WO2023125947A1 - Pharmaceutically acceptable salt of tetrahydroisoquinoline compound, and crystal form and use thereof - Google Patents

Abstract

The present invention provides a pharmaceutically acceptable salt of a compound as a PRMT5 inhibitor, a crystal form of the pharmaceutically acceptable salt, a preparation method therefor, a pharmaceutical composition comprising the pharmaceutically acceptable salt or the crystal form thereof, and the use of the pharmaceutically acceptable salt or the pharmaceutical composition comprising the pharmaceutically acceptable salt in the preparation of a drug for preventing or treating related pharmacological conditions.

Description

Pharmaceutically acceptable salts, crystal forms and uses of tetrahydroisoquinoline compounds

Cross References to Related Applications

This application claims the benefit and priority of the following 1 Chinese invention patent application, which is hereby incorporated by reference in its entirety:

Patent application No. 202111664582.X filed with the State Intellectual Property Office of China on December 30, 2021.

technical field

The present disclosure relates to the field of medicinal chemistry, in particular, to pharmaceutically acceptable salts of tetrahydroisoquinoline compounds, crystal forms of the pharmaceutically acceptable salts, preparation methods thereof, pharmaceutical compositions containing them, and uses thereof.

Background technique

Epigenetic changes are key mediators that drive and maintain the malignant phenotype of tumors. Changes in DNA methylation, histone acetylation and methylation, non-coding RNA, and post-translational modifications are all epigenetic drivers of cancer development independent of changes in DNA sequence. Arginine methylation is an important class of post-translational modifications that affect cell growth and proliferation, apoptosis, angiogenesis and metastasis by regulating transcription and post-transcriptional RNA processing. There are three types of methylarginine, namely ω-NG-monomethylarginine (MMA), ω-NG, N'G-asymmetric dimethylarginine (ADMA) and ω-NG, N'G-symmetric dimethylarginine (SDMA). This modification is catalyzed by the protein arginine methyltransferase (PRMT) family, which transfers a methyl group from S-adenosylmethionine (AdoMet) to the arginine side chain of histones and non-histones. Nine PRMT genes were annotated in the human genome, classified based on the type of methylarginine produced into type I (PRMT1, 2, 3, 4, 6 and 8), type II (PRMT5 and PRMT9) and type III enzymes ( PRMT7). PRMT5 is primarily a type II enzyme that catalyzes the symmetric dimethylation of arginine. PRMT5 was first discovered in a two-hybrid assay detecting proteins interacting with Janus tyrosine kinase (Jak2).

PRMT5 is a general transcriptional repressor that forms a complex with other transcription factors, including BRG1 and hBRM, Blimp1, and Snail. PRMT5 participates in a variety of different cellular biological processes through the methylation of a variety of substrates in the cytoplasm and nucleus, including histone H4 residue Arg3 (H4R3) and H3 residue Arg8 (H3R8). H4R3 methylation is associated with transcriptional repression, while H3R8 methylation is thought to be associated with both transcriptional activation and transcriptional repression. In addition to the direct induction of repressive histone marks by PRMT5, the enzyme's role in gene silencing is also mediated through the formation of multiple arrestin complexes, including NuRD components, HDACs, MDB proteins, and DNA methyltransferases. PRMT5 affects its substrate specificity by interacting with some binding proteins. A central component in this protein complex is MEP50. MEP50 is required for the enzymatic activity of PRMT5. The study found that PRMT5 can methylate proteins involved in RNA splicing, such as SmD3, which can be used to track the chemical activity of cellular biological PRMT5.

PRMT5 plays an important role in tumorigenesis. Studies have found that the expression of PRMT5 is upregulated in a variety of tumors, including lymphoma, lung cancer, breast cancer and colorectal cancer. In addition, PRMT5 expression was increased in mantle cell lymphoma (MCL) patient samples, and PRMT5 knockout could inhibit the proliferation of MCL cells, indicating that PRMT5 plays an important role in MCL. PRMT5 overexpression promotes cell proliferation, and in melanoma, breast cancer, and lung cancer cell lines, PRMT5 knockdown can inhibit the proliferation of these cells. Therefore, PRMT5 is a potential target for cancer therapy.

Loss of methylthioadenosine phosphorylase (MTAP) confers selective dependence on PRMT5 and its binding protein WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene CDKN2A. Cells carrying MTAP deletions have increased intracellular concentrations of methylthioadenosine (MTA, a metabolite cleaved by MTAP). In addition, MTA specifically inhibits the enzymatic activity of PRMT5. MTA or PRMT5 small-molecule inhibitors significantly inhibited the cell viability of MTAP-deficient cancer cell lines compared with MTAP-expressing cells.

Therefore, there is a need in the art to develop small molecule active compounds that can inhibit the activity of PRMT5 and treat various PRMT5-related diseases.

PCT/CN2021/103597 (application date June 30, 2021) describes a compound 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4 -Tetrahydroisoquinolin-3-yl)ethyl)-8-(2-methoxy-7-azaspiro[3.5]nonane-7-carbonyl)-1,2,3,4-tetrahydro -5H-Benzo[e][1,4]diazepine

-5-keto (compound I) hydrochloride, the study found that the compound I hydrochloride has good PRMT5 enzyme inhibition, cell proliferation inhibition and cell SDMA inhibitory activity, and good pharmacokinetic properties and liver function. Cell metabolism stability, and showed significant tumor growth inhibition in mouse subcutaneous xenograft Z-138 model, and showed a good dose-response relationship.

Contents of the invention

The present disclosure provides a pharmaceutically acceptable salt of Compound I, wherein the pharmaceutically acceptable salt is selected from monohydrochloride, hydrobromide, 1,5-naphthalene disulfonate, oxalate, citrate, Sulfate, Phosphate, L-Tartrate, L-Malate, Succinate, Adipate, Fumarate, Oxalate, Propionate, Benzoate, Acetate, Formic Acid salt or L-arginine salt, preferably monohydrochloride, hydrobromide or 1,5-naphthalene disulfonate, more preferably monohydrochloride.

In some embodiments, in the pharmaceutically acceptable salt of Compound I, the molar ratio of Compound I to acid molecules is about 1:2 to 2:1, preferably selected from 1:2, 1:1 or 2:1, More preferably 1:1.

In some embodiments, there is provided a monohydrochloride salt of Compound I, wherein the molar ratio of Compound I to hydrochloric acid is about 1:1.

In some embodiments, a hydrobromide salt of Compound I is provided.

In some embodiments, a 1,5-naphthalene disulfonate salt of Compound I is provided.

In some embodiments, the pharmaceutically acceptable salts of Compound I exist in unsolvated or solvated form.

The present disclosure also provides solid forms of the pharmaceutically acceptable salts of Compound I.

In some embodiments, the solid form of the pharmaceutically acceptable salt of Compound I is selected from amorphous or crystalline forms.

The present disclosure also provides a method for preparing a pharmaceutically acceptable salt of the aforementioned compound I, comprising: a step of forming a salt of compound I with an acid, the acid being selected from hydrochloric acid, hydrobromic acid, 1,5-naphthalene disulfonic acid, oxalic acid , citric acid, sulfuric acid, phosphoric acid, L-tartaric acid, L-malic acid, succinic acid, adipic acid, fumaric acid, oxalic acid, propionic acid, benzoic acid, acetic acid, formic acid or L-arginine, preferably hydrochloric acid, hydrogen Bromic acid or 1,5-naphthalenedisulfonic acid, more preferably hydrochloric acid.

The solvent used in the salt-forming reaction described in the present disclosure is selected from at least one of ethyl acetate, tetrahydrofuran, acetonitrile, acetone, methyl acetate, isopropanol, methanol or toluene, preferably selected from isopropanol, methyl acetate, acetic acid ethyl ester, methanol or acetone, more preferably isopropanol or ethyl acetate.

In some embodiments, the method for preparing the aforementioned pharmaceutically acceptable salt further includes at least one of the steps of volatilizing the solvent, crystallizing with stirring, filtering, washing or drying.

In some embodiments, the pharmaceutically acceptable salt of Compound I is in crystalline form, preferably hydrochloride crystal form, hydrobromide salt crystal form, 1,5-naphthalene disulfonate salt crystal form, more preferably monohydrochloride Salt crystal form.

In some embodiments, the hydrochloride crystal form of the compound I is the A crystal form of the compound I-hydrochloride, and the X-ray powder diffraction pattern of the A crystal form represented by the diffraction angle 2θ is 5.42 ± 0.2 ^o , 6.60± ^0.2o , 10.36± ^0.2o , 13.60±0.2o ^, 14.24±0.2o ^, 15.83±0.2o, ^16.62 ±0.2o ^, 17.65±0.2o, 19.29±0.2o ^, 19.86±0.2o ^, 21 .37± ^{0.2 o} and 23.88±0.2 ^o have diffraction peaks.

In some embodiments, the crystal form A has an X-ray powder diffraction pattern represented by diffraction angle 2θ at 5.42±0.2 ^° , 6.60±0.2 ^° , 9.76±0.2 ^° , 10.36±0.2 ^° , 13.00±0.2 ^° , 13.60± ^0.2o , 14.24± ^{0.2o ,} 15.83± ^0.2o , 16.62±0.2o, 17.65±0.2o, 18.28±0.2o, ^19.29 ± ^{0.2o ,} 19.86±0.2o ^, 20.61 ^± 0.2o, 21. 37± ^0.2o , 23.88±0.2 ^o , 25.08±0.2 ^o , 28.22±0.2 ^o have diffraction peaks.

In some embodiments, the crystal form A has an X-ray powder diffraction pattern represented by diffraction angle 2θ at 5.42±0.2 ^° , 6.60±0.2 ^° , 6.83±0.2 ^° , 9.76±0.2 ^° , 10.36±0.2 ^° , 13.00± ^0.2o , ^13.60 ± ^{0.2o ,} 14.24± ^0.2o , 15.27±0.2o ^, 15.83±0.2o, 16.62±0.2o, 17.30± ^{0.2o ,} 17.65±0.2o, 18.28± ^0.2o , 19. 29± ^0.2o , 19.86±0.2 ^o , ^20.61 ±0.2 ^o , 21.37±0.2 ^o , 21.88±0.2 o , 23.88±0.2 ^o , 25.08±0.2 ^o , 25.56±0.2 ^o , 26.51±0.2 ^o , 27.22±0.2 ^o , 28. 22± ^0.2o There are diffraction peaks.

In some embodiments, the crystal form A has an X-ray powder diffraction pattern represented by diffraction angle 2θ at 5.42±0.2 ^° , 6.60±0.2 ^° , 6.83±0.2 ^° , 9.76±0.2 ^° , 10.36±0.2 ^° , 13.00± ^0.2o , ^13.60 ± ^{0.2o ,} 14.24± ^0.2o , 15.27±0.2o ^, 15.83±0.2o, 16.62±0.2o, 17.30± ^{0.2o ,} 17.65±0.2o, 18.28± ^0.2o , 19. 29± ^0.2o , 19.86± ^0.2o , 20.15± ^{0.2o ,} 20.61± ^0.2o , 21.37±0.2o ^, 21.88±0.2o, 23.23±0.2o, 23.88± ^0.2o , 25.08±0.2o ^, 25.56 ^{± 0.2o} , 26. 51± ^0.2o , 27.22±0.2 ^o , 28.22±0.2 ^o , 29.42±0.2 ^o , 30.21±0.2 ^o , 30.74±0.2 ^o , 34.79±0.2 ^o , 37.74±0.2 ^o have diffraction peaks.

In some embodiments, the X-ray powder diffraction pattern of Form A is substantially as shown in FIG. 8 .

In some embodiments, the crystalline Form A has a DSC spectrum with a peak at 259.27±5.0°C.

In some embodiments, the DSC spectrum of Form A is substantially as shown in FIG. 9 .

The present disclosure also provides a method for preparing the A crystal form of compound I-hydrochloride, comprising the steps of:

1) Weighing an appropriate amount of compound I, adding it to the solvent (i), and dissolving at room temperature or heating;

2) Add aqueous hydrogen chloride solution or organic solvent solution of hydrogen chloride to crystallize.

In some embodiments, there is an optional step 3) after step 2) of the preparation method of the A crystal form, that is, a good solvent is used to dissolve the crystal obtained in step 2), and after the solution is obtained, a poor solvent is added dropwise to analyze crystal.

