Compound as well as preparation method and application thereof

Abstract

The invention provides a compound as shown in a formula (I) or a solvate, a hydrate and pharmaceutically acceptable salt thereof, a crystal form thereof, a preparation method thereof and application thereof in preparation of a medicine for treating breast cancer. The compound has good stability and oral absorbability, and has ideal pharmacokinetic characteristics and good drug development prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, in particular to a crystalline form of a novel compound, its preparation method, pharmaceutical formulations comprising it and its medical use. BACKGROUND

[0002] Ribociclib is chemically known as 7-cyclopentyl-N,N-dimethyl-2-((5-piperazin-l-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]-pyrimidine-6-carboxamide, represented by the following structural formula:

[0003]

[0004] Ribociclib is currently marketed under the brand name of Kisqnor®. The film-coated tablets contain ribociclib as succinate salt. Ribociclib is an oral, reversible, and highly selective inhibitor of CDK (cyclin-dependent kinase) 4 / 6. It is proposed for the treatment of hormone receptor (HR) positive, human epidermal growth factor receptor 2 (HER2) negative advanced breast cancer.

[0005] International (PCT) publication no. WO2010020675 Al first disclosed ribociclib and its pharmaceutically acceptable salts. Its synthesis is specifically described in WO2010020675 Al, example 74. Further, the present application discloses pharmaceutical formulations of ribociclib and its therapeutic applications. No detailed information about the final form solid state is disclosed in this document.

[0006] International (PCT) publication no. WO2012064805 Al discloses succinate salt of ribociclib and polymorphic forms of succinate salt of ribociclib, i.e. non-hydrated and hydrated forms.

[0007] US patent no. US20170342075 discloses crystalline form I of ribociclib monosuccinate salt. The present application also discloses hemisuccinate salt.

[0008] European patent no. 3156406 Al discloses crystalline forms of amorphous form of ribociclib free base. Several salts of ribociclib are disclosed, including various crystalline forms.

[0009] International (PCT) publication no. WO2016091221 Al discloses salts of ribociclib, including adipate, maleate and glycolate.

[0010] International (PCT) publication no. WO2018051280 discloses salts of ribociclib, including isethionate, oxalate, phosphoric acid, tartaric acid, acetic acid, trifluoroacetic acid, hydrobromic acid, citric acid and p-toluenesulfonic acid.

[0011] International (PCT) publication no. WO2023220640 discloses CDK protein degraders, compounds of various derivatives, and pharmaceutical compositions thereof.

[0012] International (PCT) publication no. WO2011130232 discloses a combination comprising a cyclin-dependent kinase 4 or cyclin-dependent kinase (CDK4 / 6) inhibitor and an MTOR inhibitor for the treatment of cancer.

[0013] International (PCT) publication no. WO2010020675 discloses pyrrolopyrimidine compounds as CDK inhibitors.

[0014] International (PCT) publication no. WO2021124106, WO2021124104 discloses selective CDK4 / 6 inhibitors cancer therapeutics.

[0015] International (PCT) publication no. WO2024086361 discloses molecular glue degrader compounds and uses thereof.

[0016] International (PCT) publication no. WO2022235698 discloses compounds for inhibiting or degrading a target protein, compositions including the same, methods of making the same, and methods of using the same.

[0017] According to the European CHMP assessment report (EMEA / H / C / 004213 / 0000) on , the drug substance Ribociclib succinate is in anhydrous form with low solubility and moderate permeability, which belongs to the IV class of the Biopharmaceutics Classification System (BCS). Among the four BCS classes, IV class of drug substance faces the greatest challenge in achieving sufficient bioavailability due to low solubility and poor permeability.

[0018] In addition, according to the CHMP report on , the drug substance Ribociclib succinate in the approved tablet has hygroscopicity. In line with this hygroscopicity, the CHMP report describes packaging measures commonly used to exclude moisture, such as using a “very tight” four-layer laminate to store Ribociclib succinate drug substance, and blister packaging of the drug product. In addition, The coating of the tablet includes polyvinyl alcohol, which can be used as a moisture barrier for Ribociclib succinate tablets according to WO 2016 / 166703 A1. In addition, The storage temperature of

[0019] The discovery of new derivatives of active ingredients or new polymorphs thereof offers the opportunity to improve their properties, increasing the possibilities available to the formulation expert in developing new pharmaceutical forms, drugs with a specific release profile or specific solubility.

[0020] Based on these considerations, there is still a need for new compounds and new polymorphs thereof with further improved physical and / or chemical properties. Therefore, the inventors of the present application believe it is worth exploring new pharmaceutical crystalline forms of ribociclib ester with good chemical purity and improved stability properties, which can further improve the properties of ribociclib in finished pharmaceutical products, especially improving oral bioavailability and long-term storage stability and simplifying the manufacturing process. SUMMARY

[0021] The present application, by modifying the structure of ribociclib, prepares a derivative structure with better pharmaceutical properties, aiming to at least partially solve the technical problems existing in the prior art. To this end, in one aspect of the present application, a compound represented by formula (I) or a solvate, hydrate and pharmaceutically acceptable salt thereof is provided:

[0022]

[0023] The compound is crystalline.

[0024] Preferably, the X-ray powder diffraction pattern of the crystal of the compound has characteristic peaks at diffraction angles 2θ: 8.447±0.2, 9.588±0.2, 16.913±0.2, 21.153±0.2, 21.572±0.2, 22.294±0.2.

[0025] Preferably, the X-ray powder diffraction pattern of the crystal of the compound has characteristic peaks at diffraction angles 2θ: 5.30±0.2, 7.18±0.2, 9.54±0.2, 13.72±0.2, 14.60±0.2, 16.80±0.2, 16.90±0.2, 18.46±0.2, 19.86±0.2, 20.98±0.2, 21.08±0.2, 22.40±0.2, 23.74±0.2, 25.20±0.2, 26.36±0.2, 26.40±0.2, 27.86±0.2°;

[0026] Preferably, the X-ray powder diffraction pattern of the crystal of the compound is as shown in Figure 1 It should be noted that due to different measurement conditions, the 2θ angle and relative intensity of each peak on the X-ray powder diffraction pattern will vary, generally within ±0.2° for the 2θ angle and within ±5% for the relative intensity, which are considered reasonable errors.

