CN111718340A

CN111718340A - Crystal form, preparation method and application of deuterated Palbociclib compound

Info

Publication number: CN111718340A
Application number: CN201910211031.4A
Authority: CN
Inventors: 吴豫生; 李钧; 牛成山; 梁阿朋
Original assignee: Zhejiang Tongyuankang Medicine Co ltd
Current assignee: Zhejiang Tongyuankang Medicine Co ltd
Priority date: 2019-03-20
Filing date: 2019-03-20
Publication date: 2020-09-29

Abstract

The invention belongs to the technical field of medicines, and particularly discloses several crystal forms of a deuterated Palbociclib compound, respective preparation methods and pharmaceutical applications. Through the research on normal temperature and accelerated stability and the combination of the research result on hygroscopicity, the crystal form A provided by the invention is a stable crystal form compared with other crystal forms. The crystal form A as a dominant drug crystal form can be further subjected to subsequent pharmacokinetic experiments and animal experiment researches.

Description

Crystal form, preparation method and application of deuterated Palbociclib compound

Technical Field

The invention relates to a crystal form of a deuterated Palbociclib compound, a preparation method of each crystal form and application of each crystal form in the aspect of medicines, and belongs to the technical field of medicines.

Background

Palbociclib is a novel targeted CDK4/6 inhibitor, can selectively inhibit cyclin-dependent kinases 4 and 6(CDK4/6), restore cell cycle control, and block tumor cell proliferation (J of clinical drug therapy, 2016, Vol.14, 85-88; drug evaluation, 2016, 31-37). The FDA awarded in 2013 a breakthrough therapy assertion for Palbociclib to treat advanced or metastatic estrogen receptor positive (ER +)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Palbociclib is the first FDA-approved CDK inhibitor used in combination with Letrozole (Letrozole) as the initial treatment based on endocrine therapy for post-menopausal female patients with advanced breast cancer of ER +/HER 2-. Palbociclib is an anti-breast cancer drug with a brand-new action mechanism, the clinical use effect is good after the drug is put into the market, and other indications of the drug are undergoing clinical tests (such as ovarian cancer, non-small cell lung cancer, lymphoma, multiple myeloma and the like). At present, many reports about Palbociclib exist in China, for example, Chinese patents CN1051531149A, CN104496983A, CN105418603A, CN104910149A and the like respectively report the synthesis or preparation method of non-deuterated Palbociclib.

The deuterated Palbociclib derivative is obtained by selectively deuterating Palbociclib so as to improve the pharmacokinetic property of the drug and further achieve the effects of improving the curative effect, safety and tolerance of the drug. Chinese patent (application publication number is CN104447739A) discloses a deuterated Palbociclib derivative, and also discloses a preparation method and application thereof, and the selective deuteration of Palbociclib improves the pharmacokinetic properties of the drug, thereby improving the curative effect, safety and tolerance of the drug.

The crystal form is the solid matter state existing in the medicine, the medicine crystal form research is the research on the basic state of the medicine, and the medicine crystal form solid matter more suitable for treating diseases can be possibly searched only if the crystal form state of the chemical medicine is fully and comprehensively known. The crystal form of the medicine can influence the physicochemical property of the medicine and directly influence the basis of the clinical application of the medicine for treating diseases. Different crystal forms of the same drug may have significant differences in appearance, solubility, melting point, dissolution rate, bioavailability and the like, thereby affecting the stability, bioavailability and curative effect of the drug. Therefore, the crystal form research of the deuterated Palbociclib compound has important significance for successfully developing the medicine.

In the compound of the formula (I) disclosed in the Chinese patent with application publication No. CN104447739A, the purity of the compound of the formula (I) is 99.89%, the melting point of the compound of the formula (I) is tested to be 256-257 ℃, and the X-ray powder diffraction pattern is shown in FIG. 14. In the next study, it is found that the compound of formula (i) synthesized by the preparation method of patent CN104447739A has poor stability, reduced purity, and reduced deuteration ratio, which affects the next pharmacological and pharmaceutical studies and pharmaceutical preparations, such as safety evaluation. Therefore, there is an urgent need to develop a stable and reliable crystal form of the compound shown in formula (i) to ensure the quality stability of the compound shown in formula (i), and to better apply to pharmaceutical and pharmaceutical preparations and clinical use in the future, so as to achieve the purpose of stable and controllable quality of the drug, better dissolution and higher bioavailability, thereby avoiding the safety problems such as side effects caused by the unstable production of toxic impurities due to the drug.

Disclosure of Invention

The invention provides several crystal forms of the following deuterated Palbociclib compounds, a preparation method and application of the deuterated Palbociclib compounds in the aspect of medicines.

The invention provides a crystal form A of a deuterated Palbociclib compound, wherein the structural formula of the deuterated Palbociclib compound is shown as a formula (I):

the X-ray powder diffraction of the crystal form A measured by using Cu-Kalpha rays is as follows at an angle of 2 theta (unit is degree): diffraction peaks are at 5.0 + -0.2, 7.9 + -0.2, 10.2 + -0.2, 11.5 + -0.2, and 14.0 + -0.2.

Further, form a has an X-ray powder diffraction at 2 θ angles (in °) measured using Cu-K α radiation of: diffraction peaks are also found at 15.9 + -0.2, 17.1 + -0.2, 19.7 + -0.2, 22.5 + -0.2, and 28.4 + -0.2.

Further, the X-ray powder diffraction pattern of the crystal form A measured by using Cu-K alpha rays is basically as shown in figure 1.

Further, a differential scanning calorimetry analysis chart of the crystal form A shows that the crystal form A has an endothermic peak at 260-270 ℃.

The crystal form A of the deuterated Palbociclib compound provided by the invention can be used for preparing CDK4/6 inhibitor medicines.

The crystal form B of the deuterated Palbociclib compound provided by the invention has a structural formula shown in a formula (I), and the X-ray powder diffraction of the crystal form B obtained by Cu-Kalpha ray measurement at a 2 theta angle (unit) is as follows: diffraction peaks are at 4.0 + -0.2, 4.5 + -0.2, 7.7 + -0.2, 8.8 + -0.2, 11.7 + -0.2, 12.2 + -0.2, and 13.7 + -0.2.

