CN116178433A - Salts of AXL kinase inhibitors, methods of preparation and use thereof - Google Patents

Abstract

The present invention provides a salt of the compound of formula I, a crystal form of the salt, a preparation method and application thereof. Wherein said salt is selected from methanesulfonate, benzenesulfonate, oxalate, fumarate, citrate, hippurate, hydrochloride, hydrobromide, sulfate or phosphate.

Description

AXL kinase inhibitor salts and preparation methods and uses thereof

Technical Field

The present invention belongs to the field of medical technology. The compound is an AXL kinase inhibitor, and specifically relates to a salt of the AXL inhibitor, a preparation method thereof and medical use thereof.

Background Art

Receptor tyrosine kinases (RTKs) are multidomain transmembrane proteins that can act as sensors for extracellular ligands. Ligand receptor binding induces receptor dimerization and activates its intracellular kinase domain, which in turn leads to the recruitment, phosphorylation and activation of multiple downstream signal cascades (Robinson, D.R. et al., Oncogene, 19: 5548-5557, 2000). To date, 58 RTKs have been identified in the human genome, which regulate a variety of cellular processes, including cell survival, growth, differentiation, proliferation, adhesion and movement (Segaliny, A.I. et al., J. Bone Oncol, 4: 1-12, 2015).

AXL (also known as UFO, ARK, and Tyro7) belongs to the TAM family of receptor tyrosine kinases, which also includes Mer and Tyro3. Among them, AXL and Tyro3 have the most similar gene structure, while AXL and Mer have the most similar tyrosine kinase domain amino acid sequences. Like other receptor tyrosine kinases (RTKs), the structure of the TAM family includes an extracellular domain, a transmembrane domain, and a conserved intracellular kinase domain. The extracellular domain of AXL has a unique structure that juxtaposes immunoglobulin and type III fibronectin repeat units and is reminiscent of the structure of neutral cell adhesion molecules. TAM family members have a common ligand, growth arrest-specific protein 6 (Gas6), which can bind to all TAM receptor tyrosine kinases. Binding of AXL to Gas6 will lead to receptor dimerization and AXL autophosphorylation, thereby activating multiple downstream signal transduction pathways and participating in multiple processes of tumorigenesis (Linger, RM et al., Ther. Targets, 14(10), 1073-1090, 2010; Rescigno, J et al., Oncogene, 6(10), 1909-1913, 1991).

AXL is widely expressed in normal human tissues, such as monocytes, macrophages, platelets, endothelial cells, cerebellum, heart, skeletal muscle, liver and kidney, among which myocardium and skeletal muscle have the highest expression, bone marrow CD34+ cells and stromal cells also have high expression, and normal lymphoid tissues have very low expression (Wu YM, Robinson DR, Kung HJ, Cancer Res, 64 (20), 7311-7320, 2004; hung BI et al., DNA Cell Biol, 22 (8), 533-540, 2003). In studies on many cancer cells, it was found that AXL gene was overexpressed or ectopically expressed in hematopoietic cells, stromal cells and endothelial cells. In various leukemias and most solid tumors, the overexpression of AXL kinase is particularly prominent. By inhibiting AXL receptor tyrosine kinase, the survival signal of tumor cells can be reduced, the invasion ability of tumors can be blocked, and the sensitivity of targeted drug therapy and chemotherapy can be increased. Therefore, finding effective AXL inhibitors is an important direction for the current development of tumor targeted drugs.

Summary of the invention

In one aspect, the present invention provides a pharmaceutically acceptable salt of a compound of formula I, wherein the salt is selected from an organic acid salt or an inorganic acid salt, wherein the organic acid salt is selected from one of methanesulfonate, benzenesulfonate, oxalate, fumarate, citrate and hippurate, and the inorganic acid salt is selected from one of hydrochloride, hydrobromide, sulfate or phosphate. The structure of the compound of formula I is as follows:

In some embodiments, the organic acid salt is selected from methanesulfonate.

In some embodiments, the mesylate salt is a hydrate of the mesylate salt.

In some embodiments, the mesylate salt is a dihydrate of the mesylate salt.

In some embodiments, the molar ratio of the compound of formula I to the organic acid in the organic acid salt is 1:1.

In some embodiments, the molar ratio of the compound of formula I to the inorganic acid salt is 1:1 or 1:2.

In some embodiments, the molar ratio of the compound of formula I to hydrogen chloride in the hydrochloride is 1:1 or 1:2.

In some embodiments, the molar ratio of the compound of formula I to hydrogen chloride in the hydrochloride is 1:2.

In some embodiments, the molar ratio of the compound of formula I to sulfuric acid in the sulfate salt is 1:1.

In some embodiments, the molar ratio of the compound of formula I to hydrobromic acid in the hydrobromide salt is 1:1.

In some embodiments, the molar ratio of the compound of formula I to phosphoric acid in the phosphate is 1:1.

It can be understood that the salt mentioned in the present invention is obtained by a salt-forming reaction between the compound of formula I and the corresponding acid. In the reaction, the compound of formula I is converted into a cation, which combines with the acid radical of the corresponding acid to form the salt. Therefore, the molar ratio of the compound of formula I to the acid in the present invention can be understood as the molar ratio of the cation of the compound of formula I to the acid radical of the corresponding acid in the salt.

In some typical embodiments, the present invention provides a methanesulfonate salt of a compound of formula I, wherein the molar ratio of the compound of formula I to methanesulfonic acid is 1:1, or the molar ratio of the cation of the compound of formula I to the acid radical of methanesulfonic acid is 1:1.

