CN116444496B - Pyrimidine bi-deuterated pyrazole compound and application thereof - Google Patents

Pyrimidine bi-deuterated pyrazole compound and application thereof Download PDF

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CN116444496B
CN116444496B CN202310717148.6A CN202310717148A CN116444496B CN 116444496 B CN116444496 B CN 116444496B CN 202310717148 A CN202310717148 A CN 202310717148A CN 116444496 B CN116444496 B CN 116444496B
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cancer
raf
acid
pharmaceutically acceptable
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CN116444496A (en
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刘春河
靳学健
郭丽红
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Yaokang Zhongtuo Beijing Pharmaceutical Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention discloses a compound shown in a formula I, or pharmaceutically acceptable salts, isomers, metabolites, prodrugs, solvates or hydrates thereof, a pharmaceutical composition and application thereof. The compound shown in the formula I provided by the invention has good inhibitory activity on RAF kinase, especially B-RAF, and has good therapeutic effect on cancers.

Description

Pyrimidine bi-deuterated pyrazole compound and application thereof
Technical Field
The invention belongs to the field of innovative pharmaceutical chemistry, and relates to a pyrimidine-dideutero-substituted pyrazole compound and application thereof.
Background
RAS/RAF/MEK/ERK cell signal transduction pathways play an important role in human physiology and pathology, and are involved in the processes of proliferation, differentiation and apoptosis of cells. RAF kinase is a member of RAS/RAF/MEK/ERK cell signaling pathway, and abnormal activation of RAF kinase is closely related to the occurrence and development of cancer. The RAF kinase family includes three members: A-RAF, B-RAF and C-RAF, wherein B-RAF kinase activity is strongest. B-RAF kinase mutation can lead to constitutive activation of RAS/RAF/MEK/ERK cell signaling pathway, thereby promoting tumor development and development. Studies have shown that there are varying degrees of B-RAF mutation in most malignancies, such as about 70% in melanoma, 35-70% in thyroid cancer, 5-20% in colon cancer, about 14% in liver cancer, and about 30% in ovarian cancer. Of these, the most common mutation is the substitution of valine (V) at position 600 with glutamic acid (E), B-RAF V600E . Thus, B-RAF V600E Has become an effective target point of the anti-tumor drug. Currently, a variety of B-RAFs V600E The inhibitor is applied to clinic and has better curative effect, but part of patients have drug resistance after long-term administration. Sorafenib is a non-selective RAF kinase inhibitor, which can inhibit B-RAF, C-RAF and the like, and is a multi-kinase oral inhibitor on the first market. Sorafenib alone does not work well, but has improved efficacy when combined with carboplatin, paclitaxel or temozolomide. Sorafenib is currently used to treat clear cell carcinoma and unresectable liver cancer. Vemurafenib is B-RAFV 600E Potent inhibitors of kinase type I by blocking B-RAFV 600E MEK/ERK pathway inhibition carrying B-RAF V600E The mutated melanoma cells proliferate. The onset of vemurafenib can result in some relief of the melanoma patient's condition, but the long-term efficacy of the drug is not ideal. Most drug users eventually relapse and develop drug-resistant and fatal melanoma. Darafenib is a selective B-RAF V600E Potent inhibitors of kinase I are marketed as approved by the FDA in 2013 in the united states for the treatment of non-surgical or metastatic melanoma. Darafenib also has obvious inhibition effect on other solid tumors of B-RAF mutation, such as gastrointestinal solid tumors, non-small cell lung cancer, ovarian cancer and the like. Most patients develop drug resistance for a period of time, and the duration of the therapeutic effect of dabrafenib is short. Kang Naifei Ni, on the market of FDA approval in 2018 in the United states, was combined with Mektovi (binimetinib) to treat B-RAF V600E Or B-RAF V600k Mutant unresectable or metastatic melanoma. Thus, novel B-RAF V600E The development of inhibitors is of great importance.
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 an RAF kinase inhibitor.
In some embodiments, the RAF kinase is B-RAF.
In some embodiments, the RAF kinase is B-RAF V600E 。
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 cancer.