Further, form B has an X-ray powder diffraction at 2 θ angles (in °) measured using Cu-K α radiation of: diffraction peaks are also found at 17.4 + -0.2, 18.5 + -0.2, 19.1 + -0.2, 20.3 + -0.2, 21.2 + -0.2 and 23.8 + -0.2.

Further, the X-ray powder diffraction pattern of form B measured using Cu-K α radiation is substantially as shown in fig. 3.

The crystal form B of the deuterated Palbociclib compound provided by the invention can be used for preparing CDK4/6 inhibitor medicines.

The crystal form C of the deuterated Palbociclib compound provided by the invention has a structural formula shown in a formula (I), and X-ray powder diffraction obtained by measuring Cu-Kalpha rays in the crystal form C at a 2 theta angle (unit) is as follows: diffraction peaks are found at 3.5 + -0.2, 3.8 + -0.2, 7.6 + -0.2 and 11.5 + -0.2.

Further, form C has an X-ray powder diffraction at 2 θ angles (in °) measured using Cu-K α radiation of: diffraction peaks are also found at 15.4 + -0.2, 17.4 + -0.2, 21.2 + -0.2, 23.2 + -0.2, 35.1 + -0.2.

Further, form C has an X-ray powder diffraction pattern substantially as shown in fig. 5, as measured using Cu-K α radiation.

The crystal form C of the deuterated Palbociclib compound provided by the invention can be used for preparing CDK4/6 inhibitor medicines.

The crystal form D of the deuterated Palbociclib compound provided by the invention has a structural formula shown in a formula (I), and the X-ray powder diffraction of the crystal form D obtained by Cu-Kalpha ray measurement at a 2 theta angle (unit) is as follows: diffraction peaks are at 4.0 + -0.2, 7.7 + -0.2, 8.5 + -0.2, 8.8 + -0.2, 10.5 + -0.2, 11.6 + -0.2, 12.1 + -0.2 and 13.7 + -0.2.

Further, form D has an X-ray powder diffraction at 2 θ angles (in °), measured using Cu-K α radiation, of: diffraction peaks are also found at 15.5 + -0.2, 18.5 + -0.2 and 20.4 + -0.2.

Further, form D has an X-ray powder diffraction pattern substantially as shown in fig. 7, as measured using Cu-K α radiation.

The crystal form D of the deuterated Palbociclib compound provided by the invention can be used for preparing CDK4/6 inhibitor medicines.

The active ingredients of the same drug generally exist in two or more crystal forms, referred to as drug polymorphs. Different crystal forms have different respective solubilities and dissolution rates, and the clinical treatment effect of the medicine is influenced by causing the change of in vivo bioavailability. The different crystal forms of the medicine may affect the dissolution and absorption of the medicine in vivo, and further affect the bioavailability, clinical efficacy and safety of the medicine. Meanwhile, the stability of the drug crystal form is also very important. In order to improve the bioavailability of the drug, reduce toxicity and improve the therapeutic effect, more attention is paid to the stability of the drug crystal form. The crystal form with good stability can ensure the physical and chemical stability of the medicament form in the preparation and storage processes, maintain the good solubility and bioavailability of the medicament form and ensure the equivalence among each batch of medicaments. The same drug often has a plurality of crystal forms, and the crystal form which has better treatment effect and is most suitable for clinic is called as the dominant drug crystal form at present.

The invention screens the crystal form of the deuterated Palbociclib compound shown in the formula (I) and finds out different crystal forms of the raw material medicines as much as possible. The screened solid is identified by means of powder X-ray diffraction analysis (XRPD), Differential Scanning Calorimetry (DSC), thermogravimetric analysis (TGA) and the like, and further, the crystal form of the dominant drug is determined by research and investigation of normal temperature stability, accelerated stability and hygroscopicity, so that a reference basis is provided for the selection of the crystal form of subsequent pharmacokinetic experiments and zoology experiments.

Compared with other crystal forms, the crystal form A provided by the invention is a stable crystal form and has good crystal form stability by the research on normal temperature and accelerated stability and the combination of the research result on hygroscopicity. The crystal form A as a dominant drug crystal form can be further subjected to subsequent pharmacokinetic experiments and animal experiment researches.

The crystal form A of the deuterated Palbociclib compound provided by the invention has stable physicochemical properties, and the drug accelerated stability test proves that the crystal form A of the deuterated Palbociclib compound has stable physicochemical properties, the deuterated rate can be kept unchanged, and the single impurity has no obvious change in the test period, so that the deuterated Palbociclib compound has obvious advantages in the preparation development, and is very suitable for application in the next preparation drug development aspect.

Experiments prove that the crystal form A of the deuterated Palbociclib compound provided by the invention is easy to absorb in a living body and has good stability, good blood concentration distribution in the living body and high bioavailability. Meanwhile, the physical stability of the compound preparation is good, so that the compound preparation is very beneficial to the quality control of the medicine in the later preparation development and has longer quality guarantee period, thereby leading the medicine to achieve better curative effect. The crystal form A of the deuterated Palbociclib compound provided by the invention has good metabolic stability, and the blood concentration in vivo is remarkably improved, so that a better curative effect is achieved.

Drawings

FIG. 1 is an X-ray powder diffraction pattern of the crystal form A provided by the invention measured by Cu-Ka rays;

figure 2 is a DSC-TGA profile of form a;

FIG. 3 is an X-ray powder diffraction pattern of form B provided by the present invention measured using Cu-Ka radiation;

figure 4 is a DSC-TGA profile of form B;

FIG. 5 is an X-ray powder diffraction pattern of form C provided by the present invention as measured using Cu-Ka radiation;

figure 6 is a DSC-TGA profile of form C;

FIG. 7 is an X-ray powder diffraction pattern of form D provided by the present invention measured using Cu-Ka radiation;

figure 8 is a DSC-TGA profile of form D;

figure 9 is an XRPD comparison pattern of form a before and after standing for 6 months;

figure 10 is an XRPD contrast pattern of form B before and after standing for 1 month;

figure 11 is an XRPD comparison of form C before and after standing at room temperature for 1 month;

figure 12 is an XRPD comparison of form D before and after standing at room temperature for 1 month;

FIG. 13 is an XRPD comparison pattern of form A after being exposed to 40 ℃ at 75% humidity for 1 month;

figure 14 is an X-ray powder diffraction pattern of a deuterated Palbociclib compound prepared in accordance with the present invention.

