystal form G, crystal form H, crystal form I Type, J type, K type, L type, M type, N type, O type, P type, Q type, R type, S type, T type, or Multiple types, more preferably one or more of B crystal form, N crystal form, O crystal form, and Q crystal form, most preferably B crystal form.

In the specification and claims of this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. However, in order to better understand the present disclosure, definitions and explanations of some related terms are provided below. In addition, when the definitions and explanations of terms provided in this application are inconsistent with the meanings commonly understood by those skilled in the art, the definitions and explanations of terms provided in this application shall prevail.

The “beating” mentioned in the present disclosure refers to a method of purifying substances with poor solubility in solvents but good solubility in solvents. The beating purification can remove color, change crystal form, or remove a small amount of impurities.

The “X-ray powder diffraction pattern or XRPD” mentioned in the present disclosure refers to the Bragg formula 2d sinθ=nλ (where λ is the wavelength of X-rays, and the diffraction order n is any positive integer, generally first-order diffraction Peak, n = 1), when X-rays are incident on a crystal or part of the crystal sample with a d lattice plane spacing at a grazing angle θ (the complementary angle of the incident angle, also known as the Bragg angle), then The Bragg equation can be satisfied, and this set of X-ray powder diffraction patterns can be measured.

The "X-ray powder diffraction pattern or XRPD" described in the present disclosure is a pattern obtained by using Cu-Kα radiation in an X-ray powder diffractometer.

The "differential scanning calorimetry or DSC" mentioned in the present disclosure refers to the measurement of the temperature difference and heat flow difference between the sample and the reference material during the temperature rise or constant temperature process of the sample to characterize all the physical changes and chemistry related to the thermal effect. Change, get the phase change information of the sample.

The "2θ or 2θ angle" mentioned in the present disclosure refers to the diffraction angle, θ is the Bragg angle, and the unit is ° or degree, and the error range of 2θ is ±0.3 or ±0.2 or ±0.1.

The "interplanar spacing or interplanar spacing (d value)" mentioned in this disclosure means that the spatial lattice selects three non-parallel unit vectors a, b, and c connecting two adjacent lattice points. The matrix is divided into juxtaposed parallelepiped units, called interplanar spacing. The space lattice is divided according to the determined parallelepiped unit lines to obtain a set of linear grids, which are called spatial lattices or lattices. Lattice and crystal lattice use geometric points and lines to reflect the periodicity of the crystal structure. For different crystal planes, the interplanar spacing (that is, the distance between two adjacent parallel crystal planes) is different; the unit is

Or angstrom.

The beneficial effects of the invention

The crystal form of the compound represented by formula (I) prepared by the present disclosure has high purity, good crystal form stability under the conditions of light, high temperature and high humidity, small changes in HPLC purity, high physical and chemical stability, and is more conducive to the storage of raw materials and use.

Description of the drawings

Figure 1 is the amorphous XRPD pattern of the compound represented by formula (I).

Figure 2 is the XRPD pattern of the crystal form A of the compound represented by formula (I).

Fig. 3 is an XRPD pattern of the crystal form B of the compound represented by formula (I).

Figure 4 is the XRPD pattern of the crystal form C of the compound represented by formula (I).

Figure 5 is the XRPD pattern of the crystal form D of the compound represented by formula (I).

Fig. 6 is the XRPD pattern of the crystal form E of the compound represented by formula (I).

Figure 7 is the XRPD pattern of the F crystal form of the compound represented by formula (I).

Fig. 8 is the XRPD pattern of the crystal form G of the compound represented by formula (I).

Figure 9 is the XRPD pattern of the H crystal form of the compound represented by formula (I).

Figure 10 is the XRPD pattern of the crystalline form I of the compound represented by formula (I).

Figure 11 is the XRPD pattern of the crystal form J of the compound represented by formula (I).

Figure 12 is the XRPD pattern of the K crystal form of the compound represented by formula (I).

Figure 13 is the XRPD pattern of the L crystal form of the compound represented by formula (I).

Figure 14 is the XRPD pattern of the M crystal form of the compound represented by formula (I).

Figure 15 is the XRPD pattern of the N crystal form of the compound represented by formula (I).

Figure 16 is the XRPD pattern of the O crystal form of the compound represented by formula (I).

Figure 17 is an XRPD pattern of the P crystal form of the compound represented by formula (I).

Fig. 18 is the XRPD pattern of the crystal form Q of the compound represented by formula (I).

Figure 19 is the XRPD pattern of the R crystal form of the compound represented by formula (I).

Figure 20 is a DSC chart of the crystal form A of the compound represented by formula (I).

Fig. 21 is a DSC chart of Form B of the compound represented by formula (I).

Figure 22 is a DSC chart of Form C of the compound represented by formula (I).

Fig. 23 is a DSC chart of the crystal form D of the compound represented by formula (I).

Fig. 24 is a DSC chart of Form E of the compound represented by formula (I).

Figure 25 is a DSC chart of Form F of the compound represented by formula (I).

Figure 26 is a DSC chart of Form G of the compound represented by formula (I).

Fig. 27 is a DSC chart of the crystal form H of the compound represented by formula (I).

Fig. 28 is a DSC chart of the crystal form K of the compound represented by formula (I).

Figure 29 is a DSC chart of the L crystal form of the compound represented by formula (I).

Figure 30 is a DSC chart of the M crystal form of the compound represented by formula (I).

Figure 31 is a DSC chart of the N crystal form of the compound represented by formula (I).

Figure 32 is a DSC chart of the O crystal form of the compound represented by formula (I).

Fig. 33 is a DSC chart of the crystal form Q of the compound represented by formula (I).

Figure 34 is an XRPD pattern of the S crystal form of the compound represented by formula (I).

Figure 35 is the XRPD pattern of the T crystal form of the compound represented by formula (I).

Detailed ways

The present disclosure will be explained in more detail below in conjunction with embodiments. The embodiments of the present disclosure are only used to illustrate the technical solutions of the present disclosure, and do not limit the essence and scope of the present disclosure.

Test conditions of the equipment used in the test:

1. Differential Scanning Calorimeter (DSC)

Instrument model: Mettler Toledo DSC 3+

Purge gas: nitrogen

Heating rate: 10.0℃/min

Temperature range: 25-350℃

2. X-ray Diffraction Spectrum (X-ray Powder Diffraction, XRPD)

Instrument model: BRUKER D8 DISCOVERY X-ray powder diffractometer

Ray: monochromatic Cu-Kα rays (Cu-Kα1 wavelength is

The wavelength of Cu-Kα2 is

Cu-Kα wavelength is the weighted average of Kα1 and Kα2

)

Scanning method: θ/2θ, scanning range: 3-50°, 5-50°

Voltage: 40KV, current: 40mA;

The structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS). The NMR shift (δ) is given in units of ^{10 -6 (ppm).} The NMR was measured with Bruker AVANCE-400 nuclear magnetic instrument, and the solvent was deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ OD), and the internal standard was four Methylsilane (TMS).

For MS measurement, use FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

High performance liquid chromatography (HPLC) analysis uses Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Waters HPLC e2695-2489 high pressure liquid chromatograph.

Chiral HPLC analysis and determination use Agilent 1260 DAD high performance liquid chromatograph.

The HPLC preparation uses Waters 2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson-281 preparative chromatographs.

For chiral preparation, Shimadzu LC-20AP preparative chromatograph was used.

CombiFlash rapid preparation instrument uses Combiflash Rf200 (TELEDYNE ISCO).

The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the size of the silica gel plate used for thin layer chromatography (TLC) is 0.15mm～0.2mm, and the size of the thin layer chromatography separation and purification product is 0.4mm. ~0.5mm.

The silica gel column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

The average value of ₅₀ measured kinase inhibition rate and IC NovoStar using a microplate reader (BMG, Germany).

The known starting materials of the present disclosure can be synthesized by or according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Darui Chemicals and other companies.

There is no special description in the examples, and the reaction can all be carried out under an argon atmosphere or a nitrogen atmosphere.

The argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1L.

The hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1L.

The pressure hydrogenation reaction uses Parr 3916EKX hydrogenator and Qinglan QL-500 hydrogen generator or HC2-SS hydrogenator.

