CN114315837A - Crystalline form of ERK inhibitor and preparation method thereof - Google Patents
Crystalline form of ERK inhibitor and preparation method thereof Download PDFInfo
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Abstract
The present disclosure provides a crystalline form of an ERK inhibitor and a method of preparing the same. In particular, the compound (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] is provided]A-E crystal forms of imidazole-3-ketone (formula I) and a preparation method thereof.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a crystal form of an ERK inhibitor and a preparation method thereof.
Background
Proliferation, differentiation, metabolism, and apoptosis of normal cells are strictly regulated by cell signal transduction pathways in vivo. Mitogen-activated protein kinase (MAPK) plays a very important role in signal transduction pathways, and extracellular signal regulated kinase (ERK) is a member of the MAPK family. Through the RAS-RAF-MEK-ERK step, an exogenous stimulation signal is transmitted to the ERK, the activated ERK is transferred into cell nucleus to regulate the activity of transcription factor, so as to regulate the biological functions of cell such as proliferation, differentiation and apoptosis, or participate in the regulation of cell morphology and the redistribution of cytoskeleton through phosphorylating cytoskeletal components in cytoplasm.
RAS and RAF gene mutations cause the sustained activation of the MAPK-ERK signaling pathway, promoting malignant transformation, abnormal proliferation of cells, and ultimately the production of tumors (Roberts PJ et al, Oncogene, 2007, 26(22), 3291-3310). The combination of a MEK inhibitor and a B-RAF inhibitor can further improve the tumor growth inhibition effect of the B-RAF inhibitor, and can remarkably improve the disease-free progression and the overall survival rate of melanoma patients carrying the mutations of BRAFV600E and V600K (Frederick DT et al, Clinical Cancer Research, 2013.19(5), 1225-1231). Although the combination of B-RAF/MEK inhibitors can have tumor-inhibiting effects, their therapeutic effects are transient, and the majority of patients develop drug resistance within 2-18 months, and the tumor is further worsened. The mechanism of development of B-RAF/MEK inhibitor resistance is very complex, mostly directly associated with reactivation of the ERK signaling pathway (Smalley I et al, Cancer Discovery, 2018, 8(2), 140-142). Therefore, the development of new ERK inhibitors is effective not only in patients with mutations in the MAPK signaling pathway, but also in patients with resistance to B-RAF/MEK inhibitors.
The B-RAF/MEK inhibitor can inhibit the growth of tumor and regulate the immune microenvironment of tumor. The B-RAF/MEK inhibitor can enhance the expression of tumor specific antigen, improve the recognition and killing of tumor by antigen specific T cells, and promote the migration and infiltration of immune cells. In animal models, the expression of PD-L1 in tumor tissues is enhanced after treatment with a B-RAF/MEK inhibitor, and when combined with an antibody against a checkpoint (checkpoint) molecule (e.g., PD-1 antibody, CTLA4 antibody), the tumor growth-inhibiting effect is better than that of the B-RAF/MEK inhibitor alone (Boni A et al, Cancer Research, 2010, 70(13), 5213-. Research shows that the ERK inhibitor is similar to the B-RAF/MEK inhibitor, and can play a role in regulating the tumor microenvironment when being used together with the checkpoint antibody, improve the function of cytotoxic T cells and achieve the effect of inhibiting tumor growth.
PCT/CN2020/081591 provides an ERK inhibitor with the chemical name (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one (formula I), which has been found to have a good pharmaceutical activity and is expected to provide a new treatment option for patients.
It will be appreciated by those skilled in the art that the crystal structure of the active ingredient of a drug often affects the physicochemical stability of the drug. The difference in crystallization conditions and storage conditions may lead to a change in the crystal structure of the compound, and may be accompanied by the formation of other forms of the crystal. Generally, an amorphous drug product does not have a regular crystal structure, and is often accompanied with the defects of poor product stability, fine crystallization, difficult filtration, easy agglomeration, poor flowability and the like. In view of the importance of the crystal form of the solid medicine and the stability thereof in clinical treatment, the crystal form of the compound (S) -2- (1- (3-chlorphenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidine-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazole-3-ketone is deeply researched, and the method has important significance for developing medicines which are suitable for industrial production and have good biological activity.
Disclosure of Invention
The present disclosure provides crystalline form a of the compound (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one (formula I), an X-ray powder diffraction pattern expressed in terms of diffraction angle 2 θ, having characteristic peaks at 4.040, 5.085, 11.801, 15.532, and 16.925.
In some embodiments, the compound of formula I has a crystalline form a, an X-ray powder diffraction pattern expressed in diffraction angle 2 Θ angles, and characteristic peaks at 4.040, 5.085, 11.801, 15.532, 16.925, 18.915, and 27.074.
In some embodiments, the compound of formula I has a crystalline form a, an X-ray powder diffraction pattern expressed in diffraction angle 2 Θ angles, and characteristic peaks at 4.040, 5.085, 11.801, 15.532, 16.925, 18.915, 22.000, 24.686, and 27.074.
In some embodiments, the form a of the compound of formula I has an X-ray powder diffraction pattern expressed in diffraction angle 2 Θ angles as shown in figure 1.
The present disclosure also provides a method of preparing a crystalline form a of compound I, comprising:
the method comprises the following steps:
(a) mixing (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one with a solvent (I) selected from at least one of methyl tert-butyl ether, cyclopentane, isopropyl ether, phenetole, or isopropyl acetate;
(b) pulping and crystallizing;
or, the second method:
(a) mixing (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one with a solvent (II) selected from at least one of ethyl acetate, dichloromethane, acetonitrile, trichloromethane, dioxane, butanone, tetrahydrofuran, acetone, and dissolving with stirring or heating;
(b) adding a solvent (III) and crystallizing, wherein the solvent (III) is at least one selected from isopropyl ether and methyl tert-butyl ether.
The volume (. mu.L) used for solvents (I), (II), (III) described in the present disclosure may be 1 to 200 times the amount of the compound of formula I (mg), and in non-limiting embodiments may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200 times.
The present disclosure provides a form B of the compound of formula (I) having characteristic peaks at 13.128, 13.773, 14.418, 17.645 and 19.482 in an X-ray powder diffraction pattern expressed in terms of diffraction angle 2 Θ angles.
In some embodiments, the compound of formula I form B has an X-ray powder diffraction pattern having characteristic peaks at 2 Θ angles of 13.128, 13.773, 14.418, 17.645, 19.482, 24.793, and 27.573.
