Maleate of nicotinyl alcohol ether derivative, crystal form thereof, and application thereof

Abstract

To provide a crystal form of a compound for treating diseases related to a PD-1 / PD-L1 signaling pathway, a preparation method therefor, a composition containing the crystal form, and the use of the crystal form in the preparation of a medicine for treating diseases related to a PD-1 / PD-L1 signaling pathway.SOLUTION: The present invention provides a crystal form A solid substance of isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate. When powder X-ray diffraction analysis is used and Cu target radiation experimental conditions are adopted, the position of diffraction peak, 2θ value (°) or d value (Å) and relative intensity (%) of diffraction peak have specific characteristics.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention belongs to the technical field of medicine and discloses a maleate salt of a nicotinyl alcohol ether derivative, its crystalline form and its use, i.e. isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate, its preparation method, its crystalline form, pharmaceutical composition and its use. Specifically, the present invention relates to isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate as shown in Formula I, its stereoisomers, its preparation method, its crystalline form, a composition comprising said compound or said crystalline form, and the use of said compound in the manufacture of a medicament for the treatment or prevention of diseases related to the PD-1 / PD-L1 signal pathway, such as cancer, infectious disease, autoimmune disease, etc. [Background technology]

[0002] Through in-depth research into tumor immunity, we know that the tumor microenvironment can protect tumor cells from being recognized and killed by the immune system. Immune escape of tumor cells plays a very important role in tumor development and progression. In 2013, Science magazine ranked tumor immunotherapy first among the top 10 breakthroughs, which once again made immunotherapy the "focus" of the tumor treatment field. The activation or inhibition (suppression) of immune cells is controlled by positive and negative signals. Programmed death 1 (PD-1) / PD-1 ligand (PD-L1) is a negative immune regulatory signal that inhibits the immune activity of tumor-specific CD8+ T cells and mediates immune escape.

[0003] The ability of tumor cells to escape the immune system is achieved by the binding of PD-L1 produced on their surface to the PD-1 protein on T cells. The tumor microenvironment in the body causes infiltrating T cells to highly express PD-1 molecules, and tumor cells to highly express PD-L1 and PD-L2, which are ligands of PD-1. This causes the PD-1 pathway to remain active in the tumor microenvironment, inhibiting the function of T cells, which cannot detect the tumor and cannot issue a command to the immune system to attack the tumor and kill the tumor cells. PD-1 antibodies are antibody proteins against PD-1 or PD-L1, which can block the above pathway by inhibiting the binding of PD-1 to PD-L1, partially restoring the function of T cells, and allowing these cells to continue killing tumor cells.

[0004] Recently, a series of surprising research findings have confirmed that PD1 / PD-L1 blocking antibodies have strong antitumor activity against various tumors, making them attractive. On September 4, 2014, Merck's Keytruda® (pembrolizumab) became the first PD-1 monoclonal antibody approved by the FDA for the treatment of patients with advanced or unresectable melanoma that has failed other drugs. Currently, Merck is testing the potential of Keytruda in more than 30 types of cancer, including various blood cancers, lung cancer, breast cancer, bladder cancer, gastric cancer, and head and neck cancer. On December 22, 2014, pharmaceutical giant Bristol Myers Squibb lived up to the expectations and took the lead in obtaining accelerated approval from the US Food and Drug Administration (FDA). The company's anti-cancer immunotherapy nivolumab was listed under the trade name Opdivo for the treatment of patients with unresectable or metastatic melanoma that has not responded to other treatments, making it the second PD-1 inhibitor to be listed in the United States after MSD's Keytruda. On March 4, 2015, the FDA approved nivolumab for the treatment of metastatic squamous non-small cell lung cancer whose disease has progressed during or after platinum-based chemotherapy. MSD announced data from the Phase Ib KEYNOTE-028 study of Keytruda (pembrolizumab) for the treatment of solid tumors, showing that treatment with Keytruda achieved a 28% overall response rate (ORR) in 25 patients with pleural mesothelioma (PM), with 48% of patients achieving stable disease and a disease control rate of 76%. Patients with advanced Hodgkin lymphoma (HL) who have failed currently approved drugs can achieve complete remission following treatment with MSD's Keytruda and BMS's Opdvio. At the 2015 AACR Annual Meeting, Leisha A. Emens, MD, PhD, associate professor of oncology at the Johns Hopkins Kimmel Cancer Center, reported that Roche's anti-PD-L1 monoclonal antibody MPDL3280A showed durable therapeutic activity in advanced triple-negative breast cancer.

