Crystal form of quinazoline compound and preparation method thereof

Abstract

The invention discloses a crystal form of a quinazoline compound and a preparation method of the crystal form, and particularly discloses a crystal form of a compound shown in a formula (I) and a preparation method and application of the crystal form. # imgabs0 #

Description

Crystal form of quinazoline compound and preparation method thereof

[0001] This application claims priority to:

[0002] CN202211169562.X, September 23, 2022. Technical Field

[0003] The present invention discloses a crystal form of a quinazoline compound and a preparation method thereof, and particularly discloses the crystal form of a compound of formula (I) and an application of the preparation method thereof. Background Art

[0004] RAS proteins are guanine nucleoside-binding proteins with guanosine triphosphate hydrolase (GTPase) activity. They primarily consist of three isoforms: KRAS, NRAS, and HRAS. As binary molecular switches controlled by the GDP / GTP cycle, RAS proteins can cycle between an active, GTP-bound state (GTP-RAS) and an inactive, GDP-bound state (GDP-RAS). This cycle plays a crucial regulatory role in cells and is closely associated with cell proliferation, survival, metabolism, migration, immunity, and growth.

[0005] SOS1 (Son of Sevenless 1) is a type of GEF that regulates the GDP / GTP cycle of RAS proteins. After the cell surface receptor is activated and binds to intracellular Grb2, Grb2 recruits SOS1 to the cell membrane. SOS1 then catalyzes RAS-GDP / GTP exchange, thereby activating downstream signaling pathways. Small molecule SOS1 inhibitors that bind to the catalytic site can block the binding of SOS1 to RAS proteins, thereby effectively reducing the abnormal activation of RAS downstream signaling pathways in cancer cells and playing a role in treating cancer. Currently, the only SOS1 small molecule inhibitor, BI-1701963 (WO2018115380, WO2019122129) developed by Boehringer Ingelheim, has entered Phase I clinical trials. The SOS1 inhibitor developed by Bayer (WO2018172250, WO2019201848) is still in the preclinical research stage. Recent studies have suggested that drugs targeting the RAS pathway are prone to developing resistance during clinical use. Some of this resistance stems from the negative feedback loop that occurs when ERK phosphorylation is inhibited, activating the upstream RAS pathway. This negative feedback loop is closely linked to SOS1. Therefore, the development of small molecule inhibitors of SOS1 holds great promise.

[0006] AMG-510 is a potent, orally bioavailable, selective KRAS G12C covalent inhibitor developed by Amgen for the treatment of locally advanced or metastatic non-small cell lung cancer carrying KRAS G12C mutations. Its structure is shown below:

[0007] Summary of the Invention

[0008] The present invention provides a crystalline form A of the compound of formula (I), which has an X-ray powder diffraction pattern (XRPD) having characteristic diffraction peaks at the following 2θ angles: 15.492±0.200°, 16.458±0.200°, 18.657±0.200° and 20.638±0.200°;

[0009] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 14.223±0.200°, 14.589±0.200°, 14.894±0.200°, 15.492±0.200°, 16.061±0.200°, 16.458±0.200°, 18.657±0.200° and 20.638±0.200°.

[0010] In some embodiments of the present invention, the above-mentioned crystal form A has an X-ray powder diffraction pattern, expressed as 2θ angle, comprising at least 4, 5, 6, 7 or 8 characteristic diffraction peaks selected from the following: 14.223±0.200°, 14.589±0.200°, 14.894±0.200°, 15.492±0.200°, 16.061±0.200°, 16.458±0.200°, 18.657±0.200° and 20.638±0.200°.

[0011] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 14.223±0.100°, 14.589±0.100°, 14.894±0.100°, 15.492±0.100°, 16.061±0.100°, 16.458±0.100°, 18.657±0.100° and 20.638±0.100°.

[0012] In some embodiments of the present invention, the above-mentioned crystal form A has an X-ray powder diffraction pattern, expressed as 2θ angle, comprising at least 4, 5, 6, 7 or 8 characteristic diffraction peaks selected from the following: 14.223±0.100°, 14.589±0.100°, 14.894±0.100°, 15.492±0.100°, 16.061±0.100°, 16.458±0.100°, 18.657±0.100° and 20.638±0.100°.

[0013] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.736±0.200°, 14.223±0.200°, 14.589±0.200°, 14.894±0.200°, 15.492±0.200°, 16.061±0.200°, 16.458±0.200°, 18.657±0.200°, 19.407±0.200°, 20.638±0.200°, 21.810±0.200° and 22.836±0.200°.

[0014] In some embodiments of the present invention, the above-mentioned Form A has an X-ray powder diffraction pattern, expressed as 2θ angle, comprising at least 8, 9, 10, 11 or 12 characteristic diffraction peaks selected from the following: 7.736±0.200°, 14.223±0.200°, 14.589±0.200°, 14.894±0.200°, 15.492±0.200°, 16.061±0.200°, 16.458±0.200°, 18.657±0.200°, 19.407±0.200°, 20.638±0.200°, 21.810±0.200° and 22.836±0.200°.

[0015] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.736±0.100°, 14.223±0.100°, 14.589±0.100°, 14.894±0.100°, 15.492±0.100°, 16.061±0.100°, 16.458±0.100°, 18.657±0.100°, 19.407±0.100°, 20.638±0.100°, 21.810±0.100° and 22.836±0.100°.

[0016] In some embodiments of the present invention, the above-mentioned Form A has an X-ray powder diffraction pattern, expressed as 2θ angle, comprising at least 8, 9, 10, 11 or 12 characteristic diffraction peaks selected from the following: 7.736±0.100°, 14.223±0.100°, 14.589±0.100°, 14.894±0.100°, 15.492±0.100°, 16.061±0.100°, 16.458±0.100°, 18.657±0.100°, 19.407±0.100°, 20.638±0.100°, 21.810±0.100° and 22.836±0.100°.

[0017] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.736±0.200°, 9.070±0.200°, 11.289±0.200°, 11.678±0.200°, 14.223±0.200°, 14.589±0.200°, 14.894±0 .200°, 15.492±0.200°, 16.061±0.200°, 16.458±0.200°, 18.657±0.200°, 19.407±0.200°, 20.638±0.200°, 21.085±0.200°, 21.810±0.200° and 22.836±0.200°.

[0018] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.736±0.100°, 9.070±0.100°, 11.289±0.100°, 11.678±0.100°, 14.223±0.100°, 14.589±0.100°, 14.894±0 .100°, 15.492±0.100°, 16.061±0.100°, 16.458±0.100°, 18.657±0.100°, 19.407±0.100°, 20.638±0.100°, 21.085±0.100°, 21.810±0.100° and 22.836±0.100°.

[0019] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.736±0.200°, 8.475±0.200°, 9.070±0.200°, 11.289±0.200°, 11.678±0.200°, 12.363±0.200°, 14.223±0.200°, 14.589±0.200°, 14.894±0.200°, 15.492±0.200°, 16.061±0.200°, 16.458±0.200°, 17.000±0.200°, 18. .657±0.200°, 19.030±0.200°, 19.407±0.200°, 19.882±0.200°, 20.638±0.200°, 21.085±0.200°, 21.810±0.200°, 22.836±0.200°, 23.717±0.200°, 24.147±0.200°, 24.693±0.200°, 25.311±0.200°, 26.802±0.200°, 27.462±0.200°, 28.537±0.200° and 31.264±0.200°.

