CN114315837B - Crystalline forms of ERK inhibitors and methods of making the same - Google Patents
Crystalline forms of ERK inhibitors and methods of making the same Download PDFInfo
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Abstract
The present disclosure provides crystalline forms of an ERK inhibitor and methods of making the same. Specifically, 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]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 in vivo cell signaling pathways. Mitogen-activated protein kinases (MAPKs) play a very important role in the signaling pathway, and extracellular signal-regulated kinases (extracellular signal regulated kinase, ERK) are a member of the MAPK family. Through RAS-RAF-MEK-ERK step, exogenous stimulation signal is transferred to ERK, activated ERK is transferred into cell nucleus to regulate transcription factor activity, so as to regulate cell proliferation, differentiation, apoptosis and other biological functions, or through phosphorylation of cytoskeletal component in cytosol to participate in regulation of cell morphology and redistribution of cytoskeletal.
RAS and RAF gene mutation causes continuous activation of MAPK-ERK signal pathway, promotes malignant transformation and abnormal proliferation of cells, and finally generates tumor (Roberts PJ et al, oncogene,2007, 26 (22), 3291-3310). The combination of a MEK inhibitor with a B-RAF inhibitor can further increase the tumor growth inhibiting effect of the B-RAF inhibitor and can significantly increase the disease-free progression and overall survival of melanoma patients carrying the BRAF 600E and V600K mutations (Frederick DT et al, clinical Cancer Research,2013.19 (5), 1225-1231). Although the B-RAF/MEK inhibitor combination may have a tumor-inhibiting effect, their efficacy is short-lived, and most patients develop resistance within 2-18 months, and the tumor is further exacerbated. The mechanism of resistance to B-RAF/MEK inhibitors is very complex and is mostly directly related to 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 for patients with mutations in the MAPK signaling pathway, but also for patients with B-RAF/MEK inhibitor resistance.
The B-RAF/MEK inhibitor can inhibit the growth of tumor and regulate the immune microenvironment of the tumor. The B-RAF/MEK inhibitor can enhance the expression of tumor specific antigen, improve the recognition and killing of antigen specific T cells on tumors, and promote the migration and infiltration of immune cells. In animal models, PD-L1 expression in tumor tissues is enhanced after treatment with B-RAF/MEK inhibitors, and when combined with antibodies to checkpoint (checkpoint) molecules (e.g., PD-1 antibodies, CTLA4 antibodies), they further show an effect of inhibiting tumor growth over B-RAF/MEK inhibitors alone (Boni a et al, cancer Research,2010, 70 (13), 5213-5219). Research shows that ERK inhibitor is similar to B-RAF/MEK inhibitor, and can regulate tumor microenvironment, raise the function of cytotoxic T cell and inhibit tumor growth.
PCT/CN2020/081591 provides an ERK inhibitor, which is chemically named (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 good pharmaceutical activity, and is expected to provide new therapeutic options 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 cause a change in the crystal structure of the compound, and may be accompanied by other forms of crystal form. Generally, amorphous pharmaceutical products have no regular crystal structure, and often have the defects of poor product stability, finer crystallization, difficult filtration, easy caking, poor flowability and the like. In view of the importance of solid pharmaceutical forms and their stability in clinical treatment, intensive studies of the crystalline form 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 have important significance for the development of drugs suitable for industrial production and having good bioactivity.
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), having an X-ray powder diffraction spectrum expressed in terms of diffraction angle 2θ, with characteristic peaks at 4.040, 5.085, 11.801, 15.532 and 16.925.
In some embodiments, the X-ray powder diffraction pattern of form a, diffraction angle 2θ, of the compound of formula I, has 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, form a, has an X-ray powder diffraction spectrum with diffraction angle 2θ, having 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 X-ray powder diffraction spectrum of form a of the compound of formula I, expressed in terms of diffraction angle 2θ, is shown in figure 1.
The present disclosure also provides a process for preparing a crystalline form of compound a 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 (I) selected from at least one of methyl tert-butyl ether, cyclopentane, isopropyl ether, phenetole or isopropyl acetate;
(b) Pulping and crystallizing;
alternatively, method two:
(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), stirring or heating to dissolve, wherein the solvent (II) is selected from at least one of ethyl acetate, dichloromethane, acetonitrile, chloroform, dioxane, butanone, tetrahydrofuran, and acetone;
(b) Adding a solvent (III), and crystallizing, wherein the solvent (III) is at least one selected from isopropyl ether and methyl tertiary butyl ether.
The volume (μl) used for the solvents (I), (II), (III) of the present disclosure may be 1-200 times the mass (mg) of the compound of formula I, 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 a compound of formula (I) having an X-ray powder diffraction spectrum, expressed as diffraction angle 2θ, with characteristic peaks at 13.128, 13.773, 14.418, 17.645 and 19.482.
In some embodiments, the X-ray powder diffraction pattern of form B of the compound of formula I has characteristic peaks at 2θ angles 13.128, 13.773, 14.418, 17.645, 19.482, 24.793 and 27.573.
In some embodiments, the X-ray powder diffraction spectrum of form B of the compound of formula I, expressed in terms of diffraction angle 2θ, is shown in fig. 2.
The present disclosure also provides a process for preparing crystalline form B 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 (IV), the solvent (IV) selected from at least one of ethyl acetate, ethyl propionate, acetone, n-hexane;
(b) Pulping and crystallizing;
alternatively, method two:
(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), said solvent (V) selected from at least one of acetone and butanone;
(b) N-hexane is added for crystallization.
The solvents (IV), (V) of the present disclosure may be used in a volume (μl) of 1 to 200 times the mass (mg) of the compound of formula I, 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 C of the compound of formula (I) having an X-ray powder diffraction spectrum, expressed as diffraction angle 2θ, with a characteristic peak at 17.857.