[0005] Tumor immunotherapy is considered to be the next revolution in cancer treatment after targeted therapy, but mAb drugs have some disadvantages, such as being easily degraded by proteases, being unstable in the body and not being able to be taken orally, being prone to immune cross-reactions, not being easy to control product quality and having high manufacturing technology requirements, being difficult to mass-prepare and purify, which makes manufacturing costs high, and being inconvenient because it can only be used by injection or infusion, etc. Therefore, PD1 / PD-L1 interaction small molecule inhibitors are a better choice for tumor immunotherapy.

[0006] In International Application PCT / CN2017 / 085418, the present inventors disclosed isopropyl (S)-N-[2-(pyridinyl-3-methoxy)-4-(2-bromo-3-(phenyl)benzyloxy)-5-chlorobenzyl]serinate hydrochloride and its use in the manufacture of a medicament for the prevention or treatment of diseases related to the PD-1 / PD-L1 signal pathway, such as cancer, infectious diseases, autoimmune diseases, etc. In subsequent studies, the present inventors found that the maleate salt of this compound has stronger stability and efficacy compared with its hydrochloride salt. The hydrochloride salt of Example 1 of the present invention is prepared as a comparative example in full compliance with the preparation method of International Patent Application PCT / CN2017 / 085418. Summary of the Invention [Problem to be solved by the invention]

[0007] The technical problem solved by the present invention is to provide isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate of structural formula (I), which inhibits the interaction of PD-1 / PD-L1, and its stereoisomers, preparation methods thereof, pharmaceutical compositions thereof, and uses thereof in the manufacture of medicaments for the prevention or treatment of diseases associated with the PD-1 / PD-L1 signal pathway. [Means for solving the problem]

[0008] In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0009] The first aspect of the technical solution of the present invention is to provide isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate [(S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serine acid isopropyl maleate] represented by formula (I) and its stereoisomers. [ka]

[0010] The second aspect of the technical solution of the present invention provides a crystalline form A solid material of isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate, which has the following characteristics in terms of diffraction peak position, 2θ value (°) or d value (Å), and relative intensity (%) of diffraction peak when powder X-ray diffraction analysis is used and Cu target radiation experimental conditions are adopted: [Table 1]

[0011] In the above crystalline form A solid material, the infrared spectrum shows 3059, 2984, 2841, 2761, 2519, 2170, 1988, 1968, 1807, 1741, 1716, 1623, 1602, 1580, 1505, 1481, 1460, 1446, 1425, 1401, 1389, 1368, 1309, 1262, 1242, 1205, 1171, 1111, 1095, 1069, 1040, 1004, 972, 953, 924, 884, 870, 864, 854, 824, 788, 761, 721, 703, 662 cm -1±2cm -1 is a characteristic peak position in the infrared spectrum exhibited by the crystalline form A solid material.

[0012] The above-mentioned crystalline form A solid material, when analyzed by differential scanning calorimetry, has an endothermic peak at 175°C ± 3°C in the DSC spectrum at a heating rate of 10°C / min.

[0013] The second aspect of the technical solution of the present invention also provides a mixed crystal solid material of isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate, comprising any non-zero proportion of said crystalline form A solid material of isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate.

[0014] The third aspect of the technical solution of the present invention provides a method for preparing (manufacturing) the compound in the first aspect and the crystalline form A solid substance in the second aspect. [ka] The preparation of the compound of formula (I) is as follows: reacting isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate with maleic acid in a solvent to form a salt, most preferably the solvent is isopropyl alcohol, tetrahydrofuran or acetone; Crystallizing the obtained isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate in a mixed solvent of acetone and water, the ratio of acetone to water is in the range of 200:1 to 1:1, preferably said ratio is in the range of 50:1 to 5:1, most preferably said ratio is in the range of 25:1 to 10:1. During the preparation process of the compound of formula (I) and its solvates and salts, polycrystals may appear under different crystallization conditions.