Detailed Description

The technical solution of the present invention will be explained in detail below.

Firstly, synthesis of deuterated Palbociclib compound shown as formula (I)

The preparation method is referred to patent CN104447739A, and the synthetic route is as follows:

the specific steps are as follows.

Synthesis of Compound 2

2.2g of LiAlD are taken₄100ml of diethyl ether (Et) are added₂O), cooling the system to 0 ℃; under the protection of nitrogen, slowly dropwise adding an ether solution containing 8g of cyclopentanone (compound 1), after dropwise adding, heating the system to room temperature, and continuously reacting for 1 h; after the reaction is finished, saturated NH is added₄Cl solution quenching reactionExtracting with diethyl ether for three times, mixing organic phases, drying, and concentrating to obtain 8g colorless transparent liquid as compound 2; the nmr information for compound 2 is:¹H NMR(400MHz，CDCl₃)：1.78-1.74(m,4H),1.58-1.55(m,4H)；

synthesis of Compound 3

5.2g of Compound 2 are dissolved in 100ml of Dichloromethane (DCM), and triethylamine (Et)₃N)25ml and a catalytic amount of DMAP, and cooling the system to 0 ℃; slowly dripping 15ml of MsCl under the protection of nitrogen, wherein the molar ratio of the compound 2, the hydroxyl activating reagent and the alkali reagent is 1:3:3, and reacting for 2 h; after the reaction is finished, saturated NH is dropwise added into the system₄Quenching reaction by using a Cl solution, separating liquid, washing an organic phase by using water and a saturated sodium chloride solution, drying and concentrating to obtain 10g of light reddish brown liquid which is a compound 3; the nmr information for compound 3 is:¹H NMR(400MHz,CDCl₃)：2.98(m,3H),1.98-1.86(m,4H)，1.84-1.73(m,2H),1.64-1.60(m,2H)；

synthesis of Compound 4

Under the protection of nitrogen, 10g of compound 3 and 1eq of NaN are taken₃Adding into 100ml DMF (the molar ratio of the azide group donor to the compound 3 is 1:1), heating to 80 ℃ and reacting overnight; after the reaction is finished, the system is cooled to room temperature, and H is added₂Extracting with ethyl ether, mixing organic phases, washing the organic phase with saturated sodium chloride solution, drying, and concentrating to obtain 11g of compound 4 crude product;

synthesis of Compound 5

11g of the crude compound 4 are taken and 150ml of THF and 5ml of H are added₂Adding 25g of triphenylphosphine (the molar ratio of the reducing agent to the compound 4 is 1:1) in batches into a mixed solvent prepared by mixing O, reacting for 3 hours at room temperature, dropwise adding concentrated hydrochloric acid into the system after the reaction is finished until the reaction system is weakly acidic, concentrating the system, and adding H₂O, extracting with ethyl acetate for 3 times, concentrating the water phase, adding 20ml of acetone, and filtering to obtain 4g of white solid, namely a compound 5; the nmr information for compound 5 is:¹H NMR(400MHz,D₂O)：1.94-1.89(m,2H),1.67-1.44(m,6H)；

synthesis of Compound 6Become into

Dissolving 7g of compound 5 (deuterated cyclopentylamine hydrochloride, which is generated under the action of an alkaline reagent) in 100ml of absolute ethanol, and adding 16ml of Et₃Adding 9g of 2, 4-dichloro-5-bromopyrimidine (the molar ratio of the 2, 4-dichloro-5-bromopyrimidine to the deuterated cyclopentylamine salt is 0.7:1) into the system in batches under the stirring condition, and continuing to react for 2 hours after the addition is finished; filtering after the reaction is finished, concentrating the filtrate, adding water to separate out a white solid, adding a small amount of petroleum ether into the solid, stirring for 10 minutes, and filtering to obtain 8g of a white solid which is a compound 6; the nmr information for compound 6 is:¹H NMR(400MHz,CDCl₃)：8.08(s,1H),5.44(s,1H),2.14-2.09(m,2H),1.77-1.73(m,2H),1.71-1.63(m,2H),1.50-1.42(m,2H)；

synthesis of Compound 7

Collecting 8g of Compound 6, 1eq of methyl crotonate, 5% eq of Pd (OAc)₂10% eq DABCO, 3eq K₂CO₃Adding the mixture into 50ml of DMF (the molar ratio of the compound 6 to the methyl crotonate is 1:1, the molar ratio of the main catalyst, the ligand and the compound 6 is 0.05:0.10:1, and the molar ratio of the alkali reagent to the compound 6 is 3:1), heating to 85 ℃ under the protection of nitrogen, and reacting for 3 hours; filtering after the reaction is finished, adding water into the filtrate, extracting with ethyl acetate, washing an organic phase with saturated saline solution, drying, concentrating, and purifying by column chromatography to obtain 1.8g of white solid which is a compound 7; the nmr information for compound 7 is:¹H NMR(400MHz,CDCl₃)：7.79(s,1H),5.96-5.94(d,1H,J＝1.36),4.89(s,1H),3.76(s,3H),2.41-2.40(d,3H,J＝1.28),2.11-2.06(m,2H),1.74-1.61(m,4H),1.42-1.35(m,2H)；

synthesis of Compound 8

Adding 140mg of KOH into 20ml of DMF, cooling the system to 0 ℃, slowly dropwise adding 7ml of DMF solution containing 800mg of compound 7 (the molar ratio of the alkali reagent to the compound 7 is 0.9:1) under the protection of nitrogen, and reacting for 15 min; after the reaction is finished, directly pouring the system into ice water, extracting with ethyl acetate, washing an organic phase with saturated saline solution, drying, concentrating, and purifying by column chromatography to obtain 350mg of white solid which is a compound 8; the nmr information for compound 8 is:¹H NMR(400MHz,CDCl₃)：8.74(s,1H),6.54-6.53(d,1H,J＝1.12),2.45-2.44(d,3H,J＝1.16),2.25-2.17(m,2H),2.13-2.09(m,2H),1.93-1.87(m,2H),1.69-1.65(m,2H)；

