CRYSTAL FORM OF PYRROLO HETEROCYCLIC DERIVATIVE AND PREPARATION METHOD THEREFOR

Information

  • Patent Application
  • 20230331731
  • Publication Number
    20230331731
  • Date Filed
    September 29, 2021
    2 years ago
  • Date Published
    October 19, 2023
    7 months ago
Abstract
Provided are a crystal form of a pyrrolo heterocyclic derivative and a preparation method therefor. Specifically, the present invention relates to a crystal form of a pharmaceutically acceptable salt of a compound as represented by formula (I) and a preparation method therefor. The provided crystal form of the pharmaceutically acceptable salt of the compound as represented by formula (I) has a good stability and can be better used in clinical treatment.
Description

The present application claims priority to Chinese Patent Application No. 2020110494022 filed on Sep. 29, 2020, which is incorporated herein by reference in its entirety.


TECHNICAL FIELD

The present disclosure relates to a novel crystal form of a pyrrolo heterocyclic derivative and a preparation method therefor and is in the field of pharmacy.


BACKGROUND

The proliferation, differentiation, metabolism and apoptosis of normal cells are strictly regulated by the signal transduction pathways of cells in the body. Mitogen-activated protein kinases (MAPKs) play a crucial role in the signal transduction pathways. Extracellular signal regulated kinases (ERKs) are members of the MAPK family. Through the RAS-RAF-MEK-ERK step, exogenous stimulation signals are transmitted to ERKs. The activated ERKs are transferred into nuclei to regulate the activity of transcription factors and thus the biological functions of cells such as their proliferation, differentiation and apoptosis or, by phosphorylating the cytoskeleton in the cytoplasm, to participate in the regulation of cell morphology and the redistribution of the cytoskeleton.


Mutations in the RAS and RAF genes result in the sustained activation of the MAPK-ERK signaling pathway, promoting the malignant transformation and abnormal proliferation of cells and finally leading to the development of tumors (Roberts PJ, et al., Oncogene, 2007, 26(22), 3291-3310). The combination of a MEK inhibitor and a B-RAF inhibitor can further improve the inhibitory effect of the B-RAF inhibitor on tumor growth, and can significantly improve the progression-free survival and overall survival rate of patients with melanoma containing BRAFV600E and V600K mutations (Frederick DT et al., Clinical Cancer Research, 2013.19(5), 1225-1231). Although the combination of B-RAF/MEK inhibitors has the effect of inhibiting tumors, the effect is temporary: the majority of patients will develop resistance to the drugs within 2-18 months and the tumors will further progress. The mechanism for the development of resistance to B-RAF/MEK inhibitors is very complex and, in most cases, is directly related to the reactivation of the ERK signaling pathway (Smalley I et al., Cancer Discovery, 2018, 8(2), 140-142). Therefore, the development of new ERK inhibitors would benefit not only patients containing mutations in the MAPK signaling pathway but also patients with resistance to B-RAF/MEK inhibitors.


While inhibiting tumor growth, B-RAF/MEK inhibitors have a regulatory effect on the immune microenvironment of tumors. B-RAF/MEK inhibitors can enhance the expression of tumor-specific antigens, improve the recognition and killing of tumors by antigen-specific T cells, and promote the migration and infiltration of immune cells. In animal models, the expression of PD-L1 in tumor tissues is enhanced after treatment with B-RAF/MEK inhibitors, and when used in combination with a checkpoint molecule antibody (e.g., a PD-1 antibody or CTLA4 antibody), B-RAF/MEK inhibitors show better inhibitory effects on tumor growth than them alone (Boni A et al., Cancer Research, 2010, 70(13), 5213-5219). Research shows that ERK inhibitors are similar to B-RAF/MEK inhibitors in that when used in combination with a checkpoint antibody, they have the effect of regulating the microenvironment of tumors and can promote the functioning of cytotoxic T cells, thereby achieving the effect of inhibiting tumor growth.


A number of compounds have now been developed. Among them, BVD-523 developed by BioMed Valley Discoveries Inc. is undergoing phase II clinical trials and MK-8353 by Merck & Co., Inc., as well as Astex-029 by Astex, is undergoing phase I clinical trials. The related patents include WO1999061440A1, WO2001056557A2, WO2001056993A2, WO2001057022A2, WO2002022601A1, WO2012118850A1, WO2013018733A1, WO2014179154A2, WO2015103133A1, WO2016192063A1, WO2017180817A1, and WO2018049127A1.


SUMMARY

The present disclosure provides a pharmaceutically acceptable salt of a compound of formula (I), and the pharmaceutically acceptable salt is selected from the group consisting of a fumarate, an oxalate, a succinate, a maleate, a hydrochloride, and a hydrobromide,




embedded image - (I)


The present disclosure further provides a method for preparing a pharmaceutically acceptable salt of the compound of formula (I), which comprises the step of reacting the compound of formula (I) with a fumarate, an oxalate, a succinate, a maleate, a hydrochloride, or a hydrobromide.


In an alternative embodiment, the present disclosure provides a fumarate of the compound of formula (I), wherein the compound of formula (I) and fumaric acid are in a molar ratio of 1:1 or 2:1.


The present disclosure further provides a method for preparing a fumarate of the compound of formula (I), which comprises the step of reacting the compound of formula (I) with fumaric acid.


The present disclosure further provides crystal form I of a fumarate of the compound of formula (I), wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 10.671, 22.735, 28.681, and 29.302.


The present disclosure further provides crystal form I of a fumarate of the compound of formula (I), wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 10.671, 16.228, 19.832, 22.735, 23.526, 28.681, and 29.302.


The present disclosure provides in another aspect a method for preparing crystal form I of a fumarate of the compound of formula (I), wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in a molar ratio of 1:1, the method comprising the following steps:

  • 1) dissolving the compound of formula (I) and fumaric acid in a solvent selected from ethyl acetate; and
  • 2) crystallization, wherein the compound of formula (I) and fumaric acid are in a molar ratio selected from 1:(1-1.5).


The present disclosure further provides crystal form II of a fumarate of the compound of formula (I), wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in an amount-of-substance ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 7.783, 9.151, 12.378, 14.808, 18.574, 25.755, and 26.802.


In an alternative embodiment, the present disclosure provides crystal form II of the fumarate of the compound of formula (I), wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in an amount-of-substance ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 7.783, 9.151, 10.292, 12.378, 14.808, 18.574, 19.632, 20.677, 22.305, 23.394, 25.755, 26.802, and 27.684.


In an alternative embodiment, the present disclosure provides crystal form II of the fumarate of the compound of formula (I), wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in an amount-of-substance ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 7.783, 9.151, 10.292, 12.378, 14.808, 16.386, 18.574, 19.632, 20.677, 22.305, 23.394, 24.053, 24.495, 25.755, 26.802, 27.684, 28.877, 29.681, and 31.851.


The present disclosure provides in another aspect a method for preparing crystal form II of a fumarate of the compound of formula (I), wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in a molar ratio of 1:1, the method comprising the following steps:

  • 1) dissolving the compound of formula (I) and fumaric acid in an ethanol or acetonitrile solution; and
  • 2) crystallization, wherein the compound of formula (I) and fumaric acid are in a molar ratio selected from 1:(1-1.5).


In an alternative embodiment, the present disclosure provides crystal form III of a fumarate of the compound of formula (I), wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in a molar ratio of 2:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 4.877, 10.601, 13.827, 14.867, 16.272, 18.874, 23.921, and 26.367.


In an alternative embodiment, the present disclosure provides crystal form III of the fumarate of the compound of formula (I), wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in a molar ratio of 2:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 4.877, 9.664, 10.601, 11.745, 13.827, 14.867, 16.272, 17.885, 18.874, 20.695, 21.320, 22.204, 23.921, and 26.367.


In an alternative embodiment, the present disclosure provides crystal form IV of a fumarate of the compound of formula (I), wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in a molar ratio of 2:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 4.854, 10.613, 13.692, 23.523, 24.252, and 25.925.


In an alternative embodiment, the present disclosure provides crystal form IV of the fumarate of the compound of formula (I), wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in a molar ratio of 2:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 4.854, 9.785, 10.613, 13.692, 14.794, 18.424, 19.107, 20.409, 23.523, 24.252, and 25.925.


In an alternative embodiment, the present disclosure provides crystal form IV of the fumarate of the compound of formula (I), wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in a molar ratio of 2:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 4.854, 8.650, 9.785, 10.613, 11.658, 12.449, 13.692, 14.794, 15.393, 16.257, 17.864, 18.424, 19.107, 20.409, 21.322, 22.510, 23.523, 24.252, 25.925, and 28.382.


A method for preparing crystal form IV of a fumarate of the compound of formula (I) is provided, wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in a molar ratio of 2:1; crystal form III of a fumarate of the compound of formula (I) is placed in an environment with humidity at greater than 50%.


In an alternative embodiment, the present disclosure provides an oxalate of the compound of formula (I).


The present disclosure further provides crystal form a of an oxalate of the compound of formula (I), which has characteristic peaks at 2θ angles of diffraction of 14.235, 15.983, 17.882, and 26.923 in an X-ray powder diffraction graph.


In an alternative embodiment, the present disclosure provides a maleate of the compound of formula (I), wherein the compound of formula (I) and maleic acid are in a molar ratio of 1:1.


The present disclosure further provides crystal form a of a maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 4.510, 18.170, 23.973, 24.864, 26.306, and 28.209.


In an alternative embodiment, the present disclosure provides crystal form a of the maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 4.510, 18.170, 19.959, 21.842, 23.973, 24.864, 26.306, 28.209, and 31.995.


The present disclosure provides in another aspect a method for preparing crystal form a of a maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1, the method comprising the following steps: dissolving the compound of formula (I) and maleic acid in an isopropanol solution; and 2) crystallization, wherein the compound of formula (I) and maleic acid are in a molar ratio selected from 1:(1-1.5).


The present disclosure further provides crystal form b of a maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 4.744, 10.024, 11.931, 15.969, 19.171, 25.291, and 28.006.


In an altemative embodiment, the present disclosure provides crystal form b of the maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 4.744, 10.024, 11.931, 14.420, 15.185, 15.969, 17.249, 19.171, 20.405, 24.110, 25.291, 28.006, 30.216, and 32.380.


The present disclosure provides in another aspect a method for preparing crystal form b of a maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1, the method comprising the following steps: dissolving the compound of formula (I) and maleic acid in an ethyl acetate solution; and 2) crystallization, wherein the compound of formula (I) and maleic acid are in a molar ratio selected from 1:(1-1.5).


The present disclosure further provides crystal form c of a maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 4.610, 9.840, 10.708, 11.414, 12.329, 18.485, 18.883, 19.328, and 26.160.


In an alternative embodiment, the present disclosure provides crystal form c of the maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 4.610, 9.840, 10.708, 11.414, 12.329, 14.346, 15.262, 16.655, 17.298, 18.485, 18.883, 19.328, 23.928, 26.160, and 28.389.


In an alternative embodiment, the present disclosure provides crystal form c of the maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 4.610, 9.840, 10.708, 11.414, 12.329, 12.395, 14.346, 15.262, 15.962, 16.655, 17.298, 18.485, 18.883, 19.328, 22.250, 23.928, 26.160, 28.389, and 31.162.