On the other hand, the present invention provides a crystalline form of a pharmaceutically acceptable salt of a compound of formula I, wherein the salt is selected from an organic acid salt or an inorganic acid salt, wherein the organic acid salt is selected from methanesulfonate, benzenesulfonate, oxalate, fumarate, citrate and hippurate, and the inorganic acid salt is selected from hydrochloride, hydrobromide, sulfate or phosphate.

In some embodiments, the present invention provides a crystalline form of the mesylate salt of the compound of Formula I.

In some embodiments, the present invention provides a crystalline form of a mesylate salt of the compound of formula I, whose X-ray powder diffraction pattern is shown in FIG1 .

In some embodiments, the present invention provides a crystalline form of the monohydrochloride salt of the compound of Formula I.

In some embodiments, the present invention provides a crystalline form of a monohydrochloride salt of the compound of Formula I, whose X-ray powder diffraction pattern is shown in FIG4 .

In some embodiments, the present invention provides a crystalline form of the dihydrochloride salt of the compound of Formula I.

In some embodiments, the present invention provides a crystalline form of the dihydrochloride salt of the compound of formula I, whose X-ray powder diffraction pattern is shown in FIG5 .

In some embodiments, the present invention provides a crystalline form of the phosphate salt of the compound of Formula I.

In some embodiments, the present invention provides a crystalline form of a phosphate salt of the compound of formula I, whose X-ray powder diffraction pattern is shown in FIG6 .

In some embodiments, the present invention provides a crystalline form of the hippurate salt of the compound of Formula I.

In some embodiments, the present invention provides a crystalline form of hippurate salt of the compound of formula I, whose X-ray powder diffraction pattern is shown in FIG7 .

In some embodiments, the present invention provides an amorphous form of the sulfate salt of the compound of Formula I.

In some embodiments, the present invention provides a crystalline form of the hydrobromide salt of the compound of Formula I.

In some embodiments, the present invention provides a crystalline form of a hydrobromide salt of the compound of formula I, whose X-ray powder diffraction pattern is shown in FIG9 .

In some embodiments, the present invention provides a crystalline form of the besylate salt of the compound of Formula I.

In some embodiments, the present invention provides a crystalline form of a benzenesulfonate salt of the compound of formula I, whose X-ray powder diffraction pattern is shown in FIG10 .

In some embodiments, the present invention provides a crystalline form of the oxalate salt of the compound of Formula I.

In some embodiments, the present invention provides a crystalline form of an oxalate salt of the compound of formula I, whose X-ray powder diffraction pattern is shown in FIG. 11 .

In some embodiments, the present invention provides a crystalline form of the fumarate salt of the compound of Formula I.

In some embodiments, the present invention provides a crystalline form of a fumarate salt of the compound of formula I, whose X-ray powder diffraction pattern is shown in FIG12 .

In some embodiments, the present invention provides a crystalline form of the citrate salt of the compound of Formula I.

In some embodiments, the present invention provides a crystalline form of a citrate salt of the compound of Formula I, whose X-ray powder diffraction pattern is shown in FIG13 .

On the other hand, the present invention provides a crystalline form A of the compound of formula I, whose X-ray powder diffraction pattern has diffraction peaks at 2θ of 7.6°±0.2°, 10.2°±0.2°, 17.6°±0.2°, 20.3°±0.2° and 20.9°±0.2°.

Furthermore, the crystalline form A has an X-ray powder diffraction pattern having diffraction peaks at 2θ of 4.1°±0.2°, 7.6°±0.2°, 10.2°±0.2°, 12.6°±0.2°, 13.0°±0.2°, 17.6°±0.2°, 19.7°±0.2°, 20.3°±0.2°, 20.9°±0.2° and 22.2°±0.2°.

Further, the crystalline form A has an X-ray powder diffraction pattern having diffraction peaks at 2θ of 4.1°±0.2°, 5.6°±0.2°, 7.6°±0.2°, 10.2°±0.2°, 10.9°±0.2°, 12.6°±0.2°, 13.0°±0.2°, 15.2°±0.2°, 17.6°±0.2°, 19.7°±0.2°, 20.3°±0.2°, 20.9°±0.2°, 22.2°±0.2°, 23.2°±0.2°, 24.6°±0.2°, 27.0°±0.2°, 28.8°±0.2°, 37.0°±0.2° and 37.7°±0.2°.

In some embodiments, the 2θ of the X-ray powder diffraction pattern of the crystalline form A is shown in the following table:

Table 1 X-ray powder diffraction data of Form A

In some embodiments, the X-ray powder diffraction of the crystalline form A expressed in 2θ angle has a pattern as shown in FIG. 14 .

In another aspect, the present invention provides a method for preparing a pharmaceutically acceptable salt of a compound of formula I and a crystalline form of the salt, comprising the step of forming a salt from the compound of formula I and a corresponding acid.

In some embodiments, the solvent of the reaction is selected from a mixed solvent of an alcohol solvent and an alkane solvent, a mixed solvent of a ketone solvent and an alkane solvent, a mixed solvent of an ester solvent and an alkane solvent, a mixed solvent of a nitrile-water solvent and an alkane solvent, a mixed solvent of an alkylbenzene solvent and an alkane solvent, or a mixed solvent of a halogenated hydrocarbon solvent and an alkane solvent.

In some embodiments, the alcohol solvent is selected from methanol, ethanol or isopropanol; the ketone solvent is selected from acetone or butanone; preferably acetone; the ester solvent is selected from ethyl acetate or butyl acetate; preferably ethyl acetate; the nitrile-water solvent is selected from a mixed solution of nitrile-water, and the alkane solvent is selected from n-heptane.