In some embodiments, the X-ray powder diffraction spectrum of form C of the compound of formula I, expressed in terms of diffraction angle 2θ, is shown in fig. 3.
The present disclosure also provides a process for preparing form C of a 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 (μl) used for n-pentane as described in the present disclosure may be 1-200 times the mass (mg) of the compound of formula I, 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 D-form of a compound of formula I having an X-ray powder diffraction spectrum, expressed as diffraction angle 2θ, with characteristic peaks at 4.462, 5.228, 12.476, 13.331, 13.924, 16.031 and 16.677.
In some embodiments, the compound of formula I, form D, has an X-ray powder diffraction spectrum with diffraction angles expressed in terms of 2θ, 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, form D, has an X-ray powder diffraction spectrum with diffraction angle 2θ, 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 X-ray powder diffraction spectrum of form D of the compound of formula I, expressed in terms of diffraction angle 2θ, is shown in fig. 4.
The present disclosure also provides a process for preparing 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) Isopropyl ether was added thereto, and stirred at 50℃for crystallization.
In some embodiments, the volume (μl) used for acetonitrile, isopropyl ether may be 1-200 times the mass (mg) of the compound of formula I, 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 an E-form of a compound of formula I having an X-ray powder diffraction spectrum in terms of diffraction angle 2θ, with characteristic peaks at 5.216, 11.834, 15.595 and 17.199.
In some embodiments, the X-ray powder diffraction spectrum of form E of the compound of formula I, expressed in terms of diffraction angle 2θ, is shown in fig. 5.
The present disclosure also provides a process for preparing form E of a 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) Isopropyl ether was added thereto, and stirred at 50℃for crystallization.
In some embodiments, the volume (μl) used for the dichloromethane, isopropyl ether may be 1-200 times the mass (mg) of the compound of formula I, 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 crystalline forms of the present invention further comprise steps of filtration, washing, or drying.
The present disclosure also provides a pharmaceutical composition prepared from any of the foregoing crystalline forms.
The present disclosure also provides a pharmaceutical composition comprising a crystalline form of a compound of formula I as described above or a crystalline form prepared by the process described above, and optionally from a pharmaceutically acceptable carrier, diluent or excipient.
The present disclosure also provides a method of preparing a pharmaceutical composition comprising the step of mixing a crystalline form of a compound of formula I, or a crystalline form prepared by the method described above, with a pharmaceutically acceptable carrier, diluent or excipient.
The disclosure also provides the use of the crystalline form of the compound of formula I or the crystalline form of the compound of formula I prepared by the method or the composition prepared by the method for preparing the 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 the method described above or a composition as described above or a composition prepared by the method described above in the manufacture of a medicament for the treatment or prevention of cancer, inflammation, or other proliferative diseases.
In alternative embodiments, the cancer described in the present disclosure is selected from the group consisting of 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 "2θ or 2θ angle" described in the present disclosure refers to a diffraction angle, θ is a bragg angle, and the unit is ° or degree; the error range of each characteristic peak 2 theta is +/-0.30, alternatively +/-0.20, and can be specifically-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 and 0.20.
The term "differential scanning calorimetric analysis or DSC" in the present disclosure refers to measuring the temperature difference and the heat flow difference between a sample and a reference object during the temperature rising or constant temperature process of the sample, so as to characterize all physical changes and chemical changes related to thermal effects, and obtain phase change information of the sample.
The drying temperature in the present disclosure is generally 25 to 100 ℃, preferably 40 to 70 ℃, and the drying temperature can be either normal pressure drying or reduced pressure drying.
"pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically acceptable salt or prodrug thereof, and other chemical components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.
Crystallization described in this disclosure includes, but is not limited to, stirring, cooling, concentrating, volatilizing, beating, and crystallization.
Drawings
Fig. 1: XRPD spectrum of compound a crystalline form of formula I.
Fig. 2: XRPD spectrum of compound B crystalline form of formula I.
Fig. 3: XRPD spectrum of compound C crystalline form of formula I.
Fig. 4: XRPD spectrum of compound D crystalline form of formula I.
Fig. 5: XRPD spectrum of compound E crystalline form of formula I.
Detailed Description
The present invention will be explained in more detail with reference to examples or experimental examples, which are only for illustrating the technical aspects of the present invention, and do not limit the spirit and scope of the present invention.
The reagents used in the present invention are commercially available.
Test conditions of the instrument used for the experiment in the invention:
1. differential scanning calorimeter (Differential Scanning Calorimeter DSC)
Instrument model: mettler Toledo DSC 3+
Sweep gas: nitrogen gas; nitrogen purge rate: 50mL/min
Rate of temperature rise: 10.0 ℃/min
Temperature range: 25-350 DEG C
2. X-ray powder diffraction spectrum (X-ray Powder Diffraction, XRPD)
Instrument model: bruker D8 Discover X-ray powder diffractometer
Rays: monochromatic Cu-ka radiation (λ=1.5418)
Scanning mode: θ/2θ, scan range (2θ range): 5-50 DEG
Voltage: 40kV, current: 40mA
3. Thermogravimetric analyzer (Thermogravimetric Analysis, TGA)
Instrument model: mettler Toledo TGA2
Sweep gas: nitrogen gas; nitrogen purge rate: 50mL/min
Rate of temperature rise: 10.0 ℃/min
Temperature range: 25-350 DEG C
4. DVS is dynamic moisture adsorption: SMS DVS Advantage is adopted for detection, the humidity change is 50% -95% -0% -95% -50% at 25 ℃, the step is 10% (5% in the last step), and the judgment standard is dm/dt is not more than 0.002%.
