CN116041324A

CN116041324A - Deuterated pyrazole dichlorobenzamide compound, pharmaceutical composition and application

Info

Publication number: CN116041324A
Application number: CN202211451462.6A
Authority: CN
Inventors: 郭炳华; 闫海磊; 温杰
Original assignee: Yaokang Zhongtuo Beijing Pharmaceutical Technology Co ltd
Current assignee: Yaokang Zhongtuo Beijing Pharmaceutical Technology Co ltd
Priority date: 2022-11-20
Filing date: 2022-11-20
Publication date: 2023-05-02

Abstract

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

Description

Deuterated pyrazole dichlorobenzamide compound, pharmaceutical composition and application

Technical Field

The invention belongs to the field of innovative pharmaceutical chemistry, and relates to a deuterated pyrazole dichlorobenzamide compound, a pharmaceutical composition and application.

Background

The cell division cycle is a fundamental process in life, and a series of events occurring in a cell results in the formation of two identical daughter cells. Cyclin-dependent kinases (CDKs) are an important class of serine/threonine protein kinases that are not active themselves, and must bind to cyclin (cyclin) to produce activity, catalyze substrate phosphorylation, drive phase processes of the cell cycle, and sequentially complete DNA synthesis and mitosis, leading to cell growth and proliferation. Meanwhile, CDKs can also combine with CDKs inhibitor (CDI) to play a negative regulation role, inhibit the cell cycle progress and prevent cell division. CDKs abnormalities are reported to cause proliferation, genomic and chromosomal instability, leading to human cancers, and promoting progression and aggressiveness of the cancers. Therefore, the screening and research of CDKs small molecule inhibitors has become one of the hot spot fields of tumor treatment and development of novel chemotherapeutics.

During cell division, CDK2 is a central cell cycle regulator, active from late G1 to full S phase. CDK2 is activated by binding to cyclin E1 or E2, cyclin A2, CAK complex (CDK 7, MAT1, cyclin H) phosphorylation and CDC25A inhibits removal of phosphorylation. CDK2 also phosphorylates several components of the pre-replication complex, which are necessary to initiate DNA synthesis. CDK2 plays an important role in coordinating the cell centrosome replication cycle. After DNA damage, DDR blocks cells in 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 CDK 2. Degradation with CDC25A as the target, resulting in sustained inhibition of phosphorylation of Thr14 and Tyr15 sites of CDK2 by WEE1, thus blocking entry into S phase. In addition, CDK2 regulates the 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 cleavage. CDK2 also prevents apoptosis by phosphorylating the pro-survival myeloid leukemia cell differentiation protein (MCL-1). In summary, if the cell cycle is prevented from entering the S phase, abnormal replication of DNA does not occur, whereas the G1 phase is mainly regulated by CDK2/cyclin E, so CDK2 inhibitors prevent the cell cycle from entering the S phase for DNA replication. In addition, in the whole cell cycle, CDK2/cyclin A controls the progress of S, G phase in addition to controlling the G1 phase to enter the S phase, so that CDK2 plays a very important role in the cell cycle, so that if the activity of CDK2 can be effectively inhibited, the progress of the cell cycle can be controlled, and the effect of inhibiting uncontrolled proliferation of tumor cells can be achieved. AT-7519 is currently a CDK kinase inhibitor in the early clinical research stage.

Deuterated drugs refer to the replacement of part of the hydrogen atoms in the drug molecule with deuterium. Deuterated drugs generally retain the biological activity and selectivity of the original drug due to the shape and volume of deuterium in the drug molecule, which is similar to hydrogen. Because the C-D bond is more stable than the C-H bond, the C-D bond is less likely to break during the chemical reaction of the deuterated drug, and the half-life period of the deuterated drug is prolonged. Since 2000, deuteration strategies have been widely used in drug research.

Disclosure of Invention

The invention provides a compound shown in a formula I or pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, which has the following structure:

the invention provides an application of a compound shown in a formula I or pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof in preparing a CDK kinase inhibitor.