In some embodiments, there is an optional step 3') after step 2) of the preparation method of crystal form A, that is, dissolving the crystal form obtained in step 2) in methanol and concentrating under reduced pressure to obtain compound I-hydrochloride free Shaped matter, and then dissolve the amorphous matter in solvent (ii), and then precipitate crystals by beating or adding anti-solvent dropwise.

In some embodiments, the solvent (i) in step 1) of the preparation method for crystal form A is selected from methyl acetate, ethyl acetate, acetone or isopropanol, preferably isopropanol or ethyl acetate.

In some embodiments, the volume (mL) of the solvent (i) in step 1) of the preparation method for crystal form A is 5 to 30 times, preferably 5 to 20 times, more preferably 10 to 30 times the weight of the compound (g). 15 times.

In some embodiments, an organic solvent solution of hydrogen chloride, preferably a hydrogen chloride-ethyl acetate solution, is added in step 2) of the preparation method for crystal form A.

In some embodiments, the organic solvent solution of hydrogen chloride in step 2) of the preparation method of crystal form A is added dropwise to the reaction system at -10-10°C, preferably 0-10°C, and kept for reaction.

In some embodiments, the molar amount of hydrogen chloride used in step 2) of the preparation method for crystal form A is 0.95-1.05 times the molar amount of compound I in step 1.

In some embodiments, the reaction time of step 2) of the preparation method for crystal form A is 0.5-3 hours, preferably 1-2 hours.

In some embodiments, the good solvent in step 3) of the preparation method of crystal form A is selected from methanol, dichloromethane or chloroform, preferably methanol; the poor solvent is selected from isopropanol, acetone or isopropyl ether, preferably isopropyl alcohol.

In some embodiments, crystals are prepared by beating in step 3') of the preparation method of crystal form A, and the solvent (ii) is selected from acetone, 4-methyl-2-pentanone, and 2-butanone , isopropyl ether, methyl tert-butyl ether, diethyl ether, n-propanol, isopropanol, ethanol, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, acetonitrile, tetrahydrofuran, cyclohexane, toluene , one or more of water or acetone aqueous solution, preferably ethyl acetate.

In some embodiments, in step 3') of the preparation method of crystal form A, crystals are prepared by adding anti-solvent dropwise, and the solvent (ii) is selected from dichloromethane, chloroform or methanol, preferably methanol; The anti-solvent is selected from acetone, isopropyl ether, methyl acetate, acetonitrile or tetrahydrofuran, preferably acetone.

In some embodiments, the hydrochloride crystal form of the compound I is the B crystal form of the compound I-hydrochloride, and the X-ray powder diffraction pattern of the B crystal form represented by the diffraction angle 2θ is at 5.16±0.2 ^o , 9.86± ^0.2o , 10.42± ^0.2o , 10.76±0.2o ^, 11.36±0.2o ^, 15.68±0.2o ^, 16.31±0.2o, 17.29±0.2o ^, 17.98±0.2o ^, 18.89± ^{0.2o ,} 19 .83±0.2 There are diffraction peaks at ^o and 20.99±0.2 ^o .

In some embodiments, the crystal form B has an X-ray powder diffraction pattern represented by diffraction angle 2θ at 5.16±0.2 ^° , 9.86±0.2 ^° , 10.42±0.2 ^° , 10.76±0.2 ^° , 11.36±0.2 ^° , 14.46± ^0.2o , 15.68± ^0.2o , 16.31± ^0.2o , 17.29±0.2o ^, 17.98± ^0.2o , 18.89± ^0.2o , 19.83±0.2o ^, 20.99±0.2o, 22.13 ^{± 0.2o} , 22. 72± ^0.2o , 23.61±0.2 ^o and 25.94±0.2 ^o have diffraction peaks.

In some embodiments, the crystal form B has an X-ray powder diffraction pattern represented by diffraction angle 2θ at 5.16±0.2 ^° , 6.33±0.2 ^° , 8.98±0.2 ^° , 9.86±0.2 ^° , 10.42±0.2 ^° , 10.76± ^0.2o , 11.36± ^{0.2o ,} 12.82± ^0.2o , 14.46±0.2o ^, 15.68± ^0.2o , 16.31±0.2o, 17.29± ^0.2o , 17.31±0.2o ^, 17.98±0.2o ^, 18. 89± ^0.2o , 19.30± ^0.2o , 19.83± ^{0.2o ,} 20.99± ^0.2o , 21.73±0.2o ^, 22.13±0.2o, 22.72± ^0.2o , 23.61± ^0.2o , 23.73±0.2o ^, 24.71± ^0.2o , 25. 94± ^0.2o , 26.38±0.2 ^o and 37.29±0.2 ^o have diffraction peaks.

In some embodiments, the X-ray powder diffraction pattern of Form B is substantially as shown in FIG. 11 .

In some embodiments, the DSC spectrum of Form B is substantially as shown in FIG. 12 .

In some embodiments, the hydrobromide salt crystal form of Compound I is the C crystal form, and the X-ray powder diffraction pattern of the C crystal form represented by the diffraction angle 2θ is at 6.45±0.2 ^° , 9.49±0.2 ^° , 10.01±0.2 ^o , 12.96±0.2 ^o , 13.45±0.2 ^o , 13.81±0.2 ^o , 15.50±0.2 ^o , 19.13±0.2 ^o , 20.13±0.2 o , 20.78±0.2 ^o , 23.31± ^{0.2 o .}

In some embodiments, the crystal form C has an X-ray powder diffraction pattern represented by diffraction angle 2θ at 6.45±0.2 ^° , 9.49±0.2 ^° , 10.01±0.2 ^° , 12.96±0.2 ^° , 13.45±0.2 ^° , 13.81± ^0.2o , 15.50± ^{0.2o ,} 17.30± ^0.2o , 19.13±0.2o ^, 19.46± ^0.2o , 20.13±0.2o, 20.78± ^0.2o , 23.31±0.2o ^, 24.16±0.2o ^, 24. 55± ^0.2o , 24.80±0.2 ^o , 25.62±0.2 ^o , 27.00±0.2 ^o , 28.87±0.2 ^o , 28.95±0.2 ^o have diffraction peaks.

In some embodiments, the crystal form C has an X-ray powder diffraction pattern represented by diffraction angle 2θ at 6.45±0.2 ^° , 9.49±0.2 ^° , 10.01±0.2 ^° , 12.96±0.2 ^° , 13.45±0.2 ^° , 13.81± ^0.2o , 15.50± ^0.2o , 16.29± ^0.2o , 16.95±0.2o ^, 17.30± ^0.2o , 19.13±0.2o, ^19.46 ±0.2o ^, 20.13±0.2o, 20.78± ^{0.2o ,} 23. 31± ^0.2o , 23.71± ^0.2o , 24.16± ^{0.2o ,} 24.55± ^0.2o , 24.80±0.2o ^, 25.62±0.2o, 26.05±0.2o, 27.00± ^0.2o , 27.44±0.2o ^, 28.87± ^{0.2o ,} 28. 95± ^0.2o , 30.65±0.2 ^o , 31.74±0.2 ^o , 32.78±0.2 ^o , 34.03±0.2 ^o , 34.97±0.2 ^o have diffraction peaks.

In some embodiments, the X-ray powder diffraction pattern of Form C is substantially as shown in FIG. 2 .

In some embodiments, the crystalline Form C has a DSC spectrum with a peak at 238.84±5.0°C.

In some embodiments, the DSC spectrum of Form C is substantially as shown in FIG. 3 .

In some embodiments, the 1,5-naphthalene disulfonate salt of Compound I is the D crystal form, and the X-ray powder diffraction pattern of the D crystal form represented by the diffraction angle 2θ is at 4.02±0.2 ^o , 9.89 ± ^0.2o , 11.49± ^0.2o , 12.74± ^0.2o , 15.68±0.2o, 15.69± ^{0.2o ,} 16.15±0.2o, 18.62± ^0.2o , 18.93±0.2o, 19.67± ^{0.2o ,} 19.79±0.2o ^{o ,} 21.26 There are diffraction peaks at ±0.2 ^o , 24.36±0.2 ^o , and 27.56±0.2 ^o .

In some embodiments, the crystal form D has an X-ray powder diffraction pattern represented by diffraction angle 2θ at 4.02±0.2 ^° , 8.06±0.2 ^° , 8.79±0.2 ^° , 9.89±0.2 ^° , 11.49±0.2 ^° , 12.74± ^0.2o , 13.94± ^{0.2o ,} 15.68± ^0.2o , 15.69± ^{0.2o ,} 16.15±0.2o, 17.21±0.2o, 18.62± ^0.2o , 18.93±0.2o ^, 19.67± ^0.2o , 19. 79± ^0.2o , 20.57±0.2 ^o , 21.26±0.2 ^o , 24.36±0.2 ^o , 25.64±0.2 ^o , 27.56±0.2 ^o have diffraction peaks.

In some embodiments, the crystal form D has an X-ray powder diffraction pattern represented by diffraction angle 2θ at 4.02±0.2 ^° , 8.06±0.2 ^° , 8.79±0.2 ^° , 9.89±0.2 ^° , 11.49±0.2 ^° , 12.10± ^0.2o , 12.74± ^0.2o , 13.94± ^0.2o , 14.94± ^{0.2o ,} 15.68± ^0.2o , 15.69±0.2o, 16.15± ^{0.2o ,} 17.21±0.2o, 17.63±0.2o ^, 17. 97± ^0.2o , 18.62± ^0.2o , 18.93± ^0.2o , 19.67± ^0.2o , 19.79± ^0.2o , 20.20±0.2o, 20.57± ^0.2o , ^21.26 ± ^0.2o , 22.61±0.2o ^, 23.65± ^0.2o , 24. 36± ^0.2o , 25.64±0.2 ^o , 27.56±0.2 ^o , 28.45±0.2 ^o , 32.64±0.2 ^o , 36.86±0.2 ^o , 37.78±0.2 ^o have diffraction peaks.

In some embodiments, the X-ray powder diffraction pattern of Form D is substantially as shown in FIG. 5 .

In some embodiments, the crystalline Form D has a DSC spectrum with a peak at 231.85±5.0°C.

In some embodiments, the DSC spectrum of Form D is substantially as shown in FIG. 6 .

The crystallization method of the crystal form in the present disclosure is conventional, such as crystallization from a volatile solvent, crystallization at lower temperature or crystallization at room temperature.

Further, the preparation method of the crystal form in the present disclosure also includes steps such as filtering, washing or drying.

In another aspect, the present disclosure provides a pharmaceutical composition, which comprises a pharmaceutically acceptable salt of Compound I described in the present disclosure, and pharmaceutically acceptable excipients.

In another aspect, the present disclosure provides a method for treating a disease or condition mediated by PRMT5 in a mammal, comprising administering a therapeutically effective amount of a pharmaceutically acceptable salt of Compound I of the present disclosure to a mammal in need of such treatment, preferably a human. , or a pharmaceutical composition thereof.

In another aspect, the present disclosure provides the use of a pharmaceutically acceptable salt of Compound I described in the present disclosure, or a pharmaceutical composition thereof, in the preparation of a medicament for preventing or treating a disease or disorder mediated by PRMT5.

In another aspect, the present disclosure provides the use of a pharmaceutically acceptable salt of Compound I described in the present disclosure, or a pharmaceutical composition thereof, in preventing or treating a disease or condition mediated by PRMT5.

In another aspect, the present disclosure provides a pharmaceutically acceptable salt of compound I of the present disclosure, or a pharmaceutical composition thereof, for preventing or treating a disease or condition mediated by PRMT5.

In another aspect, the present disclosure provides a pharmaceutically acceptable salt of compound I of the present disclosure, or a pharmaceutical composition thereof, for preventing or treating cancer.

In some embodiments, the disease or condition mediated by PRMT5 includes, but is not limited to, cancer.

Definitions and Explanations of Terms

Unless otherwise stated, terms used in this disclosure have the following meanings, definitions of groups and terms recorded in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, examples The definitions of specific compounds in and etc. may be combined and combined with each other arbitrarily. A specific term should not be regarded as indeterminate or unclear if there is no special definition, but should be understood according to the ordinary meaning in the art. When a trade name appears herein, it is intended to refer to its corresponding trade name or its active ingredient.