5. And (3) purity detection: high performance liquid chromatography detection; instrument model: agilent 1200DAD; chromatographic column: phenomenex kinetex EVOC18 4.6 x 250mm,5um; mobile phase a: KH (KH) 2 PO 4 Mobile phase B: acetonitrile; flow rate: 1.0mL/min; column temperature: 40 ℃; detection wavelength: 214nm.
6. The eluent system for column chromatography and the developing agent system for thin layer chromatography used for purifying the compound include: 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 -6 Units of (ppm) are given. NMR was performed using Bruker AVANCE-400 nuclear magnetic resonance apparatus with deuterated dimethyl sulfoxide (DMSO-d) 6 ) Deuterated chloroform (CDCl) 3 ) Deuterated methanol (CD) 3 OD), internal standard is Tetramethylsilane (TMS).
MS was measured using an Agilent 1200/1290DAD-6110/6120 Quadrapol MS liquid chromatography-mass spectrometry (manufacturer: agilent, MS model: 6110/6120 Quadrapol MS), waters ACQuity UPLC-QD/SQD (manufacturer: waters, MS model: waters ACQuity Qda Detector/waters SQ Detector), THERMO Ultimate 3000-Q actual (manufacturer: THERMO, MS model: THERMO Q Exactive).
Example 1: preparation 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 of formula I
The first step: (S) -2- ((tert-Butyldimethylsilanyloxy) -1- (3-chlorophenyl) ethanamine 1b
(S) -2-amino-2- (3-chlorophenyl) ethanol 1a (4 g,23.3mmol, shanghai Bi-pharmaceutical technologies Co., ltd.) was dissolved in 80mL of methylene chloride, and t-butyldimethylchlorosilane (5.2 g,35 mmol) was added under ice bath and stirred for 14 hours. Water was added thereto, 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, and the filtrate was concentrated under reduced pressure, and purified by column chromatography with eluent system C to give the title compound 1b (6.5 g), yield: 97%.
MS m/z(ESI):286.1[M+1]。
And a second step of: (S) -N- ((4-bromo-1H-pyrrol-2-yl) methyl) -2- ((tert-butyldimethylsilyl) oxy) -1- (3-chlorophenyl) ethanamine 1d
4-bromo-1H-pyrrole-2-carbaldehyde 1c (2.37 g,13.62mmol, shanghai Bi) and Compound 1b (3.9 g,13.64 mmol) were reacted for 3 hours with stirring. 100mL of methanol was added for dilution, the temperature was lowered to 0℃and sodium borohydride (516 mg,13.64 mmol) was added for reaction with stirring for 2 hours. Water was added thereto, and the reaction mixture was concentrated under reduced pressure, followed by extraction with ethyl acetate (40 mL. Times.3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and purified by column chromatography with eluent system C to give the title compound 1d (4.8 g), yield: 79%.
MS m/z(ESI):444.2[M+1]。
And a third step of: (S) -6-bromo-2- (2- ((tert-butyldimethylsilyl) oxy) -1- (3-chlorophenyl) ethyl) -1H-pyrrolo [1,2-c ] imidazol-3 (2H) -one 1e
Compound 1d (4.8 g,10.81 mmol) was dissolved in 100mL of tetrahydrofuran, N' -carbonyldiimidazole (2.45 g,15.11 mmol) was added under ice-bath stirring for 0.5 hours, sodium hydride (60%, 6271 mg,16.22 umol) was added, and the reaction was stirred 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 (4.0 g), yield: 78%.
MS m/z(ESI):469.1[M+1]。
Fourth step: (S) -2- (2- ((tert-Butyldimethylsilanyloxy) -1- (3-chlorophenyl) ethyl) -6- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrrolo [1,2-c ] imidazol-3 (2H) -one 1f
Compound 1e (4.0 g,8.51 mmol) was dissolved in 50mL of 1, 4-dioxane under argon, 4', 5' -octamethyl-2, 2 '-bis (1, 3, 2-dioxaborolane) (3.24 g,12.76 mmol), potassium acetate (3.34 g,34.04 mmol) and [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (1.24 g,1.70 mmol) were added sequentially and stirred at 90℃for 2 hours. Cooled, filtered through celite, the filtrate concentrated and purified by column chromatography with eluent system C to give the title compound 1f (2.0 g), yield: 45%.
MS m/z(ESI):517.2[M+1]。
Fifth step: 4-chloro-N- (1-methyl-1H-pyrazol-5-yl) pyrimidin-2-amine 1i
N- (1-methyl-1H-pyrazol-5-yl) carboxamide (324.82 mg,2.60mmol, prepared as disclosed in patent application "WO 2017/80979") was dissolved in 15mL of N, N-dimethylformamide, sodium hydride (60%, 311.47mg,7.79 mmol) was added at 0deg.C, the reaction was stirred for 0.5 hours, and 1g (500 mg,2.60 mmol) of 4-chloro-2- (methylsulfonyl) pyrimidine was added, and the reaction was continued for 2 hours. 20mL of water, ethyl acetate extraction (20 mL. Times.3) and concentration of the combined organic phases under reduced pressure, and purification of the resulting residue by thin layer chromatography with developer system C gave the title compound 1i (270 mg), yield: 49.6%.
MS m/z(ESI):210.3[M+1]。
Sixth step: (S) -2- (2- ((tert-Butyldimethylsilanyloxy) -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
Compound 1f (98.6 mg,0.19 mmol) was suspended in 20mL 1, 4-dioxane and 4mL water under argon, and the mixture of 4-chloro-N- (1-methyl-1H-pyrazol-5-yl) pyrimidin-2-amine 1i, [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (28 mg,0.02 mmol), cesium carbonate (124 mg,0.2 mmol) was heated to 80 ℃ and stirred for 14 hours. Cooled, filtered through celite, the filtrate was collected, extracted with ethyl acetate (20 ml×3), the organic phases were combined, concentrated under reduced pressure, and purified by column chromatography with eluent system a to give the title compound 1j (100 mg), yield: 92%.