The hydrogenation reaction is usually evacuated and filled with hydrogen, and the operation is repeated 3 times.

The microwave reaction uses the CEM Discover-S 908860 microwave reactor.

There is no special description in the examples, and the solution refers to an aqueous solution.

There are no special instructions in the examples, and the reaction temperature is room temperature, which is 20°C to 30°C.

The monitoring of the reaction progress in the examples adopts thin layer chromatography (TLC), the developing solvent used in the reaction, the eluent system of column chromatography used in the purification of the compound, and the developing reagent system of thin layer chromatography include: A: N-hexane/ethyl acetate system, B: dichloromethane/methanol system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of basic or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.

Example 1

(Ra)-17-chloro-5,13,14,22-tetramethyl-28-oxa-2,9-dithia-5,6,12,13,24-pentaazaheptane [27.6 .1.1 ^4,7 .0 ^11,15 .0 ^16,21 .0 ^20,24 .0 ^30,34] thirty-seven 1 (36), 4 (37), 6,11,14,16,18, 20,22,29,34-Undecene-23-carboxylic acid

first step

(E)-3-(3-Bromo-5-methoxyphenyl)methyl acrylate 1b

Sodium hydrogen (1.34g, 34.97mmol, 60% purity) was dissolved in tetrahydrofuran (100mL), replaced with argon three times, and methyl 2-(dimethoxyphosphoryl)acetate (6.35g, 34.87mmol) was added dropwise under ice bath. ), stirred for 30 minutes in an ice bath, and added dropwise a solution of 3-bromo-5-methoxybenzaldehyde 1a (5.00 g, 23.25 mmol) in tetrahydrofuran (30 mL), and the reaction solution was stirred and reacted at room temperature for 1 hour. Under ice bath, add ethyl acetate (100mL) and water (100mL), separate the layers, wash the organic phase with saturated sodium chloride solution (30mL×2), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure The residue obtained was purified by silica gel column chromatography with eluent system B to obtain the title product 1b (6.20 g, yield: 98.4%).

MS m/z(ESI): 271.1273.1[M+1]

Second step

Methyl 3-(3-bromo-5-methoxyphenyl)propionate 1c

1b (6.00 g, 22.1 mmol) was dissolved in methanol (75 mL) and tetrahydrofuran (75 mL) at room temperature, 5% dry rhodium carbon (600 mg) was added, and hydrogen was replaced three times. The reaction solution was stirred and reacted at room temperature for 90 minutes. The reaction solution was filtered and concentrated under reduced pressure to obtain the title product 1c (6.04 g, yield: 99.3%).

MS m/z(ESI): 273.0275.0[M+1].

third step

3-(3-bromo-5-methoxyphenyl)propionic acid 1d

Dissolve 1c (6.20g, 22.7mmol) in methanol (30mL), tetrahydrofuran (30mL) and water (30mL) at room temperature, add lithium hydroxide monohydrate (2.86g, 68.2mmol), heat at 50℃ and stir to react 1 Hour. The reaction solution was concentrated under reduced pressure, water and dichloromethane (100 mL) were added, the pH was adjusted to 2-3 with 1M HCl, and the mixed solvent (50 mL) was extracted with dichloromethane and methanol (V:V=10:1). The organic phase was washed with water (30mL×3) and saturated sodium chloride solution (30mL×2) successively, the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the title product 1d (5.80g, yield: 98.6%).

MS m/z(ESI):257.2259.2[M-1]

the fourth step

7-Bromo-5-methoxy-2,3-dihydro-1H-inden-1-one 1e-1

5-bromo-7-methoxy-2,3-dihydro-1H-inden-1-one 1e-2

1d (5.50g, 21.2mmol) was weighed into a 100mL reaction flask at room temperature, polyphosphoric acid (120g, 35.5mmol) was added, and the reaction was stirred at 95°C for 1.5 hours. Pour into ice water, add dichloromethane (200mL), separate the layers, wash the organic phase with sodium bicarbonate, water and saturated sodium chloride solution (30mL×3) successively, dry with anhydrous sodium sulfate, filter, concentrate to dryness, use The residue obtained was purified by silica gel column chromatography with eluent system B to obtain title product 1e-1 (3.50 g, yield: 68.4%) and title product 1e-2 (1.00 g, yield: 19.5%).

MS m/z(ESI): 241.1243.1[M+1]

the fifth step

6-Bromo-4-methoxy-2,3-dihydro-1H-indene 1f

1e-2 (1.00g, 4.15mmol) was dissolved in trifluoroacetic acid (10ml) at room temperature, triethylsilylhydrogen (965mg, 8.30mmol) was added, and the reaction was stirred at 80°C for 1.5 hours. The reaction solution was concentrated under reduced pressure, ethyl acetate (50mL) and water (50mL) were added, and the layers were separated. The organic phase was washed with water and saturated sodium chloride solution (30mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The obtained residue was purified by silica gel column chromatography with eluent system B to obtain the title product 1f (880 mg, yield: 93.4%).

Sixth step

6-bromo-2,3-dihydro-1H-indene-4-phenol 1g

1f (900 mg, 3.96 mmol) was dissolved in 10 mL of dichloromethane at room temperature, and 1M boron tribromide (13.9 mL, 13.9 mmol) in dichloromethane was added dropwise under an ice bath, and the reaction was stirred at room temperature for 2 hours. The reaction solution was poured into ice water, extracted with dichloromethane (50mL×2), the organic phases were combined, washed with water (30mL), saturated sodium chloride solution (30mL), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and the filtrate It was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography with eluent system B to obtain 1 g of the title product (620 mg, yield: 73.4%).

MS m/z(ESI): 211.0213.0[M-1]

Seventh step

2-ethylhexyl 3-((7-hydroxy-2,3-dihydro-1H-inden-5-yl)thio)propionate 1h

At room temperature, 1g (620mg, 2.91mmol), 2-ethylhexyl 3-mercaptopropionate (762mg, 3.49mmol), N,N-diisopropylethylamine (752mg, 5.82mmol), tris(diethylene) Benzylacetone) two palladium (133mg, 0.15mmol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (168mg, 0.29mmol) were dissolved in 20mL of dioxane, The argon was replaced three times, and the reaction was stirred at 95°C for 16 hours. After filtering through a pad of Celite, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to obtain the title product for 1 h (550 mg, yield: 53.9%).

MS m/z(ESI): 351.3[M+1]

Eighth step

6-Mercapto-2,3-dihydro-1H-indene-4-phenol 1i

Dissolve 1 h (550 mg, 1.57 mmol) in 10 mL of tetrahydrofuran under ice bath, replace argon three times, add 1M potassium tert-butoxide (5.0 mL, 5.0 mmol) in tetrahydrofuran dropwise, and stir at room temperature for 2 hours. The title product 1i is obtained, and the reaction solution is directly used for the next step.

Step 9

(3-Bromo-4-chlorophenyl)hydrazine 1k

Dissolve 3-bromo-4-chloroaniline 1j (20g, 62.58mmol, purchased from the finished product) in 96mL 25% hydrochloric acid, add dropwise a 60mL aqueous solution of sodium nitrite (7.69g, 111.46mmol) under ice bath, temperature control The temperature is less than 10°C, and the reaction is kept at 0°C for 1 hour. The above solution was added dropwise to 144 mL of stannous chloride dihydrate (98.00 g, 434.30 mmol) in 25% hydrochloric acid solution, the temperature was controlled to be less than 10°C, and the reaction was maintained at 0°C for 1 hour. After the reaction is over, add 420mL 32% sodium hydroxide dropwise under ice bath to alkalize, add 960mL water to dilute, dichloromethane extract 800mL×3, separate, the organic phase is washed with water and saturated sodium chloride each 200mL×2, anhydrous It was dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the title product 1k (19.40 g, yield: 90.42%).

MS m/z(ESI): 220.7223.0[M+1]

Tenth step

(Z)-2-(2-(3-Bromo-4-chlorophenyl)hydrazone)methyl butyrate 1l

Dissolve 1k (19.40g, 87.59mmol) in 60mL ethanol, add dropwise a 20mL ethanol solution of methyl 2-oxobutanoate (10.58g, 91.12mmol) under ice bath, and react at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, 50 mL of n-hexane was added to make a slurry and stirred, filtered, and the filter cake was collected and dried under vacuum to obtain 11 of the title product (20.00 g, yield: 71.45%).