In some embodiments, the cancer is selected from lung cancer, pancreatic cancer, bladder cancer, colon cancer, a myeloid disorder, prostate cancer, thyroid cancer, melanoma, adenocarcinoma, and ovarian, ocular, hepatic, biliary and nervous system cancer.
In some embodiments, the lung cancer, pancreatic cancer, bladder cancer, colon cancer, myeloid disorders, prostate cancer, thyroid cancer, melanoma, adenocarcinoma and ovarian, eye, liver, biliary tract and nervous system cancer associated with aberrant RAF kinase enzyme activity.
In some embodiments, the cancer is a cancer associated with aberrant RAF 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 invention provides application of a pharmaceutical composition in preparation of an RAF kinase inhibitor.
In some embodiments, the RAF kinase is B-RAF.
In some embodiments, the B-RAF is B-RAF V600E 。
The invention provides application of a pharmaceutical composition in preparing a medicament for preventing and/or treating cancer.
In some embodiments, the cancer is selected from lung cancer, pancreatic cancer, bladder cancer, colon cancer, a myeloid disorder, prostate cancer, thyroid cancer, melanoma, adenocarcinoma, and ovarian, ocular, hepatic, biliary and nervous system cancer.
In some embodiments, the lung cancer, pancreatic cancer, bladder cancer, colon cancer, myeloid disorders, prostate cancer, thyroid cancer, melanoma, adenocarcinoma and ovarian, eye, liver, biliary tract and nervous system cancer associated with aberrant RAF kinase enzyme activity.
In some embodiments, the cancer is a cancer associated with aberrant RAF kinase enzyme activity.
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 is typically accomplished byThe radiolabeled isotopes of the compounds of the invention are prepared, parenterally administered to animals, such as rats, mice, guinea pigs, monkeys, or humans, in a detectable dose (e.g., greater than about 0.5 mg/kg), for a time sufficient to allow metabolism to occur (typically about 30 seconds to 30 hours) and isolation of their conversion products 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. So long as the metabolite products are not otherwise undetectable in vivo, they are useful in assays for therapeutic dosing 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) Compounds of the invention are directed to RAF kinaseIn particular B-RAF V600E Has good inhibitory activity.
(2) The compound of the invention has high blood concentration and high oral bioavailability, and can reduce single administration dosage.
(3) 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 1
Step one: synthesis of Compound b
To a solution of compound a (78 mg, 0.47 mmol) in N, N-dimethylformamide (15 mL), potassium hydroxide (105.5 mg, 1.88 mmol) and elemental iodine (239 mg, 0.94 mmol) were added, the reaction was allowed to react at room temperature for 3 hours, the completion of the reaction was monitored by TLC, a saturated solution of sodium sulfite was added to quench the reaction, the aqueous phase was extracted with ethyl acetate (10 mL ×2), washed with water (20 mL ×2), saturated salt (20 mL) was washed with water and dried over anhydrous sodium sulfate, and the resultant was separated and purified by column chromatography to give iodo compound b (76 mg, 55%). MS (ESI, M/z) 294 (M) + +1).
Step two: synthesis of Compound c
To a deuterated acetic acid solution (8 mL) of compound b (105 mg, 0.36 mmol) was added sodium acetate (97.9 mg, 0.72 mmol), and after completion of 2 hours, the reaction was performed at room temperature for 24 hours, TLC detection was complete, and concentration under reduced pressure, column chromatography separation and purification gave compound c (42 mg, 69%). MS (ESI, M/z): 169 (M) + +1).
Step three: synthesis of Compound d
Compound c (4.0 g,24 mmol) and p-toluenesulfonic acid monohydrate (9.07 g,48 mmol) were dissolved in acetonitrileIn (150 mL), a 20mL aqueous solution of sodium nitrite (2.97 g, 43 mmol) and sodium iodide (8.0 g,48 mmol) was slowly added dropwise to the above solution at 0deg.C. Stirring was continued for 10min at 0deg.C, then transferred to room temperature, and the reaction was continued for 3h with stirring. After the reaction is completed, the reaction solution is concentrated, diluted by adding water, and the pH value of the sodium carbonate aqueous solution is adjusted to 9-10. Ethyl acetate extraction (30 mL ×3), washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating, and separating and purifying by column chromatography to obtain compound d (4.3 g, 65%). MS (ESI, M/z): 279 (M) + +1).