[0020] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.736±0.200°, 8.475±0.200°, 9.070±0.200°, 11.289±0.200°, 11.678±0.200°, 12.363±0.200°, 14.223 ±0.200°, 14.589±0.200°, 14.894±0.200°, 15.492±0.200°, 16.061±0.200°, 16.458±0.200°, 17.000±0.200°, 18.657±0.200°, 19.030±0.200°, 19.407±0. 200°, 19.882±0.200°, 20.638±0.200°, 21.085±0.200°, 21.810±0.200°, 22.836±0.200°, 23.717±0.200°, 24.147±0.200°, 24.693±0.200°, 25.311±0.20 0°, 26.802±0.200°, 27.462±0.200°, 28.537±0.200°, 30.090±0.200°, 31.264±0.200°, 32.346±0.200°, 33.429±0.200°, 35.334±0.200° and 36.567±0.200°.

[0021] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.736±0.100°, 8.475±0.100°, 9.070±0.100°, 11.289±0.100°, 11.678±0.100°, 12.363±0.100°, 14.223±0.100°, 14.589±0.100°, 14.894±0.100°, 15.492±0.100°, 16.061±0.100°, 16.458±0.100°, 17.000±0.100°, 18. .657±0.100°, 19.030±0.100°, 19.407±0.100°, 19.882±0.100°, 20.638±0.100°, 21.085±0.100°, 21.810±0.100°, 22.836±0.100°, 23.717±0.100°, 24.147±0.100°, 24.693±0.100°, 25.311±0.100°, 26.802±0.100°, 27.462±0.100°, 28.537±0.100° and 31.264±0.100°.

[0022] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.736±0.100°, 8.475±0.100°, 9.070±0.100°, 11.289±0.100°, 11.678±0.100°, 12.363±0.100°, 14.223 ±0.100°, 14.589±0.100°, 14.894±0.100°, 15.492±0.100°, 16.061±0.100°, 16.458±0.100°, 17.000±0.100°, 18.657±0.100°, 19.030±0.100°, 19.407±0. 100°, 19.882±0.100°, 20.638±0.100°, 21.085±0.100°, 21.810±0.100°, 22.836±0.100°, 23.717±0.100°, 24.147±0.100°, 24.693±0.100°, 25.311±0.10 0°, 26.802±0.100°, 27.462±0.100°, 28.537±0.100°, 30.090±0.100°, 31.264±0.100°, 32.346±0.100°, 33.429±0.100°, 35.334±0.100° and 36.567±0.100°.

[0023] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.736°, 8.475°, 9.070°, 11.289°, 11.678°, 12.363°, 14.223°, 14.589°, 14.894°, 15.492°, 16.061°, 16.458°, 17.000°, 18.657°, 19.030°, 19.407°, 19.882°, 20.638°, 21.085°, 21.810°, 22.836°, 23.717°, 24.147°, 24.693°, 25.311°, 26.802°, 27.462°, 28.537° and 31.264°.

[0024] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.736°, 8.475°, 9.070°, 11.289°, 11.678°, 12.363°, 14.223°, 14.589°, 14.894°, 15.492°, 16.061°, 16.458°, 17.000°, 18.657°, 19.03 0°, 19.407°, 19.882°, 20.638°, 21.085°, 21.810°, 22.836°, 23.717°, 24.147°, 24.693°, 25.311°, 26.802°, 27.462°, 28.537°, 30.090°, 31.264°, 32.346°, 33.429°, 35.334°, and 36.567°.

[0025] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 15.492±0.200°, 16.458±0.200°, 18.657±0.200°, and / or 7.736±0.200°, and / or 8.475±0.200°, and / or 9.070±0.200°, and / or 11.289±0.200°, and / or 11.678±0.200°, and / or 12.363±0.200°, and / or 14.223±0.200°, and / or 14.589±0.200°, and / or 14.894±0.200°, and / or 16.061±0.200°, and / or 17.000±0.200°, and / or 19.030±0.200°, and / or 19.407±0.200°, and / or or 19.882±0.200°, and / or 20.638±0.200°, and / or 21.085±0.200°, and / or 21.810±0.200°, and / or 22.836±0.200°, and / or 23.717±0.200°, and / or 24.147±0.200°, and / or 24.693±0.200°, and / or 25.311±0.200°, and / or 26.802±0.200°, and / or 27.462±0.200°, and / or 28.537±0.200°, and / or 30.090±0.200°, and / or 31.264±0.200°, and / or 32.346±0.200°, and / or 33.429±0.200°, and / or 35.334±0.200°, and / or 36.567±0.200°.

[0026] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 15.492±0.100°, 16.458±0.100°, 18.657±0.100°, and / or 7.736±0.100°, and / or 8.475±0.100°, and / or 9.070±0.100°, and / or 11.289±0.100°, and / or 11.678±0.100°, and / or 12.363±0.100°, and / or 14.223±0.100°, and / or 14.589±0.100°, and / or 14.894±0.100°, and / or 16.061±0.100°, and / or 17.000±0.100°, and / or 19.030±0.100°, and / or 19.407±0.100°, and / or or 19.882±0.100°, and / or 20.638±0.100°, and / or 21.085±0.100°, and / or 21.810±0.100°, and / or 22.836±0.100°, and / or 23.717±0.100°, and / or 24.147±0.100°, and / or 24.693±0.100°, and / or 25.311±0.100°, and / or 26.802±0.100°, and / or 27.462±0.100°, and / or 28.537±0.100°, and / or 30.090±0.100°, and / or 31.264±0.100°, and / or 32.346±0.100°, and / or 33.429±0.100°, and / or 35.334±0.100°, and / or 36.567±0.100°.

[0027] In some embodiments of the present invention, the XRPD pattern of the above-mentioned crystal form A is basically as shown in Figure 1.

[0028] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form A is shown in Table 1.

[0029] Table 1 XRPD pattern analysis data of the crystal form of compound A of formula (I)

[0030] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned crystal form A has an endothermic peak starting point at 169.0±5°C.

[0031] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned crystal form A has endothermic peak starting points at 31.6±5°C and 169.0±5°C.

[0032] In some embodiments of the present invention, the DSC spectrum of the above-mentioned crystal form A is basically as shown in Figure 2.

[0033] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned crystal form A shows a weight loss of 1.48% at 160.0±3°C.

[0034] In some embodiments of the present invention, the TGA spectrum of the above-mentioned crystal form A is basically as shown in Figure 3.

[0035] The present invention also provides a crystalline form B of the compound of formula (I), which has an X-ray powder diffraction pattern (XRPD) having characteristic diffraction peaks at the following 2θ angles: 5.932±0.200°, 12.435±0.200°, 14.091±0.200° and 16.496±0.200°;

[0036] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 5.932±0.200°, 8.551±0.200°, 10.855±0.200°, 12.435±0.200°, 14.091±0.200°, 15.171±0.200°, 16.496±0.200° and 23.682±0.200°.

[0037] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form, expressed as 2θ angle, contains at least 4, 5, 6, 7 or 8 characteristic diffraction peaks selected from the following: 5.932±0.200°, 8.551±0.200°, 10.855±0.200°, 12.435±0.200°, 14.091±0.200°, 15.171±0.200°, 16.496±0.200° and 23.682±0.200°.

[0038] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 5.932±0.200°, 6.751±0.200°, 8.551±0.200°, 9.098±0.200°, 10.855±0.200°, 12.435±0.200°, 14.091±0.200°, 15.171±0.200°, 16.496±0.200°, 19.193±0.200°, 20.501±0.200° and 23.682±0.200°.