The present disclosure provides in another aspect a method for preparing crystal form c of a maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1, the method comprising the following steps: dissolving the compound of formula (I) and maleic acid in an acetonitrile solution; and 2) crystallization, wherein the compound of formula (I) and maleic acid are in a molar ratio selected from (10-2):1.


The present disclosure further provides crystal form d of a maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 6.679, 9.346, 13.476, 14.076, 15.388, and 19.183.


In an alternative embodiment, the present disclosure provides crystal form d of the maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 6.679, 8.405, 9.346, 10.170, 13.476, 14.076, 15.388, 19.183, 21.228, and 26.247.


In an alternative embodiment, the present disclosure provides crystal form d of the maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 6.679, 8.405, 9.346, 10.170, 10.876, 13.476, 14.076, 15.388, 16.993, 19.183, 20.286, 21.228, 22.286, 23.894, 24.639, 26.247, 27.070, and 28.403.


The present disclosure further provides crystal form e of a maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 6.193, 10.009, 13.646, 17.582, 17.996, 18.758, 22.733, and 24.766.


In an alternative embodiment, the present disclosure provides crystal form e of the maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 6.193, 10.009, 13.646, 16.375, 17.582, 17.996, 18.758, 19.306, 20.181, 22.733, and 24.766.


In an alternative embodiment, the present disclosure provides crystal form e of the maleate of the compound of formula (I), wherein the maleate of the compound of formula (I) comprises the compound of formula (I) and maleic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 6.193, 10.009, 11.591, 13.646, 16.375, 17.582, 17.996, 18.758, 19.306, 20.181, 22.733, 24.766, 25.332, 26.601, 27.303, 28.859, and 31.447.


In an alternative embodiment, the present disclosure provides a succinate of the compound of formula (I).


The present disclosure further provides crystal form α of a succinate of the compound of formula (I), which has characteristic peaks at 2θ angles of diffraction of 5.640, 10.720, 13.384, 15.208, 16.244, 19.992, 21.669, 23.839, and 24.826 in an X-ray powder diffraction graph.


In an alternative embodiment, the present disclosure provides crystal form α of the succinate of the compound of formula (I), which has characteristic peaks at 2θ angles of diffraction of 5.640, 10.720, 13.384, 15.208, 16.244, 17.280, 18.020, 18.907, 19.992, 21.669, 23.839, and 24.826 in an X-ray powder diffraction graph.


The present disclosure further provides crystal form β of a succinate of the compound of formula (I), which has characteristic peaks at 2θ angles of diffraction of 5.160, 17.951, 20.618, 25.557, and 26.545 in an X-ray powder diffraction graph.


In an alternative embodiment, the present disclosure provides a hydrobromide of the compound of formula (I), wherein the compound of formula (I) and hydrobromic acid are in a molar ratio of 1:1.


The present disclosure further provides crystal form α of the hydrobromide of the compound of formula (I) described above, wherein the hydrobromide of the compound of formula (I) comprises the compound of formula (I) and hydrobromic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 6.656, 9.552, 14.110, 19.095, 20.092, 25.220, and 27.119.


In an alternative embodiment, the present disclosure provides crystal form α of the hydrobromide of the compound of formula (I), wherein the hydrobromide of the compound of formula (I) comprises the compound of formula (I) and hydrobromic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 6.656, 9.552, 13.778, 14.110, 19.095, 19.522, 20.092, 21.422, 25.220, 25.837, and 27.119.


In an alternative embodiment, the present disclosure provides crystal form α of the hydrobromide of the compound of formula (I), wherein the hydrobromide of the compound of formula (I) comprises the compound of formula (I) and hydrobromic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 6.656, 9.552, 13.778, 14.110, 19.095, 19.522, 20.092, 21.422, 22.276, 22.988, 23.653, 25.220, 25.837, 26.217, 27.119, 31.107, 31.867, 32.769, and 34.810.


In an alternative embodiment, the present disclosure provides a hydrobromide of the compound of formula (I), wherein the compound of formula (I) and hydrobromic acid are in a molar ratio of 1:2.


The present disclosure provides a method for a hydrobromide of the compound of formula (I), which comprises the step of reacting the compound of formula (I) with a hydrobromide.


The present disclosure further provides crystal form β of the hydrobromide of the compound of formula (I) described above, wherein the hydrobromide of the compound of formula (I) comprises the compound of formula (I) and hydrobromic acid in a molar ratio of 1:2 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 7.363, 12.914, 15.392, 16.865, 17.217, 19.704, 21.999, 22.515, 24.769, 27.056, and 28.236.


In an alternative embodiment, the present disclosure provides crystal form β of the hydrobromide of the compound of formula (I) described above, wherein the hydrobromide of the compound of formula (I) comprises the compound of formula (I) and hydrobromic acid in a molar ratio of 1:2 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 7.363, 12.914, 14.351, 15.392, 16.865, 17.217, 19.704, 21.999, 22.515, 24.769, 27.056, 28.236, and 29.234.


In an alternative embodiment, the present disclosure provides crystal form β of the hydrobromide of the compound of formula (I), wherein the hydrobromide of the compound of formula (I) comprises the compound of formula (I) and hydrobromic acid in a molar ratio of 1:2 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 7.363, 12.914, 14.351, 15.392, 16.865, 17.217, 19.452, 19.704, 21.999, 22.515, 23.275, 24.213, 24.769, 27.056, 28.236, 29.234, 29.764, 31.251, and 32.887.


In an alternative embodiment, the present disclosure provides crystal form γ of the hydrobromide of the compound of formula (I) described above, wherein the hydrobromide of the compound of formula (I) comprises the compound of formula (I) and hydrobromic acid in a molar ratio of 1:2 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 5.463, 16.567, 21.227, 25.784, 26.643, 28.321, and 33.788.


In an alternative embodiment, the present disclosure provides crystal form γ of the hydrobromide of the compound of formula (I) described above, wherein the hydrobromide of the compound of formula (I) comprises the compound of formula (I) and hydrobromic acid in a molar ratio of 1:2 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 5.463, 16.567, 19.733, 21.227, 22.332, 25.014, 25.784, 26.643, 28.321, 30.298, 32.170, 33.788, 36.211, and 37.939.


In an alternative embodiment, the present disclosure provides a hydrochloride of the compound of formula (I), wherein the compound of formula (I) and hydrochloric acid are in a molar ratio of 1:1.


The present disclosure further provides crystal form I of the hydrochloride of the compound of formula (I) described above, wherein the hydrochloride of the compound of formula (I) comprises the compound of formula (I) and hydrochloric acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 5.187, 9.407, 9.848, 10.388, 17.354, 18.041, and 26.234.


In an alternative embodiment, the present disclosure provides crystal form I of the hydrochloride of the compound of formula (I), wherein the hydrochloride of the compound of formula (I) comprises the compound of formula (I) and hydrochloric acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 5.187, 8.131, 9.407, 9.848, 10.388, 17.354, 18.041, 18.924, 23.830, 25.449, 26.234, 26.724, 27.362, 27.902, 29.275, and 31.581.


The present disclosure further provides crystal form II of the hydrochloride of the compound of formula (I) described above, wherein the hydrochloride of the compound of formula (I) comprises the compound of formula (I) and hydrochloric acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 13.728, 17.948, 22.678, 25.525, 27.356, and 29.543.


In an alternative embodiment, the present disclosure provides crystal form II of the hydrochloride of the compound of formula (I), wherein the hydrochloride of the compound of formula (I) comprises the compound of formula (I) and hydrochloric acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 13.728, 17.948, 20.593, 22.678, 25.525, 27.356, and 29.543.


The present disclosure further provides crystal form III of the hydrochloride of the compound of formula (I) described above, wherein the hydrochloride of the compound of formula (I) comprises the compound of formula (I) and hydrochloric acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 5.854, 8.983, 11.902, 13.181, 18.159, 20.071, 21.371, 24.180, 25.066, 26.469, 27.086, 27.729, and 28.615.


In an alternative embodiment, the present disclosure provides crystal form III of the hydrochloride of the compound of formula (I), wherein the hydrochloride of the compound of formula (I) comprises the compound of formula (I) and hydrochloric acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 5.854, 8.983, 11.902, 13.181, 16.876, 18.159, 20.071, 21.371, 22.799, 24.180, 25.066, 26.469, 27.086, 27.729, 28.615, 29.827, 30.904, 31.935, 33.571, 34.905, and 37.265.


The present disclosure further provides crystal form IV of the hydrochloride of the compound of formula (I) described above, wherein the hydrochloride of the compound of formula (I) comprises the compound of formula (I) and hydrochloric acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 5.693, 12.474, 14.474, 17.840, 22.231, 23.938, 26.866, and 29.207.


In an alternative embodiment, the present disclosure provides crystal form IV of the hydrochloride of the compound of formula (I), wherein the hydrochloride of the compound of formula (I) comprises the compound of formula (I) and hydrochloric acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 5.693, 12.474, 14.474, 17.352, 17.840, 19.596, 22.231, 23.109, 23.938, 24.573, 26.866, 27.841, 29.207, 33.452, and 34.915.


The present disclosure further provides crystal form V of the hydrochloride of the compound of formula (I) described above, wherein the hydrochloride of the compound of formula (I) comprises the compound of formula (I) and hydrochloric acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 7.476, 14.346, 15.181, 17.185, 21.606, 22.770, 24.279, 24.952, and 28.336.


In an alternative embodiment, the present disclosure provides crystal form V of the hydrochloride of the compound of formula (I), wherein the hydrochloride of the compound of formula (I) comprises the compound of formula (I) and hydrochloric acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 7.476, 9.536, 13.447, 14.346, 15.181, 17.185, 18.422, 21.606, 22.770, 24.279, 24.952, 28.336, and 29.773.


In an alternative embodiment, the present disclosure provides crystal form V of the hydrochloride of the compound of formula (I), wherein the hydrochloride of the compound of formula (I) comprises the compound of formula (I) and hydrochloric acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 7.476, 9.536, 12.349, 13.447, 14.346, 15.181, 15.639, 16.694, 17.185, 18.422, 18.859, 21.606, 22.770, 23.425, 24.279, 24.952, 26.990, 28.336, 28.772, 29.773, and 30.846.


In an alternative embodiment, for the crystal forms of the pharmaceutically acceptable salts of the compound of formula (I) described above, the 2θ angles have a margin of error of ±0.3.


In an alternative embodiment, for the crystal forms of the pharmaceutically acceptable salts of the compound of formula (I) described above, the 2θ angles have a margin of error of ±0.2.


In certain embodiments, the methods for preparing the crystal forms described herein also comprise a filtration step, a washing step, or a drying step.


The present disclosure also provides a pharmaceutical composition prepared from the pharmaceutically acceptable salt of the compound of formula (I) or the crystal form of the pharmaceutically acceptable salt described above.


The present disclosure also provides a pharmaceutical composition, which comprises the following components: i) the pharmaceutically acceptable salt of the compound of formula (I) or the crystal form of the pharmaceutically acceptable salt described above, and ii) optional pharmaceutically acceptable carriers, diluents or excipients.