The term "about" is used in this disclosure to mean approximately, around, approximately, or approximately. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the limits of the stated numerical range. Unless otherwise indicated, the term "about" is used herein to modify the upper and lower limits of the stated value by a numerical value of 10% deviation.

Unless otherwise stated, the terms "comprise", "comprise" or "comprise" and their English variants such as comprises or comprising are to be understood in an open, non-exclusive sense, ie "including but not limited to".

Reference throughout this text to "some embodiments" or "an embodiment" means that at least one embodiment includes a specific reference element, structure or feature described in relation to that embodiment. Thus, appearances of the phrase "some embodiments" or "an embodiment" in various places throughout this document are not necessarily all referring to the same embodiment. Furthermore, particular elements, structures or characteristics may be combined in any suitable manner in one or more embodiments.

The term "optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that description includes that said event or circumstance occurs and that it does not.

The room temperature mentioned in the present disclosure refers to 20±5°C.

The range "m∼n" described in the present disclosure represents an abbreviated representation of any combination of real numbers between m and n, where both m and n are real numbers. For example, the numerical range "5-30" means that 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; "10~15" means that 10, 11, 12, 13, 14 or 15 have been listed in this article, "10~15 " is just an abbreviation for these numerical combinations.

"X-ray powder diffraction pattern or XRPD pattern" described in the present disclosure refers to according to Bragg's formula 2d Sinθ=nλ (wherein, λ is the wavelength of X-ray, when using Cu-Kα ray,

The order n of diffraction is any positive integer, generally take the first-order diffraction peak, n=1), when the X-ray is incident on the crystal or part of the crystal sample at the grazing angle θ (the complementary angle of the incident angle, also known as the Bragg angle) On an atomic plane with a d-lattice plane spacing, the Bragg equation can be satisfied, and thus this group of X-ray powder diffraction patterns has been measured.

For the same crystal form of the same compound, the peak positions of their XRPD patterns are generally similar, and the relative intensity error may be large. It should also be pointed out that in the identification of mixtures, due to the decrease of content and other factors, some diffraction lines will be missing. At this time, there is no need to rely on all the diffraction peaks observed in high-purity samples, and even one diffraction peak may also affect the given is characteristic for a given crystal.

The "2θ or 2θ angle" mentioned in the present disclosure refers to the diffraction angle, θ is the Bragg angle, and the unit is ° or degree; the error range of each diffraction peak 2θ is ±0.20°.

For those skilled in the art, due to crystal defects, measurement errors and other factors, there is often a certain degree of error in the molar ratio of the disclosed compound to the acid/base molecule and the solvent molecule in the solvate, generally speaking, ±10% are within the reasonable error range. There is a degree of error that will vary with the context in which it is used, but the error will vary by no more than ±10%, preferably ±5%.

The term "therapeutically effective amount" means (i) treating or preventing a particular disease, condition or disorder, (ii) alleviating, ameliorating or eliminating one or more symptoms of a particular disease, condition or disorder, or (iii) preventing or An amount of a compound of the disclosure that delays the onset of one or more symptoms of a particular disease, condition or disorder described herein. The amount of a compound of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art according to its own knowledge and this disclosure.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without excessive Toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable excipients" refers to those excipients that have no obvious stimulating effect on the organism and will not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.

The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or pharmaceutically acceptable salts or solvates thereof and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate administration of a compound of the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, to an organism.

The pharmaceutical composition of the present disclosure can be prepared by combining the compound of the present disclosure or its pharmaceutically acceptable salt or its solvate with suitable pharmaceutically acceptable auxiliary materials, for example, it can be formulated into solid, semi-solid, liquid or gaseous preparations , such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols, etc.

Typical routes of administration of a compound of the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt or solvate thereof include, but are not limited to, oral, rectal, topical, Inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

The pharmaceutical composition of the present disclosure can be produced by methods well known in the art, such as conventional mixing methods, dissolving methods, granulating methods, emulsifying methods, freeze-drying methods and the like.

In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical compositions can be formulated by mixing the active compounds with pharmaceutically acceptable excipients well known in the art. These excipients enable the disclosed compound or its pharmaceutically acceptable salt or its solvate to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, etc. for oral administration to patients.

Solid oral compositions can be prepared by conventional methods of mixing, filling or tabletting. It can be obtained, for example, by mixing the active compound with solid excipients, optionally milling the resulting mixture, adding other suitable excipients if desired, and processing the mixture into granules to obtain tablets Or the core of the sugar coating. Suitable auxiliary materials include but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, etc.

The pharmaceutical composition may also be adapted for parenteral administration as a suitable unit dosage form of sterile solutions, suspensions or lyophilized products.

The therapeutically effective amount of the pharmaceutically acceptable salt of Compound I contained in the pharmaceutical composition of the present disclosure is selected from 0.01 mg/kg to 100 mg/kg, in single or divided doses.

Those skilled in the art recognize that measurements of XRPD peak positions and/or intensities for a given crystalline form of the same compound will vary within error limits. The 2Θ values allow for an appropriate margin of error. Usually, the margin of error is represented by "±". For example, 5.42±0.2° 2Θ means within the range of about 5.22 to 5.62° 2Θ. Depending on the sample preparation technique, the calibration technique applied to the instrument, human operator bias, etc., one skilled in the art recognizes that a suitable error range for the XRPD diffraction angle (2θ) may be about ±0.20°, ±0.15°, ±0.10° , ±0.05° or less. When used to describe an XRPD pattern, the term "substantially the same" or "substantially as shown" means comprising at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 90% Or a pattern of diffraction peaks with at least 99% of the diffraction angles within a standard deviation of ±0.2° 2Θ.

As one skilled in the art recognizes, measurements of a DSC spectrum for a given crystalline form of the same compound will vary within a margin of error. Single-peak peak values (expressed in degrees Celsius) allow for a modest margin of error. Usually, the margin of error is represented by "±". For the same crystalline form of the same compound, thermal transition temperatures and melting points are typically within about ±5.0°C in consecutive analyses. For example, a peak value of "259.27±5.0" indicates that it is in the range of 254.27 to 264.27. Depending on the sample preparation technique, the calibration technique applied to the instrument, human operator bias, etc., those skilled in the art will recognize that appropriate error ranges for unimodal peaks may be ±5.0, ±4.0, ±3.0, ±2.0 or less.

Salt forms and/or crystalline forms of the present disclosure may also be isotopically labeled. The disclosure also includes isotopically labeled disclosed compounds that are identical to those described herein, but with one or more atoms replaced by an atom of an atomic mass or mass number different from that normally found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ¹²³ I, ¹²⁵ I and ³⁶ Cl, etc.

Certain isotopically-labeled compounds of the disclosure (eg, those labeled ^{with3H and14C} ) are useful in compound and/or substrate tissue distribution assays. Tritiated ( ^ie3H ) and carbon-14 ( ^ie14C ) isotopes are especially preferred for their ease of preparation and detectability. Positron-emitting isotopes, such as ¹⁵ O, ¹³ N, ¹¹ C, and ¹⁸ F, can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the disclosure can generally be prepared by following procedures similar to those disclosed in the Schemes and/or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

Furthermore, substitution with heavier isotopes such as deuterium (i.e. ² H) may confer certain therapeutic advantages resulting from greater metabolic stability (e.g. increased in vivo half-life or reduced dosage requirements), and thus in some cases The following may be preferred, where deuterium substitution may be partial or complete, partial deuterium substitution means that at least one hydrogen is replaced by deuterium.

The compounds of the present disclosure or their pharmaceutically acceptable salts or their solvates can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, and the compounds formed by combining them with other chemical synthesis methods Embodiments and equivalent replacements known to those skilled in the art, preferred embodiments include but are not limited to the examples of the present disclosure.

The chemical reactions of the embodiments of the present disclosure are performed in suitable solvents that are suitable for the chemical changes of the present disclosure and the reagents and materials required therefor. In order to obtain the disclosed compound or its pharmaceutically acceptable salt or its solvate, it is sometimes necessary for those skilled in the art to modify or select the synthetic steps or reaction schemes based on the existing embodiments.

The test condition of the instrument used in this disclosure experiment:

1. Single crystal X-ray crystallography measurement

(1) Equipment name: single crystal diffractometer

Detector model: Bruker D8 Advance

Light source: Ga Kα

Wavelength: 1.34139

(2) Equipment name: polarized light microscope

Instrument model: Olympus BX53

Light source: LED

Eyepiece multiple: 10 times

2. X-ray Powder Diffraction (XRPD)

Instrument model: Bruker D8Focus

Rays: Cu Kα,

1.54060

Slit ( ^o ): 2.5

Scanning mode: θ/2θ, scanning range: 3-40 ^o

Dwell time (seconds): 0.12

Scan step size ( ^o 2θ): 0.01

Scanning flow rate: 5 ^o /min

Voltage: 40kV

Current: 40mA

3. Differential Scanning Calorimeter (DSC)

Instrument model: Discovery DSC2500

Purge gas: Nitrogen

Sample pan: aluminum pan, non-sealed gland

Method: linear heating

Heating rate: 10°C/min

Temperature range: 50℃～300℃

4. Thermogravimetric Analysis (TGA)

Instrument model: Discovery TA 55

Purge gas: Nitrogen

Sample tray: Platinum, open

Method: linear heating

Heating rate: starting from the initial temperature of 35°C, raise the temperature to 100°C at a rate of 20°C per minute, maintain for 5 minutes, then increase the temperature to 200°C at a rate of 20°C per minute, maintain for 1 minute, and then increase the temperature at a rate of 10°C per minute The rate of heating up to 300°C

Temperature range: 35℃～300℃

5. Dynamic Vapor Sorption (DVS)

The detection adopts DVS Intrinsic.

The pharmaceutically acceptable salts of Compound I described in the present disclosure, including its crystalline forms, have advantages in terms of physicochemical properties, preparation processability and bioavailability, and are suitable for preparing desired pharmaceutical compositions.

The compounds of the present disclosure can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art As an equivalent alternative, preferred embodiments include but are not limited to the examples of the present disclosure.

The chemical reactions of the embodiments of the present disclosure are performed in suitable solvents that are suitable for the chemical changes of the present disclosure and the reagents and materials required therefor. In order to obtain the compounds of the present disclosure, it is sometimes necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on the existing embodiments.

Description of drawings

Figure 1 is a ball-and-stick diagram of compound 1-8 single crystal.

Fig. 2 is the XRPD spectrum of compound I hydrobromide salt C crystal form.

Figure 3 is the DSC spectrum of compound I hydrobromide salt C crystal form.

Fig. 4 is the TGA spectrum of compound I hydrobromide salt C crystal form.

Figure 5 is the XRPD pattern of Compound I 1,5-naphthalene disulfonate D crystal form.

Figure 6 is the DSC spectrum of compound I 1,5-naphthalene disulfonate D crystal form.

Figure 7 is the TGA spectrum of compound I 1,5-naphthalene disulfonate D crystal form.

Fig. 8 is the XRPD pattern of compound I-hydrochloride A crystal form.

Fig. 9 is a DSC spectrum of compound I-hydrochloride A crystal form.

Figure 10 is the TGA pattern of compound I-hydrochloride A crystal form.

Figure 11 is the XRPD pattern of compound I-hydrochloride B crystal form.

Figure 12 is the DSC spectrum of compound I-hydrochloride B crystal form.

Figure 13 is the TGA pattern of compound I-hydrochloride B crystal form.

Figure 14 is the tumor growth curve of mice receiving the test compound in the Z-138 subcutaneous tumor model, in which compound 002 is the compound of Example 2.

Figure 15 is the curve of body weight change of mice receiving the test compound in the Z-138 subcutaneous tumor model, in which compound 002 is the compound of Example 2.

Detailed ways

The present disclosure will be described in detail through examples below, but it does not imply any adverse limitation to the present disclosure. The present disclosure has been described in detail herein, and its specific embodiments are also disclosed. For those skilled in the art, various changes and modifications can be made to the specific embodiments of the present disclosure without departing from the spirit and scope of the present disclosure. Improvements will be apparent. All reagents used in this disclosure are commercially available and used without further purification.

Unless otherwise specified, the ratios indicated for mixed solvents are volume mixing ratios. Unless otherwise stated, % means wt%.