In another aspect, the present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable salt of the compound of formula I.

In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

In some embodiments, the pharmaceutical composition is a solid pharmaceutical preparation suitable for oral administration, preferably a tablet or a capsule.

In another aspect, the present invention also provides a pharmaceutical composition comprising a crystalline form of a pharmaceutically acceptable salt of the compound of formula I.

In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

In some embodiments, the pharmaceutical composition is a solid pharmaceutical preparation suitable for oral administration, preferably a tablet or a capsule.

In another aspect, the present invention also provides a pharmaceutically acceptable salt of a compound of formula I or a pharmaceutical composition thereof for use as a medicament.

In another aspect, the present invention also provides a crystalline form of a pharmaceutically acceptable salt of a compound of formula I or a pharmaceutical composition thereof for use as a medicine.

On the other hand, the present invention also provides a method for preventing and/or treating AXL kinase-mediated diseases or disease states, which comprises administering a salt of the compound of formula I or a pharmaceutical composition thereof of the present invention to an individual in need thereof.

On the other hand, the present invention also provides a method for preventing and/or treating AXL kinase-mediated diseases or disease states, comprising administering the crystalline salt of the compound of formula I of the present invention or a pharmaceutical composition thereof to an individual in need thereof.

In another aspect, the present invention also provides a salt of the compound of formula I of the present invention or a pharmaceutical composition thereof for use in preventing and/or treating a disease or condition mediated by AXL kinase.

In another aspect, the present invention also provides a crystalline form of a salt of the compound of formula I of the present invention or a pharmaceutical composition thereof for preventing and/or treating a disease or disease state mediated by AXL kinase.

In some embodiments, the AXL kinase-mediated disease or condition is cancer.

In some typical embodiments, the cancer is a disease associated with hematological tumors and solid tumors.

Related definitions

Unless otherwise specified, the following terms used in the specification and claims have the following meanings:

The pharmaceutically acceptable salts of the present invention also include their hydrate forms.

The term "pharmaceutically acceptable carrier" refers to carriers that have no significant irritation to the body and do not impair the biological activity and performance of the active compound. It includes but is not limited to any diluent, disintegrant, binder, glidant, wetting agent approved by the State Food and Drug Administration for use in humans or animals.

The term "fumaric acid" refers to fumaric acid, which has the structure:

The term "alcohol solvent" refers to a substance derived from one or more hydroxyl groups (OH) replacing one or more hydrogen atoms on a C1-C6 alkane, wherein the C1-C6 alkane refers to a straight-chain or branched alkane containing 1 to 6 carbon atoms. Specific examples of alcohol solvents include, but are not limited to, methanol, ethanol, isopropanol or n-propanol.

The term "alkane solvent" refers to a straight chain, branched chain or cyclic alkane containing 5 to 7 carbon atoms, and specific examples include but are not limited to n-hexane, cyclohexane, and n-heptane.

The term "ester solvent" refers to a chain compound containing an ester group -COOR and having 3-10 carbon atoms, wherein R is a C1-C6 alkyl group, and the C1-C6 alkyl group refers to a straight-chain or branched alkane containing 1-6 carbon atoms. Specific examples of ester solvents include, but are not limited to, methyl acetate, ethyl acetate, and propyl acetate.

The term "halogenated hydrocarbon solvent" refers to a substance derived from one or more halogen atoms replacing one or more hydrogen atoms on a C1-C6 alkane, wherein the C1-C6 alkane refers to a straight chain or branched alkane containing 1 to 6 carbon atoms, and the halogen atom refers to fluorine, chlorine, bromine, or iodine. Specific examples of halogenated hydrocarbon solvents include, but are not limited to, dichloromethane or chloroform.

The term "ketone solvent" refers to a chain or cyclic compound containing a carbonyl group -CO- and having 3 to 10 carbon atoms, and specific examples include but are not limited to acetone, butanone or cyclohexanone.

The term "benzene-based solvent" refers to a solvent containing a phenyl group, and specific examples include toluene, xylene, cumene or chlorobenzene.

The term "equivalent" refers to the equivalent amount of other raw materials required according to the equivalent relationship of chemical reactions, with the basic raw material used in each step as 1 equivalent.

The “X-ray powder diffraction pattern” in the present invention is obtained by measurement using Cu-Kα radiation.

It should be noted that in X-ray powder diffraction spectra (XRPD), the diffraction spectrum obtained from a crystalline compound is often characteristic for a specific crystal, where the relative intensity of the band (especially at low angles) may vary due to the preferred orientation effect caused by differences in crystallization conditions, particle size and other measurement conditions. Therefore, the relative intensity of the diffraction peak is not characteristic for the crystal targeted. When judging whether it is the same as a known crystal, more attention should be paid to the relative position of the peak rather than their relative intensity. In addition, for any given crystal, there may be slight errors in the position of the peak, which is also well known in the field of crystallography. For example, due to temperature changes, sample movement, or instrument calibration during sample analysis, the position of the peak can move, and the measurement error of the 2θ value is sometimes about ±0.2°. Therefore, this error should be taken into account when determining each crystalline structure. In the XRPD spectrum, the peak position is usually represented by the 2θ angle or the crystal plane distance d, and there is a simple conversion relationship between the two: d=λ/2sinθ, where d represents the crystal plane distance, λ represents the wavelength of the incident X-ray, and θ is the diffraction angle. For the same type of crystals of the same compound, the peak positions of their XRPD spectra are similar overall, and the relative intensity errors may be large. It should also be pointed out that in the identification of mixtures, some diffraction lines may be missing due to factors such as decreased content. At this time, it is not necessary to rely on all the bands observed in high-purity samples, and even one band may be characteristic for a given crystal.