The present disclosure also provides a method for preparing a pharmaceutical composition, which comprises the step of mixing the component i) and component ii) described above.


The present disclosure also provides use of the pharmaceutically acceptable salt of the compound of formula (I) described above, or the crystal form of the pharmaceutically acceptable salt of the compound of formula (I) described above, or the composition described above, or a composition prepared by the method described above, in preparing a medicament for treating or preventing cancer, inflammation, or other proliferative diseases, preferably cancer; the cancer is selected from the group consisting of melanoma, liver cancer, kidney cancer, lung cancer, nasopharyngeal carcinoma, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, cervical cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma, astrocytoma, and glioma.


As used herein, “2θ or 2θ angle” refers to the angle of diffraction, and θ refers to the Bragg angle in ° or degrees; the 2θ of each characteristic peak has a margin of error of ±0.20, and the error may be -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20.


As used herein, “crystallization” includes, but is not limited to, stirring crystallization, trituration crystallization, and evaporative crystallization.


The drying described herein is generally performed at a temperature of 25-100° C., preferably 40-70° C., or may be performed under atmospheric pressure or reduced pressure.


In the present application, the step of reacting the compound of formula (I) with a fumarate, an oxalate, a succinate, a maleate, a hydrochloride, or a hydrobromide is optionally carried out in a suitable solvent, for example, an ester solvent, specifically ethyl acetate; for example, an alcohol solvent, specifically methanol, ethanol, or isopropanol; for example, a nitrile solvent, specifically acetonitrile or propionitrile; for example, a ketone solvent, specifically acetone; for example, an ether solvent, specifically tetrahydrofuran, dioxane, diethyl ether, or isopropyl ether; for example, an alkane solvent, specifically n-hexane, n-heptane, or water, or a mixed solvent of the solvents described above.





BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1 shows an XRPD graph of the amorphous form of the compound of formula (I).



FIG. 2 shows an XRPD graph of crystal form I of the fumarate of the compound of formula (I).



FIG. 3 shows an XRPD graph of crystal form II of the fumarate of the compound of formula (I).



FIG. 4 shows a DSC graph of crystal form II of the fumarate of the compound of formula (I).



FIG. 5 shows a TGA graph of crystal form II of the fumarate of the compound of formula (I).



FIG. 6 shows a DVS graph of crystal form II of the fumarate of the compound of formula (I).



FIG. 7 shows an XRPD graph of crystal form II of the fumarate of the compound of formula (I) before and after DVS.



FIG. 8 shows an XRPD graph of crystal form III of the fumarate of the compound of formula (I).



FIG. 9 shows an XRPD graph of crystal form IV of the fumarate of the compound of formula (I).



FIG. 10 shows an XRPD graph of crystal form a of the oxalate of the compound of formula (I).



FIG. 11 shows an XRPD graph of crystal form a of the maleate of the compound of formula (I).



FIG. 12 shows an XRPD graph of crystal form b of the maleate of the compound of formula (I).



FIG. 13 shows an XRPD graph of crystal form c of the maleate of the compound of formula (I).



FIG. 14 shows an XRPD graph of the amorphous form of the maleate of the compound of formula (I).



FIG. 15 shows an XRPD graph of crystal form d of the maleate of the compound of formula (I).



FIG. 16 shows an XRPD graph of crystal form e of the maleate of the compound of formula (I).



FIG. 17 shows an XRPD graph of crystal form α of the succinate of the compound of formula (I).



FIG. 18 shows an XRPD graph of crystal form β of the succinate of the compound of formula (I).



FIG. 19 shows an XRPD graph of crystal form α of the hydrobromide of the compound of formula (I).



FIG. 20 shows an XRPD graph of crystal form β of the hydrobromide of the compound of formula (I).



FIG. 21 shows an XRPD graph of crystal form γ of the hydrobromide of the compound of formula (I).



FIG. 22 shows an XRPD graph of crystal form I of the hydrochloride of the compound of formula (I).



FIG. 23 shows an XRPD graph of crystal form II of the hydrochloride of the compound of formula (I).



FIG. 24 shows an XRPD graph of crystal form III of the hydrochloride of the compound of formula (I).



FIG. 25 shows an XRPD graph of crystal form IV of the hydrochloride of the compound of formula (I).



FIG. 26 shows an XRPD graph of crystal form V of the hydrochloride of the compound of formula (I).





DETAILED DESCRIPTION

Hereinafter, the present invention will be explained in more detail with reference to the examples. The examples are only used to illustrate the technical solutions of the present invention, rather than limit the essence and scope of the present invention.


Test conditions for the instruments used in the experiment:


The structures of the compounds were determined by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectrometry (MS). NMR shifts (δ) are given in 10-6 (ppm). NMR spectra were measured on Bruker AVANCE-400 nuclear magnetic resonance instrument or Bruker AVANCE NEO 500 M, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3) and deuterated methanol (CD3OD) as solvents and tetramethylsilane (TMS) as an internal standard.


Mass spectra (MS) were measured on Agilent 1200/1290 DAD-6110/6120 Quadrupole MS liquid chromatography-mass spectrometry system (manufacturer: Agilent; MS model: 6110/6120 Quadrupole MS),


waters ACQuity UPLC-QD/SQD (manufacturer: waters; MS model: waters ACQuity Qda Detector/waters SQ Detector), and


THERMO Ultimate 3000-Q Exactive (manufacturer: THERMO; MS model: THERMO Q Exactive).


High performance liquid chromatography (HPLC) analyses were performed on Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Thermo U3000 DAD high pressure liquid chromatographs.


Chiral HPLC analyses were performed on Agilent 1260 DAD high performance liquid chromatograph.


Preparative high performance liquid chromatography was performed on Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 preparative chromatographs.


Preparative chiral chromatography was performed on Shimadzu LC-20AP preparative chromatograph.


Ion chromatography was performed on Thermo Scientific Dionex Intergrion, column model: DionexIonPacTM AS11-HC (4 µm, 4 × 250 cm). XRPD refers to X-ray powder diffraction detection: The measurement was conducted on BRUKER D8 Discovery X-ray diffractometer with a Cu anode (40 kV, 40 mA) and Cu-Kα radiation (λ = 1.5418 Å) used. Scan mode: θ/2θ, scan range (2q range): 5-50°.


DSC refers to differential scanning calorimetry: The measurement was conducted on METTLER TOLEDO DSC 3+ differential scanning calorimeter with a temperature ramp rate of 10° C./min, specific temperature ranges shown in corresponding graphs (mostly 25-300 or 25-350° C.), and a nitrogen purge rate of 50 mL/min.


TGA refers to thermogravimetric analysis: The measurement was conducted on METTLER TOLEDO TGA 2 thermogravimetric analyzer with a temperature ramp rate of 10° C./min, specific temperature ranges shown in corresponding graphs (mostly 25-300° C.), and a nitrogen purge rate of 50 mL/min.


DVS refers to dynamic vapor sorption: The measurement was conducted on SMS DVS Advantage; at 25° C., the humidity was changed as follows: 50%-95%-0%-95%-50%, in steps of 10% (5% for the last step) (specific humidity ranges are shown in corresponding graphs, and those listed here were used in most cases); the criterion was dm/dt being no greater than 0.002%.


In the examples, a solution refers to an aqueous solution unless otherwise specified.


In the examples, the reaction temperature is room temperature, i.e., 20-30° C., unless otherwise specified.


The monitoring of the reaction progress in the examples was conducted by thin layer chromatography (TLC). The developing solvent for reactions, the eluent system for column chromatography purification and the developing solvent system for thin-layer chromatography include: A: dichloromethane/methanol system. The volume ratio of the solvents was adjusted according to the polarity of the compound, or by adding a small amount of basic or acidic reagents such as triethylamine and acetic acid.


Example 1

(S)-2-(1-(3-Chlorophenyl)-2-hydroxyethyl)-6-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-1,2-dihydro-3H-pyrrolo[1,2-c]imidazol-3-one 1embedded image


Step 1

(S)-2-((tert-Butyldimethylsilyl)oxy)-1-(3-chlorophenyl)ethylamine 1b


(S)-2-Amino-2-(3-chlorophenyl)ethanol 1a (4 g, 23.3 mmol, Bide Pharmatech Ltd.) and imidazole (3.2 g, 46.6 mmol) were dissolved in 80 mL of dichloromethane, and tertbutyldimethylsilyl chloride (5.2 g, 35 mmol) was added under ice bath. The mixture was stirred for 14 h. Water was added, and extraction was performed with dichloromethane (80 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography with eluent system C to give the title compound 1b (6.5 g), yield: 97%.






MS

m
/
z



ESI


:
286.1


M+1






Step 2

(S)-N-((4-Bromo-1H-pyrrol-2-yl)methyl)-2-((tert-butyldimethylsilyl)oxy)-1-(3-chlorophenyl)ethylamine 1d


4-Bromo-1H-pyrrole-2-carbaldehyde 1c (2.37 g, 13.62 mmol, Bide Pharmatech Ltd.) and compound 1b (3.9 g, 13.64 mmol) were stirred for 3 h. The mixture was diluted with 100 mL of methanol and cooled to 0° C. Sodium borohydride (516 mg, 13.64 mmol) was added, and the mixture was stirred for 2 h. Water was added, and the reaction mixture was concentrated under reduced pressure. Water was added, and extraction was performed with ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography with eluent system C to give the title compound 1d (4.8 g), yield: 79%.






MS

m
/
z



ESI


:
444.2


M+1






Step 3

(S)-6-Bromo-2-(2-((tert-butyldimethylsilyl)oxy)-1-(3-chlorophenyl)ethyl)-1H-pyrrolo[1,2-c]imidazol-3(2H)-one 1e


Compound 1d (4.8 g, 10.81 mmol) was dissolved in 100 mL of tetrahydrofuran, and N.N′-carbonyldiimidazole (2.45 g, 15.11 mmol) was added under ice bath. The mixture was stirred for 0.5 h, and sodium hydride (60%, 621 mg, 16.22 µmol) was added. The mixture was stirred at room temperature for 14 h. Saturated ammonium chloride was added. The reaction mixture was concentrated under reduced pressure and purified by column chromatography with eluent system C to give the title compound 1e (4.0 g), yield: 78%.






MS

m
/
z



ESI


:
496.1


M+1






Step 4

(S)-2-(2-((tert-Butyldimethylsilyl)oxy)-1-(3-chlorophenyl)ethyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[1,2-c]imidazol-3(2H)-one 1f


Compound 1e (4.0 g, 8.51 mmol) was dissolved in 50 mL of 1,4-dioxane under argon atmosphere, and 4,4,4',4',5,5,5',5′-octamethyl-2,2′-bis(1,3,2-dioxaborolane) (3.24 g, 12.76 mmol), potassium acetate (3.34 g, 34.04 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (1.24 g, 1.70 mmol) were added in sequence. The mixture was stirred at 90° C. for 2 h, cooled, and filtered through celite. The filtrate was concentrated and purified by column chromatography with eluent system C to give the title compound 1f (2.0 g), yield: 45%.