Step four: synthesis of Compound f
A solution of compound d (5 g,18 mmol) in N, N-dimethylformamide diethyl acetal (50 mL) was warmed to 160℃and reacted for 20h with stirring, and concentrated to give crude compound e which was directly taken to the next reaction. The crude product of the obtained compound e (4.0 g, 12.0 mmol), guanidine hydrochloride (2.63 g, 27.6 mmol) and lithium hydroxide were added to a tube, dissolved in sec-butanol (50 mL), heated to 110℃and stirred for 20h. Cooled to room temperature, quenched with water, extracted with ethyl acetate (30 mL ×3), washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Ethyl acetate was added to the mixture to pulp, suction filtration was performed, and a cake was collected and dried in vacuo to give compound f (3.2 g, 80%). MS (ESI, M/z): 333 (M) + +1).
Step five: synthesis of Compound g
0. Sodium nitrite (314 mg, 4.55 mmol) was added in portions to a solution of compound f (500 mg, 1.52 mmol) in trifluoroacetic acid (15 mL) at C, the reaction solution was transferred to room temperature and stirred for 1h. After the completion of the reaction, the solvent was removed under reduced pressure to give a crude product, ethyl acetate (10 mL) was added to the crude product, washed with a saturated sodium hydrogencarbonate solution, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to give Compound g (). MS (ESI, M/z): 332 (M + +1).
Step six: synthesis of Compound 1
Compound g (438 mg,1.33 mmol) was added to phosphorus oxychloride (10 mL), and the reaction mixture was warmed to 110℃and stirred for 24h. After the reaction is completed, decompressing and concentratingAfter the reaction mixture was concentrated, a saturated aqueous sodium hydrogencarbonate solution was slowly added thereto, extraction was performed with ethyl acetate (5 mL ×3), washing was performed with saturated brine, drying was performed with anhydrous sodium sulfate, filtering was performed, and concentration was performed to obtain compound 1 (370 mg, 80%). MS (ESI, M/z): 350 (M) + +1)。
Example 2: synthesis of Compound I
Step one: synthesis of Compound 2
Compound 1 (1.4 g,4.01 mmol), compound 6 (0.8 g,6 mmol) and triethylamine (2.8 mL, 20 mmol) were dissolved in isopropanol (30 mL) and 1, 4-dioxane (20 mL), and the mixture was placed in a closed tube and heated to 125℃to stir for 48h. After cooling to room temperature, the solvent was concentrated under reduced pressure, and a saturated sodium hydrogencarbonate solution, ethyl acetate extraction (50. 50mL ×3), washing with saturated brine, drying over anhydrous sodium sulfate, filtration, concentration, and separation and purification by column chromatography were added to the residue to obtain compound 2 (1.1 g, 60%). MS (ESI, M/z): 446 (M) + +1)。
Step two: synthesis of Compound 3
Compound 2 (1 g,2.25 mmol) and compound 7 (1 g,2.70 mmol) were dissolved in dry toluene (7 mL), and 2M aqueous sodium carbonate (3.4. 3.4 mL) and Pd (dppf) Cl were added to the above solutions 2 (33 mg,0.045 mmol), nitrogen protection, reaction temperature was raised to 80 ℃ C. And stirred for 16h. The reaction solution was cooled to room temperature, the solvent was then removed by concentration under reduced pressure, and Compound 3 (948 mg, 75%) was obtained by column chromatography separation and purification. MS (ESI, M/z): 563 (M) + +1)。
Step three: synthesis of Compound 4
Compound 3 (900 mg,1.6 mmol) was dissolved in ethyl acetate (10 mL), EA/HCl (4 mL,4M,16 mmol) was added to the above solution, and the reaction was stirred at room temperature for 4h. After the reaction was completed, a large amount of solid was formed, suction filtration was performed, the filter cake was washed with a small amount of ethyl acetate, and vacuum drying was performed to obtain compound 4 (665 mg, 90%). MS (ESI, M/z): 463 (M) + +1)。
Step four: synthesis of Compound 5
Compound 4 (600 mg,1.30 mmol) was dissolved in 2-methyltetrahydrofuran (2.5 mL) at 20deg.C, and TEA (1.1 mL,7.5 mmol) was added to the solution. Cooling the reaction solution to 0-5 ℃, slowly dropwise adding MsCl (859 mg,7.5 mmol) into the reaction solution, heating to 18-20 ℃ after the dropwise adding, and stirring for 20min. Then, water was slowly added dropwise to the reaction solution, and stirring was continued for 10 minutes after the completion of the addition. Adjusting the pH to 6.0-6.5 by using 2N HCl; then saturated sodium bicarbonate solution is used for regulating the pH value to 7-7.5, the mixed solution is stirred for 10min, the layers are separated, and a methyl-tetrahydrofuran layer is taken and directly put into the next reaction.