In some embodiments, the CDK kinase is a CDK1 kinase, a CDK2 kinase, a CDK4 kinase, or a CDK5 kinase.

In some embodiments, the CDK kinase is a CDK2 kinase.

The invention provides application of a compound shown as I or pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof in preparing a medicament for preventing and/or treating cancers.

In some embodiments, the cancer is melanoma, liver cancer, kidney cancer, lymphoblastic 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.

In some embodiments, the melanoma, liver cancer, kidney cancer, lymphoblastic 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, lymphoblastic 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 aberrant CDK kinase enzyme activity.

In some embodiments, the cancer is a cancer associated with aberrant CDK kinase enzyme activity.

The invention provides a pharmaceutical composition, which contains a compound shown in a formula I, or pharmaceutically acceptable salts, isomers, metabolites, prodrugs, solvates or hydrates thereof, and pharmaceutically acceptable carriers or auxiliary materials.

In the pharmaceutical composition, the compound shown in the formula I or pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof is used in an amount which is effective in treatment.

The present invention provides the use of a pharmaceutical composition in the preparation of a CDK kinase inhibitor.

In some embodiments, the CDK kinase is a CDK2 kinase.

The pharmaceutical excipients can be those which are widely used in the field of pharmaceutical production. Adjuvants are used primarily to provide a safe, stable and functional pharmaceutical composition, and may also provide means for allowing the subject to dissolve at a desired rate after administration, or for promoting effective absorption of the active ingredient after administration of the composition. The pharmaceutical excipients may be inert fillers or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients can comprise one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, sizing agents, disintegrants, lubricants, anti-adherents, glidants, wetting agents, gelling agents, absorption retarders, dissolution inhibitors, enhancing agents, adsorbents, buffering agents, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

The pharmaceutical compositions of the present invention may be prepared in accordance with the disclosure using any method known to those of skill in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical compositions of the present invention may be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular). The pharmaceutical compositions of the invention may also be in controlled or delayed release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum formulations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry formulations which may 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 formulations; aerosol: such as nasal sprays or inhalants; a liquid dosage form suitable for parenteral administration; suppositories and lozenges.

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention prepared from the compounds of the present invention which have the specified substituents found herein with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts may be obtained by contacting the free form of such compounds with a sufficient amount of base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting the free form of such compounds with a sufficient amount of acid in pure solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogenphosphates, dihydrogenphosphates, sulfuric acid (forming sulfates or bisulphates), hydroiodic acid, phosphorous acid, and the like, and organic acid salts including, for example, 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, tartaric acid, methanesulfonic acid, and the like, salts of amino acids (such as arginine and the like), and salts of organic acids such as glucuronic acid.

The "pharmaceutically acceptable salts" of the present invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, the preparation of such salts is as follows: prepared via reaction of these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

The term "isomer" refers to compounds of the same chemical formula but having different arrangements of atoms.

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

Identification of metabolites typically occurs by preparing a radiolabeled isotope of a compound of the invention, parenterally administering it to an animal, such as a rat, mouse, guinea pig, monkey, or human, in a detectable dose (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 its conversion product from urine, blood, or other biological samples. These products are easy to isolate because they are labeled (others are isolated by using antibodies that are capable of binding to epitopes present in the metabolite). The metabolite structures are determined in a conventional manner, for example by MS, LC/MS or NMR analysis. In general, the analysis of metabolites is performed in the same manner as conventional drug metabolism studies known to those skilled in the art. Provided that the metabolite product is not otherwise incapable of being in vivoThey are found to be useful in assay for the administration of therapeutic doses of the compounds of the invention. The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more of the atoms comprising the compounds. For example, compounds can be labeled with radioisotopes, such as tritium @, for example ³ H) Iodine-125% ¹²⁵ I) Or C-14% ¹⁴ C) A. The invention relates to a method for producing a fibre-reinforced plastic composite All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

In addition to salt forms, the compounds provided herein exist in prodrug forms. Prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to convert to the compounds of the invention. Any compound that can be converted in vivo to provide a biologically active substance (i.e., a compound of formula I) is a prodrug within the scope and spirit of the invention. For example, compounds containing a carboxyl group can form a physiologically hydrolyzable ester that acts as a prodrug by hydrolyzing in vivo to give the compound of formula I itself. The prodrugs are preferably administered orally, as hydrolysis occurs in many cases primarily under the influence of digestive enzymes. Parenteral administration may be used when the ester itself is active or hydrolysis occurs in the blood.