[0039] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned Form B, expressed in 2θ angles, comprises at least 8, 9, 10, 11 or 12 characteristic diffraction peaks selected from the following: 5.932±0.200°, 6.751±0.200°, 8.551±0.200°, 9.098±0.200°, 10.855±0.200°, 12.435±0.200°, 14.091±0.200°, 15.171±0.200°, 16.496±0.200°, 19.193±0.200°, 20.501±0.200° and 23.682±0.200°.

[0040] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 5.932±0.200°, 6.751±0.200°, 8.551±0.200°, 9.098±0.200°, 10.186±0.200°, 10.855±0.200°, 11.710±0.200°, 12.435±0.200°, 14.091±0.200°, 15.171±0. 200°, 16.049±0.200°, 16.496±0.200°, 17.438±0.200°, 18.658±0.200°, 19.193±0.200°, 20.501±0.200°, 21.241±0.200°, 21.977±0.200°, 23.682±0.200°, 26.127±0.200°, 26.981±0.200° and 29.033±0.200°.

[0041] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned Form B has characteristic diffraction peaks at the following 2θ angles: 5.932°, 6.751°, 8.551°, 9.098°, 10.186°, 10.855°, 11.710°, 12.435°, 14.091°, 15.171°, 16.049°, 16.496°, 17.438°, 18.658°, 19.193°, 20.501°, 21.241°, 21.977°, 23.682°, 26.127°, 26.981° and 29.033°.

[0042] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 5.932±0.200°, 12.435±0.200°, 14.091±0.200°, and / or 6.751±0.200°, and / or 8.551±0.200°, and / or 9.098±0.200°, and / or 10.186±0.200°, and / or 10.855±0.200°, and / or 11.710±0.200°, and / or 12.435±0.200°, and / or 14.091±0.200°, and / or 15.171±0.200°, and / or 16.049±0.200°, and / or 16.496±0.200°, and / or 17.438±0.200°, and / or 18.658±0.200°, and / or 19.193±0.200°, and / or 20.501±0.200°, and / or 21.241±0.200°, and / or 21.977±0.200°, and / or 23.682±0.200°, and / or 26.127±0.200°, and / or 26.981±0.200°, and / or 29.033±0.200°.

[0043] In some embodiments of the present invention, the XRPD pattern of the above-mentioned Form B is basically as shown in Figure 4.

[0044] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form B is shown in Table 2.

[0045] Table 2 XRPD pattern analysis data of the crystal form of compound B of formula (I)

[0046] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned Form B has endothermic peak starting points at 64.5±5°C and 121.7±5°C.

[0047] In some embodiments of the present invention, the DSC spectrum of the above-mentioned Form B is basically as shown in Figure 5.

[0048] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned Form B shows a weight loss of 4.01% at 105.0±3°C.

[0049] In some embodiments of the present invention, the TGA spectrum of the above-mentioned B crystal form is basically as shown in Figure 6.

[0050] The present invention also provides Form C of the compound of formula (I), which has an X-ray powder diffraction pattern (XRPD) having characteristic diffraction peaks at the following 2θ angles: 13.546±0.200°, 14.908±0.200°, 15.539±0.200°, 18.230±0.200°, and 22.932±0.200°;

[0051] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 13.546±0.200°, 14.908±0.200°, 15.539±0.200°, 16.401±0.200°, 18.230±0.200°, 18.699±0.200°, 20.226±0.200° and 22.932±0.200°.

[0052] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned C crystal form, expressed as 2θ angle, contains at least 4, 5, 6, 7 or 8 characteristic diffraction peaks selected from the following: 13.546±0.200°, 14.908±0.200°, 15.539±0.200°, 16.401±0.200°, 18.230±0.200°, 18.699±0.200°, 20.226±0.200° and 22.932±0.200°.

[0053] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 7.410±0.200°, 8.474±0.200°, 13.546±0.200°, 14.122±0.200°, 14.908±0.200°, 15.539±0.200°, 16.401±0.200°, 18.230±0.200°, 18.699±0.200°, 19.665±0.200°, 20.226±0.200° and 22.932±0.200°.

[0054] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned C crystal form, expressed as 2θ angle, contains at least 8, 9, 10, 11 or 12 characteristic diffraction peaks selected from the following: 7.410±0.200°, 8.474±0.200°, 13.546±0.200°, 14.122±0.200°, 14.908±0.200°, 15.539±0.200°, 16.401±0.200°, 18.230±0.200°, 18.699±0.200°, 19.665±0.200°, 20.226±0.200° and 22.932±0.200°.

[0055] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 7.410±0.200°, 8.474±0.200°, 9.093±0.200°, 11.284±0.200°, 13.546±0.200°, 14.122±0.200°, 14.908±0. 200°, 15.539±0.200°, 16.401±0.200°, 18.230±0.200°, 18.699±0.200°, 18.911±0.200°, 19.665±0.200°, 20.226±0.200°, 21.508±0.200° and 22.932±0.200°.

[0056] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 7.410±0.100°, 8.474±0.100°, 9.093±0.100°, 11.284±0.100°, 13.546±0.100°, 14.122±0.100°, 14.908±0. 100°, 15.539±0.100°, 16.401±0.100°, 18.230±0.100°, 18.699±0.100°, 18.911±0.100°, 19.665±0.100°, 20.226±0.100°, 21.508±0.100° and 22.932±0.100°.

[0057] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 7.410±0.200°, 8.474±0.200°, 9.093±0.200°, 11.284±0.200°, 11.439±0.200°, 13.546±0.200°, 14.122±0.200°, 14.908±0.200°, 15.539±0.200°, 16.401±0.200°, 16.743±0. 200°, 17.169±0.200°, 18.230±0.200°, 18.699±0.200°, 18.911±0.200°, 19.665±0.200°, 20.226±0.200°, 21.508±0.200°, 22.932±0.200°, 23.907±0.200°, 25.111±0.200°, 27.317±0.200°, 29.113±0.200° and 31.325±0.200°.

[0058] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 7.410±0.100°, 8.474±0.100°, 9.093±0.100°, 11.284±0.100°, 11.439±0.100°, 13.546±0.100°, 14.122±0.100°, 14.908±0.100°, 15.539±0.100°, 16.401±0.100°, 16.743±0. 100°, 17.169±0.100°, 18.230±0.100°, 18.699±0.100°, 18.911±0.100°, 19.665±0.100°, 20.226±0.100°, 21.508±0.100°, 22.932±0.100°, 23.907±0.100°, 25.111±0.100°, 27.317±0.100°, 29.113±0.100° and 31.325±0.100°.

[0059] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 7.410°, 8.474°, 9.093°, 11.284°, 11.439°, 13.546°, 14.122°, 14.908°, 15.539°, 16.401°, 16.743°, 17.169°, 18.230°, 18.699°, 18.911°, 19.665°, 20.226°, 21.508°, 22.932°, 23.907°, 25.111°, 27.317°, 29.113° and 31.325°.

[0060] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 7.410±0.200°, 8.474±0.200°, 9.093±0.200°, 11.284±0.200°, 11.439±0.200°, 13.546±0.200°, 14.122±0.200°, 14.908±0.200°, 15.539±0.200°, 16.401±0.200°, 16.743±0. 200°, 17.169±0.200°, 18.230±0.200°, 18.699±0.200°, 18.911±0.200°, 19.665±0.200°, 20.226±0.200°, 21.508±0.200°, 22.932±0.200°, 23.907±0.200°, 25.111±0.200°, 27.317±0.200°, 29.113±0.200° and 31.325±0.200°.

