Maleate salt of nicotinyl alcohol ether derivative, its crystalline form, and use 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 uses thereof, namely, isopropyl (S)—N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate, its preparation method, crystalline form, pharmaceutical composition, and use thereof. Specifically, the present invention relates to isopropyl (S)—N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate represented by Formula I, its stereoisomers, preparation method, crystalline form, composition containing the compound or the crystalline form, and use of the compound in the manufacture of a medicament for the treatment or prevention of diseases associated with the PD-1 / PD-L1 signaling pathway, such as cancer, infectious disease, autoimmune disease, etc. [Background technology]

[0002] Deep research into tumor immunity has revealed that the tumor microenvironment can protect tumor cells from being recognized and killed by the immune system. Tumor cell immune evasion plays a crucial role in tumor development and progression. In 2013, Science magazine ranked tumor immunotherapy first among its top 10 breakthroughs, once again making immunotherapy a "focus" in the field of tumor treatment. 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 immunoregulatory signal that inhibits the immune activity of tumor-specific CD8+ T cells and mediates immune evasion.

[0003] Tumor cells' ability to evade 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 induces high expression of PD-1 molecules on infiltrating T cells, while tumor cells express high levels of PD-1 ligands PD-L1 and PD-L2. This results in the continued activation of the PD-1 pathway in the tumor microenvironment, inhibiting T cell function, preventing them from detecting tumors and issuing commands to the immune system to attack and kill tumor cells. PD-1 antibodies, which are antibody proteins directed against PD-1 or PD-L1, can block the pathway by inhibiting the binding of PD-1 to PD-L1, partially restoring T cell function and allowing these cells to continue killing tumor cells.

[0004] Recently, a series of surprising research findings have confirmed the potent antitumor activity of PD1 / PD-L1 inhibitory antibodies against a variety of tumors, making them attractive candidates. 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 refractory to other treatments. Currently, Merck is evaluating Keytruda's potential in over 30 cancer types, including various hematological, lung, breast, bladder, gastric, and head and neck cancers. On December 22, 2014, pharmaceutical giant Bristol-Myers Squibb delivered on this promise, becoming the first to receive accelerated approval from the U.S. Food and Drug Administration (FDA). The company's anti-cancer immunotherapy drug 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, following 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 also announced data from the Phase 1b KEYNOTE-028 trial 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 after 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, demonstrated durable therapeutic activity in advanced triple-negative breast cancer.

[0005] Tumor immunotherapy is considered the next revolution in cancer treatment after targeted therapy, but mAb drugs have drawbacks 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-reactivity, not being easy to control product quality and requiring high manufacturing technology, being difficult to prepare and purify in large quantities and resulting in high manufacturing costs, and being inconveniently available only by injection or infusion. Therefore, small molecule inhibitors of PD1 / PD-L1 interaction are a better choice for tumor immunotherapy.

[0006] In International Patent Application No. 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 associated with the PD-1 / PD-L1 signaling pathway, such as cancer, infectious diseases, and autoimmune diseases. In subsequent studies, the present inventors found that the maleate salt of this compound has stronger stability and efficacy than its hydrochloride salt. The hydrochloride salt of Example 1 of the present invention was prepared as a comparative example in accordance with the preparation method of International Patent Application No. 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, methods for preparing them, 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 signaling 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)seric acid isopropyl maleate] represented by formula (I), and its stereoisomers. [ka]

[0010] A second aspect of the technical solution of the present invention provides 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 properties in terms of diffraction peak position, 2θ value (°) or d value (Å), and relative intensity (%) of the diffraction peak when powder X-ray diffraction analysis is performed under Cu target radiation experimental conditions: [Table 1]

[0011] In the above solid crystalline form A material, infrared spectroscopy revealed the following peaks: 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] When analyzed by differential scanning calorimetry, the above-mentioned crystalline form A solid substance exhibits an endothermic peak at 175°C ± 3°C in the DSC spectrum at a heating rate of 10°C / min.

[0013] A 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 the 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 method for preparing 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 wherein the solvent is isopropyl alcohol, tetrahydrofuran or acetone; and 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, wherein the ratio of acetone to water is in the range of 200:1 to 1:1, preferably in the range of 50:1 to 5:1, and most preferably 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 according to the first aspect of the present invention or the crystalline form A solid substance according to the second aspect as an active ingredient, and a pharmaceutically acceptable carrier or excipient.