Step five: synthesis of Compound I
A2-methyltetrahydrofuran solution of Compound 5 was placed in a reaction flask, the temperature was adjusted to 15-20℃and 3N sodium hydroxide solution (2.1 mL,6.2 mmol) was added to the above solution, and the mixture was vigorously stirred at 20-23℃for 30min. Stopping stirring, removing a water layer, and adding 2N HCl into the organic layer to adjust the pH to 6.0-6.5; then the pH value of the saturated sodium bicarbonate solution is adjusted to be 8.5, and the water layer is removed. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography to give compound I. 1 H NMR (500 MHz,DMSO-d 6 ) δ 8.72 (d, J = 5.7 Hz, 1H), 7.45 – 7.36 (m, 2H), 7.36 – 7.31 (m, 2H), 6.03 (t, J = 4.6 Hz, 1H), 5.56 (d, J = 9.0 Hz, 1H), 4.83 (hept, J = 6.4 Hz, 1H), 3.90 – 3.79 (m, 1H), 3.70 (dt, J = 13.0, 4.7 Hz, 1H), 3.67 (s, 3H), 3.57 (dt, J = 13.0, 4.7 Hz, 1H), 2.89 (s, 3H), 1.44 (dd, J = 25.1, 6.4 Hz, 6H), 1.22 (d, J = 6.6 Hz, 3H). MS (ESI, m/z): 541 (M + +1)。
Example 3: B-RAF V600E Inhibition Activity test
Recombinant expressed GST-MEK was diluted to 50. Mu.g/mL with TTBS buffer and 100. Mu.L was added to a 96 Kong Guang galanin plate. The test compound solution (1. Mu.L) was added to 50. Mu.L of 50mM HEPES (pH=7.0) and 25ng of B-RAF V600E In a mixture of kinases. After the mixture was incubated at room temperature for 1 hour, 96-well glutathione treated with GST-MEK was addedTo each well of the plate, 50. Mu.L of a phosphorylation buffer was then added at 37℃and kinase phosphorylation was performed with intermittent shaking. After 30min, the reaction was stopped by rinsing with a large amount of TTBS buffer. Thereafter, a 5000-fold dilution of anti-phospho-MEK 1 (Ser 218/222)/MEK 2 (Ser 222/226) monoclonal antibody with TTBS buffer was added to 96-well plates to detect substrate phosphorylation. After 1H incubation with shaking at room temperature, goat anti-rabbit monoclonal antibody IgG (H+L) -HRP complex was added at 1:5000 to chromogenically detect the degree of substrate phosphorylation. And SuperSignal ELISA micro luminescent substrate was added after continuing the shaking culture at room temperature for a proper time. The luminescence photometer records the fluorescence signal and processes the data. Kang Naifei Ni was used as positive control.
TABLE 1 test compound pair B-RAF V600E Enzyme inhibition Activity
As shown in Table 1, compound I versus B-RAF compared to Kang Naifei Ni V600E The enzyme inhibition activity is improved significantly.