MS

m
/
z



ESI


:
517.2


M+1






Step 5

4-Chloro-N-(1-methyl-1H-pyrazol-5-yl)pyrimidin-2-amine 1i


N-(1-methyl-1H-pyrazol-5-yl)formamide 1h (324.82 mg, 2.60 mmol, prepared by the method disclosed in patent application “WO2017/80979”) was dissolved in 15 mL of N,N-dimethylformamide, and sodium hydride (60%, 311.47 mg, 7.79 mmol) was added at 0° C. The mixture was stirred for 0.5 h and, after 4-chloro-2-(methylsulfonyl)pyrimidine 1 g(500 mg, 2.60 mmol) was added, was reacted for another 2 h. Water (20 mL) was added, and extraction was performed with ethyl acetate (20 mL × 3). The organic phases were combined and concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography with developing solvent system C to give the title compound 1i (270 mg), yield: 49.6%.






MS

m
/
z



ESI


:
210.3


M+1






Step 6

(S)-2-(2-((tert-Butyldimethylsilyl)oxy)-1-(3-chlorophenyl)ethyl)-6-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-1,2-dihydro-3H-pyrrolo[1,2-c]imidazol-3(2H)-one 1j


A mixture of compound 1f (98.6 mg, 0.19 mmol), prepared 4-chloro-N-(1-methyl-1H-pyrazol-5-yl)pyrimidin-2-amine 1i, [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (28 mg, 0.02 mmol) and cesium carbonate (124 mg, 0.2 mmol) was suspended in 20 mL of 1,4-dioxane and 4 mL of water under argon atmosphere. The suspension was heated to 80° C., stirred for 14 h, cooled, and filtered through celite. The filtrate was collected and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, concentrated under reduced pressure, and purified by column chromatography with eluent system A to give the title compound 1j (100 mg), yield: 92%.






MS

m
/
z



ESI


:
564.3


M+1






Step 7

(S)-2-(1-(3-Chlorophenyl)-2-hydroxyethyl)-6-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-1,2-dihydro-3H-pyrrolo[1,2-c]imidazol-3-one 1


Compound 1j (100 mg, 0.17 mmol) was dissolved in 20 mL of dichloromethane, and 1 mL of trifluoroacetic acid was added dropwise. After addition, the mixture was stirred for 4 h. The pH was adjusted to 7 with saturated sodium bicarbonate, and extraction was performed with dichloromethane (20 mL × 2). The organic phases were combined, concentrated under reduced pressure, and purified by column chromatography with eluent system A to give the title compound 1 (15 mg), yield: 18%. The product was identified by X-ray powder diffraction as amorphous. The XRPD graph is shown in FIG. 1.






MS

m
/
z



ESI


:
450.1


M+1






1H NMR (400 MHz, CDCl3): δ 8.33 (d, 1H), 7.72 (s, 1H), 7.48 (d, 1H), 7.41-7.33 (m, 3H), 7.28-7.24 (m, 1H), 7.18 (s, 1H), 6.92 (d, 1H), 6.51 (s, 1H), 6.32 (d, 1H), 5.17 (dd, 1H), 4.46 (d, 1H), 4.32 (dd, 1H), 4.27-4.17 (m, 3H), 3.82 (s, 3H).


Biological Evaluation
Test Example 1: ERK1 Enzymatic Activity Test
I. Objective

The objective of this experiment is to measure the ability of the compound to inhibit the enzymatic activity of ERK1 and evaluate the in vitro activity of the compound according to IC50. This experiment used an ADP-Glo™ kinase assay kit. Under the action of enzyme, ADP was produced as the substrate was phosphorylated. The ADP-Glo reagent was added to remove unreacted ATP in the reaction system, and the ADP produced by the reaction was detected by a kinase detection reagent. In the presence of the compound, the inhibition rate of the compound was calculated by measuring the signal value.


II. Method

Enzyme and substrate preparation: 0.75 ng/µL ERK1 (1879-KS-010, R&D) and 100 µM substrate (AS-61777, anaspec) were prepared in buffer, and the enzyme solution and substrate solution were mixed in a ratio by volume of 2:1 for later use. ATP was diluted with buffer to 300 µM. The compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Scientific Co., Ltd.) to prepare a stock solution with an initial concentration of 20 mM, and the compound was prepared on Bravo. Finally, to each well of a 384-well plate were added in sequence 3 µL of the mixed solution of enzyme and substrate and 1 µL of the compound at different concentrations (an initial concentration of 50 µM, 4-fold dilutions). The plate was incubated at 30° C. for 10 min, and lastly, 1 µL of 300 µM ATP solution was added to each well. The plate was incubated at 30° C. for 2 h. Then 5 µL of ADP-Glo was added, and the plate was incubated at 30° C. for 40 min. Subsequently, 10 µL of kinase detection buffer was added, and the plate was incubated at 30° C. for 40 min. The 384-well plate was taken out and placed in a microplate reader (BMG labtech, PHERAstar FS) to measure chemiluminescence.


III. Data Analysis

The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5. The IC50 value of the compound was obtained. The result is shown in Table 1 below.





TABLE 1





The IC50 value of the compound of the present disclosure inhibiting the enzymatic activity of ERK1


Example No.
IC50 (nM)




1
5






Conclusion: the compound of the present disclosure has a significant inhibitory effect on the enzymatic activity of ERK1.


Test Example 2: ERK2 Enzymatic Activity Test
I. Objective

The objective of this experiment is to measure the ability of the compound to inhibit the enzymatic activity of ERK2 and evaluate the in vitro activity of the compound according to IC50. This experiment used an ADP-Glo™ kinase assay kit. Under the action of enzyme, ADP was produced as the substrate was phosphorylated. The ADP-Glo reagent was added to remove unreacted ATP in the reaction system, and the ADP produced by the reaction was detected by a kinase detection reagent. In the presence of the compound, the inhibition rate of the compound was calculated by measuring the signal value.


II. Method

Enzyme and substrate preparation: 0.75 ng/µL ERK2 (1230-KS-010, R&D) and 1500 µM substrate (custom polypeptide, GL Biochem) were prepared in buffer (40 mM Tris, 20 mM MgCl2, 0.1 mg/mL BSA, 50 µM DTT), and the enzyme solution and substrate solution were mixed in a ratio by volume of 2:1 for later use. ATP was diluted with buffer to 500 µM. The compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Scientific Co., Ltd.) to prepare a stock solution with an initial concentration of 20 mM, and the compound was prepared on Bravo. Finally, to each well of a 384-well plate were added in sequence 3 µL of the mixed solution of enzyme and substrate and 1 µL of the compound at different concentrations (an initial concentration of 50 µM, 4-fold dilutions). The plate was incubated at 30° C. for 10 min, and lastly, 1 µL of 500 µM ATP solution was added to each well. The plate was incubated at 30° C. for 2 h. Then 5 µL of ADP-Glo was added, and the plate was incubated at 30° C. for 40 min. Subsequently, 10 µL of kinase detection buffer was added, and the plate was incubated at 30° C. for 40 min. The 384-well plate was taken out and placed in a microplate reader (BMG labtech, PHERAstar FS) to measure chemiluminescence.


III. Data Analysis

The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5. The IC50 value of the compound was obtained. The result is shown in Table 2 below.





TABLE 2





The IC50 value of the compound of the present disclosure inhibiting the enzymatic activity of ERK2


Example No.
IC50 (nM)




1
7






Conclusion: the compound of the present disclosure has a significant inhibitory effect on the enzymatic activity of ERK2.


Test Example 3: Test for Inhibition of In Vitro Proliferation of Colo205 Tumor Cells by the Compound
I. Objective

The objective of this experiment is to measure the activity of the compound inhibiting the in vitro proliferation of Colo205 cells (CCL-222, ATCC). The cells were treated in vitro with different concentrations of the compound. After 3 days of culture, the proliferation of the cells was measured using CTG (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, catalog No. G7573) reagent, and the in vitro activity of the compound was evaluated according to the IC50 value.


II. Method

By way of example, a test for the inhibition of the in vitro proliferation of Colo205 cells is described below to illustrate the method of measuring the activity of the compound of the present disclosure inhibiting the in vitro proliferation of tumor cells. This method also applies to, but is not limited to, the measurement of the activity of inhibiting the in vitro proliferation of other tumor cells.


Colo205 was digested, centrifuged, and resuspended. The single cell suspension was well mixed, and the density of viable cells was adjusted with cell culture medium (RPMI1640 + 2% FBS) to 5.0 × 104 cells/mL. The suspension was added to a 96-well cell culture plate at 95 µL/well. To the peripheral wells of the 96-well plate was added only 100 µL of medium. The culture plate was incubated in an incubator for 24 h (37° C., 5% CO2).


The compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Scientific Co., Ltd.) to prepare a stock solution with an initial concentration of 20 mM. The small-molecule compound was 4-fold diluted from an initial concentration of 2 mM to 9 points. The tenth point was DMSO. To each well of another 96-well plate was added 90 µL of cell culture medium (RPMI1640 + 2% FBS), followed by 10 µL of test samples at different concentrations. The mixtures were well mixed, and 5 µL of test samples at different concentrations were then added to the cell culture plate. Two replicate wells were set for each sample. The culture plate was incubated in an incubator (37° C., 5% CO2) for 3 days. The 96-well cell culture plate was taken out, and 50 µL of CTG solution was added to each well. The plate was incubated at room temperature for 10 min and placed in a microplate reader (BMG labtech, PHERAstar FS) to measure chemiluminescence.


III. Data Analysis

The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5.


The result of this example is shown in Table 3 below.





TABLE 3





The IC50 value of the compound of the present disclosure inhibiting the in vitro proliferation of Colo205 tumor cells


Example No.
IC50 (nM)




1
62






Pharmacokinetic Evaluation
Test Example 4: Pharmacokinetic Study of the Compound of the Present Disclosure in Mice
1. Abstract

With mice as the test animals, the plasma concentration of the compounds of Examples 3, 10, 15 and 20 was determined by LC/MS/MS at different time points after intragastric administration. The pharmacokinetic behavior of the compound of the present disclosure in mice was studied and its pharmacokinetic profile was evaluated.


2. Methodology
2.1. Test Drug

The compound of Example 1.


2.2. Test Animals

Thirty-six C57 mice, female, evenly divided into 4 groups, purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd., with animal production license number: SCXK (Shanghai) 2013-0006.


2.3. Drug Preparation

A certain amount of the compound was measured out and dissolved in 5% by volume of DMSO and 5% Tween 80, and 90% normal saline was then added to prepare a 0.1 mg/mL colorless clear solution.


2.4. Administration

C57 mice were fasted overnight and then intragastrically administered the compound at a dose of 2 mg/kg and a volume of 0.2 mL/10 g.


3. Procedures

The mice were intragastrically administered the compound, and 0.1-mL blood samples were collected into heparin tubes before administration and 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 6.0 h, 8.0 h, 11.0 h, and 24.0 h after administration. Plasma was separated by centrifuging the blood at 3500 rpm for 10 min, and was stored at -20° C.


After different concentrations of the compound were injected into the mice, the plasma concentration of the compound was determined: 25 µL of mouse plasma at each time point post-dose was mixed with 50 µL of camptothecin (National Institutes for Drug Control) internal standard solution (100 ng/mL) and 200 µL of acetonitrile, and the mixture was vortexed for 5 min and centrifuged at 4000 rpm for 10 min; 4 µL of supernatant was taken for LC/MS/MS analysis.