synthesis of Compound 9

Adding 220mg of compound 8 and 270mg of NaOAc into 20ml of acetic acid, adding 270mg of liquid bromine under the protection of nitrogen (the molar ratio of a bromization reagent to the compound 8 is 2:1), and heating the system to 50 ℃ for reaction for 35 hours; after the reaction is finished, the system is cooled to room temperature, and Na is added₂SO₃The reaction was quenched with solids, the system was concentrated to remove a portion of the acetic acid, and then saturated NaHCO was added₃Extracting with ethyl acetate, washing the organic phase with saturated saline, drying, and concentrating to obtain 300mg of white solid, namely a compound 9; the nmr information for compound 9 is:¹H NMR(400MHz,CDCl₃)：8.86(s,1H),2.67(s,3H),2.22-2.11(m,4H),1.95-1.91(m,2H),1.70-1.66(m,2H)；

synthesis of Compound 10

490mg of 4- (6-aminopyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester (compound B) is added into 15ml of anhydrous toluene, the system is cooled to 0 ℃, 1.05eq of LiHMDS is added dropwise, and after the dropwise addition is finished, the temperature is raised to room temperature for continuous reaction for 30 min; after the reaction is finished, cooling the system to 0 ℃, dropwise adding 5ml of toluene solution containing 300mg of compound 9 (the molar ratio of the 4- (6-aminopyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester to the compound 9 is 2:1, and the molar ratio of the alkali reagent to the compound 9 is 1.05:1), and after the dropwise adding is finished, heating to room temperature and reacting for 40 min; after the reaction is finished, the system is cooled to 0 ℃, and saturated NH is added₄Quenching the reaction by using a Cl solution, separating liquid, concentrating an organic phase, adding ethyl acetate, and filtering to obtain 450mg of yellow solid, namely a compound 10; the nmr information for compound 10 is:¹H NMR(400MHz,CDCl₃)：8.82(s,1H),8.37(s,1H),8.19-8.16(d,1H,J＝9.04),8.05-8.04(d,1H,J＝2.76),7.35-7.32(dd,1H,J1＝2.92,J2＝9.12),3.62-3.60(t,4H,J＝4.84),3.13-3.11(t,4H,J＝4.72),2.61(s,3H),2.34-2.27(m,2H),2.12-2.07(m,2H),1.91-1.85(m,2H),1.69-1.64(m,2H),1.49(s,9H)；

synthesis of Compound 11

Under the protection of nitrogen, 450mg of compound 10 and 240mg of ethyleneButyl Ether, 10mg of Pd (OAc)₂50mg of dppf and 160mg of DIEPA are added into 20ml of n-BuOH (the molar ratio of vinyl n-butyl ether to the compound 10 is 3:1, and the molar ratio of the main catalyst, the ligand and the compound 10 is 0.05:0.10:1), and the system is heated to 95 ℃ to react for 20 hours; after the reaction is finished, concentrating the system, passing through a column (DCM/EA is 2/1), and purifying by column chromatography to obtain 300mg of yellow solid which is the compound 11; nuclear magnetic resonance information for compound 11:¹H NMR(400MHz,CDCl₃)：8.77(s,1H),8.25(s,1H),8.22-8.20(d,1H,J＝9.04),8.05-8.04(d,1H,J＝2.6),7.35-7.32(dd,1H,J1＝2.92,J2＝9.08),4.53(d,1H,J＝2.24),4.18(d,1H,J＝2.24),3.88-3.84(t,2H,J＝6.6),3.63-3.60(t,4H,J＝4.84),3.13-3.10(t,4H,J＝4.76),2.42(s,3H),2.38-2.30(m,2H),2.14-2.03(m,2H),1.87-1.81(m,2H),1.76-1.70(m,2H),1.69-1.64(m,2H),1.49(s,9H),1.47-1.39(m,2H),0.96-0.92(t,3H,J＝7.36)；

synthesis of deuterated Palbociclib compound shown as formula (I)

Adding 300mg of the compound 11 into 6ml of dichloromethane, dropwise adding about 1.1ml of concentrated hydrochloric acid at room temperature, stirring overnight, extracting 2 times with concentrated hydrochloric acid (2ml), washing the water phase once with 5ml of dichloromethane, adjusting the pH value of the water phase to 9-10 with an aqueous solution of sodium hydroxide, filtering, washing the filter cake with water, and drying to obtain 180mg of yellow solid, namely the deuterated Palbociclib compound shown in the formula (I); the nuclear magnetic resonance information of the obtained deuterated Palbociclib compound is as follows:¹H NMR(400MHz,CDCl₃)：8.82(s,1H),8.21(s,1H),8.17-8.15(d,1H,J＝9.04),8.06-8.05(d,1H,2.84),7.35-7.31(dd,1H,J12＝2.96,J13＝9.08),3.17-3.15(t,4H,J＝4.24),3.06-3.09(t,4H,J＝4.24),2.55(s,3H),2.37(s,3H),2.34-2.31(m,2H),2.08-2.01(m,2H),1.90-1.85(m,2H),1.71-1.67(m,2H)。

the X-ray powder diffraction pattern of the obtained deuterated Palbociclib compound shown in the formula (I) is shown in figure 14. As can be seen from the figure, the X-ray powder diffraction measured using Cu — K α rays is, at an angle of 2 θ: 6.0816 °, 6.728 °, 11.0176 °, 12.0783 °, 12.8951 °, 16.418 °, 17.1983 °, 18.1531 °, 19.3347 °, 19.8212 °, 21.0866 °, 21.9638 °, 22.6555 °, 23.5909 °, 24.3915 °, 26.6649 °, 28.1331 °, 30.1598 °, 30.9903 °, 33.1409 °, 35.359 °, 36.4209 °, 37.919 ° have diffraction peaks.

The melting point of the prepared deuterated Palbociclib compound is 256-257 ℃ through melting point measurement.