[0061] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 7.410±0.100°, 8.474±0.100°, 9.093±0.100°, 11.284±0.100°, 11.439±0.100°, 13.546±0.100°, 14.122±0.100°, 14.908±0.100°, 15.539±0.100°, 16.401±0.100°, 16.743±0. 100°, 17.169±0.100°, 18.230±0.100°, 18.699±0.100°, 18.911±0.100°, 19.665±0.100°, 20.226±0.100°, 21.508±0.100°, 22.932±0.100°, 23.907±0.100°, 25.111±0.100°, 27.317±0.100°, 29.113±0.100° and 31.325±0.100°.

[0062] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 13.546±0.200°, 14.908±0.200°, 15.539±0.200°, and / or 7.410±0.200°, and / or 8.474±0.200°, and / or 9.093±0.200°, and / or 11.284±0.200°, and / or or 11.439±0.200°, and / or 14.122±0.200°, and / or 16.401±0.200°, and / or 16.743±0.200°, and / or 17.169±0.200°, and / or 18.230±0.200°, and / or 18.699±0.200°, and / or 18.911±0.200°, and / or 19.665±0.200°, and / or 20.226±0.200°, and / or 21.508±0.200°, and / or 22.932±0.200°, and / or 23.907±0.200°, and / or 25.111±0.200°, and / or 27.317±0.200°, and / or 29.113±0.200°, and / or 31.325±0.200°.

[0063] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 13.546±0.100°, 14.908±0.100°, 15.539±0.100°, and / or 7.410±0.100°, and / or 8.474±0.100°, and / or 9.093±0.100°, and / or 11.284±0.100°, and / or 11.439±0.100°, and / or 14.122±0.100°, and / or 16.401±0.100°, and / or 16.743±0.100°, and / or Or 17.169±0.100°, and / or 18.230±0.100°, and / or 18.699±0.100°, and / or 18.911±0.100°, and / or 19.665±0.100°, and / or 20.226±0.100°, and / or 21.508±0.100°, and / or 22.932±0.100°, and / or 23.907±0.100°, and / or 25.111±0.100°, and / or 27.317±0.100°, and / or 29.113±0.100°, and / or 31.325±0.100°.

[0064] In some embodiments of the present invention, the XRPD pattern of the above-mentioned Form C is basically as shown in Figure 7.

[0065] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form C are shown in Table 3.

[0066] Table 3 XRPD pattern analysis data of the crystal form C of the compound of formula (I)

[0067] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned crystal form C has an endothermic peak starting point at 169.2±5°C.

[0068] In some embodiments of the present invention, the DSC spectrum of the above-mentioned crystal form C is basically as shown in Figure 8.

[0069] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned crystal form C shows a weight loss of 2.00% at 130.0±3°C.

[0070] In some embodiments of the present invention, the TGA spectrum of the above-mentioned C crystal form is basically as shown in Figure 9.

[0071] The present invention also provides the use of the crystal form of the compound of formula (I) in the preparation of a drug for treating KRAS mutant solid tumor diseases.

[0072] The present invention also provides the use of the crystal form A of the compound of formula (I) in the preparation of a drug for treating KRAS mutant solid tumor diseases.

[0073] The present invention also provides the following biological testing method for the crystalline form of the compound of formula (I):

[0074] Test method 1: In vivo efficacy evaluation of the compound in the Miapaca2 nude mouse xenograft tumor model

[0075] Cell culture:

[0076] Human pancreatic cancer cells (Miapaca2) were cultured as adherent monolayers in DMEM supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged two to three times weekly using trypsin-EDTA. When cell saturation reached 80%–90% and the desired number of cells was reached, cells were harvested, counted, and plated.

[0077] Experimental animals:

[0078] Balb / c nude mice, female, 6-7 weeks old, were purchased from Shanghai Xipul-Bikai Laboratory Animal Co., Ltd.

[0079] Model preparation:

[0080] 0.2 mL (5 × 10 6 Miapaca2 cells (with Matrigel, volume ratio of 1:1) were subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached 118 mm 3 The group dosing started at 14:00.

[0081] Tumor measurements and experimental parameters:

[0082] Tumor diameter was measured twice a week with a vernier caliper, and tumor volume was measured in cubic millimeters and calculated using the following formula: V = 0.5a × b 2 , where a and b are the long and short diameters of the tumor, respectively. The antitumor efficacy of the test compound was evaluated using the TGI (%). TGI (%) reflects the rate of tumor growth inhibition. TGI (%) = [1 – (average tumor volume of a treatment group at the end of dosing – average tumor volume of the treatment group at the start of dosing) / (average tumor volume of the solvent control group at the end of treatment – ​​average tumor volume of the solvent control group at the start of treatment)] × 100%.

[0083] Experimental conclusion: The compound of the present invention combined with AMG-510 showed excellent tumor inhibition effect in the Miapaca2 nude mouse transplant tumor model.

[0084] Technical Effects

[0085] The compound of the present invention can effectively inhibit the activity of SOS1; it also has significant inhibitory activity on the proliferation of p-ERK in DLD-1 cells and has good pharmacokinetic properties (including good oral bioavailability, oral exposure, half-life, and clearance). The compound has no significant inhibitory effect on the hERG potassium ion channel and is highly safe. The compound of the present invention has excellent tumor inhibition effects in the human pancreatic cancer Miapaca-2 xenograft tumor model. The crystalline form of the compound of formula (I) of the present invention is easily available and has good physical and chemical stability, and has high industrial application value and economic value.

[0086] Definition and Description

[0087] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0088] It must be noted that, unless the context clearly indicates otherwise or is clearly contradicted by the context, the singular forms "a," "an," "the," and similar terms as used in the disclosure of the invention as used herein and in the appended claims (especially in the appended claims) are to be construed to include both the singular and the plural, thus, for example, reference to "the compound" includes reference to one or more compounds; and so on.

[0089] The differential scanning calorimetry (DSC) of the crystal form of the present invention has experimental errors and is slightly affected by the degree of dryness of the sample. The position and peak value of the endothermic peak may vary slightly between one machine and another and between one sample and another. The numerical value of the experimental error or difference may be less than or equal to 10°C, or less than or equal to 9°C, or less than or equal to 8°C, or less than or equal to 7°C, or less than or equal to 6°C, or less than or equal to 5°C, or less than or equal to 4°C, or less than or equal to 3°C, or less than or equal to 2°C, or less than or equal to 1°C. Therefore, the peak position or peak value of the DSC endothermic peak cannot be considered absolute.

[0090] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0091] The structures of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compounds, the absolute configuration can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data on the cultured single crystal using a Bruker D8 venture diffractometer, using CuKα radiation as the light source and scanning mode: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0092] The chemical reactions of the present invention are carried out in suitable solvents that are compatible with the chemical transformations of the present invention and the reagents and materials required. To obtain the compounds of the present invention, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0093] The present invention will be described in detail below through examples, which are not intended to limit the present invention in any way.

[0094] All solvents used in the present invention were commercially available and used without further purification.

[0095] The solvent used in the present invention is commercially available.

[0096] The present invention uses the following abbreviations: Boc represents tert-butyloxycarbonyl; DCM represents dichloromethane; DMF represents N,N-dimethylformamide; THF represents tetrahydrofuran; DMSO represents dimethyl sulfoxide; EtOH represents ethanol; MeOH represents methanol; ACN (MeCN) represents acetonitrile; EtOAc represents ethyl acetate; H2O represents water; Acetone represents acetone; IPAc represents isopropyl acetate; MTBE represents methyl tert-butyl ether; 1,4-Dioxane represents 1,4-dioxane; n-Heptane represents n-heptane; i-PrOAc represents isopropyl acetate; TEA represents triethylamine; DIPEA represents diisopropylethylamine; BID represents twice daily; QD represents once daily; and po represents oral administration.