MS m/z(ESI): 318.7320.9[M+1]

Eleventh step

4-bromo-5-chloro-3-methyl-1H-indole-2-carboxylic acid methyl ester 1m

Dissolve 1 l (20.00 g, 62.58 mmol) in 200 mL of glacial acetic acid, add zinc chloride (47.00 g, 344.84 mmol), and heat the reaction at 120°C for 1 hour. The reaction solution was poured into 500 mL ice water, a white solid precipitated, filtered, and dried in vacuum to obtain the crude title product 1m (18.50 g, yield: 97.70%), which was directly subjected to the next reaction without purification.

MS m/z(ESI): 299.9301.9[M-1]

Twelfth step

4-bromo-5-chloro-1-(3-methoxy-3-oxopropyl)-3-methyl-1H-indole-2-carboxylic acid methyl ester 1n

The crude product 1m (5.00g, 16.53mmol) was added to 40mL of acetonitrile, 1,8-diazabicyclo[5.4.0]undec-7-ene (20.81g, 82.62mmol) was added under ice bath, and methyl acrylate was added dropwise Ester (2.13g, 24.74mmol), heated under reflux and stirred for 30 minutes, and added four times each of methyl acrylate (2.13g, 24.74mol). After the reaction is over, add 100mL each of water and ethyl acetate, separate the layers, and wash the organic phase with 1N HCl (30mL×2), water (30mL×2) and saturated sodium chloride (30mL×2), and dry with anhydrous sodium sulfate After filtering, the filtrate was concentrated under reduced pressure, and the residue obtained was purified by silica gel column chromatography with eluent system A to obtain the title product 1n (900 mg, yield: 14.01%).

MS m/z(ESI): 388.0390.0[M+1]

Step 13

5-chloro-1-(3-methoxy-3-oxopropyl)-4-(3-(((4-methoxybenzyl)oxy)methyl)-1,5-dimethyl -1H-pyrazol-4-yl)-3-methyl-1H-indole-2-carboxylic acid methyl ester 1p

1n (780mg, 2.01mmol), 3-(((4-methoxybenzyl)oxy)methyl)-1,5-dimethyl-4-(4,4,5,5-tetramethyl -1,3,2-dioxaborolan-2-yl)-1H-pyrazole 1o (747mg, 2.01mmol, prepared by the well-known method "WO2017182625A1") dissolved in 20mL 1,4 -In a mixed solution of dioxane and water (V:V=4:1), replace argon three times, add 1,1'-bis(di-tert-butylphosphine)ferrocene dichloropalladium (71mg, 0.10) mmol), cesium carbonate (1.31g, 4.02mmol) was added, heated to 95°C and stirred for 16 hours, concentrated under reduced pressure to remove most of the solvent, 40mL of water was added to the reaction solution, and the reaction was extracted with ethyl acetate (20mL×3), The organic phase was washed sequentially with water (30 mL), saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered to remove the desiccant, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B. The title product 1p (720 mg, yield: 64.75%) was obtained.

MS m/z(ESI): 554.2[M+1]

Step Fourteen

5-chloro-4-(3-(hydroxymethyl)-1,5-dimethyl-1H-pyrazol-4-yl)-1-(3-methoxy-3-oxopropyl)- 3-methyl-1H-indole-2-carboxylic acid methyl ester 1q

Dissolve 1p (720 mg, 1.30 mmol) in 4 mL of dichloromethane, add trifluoroacetic acid (1.48 g, 12.98 mmol) dropwise, and react at room temperature for 30 minutes. Under ice bath, add dropwise saturated sodium bicarbonate aqueous solution to the reaction solution, adjust pH=7-8, extract with dichloromethane (30mL×3), combine the organic phases, and then the organic phases with water (20mL) and saturated sodium chloride solution (20mL) washed, dried over anhydrous sodium sulfate, filtered to remove the desiccant, the filtrate was concentrated under reduced pressure, and the residue obtained was purified by silica gel column chromatography with eluent system B to obtain the title product 1q (320mg, yield: 56.75%) .

MS m/z(ESI): 434.1[M+1]

Fifteenth step

5-chloro-4-(3-(chloromethyl)-1,5-dimethyl-1H-pyrazol-4-yl)-1-(3-methoxy-3-oxopropyl)- 3-methyl-1H-indole-2-carboxylic acid methyl ester 1r

Dissolve 1q (320 mg, 0.74 mmol) in 10 mL of dichloromethane, replace with argon three times, add thionyl chloride (132 mg, 1.11 mmol) dropwise under ice bath, and react at room temperature for 30 minutes. Add 50 mL of water to the reaction solution, stir for 10 minutes, extract with dichloromethane (30 mL×2), combine the organic phases, wash with water (30 mL), saturated sodium chloride solution (30 mL), dry with anhydrous sodium sulfate, and filter to remove Desiccant, the filtrate was concentrated under reduced pressure to obtain the crude title product 1r (350mg), the product was directly subjected to the next reaction without purification

MS m/z(ESI): 452.1[M+1]

Step sixteen

5-chloro-4-(3-(iodomethyl)-1,5-dimethyl-1H-pyrazol-4-yl)-1-(3-methoxy-3-oxopropyl)- 3-methyl-1H-indole-2-carboxylic acid methyl ester 1s

The crude product 1r (350 mg, 0.77 mmol) was dissolved in 10 mL of acetonitrile, sodium iodide (232 mg, 1.55 mmol) was added, and the mixture was heated to 80° C. and stirred for reaction for 2 hours. The reaction solution was cooled to room temperature, 50mL of water was added to the reaction solution, stirred for 30 minutes, extracted with ethyl acetate (50mL×2), the organic phases were combined, washed with water (30mL), saturated sodium chloride solution (30mL), anhydrous It was dried over sodium sulfate, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure to obtain the title product 1s (370 mg yield: 87.94%).

MS m/z(ESI): 544.1[M+1]

Seventeenth step

4-(3-(((((5(((tert-butyldiphenylsilyl)oxy)methyl)-1-methyl-1H-pyrazol-3-yl)methyl)thio)methyl Yl)-1,5-dimethyl-1H-pyrazol-4-yl)-5-chloro-1-(3-methoxy-3-oxopropyl)-3-methyl-1H-indyl Dole-2-carboxylic acid methyl ester 1u

Dissolve 1s (370mg, 0.68mmol) in 10mL methanol and 2mL tetrahydrofuran, add potassium carbonate (113mg, 0.82mmol), replace argon three times, add dropwise S-((5-(((tert-butyl diphenyl) at room temperature (Silyl)oxy)methyl)-1-methyl-1H-pyrazol-3-yl)methyl)thioacetate 1t (358mg, 0.82mmol, prepared by the well-known method "WO2017182625A1" ) In 5 mL methanol solution, stirred at room temperature and reacted for 2 hours. The reaction solution was concentrated under reduced pressure to remove most of the solvent, 50mL of water was added to the reaction solution, stirred for 30 minutes, extracted with ethyl acetate (50mL×2), combined organic phases, water (30mL), saturated sodium chloride solution (30mL) Washed, dried with anhydrous sodium sulfate, filtered to remove the desiccant, the filtrate was concentrated under reduced pressure, and the residue obtained was purified by silica gel column chromatography with eluent system B to obtain the title product 1u (640mg yield: 115.77%).

MS m/z(ESI): 812.3[M+1]

Eighteenth step

5-chloro-4-(3-((((5-(hydroxymethyl)-1-methyl-1H-pyrazol-3-yl)methyl)thio)methyl)-1,5-di Methyl-1H-pyrazol-4-yl)-1-(3-methoxy-3-oxopropyl)-3-methyl-1H-indole-2-carboxylic acid methyl ester 1v

1u (640mg, 0.79mmol) was dissolved in 10mL of tetrahydrofuran, 1.0M tetrabutylammonium fluoride (0.95mL, 0.95mmol) was added dropwise, and the reaction was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure to remove most of the solvent, 50mL of water was added to the reaction solution, extracted with ethyl acetate (50mL×2), the organic phases were combined, washed with water (30mL), saturated sodium chloride solution (30mL), anhydrous The residue was dried over sodium sulfate, filtered to remove the desiccant, the filtrate was concentrated under reduced pressure, and the residue obtained was purified by silica gel column chromatography with eluent system B to obtain the title product 1v (240 mg, yield: 53.07%).