In some embodiments, the form B of the compound of formula I has an X-ray powder diffraction pattern expressed in terms of diffraction angle 2 Θ angles as shown in figure 2.
The present disclosure also provides a method of preparing a crystalline form B of compound of formula I, comprising:
the method comprises the following steps:
(a) mixing (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one with a solvent (IV) selected from at least one of ethyl acetate, ethyl propionate, acetone, n-hexane;
(b) pulping and crystallizing;
or, the second method:
(a) mixing (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one with a solvent (V) selected from at least one of acetone and butanone;
(b) adding n-hexane, and crystallizing.
The volume (. mu.L) used for the solvents (IV), (V) described in the present disclosure may be 1 to 200 times the amount of the compound of formula I (mg), and in non-limiting embodiments may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200 times.
The present disclosure provides a crystalline form C of the compound of formula (I) having an X-ray powder diffraction pattern expressed in terms of diffraction angle 2 Θ angles with a characteristic peak at 17.857.
In some embodiments, the form C of the compound of formula I has an X-ray powder diffraction pattern expressed in diffraction angle 2 Θ angles as shown in figure 3.
The present disclosure also provides a method of preparing form C of the compound of formula I, comprising:
(a) mixing (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one with n-pentane;
(b) pulping and crystallizing.
The volume (. mu.L) of n-pentane used in the present disclosure may be 1 to 200 times the amount of compound of formula I (mg), and in non-limiting embodiments may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200 times.
The present disclosure provides a crystalline form D of the compound of formula I having characteristic peaks at 4.462, 5.228, 12.476, 13.331, 13.924, 16.031, and 16.677 in an X-ray powder diffraction pattern expressed in terms of diffraction angle 2 θ.
In some embodiments, the compound of formula I has a D crystalline form, an X-ray powder diffraction pattern expressed in diffraction angle 2 Θ angles, having characteristic peaks at 4.462, 5.228, 11.503, 12.476, 13.331, 13.924, 16.031, 16.677, and 17.308.
In some embodiments, the compound of formula I has a D-form, an X-ray powder diffraction pattern expressed in diffraction angle 2 Θ angles, having characteristic peaks at 4.462, 5.228, 9.015, 10.408, 11.503, 12.476, 13.331, 13.924, 16.031, 16.677, and 17.308.
In some embodiments, the crystalline form D of the compound of formula I has an X-ray powder diffraction pattern expressed in diffraction angle 2 Θ angles as shown in figure 4.
The present disclosure also provides a method of preparing a crystalline form D of compound of formula I, comprising:
(a) mixing (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one with acetonitrile;
(b) adding isopropyl ether, stirring at 50 deg.C, and crystallizing.
In some embodiments, the volume (μ L) used by the acetonitrile, isopropyl ether may be 1-200 times the amount of compound of formula I (mg), and in non-limiting embodiments may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200 times.
The present disclosure provides crystalline form E of the compound of formula I having characteristic peaks at 5.216, 11.834, 15.595, and 17.199 in an X-ray powder diffraction pattern expressed in terms of diffraction angle 2 Θ angles.
In some embodiments, the crystalline form E of the compound of formula I has an X-ray powder diffraction pattern expressed in diffraction angle 2 Θ angles as shown in figure 5.
The present disclosure also provides a method of preparing form E of the compound of formula I, comprising:
(a) mixing (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one with dichloromethane;
(b) adding isopropyl ether, stirring at 50 deg.C, and crystallizing.
In some embodiments, the volume (. mu.L) of the methylene chloride, isopropyl ether used may be 1 to 200 times the amount of compound of formula I (mg), and in non-limiting embodiments may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200 times.
In some embodiments, the methods of preparing the crystalline form of the present invention further comprise the steps of filtering, washing, or drying.
The present disclosure also provides a pharmaceutical composition prepared from any one of the aforementioned crystalline forms.
The present disclosure also provides a pharmaceutical composition comprising a crystalline form of the compound of formula I or a crystalline form prepared by the foregoing method, and optionally a pharmaceutically acceptable carrier, diluent or excipient.
The present disclosure also provides a process for preparing a pharmaceutical composition comprising the step of mixing the crystalline form of the compound of formula I or the crystalline form prepared by the process described above with a pharmaceutically acceptable carrier, diluent or excipient.
The disclosure also provides use of a crystalline form of the compound of formula I, or a crystalline form of the compound of formula I prepared by the foregoing method, or the foregoing composition, or the composition prepared by the foregoing method, in the preparation of a medicament for inhibiting ERK.
The present disclosure also provides the use of a crystalline form of a compound of formula I as described above or a crystalline form of a compound of formula I prepared by a process as described above or a composition as prepared by a process as described above in the manufacture of a medicament for the treatment or prevention of cancer, inflammation, or other proliferative disease.
In alternative embodiments, the cancer described in the present disclosure is selected from melanoma, liver cancer, kidney cancer, lung cancer, nasopharyngeal cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, cervical cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma, astrocytoma, and glioma.
The "2 theta or 2 theta angle" referred to in this disclosure means the diffraction angle, theta being the bragg angle in degrees or degrees; the error range of each characteristic peak 2 theta is +/-0.30, optionally +/-0.20, and specifically can be-0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.
The differential scanning calorimetry or DSC in the present disclosure refers to measuring the temperature difference and the heat flow difference between the sample and the reference during the temperature rise or constant temperature process of the sample to characterize all the physical changes and chemical changes related to the thermal effect and obtain the phase change information of the sample.
In the present disclosure, the drying temperature is generally 25 ℃ to 100 ℃, preferably 40 ℃ to 70 ℃, and the drying may be performed under normal pressure or under reduced pressure.
"pharmaceutical composition" means a mixture containing one or more compounds described herein, or a physiologically acceptable salt or prodrug thereof, in admixture with other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.
The crystallization in the present disclosure includes, but is not limited to, stirring, cooling, concentrating, volatilizing, pulping and crystallizing.
Drawings
FIG. 1: an XRPD pattern of the compound A crystal form of formula I.
FIG. 2: an XRPD pattern of the compound B crystal form of formula I.
FIG. 3: an XRPD pattern of form C of compound of formula I.
FIG. 4: an XRPD pattern of form D of compound of formula I.
FIG. 5: an XRPD pattern of form E of compound of formula I.
Detailed Description
The present invention will be explained in more detail with reference to examples or experimental examples, which are only used to illustrate the technical solution of the present invention and are not intended to limit the spirit and scope of the present invention.
The reagents used in the present invention are commercially available.