[0015] A fourth aspect of the technical solution of the present invention provides a pharmaceutical composition comprising isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate and its stereoisomers in the first aspect of the present invention or crystalline form A solid substance in the second aspect as an active ingredient, and a pharma- ceutically acceptable carrier or excipient.

[0016] The present invention further relates to a pharmaceutical composition comprising the compound of the present invention as an active ingredient. The pharmaceutical composition can be prepared by a method well known in the art. Any dosage form suitable for use in humans or animals can be prepared by combining the compound of the present invention with one or more pharma- ceutically acceptable solid or liquid excipients and / or adjuvants. The content of the compound of the present invention in the pharmaceutical composition is usually 0.1-95% by weight.

[0017] The compound of the present invention or a pharmaceutical composition containing the same can be administered in a unit dosage form, and the route of administration can be oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal, intramucosal oral cavity, intraocular, intrapulmonary and airway, transdermal, intravaginal, rectal, or other enteral or parenteral.

[0018] The dosage form can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. The liquid dosage form can be a solution (including true solution and colloidal solution), an emulsion (including o / w type, w / o type and double emulsion), a suspension, an injection (including water injection, powder injection and drip), an eye drop, a nose drop, a lotion, a tincture, etc., the solid dosage form can be a tablet (including ordinary tablets, enteric-coated tablets, troches, dispersible tablets, chewable tablets, effervescent tablets, oral disintegrating tablets), a capsule (including hard capsules, soft capsules, enteric-coated capsules), a granule, a powder, a pellet, a drop pill, a suppository, a film, a patch, a gas (powder) spray, a spray, etc., and the semi-solid dosage form can be an ointment, a gel, a paste, etc.

[0019] The compounds of the present invention can be formulated into general preparations, as well as sustained release preparations, controlled release preparations, targeted preparations, various microparticle delivery systems, and the like.

[0020] In order to formulate the compounds of the present invention into tablets, a wide variety of excipients known in the art such as diluents, binders, wetting agents, disintegrants, lubricants and glidants can be used. The diluent may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent may be water, ethanol, or isopropanol, etc.; the binder may be starch syrup, dextrin, syrup (molasses), honey, glucose solution, microcrystalline cellulose, gum arabic, gelatin syrup, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrant may be dry starch, microcrystalline cellulose, low-substituted hydroxypropylcellulose, crosslinked polyvinylpyrrolidone, croscarmellose sodium, sodium starch glycolate, sodium bicarbonate, and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfonate, etc.; the lubricant and glidant may be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0021] Furthermore, tablets may be further formulated into coated tablets (coated tablets) such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets.

[0022] To prepare the dosage unit into capsules, the compound of the present invention as an active ingredient can be mixed with a diluent and a glidant, and the mixture can be directly put into a hard or soft capsule. The compound of the present invention as an active ingredient can also be formulated into granules or pellets with a diluent, a binder and a disintegrant, and then put into a hard or soft capsule. The capsules of the compound of the present invention can also be prepared using various diluents, binders, wetting agents, disintegrants or glidants for preparing tablets of the compound of the present invention.

[0023] In order to formulate the compound of the present invention into an injection, water, ethanol, isopropanol, propylene glycol or a mixture thereof may be used as a solvent, and an appropriate amount of a solubilizer (solubilizer), cosolvent, pH adjuster, and osmotic pressure adjuster commonly used in the art may be added. The solubilizer or cosolvent may be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc., the pH adjuster may be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc., and the osmotic pressure adjuster may be sodium chloride, mannitol, glucose, phosphate, acetate, etc. In addition, when preparing a freeze-dried powder injection, mannitol, glucose, etc. may be added as a proppant.

[0024] Furthermore, coloring agents, preservatives, fragrances, flavoring agents or other additives may be added to the pharmaceutical preparations, if necessary.

[0025] In order to achieve the intended purpose and enhance the therapeutic effect, the medicament or pharmaceutical composition of the present invention can be administered by any known administration method.

[0026] The dosage of the compound or pharmaceutical composition of the present invention can vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the administration route, and the dosage form. In general, the suitable daily dosage of the compound of the present invention is in the range of 0.001 to 150 mg / kg body weight, preferably in the range of 0.01 to 100 mg / kg body weight. The above dosage may be administered in one dosage unit or in divided dosage units, depending on the administration regimen including the clinical experience of the physician and the use of other therapeutic means.