4. Pharmacokinetic Parameters




TABLE 4










The pharmacokinetic parameters of the compound of the present disclosure


Example No.
Pharmacokinetic experiment in mice


Plasma concentration
Area under curve
Half-life
Residence time
Clearance
Apparent volume of distribution


Cmax (ng /mL)
AUC (ng /mL*h)
T1/2 (h)
MRT (h)
CLz/F (ml/min/k g)
Vz/F (ml/kg)




Example 1
1023
3441
3.66
3.47
9.69
3067






Conclusion: the compound of the present disclosure demonstrated a good absorption profile; it has pharmacokinetic advantages.


Example 2: Preparation of Fumarate in Crystal Form I

The amorphous compound of formula (I) (100 mg, 222.28 µmol) was added to 2 mL of ethyl acetate. The temperature was raised to 60° C., and 29 mg of fumaric acid was added. The mixture was stirred for 30 min, gradually cooled to room temperature, and stirred at room temperature for 2 h. A solid gradually precipitated. The mixture was filtered under reduced pressure, and the filter cake was collected, washed with a small amount of ethyl acetate, and dried in vacuo at 40° C. to give a solid (101.1 mg, yield: 80.4%). The 1H-NMR analysis of the product is shown below and indicates that the molar ratio of the compound of formula (I) to fumaric acid in the salt is 1:1.26.



1H NMR (400 MHz, DMSO- d6) δ 13.13 (br, 2H), 9.32 (s, 1H) 8.37 (d, 1H), 7.92 (s, 1H), 7.48 (d, 1H), 7.41-7.33 (m, 3H), 7.28-7.24 (m, 1H), 7.18 (s, 1H), 6.66 (d, 1H), 6.63 (s, 1H), 6.27 (s, 1H), 5.26 (s,1H), 5.17 (dd, 1H), 4.66 (d, 1H), 4.42 (dd, 1H), 3.94-4.06 (m, 2H), 3.82 (s, 3H).


The product was identified by X-ray powder diffraction as crystal form I. The XRPD graph is shown in FIG. 2, and the peak positions are shown in Table 5.





TABLE 5







The peak positions of the fumarate in crystal form I


Peak number
2θ(°)
d(Å)
I%




1
10.671
8.28425
37.4


2
16.228
5.45762
8.2


3
19.832
4.47310
11.3


4
22.735
3.90809
42.9


5
23.526
3.77853
18.2


6
28.681
3.11002
100.0


7
29.302
3.04553
35.0






Example 3: Preparation of Fumarate in Crystal Form II

The amorphous compound of formula (I) (100 mg, 222.28 µ µmol) was added to 2 mL of ethanol. The temperature was raised to 60° C., and 29 mg of fumaric acid was added. The mixture was stirred for 30 min, cooled to room temperature, and stirred for another 2 h. A solid gradually precipitated. The mixture was filtered under reduced pressure, and the filter cake was collected, washed with a small amount of ethanol, and dried in vacuo at 40° C. to give a solid (80 mg, yield: 63.6%). The 1H-NMR analysis of the product is shown below and indicates that the molar ratio of the compound of formula (I) to fumaric acid in the salt is 1:0.93.



1H NMR (400 MHz, DMSO- d6): δ 13.13 (br,2H), 9.32 (s,1H) 8.37 (d, 1H), 7.92 (s, 1H), 7.48 (d, 1H), 7.41-7.33 (m, 3H), 7.28-7.24 (m, 1H), 7.18 (s, 1H), 6.66 (d, 1H), 6.63 (s, 1H), 6.27 (s, 1H), 5.26 (s,1H), 5.17 (dd, 1H), 4.66 (d, 1H), 4.42 (dd, 1H), 3.94-4.06 (m, 2H), 3.82 (s, 3H).


The product was identified by X-ray powder diffraction as crystal form II. The XRPD graph is shown in FIG. 3, and the peak positions are shown in Table 6.





TABLE 6







The peak positions of the fumarate in crystal form II


Peak number
2θ(°)
d(Å)
I(%)




1
7.783
11.34974
100.00%


2
9.151
9.65566
74.50%


3
10.292
8.58777
18.80%


4
12.378
7.14527
21.90%


5
14.808
5.97759
13.40%


6
16.386
5.40528
13.50%


7
18.574
4.77319
53.80%


8
19.632
4.51839
15.60%


9
20.677
4.29231
12.20%


10
22.305
3.98243
16.30%


11
23.394
3.79950
16.90%


12
24.053
3.69687
9.50%


13
24.495
3.63113
2.80%


14
25.755
3.45636
48.90%


15
26.802
3.32358
35.00%


16
27.684
3.21974
13.80%


17
28.877
3.08937
5.20%


18
29.681
3.00750
2.50%


19
31.851
2.80732
4.80%






The DSC graph is shown in FIG. 4, where the endothermic peaks are at 58.80° C. and 161.41° C. The TGA graph is shown in FIG. 5, where a weight loss of 1.52% is shown at 25-130° C. and a weight loss of 14.54% is shown at 130-280° C.


DVS analysis shows that the sample made a moisture-absorption weight gain of about 2.38% when stored under normal conditions (i.e., 25° C., 60% RH), a moisture-absorption weight gain of about 2.75% when subjected to accelerated conditions (i.e., 70% RH), and a moisture-absorption weight gain of about 3.85% when subjected to extreme conditions (i.e., 90% RH). During the process of the RH changing from 0% to 95%, the desorption and adsorption of this sample did not coincide (see FIG. 6). After DVS analysis, the sample was re-examined for crystal form, and the result shows that the crystal form did not change (see FIG. 7).


Example 4: Preparation of Fumarate in Crystal Form III

The amorphous compound of formula (I) (100 mg, 222.28 µmol) was added to 5 mL of acetonitrile, and 27.0 mg of fumaric acid was added. The mixture was stirred at 50° C. for 12-36 h and filtered under reduced pressure. The filter cake was dried in vacuo at 40° C. The product was identified by X-ray powder diffraction as crystal form III. The XRPD graph is shown in FIG. 8, and the peak positions are shown in Table 7. NMR analysis shows that the molar ratio of the compound of formula (I) to fumaric acid in the salt is 2:1.





TABLE 7







The peak positions of the fumarate in crystal form III


Peak number
2θ(°)
d(Å)
I(%)




1
4.877
18.10485
47.10%


2
9.664
9.14464
14.40%


3
10.601
8.33869
62.00%


4
11.745
7.52842
12.10%


5
13.827
6.39948
18.00%


6
14.867
5.95380
17.70%


7
16.272
5.44278
15.50%


8
17.885
4.95538
8.50%


9
18.874
4.69798
16.00%


10
20.695
4.28849
6.90%


11
21.320
4.16428
8.00%


12
22.204
4.00034
8.90%


13
23.921
3.71693
100.00%


14
26.367
3.37746
25.80%






Example 5: Preparation of Fumarate in Crystal Form IV

A 100-mg sample of the fumarate in crystal form III was left to stand at 93% RH overnight. The product was identified by X-ray powder diffraction as crystal form IV. The XRPD graph is shown in FIG. 9, and the peak positions are shown in Table 8. The TGA graph shows that the crystal form made a weight loss of 0.73% at 30-100° C. and a weight loss of 10.89% at 120-200° C. The DSC graph shows that the crystal form has an endothermic peak at 148.3° C. NMR analysis shows that the molar ratio of the compound of formula (I) to fumaric acid in the salt is 2:1.





TABLE 8







The peak positions of the fumarate in crystal form IV


Peak number
2θ(°)
d(Å)
I(%)




1
4.854
18.18903
57.10%


2
8.650
10.21482
10.10%


3
9.785
9.03188
18.40%


4
10.613
8.32887
48.70%


5
11.658
7.58494
11.20%


6
12.449
7.10432
10.60%


7
13.692
6.46226
23.70%


8
14.794
5.98320
19.50%


9
15.393
5.75165
5.50%


10
16.257
5.44796
15.00%


11
17.864
4.96136
9.90%


12
18.424
4.81168
16.30%


13
19.107
4.64128
18.00%


14
20.409
4.34794
19.50%


15
21.322
4.16387
9.40%


16
22.510
3.94672
12.10%


17
23.523
3.77893
100.00%


18
24.252
3.66707
27.50%


19
25.925
3.43408
49.80%


20
28.382
3.14209
10.50%






Example 6: Preparation of Oxalate in Crystal Form a

100 mg of the amorphous compound of formula (I) was measured out, and 10.5 mg of oxalic acid and 5 mL of n-hexane were added. The mixture was stirred at 50° C. for 24-36 h and filtered under reduced pressure. The filter cake was dried in vacuo at 40° C. to give a solid. The product was identified by X-ray powder diffraction as crystal form a. The XRPD graph is shown in FIG. 10, and the peak positions are shown in Table 9.





TABLE 9







The peak positions of the oxalate in crystal form a


Peak number
2θ(°)
d(Å)
I(%)




1
14.235
6.21682
69.60%


2
15.983
5.54054
36.60%


3
17.882
4.95646
100.00%


4
26.923
3.30901
89.70%






Example 7: Preparation of Maleate in Crystal Form a

The amorphous compound of formula (I) (100 mg, 222.28 µmol) was added to 2 mL of isopropanol. The temperature was raised to 60° C., and 29 mg of maleic acid was added. The mixture was stirred at 60° C. for 30 min, cooled to room temperature, and stirred at room temperature for 2 h. A small amount of solid gradually precipitated. The stirring continued at room temperature overnight, and more solid precipitated. The mixture was filtered under reduced pressure, and the filter cake was collected, washed with a small amount of isopropanol, and dried in vacuo at 40° C. to give a product (90.1 mg). The 1H-NMR analysis of the product is shown below and indicates that the molar ratio of the compound of formula (I) to maleic acid in the salt is 1:0.95.



1H NMR (400 MHz, DMSO- d6): 9.33 (s,1H) 8.37 (d, 1H), 7.92 (s, 1H), 7.48 (d, 1H), 7.41-7.33 (m, 3H), 7.28-7.24 (m, 1H), 7.18 (s, 1H), 6.66 (s, 1H), 6.27 (s, 2H), 5.17 (dd, 1H), 4.66 (d, 1H), 4.42 (dd, 1H), 3.94-4.06 (m, 2H), 3.69 (s, 3H).


The product was identified by X-ray powder diffraction as crystal form a. The XRPD graph is shown in FIG. 11, and the peak positions are shown in Table 10.





TABLE 10







The peak positions of the maleate in crystal form a


Peak number
2θ(°)
d(Å)
I(%)




1
4.510
19.5785
70.90%


2
18.170
4.87846
54.00%


3
19.959
4.44501
17.10%


4
21.842
4.06581
8.80%


5
23.973
3.70899
22.10%


6
24.864
3.57811
42.40%


7
26.306
3.38517
100.00%


8
28.209
3.16096
39.90%


9
31.995
2.79505
18.40%






Example 8: Preparation of Maleate in Crystal Form b

The amorphous compound of formula (I) (100 mg, 222.28 µmol) was added to 2 mL of ethyl acetate. The temperature was raised to 60° C., and 29 mg of maleic acid was added. The mixture was stirred at 60° C. for 30 min, cooled to room temperature, and stirred at room temperature for 2 h. A solid gradually precipitated. The stirring continued at room temperature overnight. The mixture was filtered under reduced pressure, and the filter cake was collected, washed with a small amount of ethyl acetate, and dried in vacuo at 40° C. to give a product (125.1 mg).


