A deuterated pyridine amide IRAK-4 inhibitor drug and its use

Abstract

The present invention discloses a deuterated pyridinamide IRAK‑4 inhibitor, as shown in the following formula I: The present invention relates to a deuterated pyridinamide IRAK‑4 inhibitor, a pharmaceutical composition and use thereof. The deuterated pyridinamide IRAK‑4 inhibitor drug of the present invention has excellent IRAK‑4 inhibitory activity and anti-tumor activity, and can improve the pharmacokinetic properties of the IRAK‑4 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 pyridine amide compounds and uses thereof, and more specifically, to pyridine amide compounds and their use as IRAK-4 inhibitor drugs. Background Art

[0002] Interleukin-1 receptor-associated kinase 4 (IRAK-4) is a member of the intracellular serine-threonine kinase IRAK family. Other members of the kinase family include IRAK-2, IRAK-M, and IRAK-3. When the interleukin-1 receptor (IL-1R) or Toll-like receptor (TLR) binds to the ligand, IRAK-4 can mediate signal transduction and activate the expression of downstream inflammatory factors. TLRs can receive ligand signals generated by the interaction between the body and microorganisms or stimulation by endogenous substances, as well as the first wave of inflammatory signals and innate immune response signals triggered by these stimuli. TLRs play a very important role in many diseases, including infections and autoinflammatory diseases, as well as many other human diseases [4]. Like tumor necrosis factor-α (TNF-α) and other major cytokines, interleukin-1 (IL-1) is a key factor in the inflammatory pathway that can propagate and amplify signals. Since the signaling pathways mediated by TLR, IL-1R and other cytokine receptors have a cross-linking effect, IRAK-4, a key signaling factor downstream of the TLR and IL-1R inflammatory pathways, plays an important role in systemic inflammatory responses and can serve as an effective potential target for the treatment of various inflammation-related diseases.

[0003] IRAK4 has also been reported to play a key role in the development and progression of a variety of malignancies, including melanoma, Waldenstrom macroglobulinemia (WM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (T-ALL), ABC diffuse large B-cell lymphoma (ABC-DLCBL), etc. The MyD88-L256P functional enhancement mutation causes upregulation of the myddosome complex, leading to the activation of NF-κB and causing the survival and proliferation of a series of tumor cell lines. Immunohistochemistry results of melanoma biopsies showed that phosphorylated IRAK4 was highly expressed in lesion tissues. Increased mRNA expression levels of IRAK1 and IRAK4, as well as increased phosphorylation levels of IRAK1 and IRAK4, were also observed in T-ALL cells (Li Z, et al. J Clin Invest 2015; 125(3): 1081-97.). Silencing IRAK4 with shRNA or inhibiting its enzymatic activity with small molecules weakened cell proliferation in samples from T-ALL patients, indicating that signal transduction through IRAK4 is a key factor in disease progression. The above research results all confirm the potential value of IRAK4 targets in the field of tumor treatment.

[0004] At present, the research and development of IRAK-4 inhibitor drugs is a hot area both at home and abroad. Therefore, providing an IRAK-4 inhibitor drug with an effective structure will find new directions and inspirations for the treatment of cancer. Summary of the invention

[0005] The present invention provides deuterated pyridine amide IRAK-4 inhibitor compounds and pharmaceutically acceptable salts thereof, which can effectively enhance the IRAK-4 inhibitory activity and anti-tumor activity, and can also improve the pharmacokinetic properties of the IRAK-4 inhibitor, thereby reducing the dosage and possible toxic side effects.

[0006] In order to achieve the above object, the present invention provides a deuterated indole amide IRAK-4 inhibitor compound as shown in the following formula I and a pharmaceutically acceptable salt thereof:

[0007]

[0008] The deuterated indole amide IRAK-4 inhibitor compounds and pharmaceutically acceptable salts thereof described in the present invention include the following structures:

[0009]

[0010] The deuterated indole amide IRAK-4 inhibitor compound and its pharmaceutically acceptable salt described in the present invention, wherein the pharmaceutically acceptable salt is selected from methanesulfonate, maleate, hydrochloride or phosphate.