MS m/z(ESI):564.3[M+1]。
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 (100 mg,0.17 mmol) was dissolved in 20mL of dichloromethane, 1mL of trifluoroacetic acid was added dropwise, and the reaction was stirred for 4 hours after the addition. The pH was adjusted to 7 with saturated sodium bicarbonate, extracted with dichloromethane (20 mL. Times.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 (15 mg), yield: 18%. The product is detected by X-ray powder diffraction, the compound is dissolved in a tertiary butanol solvent to be amorphous, then the sample is obtained by freeze drying, and the final product is detected to be amorphous by XRPD.
MS m/z(ESI):450.1[M+1]。
1H NMR(400MHz，CDCl 3 ):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 test
1. Purpose of testing
The purpose of this experiment was to test compounds for their ability to inhibit ERK1 enzyme activity, according to IC 50 Size the compounds were evaluated for in vitro activity. ADP-Glo was used in this experiment TM The Kinase Assay Kit (Kinase Assay Kit) is used for phosphorylating a substrate under the action of an enzyme to generate ADP, adding an ADP-Glo reagent to remove unreacted ATP in a reaction system, and detecting the ADP generated by the reaction by a Kinase detection reagent (Kinase detection reagent). The inhibition of the compound is calculated by measuring the signal value in the presence of the compound.
2. Experimental method
Enzyme and substrate configuration: ERK1 (1879-KS-010, R)&D) And substrate (AS-61777, anaspec) in buffer (40mM Tris,20mM MgCl) 2 0.1mg/mL BSA, 50. Mu.M DTT) was set to 0.75 ng/. Mu.L and 100. Mu.M, respectively, and then the enzyme solution and 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, compound was dissolved in DMSO to prepare stock solution at an initial concentration of 20mM, and compound was then formulated with Bravo (SGC 120TH34702, agilent Technologies). Finally, 3. Mu.L of the substrate mixture solution and 1. Mu.L of the compound (initial concentration: 50. Mu.M, 4-fold dilution) were sequentially added to each well of 384-well plates, 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 min, followed by 10. Mu.L of kinase assay buffer (Kinase detection buffer) and at 30℃for 40 min. The 384-well plate was removed, placed in a microplate reader (BMG labtech, PHERAstar FS), and chemiluminescence was measured using the microplate reader.
3. Data analysis
Data were analyzed by treatment with Microsoft Excel, graphpad Prism 5. IC for obtaining Compounds 50 The value was 5nM. The results show that the compounds of the formula I have obvious inhibition effect on ERK1 enzyme activity.
Test example 2: ERK2 enzymatic Activity test
1. Purpose of testing
The purpose of this experiment was to test compounds for their ability to inhibit ERK2 enzyme activity, according to IC 50 Size assessmentIn vitro activity of the compounds. ADP-Glo was used in this experiment TM The Kinase Assay Kit is used for phosphorylating a substrate under the action of an enzyme and generating ADP, adding ADP-Glo Reagent to remove unreacted ATP in a reaction system, and detecting the ADP generated by the reaction by Kinase detection Reagent. The inhibition of the compound is calculated by measuring the signal value in the presence of the compound.
2. Experimental method
Enzyme and substrate configuration: ERK2 (1230-KS-010, R)&D) And substrate (custom polypeptide, jier Biochemical) in buffer (40mM Tris,20mM MgCl) 2 0.1mg/mL BSA, 50. Mu.M DTT) to 0.75ng/ul and 1500. Mu.M, then the enzyme solution and substrate solution were mixed at 2:1 to prepare a mixed solution for later use. ATP was diluted to 500uM with buffer, compound was dissolved in DMSO (dimethyl sulfoxide, shanghai Taitan technologies Co., ltd.) to prepare stock solution at initial concentration of 20mM, and then compound was formulated with Bravo. Finally, 3. Mu.L of a mixture of enzyme and substrate was added to each well of 384 well plates in sequence, 1. Mu.L of the compound at different concentrations (initial 50. Mu.M, 4-fold dilution) was incubated at 30℃for 10 minutes, and finally 1. Mu.L of 500. Mu.M 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 min, followed by 10uL of Kinase detection buffer and 30℃for 40 min. Taking out 384-well plate, placing into enzyme labeling instrument (BMG labtech, PHERAstar FS), and measuring with enzyme labeling instrument
3. Data analysis
Data were analyzed by treatment with Microsoft Excel, graphpad Prism 5. IC for obtaining Compounds 50 The value was 7nM. The results show that the compounds of the formula I have obvious inhibition effect on the activity of ERK2 enzyme.
Test example 3: in vitro proliferation inhibition test of Colo205 tumor cells by compound of formula I
1. Purpose of testing
The purpose of this experiment was to examine the inhibitory activity of the compounds on Colo205 cells (CCL-222, ATCC) proliferation in vitro. Treating cells in vitro with compounds of different concentrations, culturing for 3 days, and adopting CTG%
Luminescent Cell Viability Assay, promega, cat: g7573 The proliferation of cells is examined by the reagent and the in vitro activity of the compound is evaluated based on the IC50 value.
2. Experimental method
The following is an example of an in vitro proliferation inhibition test method for Colo205 cells, and is used to illustrate the method of the present invention for testing the in vitro proliferation inhibition activity of the compounds of the present invention on tumor cells. The method is equally applicable to, but not limited to, in vitro proliferation inhibition activity assays for other tumor cells.
