Deuterated pyrazole dichlorobenzamide compound, pharmaceutical composition and use thereof

Abstract

The present invention discloses a compound represented by formula I, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, a pharmaceutical composition and use thereof, in particular, an anti-tumor use thereof as a CDK kinase inhibitor.

Description

Technical Field

[0001] The present invention belongs to the field of innovative pharmaceutical chemistry and relates to a deuterated pyrazole dichlorobenzamide compound, a pharmaceutical composition and an application thereof. Background Art

[0002] The cell division cycle is a fundamental process in life, in which a series of events in the cell lead to the formation of two identical daughter cells. Cyclin-dependent kinases (CDKs) are an important class of serine / threonine protein kinases that are not active by themselves and must bind to cyclins to become active. They can catalyze substrate phosphorylation, drive the process of each phase of the cell cycle, complete DNA synthesis and mitosis in sequence, and cause cell growth and proliferation. At the same time, CDKs can also bind to CDK inhibitors (CDI) to exert negative regulatory effects, inhibit cell cycle progression, and prevent cell division. It is reported that abnormal CDKs can lead to proliferation, genomic and chromosomal instability, leading to human cancer, and promoting cancer progression and invasiveness. Therefore, the screening and research of CDKs small molecule inhibitors has become one of the hot areas in tumor treatment and the development of new chemotherapeutic drugs.

[0003] During cell division, CDK2 is a core cell cycle regulator that is active from late G1 phase throughout S phase. CDK2 is activated by binding to cyclin E1 or E2, cyclin A2, phosphorylation of the CAK complex (CDK7, MAT1, cyclin H), and removal of inhibitory phosphorylation of CDC25A. CDK2 also phosphorylates several components of the pre-replication complex, which is required for the initiation of DNA synthesis. CDK2 plays an important role in coordinating the centrosome replication cycle in cells. Following DNA damage, the DDR arrests cells at the G1 / S phase to repair damaged DNA and maintain genomic fidelity in daughter cells. Two mechanisms of the G1 / S DNA damage checkpoint inhibit proliferation through CDK2. Targeting CDC25A for degradation results in sustained inhibitory phosphorylation of CDK2 at Thr14 and Tyr15 by WEE1, thereby blocking its entry into S phase. Additionally, CDK2 regulates core regulatory and functional components of the apoptotic pathway. The CDK2 target protein FOXO1 plays an important role in triggering DNA damage-induced apoptosis following dsDNA breaks. CDK2 also prevents apoptosis by phosphorylating the pro-survival factor myeloid leukemia cell differentiation protein (MCL-1). In short, if the cell cycle can be prevented from entering the S phase, abnormal DNA replication will not occur, and the entry of the G1 phase into the S phase is mainly regulated by CDK2 / cyclin E, so CDK2 inhibitors can prevent the cell cycle from entering the S phase for DNA replication. In addition, throughout the cell cycle, in addition to controlling the entry of the G1 phase into the S phase, CDK2 / cyclin A also controls the progress of the S and G2 phases. It can be seen that CDK2 plays a very important role in the cell cycle, so if the activity of CDK2 can be effectively inhibited, the progress of the cell cycle can be controlled, thereby achieving the effect of inhibiting the uncontrolled proliferation of tumor cells. AT-7519 is a CDK kinase inhibitor currently in the early clinical research stage.

[0004]

[0005] Deuterated drugs refer to drugs in which some hydrogen atoms in the drug molecule are replaced with deuterium. Since the shape and volume of deuterium in the drug molecule are similar to those of hydrogen, deuterated drugs generally retain the biological activity and selectivity of the original drug. Since the CD bond is more stable than the CH bond, the CD bond of deuterated drugs is less likely to break during the chemical reaction, and its half-life will be extended. Since 2000, the deuterated strategy has been widely used in drug research. Summary of the invention

[0006] The present invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, the structure of which is as follows:

[0007]

[0008] The present invention provides a use of a compound as shown in formula I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof in the preparation of a CDK kinase inhibitor.

[0009] In some embodiments, the CDK kinase is CDK1 kinase, CDK2 kinase, CDK4 kinase and CDK5 kinase.

[0010] In some embodiments, the CDK kinase is CDK2 kinase.

[0011] The present invention provides use of a compound as shown in I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof in the preparation of a drug for preventing and / or treating cancer.

[0012] In some embodiments, the cancer is melanoma, liver cancer, kidney cancer, lymphoid leukemia, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, myelodysplastic syndrome, esophageal cancer, gastrointestinal cancer, and mesothelioma.