The 1H-NMR analysis of the product is shown below and indicates that the molar ratio of the compound of formula (I) to maleic acid in the salt is 1:0.97.


1H NMR (400 MHz, DMSO- d6): 9.33 (s,1H), 8.37 (d, 1H), 7.92 (s, 1H), 7.48 (d, 1H), 7.41-7.33 (m, 3H), 7.28-7.24 (m, 1H), 7.18 (s, 1H), 6.66 (s, 1H), 6.27 (s, 2H), 5.17 (dd, 1H), 4.66 (d, 1H), 4.42 (dd, 1H), 3.94-4.06 (m, 2H), 3.69 (s, 3H).


The product was identified by X-ray powder diffraction as crystal form b. The XRPD graph is shown in FIG. 12, and the peak positions are shown in Table 11.





TABLE 11







The peak positions of the maleate in crystal form b


Peak number
2θ(°)
d(Å)
I(%)




1
4.744
18.61048
100.00%


2
10.024
8.81670
44.50%


3
11.931
7.41168
42.80%


4
14.420
6.13735
10.30%


5
15.185
5.82993
25.90%


6
15.969
5.54536
39.60%


7
17.249
5.13667
24.20%


8
19.171
4.62586
77.40%


9
20.405
4.34883
20.10%


10
24.110
3.68827
29.90%


11
25.291
3.51869
83.10%


12
28.006
3.18345
38.40%


13
30.216
2.95538
7.20%


14
32.380
2.76265
10.10%






Example 9: Preparation of Maleate in Crystal Form c

The amorphous compound of formula (I) (100 mg) was added to 13.5 mg of maleic acid and 2 mL of acetonitrile. The mixture was stirred at 50° C. for 3 h and filtered under reduced pressure. The filter cake was dried in vacuo at 40° C. to give a solid. The 1H-NMR analysis of the product is shown below and indicates that the molar ratio of the compound of formula (I) to maleic acid in the salt is 1:1.



1H NMR (400 MHz, DMSO- d6): 9.33 (s,1H), 8.37 (d, 1H), 7.92 (s, 1H), 7.48 (d, 1H), 7.41-7.33 (m, 3H), 7.28-7.24 (m, 1H), 7.18 (s, 1H), 6.66 (s, 1H), 6.27 (s, 2H), 5.17 (dd, 1H), 4.66 (d, 1H), 4.42 (dd, 1H), 3.94-4.06 (m, 2H), 3.69 (s, 3H)


The product was identified by X-ray powder diffraction as crystal form c. The XRPD graph is shown in FIG. 13, and the peak positions are shown in Table 12.





TABLE 12







The peak positions of the maleate in crystal form c


Peak number
2θ(°)
d(Å)
I(%)




1
4.610
19.15107
100.00%


2
9.840
8.98151
44.20%


3
10.708
8.25568
63.20%


4
11.414
7.74636
45.80%


5
12.329
7.17343
54.10%


6
12.395
7.13547
42.70%


7
14.346
6.16912
20.40%


8
15.262
5.80091
25.20%


9
15.962
5.54808
16.20%


10
16.655
5.31869
22.60%


11
17.298
5.12221
20.20%


12
18.485
4.79590
61.10%


13
18.883
4.69577
42.90%


14
19.328
4.58858
42.50%


15
22.250
3.99230
5.60%


16
23.928
3.71594
23.80%


17
26.160
3.40370
69.20%


18
28.389
3.14134
23.80%


19
31.162
2.86786
4.20%






Example 10: Preparation of Maleate in Amorphous Form

The free base of the compound of formula (I) (500 mg, 1.11 mmol) was added to 10 mL of methanol, and 129 mg of maleic acid was added. The mixture was heated to 60° C., stirred for 30 min, and concentrated by rotary evaporation to give the title product (605 mg, yield: 96.2%).


The 1H-NMR analysis of the product is shown below and indicates that the molar ratio of the compound of formula (I) to maleic acid in the salt is 1:1.



1H NMR (400 MHz, DMSO- d6): 9.33 (s,1H) 8.37 (d, 1H), 7.92 (s, 1H), 7.48 (d, 1H), 7.41-7.33 (m, 3H), 7.28-7.24 (m, 1H), 7.18 (s, 1H), 6.66 (s, 1H), 6.27 (s, 2H), 5.17 (dd, 1H), 4.66 (d, 1H), 4.42 (dd, 1H), 3.94-4.06 (m, 2H), 3.69 (s, 3H)


The product was identified by X-ray powder diffraction as amorphous. The XRPD graph is shown in FIG. 14.


Example 11: Preparation of Maleate in Crystal Form d

The amorphous maleate of the compound of formula (I) (Example 10) (36 mg, 63.6 µmol) was added to 0.4 mL of toluene and triturated at room temperature for 72 h. The mixture was filtered, and the filter cake was collected and dried in vacuo at 40° C. to give the title product (26.0 mg, yield: 73.4%).


The 1H-NMR analysis of the product is shown below and indicates that the molar ratio of the compound of formula (I) to maleic acid in the salt is 1:1.


1H NMR (400 MHz, DMSO- d6): 9.31 (s,1H) 8.35 (d, 1H), 7.91 (s, 1H), 7.48 (d, 1H), 7.41-7.33 (m, 3H), 7.28-7.24 (m, 1H), 7.15 (s, 1H), 6.63 (s, 1H), 6.27 (s, 2H), 5.17 (dd, 1H), 4.63 (d, 1H), 4.42 (dd, 1H), 3.94-4.06 (m, 2H), 3.71 (s, 3H).


The product was identified by X-ray powder diffraction as crystal form d. The XRPD graph is shown in FIG. 15, and the peak positions are shown in Table 13.





TABLE 13







The peak positions of the maleate in crystal form d


Peak number
2θ(°)
d(Å)
I(%)




1
6.679
13.22322
100.0%


2
8.405
10.51102
19.70%


3
9.346
9.45469
23.30%


4
10.170
8.69093
21.10%


5
10.876
8.12845
15.10%


6
13.476
6.56514
47.70%


7
14.076
6.28693
71.20%


8
15.388
5.75338
41.60%


9
16.993
5.21367
9.00%


10
19.183
4.62300
22.60%


11
20.286
4.37398
7.10%


12
21.228
4.18215
10.50%


13
22.286
3.98581
9.70%


14
23.894
3.72115
10.70%


15
24.639
3.61029
11.70%


16
26.247
3.39268
18.10%


17
27.070
3.29132
11.80%


18
28.403
3.13979
12.20%






Example 12: Preparation of Maleate in Crystal Form e

The maleate in crystal form c was dried at 60° C. overnight to give a solid. The product was identified by X-ray powder diffraction as crystal form e. The XRPD graph is shown in FIG. 16, and the peak positions are shown in Table 14.





TABLE 14







The peak positions of the maleate in crystal form e


Peak number
2θ(°)
d(Å)
I(%)




1
6.193
14.25955
20.10%


2
10.009
8.83064
34.40%


3
11.591
7.62821
10.10%


4
13.646
6.48371
32.50%


5
16.375
5.40878
23.60%


6
17.582
5.04031
66.00%


7
17.996
4.92509
40.70%


8
18.758
4.72669
39.20%


9
19.306
4.59395
24.50%


10
20.181
4.39656
24.60%


11
22.733
3.90842
26.70%


12
24.766
3.59202
100.00%


13
25.332
3.51301
6.60%


14
26.601
3.34827
11.20%


15
27.303
3.26381
15.10%


16
28.859
3.09127
19.70%


17
31.447
2.84248
5.10%






Example 13: Preparation of Succinate in Crystal Form α

100 mg of the free base of the compound of formula (I) was measured out, and 13.8 mg of succinic acid and 5 mL of isopropyl ether were added. The mixture was stirred at 50° C. for 12-36 h and filtered under reduced pressure. The filter cake was dried in vacuo at 40° C. The product was identified as crystal form α. The XRPD graph is shown in FIG. 17, and the peak positions are shown in Table 15.





TABLE 15







The peak positions of the succinate in crystal form α


Peak number
2θ(°)
d(Å)
I(%)




1
5.640
15.65567
28.80%


2
10.720
8.24589
84.20%


3
13.384
6.61041
27.10%


4
15.208
5.82111
28.20%


5
16.244
5.45222
24.50%


6
17.280
5.12769
11.70%


7
18.020
4.91881
9.00%


8
18.907
4.68981
10.20%


9
19.992
4.43767
42.00%


10
21.669
4.09791
17.80%


11
23.839
3.72956
100.00%


12
24.826
3.58357
33.10%






Example 14: Preparation of Succinate in Crystal Form ß

100 mg of the free base of the compound of formula (I) was measured out, and 13.8 mg of succinic acid and 5 mL of acetonitrile were added. The mixture was stirred at 50° C. for 12-36 h and filtered under reduced pressure. The filter cake was dried in vacuo at 40° C. The product was identified as crystal form β. The XRPD graph is shown in FIG. 18, and the peak positions are shown in Table 16.





TABLE 16







The peak positions of the succinate in crystal form β


Peak number
2θ(°)
d(Å)
I(%)




1
5.160
17.11116
100.00%


2
17.951
4.93733
23.90%


3
20.618
4.30434
17.40%


4
25.557
3.48266
68.70%


5
26.545
3.35527
75.90%






Example 15: Preparation of Hydrobromide in Crystal Form α

100 mg of the free base of the compound of formula (I) was measured out, and 7 µL of hydrobromic acid and 5 mL of acetonitrile were added. The mixture was stirred at 50° C. for 2-36 h and filtered under reduced pressure. The filter cake was dried in vacuo at 40° C. Ion chromatography analysis shows that the bromide ion content of the product is 7.6% and the molar ratio of the compound of formula (I) to hydrobromic acid in the salt is 1:1. The product was identified as crystal form α. The XRPD graph is shown in FIG. 19, and the peak positions are shown in Table 17. The TGA graph shows that the crystal form made a weight loss of 0.27% at 30-170° C. and a weight loss of 14.47% at 190-290° C. The DSC graph shows that the crystal form has an endothermic peak at 175.85° C.