The test conditions of the apparatus used in the experiment of the present invention were as follows:
1. differential Scanning Calorimeter (DSC)
The instrument model is as follows: mettler Toledo DSC 3+
And (3) purging gas: nitrogen gas; nitrogen purging speed: 50mL/min
The heating rate is as follows: 10.0 ℃/min
Temperature range: 25-350 deg.C
2. X-ray Powder Diffraction Spectroscopy (XRPD)
The instrument model is as follows: bruker D8 Discover X-ray powder diffractometer
Ray: monochromatic Cu-ka radiation (λ ═ 1.5418)
The scanning mode is as follows: θ/2 θ, scan range (2 θ range): 5 to 50 DEG
Voltage: 40kV, current: 40mA
3. Thermogravimetric Analyzer (TGA)
The instrument model is as follows: mettler Toledo TGA2
And (3) purging gas: nitrogen gas; nitrogen purging speed: 50mL/min
The heating rate is as follows: 10.0 ℃/min
Temperature range: 25-350 deg.C
4. DVS is dynamic moisture adsorption: the detection adopts SMS DVS Advantage, the humidity change is 50% -95% -0% -95% -50% at 25 ℃, the stepping is 10% (the last step is 5%), and the judgment standard is that dm/dt is not more than 0.002%.
5. And (3) purity detection: detecting by high performance liquid chromatography; the instrument model is as follows: agilent 1200 DAD; a chromatographic column: phenomenex kinetex EVOC 184.6 × 250mm,5 um; mobile phase A: KH (Perkin Elmer)2PO4And the mobile phase B: acetonitrile; flow rate: 1.0 mL/min; column temperature: 40 ℃; detection wavelength: 214 nm.
6. The eluent system for column chromatography and the developing agent system for thin-layer chromatography used for purifying compounds comprise: a: dichloromethane/methanol system, B: n-hexane/ethyl acetate system, C: petroleum ether/ethyl acetate system.
7. The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or/and Mass Spectrometry (MS). NMR shift (. delta.) of 10-6The units in (ppm) are given. NMR was measured using a Bruker AVANCE-400 NMR spectrometer using deuterated dimethyl sulfoxide (DMSO-d)6) Deuterated chloroform (CDCl)3) Deuterated methanol (CD)3OD), internal standard Tetramethylsilane (TMS).
MS was measured using an Agilent 1200/1290DAD-6110/6120Quadrupole MS LC MS (manufacturer: Agilent, MS model: 6110/6120Quadrupole MS), waters ACQuity UPLC-QD/SQD (manufacturer: waters, MS model: waters ACQuity Qda Detector/waters SQ Detector), THERMO Ultratate 3000-Q active (manufacturer: THERMO, MS model: THERMO Q active).
Example 1: preparation of the compound of formula I (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one
The first step is as follows: (S) -2- ((tert-butyldimethylsilyl) oxy) -1- (3-chlorophenyl) ethylamine 1b
(S) -2-amino-2- (3-chlorophenyl) ethanol 1a (4g, 23.3mmol, Shanghai Biao pharmaceutical science and technology Co., Ltd.) and imidazole (3.2g, 46.6mmol) were dissolved in 80mL of dichloromethane, and tert-butyldimethylsilyl chloride (5.2g, 35mmol) was added under ice-cooling, and the reaction was stirred for 14 hours. Water was added and the mixture was extracted with methylene chloride (80 mL. times.3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure and purified by column chromatography with eluent system C to give the title compound 1b (6.5g), yield: 97 percent.
MS m/z(ESI):286.1[M+1]。
The second step is that: (S) -N- ((4-bromo-1H-pyrrol-2-yl) methyl) -2- ((tert-butyldimethylsilyl) oxy) -1- (3-chlorophenyl) ethylamine 1d
4-bromo-1H-pyrrole-2-carbaldehyde 1c (2.37g, 13.62mmol, obtained after Shanghai), compound 1b (3.9g, 13.64mmol) was stirred and reacted for 3 hours. Diluting with 100mL of methanol, cooling to 0 deg.C, adding sodium borohydride (516mg, 13.64mmol), and stirring for 2 hours. Water was added to the reaction mixture, which was concentrated under reduced pressure, and water and ethyl acetate were added to the mixture to extract the mixture (40 mL. times.3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure and purified by column chromatography with eluent system C to give the title compound 1d (4.8g), yield: 79 percent.
MS m/z(ESI):444.2[M+1]。
The third step: (S) -6-bromo-2- (2- ((tert-butyldimethylsilyl) oxy) -1- (3-chlorophenyl) ethyl) -1H-pyrrolo [1,2-c ] imidazol-3 (2H) -one 1e
Compound 1d (4.8g, 10.81mmol) was dissolved in 100mL of tetrahydrofuran, and N, N' -carbonyldiimidazole (2.45g, 15.11mmol) was added under ice-cooling and stirred for 0.5 hour, and sodium hydride (60%, 621mg, 16.22. mu. mol) was added and stirred for reaction at room temperature for 14 hours. Saturated ammonium chloride was added. The reaction solution was concentrated under reduced pressure and purified by column chromatography with eluent system C to give the title compound 1e (4.0g), yield: 78 percent.
MS m/z(ESI):469.1[M+1]。
The fourth step: (S) -2- (2- ((tert-butyldimethylsilyl) oxy) -1- (3-chlorophenyl) ethyl) -6- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrrolo [1,2-c ] imidazol-3 (2H) -one 1f
Compound 1e (4.0g, 8.51mmol) was dissolved in 50mL of 1, 4-dioxane under argon, 4,4,4 ', 4', 5,5,5 ', 5' -octamethyl-2, 2 '-bis (1,3, 2-dioxaborolan) (3.24g, 12.76mmol), potassium acetate (3.34g, 34.04mmol) and [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (1.24g, 1.70mmol) were added in that order, and stirred at 90 ℃ for 2 hours. Cooling, filtration through celite, concentration of the filtrate and purification by column chromatography with eluent system C gave the title compound 1f (2.0g), yield: 45 percent.
MS m/z(ESI):517.2[M+1]。
The fifth step: 4-chloro-N- (1-methyl-1H-pyrazol-5-yl) pyrimidin-2-amine 1i
N- (1-methyl-1H-pyrazol-5-yl) formamide (324.82mg, 2.60mmol, prepared by the method disclosed in patent application WO 2017/80979) was dissolved in 15mL of N, N-dimethylformamide, sodium hydride (60%, 311.47mg, 7.79mmol) was added at 0 ℃, the reaction was stirred for 0.5 hour, 1g (500mg, 2.60mmol) of 4-chloro-2- (methylsulfonyl) pyrimidine was added, and the reaction was continued for 2 hours. Water 20mL was added, ethyl acetate was extracted (20mL × 3), the combined organic phases were concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system C to give the title compound 1i (270mg), yield: 49.6 percent.