[0013] In some embodiments, the melanoma, liver cancer, kidney cancer, lymphoid leukemia, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, myelodysplastic syndrome, esophageal cancer, gastrointestinal cancer and mesothelioma are melanoma, liver cancer, kidney cancer, lymphoid leukemia, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, myelodysplastic syndrome, esophageal cancer, gastrointestinal cancer and mesothelioma associated with abnormal CDK kinase enzyme activity.

[0014] In some embodiments, the cancer is a cancer associated with abnormal CDK kinase enzyme activity.

[0015] The present invention provides a pharmaceutical composition, which contains a compound as shown in Formula I, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

[0016] In the pharmaceutical composition, the compound as shown in Formula I, or its pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate is used in a therapeutically effective amount.

[0017] The present invention provides use of a pharmaceutical composition in preparing a CDK kinase inhibitor.

[0018] In some embodiments, the CDK kinase is CDK1 kinase, CDK2 kinase, CDK4 kinase and CDK5 kinase.

[0019] In some embodiments, the CDK kinase is CDK2 kinase.

[0020] In some embodiments, the cancer is melanoma, liver cancer, kidney cancer, lymphoid leukemia, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, myelodysplastic syndrome, esophageal cancer, gastrointestinal cancer, and mesothelioma.

[0021] In some embodiments, the melanoma, liver cancer, kidney cancer, lymphoid leukemia, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, myelodysplastic syndrome, esophageal cancer, gastrointestinal cancer and mesothelioma are melanoma, liver cancer, kidney cancer, lymphoid leukemia, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, myelodysplastic syndrome, esophageal cancer, gastrointestinal cancer and mesothelioma associated with abnormal CDK kinase enzyme activity.

[0022] In some embodiments, the cancer is a cancer associated with abnormal CDK kinase enzyme activity.

[0023] The pharmaceutical excipients can be those widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide methods to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical excipients can be inert fillers, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipients can include one or more of the following excipients: adhesives, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0024] The pharmaceutical composition of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes.

[0025] The pharmaceutical composition of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or delayed release dosage form (e.g., liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0026] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared from compounds having specific substituents discovered by the present invention with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonate or bicarbonate), phosphoric acid (forming phosphate, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid (forming sulfate or bisulfate), hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid. Acids such as tartaric acid and methanesulfonic acid; organic acid salts also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups, and can be converted into any base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated, thereby regenerating the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubility in polar solvents.

[0027] The "pharmaceutically acceptable salts" of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. In general, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0028] The term "isomers" refers to compounds that have the same chemical formula but different arrangements of the atoms.

[0029] The term "metabolite" refers to a pharmaceutically active product produced by the in vivo metabolism of a compound of Formula I or a salt thereof. Such a product may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, glucuronidation, enzymatic cleavage, etc. of the administered compound. Therefore, the present invention includes metabolites of the compounds of the present invention, including compounds produced by a process of contacting a compound of the present invention with a mammal for a period of time sufficient to obtain a metabolite thereof.

[0030] Metabolites are typically identified by preparing radiolabeled isotopes of the compounds of the invention, administering them parenterally to animals, such as rats, mice, guinea pigs, monkeys, or humans, at detectable doses (e.g., greater than about 0.5 mg / kg), allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours), and isolating the conversion products from urine, blood, or other biological samples. These products are easily isolated because they are labeled (others are isolated by using antibodies that can bind to antigenic epitopes present in the metabolites). The metabolite structure is determined in a conventional manner, for example, by MS, LC / MS or NMR analysis. Generally, the analysis of metabolites is performed in the same manner as conventional drug metabolism studies known to those skilled in the art. As long as the metabolite products are not otherwise not found in vivo, they can be used in assays for therapeutic dosing of the compounds of the invention. The compounds of the invention may contain non-natural proportions of atomic isotopes on one or more of the atoms that make up the compound. For example, compounds may be labeled with radioisotopes, such as tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

[0031] In addition to the form of salts, the compounds provided by the present invention also exist in the form of prodrugs. The prodrugs of the compounds described herein are easily chemically changed under physiological conditions to be converted into the compounds of the present invention. Any compound that can be converted in vivo to provide a bioactive substance (i.e., a compound shown in Formula I) is a prodrug within the scope and spirit of the present invention. For example, a carboxyl-containing compound can form a physiologically hydrolyzable ester, which acts as a prodrug by being hydrolyzed in vivo to obtain the compound shown in Formula I itself. The prodrug is preferably administered orally, because hydrolysis occurs mainly under the influence of digestive enzymes in many cases. When the ester itself is active or hydrolysis occurs in the blood, parenteral administration can be used.

[0032] The positive and progressive effects of the present invention are:

[0033] (1) The compounds of the present invention have significant inhibitory activity against CDK2.