Subsequently, we carried out a number of experiments for developing a pharmaceutically stable crystal form of the compound of formula (i). First, we have tested the solubility of the compound of formula (i) in nearly 40 solvents at different temperatures (see table 1 for details), and we have found that the solubility of this compound is very different among the solvents, and that the solubility of this compound is very different among the same type of solvents, such as 1.99mg/mL at 25 ℃ in methanol, but 0.72mg/mL at 25 ℃ in ethanol, which is the same type of solvent, and almost 3-fold difference in solubility, and the difference is even greater among other similar types of solvents, such as n-propanol, isopropanol, n-butanol, trifluoroethanol, and other alcoholic solvents. We have also found that the solubility of the compound of formula (i) in non-alcoholic solvents is very close to that of alcoholic solvents, such as 0.41mg/mL at 25 ℃ in isopropanol and 0.5mg/mL at 25 ℃ in acetone, which are three types of solvents commonly used in the final API production phase of pharmaceutical development. (in the production of the API, especially to the subsequent stages of the process, the use of the first solvent is strictly limited, the use of the second solvent is limited, and the use of the third solvent is limited as much as possible. This presents a great difficulty for the search for the crystal form of the compound of formula (I). Numerous experimental attempts have been made, and various crystallization methods such as suspension crystallization, volatile crystallization, and temperature-reduced crystallization have been attempted, and various solvent systems and combinations such as a single solvent system and a mixed solvent system, and the proportion selection of each solvent have been attempted. After a number of failures and then a summary of experience from the failures, we have developed three polymorphic forms of the compound of formula (i), form B, C and D in the specification. However, in the following studies, it was found by TGA and DSC analysis that they are solvates, have poor stability, are liable to change in properties and transformation of crystal form, and are liable to deteriorate after being left for a long time. In the next study, through a large number of experiments, we surprisingly found another crystal form of the compound shown in the formula (I), namely the crystal form A in the specification, and through TGA and DSC analysis of the crystal form, the crystal form is very stable. The stability of the compound is investigated by placing at normal temperature and an accelerated experiment, the stability of the polymorphic substance is found to be very good, no crystal form change is found by XRPD research, the compound is found to be stable in purity by HPLC, HNMR, high-resolution mass spectrum and other researches, no new impurity is generated and no original impurity is changed, and the deuteration rate is stable. Finally, the crystal form is determined to be a stable crystal form for continuously promoting the research of the medicine, and the stable crystal form is used for subsequent evaluation and clinical development. After the stable and reliable crystal form A is found, a small-scale crystallization method is carried out, and after a plurality of experiments, the gram-scale amplification is realized, and a method for obtaining the polymorphic substance by using a mixed solvent of anisole and n-butanol is developed. The process then allowed stable and reliable access to form a polymorph until scale-up.

The specific crystal form study process is described in the following section.

Secondly, preparation of crystal form of deuterated Palbociclib compound shown in formula (I)

The deuterated Palbociclib compound shown in the formula (I) is taken as free base, the appearance is yellow solid, the purity is 99.89%, the water content is less than 0.5%, and no solvent residue is detected.

First, a solubility experiment was performed: about 100mg of deuterated palbociclib free base is weighed out separately and placed in a glass sample bottle, and 1ml of solvent is added. The suspension is suspended stably by magnetic stirring at room temperature (22-26 ℃) and 60 ℃ respectively, and the system is ensured to be suspension and saturated. Stirring at room temperature for 24h and at 60 ℃ for 6 h. After stirring, standing for 15min, collecting supernatant, filtering with disposable syringe with 0.22 μm nylon filter into EP tube, and detecting content by HPLC. HPLC content detection is obtained by comparing peak areas of the sample and the reference substance at a wavelength of 254nm by using C18 reversed phase liquid chromatography and methanol/acetonitrile/water as a mobile phase of a gradient method. The measured solubility data for the deuterated Palbociclib compounds are shown in table 1 below.

TABLE 1

Remarking: "-" no data was obtained due to too much solubility.

2,2, 2-trifluoroethanol, benzyl alcohol, anisole and nitrobenzene were selected as good solvents according to the solubility data of Table 1.

1. Crystal form screening method

The solid crystal form screening of the deuterated Palbociclib compound is to obtain metastable-state and stable-state crystal forms by crystallization or precipitation from a selected single solvent and a mixed solvent thereof. The adopted method comprises suspension crystallization, volatilization crystallization and cooling crystallization.

1.1 suspension crystallization

At room temperature (22 ℃ -26 ℃), under the environment of 40% -60% relative humidity, respectively weighing about 100mg of deuterated Palbociclib compound, placing the compound into a glass sample bottle, and adding 1ml of solvent. Suspending by shaking table at 60 deg.C for 72h, filtering the solution, drying the solid part at 40 deg.C for 30min, and analyzing and identifying by powder X-ray diffraction (XRPD), Differential Scanning Calorimetry (DSC) and Thermogravimetry (TGA).

1.2 volatile crystallization

Under the environment of room temperature (22-26 ℃) and 40-60% of relative humidity according to solubility classification, respectively weighing 2-10 mg of deuterated Palbociclib compound, adding 200 mu l of good solvent, heating to 60 ℃, filtering all solution/suspension into an EP tube by using a disposable syringe with a 0.22 mu m nylon filter after 10 min. Adding 200 μ l of poor solvent into the good solvent filtrate, mixing, placing in a fume hood, naturally volatilizing at room temperature under atmospheric environment, and observing whether precipitate is separated out. The precipitated solid precipitate was analyzed and characterized by powder X-ray diffraction (XRPD), Differential Scanning Calorimetry (DSC), and Thermogravimetry (TGA).

1.3 temperature reduction crystallization

Under the environment of room temperature (22-26 ℃) and 40-60% of relative humidity according to solubility classification, respectively weighing 2-10 mg of deuterated Palbociclib compound, adding 200 mu l of good solvent, heating to 60 ℃, filtering all solution/suspension into an EP tube by using a disposable syringe with a 0.22 mu m nylon filter after 10 min. Adding 200 mul of poor solvent into the good solvent filtrate, mixing uniformly, placing in a refrigerator at 4 ℃, cooling and observing whether a precipitate is separated out. The resulting solid precipitate was analyzed and characterized by powder X-ray diffraction (XRPD), Differential Scanning Calorimetry (DSC), and Thermogravimetry (TGA).