[0027] Preferably, the DSC curve of said crystalline form of the compound shows an endothermic peak with a starting temperature of ± 2°C and a peak temperature of ± 2°C, and the DSC curve of said crystalline form of the compound is as shown in Figure 1. Figure 2

[0028] In another aspect of the present application, a process for preparing the compound of formula (I) is provided, comprising the following steps:

[0029] Step 1: intermediate 1 is prepared from starting material SM1 and starting material SM2 in the presence of a non-polar solvent;

[0030] Step 2: crude product of formula (I) is prepared from the product of step 1 and rebosoxib in the presence of a solvent;

[0031] Step 3: pharmaceutical grade raw material of formula (I) is prepared from the crude product of formula (I) in the presence of a crystallization solvent, and the reaction scheme is as follows:

[0032]

[0033] Preferably, in the process for preparing the derivative of the present application, the solvent of step 1 is selected from one of dichloromethane, chloroform, ethyl acetate, isopropyl acetate, etc., preferably dichloromethane;

[0034] Preferably, in the process for preparing the derivative of the present application, the reaction solvent of step 2 is selected from N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, tetrahydrofuran, dioxane, preferably N,N-dimethylformamide;

[0035] Preferably, in the process for preparing the derivative of the present application, the crystallization solvent of step 3 is selected from one or a combination of dichloromethane, acetone, ethyl acetate, n-hexane, anhydrous ethanol, anhydrous methanol, preferably a combination of dichloromethane and acetone.

[0036] The temperature of the process for preparing the compound of the present application and the crystallization process is room temperature.

[0037] In further embodiments, the product prepared from the derivative of the present application is subjected to chromatographic spectral analysis to confirm the purity and structure, and the chromatographic analysis conditions are as follows:

[0038] Chromatographic conditions of YPK-016

[0039]

[0040] The structure and crystallization of the product are confirmed by hydrogen spectrum, carbon spectrum, mass spectrum, infrared, powder diffraction, and differential scanning.

[0041] In yet another aspect of the present application, a pharmaceutical composition is provided.

[0042] ​Preferably, the pharmaceutical composition comprises a compound or a crystalline form of a compound as previously described and a pharmaceutically acceptable carrier.

[0043] The carrier in the pharmaceutical composition is "acceptable" in the sense of being compatible with the active ingredients of the composition and not deleterious to the subject being treated (and preferably capable of stabilizing the active ingredients). One or more solubilizing agents can be used as pharmaceutical excipients for delivery of the active compounds.

[0044] The pharmaceutical compositions of the present application can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, perineural injection, axonal injection, intra-articular injection, oral administration or topical administration. In some embodiments, the pharmaceutical compositions of the present application for oral use can be in any orally acceptable dosage form including capsules, tablets, emulsions, aqueous suspensions, suppositories, sprays, inhalers, dispersions, and solutions. In the case of tablets, carriers which are used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For capsules, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added. Oral solid dosage forms can be prepared by spray-drying techniques; hot-melt extrusion strategies, micronization and nanomilling techniques. Spray or inhaler compositions can be prepared according to techniques well known in the art of pharmaceutical formulation. For example, such compositions can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. Compositions with the active compound can also be administered in the form of suppositories for rectal administration.

[0045] In some embodiments, the crystalline form of the compound or pharmaceutical composition containing the same can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenterally" as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0046] The crystalline form of the compound of the present application is a CDK4 / 6 inhibitor, which can be used in the treatment of breast cancer. The breast cancer indications include primary therapy for hormone receptor (HR) positive, human epidermal growth factor receptor 2 (HER2) negative locally advanced or metastatic breast cancer and adjuvant therapy for HR+ / HER2- high risk early stage breast cancer patients.

[0047] Another aspect of the present application also includes the use of the compound or a crystalline form of a compound of the present application or a composition containing the same in the manufacture of a medicament for the treatment of breast cancer.

[0048] The gist of the present application is:

[0049] 1) The crystal of the compound of the present application has good stability, does not change crystal during grinding, and has single crystal form stability under various solvent conditions. The crystal is packaged in a medicinal low-density polyethylene bag and a polyester / aluminum / polyethylene medicinal composite film, and under the influence of factors such as light, high temperature, and high humidity, there is no significant change in various indicators, which all meet the requirements of the quality standard, and the stability is good;

[0050] 2) The compound of the present application has high permeability and the characteristics of non-efflux transport substrate. The product has good oral absorption and bioavailability, and the difficulty of formulation development is relatively small;

[0051] 3) The compound of the present application has no hygroscopicity, high purity, and simple crystallization production process, and has no polymorphism under various solvents;

[0052] 4) The above are all the technologies that the compound of the present application does not have.

[0053] The novel derivative of the present application has a research and development code of YPK-016 of our company. BRIEF DESCRIPTION OF DRAWINGS

[0054] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0055] Figure 1 XRPD spectrum of the crystalline form I of the compound of formula (I).

[0056] Figure 2 DSC-TGA spectrum of the crystalline form I of the compound of formula (I).

[0057] Figure 3 Beagle dog plasma preliminary pharmacokinetic diagram of the compound of formula (I) and the compound of formula (II).

[0058] Figure 4 NMR spectrum of the compound of formula (I). 1 H spectrum.

[0059] Figure 5 C spectrum of the compound of formula (I). 13

[0060] Figure 6 LC-MS spectrum of the compound of formula (I). DETAILED DESCRIPTION​

[0061] The technical solutions of the present application will be described clearly and completely below in conjunction with the drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present application.

[0062] The present application will be further explained and described below in conjunction with specific implementation manners.