[0034] (2) The metabolic stability of the compounds of the present invention is significantly improved, the half-life is prolonged, and the oral bioavailability is improved, which supports oral administration.

[0035] (3) The compound has low toxicity to normal cells, indicating that it has less toxic side effects.

[0036] (4) The compounds of the present invention have good therapeutic effects on cancer. DETAILED DESCRIPTION

[0037] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0038] Example 1: Synthesis of Compound I

[0039]

[0040] Step 1: Synthesis of compound 2

[0041] Potassium hydroxide (105.5 mg, 1.88 mmol) and iodine (239 mg, 0.94 mmol) were added to a solution of compound 1 (145 mg, 0.47 mmol) in N,N-dimethylformamide (5 mL), and the mixture was reacted at room temperature for 3 hours. After TLC monitoring, the reaction was complete. A saturated sodium sulfite solution was added to quench the reaction. The aqueous phase was extracted with ethyl acetate (10 mL*2), washed with water (20 mL*2), washed with saturated salt (20 mL), dried over anhydrous sodium sulfate, and separated and purified by column chromatography to obtain compound 2 (133 mg, 65%). MS (ESI, m / z): 436 (M + +1).

[0042] Step 2: Synthesis of compound 3

[0043] Sodium acetate (97.9 mg, 0.72 mmol) was added to a deuterated acetic acid solution (8 mL) of compound 2 (157 mg, 0.36 mmol) and the mixture was added dropwise over 2 hours. The mixture was reacted at room temperature for 24 hours. The reaction was complete after TLC detection. The mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 3 (91 mg, 80%). MS (ESI, m / z): 311 (M + +1).

[0044] Step 3: Synthesis of compound 4

[0045] Compound 3 (155 mg, 0.5 mmol) was dissolved in anhydrous DMF (3 mL), and compound 5 (113 mg, 0.6 mmol), EDCI (163 mg, 0.85 mmol), HOBT (85 mg, 0.65 mmol) and TEA (0.21 mL, 1.5 mmol) were added to the above solution, and stirred at room temperature for 6 h. The reaction was stopped, and water was added to quench the reaction. The mixture was extracted with ethyl acetate for 3 times, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography to obtain compound 4 (169 mg, 70%). MS (ESI, m / z): 483 (M + +1).

[0046] Step 4: Synthesis of Compound I

[0047] Compound 4 (97 mg, 0.2 mmol) was dissolved in EA (2 mL), EA / HCl (2 mmol) was added to the above solution, and the mixture was stirred at room temperature for 3 h. After the reaction was complete, the mixture was filtered and the filter cake was collected. The filter cake was dissolved in methanol, potassium carbonate was added, and the mixture was stirred at room temperature for 30 min. The mixture was filtered and the filter cake was collected to obtain compound I. 1 H NMR (500 MHz, DMSO-d 6 )δ8.22(s,1H),7.66(d,J=9.0Hz,1H),7.42(s,2H),7.42(d,J=2.6Hz,1H),6.22(s,1H),3.83(dp,J=9.0,5. 7Hz,1H),3.00–2.91(m,2H),2.88–2.78(m,3H),1.88–1.78(m,2H),1.79–1.69(m,2H).MS(ESI,m / z):383(M + +1).

[0048] Example 2: CDK2 kinase inhibitory activity test

[0049] CDK2 kinase inhibitory activity: The inhibitory activity of the compound on CDK2 / A was determined by FRET method. CDK2 / A was obtained by purification or direct purchase of the kit. Specific method: CDK2 / A was diluted to an appropriate concentration with kinase diluent before use. The kinase reaction mixture contained CDK2 / A, peptide substrate, HEPES (pH 7.5), BRIJ-35, MgCl 2 and EDTA. CDK2phospho-peptide substrate was used as a 100% phosphorylation control, and no ATP was used as a 0% phosphorylation control. After reacting at room temperature for 1 hour, a moderately diluted Development Reagent A was added to the reaction system. The reaction was continued at room temperature for 1 hour, and Stop Reagent was added to terminate the reaction. The excitation wavelength was 400nm, and the fluorescence intensity at wavelengths of 445nm (coumarin) and 520nm (fluorescein) was detected simultaneously. The inhibition rate of the test compound was calculated according to the formula.

[0050] Table 1 Inhibitory activity of the tested compounds on CDK2 (IC 50 nM)

[0051] name CDK2 I 20 AT-7519 63

[0052] As shown in Table 1, compound I has significant inhibitory activity against CDK2, which is superior to AT-7519.