Compounds were named manually, and commercially available compounds used supplier catalog names.

Compound structures were determined by nuclear magnetic resonance (NMR) and/or mass spectroscopy (MS). The unit of NMR shift is 10 ^-6 (ppm). The solvents measured by NMR are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is _{tetramethylsilane} (TMS); concentration.

The following eluents can be mixed eluents formed by two or more solvents, and the ratio is the volume ratio of each solvent. For example, "0-10% methanol/dichloromethane" means that during the gradient elution process, the volume ratio of methanol and dichloromethane in the mixed eluent is 0:100-10:100.

Example 1: (S)-3-((S)-oxiran-2-yl)-3,4-dihydroisoquinoline-2(1H)-tert-butyl carboxylate (Intermediate 1) preparation of

Step 1: Preparation of compound b

(S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (a) (55g, 200mmol), dimethylhydroxylamine hydrochloride ( 29.4g, 300mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (91g, 240mmol) was added in a 1L single-necked bottle, Anhydrous N,N-dimethylformamide (500 mL) was added again, cooled in an ice bath after nitrogen protection, and then N,N-diisopropylethylamine (104 mL, 600 mmol) was added dropwise. The reaction solution was reacted at room temperature for 4 hours. After the reaction was complete, the excess N,N-diisopropylethylamine and N,N-dimethylformamide were removed by rotary evaporation, then cooled in an ice bath, diluted with saturated brine (1L), extracted with ethyl acetate (200mL X 2), the combined organic phases were washed with 5% aqueous sodium carbonate solution (500mL X 2), and then washed with saturated brine (500mL). Dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure and purify by silica gel column chromatography (eluent gradient: petroleum ether/ethyl acetate=2/1) to obtain the target intermediate (S)-3-(methoxy (Methyl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylic acid tert-butyl ester (b) (62 g, yield: 97%).

LCMS: Rt: 1.76 min; MS m/z (ESI): 321.3 [M+H] ⁺ .

Chiral HPLC: Rt: 3.159 min.

Step 2: Preparation of compound c

(S)-3-(methoxy(methyl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylic acid tert-butyl ester (b) (20g, 62.5mmol ) was weighed into a 500mL three-necked flask, anhydrous tetrahydrofuran (200mL) was added, cooled to -70°C, diisobutylaluminum hydride (DIBAL-H) toluene solution (1.5mol/L, 83mL, 125mmol) was slowly added dropwise, The reaction was stirred at -70°C for 1 hour. After the reaction was complete, slowly add saturated ammonium chloride solution (100 mL) at -70°C to quench, and then add 0.5 mol/L hydrochloric acid aqueous solution to dilute (200 mL). After layering, the organic phase was washed with saturated aqueous sodium chloride solution (200mL × 2), then dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (eluent gradient: sherwood oil/acetic acid Ethyl ester=8/1) purification to obtain intermediate (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-tert-butyl carboxylate (c) (15g, yield: 92%).

LCMS: Rt: 1.93 min; MS m/z (ESI): 206.1 [M-56+H] ⁺ .

Chiral HPLC: Rt: 2.018min.

Step 3: Preparation of compound d

Disperse methyltriphenylphosphine bromide (238g, 0.67mol) in anhydrous tetrahydrofuran (1.5L), cool to -70°C under nitrogen protection, slowly add sodium bis(trimethylsilyl)amide (334mL , 0.67mol), the temperature was controlled below -50°C, and after the dropwise addition was completed, it was slowly raised to room temperature and stirred for 2 hours. Re-cool to -70°C, slowly add (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-tert-butyl carboxylate (c) (87g, 0.33moL) in THF solution (200 mL), the temperature was controlled below -50°C, and after the dropwise addition was completed, it was slowly raised to room temperature overnight. After the reaction was detected by TLC, it was cooled to 0° C., quenched by adding saturated ammonium chloride solution, slowly added 1 mol/L hydrochloric acid aqueous solution to adjust the pH to 3-4, and extracted by adding ethyl acetate (500 mL). The organic phase was washed with saturated brine (200mL X 2), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was recrystallized by adding a mixed solvent (ethyl acetate/petroleum ether=1/4), and the precipitated triphenylphosphine oxide was removed by filtration, and the filtrate was concentrated and then chromatographed on a silica gel column (ethyl acetate/petroleum ether=1/8) to obtain Target product (S)-tert-butyl 3-vinyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (d) (85 g, yield: 98%).

Chiral HPLC: Rt: 1.883 min.

Step 4: Preparation of compound e

Dissolve (S)-tert-butyl 3-vinyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (d) (25.9 g, 0.1 mol) in ethyl acetate/acetonitrile (500 mL/ 500mL) solution, cooled to 0°C, added sodium periodate (32.1g, 0.15mol) and ruthenium trichloride hydrate (1.6g, 7.7mmol) aqueous solution within 10 minutes, the reaction solution was stirred at 0°C for 10 minute. TLC detects that the reaction is complete, adding saturated sodium thiosulfate solution (150mL) to quench, stirring for 30 minutes and then layering, the organic phase is washed with saturated brine (200mL × 2), dried over anhydrous sodium sulfate, and the filtrate is concentrated under reduced pressure after filtration , the residue was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether=1/2-1/1) to obtain the product (S)-3-((S)-1,2-dihydroxyethyl tert-butyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (e) (less polar product, 14 g, yield 48%). Increase the polarity of the eluent (ethyl acetate/petroleum ether=2/1-1/0) to obtain the product (S)-3-((R)-1,2-dihydroxyethyl)-3,4 -dihydroisoquinoline-2(1H)-tert-butyl carboxylate (e-1) (more polar product, 8 g, yield 28%).

Step 5: Preparation of compound f

(S)-3-((S)-1,2-dihydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-tert-butyl carboxylate (e) (14g, 0.047mol ) was dissolved in dichloromethane (150mL), triethylamine (9.90mL, 0.072mol) was added, and p-toluenesulfonyl chloride (10.0g, 0.052mol) was added in batches under stirring, and the reaction solution was stirred overnight at 40°C . The reaction solution was cooled to room temperature, washed with saturated brine (100mL × 2), and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (ethyl acetate/petroleum ether=1/4) to obtain the target product (S)-3-((S)-1-hydroxy-2-(toluenesulfonyl Oxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylic acid tert-butyl ester (f) (12.6 g, yield 59%).

Step 6: Preparation of Compound Intermediate 1

(S)-3-((S)-1-hydroxyl-2-(tosyloxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylic acid tert-butyl ester ( f) Dissolve (12.6g, 28.2mmol) in N,N-dimethylformamide (150mL), add sodium hydride (1.70g, 42.3mmol) in batches under nitrogen protection, and stir the reaction solution at 40°C for 1 hour , TLC detected that the reaction was complete. Cooled to 0°C, quenched by adding saturated brine dropwise, the reaction solution was directly purified by reverse-phase silica gel column (water/acetonitrile=50/10), the solution obtained by column chromatography was extracted with ethyl acetate (200mL X 3), and the organic phase was After drying over anhydrous sodium sulfate and filtering, the filtrate was concentrated under reduced pressure to obtain the intermediate (S)-3-((S)-oxirane-2-yl)-3,4-dihydroisoquinoline-2(1H )-tert-butylcarboxylate (Intermediate 1) (6.5 g, yield 72%).

Example 2: 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8 -(2-Methoxy-7-azaspiro[3.5]nonane-7-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepine

Preparation of -5-ketone (compound I) hydrochloride

Step 1: Preparation of Compound 1-2

4-Bromo-2-fluorobenzoic acid (1-1) (7.5g, 34.2mmol) and N-tert-butoxycarbonyl-1,2-ethylenediamine (16.4g, 102.7mmol) were added to N- Methylpyrrolidone (30mL). After the addition was complete, the temperature was raised to 120° C. for 16 hours of reaction. After the reaction was complete, the reaction solution was slowly added to water (150mL), the pH of the solution was adjusted to 5-6 with 2mol/L dilute hydrochloric acid, and then extracted with ethyl acetate (150mL×2). Combine the organic phases, wash with saturated brine (150mL×2), dry over anhydrous sodium sulfate, and filter. The filtrate was concentrated under reduced pressure to obtain the target intermediate 4-bromo-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)benzoic acid (1-2) (crude product, 11.0g, yield: 89 %).

LCMS: Rt: 1.836 min; MS m/z (ESI): 359.0 [M+H] ⁺ .

Step 2: Preparation of Compound 1-3

Add 4-bromo-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)benzoic acid (1-2) (11.0g, 30.6mmol) to 4mol/L hydrochloric acid/di Hexane solution (30 mL), and react at room temperature for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain the target intermediate 2-((2-aminoethyl)amino)-4-bromobenzoic acid (1-3) (crude product, 8 g, yield: 100%).

LCMS: Rt: 0.688 min; MS m/z (ESI): 259.0 [M+H] ⁺ .

Step 3: Preparation of Compounds 1-4

2-((2-Aminoethyl)amino)-4-bromobenzoic acid (1-3) (3g, 11.6mmol), 2-(7-azabenzotriazole)-N,N , N',N'-Tetramethyluronium hexafluorophosphate (8.8 g, 23.2 mmol) and triethylamine (4.7 g, 46.4 mmol) were added to ultra-dry N,N-dimethylformamide (20 mL) , react at room temperature for 1.5 hours. After the reaction was complete, saturated brine (100 mL) was added, extracted with ethyl acetate (100 mL X 2), the organic phase was washed with saturated brine (50 mL X 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by normal phase silica gel chromatography (eluent gradient: dichloromethane/methanol=20/1) to obtain the target intermediate 8-bromo-1,2,3,4-tetrahydro-5H-benzo[ e][1,4]diazepine

-5-Kone (1-4) (1.6 g, yield: 57%).

LCMS: Rt: 1.173 min; MS m/z (ESI): 241.0 [M+H] ⁺ .

Step 4: Preparation of Compounds 1-5

8-Bromo-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepine

-5-ketone (1-4) (1.6g, 6.6mmol), sodium cyanoborohydride (834mg, 13.3mmol) and acetaldehyde (584mg, 13.3mmol) were added to a mixed solution of acetic acid (2mL) and methanol (20mL) , react at room temperature for 1 hour. After the reaction was complete, 2mol/L dilute hydrochloric acid (1 mL) and water (50.0 mL) were added, and extracted with ethyl acetate (50.0 mL X 2). The organic phases were combined, washed with saturated brine (50 mL X 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by normal phase silica gel chromatography (eluent gradient: petroleum ether/ethyl acetate=1/2) to obtain the target intermediate 8-bromo-1-ethyl-1,2,3,4-tetrahydro -5H-Benzo[e][1,4]diazepine

-5-Kone (1-5) (1.1 g, yield: 62%).

LCMS: Rt: 1.499 min; MS m/z (ESI): 269.0 [M+H] ⁺ .

Step 5: Preparation of Compounds 1-6

8-Bromo-1-ethyl-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepine

-5-Kone(1-5) (400 mg, 1.52 mmol) was added to ultra-dry N,N-dimethylformamide (20 mL), followed by sodium hydride (90.9 mg, 2.27 mmol). After the addition was completed, the mixture was heated to 40° C. and stirred for 1 h, then the intermediate 1 (500 mg, 1.82 mmol) prepared in Example 1 was added, and reacted for 16 hours. After the reaction was complete, water (100 mL) was added and extracted with ethyl acetate (100 mL X 2). The organic phases were combined, washed with saturated brine (50 mL X 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by normal phase silica gel chromatography (eluent gradient: dichloromethane/methanol=20/1) to obtain the target intermediate (1R,10aS)-1-((8-bromo-1-ethyl-5 -Oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepine

-4-yl)methyl)-1,5,10,10a-tetrahydro-3H-oxazolo[3,4-b]isoquinolin-3-one (1-6) (150mg, yield: twenty one%).

LCMS: Rt: 1.872 min; MS m/z (ESI): 470.1 [M+H] ⁺ .