[0063] The following examples can further describe the present application, however, these examples should not be regarded as limiting the scope of the present application.

[0064] Reaction equation of Example 1:

[0065]

[0066] Material ratio:

[0067] Operation process:

[0068] Into a 250 mL three-necked flask, DMF (43 mL) and N, N-carbonyldiimidazole (1.7 g, 10.5 mmol) were added, the solution was stirred and clarified, and then cooled to 0-10 ℃, 4-(hydroxymethyl)-5-methyl-[1, 3]dioxol-2-one (1.43 g, 11 mmol, diluted with 5 mL of DMF) was added dropwise, and then the reaction was carried out at room temperature for 2 hours, after which roxilfene (4.34 g, 10 mmol) was added in batches, TLC monitoring (254 nm, DCM:MeOH = 10:1) showed that there was a clear reaction point, and roxilfene was completely reacted, 100 mL of water was added to quench the reaction at 0-5 ℃, and a clear solid was obtained, which was filtered and washed with water, and then dried and rotary evaporated to obtain 5.64 g of light yellow solid, which was purified by column chromatography (DCM:MeOH = 50:1) to obtain 1.08 g of light yellow solid, batch number: YPK016-250723-01, and the mass spectrum result of the compound +H = 591.2.

[0069] Crystallization: 0.5 g of the obtained solid was taken, 2 mL of dichloromethane was added to stir and clarify the solution, 8 mL of acetone was added, and then the mixture was naturally placed for 4 days, and then a clear solid gradually precipitated, which was filtered under reduced pressure and dried to obtain 0.48 g of white solid, batch number: YPK016-250729-01, HPLC purity: 98.03%; the melting point determined by the laboratory automatic melting point apparatus was 197.2-198.0 ℃.

[0070] The structure and purity of the product were determined by chromatography and spectroscopy, and the structure analysis is shown in detail.

[0071] Example 2

[0072]

[0073] Material ratio:

[0074]

[0075] Operation process:

[0076] 50 mL three-necked flask was added ribociclib (0.43 g, 1 mmol), DMSO (20 mL) was stirred to dissolve, the side chain (0.36 g, 1.2 mmol) was added, and the reaction was stirred at room temperature under nitrogen protection for 1 h. TLC monitoring ribociclib reaction was complete (254 nm, DCM:MeOH = 20:1), a new product point was generated, ice water (40 mL) was added to quench the reaction, ethyl acetate was extracted twice (50 mL*2), and the organic phase was dried over anhydrous sodium sulfate. After concentration under reduced pressure, 0.82 g of yellow oil was obtained. Column chromatography purification (DCM, DCM:MeOH = 40:1) gave 0.49 g of light yellow solid, which was the same as the product obtained in Example 1.

[0077] Crystallization: 0.5 g of the solid product (YPK-016) obtained above was taken, 2 mL of dichloromethane was added and stirred to dissolve, 8 mL of anhydrous ethanol was added, and it was naturally placed for 2 days. Gradually, obvious solid was precipitated. After vacuum filtration and drying, 0.47 g of white solid was obtained, batch number: YPK016-250727-01, HPLC purity: 98.23%; melting point: 197.6-198.7℃.

[0078] Example 3

[0079] 100 mL three-necked flask was added ribociclib (0.95 g, 2.2 mmol), DMSO (40 mL) was stirred to dissolve, the side chain (0.64 g, 2.2 mmol) was added, and the reaction was stirred at room temperature under nitrogen protection for 1 h. TLC monitoring ribociclib reaction was complete (254 nm, DCM:MeOH = 20:1), a new product point was generated, ice water (70 mL) was added to quench the reaction, ethyl acetate was extracted twice (100 mL*2), and the organic phase was dried over anhydrous sodium sulfate. After concentration under reduced pressure, 1.52 g of yellow oil was obtained. Column chromatography purification (DCM, DCM:MeOH = 40:1) gave 1.07 g of light yellow solid, which was detected by HPLC purity: 97.95%, batch number: YPK016-250725-01.

[0080] Example 4

[0081] Into a 100 mL flask, add the side chain (0.64 g, 2.2 mmol) to the solution of ribociclib (0.95 g, 2.2 mmol) in DCM (40 mL) and stir at room temperature. After 1 h, TLC monitoring of the reaction of ribociclib is complete (254 nm, DCM:MeOH = 20:1), and a new product spot is formed. Add ice water (70 mL) to quench the reaction, and extract twice with ethyl acetate (100 mL*2). Wash twice with brine, dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 1.56 g of yellow oil. Purify by column chromatography (DCM, DCM:MeOH = 40:1) to obtain 0.94 g of a light yellow solid, which is the same product as obtained in Example 1.

[0082] Example 5

[0083] Into a 100 mL flask, add the side chain (0.64 g, 2.2 mmol) to the solution of ribociclib (0.95 g, 2.2 mmol) in DCM (40 mL) and stir at room temperature. After 1 h, TLC monitoring of the reaction of ribociclib is complete (254 nm, DCM:MeOH = 20:1), and a new product spot is formed. Add ice water (70 mL) to quench the reaction, and extract twice with ethyl acetate (100 mL*2). Wash twice with brine, dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 1.56 g of yellow oil. Purify by column chromatography (DCM, DCM:MeOH = 40:1) to obtain 0.94 g of a light yellow solid, which is the same product as obtained in Example 1.

[0084] Example 6

[0085] Reaction equation:

[0086]

[0087] Material ratio:

[0088]

[0089] A 100 mL flask was charged with ribociclib (0.95 g, 2.2 mmol) and DCM (40 mL) and stirred at room temperature. The side chain (0.42 g, 2.2 mmol) was added and the reaction stirred at room temperature under a nitrogen atmosphere for 1 h. TLC monitoring (254 nm, DCM:MeOH = 20:1) showed that ribociclib was not fully reacted and a new product spot was formed. The reaction was quenched with ice water (70 mL) and the layers were separated. The aqueous layer was extracted once more with DCM and the combined DCM organic layers were washed twice with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 1.26 g of yellow oil. Purification by column chromatography (DCM, DCM:MeOH = 40:1) gave 0.56 g of a light yellow solid, which was the same spot as the product obtained in Example 1.