Differential Scanning Calorimetry (DSC) measures the transition temperatures of a crystal when heat is absorbed or released as a result of changes in its crystal structure or melting. For the same crystalline form of the same compound, the thermal transition temperatures and melting points are typically within about 5°C, usually within about 3°C, in consecutive analyses. When a compound is described as having a given DSC peak or melting point, it is referred to as the DSC peak or melting point ±5°C. DSC provides an auxiliary method for distinguishing different crystalline forms. Different crystal forms can be identified by their different transition temperature characteristics. It should be noted that for mixtures, the DSC peak or melting point may fluctuate over a larger range. In addition, since decomposition is associated with the melting of a substance, the melting temperature is related to the heating rate.

Thermogravimetric analysis (TGA) refers to a thermal analysis technique that measures the relationship between the mass of the sample to be tested and the temperature change under program-controlled temperature. When the substance to be tested sublimates or vaporizes during heating, it decomposes into gas or loses crystal water, causing the mass of the substance to be tested to change. At this time, the thermogravimetric curve is not a straight line but a decrease. By analyzing the thermogravimetric curve, you can know at what temperature the substance to be tested changes, and based on the lost weight, you can calculate how much mass has been lost.

The term "as shown" when referring to, for example, an XRPD pattern, a DSC pattern, or a TGA pattern includes patterns that are not necessarily identical to those depicted herein, but that fall within the limits of experimental error when considered by one skilled in the art.

Unless otherwise specified, the abbreviations of the present invention have the following meanings:

M: mol/L

mM：mmol/L

nM: nmol/L

Boc: tert-Butyloxycarbonyl

¹ H NMR: Nuclear Magnetic Resonance

MS(ESI+): Mass Spectrometry

DMSO-d ₆ : deuterated dimethyl sulfoxide

CDCl _3: deuterated chloroform

DTT: dithiothreitol

SEB: Supplemented Enzymatic Buffer

IMDM (Iscove's Modified Dulbecco's Medium): Iscove's (name) modified Dulbecco's (name) medium.

Room temperature: 25℃.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention and the technical solutions of the prior art, the following briefly introduces the drawings required for use in the embodiments and the prior art. Obviously, the drawings described below are only some embodiments of the present invention, and a person skilled in the art can also obtain other drawings based on these drawings.

Figure 1 is an X-ray powder diffraction (XRPD) spectrum of the mesylate salt of the compound of formula I;

Figure 2 is a differential scanning calorimetry (DSC) diagram of the mesylate salt of the compound of formula I;

Figure 3 is a thermogravimetric (TGA) diagram of the methanesulfonate salt of the compound of formula I;

FIG4 shows an X-ray powder diffraction (XRPD) spectrum of the monohydrochloride salt of the compound of formula I;

FIG5 shows an X-ray powder diffraction (XRPD) spectrum of the dihydrochloride salt of the compound of formula I;

FIG6 shows an X-ray powder diffraction (XRPD) spectrum of the phosphate salt of the compound of formula I;

FIG7 shows an X-ray powder diffraction (XRPD) spectrum of the hippurate salt of the compound of formula I;

FIG8 shows an X-ray powder diffraction (XRPD) spectrum of the sulfate salt of the compound of formula I;

FIG9 shows an X-ray powder diffraction (XRPD) spectrum of the hydrobromide salt of the compound of formula I;

FIG10 shows an X-ray powder diffraction (XRPD) spectrum of benzenesulfonate salt of the compound of formula I;

FIG11 shows an X-ray powder diffraction (XRPD) spectrum of the oxalate salt of the compound of formula I;

FIG12 shows an X-ray powder diffraction (XRPD) spectrum of a fumarate salt of the compound of formula I;

FIG13 shows an X-ray powder diffraction (XRPD) spectrum of a citrate salt of the compound of formula I;

FIG. 14 shows an X-ray powder diffraction (XRPD) spectrum of Form A of the compound of Formula I.

DETAILED DESCRIPTION

The present invention is described in more detail below by way of examples, but these specific descriptions are only used to illustrate the technical solution of the present invention and do not constitute any limitation to the present invention.

The test conditions of each instrument are as follows:

(1) X-ray Powder Diffraction (XRPD)

Instrument model: Bruker D2 Phaser ^2nd

(2) Thermogravimetric Analyzer (TGA)

Instrument model: TA Instruments TGA25

Purge gas: Nitrogen

Heating rate: 10℃/min

Heating range: room temperature - 300℃

Method: Place the sample in an aluminum pan, then place the aluminum pan in a platinum pan, and heat the pan from room temperature to the set temperature at a rate of 10°C/min in a nitrogen atmosphere.

(3) Differential Scanning Calorimeter (DSC)

Instrument model: TA Instruments DSC25

Purge gas: Nitrogen

Heating rate: 10℃/min

Heating range: 20-300℃

Method: The sample was placed in an aluminum pan, covered and heated from 20°C to the set temperature at a rate of 10°C/min in a nitrogen atmosphere.