Step 6: Preparation of Compounds 1-7

The intermediate (1R,10aS)-1-((8-bromo-1-ethyl-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1, 4] diazepine

-4-yl)methyl)-1,5,10,10a-tetrahydro-3H-oxazolo[3,4-b]isoquinolin-3-one (1-6) (150mg, 0.32mmol) , [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (11.7mg, 0.02mmol) and potassium acetate (94mg, 0.96mmol) were added to absolute ethanol (10mL) and replaced by CO 3 times, heated to 70°C for 3.0 hours. After the reaction was complete, it was cooled to room temperature, the reaction solution was concentrated under reduced pressure, saturated brine (50 mL) was added, and then extracted with ethyl acetate (50 mL X 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by normal phase silica gel chromatography (eluent gradient: petroleum ether/ethyl acetate=1/2) to obtain the target intermediate 1-ethyl-5-oxo-4-(((1R,10aS) -3-Carbonyl-1,5,10,10a-tetrahydro-3H-oxazolo[3,4-b]isoquinolin-1-yl)methyl)-2,3,4,5-tetrahydro -1H-Benzo[e][1,4]diazepine

-Ethyl 8-carboxylate (1-7) (130 mg, yield: 87%).

LCMS: Rt: 1.784 min; MS m/z (ESI): 464.1 [M+H] ⁺ .

Step 7: Preparation of Compounds 1-8

1-Ethyl-5-oxo-4-(((1R,10aS)-3-carbonyl-1,5,10,10a-tetrahydro-3H-oxazolo[3,4-b] Isoquinolin-1-yl)methyl)-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine

-Ethyl 8-carboxylate (1-7) (130mg, 0.28mmol) was added to a mixed solution of methanol (4mL) and water (4mL), then sodium hydroxide (90mg, 2.24mmol) was added and heated to 70°C , reacted for 16.0 hours. After the reaction was complete, the reaction system was cooled to room temperature, and Boc anhydride (122 mg, 0.56 mmol) was added to react for 1.5 hours. After the reaction was complete, the reaction system was cooled to 0°C, and the pH of the reaction solution was adjusted to 5.0 with 1mol/L hydrochloric acid aqueous solution, and then extracted with ethyl acetate (30mL X 3), the organic phase was dried with anhydrous sodium sulfate, filtered, The filtrate was concentrated under reduced pressure. The residue was subjected to reverse-phase silica gel chromatography (eluent gradient: acetonitrile/water=43%) to obtain the target intermediate 4-((R)-2-((S)-2-(tert-butoxycarbonyl)-1 ,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyl)-1-ethyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo [e][1,4]diazepine

-8-Carboxylic acid (1-8) (crude product, 60 mg, yield: 42%).

LCMS: Rt: 1.722 min; MS m/z (ESI): 510.3 [M+H] ⁺ .

Single crystal X-ray structure determination and single crystal X-ray analysis of compounds 1-8:

Single crystal preparation method: Weigh compound 1-8 (10.0 mg) into a 3 mL screw-top glass bottle, add 2 mL of methanol, stir for 5 minutes, and then the solid dissolves. Add 0.5 mL of water to the glass bottle and continue stirring for 5 minutes. The solution was filtered through a 0.22 μm microporous membrane into a 3 mL screw-top glass bottle, and the mouth of the glass bottle was covered with plastic wrap. Prick 8 small holes at the mouth of the bottle with a needle and leave it at room temperature for 7 days to obtain a single crystal of the above compound.

The obtained single crystal sample was subjected to X-ray analysis, and the test results are shown in Table 1 and Fig. 1 .

Table 1 Single crystal samples and crystal data of compounds 1-8

Through the above-mentioned X-ray crystal diffraction experiment, the chemical structure and absolute configuration of compound 1-8 can be determined as follows:

Step 8: Preparation of Compound 2-2

4-((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyl base)-1-ethyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine

-8-carboxylic acid (1-8) (100mg, 0.196mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate ( 231mg, 0.608mmol) and N,N-diisopropylethylamine (157mg, 1.216mmol) were added to ultra-dry N,N-dimethylformamide (2.5mL), then 2-methoxy-7 -Azaspiro[3.5]nonane hydrochloride (53 mg, 0.275 mmol), stirred at room temperature for 1.0 hour. After the reaction was complete, water (30 mL) was added, then extracted with ethyl acetate (30 mL X 3), the combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by normal phase silica gel chromatography (eluent: dichloromethane/methanol=20/1) to obtain the target intermediate (S)-3-((R)-2-(1-ethyl-8- (2-Methoxy-7-azaspiro[3.5]nonane-7-carbonyl)-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4 ] diazepine

-4-yl)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-tert-butyl carboxylate (2-2) (110 mg, yield: 86.7%).

LCMS: Rt: 2.062min; MS m/z (ESI): 647.3[M+H] ⁺ .

Step 9: Preparation of compound I hydrochloride

(S)-3-((R)-2-(1-ethyl-8-(2-methoxy-7-azaspiro[3.5]nonane-7-carbonyl)-5-oxo Generation-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepine

-4-yl)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-tert-butyl carboxylate (2-2) (110mg, 0.170mmol) was added to 4mol/L hydrochloric acid /dioxane (2.5mL) solution. The reaction was stirred at room temperature for 1.0 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was subjected to high performance liquid chromatography (eluent gradient:

) purification to obtain the target compound 1-ethyl-4-((R)-2-hydroxyl-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl) ethyl) -8-(2-Methoxy-7-azaspiro[3.5]nonane-7-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]di Aza

-5-Kone (compound I) hydrochloride (39.81 mg, yield: 42.8%).

¹ H NMR (400MHz, CD ₃ OD) δ7.64(d, J=7.6Hz, 1H), 7.35-7.22(m, 4H), 7.12(d, J=8Hz, 2H), 4.51-4.36(m, 2H),4.33-4.30(m,1H),4.04-3.89(m,2H),3.73-3.61(m,7H),3.51-3.50(m,1H),3.38-3.34(m,4H),3.31- 3.30(m,2H),3.22(s,3H),2.29-2.23(m,2H),1.71-1.68(m,4H),1.55(s,2H),1.17(t,J＝7.0Hz,3H) .

LCMS: Rt: 1.355min; MS m/z (ESI): 547.4[M+H] ⁺ .

Determination of the hydrochloric acid content in the obtained compound I hydrochloride by conductometric method, experimental apparatus, reagent, method and result are as follows.

Experimental instruments and reagents: high performance liquid chromatography (Shimadzu LC-20AD), conductivity detector (CDD-10Avp), electronic balance (Sartorius, MAS125P-1CE-DU), sodium chloride, p-hydroxybenzoic acid, Bis- Tris (HPLC grade, Aladdin A1910012), boric acid, ultrapure water;

Chromatographic information: Chromatographic column—Shim-pack IC-A3; guard column—Shim-pack IC-GA3; eluent—8 mmol/L p-hydroxybenzoic acid, 3.2mmol/L Bis-Tris, 50mmol/L Boric acid solution; flow rate - 1.5mL/min; column temperature - 40°C; injection volume - 50μL; conductivity detector mode - positive ion mode;

Solution preparation:

Reference solution (containing 6 μg/mL of chloride ion): Accurately measure 50 mg of sodium chloride into a 50 mL measuring bottle, add water to dissolve and dilute to the mark, shake well, precisely pipette 1.0 mL into a 100 mL measuring bottle, add water to dilute to the mark , shake well.

Test solution (0.1mg/mL): Accurately weigh 10mg of the sample to be tested into a 100mL measuring bottle, dissolve and dilute to the mark with water, and shake well.

Determination method: Accurately measure the reference substance solution and the test solution respectively, inject them into a high-performance liquid chromatograph, use the eluent as the eluent to elute, and record the chromatogram.

Experimental results: As a result, it was measured that the HCl content in the compound I hydrochloride was 10.57% (the theoretical value of the HCl content of the compound I monohydrochloride was 6.25%).

Example 3: 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8 -(2-Methoxy-7-azaspiro[3.5]nonane-7-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepine

Preparation of -5-ketone (compound I)

At room temperature, add 3.0 g of compound I hydrochloride prepared according to the method of Example 2 into a 100 mL single-necked bottle, add 12 mL of dichloromethane, add 12 mL of purified water after dissolving, and add 20% sodium hydroxide solution during stirring to adjust the pH to The pH of the aqueous phase is about 10, separate the liquids, extract the aqueous phase three times with 3ml dichloromethane, combine the organic phases, backwash the organic phase once with 10mL purified water, dry the organic phase with anhydrous sodium sulfate, and concentrate to dryness under reduced pressure , to obtain 2.5g foamy solid, that is, compound I.

¹ H NMR (400MHz,DMSO-d ₆ )δ7.49-7.41(m,1H),7.17-7.04(m,3H),7.01(d,J=5.5Hz,1H),6.90-6.79(m,2H ),5.01(d,J=5.5Hz,1H),4.03(q,J=7.1Hz,1H),3.98-3.79(m,4H),3.75(s,1H),3.63-3.35(m,6H) ,3.32-3.19(m,3H),3.18-3.12(m,2H),3.10(s,3H),2.84-2.65(m,3H),2.15(s,2H),1.70-1.39(m,6H) ,1.07(t,J=7.0Hz,3H).

Embodiment 4: Preparation of compound I hydrobromide C crystal form

Add the compound I (350mg) prepared in Example 3 into a 50mL single-necked bottle at room temperature, add isopropanol (3.5mL), stir to dissolve, then add 107.9mg of 40% hydrobromic acid aqueous solution dropwise, and stir until a large amount of solids are precipitated , the solid was suction-filtered, the filter cake was washed with 1 mL of isopropanol, and dried under vacuum at 25°C to obtain a solid (230 mg, HPLC purity: 99.19%).

¹ H NMR (400MHz, DMSO-d ₆ )δ9.21(s, 1H), 9.00(s, 1H), 7.46(d, J=7.7Hz, 1H), 7.35-7.16(m, 4H), 6.93- 6.81(m,2H),5.90(d,J＝6.0Hz,1H),4.44-4.15(m,4H),3.99-3.84(m,2H),3.52(s,5H),3.43-3.35(m, 2H),3.22-3.13(m,4H),3.10(s,3H),2.15(s,2H),1.67-1.39(m,6H),1.05(dd,J＝13.8,6.6Hz,5H).

The XRPD spectrum of the crystalline sample is shown in Figure 2, and the XRPD diffraction peak positions are shown in Table 2 below; the DSC spectrum is shown in Figure 3, and the peak is around 238.84°C; the TGA spectrum is shown in Figure 4.

Table 2

Example 5: Preparation of Compound I 1,5-Naphthalene Disulfonate Form D

Add the compound I (350 mg) prepared in Example 3 into a 50 mL single-necked bottle at room temperature, add isopropanol (3.5 mL), stir to dissolve, and dissolve 1,5-naphthalene disulfonic acid (184.8 mg) in 2.1 mL Purified water was then dropped into a one-necked bottle, stirred overnight, and the precipitated solid was suction-filtered, the filter cake was rinsed with 1 mL of isopropanol, and vacuum-dried at 25°C to obtain a solid (390 mg, HPLC purity: 99.15%).

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.22(d, J=9.9Hz, 1H), 9.00(d, J=8.8Hz, 1H), 8.86(d, J=8.8Hz, 2H), 7.93 (dd,J=7.0,1.0Hz,2H),7.52-7.37(m,3H),7.33-7.19(m,4H),6.92-6.80(m,2H),4.44-4.27(m,4H),4.04 -3.70(m,3H),3.62-3.42(m,5H),3.43-3.20(m,4H),3.14(d,J＝8.9Hz,4H),3.10(s,3H),2.15(s,2H ),1.68-1.39(m,6H),1.05(t,J＝7.0Hz,3H).

The XRPD of this crystalline sample is shown in Figure 5, and its XRPD diffraction peak positions are shown in Table 3 below; its DSC spectrum is shown in Figure 6, which has a peak around 231.85°C; its TGA spectrum is shown in Figure 7.

table 3

Embodiment 6: Preparation of compound I-hydrochloride A crystal form

Add the compound I (1.95g) prepared in Example 3 into a 100mL single-necked bottle at room temperature, add methyl acetate (30mL), stir to dissolve and place it in an ice-water bath, slowly add 1.78mL 2mol/L hydrogen chloride-acetic acid dropwise The ethyl ester solution was stirred until a large amount of solids were precipitated, and then suction-filtered, the filter cake was rinsed with 5 mL of methyl acetate, and vacuum-dried at 25°C to obtain a solid (1.87 g, HPLC purity: 98.98%).