2. Crystal form screening process

2.1 by volatile crystallization

2.1.1 Single solvent

Selecting 2,2, 2-trifluoroethanol, benzyl alcohol, anisole and nitrobenzene as solvents, respectively weighing 2mg-10mg of deuterated Palbociclib compound at room temperature (22 ℃ -26 ℃) under the environment of 40% -60% of relative humidity, adding 200 mu l of solvent, heating to 60 ℃, and filtering all solution/suspension into an EP tube by using a disposable syringe with a 0.22 mu m nylon filter after 10 min. The EP tube was placed in a fume hood open to the atmosphere at room temperature to allow natural evaporation and to observe whether or not a precipitate was precipitated. The results are shown in Table 2.

TABLE 2

Solvent(s)	Crystal form
		2,2, 2-trifluoroethanol	A
Benzyl alcohol	A
		Phenylmethyl ether	A
Nitrobenzene	A

2.1.2 Mixed solvent

Under the environment of room temperature (22-26 ℃) and 40-60% of relative humidity according to solubility classification, respectively weighing 2-10 mg of deuterated Palbociclib compound, adding 200 mu l of good solvent, heating to 60 ℃, filtering all solution/suspension into an EP tube by using a disposable syringe with a 0.22 mu m nylon filter after 10 min. Adding 200 μ l of poor solvent into the good solvent filtrate, mixing, placing in a fume hood, naturally volatilizing at room temperature under atmospheric environment, and observing whether precipitate is separated out. The results are shown in Table 3.

TABLE 3

Remarking: "-" indicates no solid was obtained.

2.2 crystallization by cooling

2.2.1 Single solvent

Selecting 2,2, 2-trifluoroethanol, benzyl alcohol, anisole and nitrobenzene as solvents, respectively weighing 2mg-10mg of deuterated Palbociclib compound at room temperature (22 ℃ -26 ℃) under the environment of 40% -60% of relative humidity, adding 200 mu l of solvent, heating to 60 ℃, and filtering all solution/suspension into an EP tube by using a disposable syringe with a 0.22 mu m nylon filter after 10 min. And (3) placing the EP pipe in a refrigerator at 4 ℃, cooling and observing whether a precipitate is separated out. The results are shown in Table 4.

TABLE 4

Solvent(s)	Crystal form
		2,2, 2-trifluoroethanol	-
Benzyl alcohol	-
		Phenylmethyl ether	-
Nitrobenzene

Remarking: "-" indicates no solid was obtained.

2.2.2 Mixed solvent

Under the environment of room temperature (22-26 ℃) and 40-60% of relative humidity according to solubility classification, respectively weighing 2-10 mg of deuterated Palbociclib compound, adding 200 mu l of good solvent, heating to 60 ℃, filtering all solution/suspension into an EP tube by using a disposable syringe with a 0.22 mu m nylon filter after 10 min. Adding 200 mul of poor solvent into the good solvent filtrate, mixing uniformly, placing in a refrigerator at 4 ℃, cooling and observing whether a precipitate is separated out. The results are shown in Table 5.

TABLE 5

Remarking: "-" indicates no solid was obtained.

2.3 suspension crystallization method

A general solvent with certain solubility and a representative poor solvent are selected to carry out suspension crystallization experiments.

At room temperature (22 ℃ -26 ℃), under the environment of 40% -60% relative humidity, respectively weighing about 100mg of deuterated Palbociclib compound, placing the compound into a glass sample bottle, and adding 1ml of solvent. Suspending at 60 deg.C for 72 hr with shaking, filtering the solution, and drying the solid part at 40 deg.C for 30 min. The results are shown in Table 6.

TABLE 6

Solvent(s)	Crystal form
		Anhydrous methanol	A
Anhydrous ethanol	A
		Isopropanol (I-propanol)	A
N-butanol	A
		Isobutanol	A
Sec-butyl alcohol	A
		1-methyl-2-butanone	A
Propylene glycol methyl ether	A
		Isopropyl benzene	A

3. Analysis of crystal form

The resulting solid sample is analyzed using a variety of detection analysis methods, such as powder X-ray diffraction (XRPD), Differential Scanning Calorimetry (DSC), and thermogravimetric analysis (TGA).

3.1 powder X-ray diffraction (XRPD)

Solid samples were analyzed using a powder X-ray diffraction analyzer (Bruker D8 advance) equipped with a LynxEye detector. The 2 theta scan angle of the sample was from 3 deg. to 40 deg., the scan step was 0.02 deg., and the tunnel voltage and current were 40kV and 40mA, respectively. Samples were tested using a zero background sample holder. The incident light is Cu-Ka spectral line, and the incident wavelength is 1.5406.

3.2 thermogravimetric analysis (TGA)

Thermogravimetric analysis was performed on the solid sample using METTLER TGA/DSC 2. Approximately 2.0mg of the sample was placed in a equilibrated platinum or aluminum sample pan and the sample mass was automatically weighed in a TGA furnace. The sample was heated to 300 ℃ at a rate of 10 ℃/min. During the test, the nitrogen flow rates to the balance chamber and sample chamber were 40mL/min and 60mL/min, respectively.

3.3 Differential Scanning Calorimetry (DSC)

The solid samples were analyzed using NETZSCH DSC200F3, the standard sample used for calibration being indium. Approximately 2.0mg of the sample was weighed accurately and placed in an aluminum sample pan and the exact mass of the sample was recorded. The sample was heated to 300 ℃ at a rate of 10 ℃/min under nitrogen (flow rate 50 mL/min).

4. Crystal form analysis results

4.1 form A

The crystal form A is an initial crystal form of the raw material deuterated palbociclib free base. The crystalline form is an anhydrate and TGA data shows weight loss < 0.5%. DSC showed a melting point between 260 ℃ and 270 ℃. The crystal form A is used as an initial crystal form and can be obtained in a plurality of single solvent and mixed solvent systems such as 2,2, 2-trifluoroethanol, benzyl alcohol and the like. Stability experiments show that the crystal form A is a stable crystal form.

Form A has an X-ray powder diffraction pattern (XRPD) and a DSC-TGA pattern which are respectively shown in figure 1 and figure 2 and are measured by using Cu-Ka rays.