[0097] Compounds are manually or Software naming, commercially available compounds use supplier catalog names.

[0098] Instrument and analysis method of the present invention

[0099] The X-ray powder diffractometer (XRPD) method 1 of the present invention has test parameters shown in Table 4.

[0100] Table 4 XRPD test parameters

[0101] The test parameters of the X-ray powder diffractometer (XRPD) method 2 of the present invention are shown in Table 5.

[0102] Table 5 XRPD test parameters

[0103] The test parameters of the differential scanning calorimeter (DSC) method of the present invention are shown in Table 6.

[0104] Table 6 DSC test parameters

[0105] Thermogravimetric analysis (TGA) method of the present invention, test parameters are shown in Table 7.

[0106] Table 7 TGA test parameters

[0107] The test parameters of the dynamic vapor sorption analysis (DVS) method of the present invention are shown in Table 8.

[0108] Table 8 DVS test parameters BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1: XRPD spectrum of the crystal form A of the compound of formula (I) using Cu-Kα radiation.

[0110] Figure 2: DSC spectrum of crystalline form A of compound of formula (I).

[0111] Figure 3: TGA spectrum of crystalline form A of compound of formula (I).

[0112] Figure 4: XRPD spectrum of the crystal form B of the compound of formula (I) using Cu-Kα radiation.

[0113] Figure 5: DSC spectrum of crystalline form B of compound of formula (I).

[0114] Figure 6: TGA spectrum of the crystalline form B of compound of formula (I).

[0115] Figure 7: XRPD spectrum of Form C of the compound of formula (I) using Cu-Kα radiation.

[0116] Figure 8: DSC spectrum of Form C of Compound of Formula (I).

[0117] Figure 9: TGA spectrum of Form C of Compound of Formula (I).

[0118] Figure 10: DVS spectrum of the crystalline form A of compound of formula (I).

[0119] Figure 11: Single crystal X-ray diffraction (SXRD) ellipsoidal diagram of the three-dimensional structure of the compound of formula (I). DETAILED DESCRIPTION

[0120] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments, but the specific implementation methods are not intended to limit the content of the present invention.

[0121] Example 1: Preparation of the hydrochloride salt of the compound of formula (I)

[0122] Synthesis route:

[0123] first step

[0124] Compound 1 (10.0 g, 49.9 mmol) was dissolved in methanol (100 mL), and aqueous formaldehyde (20.3 g, 250 mmol, purity: 37%), sodium cyanoborohydride (9.41 g, 150 mmol), and acetic acid (15.0 g, 250 mmol) were added. The mixture was stirred at 25°C for 12 hours. Water (300 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (200 mL × 3). The organic phase was washed with saturated brine (100 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (dichloromethane / methanol, 1 / 0 to 10 / 1, V / V) to obtain compound 2. 1 H NMR (400 MHz, CDCl3) δ 4.21-4.18 (m, 1H), 3.82-3.79 (m, 1H), 3.13-3.06 (m, 1H), 2.74-2.70 (m, 1H), 2.60-2.52 (m, 1H), 2.24 (s, 3H), 2.11-2.07 (m, 1H), 1.93-1.86 (m, 1H), 1.45 (s, 9H), 1.29 (d, J = 7.2 Hz, 3H). MS-ESI calculated value [M+H] + 215, measured value 215.

[0125] Step 2

[0126] Compound 2 (7.25 g, 33.8 mmol) was dissolved in dioxane (50 mL), and hydrogen chloride / dioxane solution (4 M, 67.6 mL) was added dropwise. The reaction solution was stirred at 25 °C for 12 hours, and the reaction solution was concentrated under reduced pressure to obtain the hydrochloride salt of crude compound 3.1 H NMR (400MHz, CD3OD) δ 3.82-3.73 (m, 3H), 3.74-3.66 (m, 1H), 3.66-3.31 (m, 3H), 3.02 (s, 3H), 1.47 (d, J = 7.2Hz, 3H).

[0127] Step 3

[0128] The hydrochloride of compound 3 (811 mg, 5.39 mmol) was dissolved in N,N-dimethylformamide (15 mL), and triethylamine (1.64 g, 16.2 mmol) and compound 4 (1.00 g, 2.69 mmol) were added dropwise. The mixture was stirred at 25°C for 12 hours. The mixture was filtered and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (dichloromethane / methanol, 20 / 1 to 1 / 1, V / V) to obtain compound 5. MS-ESI calculated value [M+H] + 347, measured value 347.

[0129] Step 4

[0130] Compound 5 (254 mg, 733 μmol) was dissolved in dichloromethane (5 mL), and compound 6 (333 mg, 1.10 mmol), 4-dimethylaminopyridine (8.96 mg, 73.3 μmol), and N,N-diisopropylethylamine (284 mg, 2.20 mmol) were added. The mixture was stirred at 25°C for 12 hours. Water (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (40 mL × 5). The organic phase was washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (dichloromethane / methanol, 1 / 0 to 10 / 1, V / V) to obtain compound 7. MS-ESI calculated value [M+H] + 613, measured value 613.

[0131] Step 5

[0132] Compound 7 (154 mg, 251 μmol) was dissolved in dimethyl sulfoxide (5 mL), and compound 8 (93.2 mg, 377 μmol) and triethylamine (76.3 mg, 754 μmol) were added. The mixture was stirred at 90°C for 12 hours. Water (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phase was washed with saturated brine (50 mL × 5), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated and purified by preparative high-performance liquid chromatography (column: Xtimate C18 150 × 40 mm × 5 μm; mobile phase: 0.05% aqueous hydrochloric acid-acetonitrile; gradient: acetonitrile 5%-30% over 10 min) to obtain the hydrochloride salt of compound (I).1 H NMR (400 MHz, CD3OD) δ 8.40 (s, 1H), 7.61 (t, J = 7.2 Hz, 1H), 7.42 (t, J = 6.8 Hz, 1H), 7.23 (t, J = 7.2 Hz, 1H), 7.16 (s, 1H), 6.01-5.99 (m, 1H), 4.93-4.86 (m, 1H), 4.68-4.22 (m, 1H), 4.04 (s, 3H), 3.62-3.55 (m, 4H), 3.23-3.19 (m, 1H), 2.99 (s, 3H), 2.62 (s, 3H), 1.74 (d, J = 6.8 Hz, 3H), 1.57-1.48 (m, 3H), 1.29 (s, 6H). MS-ESI calcd. [M+H] + 576, measured value 576.

[0133] Example 2: Preparation of Crystalline Form A of Compound (I)

[0134] Synthesis route:

[0135] first step

[0136] The hydrochloride salt of the compound of formula (I) (2.50 g, 4.34 mmol) was dissolved in water (100 mL) and ethyl acetate (100 mL). Saturated sodium carbonate solution was added to the solution until the pH was about 10. The mixture was extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated brine (100 mL × 1), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and lyophilized to obtain the compound of formula (I). MS-ESI calculated value [M+H] + 576, measured value 576.