[0090] Example 7

[0091] A 100 mL flask was charged with ribociclib (0.95 g, 2.2 mmol) and DCM (40 mL) and stirred at room temperature. The side chain (0.63 g, 3.3 mmol) was added and the reaction stirred at room temperature under a nitrogen atmosphere for 1 h. TLC monitoring (254 nm, DCM:MeOH = 20:1) showed that ribociclib was not fully reacted and a new product spot was formed. The reaction was quenched with ice water (70 mL) and the layers were separated. The aqueous layer was extracted once more with DCM and the combined DCM organic layers were washed twice with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 1.47 g of yellow oil. Purification by column chromatography (DCM, DCM:MeOH = 40:1) gave 0.86 g of a light yellow solid, which was the same spot as the product obtained in Example 1.

[0092] Example 8

[0093] A 100 mL flask was charged with ribociclib (0.95 g, 2.2 mmol) and DCM (40 mL) and stirred at room temperature. The side chain (0.84 g, 4.4 mmol) was added and the reaction stirred at room temperature under a nitrogen atmosphere for 1 h. TLC monitoring (254 nm, DCM:MeOH = 20:1) showed that ribociclib was fully reacted and a new product spot was formed. The reaction was quenched with ice water (70 mL) and the layers were separated. The aqueous layer was extracted once more with DCM and the combined DCM organic layers were washed twice with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 1.62 g of yellow oil. Purification by column chromatography (DCM, DCM:MeOH = 40:1) gave 1.02 g of a light yellow solid, which was the same spot as the product obtained in Example 1.

[0094] Example 9

[0095] Into a 100 mL flask, was added Zolciclib (0.95 g, 2.2 mmol) in ethyl acetate (40 mL) and stirred at room temperature. The side chain (0.84 g, 4.4 mmol) was added and the reaction was stirred at room temperature for 1 h under nitrogen protection. TLC monitoring showed that the reaction of Zolciclib was complete (254 nm, DCM:MeOH = 20:1), a new product spot was generated. The reaction was quenched by adding ice water (70 mL), and the aqueous layer was extracted once more with dichloromethane. The dichloromethane organic phase was combined, washed twice with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 1.76 g of yellow oil. Purification by column chromatography (DCM, DCM:MeOH = 40:1) gave 1.09 g of a light yellow solid, which was the same as the product obtained in Example 1.

[0096] Example 10

[0097] Into a 100 mL flask, was added Zolciclib (0.95 g, 2.2 mmol) in ethyl acetate (40 mL) and stirred at room temperature. The side chain (0.84 g, 4.4 mmol) was added and the reaction was stirred at room temperature for 1 h under nitrogen protection. TLC monitoring showed that the reaction of Zolciclib was complete (254 nm, DCM:MeOH = 20:1), a new product spot was generated. The reaction was quenched by adding ice water (70 mL), and the aqueous layer was extracted once more with dichloromethane. The dichloromethane organic phase was combined, washed twice with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 1.76 g of yellow oil. Purification by column chromatography (DCM, DCM:MeOH = 40:1) gave 1.09 g of a light yellow solid, which was the same as the product obtained in Example 1.

[0098] Example 11

[0099] YPK016 detection spectrum analysis

[0100]

[0101] 1 H-NMR (400 MHz, d-DMSO): δ [ppm] = 9.383 (s, 1H); 8.778 (s, 1H); 8.029-8.204 (m, 2H); 7.485 (dd, 1H); 6.619 (s, 1H); 4.982 (s, 2H); 4.750 (q, 1H); 3.565 (m, 4H); 3.078-3.127 (m, 10H); 2.453 (m, 2H); 2.193 (s, 3H); 1.999 (m, 4H); 1.667 (m, 2H).

[0102] Table 1: NMR hydrogen spectrum detection results of products

[0103] Into a 100 mL flask, was added Zolciclib (0.95 g, 2.2 mmol) in ethyl acetate (40 mL) and stirred at room temperature. The side chain (0.84 g, 4.4 mmol) was added and the reaction was stirred at room temperature for 1 h under nitrogen protection. TLC monitoring showed that the reaction of Zolciclib was complete (254 nm, DCM:MeOH = 20:1), a new product spot was generated. The reaction was quenched by adding ice water (70 mL), and the aqueous layer was extracted once more with dichloromethane. The dichloromethane organic phase was combined, washed twice with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 1.76 g of yellow oil. Purification by column chromatography (DCM, DCM:MeOH = 40:1) gave 1.09 g of a light yellow solid, which was the same as the product obtained in Example 1.

[0104] Table 2. NMR Carbon Spectrum Test Results of the Product

[0105]

[0106] Table 3. Mass Spectrum Test Results of the Product

[0107] Mass to charge ratio (m / z) Remarks 591.2 [[M+H] + ]]>

[0108] Table 4. Infrared Test Results of the Product

[0109]

[0110]

[0111] 5: DSC-TGA Test Results

[0112] From the DSC-TGA test pattern, it can be seen that the product does not contain crystal water or crystallization solvent, the melting point is 201.62°C, and decomposition starts at about 262°C.

[0113] 6: XRPD Test Results

[0114] XRPD shows that the product is a crystalline powder. The characteristic 2θ angle is: 8.447, 9.588, 11.784, 12.646, 13.304, 13.617, 15.066, 15.472, 16.913, 17.477, 19.025, 20.015, 20.800, 21.153, 21572, 22.294, 23.069, 23.565, 24.611, 25.457, 25.884, 26.637, 27.298, 28.062, 28.924, 29.221, 30.679. Hereinafter, this crystal form is referred to as crystal form I.

[0115] The crystalline body has good stability, and no crystal transformation occurs after grinding, high temperature for 30 days, light for 30 days, and high humidity for 30 days, respectively, and the crystal form has good stability.

