TW202241893A - Tetrahydronaphthyridine compound crystal form, salt form, and preparation method thereof - Google Patents

Abstract

A crystal form and a salt form of a tetrahydronaphthyridine compound, and preparation method thereof, and application thereof in medicine for treating related diseases.

Description

Tetrahydronaphthyridine compound crystal form, salt form and preparation method thereof

The invention relates to a tetrahydronaphthyridine compound crystal form, a salt form and a preparation method thereof, as well as its application in medicines for treating related diseases.

This application claims the following priority: CN202110343198.3, the application date is March 30, 2021; CN202111015174.1, the application date is August 31, 2021; CN2022102958433, the application date is March 23, 2022.

Programmed cell death molecule 1 (PD-1), also known as CD279, is an important immunosuppressive molecule in the CD28/CTLA-4 receptor family. It is a membrane protein containing 268 amino acid residues and is widely expressed in On the surface of various immune cells such as T cells, macrophages, and B cells, the ligands are PD-L1 and PD-L2. PD-L1 is a protein encoded by the CD274 gene and mainly expressed on the surface of tumor cells, dendritic cells and macrophages. After PD-1 and PD-L1 are combined, the PD-1/PD-L1 signaling pathway is activated, thereby inhibiting the activation of T cells, resulting in the incapacity of T cells, and contributing to the immune escape of tumor cells. PD-L2, another ligand of PD-1, is mainly expressed on the surface of dendritic cells, macrophages and B cells, and is related to inflammation and autoimmune diseases.

PD-1 negatively regulates the immune response by binding to its ligand PD-L1 and dephosphorylating multiple key molecules in the TCR signaling pathway. In healthy organisms, the activation of the PD-1/PD-L1 signaling pathway can avoid the damage of surrounding tissues caused by excessive immune responses, thereby reducing the occurrence of autoimmune diseases. However, under the induction of the tumor microenvironment, the expressions of PD-1 and PD-L1 are abnormally increased, and tumor cells can successfully evade the recognition and recognition of the organism's immune system through the combination of these PD-L1 molecules and PD-1 on T cells. attack. PD-(L)1 monoclonal antibody can block this "tumor immune escape mechanism" and restore the anti-cancer function of the patient's own immune system.

Currently, the commercially available drugs targeting the PD-1/PD-L1 pathway are monoclonal antibodies (mAbs). In 2014, the US Food and Drug Administration (FDA) approved the first two monoclonal antibodies (pembrolizumab and nivolumab) that targeted the PD-1 molecule to the market. In the following years, three additional mAbs targeting the PD-L1 molecule (atezolizumab, durvalumab, and avelumab) appeared on the market. All of the above are biomacromolecules, which themselves have significant defects, such as being easily decomposed by proteases, having poor stability in the body, and needing to be administered by injection; product quality is not easy to control, and production technology requirements are high; large-scale preparation and purification are difficult, High production cost and easy to produce immunogenicity etc. Small molecule PD-1/PD-L1 inhibitors are attracting more and more attention. Incyte’s PD-L1 small molecule inhibitor INCB86550 (WO2018119263, WO2019191707) and Gilead’s PD-L1 small molecule inhibitor GS-4224 (US20180305315, WO2019160882 ) has entered the clinical phase 2, and the small molecule PD-1/PD-L1 inhibitors of BMS benzyl phenyl ethers (WO2015034820, WO2015160641) are in the preclinical research stage. Because small molecule drugs can pass through the cell membrane to act on intracellular targets, they are convenient for storage and transportation, have low production costs, have no immunogenicity, and can usually be administered orally. Therefore, the research and development of small molecule blocking agents of PD-1/PD-L1 has broad application prospects.

。 The present invention also provides the compound of formula (II)

.

The present invention also provides crystal form A of the compound of formula (II), which is characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.19±0.20°, 7.77±0.20° and 16.22±0.20° .

In some solutions of the present invention, the above-mentioned A crystal form is characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.19±0.20°, 7.77±0.20°, 8.71±0.20°, 9.89±0.20° and 16.22±0.20°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2θ angles: 4.19±0.20°, 7.77±0.20°, 8.71±0.20°, 9.89±0.20°, 16.22±0.20°, 17.74±0.20°, 22.82±0.20°, and 25.49±0.20°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2θ angles: 4.19±0.20°, 7.77±0.20°, and/or 8.71±0.20°, and/or or 9.89±0.20°, and/or 11.47°±0.20°, and/or 14.41°±0.20°, and/or 16.22±0.20°, and/or 17.74±0.20°, and/or 19.67°±0.20°, and /or 20.98°±0.20°, and/or 22.82±0.20°, and/or 25.49±0.20°, and/or 28.15°±0.20°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2θ angles: 4.19±0.20°, 7.77±0.20°, 8.71±0.20°, 9.89±0.20°, 11.47°±0.20°, 14.41°±0.20°, 16.22±0.20°, 17.74±0.20°, 19.67°±0.20°, 20.98°±0.20°, 22.82±0.20°, 25.49±0.20°, and 28.15°±0.20°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2θ angles: 4.19±0.10°, 7.77±0.10°, 8.71±0.10°, 9.89±0.10°, 11.47°±0.10°, 14.41°±0.10°, 16.22±0.10°, 17.74±0.10°, 19.67°±0.10°, 20.98°±0.10°, 22.82±0.10°, 25.49±0.10°, and 28.15°±0.10°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2θ angles: 4.19°, 7.77°, 8.71°, 9.89°, 11.47°, 14.41°, 16.22° , 17.74°, 19.67°, 20.98°, 22.82°, 25.49°, and 28.15°.

In some solutions of the present invention, the above crystal form A has an XRPD pattern as shown in FIG. 1 .

In some solutions of the present invention, the XRPD pattern of the above crystal form A is basically as shown in FIG. 1 .

In some schemes of the present invention, the XRPD pattern analysis data of the above crystal form A are shown in Table 1: Table 1 XRPD pattern analysis data of the compound A crystal form of formula (II) serial number 2θ angle [°] peak height [cts] Left half-height width [°2θ] Interplanar spacing [Å] Relative Strength[%] 1 4.19 326.71 0.2047 21.08 41.71 2 7.77 783.29 0.2047 11.37 100.00 3 8.71 237.89 0.3582 10.15 30.37 4 9.89 307.39 0.2047 8.94 39.24 5 11.47 153.44 0.4093 7.72 19.59 6 14.41 97.20 0.3070 6.15 12.41 7 16.22 470.78 0.3582 5.47 60.10 8 17.74 163.59 0.3582 5.00 20.89 9 19.67 147.82 0.5117 4.51 18.87 10 20.98 110.52 0.6140 4.24 14.11 11 22.82 217.09 0.4605 3.90 27.72 12 25.49 358.04 0.7164 3.50 45.71 13 28.15 117.74 0.8187 3.17 15.03 .

In some solutions of the present invention, the differential scanning calorimetry curve of the above crystal form A has the onset of endothermic peaks at 31.2°C±5°C and 148.7°C±5°C.

In some solutions of the present invention, the DSC spectrum of the above crystal form A is shown in FIG. 2 .

In some solutions of the present invention, the DSC spectrum of the above crystal form A is basically as shown in FIG. 2 .

In some solutions of the present invention, the thermogravimetric analysis curve of the above-mentioned crystal form A reaches a weight loss of 1.77% at 130°C±3°C, and a weight loss of 3.32% at 160°C±3°C.

In some solutions of the present invention, the TGA spectrum of the above crystal form A is shown in FIG. 3 .

In some solutions of the present invention, the TGA spectrum of the above crystal form A is basically as shown in FIG. 3 .

， 其特徵在於，其X射線粉末繞射圖譜在下列2θ角處具有特徵繞射峰：6.52±0.20°、9.40±0.20°和14.94±0.20°。 The present invention also provides the B crystal form of the compound of formula (I)

, characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 6.52±0.20°, 9.40±0.20° and 14.94±0.20°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form B has characteristic diffraction peaks at the following 2θ angles: 6.52±0.20°, 9.40±0.20°, 14.94±0.20°, 16.10±0.20° and 22.86±0.20°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form B has characteristic diffraction peaks at the following 2θ angles: 6.52±0.20°, 9.40±0.20°, 12.93±0.20°, 14.94±0.20°, 16.10±0.20°, 19.06±0.20°, 19.89±0.20°, and 22.86±0.20°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form B has characteristic diffraction peaks at the following 2θ angles: 6.52±0.20°, 9.40±0.20°, and/or 7.47±0.20°, and/or or 11.29±0.20°, and/or 12.93±0.20°, and/or 13.90±0.20°, and/or 14.94±0.20°, and/or 16.10±0.20°, and/or 19.06±0.20°, and/or 19.89 ±0.20°, and/or 21.13±0.20°, and/or 22.86±0.20°, and/or 24.55±0.20°, and/or 25.25±0.20°, and/or 25.83±0.20°, and/or 26.23±0.20 °, and/or 27.45±0.20°, and/or 34.58±0.2°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form B has characteristic diffraction peaks at the following 2θ angles: 6.52±0.20°, 7.47±0.20°, 9.40±0.20°, 11.29±0.20°, 12.93±0.20°, 13.90±0.20°, 14.94±0.20°, 16.10±0.20°, 19.06±0.20°, 19.89±0.20°, 21.13±0.20°, 22.86±0.20°, 24.55±0.20°, 25.25±0.20°, 25.83±0.20°, 26.23±0.20°, 27.45±0.20°, and 34.58±0.20°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form B has characteristic diffraction peaks at the following 2θ angles: 6.52±0.10°, 7.47±0.10°, 9.40±0.10°, 11.29±0.10°, 12.93±0.10°, 13.90±0.10°, 14.94±0.10°, 16.10±0.10°, 19.06±0.10°, 19.89±0.10°, 21.13±0.10°, 22.86±0.10°, 24.55±0.10°, 25.25±0.10°, 25.83±0.10°, 26.23±0.10°, 27.45±0.10°, and 34.58±0.10°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form B has characteristic diffraction peaks at the following 2θ angles: 6.52°, 7.47°, 9.40°, 11.29°, 12.93°, 13.90°, 14.94° , 16.10°, 19.06°, 19.89°, 21.13°, 22.86°, 24.55°, 25.25°, 25.83°, 26.23°, 27.45°, and 34.58°.