The invention has the positive progress effects that:

(1) The compounds of the invention have very remarkable inhibitory activity on CDK 2.

(2) The compound of the invention has remarkably improved metabolic stability, prolonged half-life, improved oral bioavailability and support for oral administration.

(3) Has lower toxicity to normal cells, which indicates that the compound has less toxic and side effects.

(4) The compounds of the present invention have good therapeutic effects on cancer.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1: synthesis of Compound I

Step one: synthesis of Compound 2

To a solution of compound 1 (145 mg,0.47 mmol) in N, N-dimethylformamide (5 mL) were added potassium hydroxide (105.5 mg,1.88 mmol) and elemental iodine (239 mg,0.94 mmol), and the reaction was carried out at room temperature for 3 hours, and TLC was used to monitor the completion of the reaction, and saturated sodium sulfite solution was added to quench the reaction, and the aqueous phase was extracted with ethyl acetate (10 ml×2), washed with water (20 ml×2), dried over anhydrous sodium sulfate with saturated salt (20 mL) and concentrated column chromatography was used to separate and purify compound 2 (133 mg, 65%). MS (ESI, M/z): 436 (M) ⁺ +1).

Step two: synthesis of Compound 3

To a deuterated acetic acid solution (8 mL) of compound 2 (157 mg,0.36 mmol) was added sodium acetate (97.9 mg,0.72 mmol), and the reaction was completed for 2 hours at room temperature, and the reaction was completed by TLC, concentrated under reduced pressure, and separated and purified by column chromatography to obtain compound 3 (91 mg, 80%). MS (ESI, M/z): 311 (M) ⁺ +1).

Step three: synthesis of Compound 4

Compound 3 (155 mg,0.5 mmol) was dissolved in anhydrous DMF (3 mL), and to the above solution was added 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) and stirred at room temperature for 6h. The reaction was stopped, quenched with water, extracted 3 times with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography to give compound 4 (169 mg, 70%). MS (ESI, M/z): 483 (M) ⁺ +1).

Step four: synthesis of Compound I

Compound 4 (97 mg,0.2 mmol) was dissolved in EA (2 mL), EA/HCl (2 mmol) was added to the solution, and the reaction was stirred at room temperature for 3h. And after the reaction is complete, carrying out suction filtration, collecting a filter cake, dissolving the filter cake in methanol, adding potassium carbonate, stirring for 30min at room temperature, carrying out suction filtration, and collecting the filter cake to obtain the compound I. ¹ H NMR(500MHz,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).

Example 2: CDK2 kinase inhibition Activity assay

CDK2 kinase inhibitory activity: the inhibitory activity of compounds on CDK2/A is determined by FRET method and obtained by purification or direct purchase of a kit. The specific method comprises the following steps: CDK2/A was diluted with kinase diluent to the appropriate concentration for use. The kinase reaction mixture contains CDK2/A, peptide substrate, HEPES (pH 7.5), BRIJ-35, mgCl ₂ And EDTA. CDK2 phospho-peptide substrate was used as 100% phosphorylation control and no ATP was added as 0% phosphorylation control. After 1h of reaction at room temperature, development Reagent A was added to the reaction system in moderate dilution. The reaction was continued at room temperature for 1 hour, and the reaction was stopped by adding Stopreagent. Excitation wavelength was 400nm, and fluorescence intensities at 445nm (coumarin) and 520nm (fluorescein) were detected simultaneously. The inhibition rate of the tested compound is calculated according to the formula.