MS m/z(ESI): 574.2[M+1]

Step Nineteen

5-chloro-4-(3-((((5-(chloromethyl)-1-methyl-1H-pyrazol-3-yl)methyl)thio)methyl)-1,5-di Methyl-1H-pyrazol-4-yl)-1-(3-methoxy-3-oxopropyl)-3-methyl-1H-indole-2-carboxylic acid methyl ester 1w

Dissolve 1 v (240 mg, 0.42 mmol) in 5 mL of dichloromethane, replace with argon three times, add thionyl chloride (60 mg, 0.50 mmol) dropwise under ice bath, and react at room temperature for 30 minutes. Add 50 mL of water to the reaction solution, stir for 10 minutes, extract with dichloromethane (30 mL×2), combine the organic phases, wash with water (30 mL), saturated sodium chloride solution (30 mL), dry with anhydrous sodium sulfate, and filter to remove Desiccant, the filtrate was concentrated under reduced pressure to obtain the title product 1w (270mg), the product was directly continued to the next step without purification.

MS m/z(ESI): 592.2[M+1]

Step Twenty

5-chloro-4-(3-((((5-(((7-hydroxy-2,3-dihydro-1H-inden-5-yl)thio)methyl)-1-methyl-1H -Pyrazol-3-yl)methyl)thio)methyl)-1,5-dimethyl-1H-pyrazol-4-yl)-1-(3-methoxy-3-oxopropane Yl)-3-methyl-1H-indole-2-methyl carboxylate 1x

Dissolve 1w (250mg, 0.42mmol) in methanol (10mL) and tetrahydrofuran (3mL) at room temperature, add dropwise a step reaction solution of 0.1M 1i (6.3mL, 0.63mmol) in tetrahydrofuran under ice bath, and stir at room temperature React for 1 hour. The reaction solution was diluted with ethyl acetate (50mL), washed successively with water (30mL×3) and saturated sodium chloride solution (30mL×2), the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and used a silica gel column The resulting residue was purified by chromatography with eluent system A to obtain the title product 1x (280 mg, yield: 91.9%).

MS m/z(ESI): 722.0[M+1]

Step 21

5-chloro-4-(3-((((5-(((7-hydroxy-2,3-dihydro-1H-inden-5-yl)thio)methyl)-1-methyl-1H -Pyrazol-3-yl)methyl)thio)methyl)-1,5-dimethyl-1H-pyrazol-4-yl)-1-(3-hydroxypropyl)-3-methyl -1H-Indole-2-methyl carboxylate 1y

Dissolve 1x (280mg, 388μmol) in tetrahydrofuran (5mL) at room temperature, cool the reaction solution to 0-5°C, slowly add 1.0M borane tetrahydrofuran solution (3.9mL) dropwise, warm the reaction solution to room temperature, continue to stir the reaction 16 Hour. The reaction solution was cooled to 0-5°C, quenched with methanol, warmed to room temperature, stirred for 30 minutes, hydrochloric acid (6.0 mL, 6.0N) was added, and stirring was continued for 30 minutes. Dichloromethane and methanol (V:V==10:1) ) Extract with mixed solvent (30mL×3), combine the organic phases, wash with saturated sodium chloride solution (30mL), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and use silica gel column chromatography to determine the eluent system A The resulting residue was purified to obtain the title product 1y (130 mg, yield: 48.3%).

MS m/z(ESI): 693.9[M+1]

Step 22

17-Chloro-5,13,14,22-tetramethyl-28-oxa-2,9-dithia-5,6,12,13,24-pentaazaheptane

^{[27.6.1.1 4,7 .0 11,15 .0 16,21 .0} 20,24 .0 30,34] thirty-seven 1 (36), 4 (37), 6,11,14,16, 18,20,22,29,34-undecene-23-methyl formate 1z

1y (130mg, 187μmol) was dissolved in toluene (10mL) and tetrahydrofuran (5ml) at room temperature, tri-n-butylphosphine (189mg, 0.94mmol) was added, argon was replaced three times, azodicarboxydipiperidine ( 236mg, 0.94mmol) in toluene solution (5mL), heated at 60°C and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system A to obtain the title product 1z (100 mg, yield: 79.0%).

MS m/z(ESI): 676.0[M+1]

Step 23

(Ra)-17-Chloro-5,13,14,22-tetramethyl-28-oxa-2,9-dithia-5,6,12,13,24-pentaazaheptane

^{[27.6.1.1 4,7 .0 11,15 .0 16,21 .0} 20,24 .0 30,34] thirty-seven 1 (36), 4 (37), 6,11,14,16, 18,20,22,29,34-Undecene-23-methyl formate 1z-1

Chiral preparation of 1z (100mg, 0.84mmol) (separation conditions: CHIRALPAK ID 250*20mm, 5um; mobile phase: Hexane/EtOH/HAc=80/20/0.1(V/V/V), flow rate: 20mL/ min), collect the corresponding components and concentrate under reduced pressure to obtain the title product 1z-1 (40mg, 40mg).

MS m/z(ESI): 676.0[M+1]

Chiral HPLC analysis: retention time 7.448 minutes, chiral purity: 100% (column: CHIRALPAK ID 150*4.6mm, 5um (with guard column); mobile phase: n-hexane/ethanol/diethylamine=70/30/ 0.1(v/v/v)).

Step 24

(Ra)-17-Chloro-5,13,14,22-tetramethyl-28-oxa-2,9-dithia-5,6,12,13,24-pentaazaheptane

^{[27.6.1.1 4,7 .0 11,15 .0 16,21 .0} 20,24 .0 30,34] thirty-seven 1 (36), 4 (37), 6,11,14,16, 18,20,22,29,34-Undecene-23-carboxylic acid 1

Dissolve 1z-1 (40mg, 59μmol) in 20mL of a mixed solution of tetrahydrofuran and methanol (V:V=1:1) at room temperature, and add lithium hydroxide monohydrate (25mg, 0.60mmol) in water (5mL) , The reaction solution was heated to 50° C., and the reaction was stirred for 1 hour. Cool to room temperature and dilute with water (15mL), concentrate under reduced pressure to remove most of the organic solvents, add dilute hydrochloric acid (1.0N) dropwise to pH=2-3, use dichloromethane and methanol (V:V=10:1) Extract with mixed solvent (50mL×2), combine the organic phases, wash with water (30mL), saturated sodium chloride solution (30mL), dry with anhydrous sodium sulfate, filter to remove the desiccant, and concentrate the filtrate under reduced pressure and use HPLC Purification (Gilson GX-281, elution system: H ₂ O (10 mmol NH ₄ OAc), ACN) to obtain the title product compound 1 (10 mg).

MS m/z(ESI): 662.0[M+1]

Chiral HPLC analysis: retention time 5.609 minutes, chiral purity: 100% (column: CHIRALPAK IF 150*4.6mm, 5um with guard column; mobile phase: Hexane/EtOH/TFA=70/30/0.1(V/V /V)).

¹ H NMR (400MHz, CDCl ₃ ) δ 7.31-7.34 (m, 1H), 7.20-7.22 (m, 1H), 7.06 (s, 1H), 5.90 (s, 1H), 5.17 (s, 1H), 5.04-5.08 (m, 1H), 4.46-4.51 (m, 1H), 3.88 (s, 3H), 3.77-3.81 (m, 1H), 3.62-3.72 (m, 2H), 3.53 (s, 3H), 3.46-3.48(m, 1H), 3.32-3.36(m, 1H), 3.20-3.26(m, 1H), 3.09-3.13(m, 1H), 2.86-3.05(m, 4H), 2.69-2.72(m , 1H), 2.09-2.30 (m, 7H), 2.04 (s, 3H).

According to X-ray powder diffraction detection, compound 1 (that is, the compound represented by formula (I)) is amorphous, and the XRPD spectrum is shown in FIG. 1.