As can be seen from FIG. 1, X-ray powder diffraction peak data of form A is shown in Table 7.

TABLE 7

2θ(°)	Relative Strength (%)
		5.05	39.8
7.96	18.3
		10.25	100.0
11.52	15.2
		14.08	11.4
15.95	3.4
		17.10	7.8
19.70	5.5
		22.50	9.1
28.44	1.1

4.2 form B

TGA data shows that there is a two-stage weight loss before crystal form B melts, with a first stage weight loss of 2.0% and a second stage weight loss of 8.1%. DSC showed a melting point between 240 ℃ and 260 ℃. The crystal form B can be obtained in a mixed solvent of trifluoroethanol and propylene glycol monomethyl ether by a volatilization crystallization mode. Form B is a solvate.

Form B has an X-ray powder diffraction pattern (XRPD) and a DSC-TGA pattern as shown in figure 3 and figure 4 respectively, which are measured by using Cu-Ka rays.

As can be seen from fig. 3, X-ray powder diffraction peak data of form B is shown in table 8.

TABLE 8

2θ(°)	Relative Strength (%)
		4.09	100.0
4.54	37.7
		7.78	2.4
8.85	4.9
		11.73	4.1
12.20	3.0
		13.78	5.5
17.49	2.2
		18.58	3.2
19.17	2.9
		20.36	2.4
21.26	1.8
		23.80	1.4

4.3 form C

TGA data for form C shows that there is a two-stage weight loss prior to melting, with a first stage weight loss of 2.1% and a second stage weight loss of 12.7%. DSC showed a melting point between 195 ℃ and 215 ℃. The crystal form C can be obtained in a mixed solvent of trifluoroethanol and 4-methyl-2-pentanone by means of cooling crystallization. Form C is a solvate. Form C is a metastable form as detected by XRPD.

Form C has an X-ray powder diffraction pattern (XRPD) and a DSC-TGA pattern as measured using Cu-ka radiation as shown in figures 5 and 6, respectively.

As can be seen from fig. 5, X-ray powder diffraction peak data of form C is shown in table 9.

TABLE 9

4.4 form D

TGA data shows that there is a two-stage weight loss before melting of form D, with a first stage weight loss of 2.5% and a second stage weight loss of 8.4%. DSC showed a melting point between 230 ℃ and 250 ℃. The crystal form D can be obtained in a mixed solvent of 2,2, 2-trifluoroethanol and propylene glycol methyl ether by a cooling crystallization mode. Form D is a solvate. Form D is a metastable form as detected by XRPD.

Form D has an X-ray powder diffraction pattern (XRPD) and a DSC-TGA pattern as shown in figure 7 and figure 8 respectively, which are measured by using Cu-Ka rays.

As can be seen from fig. 7, X-ray powder diffraction peak data of form D is shown in table 10.

Watch 10

2θ(°)	Relative Strength (%)
		4.06	100.0
7.78	3.5
		8.51	1.0
8.84	2.6
		10.56	0.7
11.66	1.5
		12.18	2.6
13.77	2.1
		15.58	0.8
18.52	1.4
		20.40	1.0

5. Study of Crystal form stability

5.1 Room temperature stability Studies

The XRPD contrast pattern of form A after standing for 6m (6 months) is shown in FIG. 9. As can be seen, the crystal form remains stable.

The XRPD contrast pattern of form B after standing for 1m is shown in figure 10. As can be seen from the figure, the position of the characteristic peak of the sample is unchanged after the sample is placed, the peak intensity is obviously reduced, and the crystallinity is reduced.

The XRPD contrast pattern of form C after standing at room temperature for 1m is shown in figure 11. As can be seen from the figure, the characteristic peak position of the crystal form C is obviously changed, and the crystal form C is a metastable crystal form.

The XRPD contrast pattern of form D after standing for 1m at room temperature is shown in figure 12. As can be seen from the figure, the characteristic peak position of the crystal form D is obviously changed, and the crystal form D is a metastable crystal form.

5.2 Heat investigation

DSC-TGA heating investigation was performed on the form B which is stable in the form of 1 m. As shown in fig. 4, the crystal form B begins to lose weight after being heated at 30 ℃, and can be divided into two stages according to the weight loss rate, wherein the first stage is 30 ℃ to 130 ℃, and is a slow weight loss stage; the second stage is 130-205 deg.c and is weight loss accelerating stage lasting to 205 deg.c. The weight loss in the first stage was 2.0%, and the weight loss in the second stage was 8.1%. The crystal form is prepared from a mixed solvent of propylene glycol methyl ether and trifluoroethanol, can be deduced from a weight loss curve and the boiling point (120 ℃) of the propylene glycol methyl ether, is a solvate of the propylene glycol methyl ether, and the propylene glycol methyl ether in a crystal lattice is lost at the second stage. It is concluded from the molecular weight of deuterated palbociclib free base (448.54) and the molecular weight of propylene glycol methyl ether (90.12) that 0.5 molecule of propylene glycol methyl ether is lost during heating.

5.3 accelerated stability study

And selecting a stable crystal form A to perform accelerated stability research, placing a crystal form A sample at 40 ℃ under the condition of 75% humidity for 1m, and observing the stability of the crystal form. The XRPD contrast patterns before and after are shown in FIG. 13. As can be seen, form A remains stable.

5.4 hygroscopicity study

Stable form a was selected for hygroscopicity studies. The method comprises the following steps:

(1) taking a dry glass weighing bottle with a plug (the outer diameter is 40mm, the height is 25mm), placing the bottle in a constant temperature and humidity box with the conditions of 25 ℃ and 80% of humidity one day before the test, and precisely weighing (m 1);

(2) taking a proper amount of a test article, flatly paving the test article in the weighing bottle, wherein the thickness of the test article is about 1mm, and precisely weighing the test article (m 2);

(3) opening the weighing bottle, and placing the weighing bottle and the bottle cap under the constant temperature and humidity condition for 24 hours;

(4) covering the weighing bottle cap, and precisely weighing (m 3);

percent weight gain (m3-m2)/(m2-m1) × 100%;

according to the following test data:

m1＝18729.35mg，m2＝19197.52mg，m3＝19199.84mg；

the percentage of gain was calculated to be 0.495% by substituting the formula.

