A deuterated indazole MAPK inhibitor drug and its use

Abstract

The present invention discloses a deuterated indazole MAPK inhibitor, as shown in the following formula I: The present invention relates to a deuterated indazole MAPK inhibitor, a pharmaceutical composition and use thereof, which has excellent MAPK inhibitory activity and anti-tumor activity, and can improve the pharmacokinetic properties of the MAPK inhibitor, reduce the dosage and possible toxic side effects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to deuterated indazole MAPK inhibitor drugs and uses thereof. Background Art

[0002] The MAPK (mitogen-activated protein kinase) pathway has three levels of signal transduction: MAPK, MAPK kinase (MEK or MKK) and MAPK kinase kinase (MEKK or MKKK). These three kinases can be activated in sequence and jointly regulate a variety of important physiological / pathological effects such as cell growth, differentiation, stress, and inflammatory response. The MAPK pathway has four main branch routes: ERK, JNK, p38 / MAPK and ERK5. Among them, JNK and p38 have similar functions and are related to inflammation, apoptosis, and growth; ERK is mainly responsible for cell growth and differentiation, and its upstream signal is Ras / Raf protein. As a very critical information transmission chain in cells, the mitogen-activated protein kinase MAPK signaling pathway is widely expressed in cells. Ser / Thr protein kinases participate in a series of cell physiological activities such as cell growth, development, differentiation, and apoptosis, and are key proteins that induce tumor formation.

[0003] Currently, multiple MAPK pathway inhibitors are used to treat cancer patients, such as the drug AMG 510 targeting the KRAS G12C mutant protein, dabrafenib and vemurafenib targeting the RAF protein, and trametinib targeting the MEK protein.

[0004] Therefore, finding an effective structural MAPK pathway inhibitor will provide new directions and inspiration for cancer treatment. Summary of the invention

[0005] The present invention provides deuterated compounds of deuterated indazole MAPK inhibitors and pharmaceutically acceptable salts thereof, which can effectively enhance the MAPK inhibitory activity and anti-tumor activity, and at the same time improve the pharmacokinetic properties of the MAPK inhibitors, reduce the dosage and possible toxic side effects

[0006] In order to achieve the above object, the present invention provides a deuterated indazole compound of a MAPK inhibitor as shown in the following formula I and a pharmaceutically acceptable salt thereof:

[0007]

[0008] Wherein R1 is selected from

[0009] R2 is selected from hydrogen or

[0010] R3 is selected from fluorine or

[0011] The deuterated indazole compounds of the MAPK inhibitors of the present invention and pharmaceutically acceptable salts thereof include the following structures:

[0012]

[0013] The deuterated indazole compounds of the MAPK inhibitor of the present invention and pharmaceutically acceptable salts thereof, wherein the pharmaceutically acceptable salts are selected from methanesulfonate, maleate, hydrochloride or phosphate.

[0014] The deuterated indazole compounds and pharmaceutically acceptable salts thereof described in the present invention include applications thereof in the preparation of anti-tumor drugs.

[0015] The deuterated indazole compounds and pharmaceutically acceptable salts thereof of the present invention comprise the deuterated indazole compounds and pharmaceutically acceptable salts thereof as active ingredients and a pharmaceutically acceptable carrier.

[0016] The pharmaceutical composition of the deuterated indazole compounds and pharmaceutically acceptable salts thereof of the present invention is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0018] The present invention provides a class of deuterated pyridine amide MAPK inhibitor drugs, which have excellent MAPK inhibitory activity and anti-tumor activity, and can improve the pharmacokinetic properties of MAPK inhibitors, reduce the dosage and possible toxic side effects. DETAILED DESCRIPTION

[0019] 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.

[0020] Example 1: Synthesis of Compound 1

[0021] The synthetic route of compound 1 is as follows:

[0022]

[0023] Intermediates 1-6 can be prepared by referring to the method of patent WO2007089646;

[0024] Synthesis of intermediate 7

[0025] Potassium hydroxide (2 mmol, 4 eq) and iodine (1 mmol, 2 eq) were added to a solution of intermediate 1 (0.5 mmol) in N,N-dimethylformamide (10 mL). The mixture was reacted at room temperature for 3 hours. After the reaction was completed as monitored by TLC, 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 sodium chloride (20 mL), dried over anhydrous sodium sulfate, and concentrated by column chromatography to obtain intermediate 2 with a yield of 38%.