(4) Dynamic moisture sorption (DVS)

Instrument model: Surface Measurement System (SMS)-DVS Intrinsic

The specific instrument setting parameters are as follows:

Example 1 Preparation of (S)-(2-((5-chloro-2-((7-(pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl]amino)pyrimidin-4-yl)amino)-5-(methoxymethyl)phenyldimethylphosphine oxide

a) 2-iodo-4-(methoxymethyl)aniline

4-(Methoxymethyl)aniline (9 g), iodine (16.65 g) and sodium bicarbonate (16.53 g) were added to a solution of dichloromethane (261 mL)/water (135 mL) and stirred at 22°C for 16 h. The reaction solution was quenched with saturated sodium thiosulfate (10 ml) at room temperature. The resulting mixture was extracted with dichloromethane (3 x 100 mL), and then the combined organic layer was washed with saturated sodium chloride aqueous solution (1 x 100 mL), and the organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1/1 v/v) to obtain the title product (16 g). MS (ESI+): 264.0 (M+H).

b) 2-amino-5-(methoxymethyl)phenyl)dimethylphosphine oxide

Under nitrogen atmosphere, dimethylphosphine oxide (5.22 g) was added to a stirred solution of 2-iodo-4-(methoxymethyl)aniline (16 g, 60.82 mmol, 1.00 equivalent), potassium phosphate (14.20 g), palladium acetate (0.68 g) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (1.76 g) in N,N-dimethylformamide (224 mL), and the mixture was stirred at 120°C for 2 hours. The mixture was cooled to room temperature. The resulting mixture was filtered and the filter cake was washed with N,N-dimethylformamide (3x5 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane/methanol=20/1 v/v) to obtain the title product (12.9 g). MS (ESI+): 214.1 (M+H).

c) (2-((2,5-dichloropyrimidin-4-yl)amino)-5-(methoxymethyl)phenyl)dimethylphosphine oxide

To N,N-dimethylformamide (22 mL) were added (2-((2,5-dichloropyrimidin-4-yl)amino)-5-(methoxymethyl)phenyl)dimethylphosphine oxide (1.10 g), 2,4,5-trichloropyrimidine (1.23 g) and N,N-diisopropylethylamine (2.00 g) and stirred for 3 h at room temperature. The resulting mixture was diluted with dichloromethane (30 mL). The reaction was quenched by adding water (10 ml) at 0°C. The resulting mixture was extracted with dichloromethane (3 x 50 mL). The combined organic layers were washed with saturated sodium chloride (1 x 50 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane/methanol=20/1 v/v) to give the title product (1.28 g).

MS(ESI+):360.0(M+H).

d) (S)-(2-((5-chloro-2-((7-(pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl]amino)pyrimidin-4-yl)amino)-5-(methoxymethyl)phenyl)dimethylphosphine oxide

To isopropanol (2 mL) were added (2-((2,5-dichloropyrimidin-4-yl)amino)-5-(methoxymethyl)phenyl)dimethylphosphine oxide (50.00 mg) and (S)-7-(pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]cycloalken-2-amine (31.98 mg), followed by addition of a 1,4-dioxane solution of hydrogen chloride (10 drops, 4 M), and microwave irradiation at 130° C. for 3.5 hours. The mixture was then cooled to room temperature and concentrated under reduced pressure. The crude product was purified by reverse phase high performance liquid chromatography (column: YMC ActusTriart C18, 30*150 mm, particle size: 5 μm, mobile phase A: water (10 mmol/L ammonium bicarbonate), mobile phase B: acetonitrile, flow rate: 60 mL/min, gradient: 20% B to 50% B, 8 min, wavelength: 220 nm, retention time: 6.83 min, column temperature: 25°C) to obtain the title product (20.2 mg).

¹ H NMR (400MHz, DMSO-d ₆ , ppm): δ11.07 (s, 1H), 9.26 (s, 1H), 8.52 (d, J = 4.6Hz, 1H), 8.17 (s, 1H), 7.53 (dd,J＝14.0,2.0Hz,1H),7.44(q,J＝3.1Hz,2H),7.26(dd,J＝8.1,2.3Hz,1H),6.97(d,J＝8.1Hz,1H) , 4.42(s,2H),3.31(s,3H),3.01–2.75(m,2H),2.55(s,5H),2.50(s,2H),1.84(s,2H),1.81(s,3H ),1.77(s,3H), 1.70(q,J＝3.6,3.2Hz,4H),1.54(s,2H).MS(ESI+):554.2(M+H).

Example 2 Activity Assay

The related compounds prepared in Example 1 were subjected to related enzyme activities, cell activities, and in vivo activities

The specific structure of the positive drug 1 (BGB324) used in the activity test is as follows:

The specific structure of positive drug 2 (TP0903) is as follows:

All the above compounds were purchased from Shanghai Shenghong Biotechnology Co., Ltd.

(1) AXL kinase inhibitory activity

1. Experimental Procedure

a) Preparation and addition of AXL enzyme (Carna, 08-107): Use 1× enzyme buffer (use 200 μL Enzymatic buffer kinase 5X, 10 μL 500 mM MgCl ₂ , 10 μL 100 mM DTT, 6.26 μL 2500 nM SEB, add 773.75 μL H ₂ O, and prepare 1 ml 1× enzyme buffer.) Dilute 33.33 ng/uL of AXL enzyme to 0.027 ng/μL (1.67×, final conc. = 0.016 ng/uL), use BioTek (MultiFlo FX) automatic dispenser, add 6 μL of enzyme solution of 1.67 times the final concentration to the compound wells and positive control wells respectively; add 6 μL of 1× Enzymatic buffer to the negative control wells.

b) Compound preparation and addition: Use DMSO to dilute the compound prepared in the example and the positive drug from 10mM to 100μM, and titrate with a compound titrator (Tecan, D300e). The titrator automatically sprays the required concentration into each well. The first concentration is 1μM, 1/2log gradient dilution, a total of 8 concentrations. Centrifuge at 2500rpm for 30s and incubate at room temperature for 15min.