[0116] Example 12: Crystal Stability Test

[0117] 1 Grinding Test:

[0118] 1 g of the crystalline body of the compound of the present application was weighed into an agate mortar and ground thoroughly for half an hour. The sample was collected and weighed to obtain 0.97 g. The product was proved to be crystal form I of the compound by X-ray powder diffraction pattern, and the crystal form was not transformed under this condition.

[0119] 2 Influence Factor Test

[0120] The crystalline compound provided in Example 1 was packaged in a medicinal low-density polyethylene bag and a polyester / aluminum / polyethylene medicinal composite film, and then subjected to light irradiation (total illuminance of fluorescent lamp was not less than 1.2 x 106 Lux·hr, and near-ultraviolet lamp energy was not less than 200 w·hr / m2), high temperature (60℃), and high humidity (25℃, RH: 90%±5, high humidity for 30 days), and the results were as follows:

[0121] Table 5. Results of influence factor test

[0122]

[0123]

[0124] From the data in the table, it can be seen that the crystalline compound is stable under the conditions of high temperature, high humidity, and light irradiation. The X-ray diffraction spectrum of the compound is consistent with the initial data, and no crystal transformation occurs, indicating that the crystalline form provided by the present application has good stability and is beneficial to the storage of the drug substance.

[0125] 3. Crystal form research

[0126] The crystalline compound of formula (I) 60 mg was added to different 2 ml glass vials, and an appropriate amount of solvent was added to form a suspension or solution, and then stirred at 40-45℃ for 2 h, and the crystal was detected, and the results were as follows:

[0127] Table 6. Results of crystal form research in different solvents

[0128] No. Solvent Crystal form 1 Methanol Crystal form I 2 Ethanol Crystal form I 3 Acetone Crystal form I 4 Isopropanol Crystal form I 5 Ethyl acetate Crystal form I 6 Dichloromethane Crystal form I 7 Tetrahydrofuran Crystal form I

[0129] The results show that the crystalline compound of formula (I) is stable in the solvents in Table 6, and does not change.

[0130] Example 13. Comparison of the physical and chemical properties of the crystalline compound and ribociclib

[0131] Table 7. Comparison of the physical and chemical properties of the compound of the present application and ribociclib

[0132]

[0133] The above results show that the compound of the present application has great advantages in stability and drugability.

[0134] Example 14. Stability test

[0135] The compound of the present application will be used as a raw material for preparing a pharmaceutical grade in the future, and thus the storage problem is very critical. Other impurities can be introduced or generated during storage, which affects the use of the raw material, and thus the stability of the raw material of the present application is investigated. According to the ICH Guideline for Stability Testing of New Drug Substances and Products, the experiment is carried out by using the influence factor test.

[0136] 1 Test observation conditions: The high humidity test of the product is carried out at 25℃±2℃, RH: 75%±5%, under the conditions of removing the inner and outer packaging at high temperature (60℃) for 30 days, and light (total illumination not less than 1.2×106Lux·hr) according to the observation of removing the inner and outer packaging.

[0137] 2 Sampling detection: high temperature sampling at 5 days, 10 days and 30 days; high humidity sampling at 5 days and 10 days; light sampling at 5 days and 11 days.

[0138] 3 Observation results: see the following table

[0139] Table 8 Stability test observation results

[0140]

[0141] Conclusion: As shown in the table, the raw material of the present application can maintain good chemical stability and crystal form stability during the storage period under the relevant conditions. It is shown that the crystal form provided by the present application has good stability, which is beneficial to the storage of the raw material, and can be stored at room temperature.

[0142] Example 15 Evaluation of bidirectional permeability of Caco-2 cells

[0143] The Caco-2 cell monolayer model is a model widely used for studying small intestine absorption, which can provide comprehensive information on drug molecule absorption, distribution and transport through small intestine mucosa at the cellular level, and has good correlation with the in vivo small intestine epithelial cell absorption process of oral drugs. The Caco-2 cell line is derived from human colon adenocarcinoma cells, and its morphology, marker enzyme function expression and permeation characteristics are similar to those of the small intestine. The Caco-2 cell monolayer model is used to analyze the absorption and transport characteristics of the compound. This experiment is used to determine the bidirectional permeability of the compound of the present application and the reference compound ribociclib through the Caco-2 cell model.

[0144] 1 Reagents for test

[0145] The compound used in Example 1 of this invention (content 98.81%); the control compounds atenolol, propranolol, reboxil (synthesized by our company) and the internal standard tolbutamide were purchased from Sigma-Aldrich; MEM culture medium, Trypsin, EDTA, and fetal bovine serum (FBS) were purchased from Sigma-Aldrich; L-glutamine and sodium pyruvate were purchased from Sinopharm Reagent Group; the penicillin-streptomycin bispecific antibody was purchased from Sigma-Aldrich; and the P-glycoprotein inhibitor Elacridar (GF120918A) was purchased from TRC Canada.

[0146] 2. Caco-2 cell monolayer model absorption and transport assay method

[0147] 2.1 Cell Culture

[0148] Cell culture using 2 mmol·L -1 L-glutamine, 10% fetal bovine serum (FBS), 100 U / ml -1 Penicillin-G and 100 mg / mL -1 Streptomycin in MEM medium. Cell culture conditions were 37°C, 5% CO2, and 95% relative humidity. When cells reached 80%–90% confluence, trypsin (0.05%, w / v) / EDTA (0.02%, w / v) digestion solution was added to digest the cells and seed the plate. This experiment used passage 46 Caco-2 cells, and the cells were cultured at a concentration of 1 × 10⁻⁶ cells / mL. 5 Cell·cm -2 The medium was densely seeded into Transwell-96 well plates and incubated in a CO2 incubator for 22 days, with the medium being changed every 4 to 5 days during this period.