In some solutions of the present invention, the XRPD spectrum of the above crystal form B is shown in FIG. 4 .

In some solutions of the present invention, the XRPD spectrum of the above crystal form B is basically as shown in FIG. 4 .

In some schemes of the present invention, the XRPD pattern analysis data of the above-mentioned crystal form B are shown in Table 2: Table 2 XRPD pattern analysis data of the compound B crystal form of formula (I) serial number 2θ angle [°] peak height [cts] Left half-height width [°2θ] Interplanar spacing [Å] Relative Strength[%] 1 6.52 2070.06 0.1279 13.55 84.51 2 7.47 250.48 0.1023 11.84 10.23 3 9.40 779.53 0.1023 9.41 31.82 4 11.29 133.33 0.1535 7.84 5.44 5 12.93 729.83 0.2047 6.85 29.80 6 13.90 133.01 0.1535 6.37 5.43 7 14.94 2449.48 0.1535 5.93 100.00 8 16.10 555.67 0.1023 5.50 22.69 9 19.06 478.23 0.1535 4.66 19.52 10 19.89 405.59 0.1279 4.46 16.56 11 21.13 118.07 0.4093 4.20 4.82 12 22.86 608.64 0.1023 3.89 24.85 13 24.55 113.98 0.3070 3.63 4.65 14 25.25 157.02 0.2558 3.53 6.41 15 25.83 261.22 0.1023 3.45 10.66 16 26.23 175.59 0.1535 3.40 7.17 17 27.45 202.17 0.2558 3.25 8.25 18 34.58 62.68 0.4093 2.59 2.56 .

In some solutions of the present invention, the differential scanning calorimetry curve of the above crystal form B has an endothermic peak at 145.2°C±5°C.

In some solutions of the present invention, the DSC spectrum of the above crystal form B is shown in FIG. 5 .

In some solutions of the present invention, the DSC spectrum of the above crystal form B is basically as shown in FIG. 5 .

In some solutions of the present invention, the thermogravimetric analysis curve of the above crystal form B has a weight loss of 0.77% at 150°C±3°C.

In some solutions of the present invention, the TGA spectrum of the above crystal form B is shown in FIG. 6 .

In some solutions of the present invention, the TGA spectrum of the above crystal form B is basically as shown in FIG. 6 .

The present invention also provides a preparation method of compound B crystal form of formula (I), comprising: (a) adding the compound of formula (I) into a solvent to form a suspension; (b) Stir the suspension at 35-45°C for 8-16 hours; (c) Dry for 8 to 16 hours after filtration; Wherein, the solvent is tetrahydrofuran, ethanol, isopropanol, acetonitrile, methanol/water (1:1), ethanol/water (1:1), acetonitrile/water (1:1), isopropanol/water (1:1) :1) or water.

， 其特徵在於，其X射線粉末繞射圖譜在下列2θ角處具有特徵繞射峰：15.15±0.20°、17.50±0.20°和25.04±0.20°。 The present invention also provides the C crystal form of the compound of formula (I)

, characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 15.15±0.20°, 17.50±0.20° and 25.04±0.20°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form C has characteristic diffraction peaks at the following 2θ angles: 8.75±0.20°, 15.15±0.20°, 17.50±0.20°, 22.94±0.20° and 25.04±0.20°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form C has characteristic diffraction peaks at the following 2θ angles: 8.75±0.20°, 15.15±0.20°, 17.50±0.20°, 18.77±0.20° , 22.94±0.20°, 25.04±0.20°, 26.20±0.20°, 37.65±0.20°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form C has characteristic diffraction peaks at the following 2θ angles: 15.15±0.20°, 17.50±0.20°, and/or 8.39±0.20°, and /or 8.75±0.20°, and/or 11.32±0.20°, and/or 12.55±0.20°, and/or 13.74±0.20°, and/or 14.07±0.20°, and/or 17.03±0.20°, and/or 17.28±0.20°, and/or 18.07±0.20°, and/or 18.77±0.20°, and/or 19.70±0.20°, and/or 20.35±0.20°, and/or 21.56±0.20°, and/or 21.83± 0.20°, and/or 22.54±0.20°, and/or 22.94±0.20°, and/or 23.33±0.20°, and/or 23.74±0.20°, and/or 24.56±0.20°, and/or 25.04±0.20° , and/or 25.33±0.20°, and/or 26.20±0.20°, and/or 27.24±0.20°, and/or 27.93±0.20°, and/or 29.33±0.20°, and/or 30.75±0.20°, and /or 31.39±0.20°, and/or 32.44±0.20°, and/or 33.01±0.20°, and/or 34.07±0.20°, and/or 36.32±0.20°, and/or 37.65±0.20°, and/or 38.01±0.2°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form C has characteristic diffraction peaks at the following 2θ angles: 8.39±0.20°, 11.32±0.20°, 12.55±0.20°, 13.74±0.20° , 14.07±0.20°, 15.15±0.20°, 17.03±0.20°, 17.50±0.20°, 18.07±0.20°, 18.77±0.20°, 19.70±0.20°, 20.35±0.20°, 21.56±0.20°, 22.54±0.20° , 23.33±0.20°, 23.74±0.20°, 24.56±0.20°, 25.04±0.20°, 26.20±0.20°, 27.24±0.20°, 27.93±0.20°, 29.33±0.20°, 30.75±0.20°, 31.39±0.20° , 33.01±0.20°, 34.07±0.20°, 36.32±0.20°, 37.65±0.20°, and 38.01±0.20°.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form C has characteristic diffraction peaks at the following 2θ angles: 8.39±0.10°, 8.75±0.10°, 11.32±0.10°, 12.55±0.10° , 13.74±0.10°, 14.07±0.10°, 15.15±0.10°, 17.03±0.10°, 17.28±0.10°, 17.50±0.10°, 18.07±0.10°, 18.77±0.10°, 19.70±0.10°, 20.35±0.10° , 21.56±0.10°, 21.83±0.10°, 22.54±0.10°, 22.94±0.10°, 23.33±0.10°, 23.74±0.10°, 24.56±0.10°, 25.04±0.10°, 25.33±0.10°, 26.20±0.10° , 27.24±0.10°, 27.93±0.10°, 29.33±0.10°, 30.75±0.10°, 31.39±0.10°, 33.01±0.10°, 34.07±0.10°, 36.32±0.10°, 37.65±0.10°, and 38.01±0.10 °.

In some solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form C has characteristic diffraction peaks at the following 2θ angles: 8.39°, 8.75°, 11.32°, 12.55°, 13.74°, 14.07°, 15.15 °, 17.03°, 17.28°, 17.50°, 18.07°, 18.77°, 19.70°, 20.35°, 21.56°, 21.83°, 22.54°, 22.94°, 23.33°, 23.74°, 24.56°, 25.04°, 25.33°, 26.20°, 27.24°, 27.93°, 29.33°, 30.75°, 31.39°, 32.44°, 33.01°, 34.07°, 36.32°, 37.65°, 38.01°.

In some solutions of the present invention, the XRPD spectrum of the above crystal form C is shown in FIG. 7 .

In some solutions of the present invention, the XRPD spectrum of the above crystal form C is basically as shown in FIG. 7 .

In some schemes of the present invention, the XRPD pattern analysis data of the above crystal form C are shown in Table 3: Table 3 XRPD pattern analysis data of the compound C crystal form of formula (I) serial number 2θ angle [°] peak height [cts] Left half-height width [°2θ] Interplanar spacing [Å] Relative Strength[%] 1 8.39 616.81 0.1023 10.53 53.64 2 8.75 800.23 0.0768 10.10 69.59 3 11.32 90.92 0.1535 7.82 7.91 4 12.55 292.99 0.1279 7.05 25.48 5 13.74 212.53 0.1023 6.44 18.48 6 14.07 254.17 0.1023 6.29 22.10 7 15.15 933.32 0.1023 5.85 81.17 8 17.03 447.03 0.0768 5.21 38.88 9 17.28 542.19 0.0768 5.13 47.15 10 17.50 954.08 0.1023 5.07 82.97 11 18.07 139.60 0.1279 4.91 12.14 12 18.77 500.54 0.1023 4.73 43.53 13 19.70 128.35 0.2047 4.51 11.16 14 20.35 234.73 0.1279 4.36 20.41 15 21.56 180.35 0.1023 4.12 15.68 16 21.83 296.84 0.1023 4.07 25.82 17 22.54 591.60 0.1279 3.94 51.45 18 22.94 862.02 0.1279 3.88 74.97 19 23.33 551.90 0.1279 3.81 48.00 20 23.74 144.19 0.1535 3.75 12.54 twenty one 24.56 398.35 0.1279 3.62 34.64 twenty two 25.04 1149.85 0.1279 3.56 100.00 twenty three 25.33 226.46 0.1023 3.52 19.69 twenty four 26.20 475.63 0.1023 3.40 41.36 25 27.24 239.72 0.1023 3.27 20.85 26 27.93 172.21 0.2558 3.19 14.98 27 29.33 279.74 0.2047 3.04 24.33 28 30.75 96.21 0.3070 2.91 8.37 29 31.39 89.76 0.3070 2.85 7.81 30 32.44 114.25 0.1535 2.76 9.94 31 33.01 263.11 0.1535 2.71 22.88 32 34.07 56.02 0.3070 2.63 4.87 33 36.32 105.53 0.2047 2.47 9.18 34 37.65 612.64 0.1535 2.39 53.28 35 38.01 416.40 0.1535 2.37 36.21 .

In some solutions of the present invention, the differential scanning calorimetry curve of the above crystal form C has an endothermic peak at 184.7°C±5°C.

In some solutions of the present invention, the DSC spectrum of the above crystal form C is shown in FIG. 8 .

In some solutions of the present invention, the DSC spectrum of the above-mentioned crystal form C is basically as shown in FIG. 8 .

In some solutions of the present invention, the thermogravimetric analysis curve of the above crystal form C has a weight loss of 0.69% at 150°C±3°C.