Example 2

Weigh 3 g of the compound represented by the formula (I), add methanol to make a slurry, and filter. Add acetonitrile to the filter cake and stir, concentrate under reduced pressure to remove acetonitrile, repeat three times. Add 40 ml of water and 10 ml of acetonitrile to the residue, and freeze-dry to obtain crystal form A of the compound represented by formula (I), and its characteristic peak positions are shown in the following table:

Table 1: XRD characteristic peak positions of crystal form A

Example 3

Weigh about 10 mg of the crystal form A of the compound represented by formula (I), add 1 mL of methanol/water (volume ratio 1:1), stir and beat at room temperature, centrifuge to obtain a solid and vacuum dry to obtain the compound represented by formula (I) For crystal form B, its characteristic peak positions are shown in the table below:

Table 2: XRD characteristic peak positions of Form B

Example 4

Weigh about 10 mg of the crystal form A of the compound represented by formula (I), add 1 mL of ethanol, stir and beat at room temperature, centrifuge to obtain the solid and vacuum dry to obtain the crystal form C of the compound represented by formula (I), and its characteristic peak positions are as follows The table shows:

Table 3: XRD characteristic peak positions of Form C

Example 5

Weigh about 10 mg of crystal form A of the compound represented by formula (I), add 1.0 mL of methanol, stir to dissolve at 50°C, and cool to room temperature to obtain a solid, which is centrifuged and vacuum dried to obtain crystal form D of the compound represented by formula (I) , And its characteristic peak positions are shown in the following table:

Table 4: XRD characteristic peak positions of crystal form D

Example 6

Weigh about 10 mg of crystal form A of the compound represented by formula (I), add 1 mL of nitromethane, stir and beat at room temperature, centrifuge to obtain a solid and vacuum dry to obtain crystal form E of the compound represented by formula (I), with characteristic peaks The locations are shown in the following table:

Table 5: XRD characteristic peak positions of E crystal form

Example 7

Weigh about 10 mg of crystal form A of the compound represented by formula (I), add 1 mL of p-xylene, stir and beat at room temperature, centrifuge to obtain a solid, and then vacuum dry to obtain crystal form F of the compound represented by formula (I), with characteristic peaks The locations are shown in the following table:

Table 6: XRD characteristic peak positions of Form F

Example 8

Weigh about 10 mg of the crystal form A of the compound represented by formula (I), add 1 mL of acetonitrile, stir and beat at room temperature, centrifuge to obtain the solid and vacuum dry to obtain the crystal form G of the compound represented by formula (I), and its characteristic peak positions are as follows The table shows:

Table 7: XRD characteristic peak positions of crystal form G

Example 9

Weigh about 10 mg of the crystal form A of the compound represented by formula (I), add 1 mL of tetrahydrofuran, stir and beat at room temperature, centrifuge to obtain the solid and vacuum dry to obtain the crystal form H of the compound represented by formula (I). The characteristic peak positions are as follows The table shows:

Table 8: XRD characteristic peak positions of crystal form H

Example 10

Weigh about 10 mg of crystal form A of the compound represented by formula (I), add 0.45 mL of chloroform, stir to clear, and volatilize at room temperature to obtain a solid, to obtain crystal form I of the compound represented by formula (I), and its characteristic peak position As shown in the following table:

Table 9: XRD characteristic peak positions of Form I

Example 11

Weigh about 5 mg of the crystal form C of the compound represented by formula (I) and dissolve it in 150 μl of isopropyl acetate, add 1.0 mL of acetonitrile to precipitate a solid, centrifuge and dry under vacuum to obtain the crystal form J of the compound represented by formula (I). The characteristic peak positions are shown in the following table:

Table 10: XRD characteristic peak positions of Form J

Example 12

Weigh about 5 mg of crystal form C of the compound represented by formula (I) and dissolve it in 200 μl ethyl acetate, add 1.0 mL of acetonitrile, volatilize at room temperature to obtain a solid, and obtain crystal form K of the compound represented by formula (I), with its characteristic peak position As shown in the following table:

Table 11: XRD characteristic peak positions of crystal form K

Example 13

Weigh about 5 mg of crystal form C of the compound represented by formula (I) and dissolve it in 200 μl 2-butanone, add 1.0 mL of isopropanol, and volatilize at room temperature to obtain a solid to obtain crystal form L of the compound represented by formula (I). The characteristic peak positions are shown in the following table:

Table 12: XRD characteristic peak positions of L crystal form

Example 14

Weigh about 5 mg of crystal form C of the compound represented by formula (I) and dissolve it in 50 μl of tetrahydrofuran, add 1.0 mL of n-heptane to precipitate a solid, centrifuge and dry in vacuo to obtain crystal form M of the compound represented by formula (I). The characteristic peak positions are shown in the following table:

Table 13: XRD characteristic peak positions of crystal form M

Example 15

The crystal form B of the compound represented by formula (I) was heated at 10 degrees per minute to 170 degrees and kept for 5 minutes and then lowered to room temperature to obtain the crystal form N of the compound represented by formula (I). Its characteristic peak positions are as follows Shown:

Table 14: XRD characteristic peak positions of N crystal form

Example 16

The crystal form C of the compound represented by formula (I) was heated at 10 degrees per minute to 120 degrees and kept for 3 minutes and then lowered to room temperature to obtain crystal form O of the compound represented by formula (I). Its characteristic peak positions are shown in the table below. Show:

Table 15: XRD characteristic peak positions of O crystal form

Example 17

After placing the crystal form C of the compound represented by formula (I) at 60°C for one week, the crystal form P of the compound represented by formula (I) is obtained, and the characteristic peak positions are shown in the following table:

Table 16: XRD characteristic peak positions of P crystal form

Example 18

Weigh about 10 mg of the crystal form C of the compound represented by formula (I), add 1 mL of n-heptane, make a slurry at room temperature, and precipitate a solid, which is centrifuged to obtain the solid and dried in vacuum to obtain the crystal form Q of the compound represented by formula (I). The characteristic peak positions are shown in the following table:

Table 17: XRD characteristic peak positions of crystal form Q

Example 19

Weigh about 10 mg of the compound represented by formula (I), add 0.05 mL of NN dimethylacetamide, stir to dissolve, and volatilize at room temperature to obtain a solid, to obtain the R crystal form of the compound represented by formula (I), and its characteristic peak positions are shown in the table below Shown:

Table 18: XRD characteristic peak positions of R crystal form

Example 20

Weigh about 5 mg of the compound represented by formula (I), dissolve it in 150 μl of isopropyl acetate, add 1.0 mL of isopropanol, precipitate a solid, centrifuge and dry under vacuum to obtain crystal form C of the compound represented by formula (I).

Example 21

Weigh about 1g of the crystal form C of the compound represented by formula (I), add it to 25ml of ethanol/water (volume ratio 1:9) mixed solution, and beat at 55°C to obtain the crystal form B of the compound represented by formula (I) .

Example 22

Place each crystal sample open and flat, and examine the stability of the sample under light (4500 Lux), high temperature (40°C, 60°C), and high humidity (RH75%, RH 92.5%) conditions. The sampling period is 30 sky.

Table 19: Stability study

Example 23

The crystalline samples were placed at 4°C, 25°C/60%RH and 40°C/75%RH to investigate their stability.

Table 20: Stability of Form B

Table 21: Stability of Crystal Form C

Table 22: Stability of N crystal form

Table 23: Stability of O crystal form

Table 24: Stability of Q crystal form

Example 24 Study on hygroscopicity of crystal form

The DVS dynamic moisture adsorption analyzer was used to detect the hygroscopicity of each crystal form, and the XRPD of the samples after the DVS test of each crystal form was tested, and the XRPD results of the crystal form before and after the DVS were compared.