[0027] The compound or composition of the present invention can be administered alone or in combination with other therapeutic agents or symptom-improving agents.When the compound of the present invention exerts synergistic effects with other therapeutic agents, its dosage should be adjusted according to actual situation.

[0028] A fifth aspect of the technical solution of the present invention provides a use of isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate and its stereoisomers or crystalline form A solid material in the second aspect in the manufacture of a medicament for the prevention and / or treatment of a disease associated with the PD-1 / PD-L1 signal pathway.

[0029] The disease associated with the PD-1 / PD-L1 signal pathway is selected from the group consisting of cancer, infectious disease, and autoimmune disease. The cancer is selected from the group consisting of skin cancer, lung cancer, urinary tract tumor, blood tumor, breast cancer, glioma, digestive system tumor, reproductive system tumor, lymphoma, nervous system tumor, brain tumor, and head and neck cancer. The infectious disease is selected from the group consisting of bacterial infection and viral infection. The autoimmune disease is selected from the group consisting of organ-specific autoimmune disease and systemic autoimmune disease, and the organ-specific autoimmune disease includes chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia due to chronic atrophic gastritis, pulmonary hemorrhagic nephritic syndrome, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuropathy. The systemic autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, and autoimmune hemolytic anemia. Effect of the Invention

[0030] Compared with isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate hydrochloride, the compound of the present invention, isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate, has a stable crystal form and good stability against light irradiation, high humidity and high temperature environments, and has a high tumor inhibition rate against various tumors in a mouse subcutaneously transplanted tumor model or an NSG tumor-bearing mouse model reconstituted with a human immune system. [Brief description of the drawings]

[0031] [Figure 1] Differential scanning calorimetry / thermogravimetry spectrum of the compound of Example 1. [Diagram 2] Differential scanning calorimetry / thermogravimetry spectrum of the compound of Example 2. [Diagram 3] Powder X-ray diffraction pattern of the compound of Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present invention will be further described below in conjunction with specific examples, but the scope of the present invention is not limited thereto.

[0033] Test equipment: For nuclear magnetic resonance spectroscopy, a Bruker AVANCE III 500 high-resolution superconducting nuclear magnetic resonance spectrometer is used. For mass spectrometry, a QSTAR Elite LC / MS / MS system is used. For elemental analysis, a FLASH1112 trace element analyzer and a MX-5 part per million balance are used. For ultraviolet analysis, a Shimadzu UV-2700 UV-Visible spectrophotometer is used. For specific rotation measurements, a PE 343 polarimeter, USA is used. For powder X-ray diffraction analysis, a D8-Advance X-ray diffractometer is used. For differential scanning calorimetry / thermogravimetry (DSC / TG), a Mettler TGA / DSC3, Switzerland is used. + A thermal analyzer is used.

[0034] 1. Salt Preparation Example 1: isopropyl (S)-N-[2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-(phenyl)benzyloxy)-5-chlorobenzyl]serinate hydrochloride (this example is a comparative example, this compound is a known compound, and its preparation method is exactly the same as Example 6 of International Application PCT / CN2017 / 085418). [ka]

[0035] A 100 ml round bottom flask was charged with 598 mg of (S)-N-[2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-(phenyl)benzyloxy)-5-chlorobenzyl]serine and 60 ml of anhydrous isopropanol, and 6 ml of sulfoxide chloride and 2 drops of DMF were added while stirring in an ice-water bath. The mixture was stirred at room temperature for 2 hours and then heated to reflux until the reaction was complete. The solvent was removed under reduced pressure to give isopropyl (S)-N-[2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-(phenyl)benzyloxy)-5-chlorobenzyl]serinate hydrochloride as a white solid. 1 H NMR(400MHz,DMSO-d6) δ 9.62(s,1H,-HCl),9.40(s,1H,-HCl),9.10(s,1H,-ArH),8.86(d,1H,-ArH),8.59(d,J=7.6Hz,1H,-ArH),8.01-7.9 1(m,1H,-ArH),7.73-7.64(m,2H,-ArH),7.57-7.46(m,3H,-ArH),7.46-7.37(m,4H,-ArH),7.16(s,1H,-ArH),5.47 (s,2H,-CH2-),5.36(s,2H,-CH2-),4.92(m1H,-CH-),4.30-4.15(m,2H,-CH2-),4.05(s,1H,-CH-),3.97(dd,J=12. 0,3.0Hz,1H,-CH2-),3.84(dd,J=12.0,3.8Hz,1H,-CH2-),1.20(d,J=6.4Hz,3H,-CH3),1.18(d,J=6.4Hz,3H,-CH3). MS(FAB):640(M).