[0149] 2.2 Assessing the integrity of Caco-2 cell monolayers

[0150] The integrity of the Caco-2 cell layer was assessed using a fluorescent yellow efflux assay. Fluorescent yellow samples were collected from the apex and basal ends and analyzed using an M2 assay. e The relative fluorescence intensity was detected at 425 / 528 nm (excitation / emission) using a plate reader. The remaining solution in the cell plate was discarded by gently tapping the plate. Acetonitrile solution containing an internal standard was added to lyse the cells, and the cells were collected after gentle shaking for 5 min. All samples were vortexed and centrifuged at 20 °C for 20 min. The supernatant was diluted with ultrapure water and stored at 4 °C for analysis by ultra-high performance liquid chromatography-tandem mass spectrometry.

[0151] 2.3 Bidirectional transport test

[0152] Remove the culture medium from the culture plate and rinse the cells twice with preheated transport buffer. Add the drug solution and receiver solution to the corresponding wells of the cell plate, respectively, to begin the bidirectional transport assay: tip to base (AB) and base to tip (BA). Incubate the cell plate at 37°C, 5% CO2, and 95% relative humidity for 120 min. For T0 samples, mix the initial drug solution with the transport buffer and acetonitrile solution containing the internal standard in a specific ratio. After incubation for 120 min, collect the final sample from both the drug and receiver ends, and mix it with the transport buffer and acetonitrile solution containing the internal standard in a specific ratio.

[0153] 2.4 Test Results

[0154] The transmembrane flux of fluorescent yellow leakage markers was used as an indicator for evaluating cell integrity in the Caco-2 cell model. The experimental results are shown in the table.

[0155] Table 9. Transmittance (%) of different compounds of fluorescein

[0156]

[0157]

[0158] The results showed that the transmittance of fluorescent yellow was <1%, which met the acceptable standard, and the Caco-2 cell layer had good integrity.

[0159] The permeability of the control compound reboxil and the compound of this invention is shown in the table below.

[0160] Table 10 Permeability results of different compounds

[0161]

[0162] As a marker of permeability via the cellular bypass pathway, atenolol typically exhibits low permeability; in this assay, the mean apparent permeability coefficient (Papp(AB)) of atenolol was 0.25 × 10⁻⁶. -6 cm·s -1 As a marker of passive transcellular permeability, propranolol typically exhibits high permeability; in this experiment, the average apparent permeability coefficient (Papp(AB)) of propranolol was 23.84 × 10⁻⁶. -6 cm·s -1 .

[0163] The results show:

[0164] Without the addition of GF120918A, at dosage concentrations of 1.0, 4, and 20 μmol·L⁻¹ -1 At that time, the apparent permeability constants of the present invention in the direction from A to B were 12.893, 14.72, and 11.56 × 10⁻⁶, respectively. -6 cm·s-1 The apparent permeability constants in the direction from B to A are 6.95, 7.87, and 7.68 × 10⁻⁶, respectively. -6 cm·s -1 The efflux rates REa at the three dosage concentrations were 0.55, 0.56, and 0.67 (all <2). Therefore, no significant efflux transport was observed. This invention is unlikely to be a substrate for efflux transporters. GF120918A was added at dosage concentrations of 1.0, 4, and 20 μmol·L⁻¹. -1 At that time, the apparent permeability constants of the present invention in the direction from A to B were 14.31, 14.43, and 13.95 × 10⁻⁶, respectively. -6 cm·s -1 The apparent permeability constants in the direction from B to A are 7.83, 8.41, and 7.75 × 10⁻⁶, respectively. -6 cm·s -1 The efflux rates REi at the three dosing concentrations were 0.57, 0.58, and 0.57, respectively. Therefore, the REa / REi values ​​were 0.93, 0.93, and 1.11, respectively. The present invention achieved recoveries of 83.65%–98.97% and total recoveries of 86.83%–99.45% at different concentrations, regardless of whether GF120918A was added to the sample solution. A total recovery rate of 70% or higher was optimal. In this experiment, the present invention demonstrated sufficient recoveries under various experimental conditions. In summary, the present invention showed good recovery at test concentrations of 1.0, 4, and 20 μmol·L⁻¹. -1 At that time, it exhibited high permeability in Caco-2 cells, indicating that the tested compound was unlikely to be a substrate for efflux transporters. This demonstrates that the high permeability and non-efflux transporter characteristics of this invention suggest good oral absorption and bioavailability, and that formulation development is relatively straightforward.

[0165] Example 16: Preliminary Pharmacokinetics and Bioavailability Study of Beagle Dog Plasma

[0166] The pharmacokinetic process of a single oral dose (6.8 mg / kg of the compound of this invention (equivalent to 5.0 mg / kg of reboxil) or 5.0 mg / kg of reboxil) in dogs was investigated, and the main pharmacokinetic parameters were calculated.

[0167] Male Beagle dogs, weighing 8-10 kg, were acclimatized for one week and fasted for 12 hours before administration (with free access to water). Two dogs were assigned to each group (the groups were swapped after one week, n=4). The compound of this invention and reboxil (both suspended in 0.5% CMC-Na, 1 ml / kg body weight) were administered by gavage. Blood samples of approximately 4 ml were collected at 0 h before administration, and at 0.083, 0.25, 0.45, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after administration. Place the sample in a collection tube, transfer 200 μL to an EP tube, centrifuge at 6000 rpm and 4°C for 10 min to separate the plasma; separately, take 3 ml of whole blood and slowly add it to a 15 ml centrifuge tube containing 3 ml of canine peripheral blood mononuclear cells pre-added, centrifuge at 550 g for 25 min in a horizontal rotor centrifuge, aspirate the white cloud-like PBMC cells, wash and centrifuge to obtain cell pellet, discard the supernatant, add 200 μL of water to the pellet, disperse the cells, and store at -80°C. Accurately transfer 50 μL of plasma, process according to plasma sample processing methods, analyze by LC-MS, and record the chromatogram. Figure 3 The concentrations of the compound of the present invention and reboxil in plasma samples at different time points were calculated.