MS m/z(ESI):210.3[M+1]。
And a sixth step: (S) -2- (2- ((tert-butyldimethylsilyl) oxy) -1- (3-chlorophenyl) ethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3 (2H) -one 1j
A mixture of compound 1f (98.6mg, 0.19mmol), preformed 4-chloro-N- (1-methyl-1H-pyrazol-5-yl) pyrimidin-2-amine 1i, [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (28mg, 0.02mmol), and cesium carbonate (124mg, 0.2mmol) was suspended in 20mL of 1, 4-dioxane and 4mL of water under argon, heated to 80 deg.C, and stirred for reaction for 14 hours. Cooling, filtration through celite, collection of the filtrate, extraction with ethyl acetate (20mL × 3), combination of the organic phases, concentration under reduced pressure, purification by column chromatography with eluent system a afforded the title compound 1j (100mg), yield: 92 percent.
MS m/z(ESI):564.3[M+1]。
The seventh step: (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one I
Compound 1j (100mg, 0.17mmol) was dissolved in 20mL of dichloromethane, 1mL of trifluoroacetic acid was added dropwise thereto, and the reaction was stirred for 4 hours after the addition. The pH was adjusted to 7 with saturated sodium bicarbonate, extracted with dichloromethane (20mL × 2), the organic phases were combined, concentrated under reduced pressure, and purified by column chromatography with eluent system a to give the title compound 10(15mg), yield: 18 percent. The product is detected by X-ray powder diffraction, the compound is dissolved in tert-butanol solvent for amorphous form, and then is frozen and dried to obtain a sample, and the final product is amorphous by XRPD.
MS m/z(ESI):450.1[M+1]。
1H NMR(400MHz，CDCl3):δ8.33(d,1H)，7.72(s,1H)，7.48(d,1H)，7.41-7.33(m,3H)，7.28-7.24(m,1H)，7.18(s,1H)，6.92(d,1H)，6.51(s,1H)，6.32(d,1H)，5.17(dd,1H)，4.46(d,1H)，4.32(dd,1H)，4.27-4.17(m,3H)，3.82(s,3H)。
Test example 1: ERK1 enzymatic Activity assay
First, test purpose
The purpose of this experiment was to examine the inhibitory ability of compounds on the enzymatic activity of ERK1, according to IC50Size compounds were evaluated for in vitro activity. ADP-Glo was used for this experimentTMThe Kinase test Kit (Kinase Assay Kit) phosphorylates a substrate and generates ADP under the action of enzyme, an ADP-Glo reagent is added to remove unreacted ATP in a reaction system, and the ADP generated by the reaction is detected by a Kinase detection reagent (Kinase detection reagent). The inhibition rate of the compound is calculated by measuring the signal value in the presence of the compound.
Second, Experimental methods
Enzyme and substrate configuration: ERK1(1879-KS-010, R)&D) And substrate (AS-61777, anaspec) in buffer (40mM Tris, 20mM MgCl)20.1mg/mL BSA, 50 μ M DTT) were prepared at 0.75ng/μ L and 100 μ M, respectively, and then the enzyme solution and the substrate solution were mixed at a ratio of 2: 1 to prepare a mixed solution for later use. ATP was diluted to 300. mu.M with buffer, compounds were dissolved in DMSO to prepare a stock solution with an initial concentration of 20mM, and then Bravo (SGC120TH34702, Ag)ilent Technologies) to formulate compounds. Finally, 3. mu.L of the mixture of the substrate, 1. mu.L of the compound at different concentrations (starting at 50. mu.M, 4-fold dilution) were added sequentially to each well of the 384-well plate and incubated at 30 ℃ for 10 minutes, and finally 1. mu.L of 300. mu.M ATP solution was added to each well and incubated at 30 ℃ for 2 hours. Then 5. mu.L of ADP-Glo was added and incubated at 30 ℃ for 40 minutes, followed by 10. mu.L of Kinase detection buffer (Kinase detection buffer) and incubation at 30 ℃ for 40 minutes. The 384 well plate was removed and placed in a microplate reader (BMG labtech, PHERAStar FS) and chemiluminescence was measured using the microplate reader.
Third, data analysis
Data were processed and analyzed using Microsoft Excel, Graphpad Prism 5. To give IC of the Compound50The value was 5 nM. The results show that the compound of the formula I has obvious inhibition effect on the activity of ERK1 enzyme.
Test example 2: ERK2 enzymatic Activity assay
First, test purpose
The purpose of this experiment was to examine the inhibitory ability of compounds on the enzymatic activity of ERK2, according to IC50Size compounds were evaluated for in vitro activity. ADP-Glo was used for this experimentTMThe enzyme action of the enzyme, substrate is phosphorylated and ADP is generated, ADP-Glo Reagent is added to remove unreacted ATP in the reaction system, and the enzyme detection Reagent detects the generated ADP. The inhibition rate of the compound is calculated by measuring the signal value in the presence of the compound.
Second, Experimental methods
Enzyme and substrate configuration: ERK2(1230-KS-010, R)&D) And substrate (custom polypeptide, Gill Biochemical) in buffer (40mM Tris, 20mM MgCl)20.1mg/mL BSA, 50 μ M DTT) to 0.75ng/ul and 1500 μ M, and then the enzyme and substrate solutions were mixed at a ratio of 2: 1 to prepare a mixed solution for later use. ATP was diluted to 500uM with a buffer, and the compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Tantake, Co., Ltd.) to prepare a stock solution having an initial concentration of 20mM, and then the compound was prepared by Bravo. Finally, 3. mu.L of mixed solution of enzyme and substrate, 1. mu.L of different concentrations, were added to each well of the 384-well plateThe compound of (4-fold dilution, starting at a concentration of 50 uM) was incubated at 30 ℃ for 10 minutes, and finally 1. mu.L of 500uM ATP solution was added to each well and incubated at 30 ℃ for 2 hours. Then 5uL of ADP-Glo was added and incubated at 30 ℃ for 40 minutes, followed by 10uL of Kinase detection buffer and incubation at 30 ℃ for 40 minutes. The 384 well plate was taken out, placed in a microplate reader (BMG labtech, PHERAStar FS), and assayed by the microplate reader
Third, data analysis
Data were processed and analyzed using Microsoft Excel, Graphpad Prism 5. To give IC of the Compound50The value was 7 nM. The results show that the compound of the formula I has obvious inhibition effect on the activity of ERK2 enzyme.