[0036] Example 2: Isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate (IMMH-010) At room temperature, isopropyl (S)-N-[2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-(phenyl)benzyloxy)-5-chlorobenzyl]serinate (2.6 g) and isopropanol (9 ml) were added to a 50 ml reaction flask. The mixture was heated to 40° C., stirred for 0.5 hours, and maleic acid (0.594 g)-isopropanol (4 ml) solution was added dropwise. The temperature was controlled at 35-45° C., and after solids were precipitated, the mixture was stirred at that temperature for 0.5 hours, naturally cooled to room temperature, and stirred overnight. The next day, the mixture was filtered under suction, and the filter cake was washed with 0.5 ml of isopropanol and 0.5 ml of acetone in sequence to obtain a pale yellow solid, which is the crude product of isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate. At room temperature, the crude product and acetone (26 ml) were added to a 50 ml reaction flask, heated to reflux, and 1.4 ml of purified water was added dropwise. After complete dissolution, the mixture was subjected to hot filtration. After filtration, the filter cake was transferred to a 50 ml reaction flask, naturally cooled to room temperature, and stirred overnight for crystallization. The next day, the mixture was cooled to 5-15°C, stirred for 2 hours, and then filtered under suction. The filter cake was washed with 0.5 ml of acetone and forced air dried at 45°C to a constant weight to obtain the pure product (0.73 g) as a white solid.

[0037] 2. Confirmation of the structure of the compound of Example 2 1) Elemental analysis: (1) Test equipment: FLASH1112 trace element analyzer and MX-5 one-millionth balance. (2) Test method: Carbon, hydrogen and nitrogen were measured twice in parallel. (3) Measurement results: [Table 2]

[0038] 2) High resolution mass spectrometry: (1) Test equipment: QSTAR Elite LC / MS / MS system (2) Test conditions: ESI source (3) Actual data: [Table 3]

[0039] 3) UV absorption spectrum: (1) Test equipment: Shimadzu UV-2700 UV-Visible Spectrophotometer (2) Test method: The sample was prepared into a solution of a given concentration, and the absorption value was measured in the range of 190 to 400 nm using a 1 cm absorption cell, using the same batch of solvent as a blank control. Solvents: water-methanol (1:1), 0.1 mol / L hydrochloric acid-methanol (1:1), 0.1 mol / L sodium hydroxide-methanol (1:1) Test solution concentration: 20 μl / ml. (3) Actual data [Table 4]

[0040] 4) Infrared absorption spectrum: (1) Test equipment: British PE (Spectrum 400) infrared spectrometer (2) Test conditions: Attenuated total reflectance (ATR) infrared spectroscopy, direct powder injection method [Table 5]

[0041] 5) Nuclear magnetic resonance hydrogen and carbon spectra: (1) Test equipment: Bruker AVANCE III 500 type high-resolution superconducting nuclear magnetic resonance spectrometer (2) Test conditions: the solvent was DMSO-d6 and the internal standard was TMS. [Table 6] [Table 7]

[0042] 6) Specific rotation: (1)Test equipment: Model 343 polarimeter manufactured by PE in the United States. (2) Test method: The product of the present invention was accurately weighed, dissolved in DMSO, and quantitatively diluted to a solution containing about 50 mg per ml, and the specific rotation was measured. (3) Test temperature: 20℃ (4) Results When DMSO was used as a solvent and the measurement concentration was 50 mg / ml, the specific rotation of the compound of Example 2 was +5.5°. [α] 20 589 = +5.5° (C=5, DMSO).