Colo205 was digested, centrifuged and resuspended, and the single cell suspension was homogenized and the viable cell density was adjusted to 5.0X10 with cell culture medium (RPMI 1640+2% FBS) 4 cells/mL was added to 96-well cell culture plates at 95. Mu.l/well. 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 )。
The compound was dissolved in DMSO (dimethyl sulfoxide, shanghai Taitan technologies Co., ltd.) to prepare stock solution at an initial concentration of 20 mM. The initial concentration of the small molecule compound was 2mM, 4-fold diluted, 9 spots were diluted, and the tenth spot was DMSO. A96-well plate was additionally used, 90ul of cell culture solution (RPMI 1640+2% FBS) was added to each well, then 10ul of samples to be tested at different concentrations were added to each well, and then 5. Mu.L of samples to be tested at different concentrations were added to the cell culture plate, and two wells were multiplexed for each sample. 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 using a microplate reader (BMG labtech, PHERAstar FS).
3. Data analysis
Data were analyzed by treatment with Microsoft Excel, graphpad Prism 5. The invention is a compound IC of formula I 50 62nM.
Test example 4, mouse pharmacokinetic test of Compounds of the invention
1. Summary
The concentration of the drug in the plasma at various times after the intragastric administration of the compound of formula I in mice was determined using the LC/MS/MS method with the mice as the test animals. The pharmacokinetic behavior of the compound of the invention in mice is studied and its pharmacokinetic profile is evaluated.
2. Test protocol
2.1 test drug
A compound of formula I.
2.2 test animals
The C57 mice, 27 females, were equally divided into 3 groups, purchased from shanghai jieshi laboratory animal limited, animal production license number: SCXK (Shanghai) 2013-0006.
2.3 pharmaceutical formulation
A certain amount of the compound was weighed, dissolved in 5% by volume of DMSO and 5% tween 80, and then prepared into a colorless clear solution of 0.1mg/mL by adding 90% physiological saline.
2.4 administration of drugs
The C57 mice were fed by gastric lavage after overnight fast, and the dose was 2mg/kg and the volume was 0.2mL/10g.
3. Operation of
Mice were given by gavage, 0.1mL was collected before and after administration for 0.25,0.5,1.0,2.0,4.0,6.0,8.0, 11.0, and 24.0 hours, placed in heparinized tubes, centrifuged at 3500 rpm for 10 minutes, and plasma was isolated and stored at-20 ℃.
Determination of the content of test compounds in the plasma of mice following administration of different concentrations of drug by injection: 25. Mu.L of mouse plasma at each time after administration was taken, 50. Mu.L (100 ng/mL) of camptothecin as an internal standard solution, 200. Mu.L of acetonitrile, vortex-mixed for 5 minutes, centrifuged for 10 minutes (4000 rpm), and 4. Mu.L of supernatant was taken from the plasma sample for LC/MS/MS analysis.
4. The pharmacokinetic parameters result is as follows, and shows that the compound of the formula I has better pharmacokinetic absorption and pharmacokinetic advantage:
Example 2: preparation of crystalline form A of Compound of formula I
Amorphous compound of formula I (227 mg,1.17 mmol) was added to 50mL methyl tert-butyl ether, stirred at room temperature for 72 hours, filtered, the filter cake was collected and dried in vacuo to give the title product (371 mg, yield: 70%)
The product was defined as form a, XRPD spectrum as shown in figure 1, as measured by X-ray powder diffraction. The characteristic peak positions are shown in table 1 below. DSC spectra showed an endothermic peak at 116.28 ℃. TGA profile showed a weight loss of 5.83% before 150 ℃.
DVS testing showed that the sample had a hygroscopic gain of about 1.56% under normal storage conditions (i.e., 25 ℃, 60% rh); under accelerated experimental conditions (i.e., 70% RH), the hygroscopic gain was about 1.74%; under extreme conditions (90% RH), the hygroscopic weight gain was about 2.60%. The desorption process and adsorption process of the sample are substantially coincident during the 0% -95% RH humidity change. And (3) retesting the crystal form after DVS detection, wherein the crystal form is not transformed.
TABLE 1 peak positions for 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 crystalline form A of Compound of formula I
The compound of formula I was added to 5mL of cyclopentane in amorphous form (60 mg,0.13 mmol), stirred at room temperature for 72 hours, filtered, the filter cake collected and dried in vacuo to give the title product (58 mg, yield: 96.7%).
The product was detected by X-ray powder diffraction as form a.
Example 4: preparation of crystalline form A of Compound of formula I
The compound of formula I was added to 5mL isopropyl ether in amorphous form (64 mg,0.14 mmol), stirred at room temperature for 72 hours, filtered, the filter cake collected and dried in vacuo to give the title product (61 mg, yield: 95.3%).
The product is in the form of form A as detected by X-ray powder diffraction.
Example 5: preparation of crystalline form A of Compound of formula I
The compound of formula I was added to 5mL of phenetole in amorphous form (67 mg,0.15 mmol), stirred at room temperature for 72 hours, filtered, the filter cake collected and dried in vacuo to give the title product (40 mg, yield: 59.7%).
The product is in the form of form A as detected by X-ray powder diffraction.
Example 6: preparation of crystalline form A of Compound of formula I
The compound of formula I was added to 15mL isopropyl acetate in amorphous form (1.2 g,2.67 mmol), slurried at room temperature and stirred for 72 hours, filtered, the filter cake collected and dried in vacuo to give the title product (732 mg, yield: 61.0%).
The product is in the form of form A as detected by X-ray powder diffraction.