[0168] from Figure 3 The experimental results show that, under the investigated conditions, the plasma concentration of reboxil was lower than the reboxil concentration at the same time point within 1 hour after administration of the present invention. Pharmacokinetic parameters were calculated using the statistical moment method with DAS2.0 pharmacokinetic software, and the main parameters obtained are shown in the table below.

[0169] Table 11 Main pharmacokinetic parameters after canine administration of the compounds of this invention via gavage

[0170] Parameters Unit No. 1 No. 2 No. 3 No. 4 Mean RSD% AUC(0-t) pg-h / L 2226.182 2638.829 2971.512 5631.125 3366.912 45.738 AUC(0-∞) pg-h / L 2180.121 3244.049 3724.257 5650.212 3699.6598 39.237 MRT(0-t) h 6.724 5.621 8.507 8.074 7.2315 18.185 MRT(0-∞) h 7.231 9.031 13.242 8.922 9.6065 26.648 t1 / 2z h 4.554 8.351 10.962 5.886 7.43825 38.008 Tmax h 2 2 1 2 1.75 28.571 CLz / F L / h / kg 2.554 1.461 1.427 0.925 1.59175 43.142 Vz / F L / kg 15.328 19.386 21.635 5.212 15.39025 47.238 Zeta regression starting point -- 222 125 125 241 -- -- Cmax pg / L 508.1124 635.6241 376.3842 674.2457 548.5916 24.605

[0171] Table 12 Main pharmacokinetic parameters after canine gavage administration of reboxil

[0172] Parameters Unit No. 1 No. 2 No. 3 No. 4 Mean RSD% AUC(0-t) pg-h / L 3336.257 3468.121 5224.235 5526.031 4388.661 26.135 AUC(0-∞) pg-h / L 3229.642 3624.311 8024.901 6178.602 5264.364 42.879 MRT(0-t) h 8.125 7.333 8.902 8.045 8.10125 7.920 MRT(0-∞) h 9.246 8.408 21.522 13.434 13.1525 45.597 t1 / 2z h 7.594 6.577 18.292 8.059 10.1305 54.055 Tmax h 1 0.085 0.7 0.5 0.57125 67.187 CLz / F L / h / kg 1.553 1.655 0.824 0.905 1.23425 34.859 Vz / F L / kg 12.102 10.105 17.543 10.217 12.49175 27.938 Zeta regression starting point -- 230 240 230 235 -- -- Cmax pg / L 667.6201 1324.224 924.574 982.4548 974.71823 27.722

[0173] With AUC (0-t) Calculate the relative bioavailability of the compounds of this invention:

[0174] (AUC 0-t ) 本发明化合物 / (AUC 0-t ) 瑞波西利 =3366.912 / 4388.661 = 76.7%.

[0175] Example 17: Stability study of liver microsomes in Beagle dogs, rats, mice, and humans

[0176] This study was used to test the metabolic stability of the test substance in rat, mouse, and human liver microsomes.

[0177] Experimental operation:

[0178] 1) The test compound concentration was 0.6 μM, and the protein concentration of liver microsomes was 0.5 mg / mL, and the incubation was carried out in a 37-degree water bath;

[0179] 2) The positive controls included: testosterone (3A4 substrate), propylpyrazolone (2D6 substrate), diclofenac (2C9 substrate). The incubation conditions of the positive controls were consistent with the incubation conditions of the compounds;

[0180] 3) The reaction time points were: 0, 5, 10, 20, 30 and 60 minutes, and the reaction was terminated at the corresponding time points using a termination solution containing an internal standard. The compound was incubated with microsomes for 60 minutes without the action of the reducing coenzyme II regeneration system and served as a negative control;

[0181] 4) Each time point was a single point (n = 1);

[0182] 5) The samples were determined by LC / MS / MS, and the compound concentration was shown as the ratio of the compound peak area to the internal standard peak area (non-standard curve);

[0183] 6) Test results

[0184] Table 13 Investigation results of liver microsomal stability of different compounds

[0185] Test substance Mouse / rat / human T 1 / 2 (min) Compound of the present invention 52.26 / >188.5 / 56.03 Ribociclib 69.29 / 62.14 / 58.11

[0186] The liver microsomal stability of the compound of the present application in humans, rats and mice is obviously superior to that of ribociclib.

[0187] Example 18: Study of cytotoxicity

[0188] MTT is a yellow compound, which is a tetrazolium salt dye that can accept hydrogen atoms. The MTT colorimetric method has been widely used in many fields, such as cytotoxicity research, screening of antitumor drugs, and activity detection of bioactive factors. Its detection principle is that there is a succinate dehydrogenase in the mitochondria of living cells, which can reduce the exogenous MTT to water-insoluble blue-purple formazan crystals and deposit in the cells, but dead cells do not have this function, which is the difference between living and dead cells. Dimethyl sulfoxide is an organic solvent that can dissolve formazan in cells. The light absorption value (OD) of each well is measured by a full-wavelength enzyme-linked immunoassay instrument, which can indirectly reflect the number of living cells. When the cell number is within a certain range, the number of MTT crystals formed is proportional to the cell number. Under certain conditions, the absorbance value is inversely proportional to the cytotoxicity of the drug, i.e. the smaller the absorbance value, the fewer the living cells, and the greater the cytotoxicity of the drug.

[0189] MTT method was used to evaluate the cytotoxicity of all the target compounds synthesized. Human breast cancer cells (MDA-MB-231) and human non-small cell lung cancer cells (A549) were used as the research objects.