c) Preparation and addition of ATP and substrate: ATP (Sigma, A7699) was diluted with 1× enzyme buffer from 10 mM to 75 μM (5×) with a final concentration of 15 μM; the substrate TK Substrate 3-biotin (Cisbio, 61TK0BLC) was diluted with 1× enzyme buffer from 500 μM to 5 μM (5×) with a final concentration of 1 μM; ATP and substrate were mixed in equal volumes, and 4 μL was added to each well using a BioTek automatic dispenser; centrifuged at 2500 rpm for 30 s, and reacted at 25°C for 45 min.

d) Preparation and addition of detection reagents: Streptavidin-XL665 (Cisbio, 610SAXLG) was diluted from 16.67 μM to 250 nM (4×) with HTRF KinEASE detection buffer (cisbio), with a final concentration of 62.5 nM; TK Antibody-Cryptate (Cisbio) was diluted from 100× to 5× with HTRF KinEASE detection buffer (cisbio), with a final concentration of 1×; XL665 and Antibody were mixed in equal volumes, 10 μL was added to each well using a BioTek automatic dispenser, centrifuged at 2500 rpm for 30 s, and reacted at 25°C for 1 hour. After the reaction, detection was performed using a multi-function plate reader HTRF.

2. Data Analysis

The dose-effect curve was fitted using GraphPad Prism 5 software log (inhibitor) vs. response-Variable slope to obtain the IC ₅₀ value of the compound on AXL kinase inhibition.

The inhibition rate calculation formula is as follows:

Conversion%_sample: is the conversion rate reading of the sample;

Conversion%_min: represents the conversion rate reading of the wells without enzyme activity;

Conversion%_max: represents the conversion reading for wells with no compound inhibition.

3. The experimental results are shown in the table below.

Table 2 IC ₅₀ data of AXL inhibitory activity of compounds

(2) Detection of the inhibitory effect of compounds on cell proliferation

1. Experimental Procedure

MV-4-11 (human myelomonocytic leukemia cell line, culture medium: IMDM + 10% fetal bovine serum) was purchased from Nanjing Kebai Biotechnology Co., Ltd. and cultured in an incubator at 37°C, 5% CO _2. Cells in the logarithmic growth phase were plated in 96-well plates at cell densities of 8000 cells/well, 6000 cells/well, 2000 cells/well, 2000 cells/well and 3000 cells/well, and a blank control group was set up at the same time.

(购自Promega)，按照说明书的操作流程在Envision(PerkinElmer)上测定荧光信号，使用GraphPad Prism 5软件log(inhibitor)vs.response-Variable slope 拟合量效曲线，得到化合物对细胞增殖抑制的IC₅₀值。抑制率计算公式：The test compound and the positive drug were dissolved in dimethyl sulfoxide to prepare a 10mM stock solution, and placed in a -80°C refrigerator for long-term storage. 24 hours after cell plating, the 10mM compound stock solution was diluted with dimethyl sulfoxide to obtain a 200-fold concentration working solution (the highest concentration was 200 or 2000 μM, 3-fold gradient, a total of 10 concentrations), 3μL of each concentration was added to 197μL of complete culture medium, diluted to obtain a 3-fold concentration working solution, and then 50μL was added to 100μL of cell culture medium (the final concentration of dimethyl sulfoxide was 0.5%, v/v), and two replicate wells were set for each concentration. After 72 hours of drug treatment, 50μl of

(purchased from Promega), the fluorescence signal was measured on Envision (PerkinElmer) according to the operating procedures in the manual, and the dose-effect curve was fitted using GraphPad Prism 5 software log (inhibitor) vs. response-Variable slope to obtain the IC ₅₀ value of the compound's inhibition of cell proliferation. Inhibition rate calculation formula:

in:

Test substance signal value: mean fluorescence signal of cell+medium+compound group;

Signal value of blank group: mean value of fluorescence signal of medium group (containing 0.5% DMSO);

Signal value of negative control group: mean value of fluorescence signal of cells+culture medium group (containing 0.5% DMSO).

2. Experimental results

The IC ₅₀ (MV4-11, nM) of the anti-proliferation activity of the compound of Example 1 on MV4-11 cells is 6.97.

(3) In vivo efficacy of compound MV4-11

The test compounds and positive drugs inhibit the growth of human acute monocytic leukemia cell MV-4-11 nude mouse transplant tumor model in vivo.

1. Construction of Mouse Model

MV-4-11 cells in the logarithmic growth phase were collected, counted, and resuspended to adjust the cell concentration to 7.0×10 ⁷ cells/mL; injected into the right axilla of nude mice, 200 μL (14×10 ⁶ cells/mouse) was inoculated to establish the MV-4-11 transplant tumor model. When the tumor volume reached 100-300 mm ³ , tumor-bearing mice with good health and similar tumor volume were selected.

2. Compound configuration

The compound and the positive drug were vortexed with an appropriate solvent and ultrasonically dissolved completely, and then an appropriate volume of citric acid buffer was slowly added and vortexed to mix the liquids evenly to obtain drug administration preparations with concentrations of 0.1, 0.5, and 1 mg·mL ^-1 .

Solvent control group: PEG400 & citric acid buffer (20:80, v:v).

3. Animal Grouping and Dosing

The modeled mice were randomly divided into groups (n=6), and the relevant compounds and positive drugs were administered on the day of grouping. The experiment was terminated after 21 days or when the tumor volume of the solvent control group reached ^2000mm3 (whichever was reached first). The administration volume was 10mL·kg ^-1 . The compounds and positive drugs were administered by gavage once a day. After the start of the experiment, the tumor diameter and animal weight were measured twice a week to calculate the tumor volume.