Table 25: Hygroscopicity study

testing sample	0.0%RH-95.0%RH	20.0%RH-80.0%RH	Crystal form
Crystal Form B	0.62%	0.30% (slightly hygroscopic)	Unconverted
Crystal Form C	1.99%	1.02% (with moisture absorption)	Transform to form O
N crystal form	1.58%	1.29% (with moisture absorption)	Unconverted
O crystal form	4.71%	1.47% (with moisture absorption)	Unconverted
Q crystal form	8.90%	8.12% (with moisture absorption)	Unconverted

Example 25

Weigh about 50 mg of crystal form C of the compound represented by formula (I), add 0.5 mL of a mixed solvent of acetone and n-hexane (V/V = 1:2), beat at room temperature, filter, collect the filter cake, and dry in vacuo to obtain 23 mg The characteristic peak positions of the S crystal form of the compound represented by formula (I) are shown in the following table:

Table 26: XRD characteristic peak positions of S crystal form

Example 26

Weigh about 200 mg of the compound represented by formula (I), under the protection of argon, slowly add 10 mL of isopropanol, heat to reflux, slowly drop to room temperature and stir, filter, collect the filter cake, and vacuum dry to obtain 160 mg of formula (I) The T crystal form of the compound is shown, and its characteristic peak positions are shown in the following table:

Table 27: XRD characteristic peak positions of Form T

Test case:

Biological evaluation

Test Example 1. The binding experiment of the compound of the present disclosure and MCL-1 protein.

The following method was used to determine the binding ability of the compounds of the present disclosure to MCL-1 protein. The experimental method is briefly described as follows.

1. Experimental materials and instruments

1. His-MCL-1 protein (Shanghai Hengrui Pharmaceutical Co., Ltd., NA)

2. Biotin labeled Bim protein (R&D, 3526/1)

3. Labeled Europium cryptate anti-6His antibody (cisbio, 61HI2KLA)

4. Affinity Streptomycin Link XL665 (cisbio, 611SAXLA)

5. Binding buffer (cisbio, 62DLBDDF)

6. Detection buffer (cisbio, 62DB1FDG)

7. Microplate reader (BMG, PHERAsta)

Second, the experimental steps

MCL-1 inhibitors can bind to MCL-1 protein to prevent the binding of MCL-1 to Bim protein. In this experiment, the binding of MCL-1 and Bim protein was detected by HTRF method to evaluate the binding ability of MCL-1 inhibitor and MCL-1 protein, and the activity of the compound was evaluated according to the Ki size.

The human recombinant protein MCL-1 (sequence 171-327; NCBI ACCESSION: AAF64255) and Bim (sequence 51-76; NCBI ACCESSION: O43521) peptides are labeled with His and biotin, respectively. 0.1nM His-MCL-1, 2.5nM bio-Bim and different concentrations of small molecule compounds (the initial concentration is 10μM, 3 times the dilution of 11 concentrations, diluted in the binding buffer) mixed and incubated at room temperature for 2 hours, and then added 0.5nM labeled europium cryptate anti-6His antibody and 1.25nM affinity streptomycin linked XL665 (diluted in detection buffer). After incubating for 2 hours at room temperature, PHERAstar was used to detect the 620nm and 665nm fluorescence signals. The data is processed using GraphPad software.

3. Experimental data

The binding ability of the compound of the present disclosure with the MCL-1 protein can be determined by the above test. The measured Ki values are shown in Table 28.

Table 28: Ki for binding of compounds of the present disclosure to MCL-1 protein.

Example number	Ki/nM	Maximum inhibition rate (%)
1	0.16	100

Conclusion: The compound of the present disclosure has a strong binding ability with MCL-1 protein, and can well inhibit the binding of MCL-1 with Bim protein. The optical activity has a certain influence on the activity of the compound.

Test Example 2: Cell Proliferation Experiment

The following method evaluates the inhibitory effect of the compounds of the present disclosure on the proliferation of AMO-1 and MV-4-11 cells by detecting the content of ATP in the cells and according to the IC50. The experimental method is briefly described as follows.

1. Experimental materials and instruments

1. AMO-1, human bone marrow plasmacytoma (Nanjing Kebai, CBP60242)

2. MV-4-11, human acute monocytic leukemia cells (ATCC, CRL-9591)

3. Fetal Bovine Serum (FBS) (GIBCO, 10099)

4. RPMI1640 (GE, SH30809.01)

5. IMDM (Gibco, 12440053)

6. 2-Mercaptoethanol (sigma, 60-24-2)

7. CellTite (Promega, G7573)

8. 96-well cell culture plate (corning, 3903)

9. Trypan blue solution (Sigma, T8154-100ML)

10. Microplate reader (BMG, PHERAsta)

11. Cell counter (Shanghai Ruiyu Biotechnology Co., Ltd., IC1000)

Second, the experimental steps

AMO-1 cells were cultured in RPMI1640 medium containing 20% FBS, and MV-4-11 cells were cultured in IMDM medium containing 10% FBS. Passage 2 to 3 times a week, with a passage ratio of 1:4 or 1: 6. During passaging, transfer the cells to a centrifuge tube, centrifuge at 1200 rpm for 3 minutes, discard the supernatant medium and add fresh medium to resuspend the cells. Add 90 μL of cell suspension to the 96-well cell culture plate at a density of 1.33×10 ⁵ cells/ml, and add only 100 μL of complete medium to the periphery of the 96-well plate. The culture plate was cultured in an incubator for 24 hours (37°C, 5% CO ₂ ).

Dilute the sample to be tested with DMSO to 20 mM, and dilute to 9 concentrations sequentially by 4 times, and set blank and control wells. Take 5 μL of the test compound solution prepared as a gradient concentration and add it to 95 μL of fresh medium. Then add 10 μL of the above-mentioned drug-containing medium solution to the culture plate. The culture plate was incubated in an incubator for 3 days (37°C, 5% CO ₂ ). In a 96-well cell culture plate, add 50μL of CellTiter-Glo reagent to each well, keep it in the dark at room temperature for 5-10 minutes, read the chemiluminescence signal value in PHERAstar, and use GraphPad software to process the data.

3. Experimental data

The inhibition of the proliferation of AMO-1 and MV-4-11 cells by the compounds of the present disclosure can be determined by the above test. The measured IC ₅₀ values are shown in Table 29.

Table 29: Compound of the present disclosure proliferation IC ₅₀ values for inhibition of AMO-1 and MV-4-11 cells.

Conclusion: The compound of the present disclosure has a good cell proliferation inhibitory effect on both AMO-1 and MV-4-11.

Test Example 3. Inhibition of the compound of the present disclosure on the enzymatic activity of human liver microsomal P450 subenzymes CYP2C9 and 2C19

The enzymatic activity of the compounds of the present disclosure on human liver microsomal P450 subenzymes CYP2C9, 2C19 was determined by the following experimental method.

1. Experimental materials and instruments

1. Phosphate buffer (20×PBS, Bioengineering (Shanghai) Co., Ltd.)

2. NADPH (ACROS, A0354537)

3. Human liver microsomes (Corning Gentest, Cat No. 452161, Lot No. 6123001, 33 Donors)

4. ABI QTrap 4000 Liquid and Mass Dual-purpose Instrument (AB Sciex)

5.Inertsil C8-3 column, 4.6×50mm, 5μm (American Dima Company)

6. CYP probe substrate 2C9 (Diclofenac/4μM, SIGMA, Cat No. D6899-10G) and 2C19 ((S)-Mephenytoin/20μM, Bailingwei Technology Co., Ltd., Cat No. 303768); positive control inhibition Agents 2C9 (Sulfafenpyrazole, SIGMA, Cat No. 526-08-9) and 2C19 (Ticlopidine, SIGMA, Cat No. T6654-1G).

Second, the experimental steps

Prepare 100mM PBS buffer, use this buffer to prepare 2.5mg/mL microsome solution, 15mM MgCl ₂ and 5mM NADPH solution, dilute the 30mM stock solution with DMSO to the concentration of 10mM, 3mM, 1mM, 0.3 mM, 0.1 mM, 0.03 mM, 0.003 mM, 0 mM series of solution I, and then with phosphate buffer (PBS), the above series of solution I was diluted 200 times to obtain a series of test solution II (150, 50, 15, 5, 1.5, 0.15, 0.015, 0μM).