In some solutions of the present invention, the TGA spectrum of the above crystal form C is shown in FIG. 9 .

In some solutions of the present invention, the TGA spectrum of the above-mentioned crystal form C is basically as shown in FIG. 9 .

In some solutions of the present invention, the DVS isotherm diagram of the above crystal form C is basically as shown in FIG. 10 .

The present invention also provides a preparation method of the C crystal form of the compound of formula (I), comprising: (a) adding the compound B crystal form of formula (I) into a solvent to form a suspension; (b) Stir the suspension at 25-45°C for 8-16 hours; (c) Dry for 8 to 16 hours after filtration; Wherein, the solvent is methanol, acetone, isopropyl acetate, acetone:isopropyl acetate (2:1) and the like.

The present invention also provides a preparation method of the C crystal form of the compound of formula (I), comprising: (a) adding the crystal form B of the compound of formula (I) into a solvent to form a suspension; (b) Stir the suspension at 55-60°C for 8-16 hours; (c) Dry for 8 to 16 hours after filtration; Wherein, the solvent is acetone, isopropyl acetate and the like.

The present invention also provides the application of the above compound or crystal form A or crystal B or crystal C or the crystal form B or C prepared according to the above method in the preparation of antitumor drugs.

technical effect

The compound of the present invention has good PK property and oral absorption rate, and its crystal form is stable and has good druggability.

The compound of the present invention has a good inhibitory effect on the excessive activation of the PD-1/PD-L1 signaling pathway, and thus obtains excellent tumor growth inhibitory activity.

Definition and Description

Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A specific phrase or term should not be considered indeterminate or unclear if it is not specifically defined, but should be understood according to its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding trade name or its active ingredient.

The intermediate compound of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art of the present invention, including the specific embodiments listed below, the embodiments formed by its combination with other chemical synthesis methods, and the present invention There are equivalents known to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention.

The chemical reactions of the specific embodiments of the present invention are completed in a suitable solvent, and the solvent must be suitable for the chemical changes of the present invention and the required reagents and materials. In order to obtain the compounds of the present invention, it is sometimes necessary for those skilled in the art to which the present invention pertains to modify or select synthetic steps or reaction schemes on the basis of existing implementations.

The present invention will be specifically described by examples below, and these examples do not imply any limitation to the present invention.

All solvents used in the present invention are commercially available and used without further purification.

The following abbreviations are used in the present invention: CD ₃ OD stands for deuterated methanol; CDCl ₃ stands for deuterated chloroform.

Compounds were named manually or with ChemDraw® software, and commercially available compounds used supplier catalog names.

Powder X-ray diffraction (X-ray powder diffractometer, XRPD) method of the present invention Instrument model: Panalytical (PANalytical) X'pert ³ type X-ray diffractometer Test method: about 10 mg of sample is evenly spread on a single XRPD detection was carried out on the crystal silicon sample plate. The detailed XRPD parameters are as follows: Ray source: K-Alphal ( λ=1.54060Å), K-Alphal ( λ=1.54443Å) Phototube voltage: 45 kV, Phototube current: 40mA Measurement time: 46.665 s Scanning angle range: 3 -40 deg step width angle: 0.0263 deg

Differential Scanning Calorimeter (DSC) method of the present invention Instrument Model: TA 2500 Differential Scanning Calorimeter Test Method: Take a sample (~ 1 mg) and place it in a DSC aluminum pot for testing, at 10 ml/min N ₂ Under these conditions, heat the sample from 25°C (room temperature) to 200°C (or 250°C) at a heating rate of 10°C/min.

Thermal Gravimetric Analyzer (TGA) method of the present invention Instrument Model: TA 5500 Thermogravimetric Analyzer Test Method: Take a sample (2-5 mg) and place it in a TGA platinum pot for testing, under the condition of 10 ml/min N ₂ , at a heating rate of 10°C/min, heat the sample from room temperature to 350°C or lose 20% of its weight.

Dynamic vapor adsorption analysis (Dynamic Vapor Sorption, DVS) method of the present invention Instrument model: SMS DVS Advantage dynamic vapor adsorption instrument Test conditions: Take a sample (10-15 mg) and place it in a DVS sample tray for testing. The detailed DVS parameters are as follows: Temperature: 25°C Equilibrium: dm/dt=0.01 %/min (minimum: 10 min, longest: 180 min) Drying: 120 min at 0% RH RH (%) test step: 10% RH (%) test step range: 0% - 90% - 0% Humidity evaluation classification is as follows: Table 4 Hygroscopicity Classification ΔW% deliquescence Absorb enough water to form a liquid Very hygroscopic ΔW% ≥ 15% Hygroscopic 15% ＞ ΔW% ≥ 2% slightly hygroscopic 2% ＞ ΔW% ≥ 0.2% No or almost no hygroscopicity ΔW% ＜ 0.2% Note: ΔW% represents the moisture absorption weight gain of the test product at 25 ± 1°C and 80 ± 2% RH.

The present invention will be described in detail through examples below, but it does not imply any unfavorable limitation to the present invention. The compound of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art of the present invention, including the specific embodiments listed below, the embodiments formed by its combination with other chemical synthesis methods, and the technology of the present invention There are equivalents known to those of ordinary skill in the art, and preferred embodiments include, but are not limited to, the examples of the present invention. Various changes and modifications to the specific embodiments of the present invention will be apparent to those skilled in the art to which the present invention pertains without departing from the spirit and scope of the present invention.

Embodiment 1: the preparation of formula (I) compound and formate thereof

Synthetic Compound 1:

In the first step, compound 1-1 (100 g, 484 mmol, 1 equivalent), 1-2 (135.3 g, 533 mmol, 1.1 equivalent) were dissolved in dioxane (1000 ml), and then added to the reaction solution Potassium acetate (142.6 g, 1.45 mol, 3 eq) and dichlorobis(triphenylphosphine)palladium (10.2 g, 14.53 mmol, 0.03 eq) were added to the mixture, and stirred at 110°C for 16 hours under nitrogen protection. After the reaction was completed, filter, add the filtrate to water (800 ml), extract with isopropyl acetate (500 ml × 2), wash the organic phase with saturated brine (800 ml × 1), dry over anhydrous sodium sulfate, filter, reduce The residue concentrated under reduced pressure was diluted with isopropyl acetate (80 ml), then stirred at 20°C for 30 minutes, the mixture was filtered, and the filtrate was concentrated under reduced pressure (n-heptane:isopropyl acetate=30:1, 320 ml) Stir at room temperature for 30 minutes, filter, and dry the filter cake to obtain compound 1-3. MS-ESI calculated [M+H] ⁺ 254, found 254. ¹ H NMR (400 MHz, CD ₃ OD) δ = 7.07 - 7.01 (m, 1H), 7.00 - 6.76 (m, 2H), 1.37 (s, 12H).

In the second step, compounds 1-3 (89.3 g, 352.2 mmol, 1 equivalent), 1-4 (95.22 g, 352.2 mmol, 1 equivalent) were dissolved in N , N -dimethylformamide (800 ml), water (100 ml), then potassium carbonate (146 g, 1.06 mol, 3 equivalents), (dichlorobis(triphenylphosphine) palladium (7.42 g, 10.57 mmol, 0.03 Equivalent), stirred for 4 hours under nitrogen protection at 85 ° C. After the reaction was completed, filter, and the filtrate was added to aqueous hydrochloric acid (2 mol/liter, 2400 ml), and methyl tertiary butyl ether (1000 ml × 2 ) extraction, the organic phase was washed with saturated brine (1000 ml × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was diluted with methyl tertiary butyl ether (100 ml), and then 98% of Add concentrated sulfuric acid to the stirring solution until the pH of the solution reaches 1-2, then add (methyl tertiary butyl ether: isopropanol = 10:1, 200 ml) beating at room temperature for 30 minutes and stirring at 20°C for 60 Minutes, the mixture was filtered, the filter cake was dried and added to saturated aqueous sodium carbonate (1200 ml), extracted with dichloromethane (600 ml × 2), the organic phase was washed with saturated brine (800 ml × 2), anhydrous sodium sulfate After drying and filtering, the filtrate was concentrated under reduced pressure, the residue was stirred with isopropanol (70 ml) at room temperature for 60 minutes, and the filter cake was dried to obtain compound 1. MS-ESI calculated value [M+H] ⁺ 317, measured value 317. ¹ H NMR (400 MHz, CD ₃ OD) δ = 7.76 - 7.68 (m, 1H), 7.30 - 7.22 (m, 2H), 7.12 (t, J = 7.8 Hz, 1H), 6.90 (dd, J = 1.3 , 8.1 Hz, 1H), 6.54 (dd, J = 1.3, 7.5 Hz, 1H).

Synthetic compound 2:

In the first step, compound 2-1 (100 g, 723.99 mmol, 1 eq) was dissolved in toluene (1000 ml), then compound 2-2 (168.13 g, 1.45 mol, 160.13 ml, 2 eq) was added, 20 Stir at 100°C for 15 minutes, then add phosphorus oxychloride (333.03 g, 2.17 mol, 201.84 ml, 3 equivalents) dropwise, heat to 100°C and stir for 2 hours. After the reaction is complete, distill phosphorus oxychloride and toluene under reduced pressure at 100°C until the reaction solution becomes very viscous, add 1000 ml of ethyl acetate to dilute, slowly pour into 6 liters of water and stir for 10 minutes, add sodium carbonate to adjust the pH to 7, then extracted with ethyl acetate (1000 ml × 3), the combined organic phase was washed with brine (1000 ml × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product by silica gel chromatography Compound 2-3 was obtained by column purification (petroleum ether: ethyl acetate = 20:1-2:1). MS-ESI calculated [M+H] ⁺ 237, found 237. ¹ H NMR (400 MHz, CDCl ₃ ) δ = 9.76 (s, 1H), 8.84 (d, J = 5.8 Hz, 1H), 8.48 (s, 1H), 8.09 (d, J = 5.9 Hz, 1H), 4.61 (q, J = 7.2 Hz, 2H), 1.53 (t, J = 7.2 Hz, 3H).