4. Data Analysis

The formula for calculating tumor volume (TV) is: tumor volume (mm ³ ) = l × w ² /2,

Wherein, l represents the long diameter of the tumor (mm); w represents the short diameter of the tumor (mm).

The relative tumor volume (RTV) was calculated as follows: RTV = TV _t / TV _initial

Wherein, TV _initial is the tumor volume measured at the time of group administration; TV _t is the tumor volume measured at each time during the administration period.

The calculation formula of tumor growth inhibition rate TGI (%) is: TGI = 100% × [1 - (TV _t(T) - TV _initial(T) )/ (TV _t(C) - TV _initial(C) )]

Wherein, TV _t(T) represents the tumor volume of the treatment group measured each time; TV _initial(T) represents the tumor volume of the treatment group at the time of group administration; TV _t(C) represents the tumor volume of the solvent control group measured each time; TV _initial(C) represents the tumor volume of the solvent control group at the time of group administration.

The calculation formula of relative tumor proliferation rate (%T/C) is: %T/C = 100% × (RTV _T /RTV _C )

Among them, RTV _T represents the RTV of the treatment group; RTV _C represents the RTV of the solvent control group.

The experimental data were calculated and statistically processed using Microsoft Office Excel 2007 software.

5. The experimental results are as follows:

Table 3 In vivo efficacy of compounds

Note: The experimental data in the table are the relevant data obtained at the end of the experiment (the end of the experiment is defined as: the end of the experiment after ²¹ days or when the tumor volume of the solvent control group reaches 2000mm3 (whichever is reached first)).

(4) Pharmacokinetic study of the compound in ICR mice

1. Preparation of compound for oral administration

Each compound was prepared in DMSO to a 10 mg/mL stock solution.

Mixed solvent preparation: Tween 80:PEG400:Water=1:9:90 (v/v/v)

Accurately pipette 450 μl of 10 mg/mL DMSO stock solution of the compound into glass bottles, add appropriate volumes of DMSO and mixed solvent, the ratio of the solvent in the final preparation is DMSO: mixed solvent (v/v) = 10:90, vortex (or ultrasound) to disperse evenly, and obtain 4.5 mL of dosing solution with a concentration of 1 mg/mL.

2. Experimental plan

Male ICR mice aged 6 to 10 weeks (source: Weitong Lihua Experimental Animal Technology Co., Ltd.) were selected, with 6 mice in each group. The mice were fasted overnight and fed 4 hours after administration. On the day of the experiment, the mice were gavaged with 10 mg kg ^-1 compound test solution. After administration, about 100 μL of blood was collected from the eye sockets of the mice at 0, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h, and placed in EDTA-K ₂ anticoagulant tubes. The whole blood sample was centrifuged at 1500-1600 g for 10 min, and the separated plasma was stored in a refrigerator at -40 to -20 ° C for biological sample analysis. The blood drug concentration was determined by LC-MS/MS method.

3. Data Analysis and Results

The non-compartmental model in Pharsight Phoenix 7.0 was used to calculate the pharmacokinetic parameters. The specific results are shown in the table below.

Table 4: Pharmacokinetic results of compounds in mice

Compound Cmax(ng/mL) Tmax(h) AUC _0-24 (ng.h/mL) T1/2(h) Example 1 324 2.17 1300 1.35 Positive drug 2 (TP-0903) 26.8 0.25 52.2 1.20

Example 3 Preparation of salts and crystal forms of compounds of formula I

Take about 50 mg of free base, i.e., compound of formula 1, and 1.05 equivalents of acid (hydrochloric acid with a molar ratio of acid to free base of 2.10), add 1 mL of solvent and stir at room temperature for 2 days. The obtained clear solution is crystallized by stirring at 5°C and slowly evaporating, and the solid is separated by centrifugation and dried under forced air or reduced pressure at 40°C for 2-5 hours for XRPD characterization.

Table 5 Salt formation results of compounds of formula I

Figures 4-13 are the X-ray powder diffraction (XRPD) spectra of hydrochloride, dihydrochloride, phosphate, hippurate, sulfate, hydrobromide, benzenesulfonate, oxalate, fumarate, and citrate, respectively.

Example 4 Preparation of Methanesulfonate

To a 20-mL glass vial, (S)-(2-((5-chloro-2-((7-(pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)amino))pyrimidin-4-yl)amino)-5-(methoxymethyl)phenyldimethylphosphine oxide (50 mg), toluene (1 mL) and methanesulfonic acid (10 mg) were added successively, and the reaction was stirred at room temperature for 2 hours to obtain a suspension state of (S)-(2-((5-chloro-2-((7-(pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)amino))pyrimidin-4-yl)amino)-5-(methoxymethyl)phenyldimethylphosphine oxide methanesulfonate crystalline form.

Example 5 Preparation of Methanesulfonate

To a 20-mL glass vial, (S)-(2-((5-chloro-2-((7-(pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)amino))pyrimidin-4-yl)amino)-5-(methoxymethyl)phenyldimethylphosphine oxide (1 g), ethyl acetate (20 mL) and methanesulfonic acid (172.8 mg) were added successively, and the reaction was stirred at room temperature for 10 minutes, and then the mesylate crystals prepared in Example 4 were added as seeds (5 mg) and stirred for 30 minutes. To a glass vial, ethyl acetate (20 mL), acetone (10 mL) and crystal form A seeds (10 mg) were added successively and stirred for crystallization for 20 minutes. The mixture was filtered and the wet cake was dried under vacuum at 40° C. for 20 hours to obtain (S)-(2-((5-chloro-2-((7-(pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)amino))pyrimidin-4-yl)amino)-5-(methoxymethyl)phenyldimethylphosphine oxide methanesulfonate crystalline form in the form of a light yellow solid powder.