[0043] 3. Crystal Form Analysis of the Compound of Example 2 1) Powder X-ray diffraction analysis: (1) Test equipment: D8-Advance X-ray diffractometer (2) Test conditions: operating voltage: 40 kV, operating current: 40 mA, Cu target, scanning speed: 0.02 degrees / step, data acquisition time: 0.1 seconds / step. (3) Test results (see Figure 3): [Table 8(1)] [Table 8(2)] (4) Analysis: Powder X-ray diffraction analysis showed that the compound of Example 2 was a crystalline substance. 2) Differential Scanning Calorimetry / Thermogravimetry (DSC / TG): (1) Test equipment: TGA / DSC3 from Mettler, Switzerland + thermal analyzer (2) Parameter setting: start temperature: 35℃; end temperature: 250℃; heating rate: 10℃ / min. (3) Actual data: DSC: Peak temperature: 174.68°C (endothermic). TGA: Weight loss began at about 170°C, and weight loss became evident at 175°C. (4) Analysis: DSC showed an endothermic peak at 174.68°C, which should be attributed to the heat absorbed from melting the compound of Example 2. TGA showed essentially no change in the thermogravimetric curve and no weight loss up to 160°C, suggesting that the compound of Example 2 did not contain a crystallization solvent. Upon increasing the temperature to the apex of the DSC endothermic peak (about 175°C), weight loss became evident, which was associated with the melting decomposition of the sample. This DSC endothermic peak was the decomposition point of the compound of Example 2.

[0044] 4. Comparison of stability between the compound of Example 1 and the compound of Example 2 1) Stability of the compound of Example 1 (1) Physical and chemical properties Appearance: Off-white powder Melting point: 119.36°C (DSC method) (see Figure 1) Purity: 99.5% (HPLC standardization method) logP=2.4 (2) Influence factor testing [Table 9]

[0045] The compound of Example 1 shows no obvious changes in appearance, melting point and impurity content even after being placed under high temperature of 60°C for 5 days, which suggests that the compound of Example 1 is stable under high temperature conditions. After being placed under high humidity of RH92.5% for 5 days, the compound shows serious moisture absorption and becomes a colorless highly viscous liquid, but there is no change in related substances. Under lighting conditions, the compound becomes a transparent bulk, and its impurity content increases to 4.0%, which suggests that the compound is unstable under lighting conditions.

[0046] 2) Stability of the compound of Example 2 (1) Physical and chemical properties Appearance: White solid Melting point: 174.68℃ (DSC method) (see Figure 2) Purity: 98.7% (HPLC standardization method) logP=3.2 (2) Influence factor testing [Table 10]

[0047] The compound of Example 2 was stable even under conditions of light, high temperature and high humidity.

[0048] 5. Comparison of in vivo antitumor effects of the compound of Example 1 and the compound of Example 2 against mouse melanoma B16F10 Objective of the experiment: The in vivo antitumor effects of the compound of Example 1 and the compound of Example 2 as PD-L1 inhibitors against mouse melanoma B16F10 were evaluated in a mouse subcutaneously transplanted tumor model. Experimental Solutions: Animal grouping: The experimental animals were divided into a solvent control group, a cyclophosphamide 80 mg / kg group (CTX), a compound of Example 1 5 mg / kg group and a compound of Example 2 5 mg / kg group and a compound of Example 2 10 mg / kg group, respectively. Experimental procedure: Subcultured B16F10 tumor cells were homogenized, washed twice with sterile normal saline, counted, and the cell concentration of the tumor cell suspension was adjusted to 9 × 10 in normal saline. 6 The cell suspension was adjusted to 0.2 ml / ml. 0.2 ml of this cell suspension was inoculated into the right armpit of C57BL / 6J mice, and the day was recorded as day 0. The day after inoculation, the animals were randomly divided into groups, weighed, and dosed. Mice in the vehicle control group were orally administered 0.5% CMC daily. Cyclophosphamide was administered intraperitoneally. The compounds to be tested were orally administered once daily. During the course of treatment, the animals were weighed and sacrificed. The tumor tissues were excised, weighed, and photographed. Finally, the tumor inhibition rate was calculated to evaluate the strength of the antitumor effect. Calculation and statistical methods: Tumor growth inhibition rate TGI (%): TGI=(1-T / C)×100 (T: tumor weight in the treatment group, C: tumor weight in the negative control group). Statistical methods: Graphpad was used for statistical analysis of the data, and a one-way ANOVA test was used.* P < 0.05, ** P < 0.01, *** P<0.001.