In the second step, compound 2-3 (40 g, 169.02 mmol, 1 eq) was dissolved in methanol (400 ml), and methanesulfonic acid (97.47 g, 1.01 mol, 72.20 ml, 6 eq) was slowly added dropwise. After heating to 85°C, it was stirred for 3 hours. After the reaction was completed, cool to room temperature, then add solid sodium acetate at 0°C to adjust the pH to 7, add 2000 ml of water to dilute, stir at room temperature for 30 minutes, filter, and dry the filter cake to obtain compound 2-4. MS-ESI calculated [M+H] ⁺ 219, found 219. ¹ H NMR (400 MHz, CDCl ₃ ) δ = 9.65 (s, 1H), 8.70 (d, J = 5.7 Hz, 1H), 8.04 (d, J = 5.7 Hz, 1H), 7.76 (s, 1H), 4.19 (s, 3H), 4.13 (s, 3H).

In the third step, compound 2-4 (25 g, 114.57 mmol, 1 equivalent) was dissolved in methanol (250 ml), and then sulfuric acid (34.40 g, 343.71 mmol, 18.70 ml, 98% purity, 3 equivalent), and then wet palladium carbon (2.5 g, 11.46 mmol, 10% purity, 0.1 equivalent) was added under the protection of nitrogen. After the reaction was completed, it was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 2-5. MS-ESI calculated [M+H] ⁺ 223, found 223.

In the fourth step, compound 2-5 (40 g, 62.44 mmol, 1 equivalent, sulfate salt, crude product) was dissolved in dichloromethane (300 ml), and 1.8-diazabicyclo[5.4.0 ] Undecane-7-ene (38.02 g, 249.75 mmol, 37.65 ml, 2 eq), and then compound 2-6 (23.94 g, 137.36 mmol, 26.17 ml, 1.1 eq), was added at 20°C After stirring for 0.5 hour, sodium acetate borohydride (79.40 g, 374.63 mmol, 3 equiv) was added and stirred at 20°C for 1.5 hours. After the reaction is complete, pour the reaction solution into 600 ml of water, adjust the pH to 7 with sodium bicarbonate, extract with dichloromethane (300 ml × 3), and wash the combined organic phase with saturated brine (300 ml × 2) , and dried over anhydrous sodium sulfate. The crude product obtained by filtration and concentration under reduced pressure was subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 10:1-2:1) and preparative high performance liquid chromatography (chromatographic column: Kromasil Eternity XT 250 × 80 mm × 10 microns; mobile Phase: mobile phase A: 10 millimolar ammonium bicarbonate aqueous solution; mobile phase B: acetonitrile; B%: 50%-80%, 20 minutes) Compound 2 was purified and isolated. MS-ESI calculated [M+H] ⁺ 381, found 381. ¹ H NMR (400 MHz, CD ₃ OD) δ = 7.48 (s, 1H), 3.88 (s, 3H), 3.86 (s, 3H), 3.79 (t, J = 5.9 Hz, 2H), 3.68 (s, 2H), 2.84 - 2.76 (m, 2H), 2.73 - 2.68 (m, 2H), 2.66 (t, J = 5.9 Hz, 2H), 0.82 (s, 9H), 0.01 (br s, 6H).

Synthesis of Compound 5:

In the first step, compound 5-1 (200 g, 980.27 mmol) was dissolved in ethylene glycol dimethyl ether (600 ml) and toluene (600 ml), and copper iodide (37.34 g, 196.05 Millimoles), then add potassium iodide (325.45 g, 1.96 moles), replace nitrogen three times, set up a tail gas absorption device, heat the reaction solution to 60°C, and slowly add isoamyl nitrite dropwise after the internal temperature rises to 60°C ester (660 ml, 4.90 mol), and the reaction solution was reacted at room temperature for 2 hours. After the reaction was completed, the reaction solution was filtered with diatomaceous earth, the filtrate was poured into ice (1000 ml), and after stirring for 20 minutes, saturated ammonium chloride solution (1000 ml) was added, and stirred for 30 minutes, and the mixture was washed with ethyl acetate (2000 ml) for extraction and separation, the organic phase was washed with saturated sodium sulfite (2000 ml), washed with saturated brine (2000 ml), dried over sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was stirred with 120 ml of methyl tertiary butyl ether and 120 ml of n-heptane, filtered, and the filter cake was dried to obtain compound 5-2. MS-ESI calculated [M+H] ⁺ 315, found 315.

second step Compound 5-2 (60 g, 190.53 mmol) was dissolved in 2-methyltetrahydrofuran (800 ml), nitrogen was replaced three times, the temperature was lowered to -40°C, and the iso Propylmagnesium chloride lithium chloride (1.3 mol/L, 1 ml), stirred at -40°C for 0.5 hours, then added dropwise N,N-dimethylamide (73.3 ml, 952.67 mmol), and the reaction solution was The reaction was carried out at room temperature for 3 hours. After the reaction was completed, the reaction solution was quenched by pouring ice hydrochloric acid (1 mol/L, 800 ml), extracted and separated, the aqueous phase was extracted with methyl tertiary butyl ether (400 ml × 2), the organic phases were combined and washed with saturated It was washed with brine (400 ml×2), dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 5-3.

The third step Dissolve compound 5-4 (10.59 g, 85.71 mmol) in 2-methyltetrahydrofuran (150 ml), add potassium carbonate (11.85 g, 85.71 mmol), then add compound 5-3 (15.5 g, 71.42 mmol), and after stirring for 0.5 hours, sodium acetate borohydride (22.71 g, 107.13 mmol) was added in portions. The reaction solution was reacted at 20° C. for 1 hour. The reaction solution was quenched by pouring glacial hydrochloric acid (1 mol/L, 200 mL), extracted and separated, the aqueous phase was adjusted to pH=8 with solid sodium carbonate, extracted with ethyl acetate (100 mL×2), and the organic phase was saturated with Wash with brine (100ml×2), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The crude product was prepared by high performance liquid chromatography (column: Xtimate C18 250×50 mm×10 microns; mobile phase: mobile phase A: 0.05% volume fraction ammonia solution; mobile phase B: acetonitrile; B%: 15%-45%, 18 minutes) to isolate compound 5. MS-ESI calculated [M+H] ⁺ 288, found 288. ¹ H NMR (400 MHz, CD ₃ OD) δ = 8.23 - 8.19 (m, 1H), 4.08 - 4.04 (m, 1H), 4.01 - 3.99 (m, 3H), 3.78 (s, 2H), 3.73 - 3.69 (m, H), 3.26 - 3.24 (m, 3H), 3.19 - 3.15 (m, 2H).

Synthetic formula (I) compound:

In the first step, 14 liters of anhydrous 2-methyltetrahydrofuran were added to the reaction kettle at 20-25°C, and then 974.32 grams of compound 2 (1.05 equivalents) and 720.00 grams of compound 1 (1.00 equivalents) were added. Nitrogen replacement for 5-10 minutes, then adjust the temperature of the reactor to 0-5°C, and slowly add 6.67 liters of tetrahydrofuran solution of bis(trimethylsilyl)amide lithium (1 mol/liter, 3 equivalents) dropwise to the reaction After the dropwise addition, stir at 0-5°C for 1 hour. At 0-5°C, slowly pump the reaction liquid into a reaction kettle containing 14 liters of saturated ammonium chloride aqueous solution in batches, separate the liquids, collect the aqueous phase and the organic phase, wash with 14 liters of saturated saline, and collect the water phase and the organic phase, the organic phase was dried, filtered, and concentrated to give a crude solid. At 20-25°C, add 4.2 liters of methanol into the reaction kettle, then add the solid crude product and continue stirring for 30 minutes. After filtering, the filter cake was washed twice with methanol, and the filter cake was vacuum-dried to constant weight to obtain compound 3. MS-ESI calculated [M+H] ⁺ 666, found 666. ¹ H NMR (400 MHz, CDCl ₃ ) δ= 10.64 - 10.90 (m, 1 H), 8.63 (m, 1 H), 7.50 - 7.68 (m, 2 H) 7.33 (t, J=7.95 Hz, 1 H ) 7.12 - 7.20 (m, 2H) 6.94 (m, 1H) 3.90 (s, 3H) 3.67 - 3.83 (m, 4H) 2.61 - 2.85 (m, 6H) 0.83 (s, 9H) 0.00 (s, 6 H).