Example 7: preparation of crystalline form A of Compound of formula I
10mg of the compound of formula I is amorphous, 0.3mL of dichloromethane is added for dissolution and clarification, 0.6mL of isopropyl ether is added, the initial temperature is 60 ℃ for stirring for 2 hours, the temperature is slowly reduced to 20 ℃, the temperature is constant, the centrifugation and the vacuum drying are carried out, the title product is obtained, and the product is the crystal form A through X-ray powder diffraction detection.
Example 8: preparation of crystalline form A of Compound 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 mixture is stirred for 2 hours at the initial temperature of 60 ℃, the temperature is slowly reduced to 20 ℃, the temperature is constant, the mixture is centrifuged, and the vacuum drying is carried out to obtain the title product, and the product is the crystal form A through X-ray powder diffraction detection.
Example 9: preparation of crystalline form A of Compound of formula I
10mg of the compound of formula I is amorphous, 50. Mu.L of ethyl acetate is added for dissolution, 1mL of isopropyl ether is added, stirring is carried out at normal temperature or 50 ℃ overnight, centrifugation is carried out, and the title product is obtained by vacuum drying, and is detected by X-ray powder diffraction, and the product is in a crystal form A.
Example 10: preparation of crystalline form A of Compound of formula I
10mg of the compound of formula I is amorphous, 50. Mu.L of dioxane is added for dissolution, 1mL of methyl tertiary butyl ether is added, stirring is carried out at 50 ℃ overnight, centrifugation is carried out, vacuum drying is carried out to obtain the title product, and the product is the crystal form A detected by X-ray powder diffraction.
Example 11: preparation of crystalline form A of Compound of formula I
10mg of the compound of formula I is amorphous, 50. Mu.L of ethyl acetate are added for dissolution, 1mL of methyl tert-butyl ether are added, stirring is carried out overnight, centrifugation is carried out, and vacuum drying is carried out to obtain the title product, which is the crystalline form A, detected by X-ray powder diffraction.
Example 12: preparation of crystalline form A of Compound of formula I
10mg of the compound of formula I is amorphous, 50. Mu.L of butanone is added for dissolution, 1mL of methyl tertiary butyl ether is added, stirring is carried out at 50 ℃ overnight, centrifugation is carried out, and vacuum drying is carried out to obtain the title product, which is the crystalline form A, detected by X-ray powder diffraction.
Example 13: preparation of crystalline form A of Compound of formula I
10mg of the compound of formula I is amorphous, 50. Mu.L of tetrahydrofuran is added for dissolution, 1mL of methyl tert-butyl ether is added, stirring is carried out at 50 ℃ overnight, centrifugation is carried out, and the title product is obtained by vacuum drying, and is detected by X-ray powder diffraction and is in the form of a crystal form A.
Example 14: preparation of crystalline form A of Compound of formula I
10mg of the compound of formula I is amorphous, 50. Mu.L of tetrahydrofuran is added for dissolution, 1mL of isopropyl ether is added, stirring is carried out at 50 ℃ overnight, centrifugation is carried out, and vacuum drying is carried out to obtain the title product, which is the crystalline form A, detected by X-ray powder diffraction.
Example 15: preparation of crystalline form A of Compound of formula I
10mg of the compound of formula I is amorphous, 50. Mu.L of acetone is added for dissolution, 1mL of isopropyl ether is added, stirring is carried out at 50 ℃ overnight, centrifugation is carried out, vacuum drying is carried out to obtain the title product, and the product is the crystal form A detected by X-ray powder diffraction.
Example 16: preparation of crystalline form A of Compound 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 is carried out, and vacuum drying is carried out to obtain the title product, which is the crystal form A, through X-ray powder diffraction detection.
Example 17: preparation of crystalline form B of Compound of formula I
The compound of formula I was added amorphous (60 mg,0.13 mmol) to a mixture of 1mL acetone and n-hexane (V: v=1:3), stirred at room temperature for 72 hours, filtered, the filter cake was collected and dried in vacuo to give the title product (47 mg, yield: 78.3%).
The product was defined as form B, XRPD pattern as shown in figure 2, as measured by X-ray powder diffraction.
DSC spectrum shows endothermic peak value of 123.81 ℃; TGA profile showed a weight loss of 1.72% before 150 ℃.
DVS testing showed that the sample had a hygroscopic gain of about 0.926% under normal storage conditions (i.e., 25 ℃, 60% rh); under accelerated experimental conditions (i.e., 70% rh), the hygroscopic gain was about 1.021%; under extreme conditions (90% RH), the hygroscopic gain was about 1.432%. The desorption process and adsorption process of the sample are substantially coincident during the 0% -95% RH humidity change. And (3) retesting the crystal form after DVS detection, 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 crystalline form B of Compound of formula I
The compound of formula I was added to 0.5mL of ethyl acetate in amorphous form (55 mg,0.12 mmol), stirred at room temperature for 72 hours, filtered, and the filter cake was collected and dried in vacuo to give the title product (3 mg, yield: 6.4%).
The product is in the form of crystal B by X-ray powder diffraction detection.
Example 19: preparation of crystalline form B of Compound of formula I
The compound of formula I was added to 8mL of ethyl propionate in amorphous form (1.2 g,2.67 mmol), stirred at room temperature for 144 hours, filtered, and the filter cake was collected and dried in vacuo to give the title product (0.75 g, yield: 62.5%).
The product is in the form of crystal B by X-ray powder diffraction detection.
Example 20: preparation of crystalline form B of Compound of formula I
10mg of the compound of formula I was dissolved by adding 50. Mu.L of butanone, 1mL of n-hexane was added, stirred overnight at room temperature, centrifuged, and dried in vacuo to give the title product as form B as detected by X-ray powder diffraction.