[0049] Experimental Results After administration, the animals were killed and the tumor weights were measured. The antitumor effects of the compounds of Example 1 and Example 2 against mouse melanoma B16F10 are shown in the table below.

[0050] [Table 11]

[0051] [Table 12]

[0052] 6. Comparison of in vivo antitumor effects of the compound of Example 1 and the compound of Example 2 against mouse colon cancer MC38 Objective of the experiment: The in vivo antitumor effects of the compound of Example 1 and the compound of Example 2 as PD-L1 inhibitors against mouse colon cancer MC38 were evaluated in a mouse subcutaneously transplanted tumor model. Experimental Solutions: Animal grouping: The experimental animals were divided into a solvent control group, a cyclophosphamide 80 mg / kg group (CTX), a compound of Example 1 5 mg / kg group and a compound of Example 2 5 mg / kg group and a compound of Example 2 10 mg / kg group, respectively. Experimental procedure: Subcultured MC38 tumor cells were homogenized, washed twice with sterile normal saline, counted, and the cell concentration of the tumor cell suspension was adjusted to 9 × 10 in normal saline. 6The cell suspension was adjusted to 0.1 ml / ml, and 0.2 ml of this cell suspension was inoculated into the right armpit of C57BL / 6J mice, and the day was recorded as day 0. The day after inoculation, the animals were randomly divided into groups, weighed, and dosed. Mice in the vehicle control group were orally administered 0.5% CMC daily. Cyclophosphamide was administered intraperitoneally. The compounds to be tested were orally administered once daily. During the course of treatment, the animals were weighed and sacrificed. The tumor tissues were excised, weighed, and photographed. Finally, the tumor inhibition rate was calculated to evaluate the strength of the antitumor effect. Calculation and statistical methods: Tumor growth inhibition rate TGI (%): TGI=(1-T / C)×100 (T: tumor weight in the treatment group, C: tumor weight in the negative control group). Statistical methods: Graphpad was used for statistical analysis of data, and one-way ANOVA test was used. * P < 0.05, ** P < 0.01, *** P<0.001. Test Results: After administration, the animals were killed and the tumor weights were measured. The antitumor effects of the compounds of Example 1 and Example 2 against mouse colon cancer MC38 are shown in the table below.

[0053] [Table 13]

[0054] [Table 14]

[0055] 7. Comparison of antitumor effects of the compound of Example 1 and the compound of Example 2 against human lung cancer NCI-H460 model Objective of the experiment: The in vivo antitumor efficacy of the compound of Example 1 and the compound of Example 2 as PD-L1 inhibitors was evaluated in the NCI-H460 model of human lung cancer in NSG tumor-bearing mice reconstituted with a human immune system. Experimental Solutions: Animal grouping: The experimental animals were divided into a solvent control group, a cyclophosphamide 80 mg / kg group (CTX), a compound of Example 1 5 mg / kg group and a compound of Example 2 10 mg / kg group, respectively. Experimental procedure: Fresh human leukocytes were isolated to obtain PBMCs, which were then inoculated into NSG mice via the tail vein, with each mouse receiving 1 × 10 7 On day 3, NCI-H460 tumor cells were inoculated into the armpits of the mice, and 1 × 10 6 The tumors were 100-300 mm 3 After the mice grew to 100 mm, they were divided into groups and administered the treatments. The mice in the vehicle control group were orally administered 0.5% CMC daily. Cyclophosphamide was administered intraperitoneally. The compounds to be tested were orally administered once daily. During the treatment process, the animals were weighed and sacrificed. The tumor tissues were excised, weighed, and photographed. Finally, the tumor inhibition rate was calculated to evaluate the strength of the antitumor effect. Calculation and statistical methods: Tumor growth inhibition rate TGI (%): TGI=(1-T / C)×100 (T: tumor weight in the treatment group, C: tumor weight in the negative control group). Statistical methods: Graphpad was used for statistical analysis of data, and one-way ANOVA test was used. * P < 0.05, ** P < 0.01, *** P<0.001. Test Results: After administration, the animals were killed and the tumor weights were measured. The antitumor effects of the compounds of Example 1 and Example 2 against NCI-H460 are shown in the table below. [Table 15]