A weight gain of less than 2% but greater than 0.2% is slightly hygroscopic according to the hygroscopicity criteria described in the pharmacopoeia. Thus form a is slightly hygroscopic.

6. Conclusion of Crystal form research

The invention comprehensively and deeply researches the polymorphism of the raw material drug deuterated palbociclib free base and screens the possible crystal forms of the raw material drug under different conditions. The polymorphic forms of the raw material medicines are screened by various ways, including solvent screening, suspension crystal transformation, volatilization crystallization, cooling crystallization and the like, and the crystal forms A to D are screened out. And judging that the crystal form A is a stable crystal form by normal temperature and accelerated stability research and combining hygroscopicity data. The crystal form A is recommended to be used as a dominant drug crystal form, and subsequent pharmacokinetic experiments and zoology experiments are carried out.

TABLE 11

7. Process for the preparation of polymorphic form A on gram scale

The deuterated Palbociclib compound (2g) shown in formula (I) is taken as free base, and the compound is yellow solid in appearance, has the purity of 99.89%, has the water content of less than 0.5%, and has no solvent residue. Dispersing into a mixed solution of anisole (48ml) and n-butanol (32ml), heating to 105 ℃ for complete dissolution, slowly cooling (cooling rate of 2 ℃/15 min) to 70 ℃, keeping the solution turbid, continuously slowly cooling (cooling rate of 1 ℃/20 min) to 58 ℃ for heat preservation overnight, then slowly cooling (cooling rate of 2 ℃/15 min) to 20 ℃, and filtering to obtain 1.45g of solid. Vacuum drying to obtain the polymorphic substance of the deuterated Palbociclib compound crystal form A shown in the formula (I). The X-ray powder diffraction pattern is in accordance with FIG. 1.

In the present invention, the error range of the 2 θ angle value of the X-ray powder diffraction peak is. + -. 0.2 °. It should be understood that the 2 θ values of the X-ray powder diffraction pattern may vary slightly from machine to machine and from sample to sample, and that the numerical ranges may differ by within ± 0.2 units, and therefore the values quoted are not to be interpreted as absolute values.

The crystal form A provided by the invention has stable physicochemical property and is very suitable for application in the aspects of research and development of medicines such as preparations in the future.

The crystal form A-crystal form D provided by the invention can be used for preparing CDK4/6 inhibitor medicines, especially the crystal form A.

Claims

1. A crystal form A of a deuterated Palbociclib compound is characterized in that the structural formula of the deuterated Palbociclib compound is shown as a formula (I):

2. Form a of the deuterated Palbociclib compound according to claim 1, characterized in that the X-ray powder diffraction measured with Cu-ka radiation at 2 Θ angles (in °): diffraction peaks are at 15.9 + -0.2, 17.1 + -0.2, 19.7 + -0.2, 22.5 + -0.2, and 28.4 + -0.2.

3. The crystalline form A of the deuterated Palbociclib compound according to claim 1 or 2, characterized in that the differential scanning calorimetry analysis pattern of the crystalline form A shows an endothermic peak at 260-270 ℃.

4. Use of the crystalline form a of the deuterated Palbociclib compound of any one of claims 1-3 in the preparation of a CDK4/6 inhibitor medicament.

5. A crystal form B of a deuterated Palbociclib compound is characterized in that the structural formula of the deuterated Palbociclib compound is shown as a formula (I):

the X-ray powder diffraction of the crystal form B measured by using Cu-Kalpha rays is as follows at an angle of 2 theta (unit is degree): diffraction peaks are at 4.0 + -0.2, 4.5 + -0.2, 7.7 + -0.2, 8.8 + -0.2, 11.7 + -0.2, 12.2 + -0.2, and 13.7 + -0.2.

6. Form B of the deuterated Palbociclib compound according to claim 5, characterized in that the X-ray powder diffraction measured using Cu-ka radiation at 2 Θ angles (in °) is: diffraction peaks are at 17.4 + -0.2, 18.5 + -0.2, 19.1 + -0.2, 20.3 + -0.2, 21.2 + -0.2 and 23.8 + -0.2.

7. Use of the crystalline form B of the deuterated Palbociclib compound of claim 5 or 6 in the preparation of a CDK4/6 inhibitor medicament.

8. A crystal form C of a deuterated Palbociclib compound is characterized in that the structural formula of the deuterated Palbociclib compound is shown as a formula (I):

the X-ray powder diffraction of the crystal form C measured by using Cu-Kalpha rays is as follows at an angle of 2 theta (unit is degree): diffraction peaks are found at 3.5 + -0.2, 3.8 + -0.2, 7.6 + -0.2 and 11.5 + -0.2.

9. Form C of the deuterated Palbociclib compound according to claim 8, characterized in that the X-ray powder diffraction measured using Cu-ka radiation at 2 Θ angles (in °) is: diffraction peaks are at 15.4 + -0.2, 17.4 + -0.2, 21.2 + -0.2, 23.2 + -0.2, and 35.1 + -0.2.

10. Use of the crystalline form C of the deuterated Palbociclib compound of claim 8 or 9 in the preparation of a CDK4/6 inhibitor medicament.

11. A crystal form D of a deuterated Palbociclib compound is characterized in that the structural formula of the deuterated Palbociclib compound is shown as a formula (I):

the X-ray powder diffraction of the crystal form D measured by using Cu-Kalpha rays is as follows at an angle of 2 theta (unit is degree): diffraction peaks are at 4.0 + -0.2, 7.7 + -0.2, 8.5 + -0.2, 8.8 + -0.2, 10.5 + -0.2, 11.6 + -0.2, 12.1 + -0.2 and 13.7 + -0.2.

12. Form D of the deuterated Palbociclib compound according to claim 11, wherein the X-ray powder diffraction at 2 Θ angles (in °), measured using Cu-ka radiation, of form D is: diffraction peaks are at 15.5 + -0.2, 18.5 + -0.2 and 20.4 + -0.2.

13. Use of the crystalline form D of the deuterated Palbociclib compound of claim 11 or 12 in the preparation of a CDK4/6 inhibitor medicament.