TABLE 17







The peak positions of the hydrobromide in crystal form α


Peak number
2θ(°)
d(Å)
I(%)




1
6.656
13.26907
20.20%


2
9.552
9.25152
14.60%


3
13.778
6.42219
19.50%


4
14.110
6.27166
49.90%


5
19.095
4.64409
43.20%


6
19.522
4.54340
14.50%


7
20.092
4.41583
46.40%


8
21.422
4.14470
12.30%


9
22.276
3.98759
10.60%


10
22.988
3.86564
6.70%


11
23.653
3.75850
9.70%


12
25.220
3.52844
100.00%


13
25.837
3.44553
14.50%


14
26.217
3.39647
7.60%


15
27.119
3.28551
40.40%


16
31.107
2.87277
8.60%


17
31.867
2.80600
6.90%


18
32.769
2.73079
8.20%


19
34.810
2.57517
6.40%






Example 16: Preparation of Hydrobromide in Crystal Form ß

100 mg of the free base of the compound of formula (I) was measured out, and 14 µL of hydrobromic acid and 5 mL of acetonitrile were added. The mixture was stirred at 50° C. for 2 h and filtered under reduced pressure. The filter cake was dried in vacuo at 40° C. Ion chromatography analysis shows that the bromide ion content of the product is 14.3% and the molar ratio of the compound of formula (I) to hydrobromic acid in the salt is 1:2. The product was identified as crystal form β. The XRPD graph is shown in FIG. 20, and the peak positions are shown in Table 18. The TGAgraph shows that the crystal form made a weight loss of 0.58% at 30-130° C. and a weight loss of 17.26% at 150-290° C. The DSC graph shows that the crystal form has an endothermic peak at 213.86° C.





TABLE 18







The peak positions of the hydrobromide in crystal form β


Peak number
2θ(°)
d(Å)
I(%)




1
7.363
11.99692
23.00%


2
12.914
6.84991
21.30%


3
14.351
6.16695
17.90%


4
15.392
5.75220
23.60%


5
16.865
5.25297
40.50%


6
17.217
5.14629
28.60%


7
19.452
4.55974
19.40%


8
19.704
4.50185
32.40%


9
21.999
4.03719
51.50%


10
22.515
3.94590
31.70%


11
23.275
3.81865
22.60%


12
24.213
3.67275
8.60%


13
24.769
3.59161
100.00%


14
27.056
3.29296
36.40%


15
28.236
3.15797
32.70%


16
29.234
3.05243
26.80%


17
29.764
2.99924
15.00%


18
31.251
2.85985
19.60%


19
32.887
2.72127
9.30%






Example 17: Preparation of Hydrobromide in Crystal Form γ

100 mg of the free base of the compound of formula (I) was measured out, and 14 µL of hydrobromic acid and 5 mL of acetonitrile were added. The mixture was stirred at 50° C. for 36 h and filtered under reduced pressure. The filter cake was dried in vacuo at 40° C. for 1 h. The product was identified as crystal form γ. The XRPD graph is shown in FIG. 21, and the peak positions are shown in Table 19.





TABLE 19







The peak positions of the hydrobromide in crystal form γ


Peak number
2θ(°)
d(Å)
I(%)




1
5.463
16.16374
41.70%


2
16.567
5.34670
100.00%


3
19.733
4.49534
9.50%


4
21.227
4.18229
66.70%


5
22.332
3.97776
7.70%


6
25.014
3.55697
14.10%


7
25.784
3.45244
28.00%


8
26.643
3.34307
56.10%


9
28.321
3.14868
38.50%


10
30.298
2.94759
11.60%


11
32.170
2.78026
12.50%


12
33.788
2.65074
30.00%


13
36.211
2.47872
8.10%


14
37.939
2.36970
14.30%






Example 18: Preparation of Hydrochloride in Crystal Form I

100 mg of the free base of the compound of formula (I) was measured out, and 20 µL of hydrochloric acid and 5 mL of acetonitrile were added. The mixture was stirred at 50° C. for 36 h and filtered under reduced pressure. The filter cake was dried in vacuo at 40° C. Ion chromatography analysis shows that the chloride ion content of the product is 6.52%, which indicates that the molar ratio of the compound of formula (I) to hydrochloric acid in the salt is 1:1. The product was identified as crystal form I. The XRPD graph is shown in FIG. 22, and the peak positions are shown in Table 20.





TABLE 20







The peak positions of the hydrochloride in crystal form I


Peak number
2θ(°)
d(Å)
I(%)




1
5.187
17.02196
26.50%


2
8.131
10.86513
15.70%


3
9.407
9.39446
55.90%


4
9.848
8.97424
100.00%


5
10.388
8.50920
33.80%


6
17.354
5.10590
42.80%


7
18.041
4.91303
67.20%


8
18.924
4.68572
18.90%


9
23.830
3.73100
11.40%


10
25.449
3.49720
8.70%


11
26.234
3.39431
42.80%


12
26.724
3.33310
31.80%


13
27.362
3.25684
19.80%


14
27.902
3.19507
16.10%


15
29.275
3.04820
10.50%


16
31.581
2.83071
6.90%






Example 19: Preparation of Hydrochloride in Crystal Form II

100 mg of the free base of the compound of formula (I) was measured out, and 20 µL of hydrochloric acid and 5 mL of ethanol were added. The mixture was made clear by being stirred at 50° C. for 1 h, and was cooled at room temperature for 1 h, stirred at 25° C. for 36 h, and filtered under reduced pressure. The filter cake was dried in vacuo at 40° C. Ion chromatography analysis shows that the chloride ion content of the product is 6.88%, which indicates that the molar ratio of the compound of formula (I) to hydrochloric acid in the salt is 1:1. The product was identified as crystal form II. The XRPD graph is shown in FIG. 23, and the peak positions are shown in Table 21.





TABLE 21







The peak positions of the hydrochloride in crystal form II


Peak number
2θ(°)
d(Å)
I(%)




1
13.728
6.44541
49.10%


2
17.948
4.93813
17.30%


3
20.593
4.30962
6.90%


4
22.678
3.9179
18.10%


5
25.525
3.4869
53.70%


6
27.356
3.25757
100.00%


7
29.543
3.02125
13.20%






Example 20: Preparation of Hydrochloride in Crystal Form III

100 mg of the free base of the compound of formula (I) was measured out, and 10 µL of hydrochloric acid and 5 mL of ethanol were added. The mixture was made clear by being stirred at 50° C. for 1 h, and was cooled at room temperature for 1 h, stirred at 25° C. for 36 h, and filtered under reduced pressure. The filter cake was dried in vacuo at 40° C. Ion chromatography analysis shows that the chloride ion content of the product is 6.82%, which indicates that the molar ratio of the compound of formula (I) to hydrochloric acid in the salt is 1:1. The product was identified as crystal form III. The XRPD graph is shown in FIG. 24, and the peak positions are shown in Table 22.





TABLE 22







The peak positions of the hydrochloride in crystal form III


eak number
2θ(°)
d(Å)
I(%)




1
5.854
15.08568
86.60%


2
8.983
9.83680
93.70%


3
11.902
7.42951
72.70%


4
13.181
6.71157
36.30%


5
16.876
5.24937
28.10%


6
18.159
4.88131
68.30%


7
20.071
4.42037
100.00%


8
21.371
4.15444
47.30%


9
22.799
3.89724
20.30%


10
24.180
3.67779
43.60%


11
25.066
3.54969
48.30%


12
26.469
3.36471
36.10%


13
27.086
3.28943
53.30%


14
27.729
3.21454
33.70%


15
28.615
3.11703
51.90%


16
29.827
2.99309
15.20%


17
30.904
2.89118
17.50%


18
31.935
2.80013
18.00%


19
33.571
2.66731
9.70%


20
34.905
2.56839
11.60%


21
37.265
2.41101
12.10%






Example 21: Preparation of Hydrochloride in Crystal Form IV

The hydrochloride in crystal form I was heated to 135° C. (10 K/min). The product was identified as crystal form IV. The XRPD graph is shown in FIG. 25, and the peak positions are shown in Table 23.





TABLE 23







The peak positions of the hydrochloride in crystal form IV


Peak number
2θ(°)
d(Å)
I(%)




1
5.693
15.51271
51.70%


2
12.474
7.09045
100.00%


3
14.474
6.11478
37.20%


4
17.352
5.10641
38.80%


5
17.840
4.96786
92.80%


6
19.596
4.52641
18.00%


7
22.231
3.99561
29.50%


8
23.109
3.84572
15.70%


9
23.938
3.71433
29.70%


10
24.573
3.61988
12.50%


11
26.866
3.31591
51.00%


12
27.841
3.20188
21.70%


13
29.207
3.05516
25.80%


14
33.452
2.67658
14.10%


15
34.915
2.56767
12.20%






Example 22: Preparation of Hydrochloride in Crystal Form V

100 mg of the free base of the compound of formula (I) was measured out, and 40 µL of hydrochloric acid and 10 mL of ethanol were added. The mixture was made clear by being stirred at 50° C. for 1 h, and was stirred at 25° C. for 3-12 h and filtered under reduced pressure. The filter cake was dried in vacuo at 40° C. The product was identified as crystal form V. The XRPD graph is shown in FIG. 26, and the peak positions are shown in Table 24.





TABLE 24







The peak positions of the hydrochloride in crystal form V


Peak number
2θ(°)
d(Å)
I(%)




1
7.476
11.8152
23.9%


2
9.536
9.26702
19.4%


3
12.349
7.16162
16.6%


4
13.447
6.57921
21.5%


5
14.346
6.16915
74.0%


6
15.181
5.83170
72.9%


7
15.639
5.66169
11.6%


8
16.694
5.30619
16.2%


9
17.185
5.15564
17.3%


10
17.658
5.01861
9.6%


11
17.877
4.95782
6.1%


12
18.422
4.81217
24.2%


13
18.859
4.70175
19.9%


14
20.769
4.27348
6.4%


15
21.606
4.10983
35.5%


16
22.770
3.90227
29.4%


17
23.425
3.79464
30.4%


18
24.279
3.66292
100.0%


19
24.952
3.56563
41.2%


20
26.239
3.39365
1.8%


21
26.990
3.30093
11.3%


22
27.754
3.21178
7.7%


23
28.336
3.14711
23.1%


24
28.772
3.10034
13.7%


25
29.773
2.99840
20.9%


26
30.846
2.89648
18.5%


27
33.089
2.70507
5.2%


28
34.183
2.62095
8.4%


29
36.039
2.49016
4.4%


30
39.987
2.25291
2.7%






Example 23. Influencing Factor Stability Study

The hydrobromide in crystal form α, the hydrobromide in crystal form β, the fumarate in crystal form II, and the fumarate in crystal form IV were left to stand open, and their stability under lighting conditions (4500 Lux), high-temperature conditions (40° C. and 60° C.), high-humidity conditions (75% RH and 92.5% RH) was investigated in a period of 30 days.