[0026] Synthesis of intermediate 8

[0027] Sodium acetate (1 mmol, 2 eq) was added to a deuterated acetic acid solution (8 mL) of intermediate 2 (0.5 mmol) and the mixture was added dropwise over 2 hours. The mixture was reacted at room temperature for 24 hours. The reaction was completed by TLC. The mixture was concentrated under reduced pressure and column chromatography was performed to obtain intermediate 8 in a yield of 41%.

[0028] Example 1 Synthesis of Compounds

[0029] Intermediate 8 (0.5 mmol) was dissolved in DCM (2 mL), TMSI (0.8 mmol) was added, and the reaction was stirred at room temperature. The reaction was completed by TLC detection. Water was added and extracted with dichloromethane. The organic phase was collected, washed once with water and once with saturated brine. The organic phase was concentrated and dissolved in methanol. Sodium borohydride (1 mmmol) was added at 0°C. After continuing the reaction for one hour, water was added to quench the reaction, dichloromethane was added to extract the reaction, and the organic phase was concentrated. Example 1 compound was obtained by column chromatography. 1 H NMR (500MHz, Chloroform-d) δ8.22 (d, J=8.5Hz, 1H), 7.77 (d, J=2.7Hz, 1H), 7.44 (s , 1H), 7.36-7.27 (m, 2H), 7.27-7.16 (m, 3H), 6.98 (td, J=8.3, 2.6Hz, 2H), 6.89-6.7 3 (m, 2H), 6.51 (t, J=6.2Hz, 1H), 4.53 (dd, J=6.1, 1.0Hz, 2H), 4.23 (t, J=6.7Hz, 2H) , 3.99 (q, J=6.9Hz, 2H), 3.77 (t, J=7.3Hz, 1H), 2.36 (d, J=0.9Hz, 3H), 1.37 (s, 9H).

[0030] Example 2: Synthesis of Compound 2

[0031]

[0032] Referring to the synthesis method of the compound in Example 1, the compound in Example 2 can be prepared with a yield of 38%. 1H NMR (500MHz, Chloroform-d) δ 8.66 (s, 1H), 8.41 (d, J = 9.3Hz, 1H), 7.75 (s, 1H), 7.16-7.00 (m, 5H), 3.91 (d, J = 3.7Hz, 2H), 3.82 (dt, J = 9.3, 5.9Hz, 1 H), 2.65-2.48 (m, 2H), 2.22 (s, 6H), 2.11 (dqq, J=13.9, 6.9, 3.4Hz, 1H), 1 .93 (td, J=7.3, 5.9Hz, 2H), 1.10 (d, J=6.8Hz, 3H), 1.05 (d, J=7.0Hz, 3H).

[0033] Test Example 1: MAPK kinase inhibitory activity test

[0034] Add 100 μL of 5 nM activated p38a enzyme and 1 μM ATF-2 (activating transcription factor 2 fusion protein) to a 384-well plate. At room temperature, add 25 mM HEPES (pH 7.4), 100 μM vanadate, 1 mM DTT, 10 mM MgCl and 10 μM [γ-33P]-ATP. Add 25% TCA, continue incubation for 30-40 minutes, and transfer directly to the GF-B membrane filter plate. Wash the filter twice with 0.5% phosphoric acid. After washing, add approximately 30 μL of scintillant to the wells and read in a liquid scintillation counter (Packard Top Count HTS).

[0035] Compound <![CDATA[IC 50 (nM)]]> Compound 1 0.35 Compound 2 0.27 ARRY-614 2.1

[0036] As can be seen from the above table, Example compounds 1 and 2 have excellent MAPK inhibitory activity. Compared with the positive drug ARRY-614, the inhibitory activity of Example compounds 1 and 2 is significantly better than that of the positive drug.

[0037] Test Example 2: Anti-lung adenocarcinoma A549 activity experiment

[0038] The inhibition rate of the target compound was investigated using the lung adenocarcinoma A549 cell line, and the IC of each compound was determined using the MTT method with ARRY-614 as a positive control. 50 The succinate dehydrogenase in the mitochondria of living cells can reduce exogenous tetrazolium bromide (MTT) to insoluble blue-purple crystals and deposit them in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the purple crystals in the cells, and the absorbance value detected at a wavelength of 570nm using an enzyme-linked immunosorbent assay can reflect the number of living cells.

[0039] (I) Experimental materials and equipment

[0040] Cell line: Lung adenocarcinoma A549 cell line.