[0016] The present invention further relates to pharmaceutical compositions containing the compounds of the present invention as an active ingredient. Such pharmaceutical compositions can be prepared by methods well known in the art. By combining the compounds of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants, any dosage form suitable for use in humans or animals can be prepared. The content of the compound of the present invention in such pharmaceutical compositions is usually 0.1 to 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, oral mucosal, intraocular, pulmonary and airway, transdermal, intravaginal, rectal, or other enteral or parenteral.

[0018] The dosage form may be a liquid dosage form, a solid dosage form, or a semi-solid dosage form. Liquid dosage forms may be solutions (including true solutions and colloidal solutions), emulsions (including oil-in-water, water-in-oil, and double emulsions), suspensions, injections (including water injections, powder injections, and drip infusions), eye drops, nasal drops, lotions, tinctures, etc. Solid dosage forms may be tablets (including regular tablets, enteric-coated tablets, troches, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, pellets, drop pills, suppositories, films, patches, gas (powder) sprays, sprays, etc. Semi-solid dosage forms may be ointments, gels, pastes, etc.

[0019] The compounds of the present invention can be formulated into general formulations as well as sustained-release formulations, controlled-release formulations, targeted formulations, 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, 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, cross-linked polyvinylpyrrolidone, croscarmellose sodium, sodium starch glycolate, sodium bicarbonate, citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfonate, etc.; and the lubricant and glidant may be talc, silica, stearates, 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, and enteric-coated tablets, or double-layered tablets and multi-layered tablets.

[0022] To prepare a dosage unit into a capsule, 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 placed in a hard or soft capsule.The compound of the present invention as an active ingredient can also be formulated into granules or pellets together with a diluent, a binder, and a disintegrant, and then placed in a hard or soft capsule.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] 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 appropriate amounts 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.; the osmotic pressure adjuster may be sodium chloride, mannitol, glucose, phosphate, acetate, etc.; and when preparing a lyophilized powder injection, mannitol, glucose, etc. may be added as a proppant.

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

[0025] 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 over a wide range 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, the dosage form, etc. In general, a suitable daily dosage of the compound of the present invention is in the range of 0.001 to 150 mg / kg body weight, preferably 0.01 to 100 mg / kg body weight. The dosage may be administered in a single 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 compounds or compositions of the present invention can be administered alone or in combination with other therapeutic or symptom-ameliorating agents. When the compounds of the present invention exert synergistic effects with other therapeutic agents, the dosage should be adjusted according to the actual situation.

[0028] A fifth aspect of the technical solution of the present invention provides use of isopropyl (S)—N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate and its stereoisomers or the 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 signaling 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, hematological 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 diseases and systemic autoimmune diseases, including 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. [Effects 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 crystalline form and good stability against light irradiation, high humidity, and high temperature environments. It also 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 explanation of the drawings]

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

[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 an MX-5 part-per-million balance are used. For ultraviolet analysis, a Shimadzu UV-2700 UV-visible spectrophotometer is used. For specific rotation measurement, 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, and this compound is a known compound. The preparation method is exactly the same as Example 6 of International Application No. 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 chlorosulfoxide 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 evaporated 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 and stirred for 0.5 hours, and then a solution of maleic acid (0.594 g) in isopropanol (4 ml) was added dropwise. The temperature was controlled at 35-45°C to precipitate a solid. The mixture was then 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 successively with 0.5 ml of isopropanol and 0.5 ml of acetone to obtain a pale yellow solid, which was 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: trace element analyzer FLASH1112 and millionth balance MX-5. (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 measurement 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 predetermined 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 measurement 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 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°C (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: Swiss Mettler TGA / DSC3 + thermal analyzer (2) Parameter setting: starting temperature: 35°C; ending temperature: 250°C; heating rate: 10°C / min. (3) Measurement data: DSC: Peak temperature: 174.68°C (endothermic). TGA: Weight loss began at around 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 the melting of the compound of Example 2. TGA showed that there was 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. As the temperature increased to the apex of the DSC endothermic peak (approximately 175°C), a weight loss became apparent, 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 test [Table 9]