In the second step, at 20-25°C, add 13 liters of anhydrous dioxane into the reactor, then add 1306.10 grams of compound 3 (1.00 equivalents), 598.03 grams of double pinacol borate (1.24 equivalents), 577.81 Add 1 gram of potassium acetate (3.10 equivalents) and 13.77 grams of dichlorobis(triphenylphosphine) palladium (0.01 equivalents) into the reactor, replace with nitrogen for 5 to 10 minutes, then adjust the temperature of the reactor to 100 to 110 ° C, and Stirring at ~110°C for 12 hours gave the crude compound 4, which was directly used in the next step. Adjust the temperature of the reaction kettle to 20-25°C, add 3.25 liters of water into the reaction kettle, then add 577.81 grams of compound 5 (1.01 equivalents) and 790.97 grams of potassium carbonate (3.01 equivalents) into the reaction kettle. Replace with nitrogen for 5-10 minutes, add 13.72 g of dichlorobis(triphenylphosphine) palladium (0.01 equivalent) into the reaction kettle, then adjust the temperature of the reaction kettle to 70-75°C and stir for 12 hours at 70-75°C . The reaction solution was cooled to room temperature, filtered, the filter cake was washed with 13 liters of ethyl acetate, and 9.75 liters of water was added to the combined filtrate, and the liquid was separated. The organic phase and the water phase were collected respectively, and the water phase was washed with 6.5 liters of ethyl acetate, and the liquid was separated. Collect the organic phase. The combined organic phases were washed with 13 liters of 10% citric acid aqueous solution, separated, the aqueous phase and the organic phase were collected respectively, and the aqueous phase was extracted twice with 13 liters of ethyl acetate. The organic phases were combined, washed with 6.5 liters of water, separated, and the aqueous phase and the organic phase were collected respectively. Combine the water phases, adjust the pH to 9 with 4 mol/L sodium hydroxide aqueous solution, add 13 liters of ethyl acetate, separate the liquids, collect the water phase and the organic phase respectively, add 6.5 liters of ethyl acetate to the water phase, and separate the liquids , respectively collect the aqueous phase and the organic phase, combine the organic phases, wash with 13 liters of saturated brine, wash twice, and collect the organic phases. The organic phase was dried, filtered and concentrated to give the crude product. At 20-25°C, dissolve the crude product in 18 liters of ethyl acetate and pour it into the reaction kettle, add palladium-removing modified silica gel into the reaction kettle, and stir at 65-70°C for 16 hours, then filter the reaction solution, The filter cake was washed with 36 liters of ethyl acetate, the washing solution was spin-dried and combined with the filtrate, and the palladium removal operation was repeated once, and the filtrate was concentrated to obtain the crude compound 6. Add 14.6 liters of acetone to the reaction kettle, pour the above crude product until completely dissolved, then dissolve 607.95 grams of anhydrous oxalic acid (1.70 equivalents) in 2.92 liters of acetone, slowly add to the above solution in batches, and finally add 5.84 liters of acetonitrile , stirred at 20-25° C. for 1 hour, filtered, the filter cake was washed with 14.6 liters of acetonitrile, and the filter cake was vacuum-dried to constant weight to obtain the oxalate salt of compound 6. MS-ESI calculated [M+H] ⁺ 793, found 793. ¹ H NMR (400 MHz, CD ₃ OD) δ= 8.57 - 8.67 (m, 1 H), 8.48 (s, 1 H), 7.82 (s, 1 H), 7.72 (m, 1 H), 7.58 (s , 1 H), 7.48 (br d, J=13.08 Hz, 2 H), 7.18 (s, 1 H), 4.72 (s, 2 H), 4.56 - 4.66 (m, 4 H), 4.39 (br t, J=5.26 Hz, 1 H), 4.19 - 4.28 (m, 2 H), 4.05 - 4.14 (m, 8 H), 3.67 (br t, J=6.17 Hz, 2 H), 3.43 - 3.50 (m, 2 H), 3.36 (s, 3 H), 3.11 (s, 2 H), 0.92 (s, 8 H) 0.13 (s, 6 H).

The third step is at 20-25°C, add 7.5 liters of anhydrous tetrahydrofuran and 22.5 liters of water into the reaction kettle, then add 3000.00 g of compound 6 oxalate (1.00 equivalent) and 1349.50 g of anhydrous oxalic acid (5.00 equivalent) °C, stirred for 12 hours. The reaction solution was extracted twice with 30 liters of methyl tertiary butyl ether, separated, the aqueous phase and the organic phase were collected respectively, the organic phases were combined, washed with 15 liters of water, the liquids were separated, and the aqueous phase and the organic phase were collected respectively. Combine the aqueous phases, adjust the pH to 9 with 4 mol/liter sodium hydroxide aqueous solution, add 45 liters of 2-methyltetrahydrofuran, separate the liquids, collect the aqueous phase and the organic phase, and add 15 liters of 2-methyltetrahydrofuran to the aqueous phase. Tetrahydrofuran, liquid separation, respectively collect the water phase and the organic phase, combine the organic phase, wash twice with 30 liters of saturated brine, and collect the organic phase. The organic phase was dried, filtered and concentrated to crude product. At 20-25°C, dissolve the above solid in 14 liters of anhydrous tetrahydrofuran and 6 liters of ethyl acetate into the reaction kettle, then add palladium-removed modified silica gel into the reaction kettle, and stir for 12 hours at 20-25°C , then the reaction solution was filtered, spin-dried, the filtrate was concentrated to obtain a crude product, and vacuum-dried to constant weight to obtain the compound of formula (I). MS-ESI calculated [M+H] ⁺ 679, found 679. ¹ H NMR (400 MHz, CD ₃ OD) δ= 8.60 (m, 1 H), 8.40 (s, 1 H), 7.64 - 7.76 (m, 2 H), 7.54 (t, J=7.64 Hz, 1 H ), 7.40 - 7.50 (m, 2H), 7.15 (m, 1H), 4.07 - 4.13 (m, 1H), 4.03 (d, J=12.96 Hz, 6H), 3.89 (s, 2H) , 3.74 - 3.83 (m, 6 H), 3.26 (s, 3 H), 3.19 - 3.24 (m, 2 H), 2.86 (m, 4 H), 2.76 (t, J=5.87 Hz, 2 H).

將化合物6的粗品（68 毫克，85.66微莫耳）溶於二氯甲烷（3 毫升），加入氯化氫的乙酸乙酯溶液（4莫耳/升，1.5毫升），室溫25℃攪拌0.5小時。反應完畢後，濃縮。粗品經製備高效液相色譜（色譜柱：Phenomenex Gemini-NX C18 75×30毫米×3微米；流動相：流動相A：體積分數0.225%甲酸水溶液；流動相B：乙腈；B%：10%-40%，7分鐘）分離得到式（I）化合物的甲酸鹽。MS-ESI 計算值[M+H] ⁺679，實測值679。 ¹H NMR (400 MHz, CD ₃OD) δ = 8.58 - 8.65 (m, 1 H), 8.49 (s, 1 H) 8.43 (br s, 1 H), 7.70 - 7.78 (m, 2 H), 7.59 (t, J= 7.63 Hz, 1 H), 7.45 - 7.53 (m, 2 H), 7.18 (dd, J= 7.63, 1.38 Hz, 1 H), 4.52 (s, 2 H), 4.30 - 4.41 (m, 3 H), 4.12 (s, 3 H), 4.06 (s, 3 H), 3.94 - 4.01 (m, 4 H), 3.81 - 3.87 (m, 2 H), 3.35 - 3.37 (m, 3 H), 3.02 - 3.07 (m, 2 H), 2.91 (q, J= 5.38 Hz, 4 H)。 Synthesis of the formate salt of the compound of formula (I):

The crude compound 6 (68 mg, 85.66 micromole) was dissolved in dichloromethane (3 mL), added with a solution of hydrogen chloride in ethyl acetate (4 mol/L, 1.5 mL), and stirred at room temperature 25°C for 0.5 hour. After the reaction was completed, it was concentrated. The crude product was prepared by high-performance liquid chromatography (column: Phenomenex Gemini-NX C18 75 × 30 mm × 3 microns; mobile phase: mobile phase A: 0.225% volume fraction of formic acid aqueous solution; mobile phase B: acetonitrile; B%: 10%- 40%, 7 minutes) to isolate the formate salt of the compound of formula (I). MS-ESI calculated [M+H] ⁺ 679, found 679. ¹ H NMR (400 MHz, CD ₃ OD) δ = 8.58 - 8.65 (m, 1 H), 8.49 (s, 1 H) 8.43 (br s, 1 H), 7.70 - 7.78 (m, 2 H), 7.59 (t, J = 7.63 Hz, 1 H), 7.45 - 7.53 (m, 2 H), 7.18 (dd, J = 7.63, 1.38 Hz, 1 H), 4.52 (s, 2 H), 4.30 - 4.41 (m , 3 H), 4.12 (s, 3 H), 4.06 (s, 3 H), 3.94 - 4.01 (m, 4 H), 3.81 - 3.87 (m, 2 H), 3.35 - 3.37 (m, 3 H) , 3.02 - 3.07 (m, 2 H), 2.91 (q, J = 5.38 Hz, 4 H).

Embodiment 2: Preparation of Formula (I) Compound B Crystal Form

Weigh 100 mg of the compound of formula (I) into a 4.0 ml glass vial, and add an appropriate amount of ethanol to form a suspension. After adding magnets, put the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and dry the residual solid sample in a vacuum oven (30°C) overnight to obtain Form B of the compound of formula (I).

Weigh 100 mg of the compound of formula (I) into a 4.0 ml glass vial, and add an appropriate amount of isopropanol to form a suspension. After adding magnets, put the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and dry the residual solid sample in a vacuum oven (30°C) overnight to obtain Form B of the compound of formula (I).

Weigh 100 mg of the compound of formula (I) into a 4.0 ml glass vial, and add an appropriate amount of acetonitrile to form a suspension. After adding magnets, put the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and dry the residual solid sample in a vacuum oven (30°C) overnight to obtain Form B of the compound of formula (I).

Weigh 100 mg of the compound of formula (I) into a 4.0 ml glass vial, and add an appropriate amount of methanol/water (1:1) to make a suspension. After adding magnets, put the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and dry the residual solid sample in a vacuum oven (30°C) overnight to obtain Form B of the compound of formula (I).

Weigh 100 mg of the compound of formula (I) into a 4.0 ml glass vial, and add an appropriate amount of acetonitrile/water (1:1) to make a suspension. After adding magnets, put the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and dry the residual solid sample in a vacuum oven (30°C) overnight to obtain Form B of the compound of formula (I).

Weigh 100 mg of the compound of formula (I) into a 4.0 ml glass vial, and add an appropriate amount of isopropanol/water (1:1) to make a suspension. After adding magnets, put the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and dry the residual solid sample in a vacuum oven (30°C) overnight to obtain Form B of the compound of formula (I).

Add 9 liters of acetone to the reaction kettle, then add the crude product of the compound of formula (I), and stir for 24 hours at 20-25°C. After filtering, the filter cake was washed twice with 1.8 liters of acetone, and the filter cake was vacuum-dried to constant weight to obtain the crystal form B of the compound of formula (I). MS-ESI calculated [M+H] ⁺ 679, found 679. ¹ H NMR (400 MHz, CD ₃ OD) δ= 8.60 (m, 1 H), 8.40 (s, 1 H), 7.64 - 7.76 (m, 2 H), 7.54 (t, J=7.64 Hz, 1 H ), 7.40 - 7.50 (m, 2H), 7.15 (m, 1H), 4.07 - 4.13 (m, 1H), 4.03 (d, J=12.96 Hz, 6H), 3.89 (s, 2H) , 3.74 - 3.83 (m, 6 H), 3.26 (s, 3 H), 3.19 - 3.24 (m, 2 H), 2.86 (m, 4 H), 2.76 (t, J=5.87 Hz, 2 H).