[0190] Experimental specific steps:

[0191] MDA-MB-231 and A549 cells in the logarithmic growth phase were inoculated in a 96-well plate at a density of 1×105 cells / mL and allowed to adhere overnight. After the cells adhered, the drug was tested. First, the target compound was dissolved in dimethyl sulfoxide to prepare a 2 mmol / L solution. Then it was diluted with culture medium to 1.25, 2.5, 5, 10, 20, and 40 μmol / L, respectively. In addition, cells without drug treatment were used as a negative control group. Different concentrations of drugs were added to the test wells, and the cells were incubated in a 37°C incubator for 72 h. Then 10 μL of 5 mg / mL MTT solution was added and incubated for another 4 h. After 4 h, the supernatant was removed, 150 μL of DMSO was added to replace the culture medium, and the formazan was fully dissolved. The OD value of each well was measured at 490 nm wavelength using a full-wavelength enzyme marker, the cell growth inhibition rate was calculated, and the IC50 value required was calculated by SPSS18 software or formula.

[0192] The inhibition rate calculation formula is as follows:

[0193] Inhibition rate % = (blank group OD value - drug group OD value) / blank group OD value × 100%

[0194] The IC50 value is the average value ± SD of three independent experiments

[0195] The results are shown in the following table:

[0196] Table 14 Anti-proliferative activity of compounds on MDA-MB-231 and A549 cell lines

[0197] Compound MDA-MB-231 A549 The present invention 3.87±1.05 pM 2.31±1.01 pM Ribociclib 22.46±1.60 pM 17.94±1.35 pM

[0198] The results show that the compounds of the present application all have very good anti-proliferation effect. This shows that the modification of the piperazine position of ribociclib does not change the key interaction with the target protein.

[0199] Example 19: Study on CDK4 / 6 / 9 / 12 enzyme inhibition activity

[0200] CDK4 / 6 is very important for the progress of cell cycle, ribociclib prevents the phosphorylation of Rb protein, selectively prevents the formation of CDK4 / 6-cyclinD1 complex, thereby inhibiting the activity of CDK4 / 6, leading to the arrest of G1 phase. We are a derivative of ribociclib, so we speculate that the target compound should have a similar mechanism of action as ribociclib, and can selectively inhibit the activity of CDK4 / 6.

[0201] Experimental steps: The application is dissolved in 100% DMSO to prepare a 250.0 μmol / L stock solution. Dilute with 1xkinase base buffer to prepare 1, 10, 50, 250, 1250 nM (test final concentration) solutions. Select blank DMSO and ribociclib as negative and positive controls, respectively. Use the dispenser Echo 550 to transfer the test compound solution to the target plate, add the test compound solution to each well of the 96-well plate, then add 0.1 μg / mL enzyme solution 10.0 μL to each well, and incubate at room temperature for 10 min. Next, add 10.0 μL of substrate and ATP mixture to each well, and incubate in a 37°C incubator for 60 min. Add 25.0 μL of reaction termination buffer to each well. Read the data with a Caliper instrument, calculate the inhibition rate, and plot the regression curve to measure the IC50 value. The results are as follows:

[0202] Table 15 Study of the inhibitory activity of different compounds on CDK4 / 6 / 9 / 12 enzymes

[0203]

[0204] The results show that the positive control ribociclib shows strong inhibitory activity on CDK4 and 6, with IC50 values of 10 nM and 39 nM, respectively, and almost no activity on CDK9 and 12, indicating that it is a CDK4 / 6 selective inhibitor. The IC50 values of the application on CDK4 / 6 are all less than that of ribociclib, and its activity is significantly improved compared with ribociclib.

[0205] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A compound represented by formula (I) or a solvate, hydrate or pharmaceutically acceptable salt thereof:

2. The compound of claim 1, wherein: The compound is a crystal, and the X-ray powder diffraction pattern of the crystal has characteristic peaks at diffraction angles 2θ: 8.447±0.2, 9.588±0.2, 16.913±0.2, 21.153±0.2, 21.572±0.2, 22.294±0.

2.

3. The crystal of claim 2, wherein The X-ray powder diffraction pattern of the crystal has characteristic peaks at diffraction angles 2θ: 8.447±0.2, 9.588±0.2, 11.784±0.2, 12.646±0.2, 13.304±0.2, 13.617±0.2, 15.066±0.2, 15.472±0.2, 16.913±0.2, 17.477±0.2, 19.025±0.2, 20.015±0.2, 20.800±0.2, 21.153±0.2, 21.572±0.2, 22.294±0.2, 23.069±0.2, 23.565±0.2, 24.611±0.2, 25.457±0.2, 25.884±0.2, 26.637±0.2, 27.298±0.2, 28.062±0.2, 28.924±0.2, 29.221±0.2, 30.679±0.

2.

4. The crystal of claim 3, wherein The X-ray powder diffraction pattern of the crystal is shown in Figure 1.

5. The crystal of claim 2, wherein The DSC curve of the crystal shows that the starting temperature of the endothermic peak is 194.25±2℃, and the peak temperature is 201.62±2℃.

6. A process for the preparation of a compound according to claim 1, characterized in that: The method comprises the following steps: Step 1: intermediate 1 is prepared from starting material SM1 and starting material SM2 in the presence of a non-polar solvent; Step 2: crude product of formula (I) is prepared from the product of step 1 and ribociclib in the presence of a solvent; Step 3: pharmaceutical-grade raw material of formula (I) is prepared from the crude product of formula (I) in the presence of a crystallization solvent, and the reaction scheme is as follows:

7. The method for preparing the compound according to claim 6, characterized in that: The solvent of step 1 is selected from one or more of dichloromethane, chloroform, ethyl acetate, isopropyl acetate, etc., and dichloromethane is preferred; the reaction solvent of step 2 is selected from one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, tetrahydrofuran, dioxane, and N,N-dimethylformamide is preferred; the crystallization solvent of step 3 is selected from one or more of dichloromethane, acetone, ethyl acetate, n-hexane, anhydrous ethanol, anhydrous methanol, and a combination of dichloromethane and acetone is preferred.

8. The process of claim 7, wherein: The temperature of the crystallization process is room temperature.

9. A pharmaceutical composition comprising the compound of claim 1 or the crystal of any one of claims 2-5, and a pharmaceutically acceptable carrier.

10. Use of the compound of claim 1 or the crystal of any one of claims 2-5 or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating breast cancer.

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