¹ H NMR (400MHz, DMSO-d ₆ )δ9.73(s, 1H), 9.04(s, 1H), 7.45(d, J=7.7Hz, 1H), 7.26(dd, J=7.7, 4.4Hz, 4H),6.91-6.81(m,2H),5.94(d,J=5.7Hz,1H),4.28(d,J=16.0Hz,3H),3.99(dd,J=13.8,6.1Hz,1H), 3.84(s,1H),3.48(d,J=30.3Hz,5H),3.39(dd,J=13.4,7.0Hz,2H),3.32-3.23(m,2H),3.22-3.12(m,5H) ,3.10(s,3H),2.15(s,2H),1.67-1.40(m,6H),1.04(t,J=7.0Hz,3H).

The content of hydrochloric acid in crystal form A was determined by potentiometric titration, and it was determined that the molar ratio of compound I to HCl in the salt was about 1:1.

The experimental instruments, reagents and methods for determining the content of hydrochloric acid by potentiometric titration are as follows.

Experimental instruments and reagents: potentiometric titrator (Metrohm, model 888), electronic balance (Mettler Toledo, model XS205DU); methanol (chromatographic grade, diluted to 60% methanol as solvent and blank), 0.1mol/L sodium hydroxide titration solution (Nanjing Chemical Reagent Co., Ltd., batch number 210926428W);

Titration parameters:

Titration process:

Blank titration: Measure 50 mL of solvent (60% methanol) into a 100 mL beaker, and titrate to the end point with sodium hydroxide titration solution (0.1 mol/L).

Titration of the sample to be tested: Accurately weigh 300 mg of the sample to be tested, place it in a 100 mL beaker, add 50 mL of solvent (60% methanol), and titrate to the end point with sodium hydroxide titration solution (0.1 mol/L).

The hydrochloric acid content is calculated by the following formula:

Remark:

V ₀ : blank consumes the volume of sodium hydroxide titration solution, unit (mL);

V: the volume of sodium hydroxide titration solution consumed by the sample to be tested, unit (mL);

C: concentration of sodium hydroxide titration solution, unit (mol/L);

m: the weighing amount of the sample to be tested, unit (mg);

3.646: titer of sodium hydroxide titration solution, mg/mL.

The XRPD of the crystalline sample is shown in Figure 8, and the XRPD diffraction peak positions are shown in Table 4 below; the DSC spectrum is shown in Figure 9, and the peak is around 259.27°C; the TGA spectrum is shown in Figure 10.

Table 4

Embodiment 7: Preparation of compound I-hydrochloride A crystal form

Add the compound I (500mg) prepared in Example 3 into a 50mL single-necked bottle at room temperature, add ethyl acetate (5mL), stir to dissolve, place it in an ice-water bath, and slowly add 445 μL 2mol/L hydrogen chloride-ethyl acetate dropwise The solution was stirred until a large amount of solids were precipitated, and then suction-filtered, the filter cake was rinsed with 1 mL of ethyl acetate, and vacuum-dried at 25°C to obtain a solid (429 mg).

Embodiment 8: Preparation of compound I-hydrochloride A crystal form

Add the compound I (250mg) prepared in Example 3 into a 50mL single-necked bottle at room temperature, add acetone (2.5mL), stir to dissolve, place in an ice-water bath, and slowly add 220 μL of 2mol/L hydrogen chloride-ethyl acetate solution dropwise , stirred until a large amount of solids were precipitated, then filtered with suction, rinsed the filter cake with 1 mL of acetone, and dried in vacuo at 25°C to obtain a solid (166 mg).

Embodiment 9: Preparation of compound I-hydrochloride A crystal form

Add the compound I (4.31g) prepared in Example 3 into a 500mL single-necked bottle at room temperature, add isopropanol (43mL), stir to dissolve and place it in an ice-water bath, slowly add 3.9mL 2mol/L hydrogen chloride-acetic acid dropwise Ethyl ester solution, stirred until a large amount of solids were precipitated, then filtered with suction, rinsed the filter cake with 5 mL of isopropanol, and dried under vacuum at 25°C to obtain a solid (3.74 g).

Embodiment 10: Preparation of compound I-hydrochloride amorphous

Add the compound I-hydrochloride A crystal form (300mg) prepared in Example 6 into methanol (10mL) at room temperature, stir to dissolve, and concentrate under reduced pressure to obtain a foamy solid, which is detected as compound I-hydrochloride by XRPD Amorphous.

Example 11: Preparation of compound I-hydrochloride A crystal form

Add compound I-amorphous hydrochloride (10mg) prepared in Example 10 into a 5mL centrifuge tube at room temperature, add methyl acetate (100μL) to form a suspension, beat at room temperature overnight, and filter the solid with suction. Wash with 1 mL of methyl acetate and dry under vacuum at 25°C to give a solid (8 mg). It was detected as crystal form A by XRPD.

Example 12: Preparation of compound I-hydrochloride A crystal form

At room temperature, add the compound I-hydrochloride amorphous (20 mg) prepared in Example 10 into a 4.5 mL centrifuge tube, add methanol (200 μL), dissolve and add acetone dropwise until solid precipitates, stir for several hours The precipitated solid was suction filtered, the filter cake was washed with 1 mL of acetone, and dried under vacuum at 25°C to obtain a solid (12 mg).

Example 13: Preparation of compound I-hydrochloride B crystal form

At room temperature, add Compound I-amorphous hydrochloride (20 mg) prepared in Example 10 into a 4.5 mL centrifuge tube, add 99% aqueous methanol (200 μL), dissolve and add methyl acetate (1.2 mL) dropwise A solid was precipitated, and after stirring for several hours, the precipitated solid was suction-filtered, the filter cake was washed with 1 mL of methyl acetate, and dried in vacuo at 25°C to obtain a solid (14 mg).

The XRPD of the crystalline sample is shown in Figure 11, and its XRPD diffraction peak positions are shown in Table 5 below; its DSC spectrum is shown in Figure 12; its TGA spectrum is shown in Figure 13.

table 5

Test Test Example 1: PRMT5 Enzymatic Activity Inhibition Experiment

Materials: PRMT5/MEP50 protein was purchased from BPS bioscience (USA); histone H4 peptide (Histone H4 Peptide) substrate was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; Anti-Histone H4 (symmetric dimethyl R3) antibody-ChIP Grade was purchased from Abcam (US); S-(5'-adenosyl)-L-methionine chloride dihydrochloride was purchased from Sigma (US); 384-well plate, AlphaScreen streptavidin AlphaScreen Streptavidin Donor beads, AlphaScreen Protein A Acceptor beads and Envision 2104 multi-label Reader were purchased from PerkinElmer Instruments Co., Ltd. (USA) Echo 550 pipette (Echo 550 Liquid Handler) was purchased from Labcyte (U.S.).

Detection of enzymatic activity: the compound was injected into a 384-well plate by Echo, so that the final concentration was 0-1000 nM (initial concentration 1000 nM, 3-fold dilution, 10 points), and the DMSO content was 0.5%. Add 10 μL 2X PRMT5/MEP50 solution to each well and incubate at room temperature for 30 minutes. Add 10 μL 2X PRMT5/MEP50 substrate solution to each well to start the reaction, and incubate at room temperature for 60 minutes. Prepare 6X detection reagent containing AlphaScreen Protein A Acceptor beads and Anti-Histone H4 (symmetric dimethyl R3) antibody, add 5 μL to each well, and incubate at room temperature for 60 minutes. Prepare 6X detection reagent containing AlphaScreen Streptavidin Donor beads, add 5 μL to each well, and incubate at room temperature for 60 minutes. Envision detection signal value. The test results are shown in Table 6.

Test Test Example 2: Experiment of Inhibitory Activity of Compounds on Tumor Cell Proliferation

Materials and cells: Z-138 cells were purchased from ATCC (US); IMDM medium and penicillin-streptomycin were purchased from Sigma (US); horse serum was purchased from Hyclone (US); 96-well plates were purchased from Corning ( U.S.); Cell-Titer Glo reagent was purchased from Promega (U.S.).

Cell culture: Z-138 cells were cultured in IMDM medium containing 10% horse serum + 1% penicillin-streptomycin at 37°C and 5% CO ₂ . Cells in the logarithmic growth phase can be used for experiments.

Detection of cell proliferation activity: Cell-Titer Glo reagent was used to detect the proliferation inhibitory activity of the compound on Z-138 cells. Adjust the cell concentration, inoculate 96-well plates with 180 μL per well (500/well), and place them at 37° C. and 5% CO ₂ to equilibrate for 10-15 minutes. 20 μL of compound-containing culture solution was added to each well to make the final concentration 0-300 nM (initial concentration 300 nM, 3-fold dilution, 10 points), and the DMSO content was 0.1%. Cell plates were incubated at 37°C, 5% CO ₂ for 8 days. The medium was changed on the fourth day: 100 μL of the supernatant was slowly aspirated, and 100 μL of fresh culture medium containing the compound was added to keep the concentration of the compound unchanged. Cell viability was detected by Cell-Titer Glo reagent. The test results are shown in Table 6.

Test Test Example 3: Compound's inhibitory activity experiment on SDMA

Materials and cells: Z-138 cells were purchased from ATCC (US); IMDM medium and penicillin-streptomycin were purchased from Sigma (US); horse serum was purchased from Hyclone (US); Hoechst antibody was purchased from Invitrogen (US) ); Alexa Fluor 488 goat anti-rabbit IgG antibody was purchased from Invitrogen (U.S.); Anti-dimethyl-Arginine symmetric (SYM11) antibody was purchased from Merck (U.S.); DPBS was purchased from Gibco (U.S.); From Cell signaling technology company (USA); paraformaldehyde was purchased from Beijing Suo Laibao Technology Co., Ltd.; 384-well plate and Echo 550 Liquid Handler were purchased from Labcyte Company (USA); ImageXpress Nano was purchased from Molecular Devices company (USA).

Cell culture: Z-138 cells were cultured in IMDM medium containing 10% horse serum + 1% penicillin-streptomycin at 37°C and 5% CO ₂ . Cells in the logarithmic growth phase can be used for experiments.

Immunofluorescence detection: Immunofluorescence was used to detect the effect of compounds on SDMA in Z-138 cells. The cell concentration was adjusted to 1*10 ⁵ /mL, 40 μL per well was inoculated into a 384-well plate (4000/well), and placed at 37° C., 5% CO ₂ to equilibrate for 10-15 minutes. The compound was injected into a 384-well plate by Echo, so that the final concentration was 0-300 nM (initial concentration 300 nM, 3-fold dilution, 10 points), and the DMSO content was 0.1%. Cell plates were incubated at 37°C, 5% CO ₂ for 2 days. Add 40 μL of 8% paraformaldehyde to each well and incubate at room temperature for 30 minutes. Discard the supernatant, wash the plate with DPBS, add 40 μL of 0.5% PBST (phosphate Tween buffer) to each well, and incubate at room temperature for 60 minutes. Discard the supernatant, wash the plate with 0.05% PBST, add 40 μL of blocking solution (1% Nonfat milk in 0.05% PBST) to each well, and incubate at room temperature for 60 minutes. Discard the supernatant, add 20 μL primary antibody (SYM11, 1:500 dilution in blocking solution) to each well, and leave overnight at 4°C. Discard the supernatant, wash the plate with 0.05% PBST, add 20 μL of secondary antibody (diluted in Alexa Fluor 488goat anti-rabbit 1:1000 and Hoechst 1:5000 in blocking solution) to each well, and incubate at room temperature for 60 minutes. Discard the supernatant, wash the plate with 0.05% PBST, and detect the fluorescence intensity with ImageXpress Nano. The test results are shown in Table 6.

Table 6

Test Example 4: Pharmacokinetic experiment in mice

Experimental materials: CB17-SCID mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.; DMSO, HP-β-CD (hydroxypropyl-β-cyclodextrin), MC (methylcellulose), and acetonitrile were purchased from From Merck (USA), K ₂ EDTA anticoagulant tube was purchased from Jiangsu Xinkang Medical Instrument Co., Ltd.