[0041] Drugs and reagents: MTT and dimethyl sulfoxide (DMSO) were purchased from Sigma; trypsin, newborn calf serum, fetal bovine serum, RPMI-1640, DMEM and penicillin-streptomycin were purchased from Gibco; other reagents were purchased from Sigma unless otherwise specified. Instruments CO2 incubator (Thermo), microplate reader (Thermo) and fluorescence inverted microscope (Olympus) were purchased from Sigma.

[0042] (II) Experimental methods

[0043] Cell culture method: cells were cultured in a conventional manner, and cells in the logarithmic growth phase were used in the experiment. Cell growth assay (MTT) method: cells in the logarithmic growth phase were used to make 5×10 4 100 μL / mL cell suspension was inoculated in a 96-well plate, and each well was incubated in a 5% CO2, 37°C constant temperature incubator overnight. Then, the culture medium containing different drug concentration gradients was replaced, and each well was incubated for 72 hours. 5 mg / mL MTT (30 μL / well) was added, and the culture medium was incubated in a constant temperature incubator for 4 hours. The culture medium was discarded, and DMSO (100 μL / well) was added. After shaking for 10 seconds, the absorbance was detected at a wavelength of 570 nm using an enzyme marker, and the inhibition rate was calculated.

[0044] (III) Experimental results

[0045] Each concentration gradient was repeated three times, and the IC of the compound was calculated using Sigmaplot 5.1 software, with concentration as the horizontal axis and inhibition rate as the vertical axis. 50 value.

[0046] Compound <![CDATA[IC 50 (μM)]]> Compound 1 4.1 Compound 2 3.9 ARRY-614 27.5

[0047] As can be seen from the above table, Example compounds 1 and 2 have strong inhibitory activity against human lung cancer A549 cells. Compared with the positive drug ARRY-614, the inhibitory activity of Example compounds 1 and 2 is significantly better than that of the positive drug.

[0048] Test Example 3: Pharmacokinetics of Compounds

[0049] 1. Experimental instruments and materials

[0050] High-speed refrigerated centrifuge, vortex oscillator (Vortex Genius3), high-speed centrifuge (Eppendorf5415D), disposable syringes, pipettes (Eppendorf), SD male rats used in the experiment were purchased from Yangzhou University, EDTA-K2 vacuum blood collection tubes, and normal saline. All rats in the oral group fasted for 12 hours before administration, and had free access to water and food during administration.

[0051] (II) Experimental steps

[0052] Example 1 or intermediate 1 was dissolved in DMSO / solutol / water (10 / 10 / 80) to prepare a clear solution. The compound was administered orally at a dose of 25 mg / kg, and the compound was administered by tail vein at a dose of 5 mg / kg. At 2 min, 10 min, 30 min, 1 h, 2 h, 3 h, 5 h, 8 h, 12 h, 16 h, and 24 h after tail vein administration, 0.5 mL of blood was continuously taken from the venous plexus of the fundus into a heparin tube. At 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 5 h, 8 h, 12 h, 16 h, and 24 h after oral administration, 0.5 mL of blood was continuously taken from the venous plexus of the fundus into a heparin tube. After the sample was centrifuged at 8000 r and 4 ° C for 10 min, 0.15 mL of upper plasma was taken and stored at -20 ° C, and then LC-MS / MS analysis was performed. The data was analyzed by WinNolin non-compartmental model to obtain key pharmacokinetic parameters.

[0053] (III) Experimental results

[0054] Table 1 Pharmacokinetic parameters

[0055]

[0056] Compared with the positive drug ARRY-614, the half-life and peak concentration of Example 1 after oral administration are significantly improved, and the oral bioavailability is also significantly improved, which can effectively improve the dosage and thus reduce the toxic side effects of high-dose administration.

[0057] 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 deuterated indazole MAPK inhibitor compound and a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from the following structures: 。 2. The deuterated indazole compound and its pharmaceutically acceptable salt according to claim 1, characterized in that The pharmaceutically acceptable salt is selected from methanesulfonate, maleate, hydrochloride or phosphate.

3. Use of the deuterated indazole compound and a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-tumor drug, wherein the anti-tumor target is MAPK.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition is composed of the deuterated indazole compound and a pharmaceutically acceptable salt thereof as claimed in claim 1 as an active ingredient and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral solutions, inhalants, ointments, suppositories or patches.

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