[0053] Example 3: Antiproliferative Activity Test

[0054] The cell anti-proliferation activity detection adopts CTG luminescence method. ATP is an essential factor for maintaining normal cell life activities and a key indicator of the metabolism of living cells. It can truly reflect the state and number of living cells. During the test, CellTiter-GloTM reagent is added to the culture medium and the luminescence value is measured. The luminescence value is proportional to the ATP content, so the number of living cells can be detected by measuring the ATP content.

[0055] The specific experimental steps are as follows:

[0056] 1. Compound configuration:

[0057] 1) Prepare the compound at a stock concentration of 10 mM using DMSO;

[0058] 2) The compound was diluted twice at a top dose of 10 mM (100% DMSO) for a total of ten points, with two replicate wells for each concentration;

[0059] 3) The compound was diluted 100-fold using the cell-specific culture medium to make the compound concentration reach the top dose of 100 μM (1% DMSO).

[0060] 2. Cell plating:

[0061] 1) The cell plating density is 5000 cells / well, and the cells are plated overnight in a volume of 20 μL;

[0062] 2) Add 20 μL of the compound to be tested to the 96-well plate, so that each well contains 40 μL and the final concentration of the compound is 50 μM (0.5% DMSO). After the addition of the drug, incubate at 37° C., 5% CO 2 for 72 h.

[0063] 3. Cell detection: Add 20 μL CTG reagent to each well, incubate for 20 min, and use the Luminescence program for detection.

[0064] 4. Data processing: Graphpad software was used to calculate IC 50 value.

[0065] Table 2 Anti-cell proliferation activity test results of the tested compounds (IC 50 nM)

[0066] name A2780 HCT116 MCF-7 MRC5 I 120 25 15 ＞1000 AT-7519 350 83 56 ＞1000

[0067] As shown in Table 2, compound I has significant anti-proliferative activity against tumor cells such as ovarian cancer A2780, colon cancer HCT116, and breast cancer MCF-7, and is superior to the positive control AT-7519. In addition, compound I has less toxicity to normal fibroblast cells MRC5.

[0068] Example 4: Detection of pharmacokinetic properties of test compounds

[0069] BALB / c mice were selected and the drug was administered orally (10 mg / kg) or intravenously (2 mg / kg). At 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 10 h, and 24 h after administration, blood was continuously collected from the fundus venous plexus and placed in EP tubes containing heparin. The blood was centrifuged and the upper plasma was collected for LC-MS / MS analysis. Based on the blood drug concentration-time data obtained from the test, WinNonlin software was used to calculate the pharmacokinetic parameters and the oral bioavailability.

[0070] The results of the study showed that AT-7519 could not be administered orally due to its low oral bioavailability (<1%), and its intravenous half-life was 0.7h; the pharmacokinetic properties of compound I were improved, with its oral bioavailability increased to 10% and its intravenous half-life extended to 2.1h.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound as shown in formula I or a pharmaceutically acceptable salt thereof, whose structure is as follows: 。 2. A pharmaceutical composition, It is characterized in that The pharmaceutical composition contains a therapeutically effective amount of the compound of formula I as claimed in claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. Use of the compound of formula I or a pharmaceutically acceptable salt thereof as claimed in claim 1, or the pharmaceutical composition as claimed in claim 2 in the preparation of a CDK kinase inhibitor.

4. The use according to claim 3, It is characterized in that The CDK kinase is CDK1 kinase, CDK2 kinase, CDK4 kinase or CDK5 kinase.

5. The use according to claim 4, It is characterized in that The CDK kinase is CDK2 kinase.

6. Use of the compound of formula I or a pharmaceutically acceptable salt thereof as claimed in claim 1, or the pharmaceutical composition as claimed in claim 2, in the preparation of a medicament for treating and / or preventing cancer.

7. The use according to claim 6, It is characterized in that The cancer is melanoma, liver cancer, kidney cancer, lymphatic leukemia, non-Hodgkin's lymphoma, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, myelodysplastic syndrome, esophageal cancer, gastrointestinal cancer or mesothelioma.

8. The use according to claim 7, It is characterized in that The melanoma, liver cancer, kidney cancer, lymphatic leukemia, non-Hodgkin's lymphoma, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, myelodysplastic syndrome, esophageal cancer, gastrointestinal cancer and mesothelioma are melanoma, liver cancer, kidney cancer, lymphatic leukemia, non-Hodgkin's lymphoma, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, myelodysplastic syndrome, esophageal cancer, gastrointestinal cancer or mesothelioma associated with abnormal CDK kinase enzyme activity.

9. The use according to claim 6, It is characterized in that The cancer is a cancer associated with abnormal CDK kinase enzyme activity.

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