Test example 3: in vitro Colo205 tumor cell proliferation inhibition assay with Compounds of formula I
First, test purpose
The purpose of this experiment was to examine the inhibitory activity of compounds on the proliferation of Colo205 cells (CCL-222, ATCC) in vitro. Treating cells with different concentrations of compounds in vitro, culturing for 3 days, and culturing with CTG: (
Lumi-nescent Cell visual Assay, Promega, cat #: g7573) The proliferation of cells was measured by the reagents and the in vitro activity of the compound was assessed according to the IC50 value.
Second, Experimental methods
In the following, the method for testing the inhibition of proliferation of Colo205 cells in vitro is used as an example to illustrate the method for testing the inhibition of proliferation of tumor cells in vitro by the compound of the present invention. The method is equally applicable, but not limited to, testing for proliferation inhibitory activity in vitro on other tumor cells.
Digesting Colo205, centrifuging, resuspending, mixing single cell suspension, adjusting viable cell density to 5.0 × 10 with cell culture medium (RPMI1640+ 2% FBS)4cells/mL, 95. mu.l/well in 96 well cell culture plates. Only 100ul of medium was added to the peripheral wells of the 96-well plate. The plates were incubated in an incubator for 24 hours (37 ℃, 5% CO)2)。
With DMSO (dimethyl)Sulfoxide, Shanghai Tantake technologies, Ltd.) dissolved compound to prepare a stock solution with an initial concentration of 20 mM. The initial concentration of the small molecular compound is 2mM, 4 times of dilution is carried out, 9 points are diluted, the tenth point is DMSO, another 96-well plate is taken, 90ul of cell culture solution (RPMI1640+ 2% FBS) is added into each well, 10ul of samples to be detected with different concentrations are added into each well, the samples to be detected are mixed evenly, 5 mu L of samples to be detected with different concentrations are added into the cell culture plate, and each sample has two multiple wells. The plates were incubated in an incubator for 3 days (37 ℃, 5% CO)2). The 96-well cell culture plate was removed, 50. mu.L of CTG solution was added to each well, and incubated at room temperature for 10 minutes. Chemiluminescence was measured in a microplate reader (BMG labtech, PHERAStar FS).
Third, data analysis
Data were processed and analyzed using Microsoft Excel, Graphpad Prism 5. The compounds of the formula I IC according to the invention50Was 62 nM.
Test example 4 mouse pharmacokinetic testing of the Compound of the invention
1. Abstract
The drug concentration in plasma of mice at different times after gavage administration of the compound of formula I was determined by LC/MS/MS method using mice as test animals. The pharmacokinetic behavior of the compounds of the invention in mice was studied and their pharmacokinetic profile was evaluated.
2. Test protocol
2.1 test drugs
A compound of formula I.
2.2 test animals
27C 57 mice, female, were divided into 3 groups on average, purchased from shanghai jequirity laboratory animals ltd, animal production license number: SCXK (Shanghai) 2013 and 0006.
2.3 pharmaceutical formulation
A certain amount of the compound was weighed, dissolved by adding 5% by volume of DMSO and 5% Tween 80, and then prepared into a colorless clear solution of 0.1mg/mL with 90% physiological saline.
2.4 administration
C57 mice were fasted overnight and then gavaged at 2mg/kg for each dose and at 0.2mL/10g for each volume.
3. Operation of
The mouse is administrated by gastric lavage, blood is collected for 0.1mL before and after administration at 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0 and 24.0 hours, the blood is placed in a heparinized test tube, blood plasma is separated after centrifugation at 3500 rpm for 10 minutes, and the blood plasma is stored at the temperature of minus 20 ℃.
Determination of the content of the test compound in the plasma of mice administered with different concentrations of the drug by injection: mu.L of mouse plasma at each time after administration was taken, 50. mu.L of camptothecin as an internal standard solution (100ng/mL) and 200. mu.L of acetonitrile were added, vortex mixed for 5 minutes, centrifuged for 10 minutes (4000 rpm), and 4. mu.L of supernatant was taken from the plasma samples for LC/MS/MS analysis.
4. The pharmacokinetic parameter results are as follows, showing that the compounds of formula I of the present disclosure have better pharmacokinetic absorption with pharmacokinetic advantages:
example 2: preparation of compound A crystal form of formula I
Amorphous compound of formula I (527mg, 1.17mmol) was added to 50mL of methyl t-butyl ether, slurried at room temperature and stirred for 72 hours, filtered, the filter cake was collected and dried in vacuo to give the title product (371mg, yield: 70%)
The product is defined as form A by X-ray powder diffraction detection, and an XRPD spectrum is shown in figure 1. The characteristic peak positions thereof are shown in table 1 below. The DSC spectrum showed an endothermic peak at 116.28 ℃. The TGA spectrum showed a loss of 5.83% before 150 ℃.
DVS testing showed that under normal storage conditions (i.e., 25 ℃, 60% RH), the sample had a moisture absorption weight gain of about 1.56%; under accelerated test conditions (i.e., 70% RH), the moisture pick-up weight is about 1.74%; under extreme conditions (90% RH), the hygroscopic weight gain was about 2.60%. The desorption process and the adsorption process of the sample substantially coincide during the humidity change of 0% -95% RH. And (4) after DVS detection, re-testing the crystal form, wherein the crystal form is not transformed.
TABLE 1 peak position of form A
| Peak number | 2 theta value [ ° or degree] | Relative strength% |
| 1 | 4.040 | 70.9 |
| 2 | 5.085 | 100.0 |
| 3 | 11.801 | 25.9 |
| 4 | 15.532 | 46.0 |
| 5 | 16.925 | 30.3 |
| 6 | 18.915 | 12.6 |
| 7 | 22.000 | 12.3 |
| 8 | 24.686 | 10.4 |
| 9 | 27.074 | 13.8 |
Example 3: preparation of compound A crystal form of formula I
Amorphous compound of formula I (60mg, 0.13mmol) was added to 5mL cyclopentane and slurried at room temperature with stirring for 72 hours, filtered, the filter cake was collected and dried in vacuo to give the title product (58mg, yield: 96.7%).
The product is a crystal form A through X-ray powder diffraction detection.
Example 4: preparation of compound A crystal form of formula I
Amorphous form of the compound of formula I (64mg, 0.14mmol) was added to 5mL isopropyl ether, slurried at room temperature with stirring for 72 hours, filtered, the filter cake was collected and dried in vacuo to give the title product (61mg, yield: 95.3%).