Experimental method: 6 female CB17-SCID mice (20-30g, 4-6 weeks) were randomly divided into 2 groups, 3 mice in each group. Group 1 was given the compound by tail vein injection, the dose was 2mg/kg, the vehicle was 5% DMSO+95% 10% HP-β-CD aqueous solution, the second group was orally administered the compound, the dose was 10mg/kg, the vehicle was 0.5% MC aqueous solution. Animals were fed and watered normally before the experiment. Venous blood was collected from mice in each group before administration and at 0.083 (intravenous injection group only), 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. The collected whole blood samples were placed in K ₂ EDTA anticoagulant tubes, centrifuged for 5 minutes (4000 rpm, 4° C.) and the plasma was collected for testing.

Take 10 μL of mouse plasma sample, add 150 μL of acetonitrile solvent (which contains internal standard compound) to precipitate protein, vortex for 0.5 min, centrifuge (4700 rpm, 4 ° C) for 15 min, and supernatant with 0.05% (v/v) formic acid Dilute 2 times with water, inject 3 μL into LC-MS/MS system (AB Sciex Triple Quad 6500+) for quantitative detection. When determining the sample concentration, the CB17-SCID mouse plasma standard curve (linear range: 0.5-1000ng/mL) and quality control samples were followed. For the preparation of 10-fold diluted samples, take 2 μL of mouse plasma samples and add 18 μL of blank plasma, vortex for 0.5 min, add 300 μL of acetonitrile solvent (including internal standard compounds) to precipitate proteins, and the rest of the processing steps are the same as for undiluted samples.

The pharmacokinetic test results are shown in Table 7.

Table 7 Mouse pharmacokinetic test results

Test Example 5: Hepatocyte Metabolic Stability Test

Experimental materials: human hepatocytes were purchased from Biopredic Company; mouse hepatocytes were purchased from BioIVT Company; acetonitrile and methanol were purchased from Merck Company; AOPI stain was purchased from Nexcelom Company; dexamethasone was purchased from NIFDC Company; Bao Technology Co., Ltd.; DPBS (10x), GlutaMAX ^TM -1 (100x) and human recombinant insulin were purchased from Gibco by Life Technologies; fetal bovine serum was purchased from Corning; formic acid was purchased from DIKMAPURE; Isotonic Percoll was purchased from GE Healthcare; Alprazolam was purchased from Supelco; caffeine was purchased from ChromaDex.inc; HEPES, tolbutamide and Williams' Medium E were purchased from Sigma.

Experiment preparation:

Prepare a high-concentration stock solution from the powder of the test substance in DMSO, and dilute to a working solution of 100 μM with acetonitrile before use, and the final concentration of the test substance is 1 μM.

The specific preparation information of the liver cell resuscitation solution is shown in Table 8 below. Mix 49.5mL Williams'E Medium and 0.5mL GlutaMAX as incubation solution. Preheat the liver cell resuscitation solution and incubation solution in a 37°C water bath for at least 15 minutes before use. Take a tube of cryopreserved hepatocytes and make sure the hepatocytes are still cryogenically frozen before resuscitating. The hepatocytes were quickly placed in a 37°C water bath and shaken until all ice crystals were dispersed, sprayed with 70% ethanol and transferred to a biological safety cabinet. The contents of the hepatocyte tubules were poured into a centrifuge tube containing 50 mL of resuscitation medium, which was centrifuged at 100 g for 10 minutes. After centrifugation, aspirate the recovery medium and add enough incubation medium to obtain a cell suspension with a cell density of about 1.5×10 ⁶ cells/mL. Use Cellometer Vision to count liver cells and determine the density of live cells. The survival rate of liver cells must be greater than 75%. Dilute the hepatocyte suspension with incubation medium to a viable cell density of 0.5× ¹⁰⁶ viable cells/mL.

Table 8 Preparation of liver cell resuscitation solution

experimental method:

Transfer 247.5 μL of live cell (human hepatocytes or mouse hepatocytes) suspension or culture medium to a 96-well deep-well plate, and place the deep-well plate in a vortex-topped incubator to preheat for 10 minutes. All samples were incubated in double parallel. Add 2.5 μL of 100 μM test substance to each well to initiate the reaction, and put the deep well plate back on the incubator vortexer. Incubate the samples at 0, 15, 30, 60, 90 and 120 minutes respectively, take 25 μL of the suspension, add 125 μL of acetonitrile containing internal standard (100 nM alprazolam, 200 nM caffeine, 100 nM tolbutamide) to terminate the reaction. Vortex for 10 minutes, centrifuge at 3220g, 4°C for 30 minutes, transfer 100 μL of supernatant to the sample plate after centrifugation, add 150 μL of pure water and mix well for LC-MS/MS analysis.

All data calculations were performed by Microsoft Excel software. Peak areas were detected by extracted ion spectra. The in vitro half-life (t _1/2 ) of the parent drug was determined by linear fitting the natural logarithm of the percent elimination of the parent drug with time.

In vitro half-life (t _1/2 ) is calculated by the slope:

in vitro t _1/2 ＝0.693/k

The experimental results are shown in Table 9.

Table 9 Hepatocyte metabolic stability test results

compound	Person t 1/2 (min)	Rat 1/2 (min)
Example 2	252.37	94.62

Test Test Example 6: Drug efficacy experiment in mice

Experimental materials: Z138 cells were purchased from ATCC; IMDM medium, penicillin and streptomycin and 0.25% trypsin-EDTA were purchased from Gibco; horse serum and PBS were purchased from Hyclone; Matrigel was purchased from Corning.

Animal information: CB17-SCID mice, female, 6-7 weeks old, weighing about 14-20 grams, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The mice were raised in an SPF-grade environment, and each cage was individually All animals had free access to a standard certified commercial laboratory diet and water ad libitum.

experimental method:

Cell culture: human mantle cell lymphoma Z-138 cell line was cultured in vitro, the culture condition was adding 10% horse serum, 1% penicillin and streptomycin solution to IMDM (cell culture medium), 37°C, 5% _CO2 incubator. Routine digestion with 0.25% trypsin-EDTA digestion solution was performed twice a week for subculture. When the cell saturation is 85%-90% and the number reaches the requirement, collect the cells and count them.

Cell inoculation: 0.1 mL/(containing 1×10 ⁷ ) Z-138 cell suspension (PBS:Matrigel=1:1) was subcutaneously inoculated on the right back of each mouse. On the 18th day after inoculation, when the average volume of the measured tumor reached about 125 mm ³ , the grouping method was randomly stratified and administered according to the tumor volume and animal body weight. PBS is phosphate-buffered saline without calcium and magnesium ions, and Matrigel is Matrigel.

Administration: The dosage of the compound of Example 2 is 1.5mg/kg, 5mg/kg or 15mg/kg, PO, administered twice a day (BID) x 3 weeks. 6 mice per group.

Tumor measurements and experimental indicators:

Tumor diameters were measured twice a week with vernier calipers. The formula for calculating the tumor volume is: V=0.5a x b ² , where a and b represent the long diameter and short diameter of the tumor, respectively. Mouse body weights were measured twice a week.

The antitumor efficacy of compounds was evaluated by tumor growth inhibition rate TGI (%).

TGI (%)=[(1-(average tumor volume at the end of administration of a certain treatment group-average tumor volume at the beginning of administration of this treatment group)/(average tumor volume at the end of treatment of the solvent control group-at the beginning of treatment of the solvent control group Mean tumor volume)] x 100%.

Experimental results:

See Table 10, Figure 14 and Figure 15. No mice became ill or died during the experiment.

Table 10 Z-138 subcutaneous tumor model tumor volume

Experimental results:

In the Z-138 model of subcutaneous tumor transplantation in mice, the compound of Example 2 of the present disclosure had a significant inhibitory effect on tumor growth when administered twice a day at 1.5 mg/kg, 5 mg/kg and 15 mg/kg, and showed a better Dose-response relationship, administration of 15mg/kg twice a day has the effect of shrinking tumors. The compound of Example 2 did not significantly affect the body weight of the mice at the dose tried in this pharmacodynamic experiment, nor did it cause any death of the mice, and the mice could tolerate it.

Test Test Example 7: Hygroscopicity Study

(1) Experimental instrument: dynamic water adsorption instrument DVS Intrinsic;

(2) Experimental conditions: take samples (appropriate amount) and place them in the DVS sample tray for testing.

(3) DVS parameters:

Temperature: 25°C;

Balance: dm/dt≤0.002%/min

RH(%) test rung: 10%

RH (%) test step range: 0%-90%-0%.

Hygroscopicity evaluation standard (according to the description of hygroscopicity characteristics and the definition of hygroscopicity weight gain in "9103 Drug Hygroscopicity Guiding Principles" in "Chinese Pharmacopoeia" 2015 Edition IV):

Hygroscopicity Classification	Moisture absorption weight gain*(ΔW%)
deliquescence	Absorb enough water to form a liquid
Very hygroscopic	15%≤ΔW%
Hygroscopic	2%≤ΔW%<15%
slightly hygroscopic	0.2%≤ΔW%<2%
No or almost no hygroscopicity	ΔW%<0.2%

*Weight gain on moisture absorption at 25±1°C and 80±2% RH.

(4) Experimental results:

The sample of Example 4 (Crystal Form C) has a moisture absorption weight gain of 2.072% under the condition of 80.0% RH, and has hygroscopicity.

The sample of Example 5 (crystal form D) has a moisture absorption weight gain of 14.470% under the condition of 80.0% RH, and has hygroscopicity.

The sample of Example 6 (crystal form A) has a hygroscopic weight gain of 2.318% under the condition of 80.0% RH, and has hygroscopicity.

Test Test Example 8: Saturation Solubility Determination

1. Determination of Apparent Solubility

Test instruments and reagents: pH meter (model: 914PH; brand: Metrohm), electronic balance (model: XS205DU; brand: METTLER TOLEDO), constant temperature water bath oscillator (model: DHSZ-300A; brand: Taicang Wenerhuijin); Concentrated hydrochloric acid, potassium dihydrogen phosphate, phosphoric acid, sodium hydroxide, sodium acetate trihydrate, glacial acetic acid, 80% aqueous methanol.

Test operation:

1. Medium preparation:

pH1.0 buffer solution: take 9mL of concentrated hydrochloric acid, add purified water to dilute to 1L, stir well, and measure pH;

pH4.5 buffer solution: Weigh 2.99g of sodium acetate trihydrate and 1.68g of glacial acetic acid, add purified water to dilute to 1L, stir well, and measure pH;

pH6.8 buffer solution: Weigh 6.805g of potassium dihydrogen phosphate and 0.896g of sodium hydroxide, add purified water to dilute to 1L, stir well, and measure pH.

2. Determination of apparent solubility: Weigh about 50mg of the sample to be tested into a 50mL measuring bottle, and weigh 3 parts in parallel; add 10mL of each of the above three media to the volumetric flask, observe the solution phenomenon, and place in a constant temperature water bath oscillator at 37°C Shake on the top, observe the phenomenon of the solution at 2h and 24h; sample 3mL, centrifuge to get the supernatant, as the test solution. Weigh the sample to be tested, dissolve it with 80% aqueous methanol and dilute it to a solution containing about 0.2 mg per 1 mL, as the reference solution. Using high-performance liquid chromatography, measure 5 μL each of the reference substance solution and the test solution, inject it into the liquid chromatograph, record the chromatogram, and calculate the content of this product by the peak area according to the external standard method.

2. Determination of Saturation Solubility in Biological Media

Test instruments and reagents: high performance liquid chromatography (model: Agilent 1260; brand: Agilent), constant temperature water bath oscillator (model: DHSZ-300A; brand: Taicang Wenerhuijin), pH meter (model: 914PH; brand Metrohm ); sodium chloride, sodium hydroxide (partially prepared as 1mol/L sodium hydroxide solution for use), concentrated hydrochloric acid (diluted to 1mol/L hydrochloric acid for use), sodium acetate, glacial acetic acid, anhydrous sodium dihydrogen phosphate , Pure Milk, FaSSGF/FaSSIF Powder.

Test operation:

1. Medium preparation:

pH5.8 medium: appropriate amount of purified water.

Preparation of fasting gastric juice (FaSSGF): Weigh 2.0g of sodium chloride, add 900mL of water to dissolve, adjust the pH value to 1.6 with 1mol/L hydrochloric acid solution, and dilute to 1000mL with water to obtain a buffer solution; weigh about 15mg of FaSSGF powder to In a 250mL measuring bottle, add 125mL of buffer solution and stir to dissolve, dilute to the mark with buffer solution, and let it stand still.