TABLE 1 inhibitory Activity of test Compounds against CDK2 (IC ₅₀ nM)

Name of the name	CDK2
		I	20
AT-7519	63

As shown in table 1, compound I has significant inhibitory activity on CDK2, superior to AT-7519.

Example 3: antiproliferative activity assay

Cell antiproliferative activity was detected using CTG luminescence. ATP is an essential factor for maintaining normal cell vital activity, is a key index of metabolism of living cells, and 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 and is proportional to the ATP content, so that the number of living cells can be detected by measuring the ATP content.

The specific experimental operation steps are as follows:

1. compound configuration:

1) Compounds were formulated using DMSO to a storage concentration of 10 mM;

2) The compounds were diluted twice at the highest concentration point at 10mM top dose (100% DMSO) for ten points, two duplicate wells were set for each concentration;

3) The compound was diluted 100-fold using the cell-corresponding medium to give a compound concentration of 100. Mu.M top dose (1% DMSO).

2. Cell plating:

1) Cell plating density was 5000cells/well, cell plating was performed overnight, and the volume was 20. Mu.L;

2) To a 96-well plate, 20. Mu.L of test compound was added, 40. Mu.L of each well, and the final concentration of top dose of compound was 50. Mu.M (0.5% DMSO). After the completion of the dosing, 5% CO2 was incubated at 37℃for 72h.

3. Cell detection: mu.L of CTG reagent was added to each well and incubated for 20min for detection using the program Luminescence.

4. And (3) data processing: calculation of IC using Graphpad software ₅₀ Values.

TABLE 2 test results of anti-cell proliferation Activity of test Compounds (IC ₅₀ nM)

Name of the name	A2780	HCT116	MCF-7	MRC5
					I	120	25	15	＞1000
AT-7519	350	83	56	＞1000

As shown in table 2, compound I has significant antiproliferative activity against tumor cells such as ovarian cancer a2780, colon cancer HCT116, breast cancer MCF-7, and the like, and is superior to the positive control AT-7519. In addition, compound I was less toxic to normal fibroblast cells MRC 5.

Example 4: test compound pharmacokinetic property detection

BALB/c mice are selected for oral administration (10 mg/kg) or intravenous injection (2 mg/kg), 5min,15min,30min,1h,2h,4h,8h,10h and 24h after administration, blood is continuously taken from the eyeground venous plexus and placed in an EP tube containing heparin, and LC-MS/MS analysis is carried out on centrifugations and upper plasma, and according to blood concentration-time data obtained by testing, pharmacokinetic parameters are calculated by adopting WinNonlin software, and the oral bioavailability is calculated.

The research result shows that the AT-7519 can not be orally administrated because of low oral bioavailability (< 1%), and the intravenous injection half-life is 0.7h; the pharmacokinetic property of the compound I is improved, the oral bioavailability is improved to 10%, and the intravenous injection half life is prolonged to 2.1h.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, having the structure:

2. a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I according to claim 1, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, and a pharmaceutically acceptable carrier or adjuvant.

3. Use of a compound of formula I as defined in claim 1, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, or a pharmaceutical composition as defined in claim 2, in the preparation of a CDK kinase inhibitor.

4. Use according to claim 3 wherein the CDK kinase is a CDK1 kinase, a CDK2 kinase, a CDK4 kinase or a CDK5 kinase.

5. The use according to claim 4 wherein the CDK kinase is a CDK2 kinase.

6. Use of a compound of formula I as defined in claim 1, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, or a pharmaceutical composition as defined in claim 2, for the manufacture of a medicament for the treatment and/or prophylaxis of cancer.

7. The use according to claim 6, wherein the cancer is melanoma, liver cancer, kidney cancer, lymphocytic 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.

8. The use according to claim 7 wherein the melanoma, liver cancer, kidney cancer, lymphoblastic 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, lymphoblastic 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 associated with aberrant CDK kinase enzyme activity.

9. The use according to claim 6 wherein the cancer is a cancer associated with aberrant CDK kinase enzyme activity.