[0137] Step 2

[0138] The compound of formula (I) (150 mg, 261 μmol) was added to n-heptane (1.5 mL). The reaction solution was stirred at 50°C for 24 hours, then at 25°C for 2 hours, filtered, and the filter cake was washed with n-heptane (3 mL). The mixture was dried under vacuum at 50°C to obtain Form A of the compound of formula (I). 1H NMR (400MHz, CD3OD) δ8.05(s,1H),7.51(t,J=6.8Hz,1H),7.35(t,J=6.8Hz,1H),7.13(t,J=7.6Hz,1H),7.09(s, 1H), 5.88-5.78 (m, 1H), 4.79-4.00 (m, 2H), 3.94 (s, 3H), 3.48-3.32 (m, 1H), 2.95-2.76 (m, 2H), 2.41 (s, 3H), 2.32 (s, 3H), 2.29-2.21 (m, 1H), 2.14-2.00 (m, 1H), 1.63 (d, J = 6.8 Hz, 3H), 1.42 (s, 3H), 1.33-1.22 (m, 6H). MS-ESI calculated value [M+H] + 576, found 576. The XRPD of the crystalline form A of the compound of formula (I) was tested using the corresponding method 1, and the XRPD, DSC, TGA, and DVS test results are shown in Figures 1, 2, 3, and 10, respectively.

[0139] The n-heptane solvent in the second step was replaced with the solvents listed in Table 9 to obtain Form A of the compound of formula (I). The experimental results are shown in Table 9.

[0140] Table 9 50℃ suspension stirring test

[0141] Example 3: Preparation of Crystalline Form B of Compound (I)

[0142] The compound of formula (I) (150 mg, 261 μmol) was added to water (1.5 mL). The reaction solution was stirred at 50°C for 24 hours and then at 25°C for 2 hours. Solid precipitated in the reaction solution, which was filtered. The filter cake was washed with water (3 mL) and dried in vacuo at 50°C to obtain Form B of the compound of formula (I). 1 H NMR(400MHz,CD3OD)δ8.05(s,1H),7.50(t,J=6.8Hz,1H),7.35(t,J=6.8Hz,1H),7 .13(t,J=7.8Hz,1H),7.09(s,1H),5.87-5.79(m,1H),4.72-4.03(m,2H),3.94(s, 3H),3.50-3.32(m,1H),2.92-2.76(m,2H),2.41(s,3H),2.32(s,3H),2.29-2.23( m,1H),2.13-2.01(m,1H),1.62(d,J=7.2Hz,3H),1.43(s,3H),1.35-1.24(m,6H). MS-ESI calculated value [M+H] +576, found 576. The XRPD of the crystalline form B of the compound of formula (I) was tested using the corresponding method 2, and the XRPD, DSC, and TGA test results are shown in Figures 4, 5, and 6, respectively.

[0143] Example 4: Preparation of Crystalline Form C of Compound of Formula (I)

[0144] The compound of formula (I) (300 mg, 521 μmol) was dissolved in isopropyl acetate (1.5 mL), and the reaction solution was stirred at 25° C. for 12 hours. Solid precipitated in the reaction solution, which was filtered and the filter cake was dried in a forced air dryer at 150° C. to obtain Form C of the compound of formula (I). 1 H NMR(400MHz,CD3OD)δ8.04(s,1H),7.49(t,J=6.8Hz,1H),7.38-7.31(m,1H),7.12(t, J=7.8Hz,1H),7.09(s,1H),5.86-5.78(m,1H),4.61-3.98(m,2H),3.94(s,3H),3.48-3 .32 (m, 1H), 2.92-2.85 (m, 1H), 2.83-2.75 (m, 1H), 2.41 (s, 3H), 2.31 (s, 3H), 2.29-2.23 (m, 1H), 2.12-2.01 (m, 1H), 1.62 (d, J = 7.2 Hz, 3H), 1.43 (s, 3H), 1.33-1.27 (m, 6H). MS-ESI calculated value [M+H] + 576, found 576. The XRPD of the crystalline form C of the compound of formula (I) was tested using the corresponding method 2, and the XRPD, DSC, and TGA test results are shown in Figures 7, 8, and 9, respectively.

[0145] Example 5: Solid Preliminary Stability Test of Crystalline Form A of Compound of Formula (I)

[0146] According to the "Guidelines for Stability Testing of APIs and Preparations" (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 9001), the stability of the crystalline form of compound A of formula (I) was investigated under high temperature (60°C, open), high humidity (25°C / relative humidity 92.5%, open), strong light (5000lx and ultraviolet intensity 90μw / cm 2 , open) and pre-stability under high temperature and high humidity conditions (40℃ / relative humidity 75% and 60℃ / relative humidity 75% or 30℃ / relative humidity 65%, open).

[0147] Weigh about 30 mg of the crystalline form A of the compound of formula (I), place it in a dry and clean glass bottle, spread it into a thin layer, and use it as a test sample. Place it under influencing factors and accelerated conditions, and the sample is completely exposed. Sampling and analysis are performed on 5 days and 10 days under high temperature and high humidity, and on 1 month, 2 months, and 3 months under accelerated conditions. The samples placed under light conditions are completely exposed and placed at room temperature. XRPD was performed on the samples placed under different conditions, and the test results were compared with the initial test results on day 0. The results are shown in Table 10 below, which show that the crystalline form A of the compound of formula (I) is a stable crystalline form.

[0148] Table 10 Preliminary stability results of the crystal form of compound A of formula (I)

[0149] Example 6: Hygroscopicity study of the crystalline form of compound A of formula (I)

[0150] Experimental Materials:

[0151] Dynamic Vapor Adsorption Analyzer

[0152] Experimental methods:

[0153] About 10-20 mg of Form A of the compound of formula (I) was weighed and placed in a DVS sample tray for testing.

[0154] The classification of moisture absorption evaluation is shown in Table 11:

[0155] Table 11 Moisture absorption evaluation classification table Note: ΔW% indicates the weight gain of the test sample at 25±1℃ and 80±2%RH.

[0156] Experimental results:

[0157] The DVS spectrum of Form A of Compound of Formula (I) is shown in Figure 10. The DVS results show that the sample absorbed 0.3815% moisture at 25°C / 80% RH, indicating that the sample is slightly hygroscopic. After completing the DVS test (0-95-0% RH), the sample was exposed to air and analyzed by XRPD. The results showed that the crystalline form did not change before and after the DVS test.

[0158] Experimental conclusion:

[0159] The crystal form A of the compound of formula (I) is slightly hygroscopic at 25±1° C. and 80±2% RH, and the crystal form remains unchanged.

[0160] Example 7: Single crystal X-ray diffraction analysis of the compound of formula (I)

[0161] 1. Instrument Parameters and Data Collection

[0162] Manufacturer: Bruker;

[0163] Instrument model: Bruker D8 VENTURE

[0164] X-ray light source: high-intensity micro-focus rotating anode light source, Cu target;

[0165] Power: 2.5kW;

[0166] Tube voltage: 50kV;

[0167] Tube current: 45mA;

[0168] Goniometer: Four-axis (Kappa, ω, 2θ, ) goniometer;

[0169] Detector: Large-area photon type II detector, with an effective area of ​​14cm×10cm. The distance from the detector to the sample is automatically adjusted by a motor.

[0170] 2. Crystal Cultivation

[0171] 10 mg of Form A of the compound of formula (I) was added to 2 mL of acetonitrile and stirred until the sample was completely dissolved. The sample supernatant was placed in a 4 mL semi-sealed sample vial and slowly evaporated at room temperature. Colorless flaky crystals were obtained after two weeks.

[0172] 3. Test results

[0173] The basic structural information of the compound is: Molecular formula 2(C 29 H 36 F3N5O4)·CH3CN, crystal system is orthorhombic, space group P212121, wavelength The unit cell parameters are α=β=γ=90°, unit cell volume

[0174] 4. Conclusion

[0175] Single crystal data showed that the single crystal was the acetonitrile complex of the compound of formula (I). The single crystal SXRD ellipsoidal structure is shown in Figure 11. The results showed that C12 and C18 in the figure were in R,R configuration.