Example 21: preparation of crystalline form C of Compound of formula I
69mg of the compound of formula I was added amorphous to 5mL of n-pentane and slurried at room temperature for 3 days. Filtration gave 67mg of a pale yellow solid, which was detected by X-ray powder diffraction and was defined as form C. The XRPD spectrum of this form C sample is shown in fig. 3, and the characteristic peak positions are shown in table 3 below. DSC spectra showed endothermic peaks at 64.13℃and 93.98 ℃; TGA spectrum shows a weight loss of 3.18% at 30-230 ℃, 32.32% at 230-340 ℃.
TABLE 3 peak positions for form C
| Peak number | 2 theta value [ ° or degree] | Relative strength% |
| 1 | 17.857 | 100.0 |
Example 22: preparation of crystalline form D of Compound of formula I
100mg of the compound shown in the formula I is amorphous, 1mL of acetonitrile is added for dissolution, 10mL of isopropyl ether is added, stirring is carried out at 50 ℃ overnight, centrifugation and vacuum drying are carried out, and the target product is defined as a D crystal form through X-ray powder diffraction detection. The XRPD spectrum of this form D sample is shown in fig. 4, and the characteristic peak positions are shown in table 4 below. DSC spectrum shows that the peak value of the endothermic peak is 125.32 ℃; TGA profile shows a weight loss of 2.79% at 30 ℃ -120 ℃.
Table 4.D peak positions of crystalline forms
| 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 crystalline form E of Compound of formula I
10mg of the compound shown in the formula I is amorphous, 50 mu L of dichloromethane is added for dissolution, 1mL of isopropyl ether is added, stirring is carried out at 50 ℃ overnight, centrifugation is carried out, vacuum drying is carried out, and the target product is defined as E crystal form through X-ray powder diffraction detection. The XRPD spectrum of this form E sample is shown in fig. 5, and the characteristic peak positions are shown in table 5 below. DSC spectrum shows that the endothermic peak is 116.28 ℃; TGA spectrum shows that the weight loss is 0.81% at 0-60 ℃ and 4.77% at 60-140 ℃.
Table 5. Peak positions of form e
| Peak number | 2 theta value [ ° or degree] | Relative strength% |
| 1 | 5.216 | 98.0 |
| 2 | 11.834 | 85.7 |
| 3 | 15.595 | 100.0 |
| 4 | 17.199 | 58.2 |
Example 24: stability study of influence factors of compound A of formula I
The form a (purity 99.22%) of the compound of formula I prepared with reference to the previous examples was subjected to experimental investigation of stability of influencing factors under the experimental conditions of table 6, and the results are shown in table 6 below.
TABLE 6 stability of form factor influencing
Experimental results show that the crystal form A of the compound 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 crystal form A sample has good physical and chemical stability.
Example 25: stability study of influence factor of B crystal form of compound of formula I
The form B (purity 98.97%) of the compound of formula I prepared with reference to the previous examples was subjected to experimental investigation of stability of influencing factors under the experimental conditions of table 7, and the results are shown in table 7 below.
TABLE 7 stability of form factor of influence
Experimental results show that the crystal form B of the compound in the formula I has good physical and chemical stability when being 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.
Example 26: stability study of influence factor of D crystal form of compound of formula I
The D crystal form (purity 99.58%) of the compound of formula I prepared with reference to the foregoing examples was subjected to experimental investigation of stability of influencing factors under the experimental conditions of table 8, and the results are shown in table 8 below.
TABLE 8 stability of form D influencing factors
Experimental results show that the crystal form D of the compound in the formula I has good physical and chemical stability 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 for 30 days.
Example 27: stability study of influencing factors of E crystal form of compound of formula I
The E-form (purity 99.43%) of the compound of formula I prepared with reference to the previous examples was subjected to experimental investigation of stability of influencing factors under the experimental conditions of table 9, and the results are shown in table 9 below.
Table 9.E stability of form factor
Experimental results show that the E crystal form of the compound in the formula I has good physical and chemical stability 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 for 30 days.
Example 28: stability study of Compound A Crystal form of formula I under Long-term acceleration condition
The stability of the A crystal form of the compound of formula I prepared by reference to the previous examples was examined under conditions of light shielding at 25 ℃,60% RH and 40 ℃,75% RH, and the results are shown in Table 10.
Table 10 stability of form a under long term acceleration conditions
The base A crystal form has good physical and chemical property stability after being placed for 1 month under the condition of long-term acceleration stability.
Example 29: stability study of Compound B Crystal form of formula I under Long-term acceleration condition
The stability of the B crystal form of the compound of formula I prepared by reference to the previous examples was examined under conditions of light shielding at 25 ℃,60% RH and 40 ℃,75% RH, and the results are shown in Table 11:
table 11 stability of form b under long term acceleration conditions
The results show that the compound of the formula I, the crystal form B, has good physicochemical stability after being placed for 6 months under the conditions of long term (25 ℃,60% RH) and acceleration (40 ℃ and 75% RH).
Example 30: stability study of Compound D form of formula I under Long-term acceleration
The stability of the base D crystal form was examined under conditions of 5℃at 25℃at 60% RH and 40℃at 75% RH, respectively, and the results are shown in Table 12:
table 12. Stability of form d under long term acceleration
Experiments show that the compound D of the formula I has better physicochemical stability after being placed for 3 months under the condition of long-term acceleration stability.
Example 31: stability study of Compound E Crystal form of formula I under Long-term acceleration condition
The stability of base E crystal forms was examined under conditions of 25 ℃,60% RH and 40 ℃,75% RH, and the results are shown in Table 13:
TABLE 13 stability of E Crystal form under Long term acceleration
Experiments show that the compound E crystal form of the formula I has better physicochemical stability after being placed for 1 month under the condition of long-term acceleration stability.