The product is crystal form A through X-ray powder diffraction detection.
Example 5: preparation of compound A crystal form of formula I
Amorphous compound of formula I (67mg, 0.15mmol) was added to 5mL of phenetole, slurried at room temperature with stirring for 72 hours, filtered, the filter cake was collected and dried in vacuo to give the title product (40mg, yield: 59.7%).
The product is crystal form A through X-ray powder diffraction detection.
Example 6: preparation of compound A crystal form of formula I
Amorphous form of the compound of formula I (1.2g, 2.67mmol) was added to 15mL isopropyl acetate, slurried at room temperature for 72 hours, filtered, the filter cake collected, and dried in vacuo to give the title product (732mg, yield: 61.0%).
The product is crystal form A through X-ray powder diffraction detection.
Example 7: preparation of compound A crystal form of formula I
10mg of the compound of formula I is amorphous, 0.3mL of dichloromethane is added to dissolve and clarify, 0.6mL of isopropyl ether is added, the initial temperature is 60 ℃, stirring is carried out for 2h, the temperature is slowly reduced to 20 ℃, the constant temperature is kept, centrifugation and vacuum drying are carried out to obtain the title product, and the product is detected by X-ray powder diffraction and is the crystal form A.
Example 8: preparation of compound A crystal form of formula I
10mg of the compound of formula I is amorphous, 0.2mL of acetonitrile is added for dissolution and clarification, 0.6mL of isopropyl ether is added, the initial temperature is 60 ℃, the mixture is stirred for 2h, the temperature is slowly reduced to 20 ℃, the constant temperature is kept, centrifugation and vacuum drying are carried out to obtain the title product, and the product is detected by X-ray powder diffraction and is the crystal form A.
Example 9: preparation of compound A crystal form of formula I
10mg of the compound of formula I is amorphous, dissolved in 50. mu.L of ethyl acetate, added with 1mL of isopropyl ether, stirred overnight at room temperature or 50 ℃, centrifuged, and dried under vacuum to give the title product, which is crystalline form A as detected by X-ray powder diffraction.
Example 10: preparation of compound A crystal form of formula I
10mg of the compound of formula I was amorphous, dissolved in 50. mu.L dioxane, added 1mL methyl tert-butyl ether, stirred overnight at 50 ℃, centrifuged, and dried under vacuum to give the title product as form A by X-ray powder diffraction.
Example 11: preparation of compound A crystal form of formula I
10mg of the compound of formula I was amorphous, dissolved in 50. mu.L of ethyl acetate, added 1mL of methyl tert-butyl ether, stirred overnight, centrifuged, and dried under vacuum to give the title product as form A by X-ray powder diffraction.
Example 12: preparation of compound A crystal form of formula I
10mg of the compound of formula I was amorphous, dissolved in 50. mu.L of butanone, added 1mL of methyl tert-butyl ether, stirred overnight at 50 ℃, centrifuged, and dried under vacuum to give the title product as form A by X-ray powder diffraction.
Example 13: preparation of compound A crystal form of formula I
10mg of the compound of formula I was amorphous, dissolved in 50. mu.L tetrahydrofuran, added 1mL of methyl tert-butyl ether, stirred overnight at 50 ℃, centrifuged, and dried under vacuum to give the title product as form A by X-ray powder diffraction.
Example 14: preparation of compound A crystal form of formula I
10mg of the compound of formula I is amorphous, dissolved in 50. mu.L of tetrahydrofuran, added 1mL of isopropyl ether, stirred overnight at 50 ℃, centrifuged, and dried under vacuum to give the title product, which is crystalline form A as detected by X-ray powder diffraction.
Example 15: preparation of compound A crystal form of formula I
10mg of the compound of formula I is amorphous, dissolved in 50. mu.L of acetone, added 1mL of isopropyl ether, stirred overnight at 50 ℃, centrifuged, and dried under vacuum to give the title product, which is form A as detected by X-ray powder diffraction.
Example 16: preparation of compound A crystal form of formula I
10mg of the compound of formula I is amorphous, 50. mu.L of dioxane is added for dissolution, 1mL of isopropyl ether is added, stirring is carried out at 50 ℃ overnight, centrifugation and vacuum drying are carried out to obtain the title product, and the product is detected by X-ray powder diffraction to be crystal form A.
Example 17: preparation of compound B crystal form of formula I
Amorphous form of the compound represented by the formula I (60mg, 0.13mmol) was added to a mixed solvent of 1mL of acetone and n-hexane (V: V ═ 1:3), and the mixture was stirred at room temperature for 72 hours, filtered, the cake was collected and dried in vacuo to obtain the title product (47mg, yield: 78.3%).
The product is defined as form B by X-ray powder diffraction detection, and the XRPD spectrum is shown in figure 2.
The DSC spectrum shows that the peak value of the endothermic peak is 123.81 ℃; the TGA spectrum showed a loss of weight of 1.72% before 150 ℃.
The DVS assay showed that under normal storage conditions (i.e., 25 ℃, 60% RH), the sample had a moisture absorption weight gain of about 0.926%; under accelerated test conditions (i.e., 70% RH), the moisture pick-up weight gain was about 1.021%; under extreme conditions (90% RH), the hygroscopic weight gain was about 1.432%. The desorption process and the adsorption process of the sample substantially coincide during the humidity change of 0% -95% RH. And (4) after DVS detection, re-testing the crystal form, wherein the crystal form is not transformed.
TABLE 2 peak positions of form B
| Peak number | 2 theta value [ ° or degree] | Relative strength% |
| 1 | 13.128 | 100 |
| 2 | 13.773 | 82.1 |
| 3 | 14.418 | 65.8 |
| 4 | 17.645 | 98.8 |
| 5 | 19.482 | 58.5 |
| 6 | 24.793 | 43.4 |
| 7 | 27.573 | 43.7 |
Example 18: preparation of compound B crystal form of formula I
Amorphous compound of formula I (55mg, 0.12mmol) was added to 0.5mL ethyl acetate, slurried at room temperature for 72 hours, filtered, the filter cake was collected and dried in vacuo to give the title product (3mg, yield: 6.4%).
The product is crystal form B by X-ray powder diffraction detection.
Example 19: preparation of compound B crystal form of formula I
Amorphous form of the compound of formula I (1.2g, 2.67mmol) was added to 8mL ethyl propionate, slurried at room temperature for 144 hours, filtered, the filter cake collected, and dried in vacuo to give the title product (0.75g, yield: 62.5%).