¹ H NMR (400MHz, DMSO-d6): 11.11 (s, 1H); 9.46 (br, 1H); 9.36 (s, 1H); 8.58-8.50 (m, 1H); 8.19 (s, 1H); 7.60- 7.43, (m, 3H); 7.37 (dd, J = 8.2, 2.2Hz, 1H); 7.04 (d, J = 8.2Hz, 1H); 4.44 (s, 2H); 3.53-3.46 (m, 3H); 3.33(s, 3H); 3.15 (s, 2H); 2.82-2.62 (m, 4H); 2.32-2.29 (m, 5H); 1.99 (s, 2H); 1.89-1.75 (m, 8H); 1.40 (q, J＝ 12.3Hz,2H).

Its XRD spectrum is shown in Figure 1, its DSC spectrum is shown in Figure 2, and its TGA spectrum is shown in Figure 3.

Example 6 Preparation of Form A of the Compound of Formula I

In a 3 mL glass bottle, 200 mg of S)-(2-((5-chloro-2-((7-(pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl]amino)pyrimidin-4-yl)amino)-5-(methoxymethyl)phenyldimethylphosphine oxide prepared according to the method of Example 1 and 2 mL of purified water were added successively. After magnetic stirring at room temperature for 6 hours, the sample was centrifuged, and the wet sample was placed at 40° C. and dried under reduced pressure for 21 hours to obtain 176 mg of Form A with a yield of 88.0%. The XRD spectrum is shown in Figure 14, and the X-ray powder diffraction pattern data is shown in Table 6.

Table 6 X-ray powder diffraction pattern data of free base form A

Claims (10)

1. A pharmaceutically acceptable salt of a compound of formula I selected from the group consisting of organic acid salts selected from one of methanesulfonic acid salts, benzenesulfonic acid salts, oxalic acid salts, fumaric acid salts, citric acid salts, and hippuric acid salts, or inorganic acid salts selected from one of hydrochloric acid salts, hydrobromic acid salts, sulfuric acid salts, or phosphoric acid salts, the compound of formula I having the structure:

2. the salt according to claim 1, wherein the organic acid is selected from methanesulfonic acid.

3. The mesylate salt of a compound of formula I according to claim 2, which is in the form of a hydrate, in particular dihydrate.

4. The salt of the compound of formula I according to claim 1, the molar ratio of the compound of formula I to the organic acid of the organic acid salt being 1:1.

5. the salt of the compound of formula I according to claim 1, wherein the molar ratio of the compound of formula I to the mineral acid in the mineral acid salt is 1:1 or 1:2, further the molar ratio of the compound of formula I to hydrogen chloride in the hydrochloride is 1:1 or 1:2.

6. a crystalline form of a pharmaceutically acceptable salt of a compound of formula I, said salt being selected from the group consisting of organic acid salts selected from the group consisting of methanesulfonic acid salts, benzenesulfonic acid salts, oxalic acid salts, fumaric acid salts, citric acid salts and hippuric acid salts, or inorganic acid salts selected from the group consisting of hydrochloric acid salts, hydrobromic acid salts, sulfuric acid salts or phosphoric acid salts.

7. The crystalline form of a salt of a compound of formula I according to claim 6, which salt is a mesylate salt, further having an X-ray powder diffraction pattern as shown in figure 1.

8. A crystalline form of a compound of formula I having an X-ray powder diffraction pattern with diffraction peaks at 7.6 ° ± 0.2 °, 10.2 ° ± 0.2 °, 17.6 ° ± 0.2 °, 20.3 ° ± 0.2 ° and 20.9 ° ± 0.2 ° in 2Θ; further X-ray powder diffraction patterns have diffraction peaks at 2θ of 4.1°±0.2°, 7.6°±0.2°, 10.2°±0.2°, 12.6°±0.2°, 13.0°±0.2°, 17.6°±0.2°, 19.7°±0.2°, 20.3°±0.2°, 20.9°±0.2° and 22.2°±0.2°; further, the X-ray powder diffraction pattern thereof has diffraction peaks at 4.1 ° ± 0.2 °, 5.6 ° ± 0.2 °, 7.6 ° ± 0.2 °, 10.2 ° ± 0.2 °, 10.9 ° ± 0.2 °, 12.6 ° ± 0.2 °, 13.0 ° ± 0.2 °, 15.2 ° ± 0.2 °, 17.6 ° ± 0.2 °, 19.7 ° ± 0.2 °, 20.3 ° ± 0.2 °, 20.9 ° ± 0.2 °, 22.2 ° ± 0.2 °, 23.2 ° ± 0.2 °, 24.6 ° ± 0.2 °, 27.0 ° ± 0.2 °, 28.8 ° ± 0.2 °, 37.0 ° ± 0.2 ° and 37.7 ° ± 0.2 °, and the further X-ray powder diffraction pattern expressed in terms of 2θ angle has the pattern as shown in fig. 14.

9. A process for the preparation of pharmaceutically acceptable salts and crystalline forms of the salts of the compounds of formula I, comprising the step of salifying the compounds of formula I with the corresponding acids.

10. A pharmaceutical composition of a pharmaceutically acceptable salt of a compound of formula I.

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