Take 2.5mg/mL microsome solution, 20μM diclofenac (2C9) or 100μM ((S) _{-mephenytoin (2C19) working solution, MgCl 2} solution and compound working solution (150, 50, 15, 5, 1.5) respectively. , 0.15, 0.015, 0μM, each concentration set corresponding reaction system) 20μL each, mix well. The positive control group uses the same concentration of sulfaphenazole (2C9) or ticlopidine (2C19) instead of the compound. At the same time, 5mM Pre-incubate the NADPH solution at 37°C for 5 minutes. After 5 minutes, add 20μL of NADPH to each well, start the reaction, and incubate for 30 minutes. All incubation samples are set as dual samples. After 30 minutes, add 250μL of internal standard to all samples Acetonitrile, mix well, shake at 800 rpm for 10 minutes, then centrifuge at 3700 rpm for 10 minutes. Take 165 μL of supernatant and transfer to LC-MS/MS for analysis.

_{The values are calculated by Graphpad Prism to obtain the IC 50} value of the drug against the metabolic sites of CYP2C9 diclofenac and 2C19(S)-mephenytoin, shown in Table 30.

Table 30: Compound of the present disclosure diclofenac CYP2C9 and 2C19 (S) - IC ₅₀ value of metabolic mephenytoin site

Example number	2C9 IC 50 (μM)	2C19 IC 50 (μM)
1	16.6	＞30

Conclusion: The compound of the present disclosure has no inhibitory effect on the metabolism sites of human liver microsomes CYP2C9 diclofenac and 2C19(S)-mephenytoin, and shows better safety in drug interactions, suggesting that compound-based CYP2C9 Metabolic drug interactions caused by the inhibition of the metabolic sites of diclofenac and 2C19(S)-mephenytoin.

Claims (51)

1. A crystal form of the compound represented by formula (I),

   
2. A crystal form A of a compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 5.999, 7.002, 9.760, 11.061, 11.645, 12.118, 13.700, 16.517, 17.717, 19.406, 19.852, 20.554, 21.523 , 22.279, 23.007, 23.510, 24.018, 24.890, 25.614, 26.618, 27.637, 28.283, 28.932, 31.283 have characteristic peaks,

   
3. The X-ray powder diffraction pattern of the crystal form A of the compound represented by formula (I) according to claim 2 is shown in FIG. 2.
4. A crystal form B of a compound represented by formula (I), and its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 5.281, 8.322, 10.667, 12.483, 14.297, 17.295, 18.241, 19.934, and 27.042.

   
5. The crystal form B of the compound represented by formula (I) according to claim 4, and its X-ray powder diffraction pattern at 2θ angles is 5.281, 6.901, 8.322, 10.667, 12.483, 14.297, 15.175, 17.295, 18.241, 18.744, 19.188, 19.934, 20.679, 20.931, 22.075, 22.517, 23.137, 23.839, 25.249, 26.496, 27.042, 27.828, 29.134, 30.526, 31.518, 32.762, 33.417, 34.713, 37.933, 38.135, 48.442 have characteristic peaks.
6. The X-ray powder diffraction pattern of the crystal form B of the compound represented by formula (I) according to claim 4 is shown in FIG. 3.
7. A crystal form C of a compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 7.000, 9.202, 10.827, 12.192, 13.421, 14.204, 14.751, 14.938, 16.097, 18.268, 19.438, 21.026, 21.374 , 22.303, 23.161, 23.748, 24.682, 25.853, 29.444 have characteristic peaks,

   
8. The X-ray powder diffraction pattern of the crystal form C of the compound represented by formula (I) according to claim 7 is shown in FIG. 4.
9. A crystal form D of a compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 6.737, 8.298, 9.742, 11.242, 11.887, 12.954, 13.585, 14.115, 15.047, 16.243, 16.766, 17.925, 18.183 , 18.767, 19.693, 20.366, 21.067, 22.181, 22.640, 22.962, 23.833, 24.469, 24.890, 25.254, 25.938, 26.555, 27.711, 28.258, 28.507, 29.235, 30.094, 30.363, 30.727, 31.341, 33.015, 33.288, 34.508 , 39.325, 40.039, 41.734, 42.711 have characteristic peaks,

   
10. The X-ray powder diffraction pattern of the crystal form D of the compound represented by formula (I) according to claim 9 is shown in FIG. 5.
11. A crystal form E of a compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 5.967, 6.999, 9.626, 10.105, 11.043, 11.617, 12.753, 13.239, 13.641, 14.988, 15.486, 16.437, 16.796 There are characteristic peaks at, 17.659, 18.046, 19.326, 19.820, 21.314, 21.775, 22.162, 22.896, 23.333, 23.999, 24.305, 25.907, 26.563, 26.976, 27.526, 28.113, 28.988, 29.984, 31.165.

    
12. The crystal form E of the compound represented by formula (I) according to claim 11, and its X-ray powder diffraction pattern is shown in FIG. 6.
13. A crystal form F of a compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 10.118, 10.726, 11.073, 11.875, 12.361, 12.944, 14.098, 14.697, 15.197, 15.585, 16.597, 17.494, 18.577 There are characteristic peaks at, 18.880, 19.684, 19.837, 20.481, 21.682, 22.575, 23.073, 23.653, 24.166, 25.480, 26.315, 27.880, 28.511, 30.314, 30.868, 34.194, 40.719,

    
14. The X-ray powder diffraction pattern of the F crystal form of the compound represented by formula (I) according to claim 13 is shown in FIG. 7.
15. A crystal form G of a compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 5.934, 6.937, 9.695, 10.983, 12.020, 13.604, 16.396, 16.547, 17.623, 19.309, 19.797, 21.438, 22.107 , 22.954, 23.501, 23.880, 25.648, 26.542, 27.559, 28.142, 31.182 have characteristic peaks,

    
16. The X-ray powder diffraction pattern of the crystal form G of the compound represented by formula (I) according to claim 15 is shown in FIG. 8.
17. A crystal form H of a compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 7.718, 8.594, 13.426, 14.282, 14.788, 15.886, 16.517, 17.384, 18.271, 19.160, 20.587, 21.144, 22.083 , 22.743, 23.314, 24.215, 25.834, 28.090, 30.658 have characteristic peaks,

    
18. The X-ray powder diffraction pattern of the crystal form H of the compound represented by formula (I) according to claim 17 is shown in FIG. 9.
19. A crystal form I of a compound represented by formula (I), and its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 8.508, 10.914, 12.724, 14.787, and 16.260,

    
20. The X-ray powder diffraction pattern of the crystal form I of the compound represented by formula (I) according to claim 19 is shown in FIG. 10.
21. A crystal form J of a compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 5.743, 10.683, 11.526, 13.219, 14.351, 15.821, 16.164, 17.091, 17.333, 18.566, 21.356, 23.311, 25.193 , 26.034, 26.702, 27.038, 29.344, 29.977, 33.657, 35.309 have characteristic peaks,

    
22. The crystal form J of the compound represented by formula (I) according to claim 21, and its X-ray powder diffraction pattern is shown in FIG. 11.
23. A crystal form K of a compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 5.838, 11.996, 13.592, 19.262, 19.745, 22.060, 22.912, 24.807, 25.774, 27.235, 27.627, 28.188, 30.500 , There are characteristic peaks at 31.247,

    
24. The crystal form K of the compound represented by formula (I) according to claim 23, and its X-ray powder diffraction pattern is shown in FIG. 12.
25. A crystal form L of the compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 6.878, 9.075, 10.695, 12.040, 13.241, 13.960, 15.964, 19.283, 20.308, 21.028, 21.888, 23.326, 24.236 , 24.656, 25.568, 25.922, 28.155, 29.374, 31.777, 36.458, 40.122, 43.686, 47.006 have characteristic peaks,

    
26. The X-ray powder diffraction pattern of the L crystal form of the compound represented by formula (I) according to claim 25 is shown in FIG. 13.
27. A crystal form M of a compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 6.362, 8.845, 9.799, 10.421, 10.824, 12.128, 12.920, 14.178, 15.321, 16.941, 17.376, 17.940, 18.693 There are characteristic peaks at, 19.853, 21.125, 22.604, 23.628, 24.456, 25.143, 25.748, 26.517, 27.482, 28.435, 29.464, 30.068, 32.062,

    
28. The X-ray powder diffraction pattern of the M crystal form of the compound represented by formula (I) according to claim 27 is shown in FIG. 14.
29. The N crystal form of a compound represented by formula (I) has characteristic peaks in its X-ray powder diffraction pattern at 2θ angles of 5.215, 8.261, 10.555, 12.382, 13.903, 16.676, 17.359, 18.083, and 20.288.