Embodiment 3: Research on the salt form of the compound of formula (I)

About 100 mg of the compound of formula (I) was taken, dissolved in 0.5 ml of solvent, and then the acid (1.05 equivalents) was slowly added under stirring. After stirring at 25°C for 0.5 hour, a solid precipitated out and was filtered. The residual solid sample was dried overnight in a vacuum oven (30°C) to obtain the salt of the compound of formula (I). The properties of the specific compounds are as follows: Table 5 serial number acid Isopropanol ethyl acetate character Is it easy to absorb moisture character hygroscopicity 1 sulfuric acid White powder Severe moisture absorption White powder slightly hygroscopic 2 phosphoric acid light yellow powder Obvious moisture absorption light yellow powder Obvious moisture absorption 3 hydrochloric acid - - light yellow powder Obvious moisture absorption 4 formic acid - - light yellow powder slightly hygroscopic 5 fumaric acid light yellow powder slightly hygroscopic White powder No obvious moisture absorption 6 maleic acid Oil - Oil - 7 L-Tartrate yellow powder Obvious moisture absorption yellow powder Obvious moisture absorption 8 D-Tartrate - - yellow powder Severe moisture absorption

Embodiment 4: the preparation of formula (II) compound

2.01 g of the compound of formula (I) was taken, dissolved in 20 ml of ethyl acetate, and then fumaric acid (675 mg, 2.01 equivalents) was slowly added with stirring. After stirring at 25°C for 16 hours, a solid was precipitated, filtered, and the residual solid sample was dried overnight in a vacuum oven (30°C) to obtain a compound of formula (II). ¹ H NMR (400 MHz, CD ₃ OD) δ = 8.62 (dd, J = 1.5, 8.2 Hz, 1H), 8.50 (s, 1H), 7.79 (s, 1H), 7.73 (dd, J = 1.6, 7.7 Hz, 1H), 7.59 (t, J = 7.7 Hz, 1H), 7.54 - 7.43 (m, 2H), 7.18 (dd, J = 1.5, 7.7 Hz, 1H), 6.70 (s, 4H), 4.67 (s , 2H), 4.53 (dd, J = 6.4, 11.7 Hz, 2H), 4.44 - 4.34 (m, 1H), 4.23 (s, 2H), 4.13 (s, 4H), 4.07 (s, 3H), 3.91 ( t, J = 5.4 Hz, 2H), 3.38 (s, 3H), 3.14 (t, J = 5.5 Hz, 2H), 3.01 (t, J = 6.1 Hz, 2H).

Embodiment 5: Preparation of the crystal form of compound A of formula (II)

Weigh 100 mg of the compound of formula (II) into a 4.0 ml glass vial, and add an appropriate amount of acetone to form a suspension. After adding magnets, place the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and place the residual solid sample in a vacuum oven (30°C) to dry overnight , to obtain the A crystal form of the compound of formula (II).

Weigh 100 mg of the compound of formula (II) into a 4.0 ml glass vial, and add an appropriate amount of ethanol to form a suspension. After adding magnets, place the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and place the residual solid sample in a vacuum oven (30°C) to dry Overnight, crystal form A of the compound of formula (II) was obtained.

Weigh 100 mg of compound fumarate of formula (II) into a 4.0 ml glass vial, and add an appropriate amount of ethyl acetate to form a suspension. After adding magnets, place the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and place the residual solid sample in a vacuum oven (30°C) to dry Overnight, crystal form A of the compound of formula (II) was obtained.

Weigh 100 mg of the compound of formula (II) into a 4.0 ml glass vial, and add an appropriate amount of methyl tertiary butyl ether to form a suspension. After adding magnets, place the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and place the residual solid sample in a vacuum oven (30°C) to dry Overnight, crystal form A of the compound of formula (II) was obtained.

Weigh 100 mg of the compound of formula (II) into a 4.0 ml glass vial, and add an appropriate amount of acetonitrile to form a suspension. After adding magnets, place the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and place the residual solid sample in a vacuum oven (30°C) to dry Overnight, crystal form A of the compound of formula (II) was obtained.

Weigh 100 mg of the compound of formula (II) into a 4.0 ml glass vial, and add an appropriate amount of n-heptane to form a suspension. After adding magnets, place the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and place the residual solid sample in a vacuum oven (30°C) to dry Overnight, crystal form A of the compound of formula (II) was obtained.

Embodiment 6: Preparation of crystal form C of compound of formula (I)

Weigh 100 mg of compound B crystal form of formula (I) into a 4.0 ml glass vial, and add an appropriate amount of methanol to form a suspension. After adding magnets, put the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and dry the residual solid sample in a vacuum oven (30°C) overnight to obtain Form C of the compound of formula (I).

Weigh 100 mg of compound B crystal form of formula (I) into a 4.0 ml glass vial, and add an appropriate amount of acetone to form a suspension. After adding magnets, put the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and dry the residual solid sample in a vacuum oven (30°C) overnight to obtain Form C of the compound of formula (I).

Weigh 100 mg of compound B crystal form of formula (I) into a 4.0 ml glass vial, and add an appropriate amount of isopropyl acetate to form a suspension. After adding magnets, put the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and dry the residual solid sample in a vacuum oven (30°C) overnight to obtain Form C of the compound of formula (I).

Add 10 liters of isopropyl acetate to the reaction kettle, then add 1.32 kg of the crystal form of compound B of formula (I), and stir for 72 hours at 20-25°C. After filtering, the filter cake was washed twice with 2 liters of acetone, and the filter cake was vacuum-dried to constant weight to obtain the C crystal form of the compound of formula (I). MS-ESI calculated [M+H] ⁺ 679, found 679. ¹ H NMR (400 MHz, CD ₃ OD) δ = 8.60 (m, 1 H), 8.40 (s, 1 H), 7.65 - 7.77 (m, 2 H), 7.54 (t, J=7.64 Hz, 1 H ) 7.38 - 7.50 (m, 2 H), 7.15 (m, 1 H), 4.09 (t, J=5.81 Hz, 1 H), 4.04 (d, J=12.72 Hz, 6 H), 3.90 (s, 2 H), 3.74 - 3.83 (m, 6 H), 3.26 (s, 3 H), 3.19 - 3.25 (m, 2 H), 2.86 (m, 4 H), 2.76 (t, J=5.87 Hz, 2 H ).

Example 7: Solvent Stability Study of Form C of Compound C of Formula (I)

Weigh 100 mg of formula (I) compound C crystal form into a 4.0 ml glass vial, add an appropriate amount of solvent to make it into a suspension (in order to ensure that the sample is suspended as much as possible, during the experiment, the amount of compound and amount of solvent or even change the container used in the experiment). After adding magnets, place the above suspension sample on a magnetic heating stirrer (40°C) for testing, stir overnight at 40°C and then centrifuge, and place the residual solid sample in a vacuum drying oven (30°C) to dry overnight and detect (XRPD) The crystalline form of a solid was obtained. The test results are as follows: Table 6 solvent crystal form solvent crystal form Methanol Form C Methyl tertiary butyl ether Form C ethanol Form C Acetone: Isopropyl acetate (2:1) Form C ethyl acetate Form C Methanol: water (3:1) Form C acetone Form C Ethanol: water (1:1) Form C Acetonitrile Form C Ethanol: water (3:1) Form C 2-Methyltetrahydrofuran Form C Acetone:water (2:1) Form C Isopropyl acetate Form C Isopropanol: water (1:1) Form C

Conclusion: The crystal form C of the compound of formula (I) has good stability in different solvents.

Example 8: Research on Hygroscopicity of Form C of Compound C of Formula (I) Experimental Materials: SMS DVS Advantage Dynamic Vapor Sorbent experimental method: 10-30 mg of compound C crystal form of formula (I) was placed in a DVS sample tray for testing. Experimental results: The DVS spectrum of the crystal form C of the compound of formula (I) is shown in Figure 10, △W=0.33%. Experimental results: The hygroscopic weight gain of compound C crystal form of formula (I) at 25°C and 80% RH is 0.33%, which is slightly hygroscopic.

Example 9: Solid Stability Test of Formula (I) Compound B Crystal Form and C Crystal Form

According to the "Guiding Principles for Stability Tests of APIs and Preparations" (Chinese Pharmacopoeia 2015 Edition Four General Rules 9001), the crystal form A of compound III was investigated at high temperature (60°C, open), high humidity (room temperature/relative humidity 92.5%, exposure) and light (total illuminance=1.2×10 ⁶ Lux•hr/near ultraviolet=200w•hr/m ² , exposure) conditions.

Weigh 10 mg of compound B or C of formula (I) respectively, place them on the bottom of the glass sample bottle, and spread them into a thin layer. For samples placed under high temperature (60°C) and high humidity (relative humidity 92.5%RH) conditions, seal the bottle mouth with aluminum foil, and pierce some small holes on the aluminum foil to ensure that the sample can fully contact with the ambient air, and place it in a corresponding constant temperature In the constant humidity box; the light sample (open, not covered with aluminum foil) and the light control substance (the entire sample bottle is covered with aluminum foil) are placed in the light box. Two samples were weighed at each time point as formal test samples. In addition, about 50 mg of the crystal form B or C of the compound of formula (I) was weighed for the XRPD test. The sample bottle was wrapped with aluminum foil and punched with small holes, and placed in the corresponding constant temperature and humidity box. Samples were collected on days 5 and 10 for testing (XRPD), and the test results were compared with the initial test results on day 0. The solid stability test results of compound B or C of formula (I) are as follows: Table 7 Test conditions point in time crystal form - 0 days Form B Form C High temperature (60°C, open) 5 days Form B Form C 10 days Form B Form C High humidity (92.5% relative humidity, open) 5 days Form B Form C 10 days Form B Form C Illumination (total illuminance=1.2×10 ⁶ Lux•hr/near ultraviolet=200w•hr/m ² , exposure) 5 days Form B Form C 10 days Form B Form C

Conclusion: Both the crystal form B and the crystal form C of the compound of formula (I) have good stability under the conditions of high temperature, high humidity and strong light.