[0176] Biological activity:

[0177] Experimental Example 1: KRAS(G12C) and SOS1 binding experiment

[0178] Experimental principle:

[0179] Small molecule compounds bind to the catalytic site of SOS1, inhibiting the binding of SOS1 to KRAS(G12C). When the binding of fluorescently labeled SOS1 to fluorescently labeled KRAS(G12C) is inhibited, the emitted fluorescence changes. By measuring this fluorescence change, the ability of small molecules to inhibit the binding of SOS1 to KRAS(G12C) can be tested. A homogeneous time-resolved fluorescence (HTRF) binding assay was used to test the ability of the compounds of the present invention to inhibit the binding of SOS1 to KRAS(G12C).

[0180] Experimental Materials:

[0181] KRAS (G12C) protein was expressed and purified by Wuhan Pujian Biotechnology Co., Ltd., SOS1 exchange domin (564-1049) protein (Hμman recombinant) was purchased from Cytoskeleton, and Mab Antibody 6HIS-XL665 and Mab Antibody GST-Eμcryptate were purchased from Cisbio. A multifunctional microplate reader Nivo5 was purchased from PerkinElmer.

[0182] Experimental methods:

[0183] 1X buffer preparation (prepared and used immediately): Hepes: 5mM; NaCl: 150mM; EDTA: 10mM; Igepal: 0.0025%; KF: 100mM; DTT: 1mM; BSA: 0.05%;

[0184] The test compound was diluted 5-fold with DMSO using a dispenser to the eighth concentration, that is, from 1 mM to 0.064 μM.

[0185] Dilute the test compound to a 2% DMSO working solution using 1X buffer. Add 5 μL / well to the corresponding wells, with a concentration gradient from 20 μM to 0.00128 nM. Perform the experiment in duplicate. Centrifuge at 1000 rpm for 1 minute.

[0186] Use 1X buffer to prepare a mixed working solution of KRAS (G12C) (200nM) and Mab Anti GST-Eμcryptate (1ng / μL), incubate the mixed working solution at 25°C for 5 minutes, and add 2.5μL / well to the corresponding wells.

[0187] A working solution of SOS1 (80 nM) and Mab Antibody 6HIS-XL665 (8 g / μL) was prepared in 1X buffer and added to the corresponding wells at 2.5 μL / well. In blank wells, 2.5 μL of the Mab Antibody 6HIS-XL665 (8 g / μL) dilution was added. The final compound concentration gradient was diluted from 10 μM to 0.64 nM: KRAS (G12C) (500 nM), Mab Antibody GST-Eu cryptate (0.25 ng / μL), SOS1 (20 nM), and Mab Antibody 6HIS-XL665 (2 g / μL). The reaction system was incubated at 25°C for 60 minutes. After completion of the reaction, HTRF was read using a multilabel analyzer.

[0188] Data Analysis:

[0189] The raw data were converted into inhibition rate, IC using the equation (Sample-Min) / (Max-Min)×100%. 50 The value of can be obtained by four-parameter curve fitting (derived using the log(inhibitor) vs. response--Variable slope mode in GraphPad Prism). The results of the inhibitory activity test of the compounds of the present invention on the binding of KRAS (G12C) and SOS1 are shown in Table 12.

[0190] Table 12 IC values ​​of the compounds of the present invention for binding to KRAS (G12C) and SOS1 50 Value test results

[0191] Experimental conclusion: The compound of the present invention has a significant inhibitory effect on the binding of KRAS (G12C) and SOS1.

[0192] Experimental Example 2: H358 cell 3D proliferation inhibition activity test

[0193] Experimental principle:

[0194] In H358 cells harboring the KRAS (G12C) mutation, the KRAS signaling pathway is abnormally activated. Small molecule SOS1 inhibitors inhibit SOS1 binding to RAS proteins, reducing its GEF activity and decreasing the ratio of activated RAS-GTP. This further downregulates phosphorylation of the MEK / ERK pathway downstream of RAS, thereby inhibiting cell proliferation. Small molecules were co-cultured with H358 cells in 3D space, and cellular readouts were used to indirectly assess the inhibitory activity of SOS1 inhibitors on H358 cell proliferation.

[0195] Experimental Materials:

[0196] RPMI1640 medium, fetal bovine serum, and penicillin / streptomycin antibiotics were purchased from Vicente, and low-melting-point agarose was purchased from Sigma. Almar blue reagent was purchased from Invitrogen. NCI-H358 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Nivo multi-label analyzer (PerkinElmer).

[0197] Experimental methods:

[0198] H358 cells were seeded in a 96-well U-shaped plate. Low-melting-point agarose was first prepared into a 2% stock solution. When used, the agarose stock solution was first heated in a microwave oven to completely melt it, and then placed in a 42°C water bath to keep the agarose in a liquid state. The gel was added to the serum-containing culture medium to prepare a gel concentration of 0.6% as the bottom layer gel, and 50 μL was spread into each well of the 96-well U-shaped plate. After the bottom layer gel solidified, 2% gel was added to the cell-containing culture medium to prepare a cell-containing top layer gel with a gel concentration of 0.4%. The cell density was 4×10 4 Cells / ml were added to a 96-well U-shaped plate covered with bottom layer gel at a density of 3000 cells per well. After the top layer gel solidified, the cell plate was placed in a CO2 incubator for overnight culture.

[0199] On the day of compound addition, add 85 μL of liquid culture medium to the 96-well U-shaped plate containing the cells. Use a dispenser to dilute the test compound 3-fold to the ninth concentration, from 6 mM to 0.9 μM, in duplicate. Add 97 μL of culture medium to the middle plate. Transfer 2.5 μL of the serially diluted compound to each well of the middle plate, mixing thoroughly before transferring 40 μL to each well of the cell plate. The concentration of the compound transferred to the cell plate ranges from 30 μM to 4.5 nM. Incubate the cell plate in a CO2 incubator for 7 days. On the eighth day, use a dispenser to dilute the test compound 3-fold to the ninth concentration, from 6 mM to 0.9 μM, in duplicate. Add 198 μL of culture medium to the middle plate. Then, transfer 2 μL of the serially diluted compound per well to the first middle plate, following the corresponding position. Then, add 100 μL of culture medium to the second middle plate, add 100 μL of the mixed compound from the first middle plate, mix thoroughly, and transfer 40 μL per well to the cell plate. The concentration of the compound transferred to the cell plate ranges from 30 μM to 4.5 nM. The cell plate is incubated in a CO2 incubator for an additional 7 days. After incubating the cells with the compound for 14 days, add 20 μL of Almar blue detection reagent per well to the cell plate. The plate with dye is shaken on a horizontal shaker for 15 minutes, then incubated at room temperature for 5 hours to allow the luminescence signal to stabilize. The results are read using a multilabel analyzer.

[0200] Data Analysis:

[0201] The raw data were converted into inhibition rate, IC using the equation (Sample-Min) / (Max-Min)×100%. 50 The value can be obtained by four-parameter curve fitting (obtained in "log(inhibitor)vs.response--Variable slope" mode in GraphPad Prism).

[0202] Experimental conclusion: The compound of the present invention can inhibit the proliferation of H358 cells under 3D conditions.