The product is crystal form B by X-ray powder diffraction detection.
Example 20: preparation of compound B crystal form of formula I
Dissolving 10mg of the compound shown in the formula I in 50 mu L of butanone in an amorphous state, adding 1mL of n-hexane, stirring at normal temperature overnight, centrifuging, and drying in vacuum to obtain the title product, wherein the product is detected by X-ray powder diffraction to be crystal form B.
Example 21: preparation of compound C crystal form of formula I
69mg of the compound of the formula I are added in amorphous form to 5mL of n-pentane and slurried at room temperature for 3 days. Filtering to obtain a pale yellow solid 67mg, and detecting by X-ray powder diffraction, wherein the product is defined as a C crystal form. The XRPD pattern of the form C sample is shown in fig. 3, with characteristic peak positions shown in table 3 below. The DSC spectrum shows that the peak values of the endothermic peaks are 64.13 ℃ and 93.98 ℃; the TGA spectrogram shows that the weight loss is 3.18 percent at the temperature of 30-230 ℃ and 32.32 percent at the temperature of 230-340 ℃.
TABLE 3 peak position of form C
| Peak number | 2 theta value [ ° or degree] | Relative strength% |
| 1 | 17.857 | 100.0 |
Example 22: preparation of compound D crystal form of formula I
And (3) adding 1mL of acetonitrile into 100mg of a compound shown as the formula I to dissolve the amorphous compound, adding 10mL of isopropyl ether, stirring at 50 ℃ overnight, centrifuging, drying in vacuum to obtain a target product, and detecting by X-ray powder diffraction, wherein the product is defined as a D crystal form. The XRPD pattern of the form D sample is shown in fig. 4, with characteristic peak positions shown in table 4 below. The DSC spectrum shows that the peak value of the endothermic peak is 125.32 ℃; the TGA spectrum shows that the weight loss is 2.79 percent at the temperature of 30-120 ℃.
TABLE 4 peak position of form D
| Peak number | 2 theta value [ ° or degree] | Relative strength% |
| 1 | 4.462 | 63.3 |
| 2 | 5.228 | 100.0 |
| 3 | 7.224 | 11.6 |
| 4 | 9.015 | 13.5 |
| 5 | 10.408 | 14.7 |
| 6 | 11.503 | 23.1 |
| 7 | 12.476 | 25.8 |
| 8 | 13.331 | 38.9 |
| 9 | 13.924 | 38.1 |
| 10 | 14.836 | 13.6 |
| 11 | 16.031 | 68.5 |
| 12 | 16.677 | 35.4 |
| 13 | 17.308 | 18.1 |
| 14 | 18.915 | 11.8 |
| 15 | 24.487 | 9.8 |
Example 23: preparation of Compound E Crystal form of formula I
And (3) adding 50 mu L of dichloromethane into 10mg of the compound shown in the formula I to dissolve the amorphous compound, adding 1mL of isopropyl ether, stirring overnight at 50 ℃, centrifuging, drying in vacuum to obtain a target product, and detecting by X-ray powder diffraction, wherein the product is defined as an E crystal form. The XRPD pattern of the E form sample is shown in fig. 5, with characteristic peak positions shown in table 5 below. The DSC spectrum shows that the peak value of the endothermic peak is 116.28 ℃; the TGA spectrogram shows that the weight loss is 0.81 percent at the temperature of 0-60 ℃ and 4.77 percent at the temperature of 60-140 ℃.
TABLE 5 peak position of the E crystal form
Example 24: study on stability of crystal form influencing factors of compound A of formula I
The compound of formula I, prepared according to the previous examples, as form a (99.22% pure) was subjected to the conditions of the experiment of table 6 for stability test and the results are shown in table 6 below.
TABLE 6 stability of the influencing factors of the form A
The experimental result shows that the crystal form A of the compound shown in the formula I is placed for 30 days under the conditions of illumination, high temperature of 40 ℃, high temperature of 60 ℃, high humidity of 75% RH and high humidity of 92.5% RH, and a crystal form A sample has good physical and chemical stability.
Example 25: research on stability of crystal form B influencing factor of compound I
The compound of formula I in form B (98.97% pure) prepared according to the previous examples was subjected to the experimental conditions of table 7 for stability test and the results are shown in table 7 below.
TABLE 7 stability of the influencing factors of the form B
The experimental result shows that the crystal form B of the compound shown in the formula I is placed for 30 days under the conditions of illumination, high temperature of 40 ℃, high temperature of 60 ℃, high humidity of 75% RH and high humidity of 92.5% RH, and a crystal form B sample has good physical and chemical stability.
Example 26: study on stability of crystal form D influencing factor of compound of formula I
The compound of formula I in form D (99.58% pure) prepared by reference to the foregoing examples was subjected to the experimental conditions of table 8 for stability testing, and the results are shown in table 8 below.
TABLE 8 stability of the influencing factors of the D form
The experimental result shows that the crystal form D of the compound shown in the formula I is placed for 30 days under the conditions of illumination, high temperature of 40 ℃, high temperature of 60 ℃, high humidity of 75% RH and high humidity of 92.5% RH, and the sample of the crystal form D has good physical and chemical stability.
Example 27: study on stability of crystal form influencing factor of compound E in formula I
The compound of formula I, prepared according to the previous examples, as form E (99.43% pure), was subjected to the conditions of the experiment of table 9 for stability test and the results are shown in table 9 below.
The experimental result shows that the crystal form E of the compound shown in the formula I is placed for 30 days under the conditions of illumination, high temperature of 40 ℃, high temperature of 60 ℃, high humidity of 75% RH and high humidity of 92.5% RH, and the sample of the crystal form E has good physical and chemical stability.
Example 28: stability study of crystal form A of compound I under long-term acceleration condition
The compound of formula I in crystal form a prepared according to the previous examples was left at 25 ℃, 60% RH and 40 ℃, 75% RH, respectively, and the stability was examined under dark conditions, with the results shown in table 10.
TABLE 10 stability of form A under long-term acceleration conditions
The physical and chemical properties of the base A crystal form are good when the crystal form is placed for 1 month under the condition of long-term accelerated stability.
Example 29: stability study of B crystal form of compound of formula I under long-term acceleration condition
The compound of formula I prepared according to the previous examples in form B was left alone at 25 ℃, 60% RH and 40 ℃, 75% RH, respectively, and the stability was checked in the dark, with the results shown in table 11:
TABLE 11 stability of form B under long-term acceleration conditions
The results indicate that the sample of form B of the compound of formula I is physically and chemically stable for 6 months under long-term (25 ℃, 60% RH), accelerated (40 ℃, 75% RH) conditions.