[0056] In summary, the experimental results showed the following: In a subcutaneously transplanted tumor model of the highly metastatic mouse melanoma strain B16F10, the tumor inhibition rates of the compound of Example 2 at daily oral doses of 5 mg / kg and 10 mg / kg were 40% and 55%, respectively, while the tumor inhibition rates of the compound of Example 1 at the same doses were 11% and 13%, respectively. In a subcutaneously transplanted tumor model of mouse colon cancer MC38, the tumor inhibition rates of the compound of Example 2 at daily oral doses of 5 mg / kg and 10 mg / kg were 75% and 57%, respectively, while the tumor inhibition rates of the compound of Example 1 at the same doses were 18% and 27%, respectively. In NSG tumor-bearing mice (NCI-H460) with reconstituted human immune systems, the tumor inhibition rate of the compound of Example 2 at a daily oral dose of 15 mg / kg was superior to that of the compound of Example 1 (tumor inhibition rate: 30.6% vs. 24.7%).

[0057] [Table 16]

Claims

1. Isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate represented by formula (I) and its stereoisomers. 【Chemistry 1】

2. A solid material of isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate, crystalline form A, which has the following characteristics in terms of diffraction peak position, 2θ value (°) or d value (Å), and relative intensity (%) of diffraction peak when powder X-ray diffraction analysis is performed under Cu target radiation experimental conditions. 【Table 1】

3. When analyzed by infrared spectroscopy, the following peaks were observed: 3059, 2984, 2841, 2761, 2519, 2170, 1988, 1968, 1807, 1741, 1716, 1623, 1602, 1580, 1505, 1481, 1460, 1446, 1425, 1401, 1389, 1368, 1309, 1262, 1242, 1205, 1171, 1111, 1095, 1069, 1040, 1004, 972, 953, 924, 884, 870, 864, 854, 824, 788, 761, 721, 703, and 662 cm －１ ±2cm －１ isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate crystalline form A solid material according to claim 2, characterized in that the absorption peak of

4. The solid material of crystalline form A of isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate according to claim 2, characterized in that, when analyzed by differential scanning calorimetry, a DSC spectrum at a heating rate of 10°C / min shows an endothermic peak at 175°C±3°C.

5. A mixed crystal solid material of isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate, characterized in that it contains any non-zero proportion of the crystalline form A solid material of isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate according to claim 2.

6. A process for preparing isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate according to claim 1 or crystalline form A solid material according to claim 2, comprising the steps of: reacting isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate with maleic acid in a solvent to form a salt, preferably the solvent is isopropyl alcohol, tetrahydrofuran or acetone; crystallizing the obtained isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate in a mixed solvent of acetone and water, the ratio of acetone to water being in the range of 200:1 to 1:1, preferably in the range of 50:1 to 5:1, more preferably in the range of 25:1 to 10:1; The method according to claim 1, further comprising:

7. A pharmaceutical composition comprising the isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate and its stereoisomers according to claim 1, or the crystalline form A solid substance according to any one of claims 2 to 4, or the mixed crystal solid substance according to claim 5 as an active ingredient, and a pharma- ceutically acceptable carrier or excipient thereof.

8. Use of isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate and its stereoisomers according to claim 1, or the crystalline form A solid material according to any one of claims 2 to 4, or the mixed crystal solid material according to claim 5, in the manufacture of a medicament for the prevention and / or treatment of a disease associated with the PD-1 / PD-L1 signal pathway.

9. The use according to claim 8, wherein the disease associated with the PD-1 / PD-L1 signal pathway is selected from cancer, infectious diseases and autoimmune diseases.

10. 10. The use according to claim 9, wherein the cancer is selected from skin cancer, lung cancer, urinary system tumors, blood tumors, breast cancer, glioma, digestive system tumors, reproductive system tumors, lymphoma, nervous system tumors, brain tumors, and head and neck cancer; the infectious disease is selected from bacterial infections and viral infections; the autoimmune disease is selected from organ-specific autoimmune diseases and systemic autoimmune diseases, the organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia due to chronic atrophic gastritis, pulmonary hemorrhagic nephritic syndrome, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuropathy; and the systemic autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, and autoimmune hemolytic anemia.

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