[0203] Experimental Example 3: DLD-1 cell p-ERK proliferation inhibition activity test

[0204] Experimental Materials:

[0205] DLD-1 cells were purchased from Nanjing Kebai; 1640 culture medium was purchased from Biological Industries; fetal bovine serum was purchased from Biosera; Advanced Phospho-ERK1 / 2 (THR202 / TYR204) KIT was purchased from Cisbio, and its composition is shown in Table 13.

[0206] Table 13 Advanced Phospho-ERK1 / 2 (THR202 / TYR204) KIT ingredients

[0207] Experimental methods:

[0208] DLD-1 cells were seeded in a transparent 96-well cell culture plate, with 80 μL of cell suspension per well, and each well contained 8,000 DLD-1 cells. The cell plate was placed in a CO2 incubator and incubated overnight at 37°C.

[0209] Dilute the test compound to 2 mM with 100% DMSO as the first concentration, then pipette a further 5-fold dilution to the eighth concentration, from 2 mM to 0.026 μM. Add 2 μL of compound to 78 μL of cell starvation medium and mix thoroughly. Then, add 20 μL of compound solution to the corresponding well of the cell plate. Return the cell plate to the CO2 incubator and incubate for another 1 hour. At this point, the compound concentration ranges from 10 μM to 0.128 nM, and the DMSO concentration is 0.5%.

[0210] After the incubation, discard the cell supernatant and add 50 μL of cell lysis buffer to each well, and incubate at room temperature with shaking for 30 minutes;

[0211] Dilute Phospho-ERK1 / 2 Eu Cryptate antibody and Phospho-ERK1 / 2 d2 antibody 20-fold using detection buffer;

[0212] Transfer 16 μL of cell lysate supernatant to each well of a new 384-well white microplate, then add 2 μL of Phospho-ERK1 / 2 Eu Cryptate antibody dilution and 2 μL of Phospho-ERK1 / 2 d2 antibody dilution, and incubate at room temperature for 4 hours.

[0213] After incubation, use a multi-label analyzer to read HTRF excitation: 320nm, emission: 615nm, 665nm.

[0214] Data Analysis:

[0215] The raw data were converted into inhibition rate, IC, using the equation (Sample-Min) / (Max-Min)*100%. 50 The value can be obtained by four-parameter curve fitting (obtained by log(inhibitor) vs.response--Variable slope mode in GraphPad Prism).

[0216] Max well: Positive control well reading value is 1X lysate

[0217] Min well: negative control well reading value is 0.5% DMSO cell well cell lysate

[0218] Table 14 shows the results of the inhibitory activity test of the compounds of the present invention on p-ERK in DLD-1 cells.

[0219] Table 14 IC values ​​of the compounds of the present invention on the proliferation of p-ERK in DLD-1 cells 50 Value test results

[0220] Experimental conclusion: The compound of the present invention has a significant inhibitory effect on the proliferation of p-ERK in DLD-1 cells.

[0221] Experimental Example 4: Pharmacokinetic Evaluation of Compounds

[0222] Experimental Materials:

[0223] Balb / c mice (male, Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.)

[0224] Experimental operation:

[0225] The pharmacokinetic profile of the compound following intravenous and oral administration in rodents was tested using a standard protocol. The candidate compound was prepared as a clear solution and administered to mice as a single intravenous and oral dose. The intravenous and oral vehicles used were a mixture of 5% dimethyl sulfoxide, 5% solutol, and 90% water. Four male Balb / c mice were used in this study. Two mice received a 10 mg / kg intravenous dose, and plasma samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration. Two mice received a 50 mg / kg oral gavage dose, and plasma samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after administration. Blood samples were placed on ice and centrifuged within 1 hour to separate plasma (6000 g, 3 minutes, 2-8°C). Plasma samples were stored at -80°C prior to analysis. LC-MS / MS analysis was used to quantitatively analyze the blood drug concentration and calculate the pharmacokinetic parameters, such as peak concentration (C max ), clearance (CL), half-life (T 1 / 2 ), tissue distribution (Vdss), area under the drug-time curve (AUC 0-last ), bioavailability (F), etc.

[0226] The experimental results are shown in Table 15:

[0227] Table 15 Pharmacokinetic test results of the compounds of the present invention

[0228] Experimental conclusion: The compounds of the present invention have good pharmacokinetic properties, including good oral bioavailability, oral exposure, half-life and clearance rate.

Claims

1. Form A of the compound of formula (I), having an X-ray powder diffraction pattern (XRPD) with characteristic diffraction peaks at the following 2θ angles: 15.492±0.200°, 16.458±0.200°, 18.657±0.200°, and 20.638±0.200°; 2. The crystal form A according to claim 1, has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 14.223±0.200°, 14.589±0.200°, 14.894±0.200°, 15.492±0.200°, 16.061±0.200°, 16.458±0.200°, 18.657±0.200° and 20.638±0.200°.

3. The crystal form A according to claim 2, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.736±0.200°, 14.223±0.200°, 14.589±0.200°, 14.894±0.200°, 15.492±0.200°, 16.061±0.200°, 16.458±0.200°, 18.657±0.200°, 19.407±0.200°, 20.638±0.200°, 21.810±0.200° and 22.836±0.200°.

4. The crystal form A according to claim 3, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.736±0.100°, 9.070±0.100°, 11.289±0.100°, 11.678±0.100°, 14.223±0.100°, 14.589±0.100°, 14.894±0.100° .100°, 15.492±0.100°, 16.061±0.100°, 16.458±0.100°, 18.657±0.100°, 19.407±0.100°, 20.638±0.100°, 21.085±0.100°, 21.810±0.100° and 22.836±0.100°.

5. The crystal form A according to claim 4, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.736±0.200°, 8.475±0.200°, 9.070±0.200°, 11.289±0.200°, 11.678±0.200°, 12.363±0.200°, 14.223±0.200°, 14.589±0.200°, 14.894±0.200°, 15.492±0.200°, 16.061±0.200°, 16.458±0.200°, 17.000±0.200°, 18. The peak values ​​were 8.657±0.200°, 19.030±0.200°, 19.407±0.200°, 19.882±0.200°, 20.638±0.200°, 21.085±0.200°, 21.810±0.200°, 22.836±0.200°, 23.717±0.200°, 24.147±0.200°, 24.693±0.200°, 25.311±0.200°, 26.802±0.200°, 27.462±0.200°, 28.537±0.200° and 31.264±0.200°.

6. The crystal form A according to claim 5, whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.736°, 8.475°, 9.070°, 11.289°, 11.678°, 12.363°, 14.223°, 14.589°, 14.894°, 15.492°, 16.061°, 16.458°, 17.000°, 18.657°, 19.030°, 19.407°, 19.882°, 20.638°, 21.085°, 21.810°, 22.836°, 23.717°, 24.147°, 24.693°, 25.311°, 26.802°, 27.462°, 28.537° and 31.264°.

7. The XRPD pattern of Form A of the compound of formula (I) is substantially as shown in Figure 1.

8. The crystal form A according to any one of claims 1 to 7, wherein its differential scanning calorimetry (DSC) curve has an endothermic peak starting point at 169.0±5°C.

9. The crystal form A according to claim 8, whose DSC spectrum is substantially as shown in Figure 2.

10. The crystal form A according to any one of claims 1 to 7, wherein the thermogravimetric analysis (TGA) curve thereof shows a weight loss of 1.48% at 160.0±3°C.

11. The crystal form A according to claim 10, whose TGA spectrum is substantially as shown in FIG3.

12. Use of the crystal form A of the compound of formula (I) according to any one of claims 1 to 11 in the preparation of a medicament for treating KRAS mutant solid tumor diseases.

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