[0045] The compound of Example 1 showed no obvious changes in appearance, melting point, or impurity content even after being exposed to a high temperature of 60°C for 5 days, suggesting that the compound of Example 1 is stable under high temperature conditions. After being exposed to a high humidity of RH 92.5% for 5 days, the compound exhibited severe moisture absorption and turned into a colorless, highly viscous liquid, but there was no change in related substances. Under lighting conditions, the compound turned into a transparent bulk, and its impurity content increased to 4.0%, suggesting 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°C (DSC method) (see Figure 2) Purity: 98.7% (HPLC standardization method) logP=3.2 (2) Influence factor test [Table 10]

[0047] The compound of Example 2 was stable 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 Experimental Objective: The in vivo antitumor effects of the compounds of Example 1 and 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 pulverized with a homogenizer, 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 administered. Mice in the vehicle control group were orally administered 0.5% CMC daily. Cyclophosphamide was administered intraperitoneally. The test compounds were orally administered once daily. During the treatment process, the animals were weighed and sacrificed. 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.

[0049] Experimental results After administration, the animals were sacrificed and the tumor weights were measured.The antitumor effects of the compounds of Example 1 and Example 2 on 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 Experimental Objective: The in vivo antitumor effects of the compounds of Example 1 and 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 pulverized with a homogenizer, washed twice with sterile normal saline, and counted. The cell concentration of the tumor cell suspension was adjusted to 9 × 10 in normal saline. 6The cell suspension was adjusted to 1 / ml, and 0.2 ml of this cell suspension was inoculated into the right armpit of C57BL / 6J mice, with the day recorded as day 0. The day after inoculation, the animals were randomly divided into groups, weighed, and administered. Mice in the vehicle control group were orally administered 0.5% CMC daily. Cyclophosphamide was administered intraperitoneally. The test compounds were orally administered once daily. During the treatment process, the animals were weighed and sacrificed. 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 sacrificed and the tumor weights were measured.The antitumor effects of the compounds of Example 1 and Example 2 on 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 on the human lung cancer NCI-H460 model Experimental Objective: 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, and a compound of Example 2 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. Each mouse received 1 × 10 7 On day 3, NCI-H460 tumor cells were inoculated into the armpits of the mice, and 1 × 10 cells were inoculated into each mouse. 6 The tumors were 100-300 mm 3 After the mice reached maturity, they were divided into groups and administered the treatments. Mice in the vehicle control group were orally administered 0.5% CMC daily. Cyclophosphamide was administered intraperitoneally. The test compounds were orally administered once daily. During the treatment process, the animals were weighed and sacrificed. 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 sacrificed and the tumor weights were measured. The antitumor effects of the compounds of Example 1 and Example 2 on 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 MC38 colon cancer in mice, 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) in which the human immune system had been reconstituted, 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. Crystal A of isopropyl (S)—N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate, wherein when powder X-ray diffraction analysis is performed using Cu target radiation experimental conditions, the diffraction peak positions, 2θ values ​​(°) or d values ​​(Å), and relative intensities (%) of the diffraction peaks have the following properties: Table 1

2. 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 is a characteristic peak in the infrared spectrum exhibited by Crystal A.

3. Crystal A of isopropyl (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl)serinate maleate according to claim 1, characterized in that, when analyzed by differential scanning calorimetry, an endothermic peak is present at 175°C ± 3°C in a DSC spectrum obtained at a heating rate of 10°C / min.

4. A method for preparing Crystal A according to claim 1, comprising: 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; 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, wherein the ratio of acetone to water is in the range of 200:1 to 1:1; A method comprising:

5. A pharmaceutical composition comprising Crystal A according to any one of claims 1 to 3 as an active ingredient and a pharmaceutically acceptable carrier or excipient therefor.

6. Use of Crystal A according to any one of claims 1 to 3 in the manufacture of a medicament for the prevention and / or treatment of a disease associated with the PD-1 / PD-L1 signal pathway.

7. The use according to claim 6, wherein the disease associated with the PD-1 / PD-L1 signal pathway is selected from the group consisting of cancer, infectious diseases, and autoimmune diseases.

8. The use according to claim 7, 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, wherein 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 wherein 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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