Example 4: 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 stock concentration of 10 mM;
2) The compound was diluted twice at the highest concentration point with top dose of 10 mM (100% DMSO)
Ten points are added, and two complex holes are arranged for each concentration;
3) The compound was diluted 100-fold with 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 dosing was completed, 72 h was incubated with 5% CO2 at 37 ℃.
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 anti-cell proliferation Activity of test Compounds
As shown in Table 2, the pair of compounds I carries B-RAF compared to Kang Naifei Ni V600E Has more remarkable inhibition activity.
Example 5: a549 Analysis of p38 selectivity of test Compound for p38 by the Bright-Glo reporter Gene of the p38 alpha MAP kinase
A549 cells were stably transfected with the reporter gene pGL3-IL8-Luc driven by the IL-8 promoter. The cells were packed in 4X 10 cells 5 spots/mL plates were fixed on 384-well plates (40. Mu.L/well, 5% CD-FBS, 1 XP/S, DEME) and incubated overnight at 37 ℃. Test compounds were serially diluted in DMSO, and then 50nL of test solution was added to the incubation. After 30min incubation with the test compound, the cells were stimulated with 1ng/mL IL-1β (10. Mu.L per well of 5ng/mL solution). Bright-Glo (25. Mu.L/well) was added to determine luciferase expression after 7-8 hours of stimulation.
A375-luc cells (1500/50. Mu.L) engineered to express luciferase were spotted onto 384-well white transparent floors in MEM medium containing 10% FBS. Test compounds were serially diluted in DMSO and then used in automated needle formatThe tool was transferred into cells (100 nL). Cells were incubated at 25℃for 2d, then Bright-Glo (25. Mu.L/well) was added to each well, and fluorescence values were read. IC for each compound 50 The XL Fit data analysis software was used to calculate by nonlinear regression.
TABLE 3 Selectivity of test Compounds for p38
As shown in Table 3, compound B-RAF was found to be at the cellular level compared to Kang Naifei Ni V600E Has better inhibition activity and better selectivity to p38 kinase.
Example 6: test compound pharmacokinetic property detection
Male SD rats were selected for oral (10 mg/kg) or intravenous (2 mg/kg) administration, and after 5min,15min,30min,1h,2 h,4 h,8 h,10 h,24 h administration, blood was continuously taken from the ocular fundus venous plexus and placed in an EP tube containing heparin, centrifuged, and the upper plasma was taken for LC-MS/MS analysis, and based on the blood concentration-time data obtained by the test, pharmacokinetic parameters were calculated using WinNonlin software.
Experimental results show that the half-life of Kang Naifei is 4.2h, the oral bioavailability is 30%, and the half-life of the compound I is obviously prolonged to 8.9 h, and the oral bioavailability is obviously improved to 64%.
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 (5)

1. A compound of formula I or a pharmaceutically acceptable salt thereof, having the structure:
。
2. a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier or adjuvant.
3. A compound of formula I as defined in claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined in claim 2, for use in the preparation of B-RAF V600E Use of a kinase inhibitor.
4. Preparation of a compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, or a pharmaceutical composition according to claim 2 for use in the treatment and/or prophylaxis of B-RAF V600E Use of a kinase enzyme activity abnormality related cancer drug.
5. The use according to claim 4, wherein the cancer is selected from lung cancer, pancreatic cancer, bladder cancer, colon cancer, prostate cancer, thyroid cancer, melanoma and ovarian, ocular, hepatic, biliary tract or nervous system cancer.
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US5286872A (en) * 1991-05-21 1994-02-15 Fuji Film Co., Ltd. Deuterated pyrazole compound
CN103958507A (en) * 2011-09-30 2014-07-30 沃泰克斯药物股份有限公司 Compounds useful as inhibitors of ATR kinase
CN110256408A (en) * 2018-07-12 2019-09-20 深圳市塔吉瑞生物医药有限公司 A kind of diaryl pyrazole azole compounds and the composition comprising the compound and application thereof
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