Claims (26)
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, having an X-ray powder diffraction spectrum expressed as diffraction angle 2θ, with characteristic peaks at 4.040, 5.085, 11.801, 15.532 and 16.925.
2. Form a of claim 1, having an X-ray powder diffraction spectrum in terms of diffraction angle 2Θ, having characteristic peaks at 4.040, 5.085, 11.801, 15.532, 16.925, 18.915, and 27.074.
3. Form a of claim 2, having an X-ray powder diffraction spectrum in terms of diffraction angle 2Θ, having characteristic peaks at 4.040, 5.085, 11.801, 15.532, 16.925, 18.915, 22.000, 24.686, and 27.074.
4. The form a of claim 3, wherein the X-ray powder diffraction spectrum expressed as diffraction angle 2Θ is shown in figure 1.
5. A process for preparing form a of any one of claims 1 to 4 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;
alternatively, method two:
(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), stirring or heating to dissolve, wherein the solvent (II) is selected from at least one of ethyl acetate, dichloromethane, acetonitrile, chloroform, dioxane, butanone, tetrahydrofuran or acetone;
(b) Adding a solvent (III), and crystallizing, wherein the solvent (III) is at least one selected from isopropyl ether and methyl tertiary butyl ether.
6. Crystalline form B 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, having an X-ray powder diffraction spectrum expressed as diffraction angle 2θ, with characteristic peaks at 13.128, 13.773, 14.418, 17.645 and 19.482.
7. The form B of claim 6, wherein the X-ray powder diffraction pattern in terms of diffraction angle 2Θ has characteristic peaks at 13.128, 13.773, 14.418, 17.645, 19.482, 24.793, and 27.573.
8. Form B of claim 7, having an X-ray powder diffraction spectrum in terms of diffraction angle 2Θ as shown in fig. 2.
9. A process for preparing form B of any one of claims 6 to 8 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), the solvent (IV) selected from at least one of ethyl acetate, ethyl propionate, acetone, n-hexane;
(b) Pulping and crystallizing;
alternatively, method two:
(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), said solvent (V) selected from at least one of acetone and butanone;
(b) N-hexane is added for crystallization.
10. Crystalline form C 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, having a characteristic peak at 17.857 in the X-ray powder diffraction spectrum expressed as angle 2θ.
11. Form C of claim 10, having an X-ray powder diffraction spectrum in terms of diffraction angle 2Θ as shown in fig. 3.
12. 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, having an X-ray powder diffraction spectrum, expressed as diffraction angle 2θ, with characteristic peaks at 4.462, 5.228, 12.476, 13.331, 13.924, 16.031 and 16.677.
13. The form D of claim 12, having an X-ray powder diffraction spectrum in terms of diffraction angle 2Θ, having characteristic peaks at 4.462, 5.228, 11.503, 12.476, 13.331, 13.924, 16.031, 16.677, and 17.308.
14. The form D of claim 13, having an X-ray powder diffraction spectrum in terms of diffraction angle 2Θ, 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.
15. Form D according to claim 14, having an X-ray powder diffraction spectrum in terms of diffraction angle 2Θ as shown in figure 4.
16. A process for preparing the form D of any one of claims 12 to 15 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) Isopropyl ether was added thereto, and stirred for crystallization.
17. Crystalline form E 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, having an X-ray powder diffraction spectrum expressed as diffraction angle 2θ, with characteristic peaks at 5.216, 11.834, 15.595 and 17.199.
18. The form E of claim 17, having an X-ray powder diffraction spectrum in terms of diffraction angle 2Θ as shown in fig. 5.
19. The crystalline form of any one of claims 1 to 4, 7 to 8, 10 to 11, 12 to 15, 17 to 18, the 2Θ angle error range is ± 0.30.
20. The crystalline form of claim 19, wherein the 2Θ angle error range is ± 0.20.
21. A pharmaceutical composition prepared from the crystalline form of any one of claims 1 to 4, 7 to 8, 10 to 11, 12 to 15, 17 to 20.
22. A pharmaceutical composition comprising a crystalline form according to any one of claims 1 to 4, 7 to 8, 10 to 11, 12 to 15, 17 to 20, or a crystalline form prepared by the process of claim 5, 9 or 16, and optionally from a pharmaceutically acceptable carrier, diluent or excipient.
23. A process for the preparation of a pharmaceutical composition comprising the step of mixing a crystalline form according to any one of claims 1 to 4, 7 to 8, 10 to 11, 12 to 15, 17 to 20 or a crystalline form prepared by a process according to any one of claims 5, 9 or 16 with a pharmaceutically acceptable carrier, diluent or excipient.
24. Use of a crystalline form according to any one of claims 1 to 4, 7 to 8, 10 to 11, 12 to 15, 17 to 20 or a crystalline form prepared by a process according to any one of claims 5, 9 or 16 or a composition according to any one of claims 21 or 22 or a composition prepared by a process according to claim 23 in the manufacture of a medicament for inhibiting ERK.
25. Use of a crystalline form according to any one of claims 1 to 4, 7 to 8, 10 to 11, 12 to 15, 17 to 20 or a crystalline form prepared by a process according to claim 5, 9 or 16 or a composition according to claim 21 or 22 or a composition prepared by a process according to claim 23 in the manufacture of a medicament for the treatment or prophylaxis of cancer, inflammation, or other proliferative disorders.
26. The use of claim 25, wherein the cancer is selected from melanoma, liver cancer, kidney cancer, lung cancer, nasopharyngeal cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma, astrocytoma, and glioma.
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