Example 30: stability study of form D of compound of formula I under long-term acceleration conditions
The base D crystal form was examined for stability under the conditions of 5 ℃, 25 ℃, 60% RH, and 40 ℃, 75% RH, respectively, and the results are shown in Table 12:
TABLE 12 stability of form D under long-term acceleration conditions
Experiments show that the physical and chemical stability of the crystal form D of the compound in the formula I is better after being placed for 3 months under the condition of long-term accelerated stability.
Example 31: stability study of E crystal form of compound of formula I under long-term acceleration condition
The base E crystal form was subjected to conditions of 25 ℃, 60% RH and 40 ℃, 75% RH, respectively, to examine the stability, and the results are shown in Table 13:
TABLE 13 stability of form E under long-term acceleration conditions
Experiments show that the compound E crystal form of the formula I has better physical and chemical stability after being placed for 1 month under the condition of long-term accelerated stability.
Claims (14)
1. Crystalline form A of the compound (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one, an X-ray powder diffraction pattern expressed in diffraction angle 2 theta angles, x-ray powder diffraction patterns having characteristic peaks at 4.040, 5.085, 11.801, 15.532 and 16.925, preferably at 4.040, 5.085, 11.801, 15.532, 16.925, 18.915 and 27.074, more preferably at 4.040, 5.085, 11.801, 15.532, 16.925, 18.915, 22.000, 24.686 and 27.074, and most preferably at diffraction angle 2 θ are shown in fig. 1.
2. A process for preparing the crystalline form a of claim 1, comprising:
the method comprises the following steps:
(a) mixing (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one with a solvent (I) selected from at least one of methyl tert-butyl ether, cyclopentane, isopropyl ether, phenetole, or isopropyl acetate;
(b) pulping and crystallizing;
or, the second method:
(a) mixing (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one with a solvent (II) selected from at least one of ethyl acetate, dichloromethane, acetonitrile, trichloromethane, dioxane, butanone, tetrahydrofuran, or acetone, and dissolving with stirring or heating;
(b) adding a solvent (III) and crystallizing, wherein the solvent (III) is at least one selected from isopropyl ether and methyl tert-butyl ether.
3. The compound (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one in crystal form B has an X-ray powder diffraction pattern expressed in terms of diffraction angle 2 θ, having characteristic peaks at 13.128, 13.773, 14.418, 17.645 and 19.482, preferably characteristic peaks at 13.128, 13.773, 14.418, 17.645, 19.482, 24.793 and 27.573, more preferably an X-ray powder diffraction pattern expressed in terms of diffraction angle 2 θ as shown in fig. 2.
4. A process for preparing the form B of claim 3, comprising:
the method comprises the following steps:
(a) mixing (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one with a solvent (IV) selected from at least one of ethyl acetate, ethyl propionate, acetone, n-hexane;
(b) pulping and crystallizing;
or, the second method:
(a) mixing (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one with a solvent (V) selected from at least one of acetone and butanone;
(b) adding n-hexane, and crystallizing.
5. The compound (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-C ] imidazol-3-one in crystalline form C has an X-ray powder diffraction pattern expressed in terms of diffraction angle 2 theta, with a characteristic peak at 17.857, preferably an X-ray powder diffraction pattern expressed in terms of diffraction angle 2 theta as shown in fig. 3.
6.A crystalline form D of the compound (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one, an X-ray powder diffraction pattern expressed in terms of diffraction angle 2 θ, an X-ray powder diffraction pattern having characteristic peaks at 4.462, 5.228, 12.476, 13.331, 13.924, 16.031 and 16.677, preferably at 4.462, 5.228, 11.503, 12.476, 13.331, 13.924, 16.031, 16.677 and 17.308, more preferably at 4.462, 5.228, 9.015, 10.408, 11.503, 12.476, 13.331, 13.924, 16.031, 16.677 and 17.308, and most preferably expressed in a diffraction angle 2 θ angle is shown in fig. 4.
7. A process for preparing the form D of claim 6, comprising:
(a) mixing (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one with acetonitrile;
(b) adding isopropyl ether, stirring and crystallizing.
8. The compound (S) -2- (1- (3-chlorophenyl) -2-hydroxyethyl) -6- (2- ((1-methyl-1H-pyrazol-5-yl) amino) pyrimidin-4-yl) -1, 2-dihydro-3H-pyrrolo [1,2-c ] imidazol-3-one in crystal form E has an X-ray powder diffraction pattern expressed in terms of diffraction angle 2 theta with characteristic peaks at 5.216, 11.834, 15.595 and 17.199, preferably an X-ray powder diffraction pattern expressed in terms of diffraction angle 2 theta as shown in fig. 5.
9. The crystalline form according to any one of claims 1,3, 5, 6 or 8, the 2 Θ angle error range being ± 0.30, preferably ± 0.20.
10.A pharmaceutical composition prepared from the crystalline form of any one of claims 1,3, 5, 6, 8, or 9.
11. A pharmaceutical composition comprising the crystalline form of any one of claims 1,3, 5, 6, 8 or 9 or prepared by the process of claim 2, 4 or 7, and optionally a pharmaceutically acceptable carrier, diluent or excipient.
12. A process for the preparation of a pharmaceutical composition comprising the step of admixing the crystalline form of any one of claims 1,3, 5, 6, 8 or 9 or prepared by the process of any one of claims 2, 4 or 7 with a pharmaceutically acceptable carrier, diluent or excipient.
13. Use of the crystalline form according to any one of claims 1,3, 5, 6, 8 or 9, or the crystalline form prepared by the process of any one of claims 2, 4 or 7, or the composition of any one of claims 10 or 11, or the composition prepared by the process of claim 12, for the preparation of a medicament for inhibiting ERK.
14. Use of the crystalline form of any one of claims 1,3, 5, 6, 8 or 9, the crystalline form prepared by the process of claim 2, 4 or 7, or the composition of claim 10 or 11, or the composition prepared by the process of claim 12, for the manufacture of a medicament for the treatment or prevention of cancer, inflammation, or other proliferative disease, preferably cancer; the cancer is selected from melanoma, liver cancer, kidney cancer, lung cancer, nasopharyngeal carcinoma, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, cervical cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma, astrocytoma, and glioma.
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| WO2023185869A1 (en) * | 2022-03-28 | 2023-10-05 | 江苏恒瑞医药股份有限公司 | Crystalline form of fumarate of pyrroloheterocyclic derivative and preparation method therefor |
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