    
30. The N crystal form of the compound represented by formula (I) according to claim 29, and its X-ray powder diffraction pattern at 2θ angles is 5.215, 8.261, 10.555, 12.382, 13.903, 14.528, 16.676, 17.359, 18.083, 18.284, There are characteristic peaks at 20.288, 22.343, 23.296, 23.777, 26.812, 28.196, 29.067, 30.910, 32.931.
31. The N crystal form of the compound represented by formula (I) according to claim 29, and its X-ray powder diffraction pattern is shown in FIG. 15.
32. A crystal form O of the compound represented by formula (I), and its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 7.026, 9.353, 11.154, 12.308, 15.113, 15.543, 16.396, 18.634, 18.980, and 21.526.

    
33. The O crystal form of the compound represented by formula (I) according to claim 32, and its X-ray powder diffraction pattern at 2θ angles is 7.026, 9.353, 11.154, 12.308, 15.113, 15.543, 16.396, 16.717, 17.734, 18.634, There are characteristic peaks at 18.980, 19.881, 20.897, 21.526, 21.750, 22.397, 23.206, 24.024, 25.173, 25.880, 26.643, 27.878, 27.962, 28.681, 29.495, 30.026, 30.891, 31.476, 32.653.
34. The X-ray powder diffraction pattern of the O crystal form of the compound represented by formula (I) according to claim 32 is shown in FIG. 16.
35. A crystal form P of a compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 7.085, 8.452, 9.395, 10.398, 10.701, 11.198, 12.375, 12.598, 13.134, 14.225, 15.163, 15.519, 16.400 , 16.804, 17.488, 18.113, 18.685, 19.055, 20.905, 21.530, 21.845, 22.403, 23.520, 24.047, 25.241, 25.853, 26.153, 26.638, 27.164, 27.458, 27.795, 28.679, 29.437, 30.068, 31.466, 32.005, 32.678 Characteristic peaks,

    
36. The X-ray powder diffraction pattern of the P crystal form of the compound represented by formula (I) according to claim 35 is shown in FIG. 17.
37. A crystal form Q of the compound represented by formula (I), and its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 7.441, 11.821, 13.781, 14.683, 16.886, 17.614, 18.673, 19.838, and 20.495.

    
38. The crystal form Q of the compound represented by formula (I) according to claim 37, and its X-ray powder diffraction pattern at 2θ angles is 7.441, 8.381, 10.030, 11.125, 11.821, 13.075, 13.781, 14.683, 16.277, 16.886, There are characteristic peaks at 17.614, 18.673, 19.838, 20.495, 22.155, 22.724, 23.464, 23.938, 24.366, 24.975, 26.051, 26.406, 27.121, 27.963, 28.435, 31.415, 32.384, 33.183, 34.657.
39. The X-ray powder diffraction pattern of the crystal form Q of the compound represented by formula (I) according to claim 37 is shown in FIG. 18.
40. A crystal form R of a compound represented by formula (I), and its X-ray powder diffraction pattern at 2θ angles is 6.747, 7.763, 9.143, 11.137, 11.942, 13.115, 13.584, 14.113, 15.042, 16.240, 17.467, 17.983, 18.741 , 19.343, 19.643, 21.045, 22.343, 23.785, 24.101, 24.646, 25.543, 26.822, 27.398, 27.926, 28.570, 29.164, 30.912, 31.584, 32.426, 33.352 have characteristic peaks,

    
41. The X-ray powder diffraction pattern of the R crystal form of the compound represented by formula (I) according to claim 40 is shown in FIG. 19.
42. The crystal form of the compound represented by formula (I) according to any one of claims 1-41, wherein the error range of the 2θ angle is ±0.2.
43. A method for preparing crystal form B of the compound represented by formula (I) according to any one of claims 4-6, the method comprising: mixing the compound represented by formula (I) with an appropriate amount of solvent to volatilize the crystal form , The solvent is one or more of isopropyl acetate, methyl isobutyl ketone, isoamyl alcohol, and butyl acetate; or,

    The compound represented by the formula (I) is mixed with an appropriate amount of solvent to be slurried, and the solid is separated. The solvent is methanol/water, ethanol/water, ethyl acetate/n-heptane.
44. A method for preparing the N crystal form of the compound represented by formula (I) according to any one of claims 29-31, the method comprising: heating the B crystal form of the compound represented by formula (I) to a temperature greater than 120 ℃, then cool down and crystallize.
45. A method for preparing the crystal form O of the compound represented by formula (I) according to any one of claims 32-34, the method comprising: heating the crystal form C of the compound represented by formula (I) to greater than 90 ℃, then cool down and crystallize.
46. A method for preparing the Q crystal form of the compound represented by formula (I) according to any one of claims 37-39, the method comprising: mixing and beating the compound represented by formula (I) with an appropriate amount of solvent, and separating Solid, the solvent is one or more of methylene chloride and n-heptane; or

    The compound represented by the formula (I) is mixed with a solvent, dissolved at elevated temperature, and crystals are precipitated and separated at a lower temperature. The solvent is acetone.
47. A pharmaceutical composition comprising the crystal form of the compound represented by formula (I) according to any one of claims 1-42, and one or more pharmaceutically acceptable carriers or excipients, preferably The crystal form is selected from crystal form A, crystal B, crystal C, crystal D, crystal E, crystal F, crystal G, crystal H, crystal I, crystal J, crystal K, One or more of L crystal type, M crystal type, N crystal type, O crystal type, P crystal type, Q crystal type, and R crystal type.
48. A pharmaceutical composition prepared by the crystal form of the compound represented by formula (I) according to any one of claims 1-42 and one or more pharmaceutically acceptable carriers or excipients Preferably, the crystal form is selected from crystal form A, crystal B, crystal C, crystal D, crystal E, crystal F, crystal G, crystal H, crystal I, crystal J, crystal K One or more of crystal type, L crystal type, M crystal type, N crystal type, O crystal type, P crystal type, Q crystal type, and R crystal type.
49. A use of the crystal form of the compound represented by formula (I) according to any one of claims 1-42 or the pharmaceutical composition according to claims 47 and 48 in the preparation of a medicament for inhibiting MCL-1, Preferably, the crystal form is selected from the group consisting of crystal form A, crystal form B, crystal form C, crystal form D, crystal form E, crystal form F, crystal form G, crystal form H, crystal form I, crystal form J, crystal form K , L crystal form, M crystal form, N crystal form, O crystal form, P crystal form, Q crystal form, R crystal form one or more.
50. A preparation method of a pharmaceutical composition, comprising the step of mixing the crystal form of the compound represented by formula (I) according to any one of claims 1-42 with a pharmaceutically acceptable carrier, preferably the crystal form Selected from crystal form A, crystal B, crystal C, crystal D, crystal E, crystal F, crystal G, crystal H, crystal I, crystal J, crystal K, crystal L, One or more of M crystal form, N crystal form, O crystal form, P crystal form, Q crystal form, and R crystal form.
51. A crystal form of the compound represented by formula (I) according to any one of claims 1-42 or the pharmaceutical composition according to claims 47 and 48 is prepared for the treatment or prevention of tumors, autoimmune diseases or For use in drugs for immune system diseases, the tumors are preferably selected from bladder cancer, brain tumors, breast cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, leukemia, kidney cancer, colon cancer, rectal cancer, and colon Rectal cancer, esophageal cancer, liver cancer, stomach cancer, head and neck cancer, skin cancer, lymphoma, pancreatic cancer, melanoma, myeloma, bone cancer, neuroblastoma, glioma, sarcoma, lung cancer, lung cancer, thyroid cancer, and prostate Cancer, preferably the crystal form is selected from crystal form A, crystal form B, crystal form C, crystal form D, crystal form E, crystal form F, crystal form G, crystal form H, crystal form I, crystal form J, crystal form K One or more of crystal form, L crystal form, M crystal form, N crystal form, O crystal form, P crystal form, Q crystal form, and R crystal form.

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