TABLE 25








The influencing factor stability data of the hydrobromide in crystal form α


Conditions Time (days)
Hydrobromide in crystal form α


Color and appearance
Main peak purity (%)
Crystal form




40° C.
7
Off-white solid
99.42
Did not change


14
Off-white solid
99.32
Did not change


30
Off-white solid
99.28
Did not change


7
Off-white solid
99.38
Did not change


60° C.
14
Off-white solid
98.99
Did not change


30
Off-white solid
99.02
Did not change


7
Off-white solid
99.41
Did not change


75% RH
14
Off-white solid
99.30
Did not change


30
Off-white solid
99.31
Did not change


7
Off-white solid
99.39
Did not change


92.5% RH
14
Off-white solid
99.42
Did not change


30
Off-white solid
99.28
Did not change


7
Off-white solid
99.09
Did not change


4500 Lux
14
Off-white solid
98.94
Did not change


30
Off-white solid
98.50
Did not change









TABLE 26








The influencing factor stability data of the hydrobromide in crystal form β


Conditions
Time (days)
Hydrobromide in crystal form β


Color and appearance
Main peak purity (%)
Crystal form




Initial
0
Off-white solid
99.12
Crystal form β


7
Off-white solid
99.13
Did not change


40° C.
14
Off-white solid
99.07
Did not change


30
Off-white solid
98.96
Did not change


7
Off-white solid
98.82
Did not change


60° C.
14
Off-white solid
98.82
Did not change


30
Off-white solid
98.88
Did not change


7
Off-white solid
99.13
Did not change


75% RH
14
Off-white solid
99.11
Did not change


30
Off-white solid
99.11
Did not change


7
Off-white solid
99.09
Did not change


92.5% RH
14
Off-white solid
99.15
Did not change


30
Off-white solid
98.93
Did not change


4500 Lux
7
Off-white solid
98.99
Did not change


14
Off-white solid
98.54
Did not change


30
Off-white solid
98.28
Did not change









TABLE 27








The results of the 30-day influencing factor study for the fumarate in crystal form II


Conditions
Time (days)
Fumarate in crystal form II


Color and appearance
Purity (%)
Weight gain (%)




Initial
0
White solid
99.7



5
White solid
99.6



4500 Lux
10
White solid
99.6



30
White solid
98.4



5
White solid
99.6



40° C.
10
White solid
99.5



30
White solid
99.2



5
White solid
99.5



60° C.
10
White solid
99.5



30
White solid
98.0



5
White solid
99.7
0.54


RH 75%
10
White solid
99.7
1.03


30
White solid
99.7
1.42


5
White solid
99.7
1.16


RH 90%
10
White solid
99.7
2.77


30
White solid
99.7
3.97









TABLE 28








The influencing factor stability data of the fumarate in crystal form IV


Conditions
Time (days)
Fumarate in crystal form IV


Color and appearance
Main peak purity
Crystal form


(%)




Initial
0
Pale yellow solid
99.47
Crystal form IV



7
Pale yellow solid
99.46
Did not change


40° C.
14
Pale yellow solid
99.47
Did not change



30
Pale yellow solid
99.26
Did not change


60° C.
7
Pale yellow solid
99.45
Did not change


75% RH
14
Pale yellow solid
99.46
Did not change


30
Pale yellow solid
99.23
Did not change


7
Pale yellow solid
99.52
Did not change


14
Pale yellow solid
99.43
Did not change


30
Pale yellow solid
99.36
Did not change


7
Pale yellow solid
99.47
Did not change


92.5% RH
14
Pale yellow solid
99.45
Did not change


30
Pale yellow solid
99.40
Did not change


7
Pale yellow solid
99.43
Did not change


4500 Lux
14
Pale yellow solid
99.29
Did not change


30
Pale yellow solid
99.19
Did not change






Conclusion: the influencing factor experiment shows that the hydrobromide in crystal form α, the hydrobromide in crystal form β, the fumarate in crystal form II, and the fumarate in crystal form IV have relatively good physical and chemical stability under high-temperature conditions (40° C. and 60° C.) and high-humidity conditions (75% and 92.5%).


Example 24. Long-Term/Accelerated Stability Study

1. The hydrobromide in crystal form α was left to stand under 25° C., 60% RH conditions and 40° C., 75% RH conditions to investigate its stability.





TABLE 29








Sample
Conditions of sitting
Purity (%)
Purity (%)
Crystal form


Initial
1 month






25° C., 60%RH
99.43
99.33
α





40° C., 75%RH
99.24
α






The long-term/accelerated stability study shows that the hydrobromide in crystal form α has good physical and chemical stability when left to stand under long-term and accelerated stability conditions for 1 month.


2. The hydrobromide in crystal form β was left to stand under 25° C., 60% RH conditions and 40° C., 75% RH conditions to investigate its stability.





TABLE 30








Sample
Conditions of sitting
Purity (%)
Purity (%)
Crystal form




Initial
1 month






25° C., 60%RH
99.17
98.99
β


40° C., 75%RH
99.00
β






The long-term/accelerated stability study shows that the hydrobromide in crystal form β has good physical and chemical stability when left to stand under long-term and accelerated stability conditions for 1 month.


3. The fumarate in crystal form IV was left to stand under 25° C., 60% RH conditions and 40° C., 75% RH conditions to investigate its stability.





TABLE 31









Conditions of sitting
Purity (%)
Purity (%)
Crystal form


Sample

Initial
1 month






25° C., 60%RH
99.46
99.40
IV


40° C., 75%RH

99.34
IV






The long-term/accelerated stability study shows that the fumarate in crystal form IV has good physical and chemical stability when left to stand under long-term and accelerated stability conditions for 1 month.


4. The fumarate in crystal form II was subjected to 6-month long-term (25° C., 60% RH) and accelerated (40° C., 75% RH) stability studies. The results are shown in the table below.





TABLE 32











The results of the long-term and accelerated stability studies for the fumarate in crystal form II


Sample
Conditions of sitting
Initial
Purity (%) 1 month
Purity (%) 2 months
Purity (%) 3 months
Purity (%) 6 months
Crystal form




Crystal
25° C., 60%RH
99.7
99.7
99.7
99.7
99.7
II


form II
40° C., 75%RH
99.7
99.7
99.7
99.7
99.7
II






Conclusion: the results show that the crystal form II sample of the fumarate of the compound of formula 1 has good physical and chemical stability when left to stand under long-term (25° C., 60% RH) and accelerated (40° C., 75% RH) conditions for 6 months.


Example 25: Preparation of Fumarate in Amorphous Form

500 mg of the free base of the compound of formula (I) was dissolved in 10 mL of methanol, and 1 eq of fumaric acid and 60 acid were added. The mixture was stirred for 30 min and concentrated to dryness by rotary evaporation to give a solid. The solid was identified as amorphous by XRPD.

Claims
  • 1. A pharmaceutically acceptable salt of a compound of formula (I), wherein the pharmaceutically acceptable salt is selected from the group consisting of a fumarate, an oxalate, a succinate, a maleate, a hydrochloride, a malate, and a hydrobromide, .
  • 2. The fumarate of the compound of formula (I) according to claim 1, wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in a molar ratio of 1:1 or 2:1.
  • 3. (canceled)
  • 4. A crystal form II of a fumarate of a compound of formula (I) according to claim 2, wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in an amount-of-substance ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 7.783, 9.151, 12.378, 14.808, 18.574, 25.755, and 26.802, wherein the 2θ angles have a margin of error of ± 0.2.
  • 5. (canceled)
  • 6. A crystal form IV of a fumarate of the compound of formula (I) according to claim 2, wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in a molar ratio of 2:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 4.854, 10.613, 13.692, 23.523, 24.252, and 25.925, wherein the 2θ angles have a margin of error of ± 0.2.
  • 7. A The hydrobromide of the compound of formula (I) according to claim 1, wherein the compound of formula (I) and hydrobromic acid are in a molar ratio of 1:1 or 1:2.
  • 8. A crystal form α of a hydrobromide of a compound of formula (I) according to claim 7, wherein the hydrobromide of the compound of formula (I) comprises the compound of formula (I) and hydrobromic acid in a molar ratio of 1:1 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 6.656, 9.552, 14.110, 19.095, 20.092, 25.220, and 27.119, wherein the 2θ angles have a margin of error of ± 0.2.
  • 9. A crystal form β of a hydrobromide of a compound of formula (I) according to claim 7, wherein the hydrobromide of the compound of formula (I) comprises the compound of formula (I) and hydrobromic acid in a molar ratio of 1:2 and, in an X-ray powder diffraction graph, has characteristic peaks at 2θ angles of diffraction of 7.363, 12.914, 15.392, 16.865, 17.217, 19.704, 21.999, 22.515, 24.769, 27.056, and 28.236, wherein the 2θ angles have a margin of error of ± 0.2.
  • 10-14. (canceled)
  • 15. The fumarate of the compound of formula (I) according to claim 2, wherein the fumarate of the compound of formula (I) comprises the compound of formula (I) and fumaric acid in a molar ratio of 2:1.
  • 16. A method for preparing the pharmaceutically acceptable salt of the compound of formula (I) according to claim 1, which comprises the step of reacting the compound of formula (I) with a fumaric acid, oxalic acid, succinic acid, maleic acid, hydrochloric acid, or hydrobromic acid.
  • 17. The method according to claim 16, wherein the step of reacting the compound of formula (I) with a fumaric acid, oxalic acid, succinic acid, maleic acid, hydrochloric acid, or hydrobromic acid carried out in a solvent, and the solvent select from ester solvent, alcohol solvent, nitrile solvent, ketone solvent, ether solvent, alkane solvent, water or a mixed solvent of the solvents described above, said ester solvent is ethyl acetate, said alcohol solvent is methanol, ethanol, or isopropanol, said nitrile solvent is acetonitrile, propionitrile, said ketone solvent is acetone, said ether solvent is tetrahydrofuran, dioxane, diethyl ether, or isopropyl ether, said alkane solvent is n-hexane, n-heptane.
  • 18. A method for preparing the fumarate of the compound of formula (I) according to claim 15, comprising a step of reacting the compound of formula (I) with fumaric acid.
  • 19. The method according to claim 18, wherein the step of reacting the compound of formula (I) with a fumaric acid carried out in a solvent, and the solvent select from ester solvent, alcohol solvent, nitrile solvent, ketone solvent, ether solvent, alkane solvent, water or a mixed solvent of the solvents described above, said ester solvent is ethyl acetate, said alcohol solvent is methanol, ethanol, or isopropanol, said nitrile solvent is acetonitrile, propionitrile, said ketone solvent is acetone, said ether solvent is tetrahydrofuran, dioxane, diethyl ether, or isopropyl ether, said alkane solvent is n-hexane, n-heptane.
  • 20. A pharmaceutical composition, comprising the following components: i) the pharmaceutically acceptable salt of the compound of formula (I) according to claim 1;ii) one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • 21. A method for treating or preventing cancer, inflammation, or other proliferative diseases in a subject in need thereof comprising administering to the subject the pharmaceutically acceptable salt of the compound of formula (I) according to claim 1.
  • 22. The method according to claim 21, wherein the cancer is selected from the group consisting of melanoma, liver cancer, kidney cancer, lung cancer, nasopharyngeal carcinoma, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, cervical cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma, astrocytoma, and glioma.
  • 23. A pharmaceutical composition, comprising the following components: i) the fumarate of the compound of formula (I) of claim 15;ii) one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • 24. A method for treating or preventing cancer, inflammation, or other proliferative diseases in a subject in need thereof comprising administering to the subject the fumarate of the compound of formula (I) according to claim 15.
  • 25. The method according to claim 24, wherein the cancer is selected from the group consisting of melanoma, liver cancer, kidney cancer, lung cancer, nasopharyngeal carcinoma, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, cervical cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma, astrocytoma, and glioma.
Priority Claims (1)
Number Date Country Kind
202011049402.2 Sep 2020 CN national
PCT Information
Filing Document Filing Date Country Kind
PCT/CN2021/121641 9/29/2021 WO