Postprandial gastric juice (FeSSGF) preparation: Weigh 13.85g of sodium chloride, 2.441g of sodium acetate, 1.028g of glacial acetic acid, add 1000mL of water to dissolve, adjust the pH value to 1mol/L hydrochloric acid solution or 1mol/L sodium hydroxide solution 5.0 to obtain the buffer solution; take 100mL of pure milk and 100mL of the buffer solution, mix well, adjust the pH value to 5.0, and obtain the solution.

Fasting intestinal fluid (FaSSIF) preparation: Weigh 0.42g sodium hydroxide, 6.19g sodium chloride, 3.44g anhydrous sodium dihydrogen phosphate, add 900mL water to dissolve, and use 1mol/L hydrochloric acid solution or 1mol/L sodium hydroxide solution Adjust the pH value to 6.5, then dilute to 1000mL with water to obtain the buffer; weigh about 112mg of FaSSIF powder into a 50mL measuring bottle, add 25mL of buffer and stir to dissolve, dilute to the mark with buffer, and stand for 2 hours to obtain .

Preparation of postprandial intestinal fluid (FeSSIF): Weigh 4.04g of sodium hydroxide, 11.87g of sodium chloride, and 8.65g of glacial acetic acid, add 900mL of water to dissolve, and adjust the pH value with 1mol/L hydrochloric acid solution or 1mol/L sodium hydroxide solution to 5.0, then diluted with water to 1000mL to obtain the buffer solution; weigh about 560mg of FaSSIF powder into a 50mL measuring bottle, add 25mL buffer solution and stir to dissolve, dilute to the mark with buffer solution, and stand still to obtain the solution.

2. Determination of saturation solubility: Weigh about 70mg of the test product into a 50mL measuring bottle, and weigh 5 parts in parallel; add 10mL of the above-mentioned 5 kinds of media to the volumetric flask respectively, observe the solution phenomenon, and place it on a constant temperature water bath shaker at 37°C Shake, and observe the solution phenomenon at 2h, 8h and 24h; sample 3mL, centrifuge, and take the supernatant as the test solution. Weigh the sample to be tested, dissolve it with 80% aqueous methanol and dilute it to a solution containing about 0.2 mg per 1 mL, as the reference solution. Using high-performance liquid chromatography, measure 5 μL each of the reference substance solution and the test solution, inject it into the liquid chromatograph, record the chromatogram, and calculate the content of this product by the peak area according to the external standard method.

The results of the above apparent solubility measurements and the saturation solubility measurements in biological media are shown in Table 11 below.

Table 11 Solubility determination results of crystal form A in different pH buffers

Similarly, according to the above-mentioned apparent solubility determination method, the solubility of Compound I obtained in Example 3 was measured, and its solubility in purified water was measured to be less than 0.5 mg/mL.

Test Test Example 9: Influencing Factor Test

Compound I and the A crystal form, C crystal form and D crystal form of compound I were packed in a double-layer polyethylene bag plus a layer of aluminum foil bag. Stability under nitrogen conditions.

Related substances HPLC analysis method:

Chromatographic column: YMC-Pack Pro C18 150×4.6mm, 3μm

Column temperature: 35°C

Flow rate: 1.0ml/min

Detection wavelength: 240nm

Injection volume: 5μl

Mobile phase: A: 0.1% trifluoroacetic acid in water; B: acetonitrile

gradient:

time (min)	Mobile phase B(%)	Mobile phase A(%)
0	15	85
18	40	60
28	85	15
35	85	15
35.10	15	85
40	15	85

Diluent: 90% Methanol

The results are shown in Table 12 and Table 13.

Table 12 Test results of influencing factors under storage conditions of 40°C and 60°C

Note: "-" means not determined.

Table 13 Test results of influencing factors under storage conditions at 25°C

Note: "-" means undetermined; ^a The storage time is 1 month.

In addition, the purity of Compound I packaged in double-layer polyethylene bags and aluminum foil dropped from 97.4% at 0 to 94.8% at 60°C without nitrogen filling for 3 days; Under the same storage conditions, the purity of the crystal form only decreased from 98.97% at 0 to 98.92%. Therefore, compound I has better thermal stability after salt formation.

Similarly, the crystal form B of compound I was packaged in a double-layer polyethylene bag plus aluminum foil, and its stability at 30°C, 40°C, and nitrogen-filled or non-nitrogen-filled conditions in the bag was also investigated. The results are shown in Table 14.

Table 14 B crystal form influence factor test result

It can be seen from Table 14 that the impurity content of Form B increases significantly at 30°C and 40°C without nitrogen filling, compared with the results of Form A, Form C and Form D placed at 40°C , indicating that under this storage condition, the chemical stability of Form B is slightly worse.

The embodiments of the present disclosure have been described above. However, the present disclosure is not limited to the above-mentioned embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included within the protection scope of the present disclosure.

Claims (21)

1. A pharmaceutically acceptable salt of compound I, wherein the pharmaceutically acceptable salt is selected from monohydrochloride, hydrobromide, 1,5-naphthalene disulfonate, oxalate, citrate, sulfate, phosphate , L-tartrate, L-malate, succinate, adipate, fumarate, oxalate, propionate, benzoate, acetate, formate or L-arginate Amino acid salt, preferably monohydrochloride, hydrobromide or 1,5-naphthalene disulfonate, more preferably monohydrochloride

   
2. The pharmaceutically acceptable salt of compound I according to claim 1, wherein the molar ratio of the compound to the acid molecule is about 1:2 to 2:1, preferably selected from 1:2, 1:1 or 2:1, More preferably 1:1.
3. The pharmaceutically acceptable salt of compound I according to claim 1 or 2, wherein said pharmaceutically acceptable salt is selected from the monohydrochloride of compound I, the hydrobromide of compound I or the 1,5-naphthalene of compound I Disulfonate.
4. The pharmaceutically acceptable salt of compound I according to claim 3, wherein said pharmaceutically acceptable salt is in amorphous or crystalline form, preferably in crystalline form.
5. The method for preparing the pharmaceutically acceptable salt of compound I as claimed in claim 1 or 2, comprises: the step of compound I and acid salt formation, and described acid is selected from hydrochloric acid, hydrobromic acid, 1,5-naphthalene disulfonic acid , oxalic acid, citric acid, sulfuric acid, phosphoric acid, L-tartaric acid, L-malic acid, succinic acid, adipic acid, fumaric acid, oxalic acid, propionic acid, benzoic acid, acetic acid, formic acid or L-arginine, preferably hydrochloric acid , hydrobromic acid or 1,5-naphthalene disulfonic acid, more preferably hydrochloric acid; The solvent used in the salt-forming reaction is selected from ethyl acetate, tetrahydrofuran, acetonitrile, acetone, methyl acetate, isopropanol, methanol or toluene At least one, preferably selected from isopropanol, methyl acetate, ethyl acetate, methanol or acetone, more preferably isopropanol or ethyl acetate.
6. The pharmaceutically acceptable salt of compound I according to claim 4, wherein the pharmaceutically acceptable salt is the A crystal form of compound I hydrochloride, and the X-ray powder diffraction of the A crystal form represented by diffraction angle 2θ Spectrum at 5.42±0.2°, 6.60±0.2°, 10.36±0.2°, 13.60±0.2°, 14.24±0.2°, 15.83±0.2°, 16.62±0.2°, 17.65±0.2°, 19.29±0.2°, 19.86± There are diffraction peaks at 0.2°, 21.37±0.2°, and 23.88±0.2°.
7. The pharmaceutically acceptable salt of Compound I according to claim 6, wherein the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the A crystal form is at 5.42±0.2°, 6.60±0.2°, 9.76±0.2°, 10.36±0.2°, 13.00±0.2°, 13.60±0.2°, 14.24±0.2°, 15.83±0.2°, 16.62±0.2°, 17.65±0.2°, 18.28±0.2°, 19.29±0.2°, 19.86±0.2°, There are diffraction peaks at 20.61±0.2°, 21.37±0.2°, 23.88±0.2°, 25.08±0.2°, 28.22±0.2°.
8. The pharmaceutically acceptable salt of compound I according to claim 6 or 7, wherein the X-ray powder diffraction pattern of the crystal form A is substantially as shown in FIG. 8 .
9. The pharmaceutically acceptable salt of Compound I according to any one of claims 6-8, wherein the crystal form A has a DSC spectrum with a peak at 259.27±5.0°C.
10. The method for preparing the pharmaceutically acceptable salt of compound I as described in any one of claims 6-9, comprises the steps:

    1) Weighing an appropriate amount of compound I, adding it to the solvent (i), and dissolving at room temperature or heating;

    2) Add aqueous hydrogen chloride solution or organic solvent solution of hydrogen chloride to crystallize.
11. The preparation method according to claim 10, wherein the solvent (i) is selected from methyl acetate, ethyl acetate, acetone or isopropanol, preferably isopropanol or ethyl acetate.
12. The preparation method according to claim 10 or 11, wherein there is an optional step 3) after the step 2), that is, using a good solvent to dissolve the crystal obtained in step 2), and then adding the poor solvent dropwise after obtaining the solution, Crystallization.
13. The preparation method according to claim 10 or 11, wherein there is an optional step 3') after the step 2), that is, dissolving the crystal form obtained in step 2) in methanol and concentrating under reduced pressure to obtain Compound I-hydrochloride Amorphous substance, and then dissolve the amorphous substance in solvent (ii), and then precipitate crystals by beating or adding anti-solvent dropwise.
14. The pharmaceutically acceptable salt of compound I according to claim 4, wherein the pharmaceutically acceptable salt is the B crystal form of compound I-hydrochloride, and the X-ray powder diffraction of the B crystal form represented by diffraction angle 2θ Spectrum, at 5.16±0.2°, 9.86±0.2°, 10.42±0.2°, 10.76±0.2°, 11.36±0.2°, 15.68±0.2°, 16.31±0.2°, 17.29±0.2°, 17.98±0.2°, 18.89± There are diffraction peaks at 0.2°, 19.83±0.2°, and 20.99±0.2°.
15. The pharmaceutically acceptable salt of compound I according to claim 14, wherein the X-ray powder diffraction pattern of the crystal form B is substantially as shown in FIG. 11 .
16. The pharmaceutically acceptable salt of compound I according to claim 4, wherein the pharmaceutically acceptable salt is the C crystal form of compound I hydrobromide, the X-ray powder diffraction of the C crystal form represented by diffraction angle 2θ Spectrum at 6.45±0.2°, 9.49±0.2°, 10.01±0.2°, 12.96±0.2°, 13.45±0.2°, 13.81±0.2°, 15.50±0.2°, 19.13±0.2°, 20.13±0.2°, 20.78± There are diffraction peaks at 0.2° and 23.31±0.2°.
17. The pharmaceutically acceptable salt of compound I according to claim 16, wherein the X-ray powder diffraction pattern of the crystal form C is substantially as shown in FIG. 2 .
18. The pharmaceutically acceptable salt of Compound I according to claim 4, wherein the pharmaceutically acceptable salt is the D crystal form of 1,5-naphthalene disulfonate of Compound I, and the D crystal form is represented by diffraction angle 2θ X-ray powder diffraction pattern at 4.02±0.2°, 9.89±0.2°, 11.49±0.2°, 12.74±0.2°, 15.68±0.2°, 15.69±0.2°, 16.15±0.2°, 18.62±0.2°, 18.93 There are diffraction peaks at ±0.2°, 19.67±0.2°, 19.79±0.2°, 21.26±0.2°, 24.36±0.2°, 27.56±0.2°.
19. The pharmaceutically acceptable salt of compound I according to claim 18, wherein the X-ray powder diffraction pattern of the D crystal form is substantially as shown in FIG. 5 .
20. A pharmaceutical composition, containing the pharmaceutically acceptable salt of compound I according to any one of claims 1-4, 6-9, 14-19, and pharmaceutically acceptable auxiliary materials.
21. The pharmaceutically acceptable salt of compound I described in any one of claims 1-4, 6-9, 14-19 or the pharmaceutical composition described in claim 20 are used in the preparation of prevention or treatment of diseases or diseases mediated by PRMT5 Use in medicine; Preferably, the disease or disease mediated by PRMT5 is cancer.

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