Experimental example 1: PD-1/PD-L1 homogeneous time-resolved fluorescence binding experiment

Experimental principle: Small molecule compounds can competitively inhibit the binding of PD-1 and PD-L1 by binding to PD-L1; when the PD-1 molecule as a donor is very close to the PD-L1 molecule as an acceptor, the donor molecule will The energy is transmitted to the receptor molecule, which in turn causes the receptor molecule to emit fluorescence; by detecting the intensity of the fluorescence, the ability of the small molecule to prevent the combination of PD-L1 and PD-1 can be tested. A Homogenouse Time-Resolved Fluorescence (HTRF) binding assay was used to detect the ability of the compounds of the present invention to inhibit the mutual binding of PD-1/PD-L1.

Experimental Materials: PD-1/PD-L1 TR-FRET Detection Kit was purchased from BPS Biosciences. Nivo Multilabel Analyzer (PerkinElmer).

experimental method: Use the buffer in the kit to dilute PD1-Eu, Dye-labeled acceptor, PD-L1-biotin and the test compound. The compound to be tested was diluted 5 times to the 8th concentration with a discharge gun, that is, diluted from 40 micromolar to 0.5 nanomolar, and the DMSO concentration was 4%, and a double-well experiment was set up. Add 5 micromolar inhibitor concentration gradients to the microplate, add 5 microliters of buffer containing 4% DMSO to the maximum (Max) signal well and minimum (Min) signal well, 5 microliters of PD-L1-bio PD-L1-biotin (60 nmol), only 5 microliters of buffer was added to the Min signal well, and incubated at 25 degrees for 20 minutes. After the incubation, 5 μl of diluted PD1-Eu (10 nM) and 5 μl of diluted Dye-labeled acceptor were added to each well. The reaction system was placed at 25°C for 90 minutes. After the reaction, the TR-FRET signal was read using a multi-label analyzer.

Data analysis: Use the equation (sample-Min)/(Max-Min)×100% to convert the original data into inhibition rate, and the value of IC ₅₀ can be obtained by curve fitting with four parameters (log(inhibitor) in GraphPad Prism vs. response -- derived from Variable slope mode). Table 8 provides the inhibitory activity of the compounds of the present invention on PD-1/PD-L1 binding. Table 8 IC50 test results of compounds of the present invention binding to PD-1/PD-L1 test compound IC50(nM) Formate salt of compound of formula (I) 4.61

Experimental conclusion: the compound of formula (I) has a significant inhibitory effect on the combination of PD-1/PD-L1.

Experimental example 2: Using MDA-MR-231 cells to detect the effect of compounds on the expression level of PD-L1

Experimental principle: The use of a triple-negative breast cancer cell line (MDA-MB-231) is an indirect method to assess PD-L1 endocytosis. PD-L1 molecules on the cell surface can be degraded through lysosome and proteasome pathways, adding small molecule inhibitors can induce PD-L1 endocytosis. After co-incubating small molecules with MDA-MB-231 cells for 24 hours, using flow cytometry (Fluorescence-activated Cell Sorting, FACS) to detect the content of PD-L1 on the cell surface can indirectly reflect the effect of small molecules on PD-L1 endocytosis. Effect. Flow cytometry (FACS) was used to detect the effect of the compound of the present invention on the expression level of PD-L1 in MDA-MR-231 cells.

Experimental Materials: Phosphate buffered saline (DPBS), 1640 medium, penicillin-streptomycin, fetal bovine serum, non-essential amino acids, β-mercaptoethanol (2-ME), human interferon-γ, LIVE/DEAD staining solution, staining staining buffer, Fixation buffer, 0.25% trypsin, EDTA, anti-human PD-L1 (Anti-human PD-L1), isotype control anti-human PD-L1 (Anti-human PD-L1 Isotype). 1640 complete medium configuration: add 50 ml of fetal bovine serum, 5 ml of non-essential amino acids, 5 ml of penicillin-streptomycin and 0.5 ml of β-liter mercaptoethanol to 439.5 ml of 1640 medium, and mix well. 10mM EDTA configuration: Add 1ml of 0.5M EDTA to 49ml of DPBS and mix well.

Experimental steps: 1) MDA-MB-231 cell counting and plating: take out the culture flask, remove the medium and wash once with phosphate buffered saline (DPBS). After rinsing, add 3 ml of 0.25% trypsin to the culture bottle and place it in a 37°C incubator for 1.5 minutes. Take out the culture bottle and add 9 ml of 1640 complete medium to terminate the reaction, transfer the cells to a 50 ml centrifuge tube, and centrifuge at 1000 rpm at 37°C for 5 minutes. According to the number of cells, add an appropriate volume of culture medium to resuspend the cells, and count them with a cell counter. Adjust the cell concentration to ⁵ x 10 cells/mL with culture medium. Plating: 200 microliters of cell suspension was added to each well of a 96-well plate, so that the number of cells in each well was 1×10 ⁵ . Place in an incubator overnight. 2) Drug incubation: Prepare 100X compound dilution solution and dilute the drug in a 5-fold gradient. Add 2 microliters of each 100X compound solution to each well of cells. Incubate in a 37°C incubator for 24 hours. 3) PD-L1 cell staining and FACS detection: Take out the culture plate and discard the upper culture medium. Wash once with 200 μl 1XPBS. Add 100 microliters of EDTA (final concentration: 10 mM) and incubate at 37°C for 10 minutes. After centrifugation at 1500 rpm for 5 minutes, wash once with 200 microliters of staining solution. Staining: Dilute anti-human PD-L1 (2 microliters per well) and LIVE/DEAD staining solution (1:1000) in staining solution, add 50 microliters to each well, and stain at 4°C for 30 minutes. Wash twice with 200 μl staining solution. Fixation: Add 100 microliters of fixative to each well, and fix at 4°C for 15 minutes. Wash twice with 200 μl staining solution. 150 µl of resuspended cells. FACS detection. Table 9 shows the test results of the effect of the compounds of the present invention on the expression level of PD-L1 in MDA-MR-231 cells. Table 9 The test results of the impact of the compounds of the present invention on the expression level of PD-L1 in MDA-MR-231 cells test compound IC50(nM) Formate salt of compound of formula (I) 5.85

Experimental conclusion: the compound of formula (I) has a significant inhibitory effect on the expression level of PD-L1 in MDA-MR-231 cells.

Experimental example 3: NFAT activity test

Experimental principle: The surface of engineered T cells expresses PD-1 molecule and T cell receptor (TCR), and after co-culture with engineered antigen-presenting cells (APC), it can activate the NFAT signaling pathway of T cells. Expression of PD-L1 molecules on APCs can effectively weaken the NFAT signaling pathway in T cells; the use of PD-L1 inhibitors can effectively block the PD-1/PD-L1 regulatory mechanism, thereby reversing the weakened NFAT signaling pathway. After the small molecule was pretreated with APC, it was co-cultured with T cells, and then the expression of luciferase was detected to indirectly reflect the activation degree of the NFAT pathway in T cells.

Experimental Materials: PD-1/PD-L1 NFAT detection kit was purchased from BPS Biosciences. Birght-Glo reagent was purchased from Promega. Nivo Multilabel Analyzer (PerkinElmer).

Experimental method: Spread TCR Activator/PD-L1 CHO cells with a growth confluence of 80% into the plate at 35,000 cells per well and put them in a 37°C cell culture incubator overnight; dilute the compound to be tested 5 times with a row gun To the eighth concentration, that is, from 20 micromole to 0.25 nanomole, the DMSO concentration is 2%, and a double-well experiment is set up. Discard the TCR Activator/PD-L1 CHO cell supernatant, add 50 μl compound working solution to each well, and incubate at 37°C for 30 minutes; after the incubation, add 50 μl PD- ¹ / NFAT Reporter-Jurkat cell suspension, incubated at 37°C for 5 hours. After the incubation, add 100 microliters of Bright-Glo to each well, mix well and read the chemiluminescence signal with a Nivo multi-label analyzer.

Data analysis: Use the equation (sample-Min)/(Max-Min)×100% to convert the original data into inhibition rate, and the value of EC ₅₀ can be obtained by curve fitting with four parameters (log(inhibitor) in GraphPad Prism vs. response -- derived from Variable slope mode). Table 10 shows the test results of the compounds of the present invention on the degree of activation of the NFAT pathway in T cells. Table 10 The test results of the compounds of the present invention on the degree of activation of the NFAT pathway in T cells test compound EC50(nM) Maximum effect (fold induction) Formate salt of compound of formula (I) 41.45 5.05

Experimental conclusion: the compound of formula (I) can inhibit the interaction of PD-1/PD-L1 at the cellular level, thereby significantly activating the NFAT signaling pathway of T cells.

Experimental Example 4: Pharmacokinetic Test

Purpose of the experiment: To study the pharmacokinetics of compounds in C57BL/6 mice

Experimental materials: C57BL/6 mice (male, 8 weeks old, weighing 25g-30g)

Experimental operation: test the pharmacokinetic characteristics of rodents after intravenous (IV) and oral (PO) administration of the compound according to the standard protocol. Oral administration. Both intravenous and oral vehicles are 5% DMSO/5% polyethylene glycol 15-hydroxystearate (Solutol)/90% aqueous solution. The project used four male C57BL/6 mice, two mice were administered intravenously, the dose was 1 mg/kg, and 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8 were collected after administration , 24-hour plasma samples, and the other two mice were orally administered with a dose of 10 mg/kg, and plasma samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration . Collect whole blood samples within 24 hours, centrifuge at 3000 g for 15 minutes, separate supernatant to obtain plasma samples, add acetonitrile solution containing internal standard to precipitate protein, mix well and centrifuge to take supernatant for injection, and use LC-MS/MS analysis method Quantitative analysis of blood drug concentration, and calculation of pharmacokinetic parameters, such as peak concentration (C _max ), clearance (CL), half-life (T _1/2 ), tissue distribution (V _dss ), area under the drug-time curve (AUC _{0 -last} ), bioavailability (F) etc.

The pharmacokinetic parameters of the compounds of the present invention in mice are shown in Table 11 below. Table 11 Pharmacokinetic test results compound Formate salt of compound of formula (I) Peak concentration Cmax (nM) 370 Clearance CL (mL/min/kg) 18.5 Tissue distribution Vdss (L/kg) 36.8 Half-life T1/2 (IV, h) 26.3 Area under drug time curve AUC0-last PO (nM.hr) 5753 Bioavailability F (%) 80.8

Experimental conclusion: the compound of formula (I) has good pharmacokinetic properties, including good oral bioavailability, oral exposure, half-life and clearance rate, etc.

Experimental Example 5: Pharmacodynamic evaluation of compounds in C57BL/6-hPDL1 mouse colorectal cancer MC38-hPDL1 subcutaneous transplantation model

The purpose of the experiment: To evaluate the anti-tumor effect of the compound on mouse colorectal cancer cell MC38-hPDL1 transplanted into humanized mice C57BL/6-hPDL1.

Experimental Design: Table 12 group N G dose (mpk) way frequency G1 8 Vehicle (blank group) - po BID G2 8 Tecentriq 5 ip Q3D G3 8 Formate salt of compound of formula (I) 3 po BID G4 8 Formate salt of compound of formula (I) 10 po BID G5 8 Formate salt of compound of formula (I) 30 po BID G6 8 Formate salt of compound of formula (I) 60 po QD Remarks: G: Group; N: number of animals; po: oral administration; ip: intraperitoneal injection; BID: twice a day; Q3D: once every three days; QD: once a day.

Experimental Animals: Table 13 species mouse strain C57BL/6-hPDL1 level SPF level week age 4.29～5.71 gender female

Experimental method: 1. Tumor cell inoculation Experimental cell: Mouse colon cancer cell MC38-hPDL1 was revived, and the passage of revival was Pn+6. On the day of inoculation, collect MC38-hPDL1 cells in the logarithmic growth phase, remove the culture medium and wash twice with PBS before inoculation (the survival rates of MC38-hPDL1 cells before and after tumor loading were: 98.6% and 98.0%), the inoculum volume : 1×106/100 μL/mouse, inoculation position: right forelimb of mouse. 2. Group administration On the 7th day after inoculation, when the average tumor volume reached 85.81 mm ³ , 48 mice were randomly divided into 6 groups according to the tumor volume, with 8 mice in each group. The day of grouping was defined as D0 day, and administration began on D0 day. 3. Drug preparation and administration volume: adjusted according to mouse body weight (mouse administration volume = 10 μL/g × mouse body weight (g)) 4. Experimental observation and data collection After administration, on D0, D3, D5 , D7, D10, D12, D14, D17, D19, D21, D24 days to observe the tumor size and weigh the body weight of the mice. The calculation method of tumor volume is: tumor volume (mm3) = 0.5 × (tumor long diameter × tumor short diameter2). 5. At the end of the experiment, at the end of the experiment, analyze the following indicators: 1) Tumor volume change (TGItv); 2) Average body weight change; TGITV (relative tumor inhibition rate) calculation formula: TGItv (%) = [1-(meanTVtn-meanTVt0) /(meanTVvn-mean TVv0)]×100% meanTVtn: the average tumor volume of a certain treatment group measured on day n meanTVt0: the average tumor volume of a certain treatment group measured on day 0 meanTVvn: the solvent control group on day n Mean tumor volume at the time of measurement mean TVv0: Mean tumor volume of the solvent control group at day 0 when measured

Experimental results: The evaluation of the antitumor efficacy of the compound of the present invention on the subcutaneous transplantation model of colorectal cancer MC38-hPDL1 in C57BL/6-hPDL1 mice (calculated based on the tumor volume on the 24th day after administration) and the average body weight of the mice The impacts are shown in Tables 14 and 15 respectively: Table 14 G Dose (mg/kg) way frequency Tumor volume (mm3) (Day 24) Relative Tumor Inhibition Rate (TGI) (%) Vehicle (blank group) - po BID 1711.43±288.64 -- Formate salt of compound of formula (I) 3 po BID 717.79±223.17 61.12% Formate salt of compound of formula (I) 10 po BID 670.57±207.11 64.02% Formate salt of compound of formula (I) 30 po BID 580.07±173.73 69.59% Formate salt of compound of formula (I) 60 po QD 228.6±82.83 91.21% Remarks: Data are expressed as mean ± SEM. Table 15 G Average body weight (g) before administration (day 0) Average body weight on the 24th day of administration (g) Vehicle (blank group) 18.29±0.27 20.01±0.35 Formate salt of compound of formula (I) 18.08±0.32 18.91±0.38 Formate salt of compound of formula (I) 18.44±0.33 18.94±0.50 Formate salt of compound of formula (I) 18.64±0.28 19.41±0.43 Formate salt of compound of formula (I) 18.89±0.33 20.48±0.38 Remarks: Data are expressed as mean ± SEM.

Experimental conclusion: the compound of the present invention has an excellent tumor-inhibiting effect on the MC38-hPDL1 subcutaneous transplantation model of colorectal cancer in C57BL/6-hPDL1 mice, and there is no significant decrease in animal body weight during administration, and the tolerance is good.

Figure 1 is the XRPD spectrum of (II) Compound A crystal form. Figure 2 is the DSC spectrum of (II) Compound A crystal form. Figure 3 is the TGA spectrum of (II) Compound A crystal form. Figure 4 is the XRPD spectrum of (I) Compound B crystal form. Figure 5 is the DSC spectrum of (I) Compound B crystal form. Figure 6 is the TGA spectrum of (I) Compound B crystal form. Figure 7 is the XRPD spectrum of (I) Compound C crystal form. Figure 8 is the DSC spectrum of (I) Compound C crystal form. Figure 9 is the TGA spectrum of (I) Compound C crystal form. Figure 10 is the DVS isotherm spectrum of (I) Compound C crystal form.

Claims (28)

A crystal form of a compound of formula (II)

, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.19±0.20°, 7.77±0.20° and 16.22±0.20°. According to the crystal form A described in Claim 1, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.19±0.20°, 7.77±0.20°, 8.71±0.20°, 9.89±0.20° and 16.22 ±0.20°. According to the crystal form A described in Claim 2, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.19±0.20°, 7.77±0.20°, 8.71±0.20°, 9.89±0.20°, 16.22 ±0.20°, 17.74±0.20°, 22.82±0.20°, and 25.49±0.20°. According to the crystal form A described in Claim 3, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.19°, 7.77°, 8.71°, 9.89°, 11.47°, 14.41°, 16.22°, 17.74°, 19.67°, 20.98°, 22.82°, 25.49°, and 28.15°. According to the crystal form A described in Claim 4, its XRPD spectrum is shown in Figure 1 . According to the crystal form A described in any one of claims 1-5, its differential scanning calorimetry curve has the start points of endothermic peaks at 31.2°C±5°C and 148.7°C±5°C. According to the crystal form A described in Claim 6, its DSC spectrum is shown in FIG. 2 . According to the crystal form A described in any one of claims 1 to 5, its thermogravimetric analysis curve has a weight loss of 1.77% at 130°C±3°C, and a weight loss of 3.32% at 160°C±3°C. According to the crystal form A described in Claim 8, its TGA spectrum is shown in FIG. 3 . A crystal form B of a compound of formula (I)

, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 6.52±0.20°, 9.40±0.20° and 14.94±0.20°. According to the crystal form B described in Claim 10, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 6.52±0.20°, 9.40±0.20°, 14.94±0.20°, 16.10±0.20° and 22.86 ±0.20°. According to the B crystal form described in claim item 11, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 6.52±0.20°, 9.40±0.20°, 12.93±0.20°, 14.94±0.20°, 16.10 ±0.20°, 19.06±0.20°, 19.89±0.20°, and 22.86±0.20°. According to the crystal form B described in Claim 12, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 6.52°, 7.47°, 9.40°, 11.29°, 12.93°, 13.90°, 14.94°, 16.10°, 19.06°, 19.89°, 21.13°, 22.86°, 24.55°, 25.25°, 25.83°, 26.23°, 27.45°, and 34.58°. According to the crystal form B described in Claim 13, its XRPD spectrum is shown in Figure 4 . According to the B crystal form described in any one of claims 10-14, its differential scanning calorimetry curve has an endothermic peak start point at 145.2°C±5°C. According to the crystal form B described in Claim 15, its DSC spectrum is shown in FIG. 5 . According to the crystal form B described in any one of claims 10-14, its thermogravimetric analysis curve has a weight loss of 0.77% at 150°C±3°C. According to the crystal form B described in Claim 17, its TGA spectrum is shown in Figure 6 . A crystal form C of a compound of formula (I)

, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 15.15±0.20°, 17.50±0.20° and 25.04±0.20°. According to the C crystal form described in Claim 19, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.75±0.20°, 15.15±0.20°, 17.50±0.20°, 22.94±0.20° and 25.04 ±0.20°. According to the C crystal form described in Claim 20, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.75±0.20°, 15.15±0.20°, 17.50±0.20°, 18.77±0.20°, 22.94 ±0.20°, 25.04±0.20°, 26.20±0.20°, 37.65±0.20°. According to the crystal form C described in Claim 21, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.39°, 8.75°, 11.32°, 12.55°, 13.74°, 14.07°, 15.15°, 17.03°, 17.28°, 17.50°, 18.07°, 18.77°, 19.70°, 20.35°, 21.56°, 21.83°, 22.54°, 22.94°, 23.33°, 23.74°, 24.56°, 25.04°, 25.33°, 26.20° , 27.24°, 27.93°, 29.33°, 30.75°, 31.39°, 32.44°, 33.01°, 34.07°, 36.32°, 37.65°, 38.01°. According to the crystal form C described in claim 22, its XRPD spectrum is shown in Figure 7 . According to the crystal form C according to any one of claims 19-23, its differential scanning calorimetry curve has an endothermic peak start point at 184.5°C±5°C. According to the C crystal form described in Claim 24, its DSC spectrum is shown in Figure 8 . According to the C crystal form described in any one of claims 19-23, its thermogravimetric analysis curve has a weight loss of 0.69% at 150°C±3°C. According to the C crystal form described in Claim 26, its TGA spectrum is shown in Figure 9 . According to the crystal form A described in any one of claim items 1-9 or the crystal form B according to any one of claim items 10-18 or the crystal form C according to any one of claim items 19-27 in the preparation of anti- Application in tumor medicine.

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