[0011] The deuterated pyridine amide compounds and pharmaceutically acceptable salts thereof of the present invention include their use in preparing anti-tumor drugs.

[0012] The deuterated pyridine amide compounds and pharmaceutically acceptable salts thereof of the present invention comprise the deuterated pyridine amide compounds and pharmaceutically acceptable salts thereof as active ingredients and a pharmaceutically acceptable carrier.

[0013] The pharmaceutical composition of the deuterated pyridine amide compound and a pharmaceutically acceptable salt thereof of the present invention is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0015] The present invention provides a class of deuterated pyridine amide IRAK-4 inhibitor drugs, which have excellent IRAK-4 inhibitory activity and anti-tumor activity, and can also improve the pharmacokinetic properties of IRAK-4 inhibitors, reduce the dosage and possible toxic side effects. DETAILED DESCRIPTION

[0016] 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.

[0017] Example 1: Synthesis of Compound 1

[0018] The synthetic route of compound 1 is as follows:

[0019]

[0020] Among them, intermediate 1 can be prepared by referring to the method of patent WO2017186693;

[0021] Intermediate 2 (1 mmol) was dissolved in 20 mL of DMF, potassium carbonate (3 mmol) and potassium iodide (0.5 mmol) were added with stirring, and the mixture was stirred at room temperature for half an hour. (4-bromo-2-methylbutyl-2-yl)oxy) (tert-butyl) dimethylsilane (1.5 mmol) was added, and stirring was continued for 24 hours. Water was added, and the mixture was extracted with ethyl acetate. The organic phase was collected, washed with saturated brine, and the organic phase was concentrated and subjected to column chromatography to obtain Intermediate 2.

[0022] To a solution of intermediate 2 (0.5 mmol) in N,N-dimethylformamide (10 mL) were added potassium hydroxide (2 mmol, 4 eq) and iodine (1 mmol, 2 eq), and 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 3.

[0023] Sodium acetate (1 mmol, 2 eq) was added to a solution of intermediate 3 (0.5 mmol) in deuterated acetic acid (8 mL) over 2 hours. The mixture was reacted at room temperature for 24 hours. The reaction was complete when detected by TLC. The mixture was concentrated under reduced pressure and column chromatography was performed to obtain intermediate 4.

[0024] The intermediate 4 (1 mmol) was dissolved in methanol (10 mL), palladium carbon (10% mol) was added, and the mixture was stirred at room temperature for three hours under standard hydrogen pressure, filtered through celite, and the organic phase was concentrated and subjected to column chromatography to obtain the intermediate 5.

[0025] Intermediate 6 (1 mmol) was dissolved in THF (20 mL), HATU (2 mmol) and DIPEA (2 mmol) were added, and stirring was continued at room temperature for half an hour. Intermediate 5 (1 mmol) was added and stirring was continued overnight. Water was added, the mixture was filtered, and the solid was dried to obtain Intermediate 7.

[0026] Intermediate 7 was dissolved in 2 mL of trifluoroacetic acid and stirred at room temperature for half an hour. The solvent was concentrated and the compound of Example 1 was obtained by column chromatography. 1 H NMR (500MHz, Chloroform-d) δ9.12 (s, 1H), 7.91 (ddd, J=11.9, 7.9, 1.2Hz, 2H), 7.72 (t, J=7.8Hz, 1H), 7.67 (s, 1H), 5.21 (s, 1H), 4.08 (t, J=7.3Hz, 2H), 3.66 (s, 1H), 1.88 (t, J=7.2Hz, 2H), 1.70 (s, 6H), 1.24 (s, 6H).

[0027] Example 2: Synthesis of Compound 2

[0028]

[0029] Referring to the synthesis method of the compound in Example 1, the compound in Example 2 can be obtained with a yield of 46%. 1H NMR (500MHz, Chloroform-d) δ9.11 (s, 1H), 7.91 (ddd, J=11.9, 7.9, 1.2Hz, 2H), 7.72 (t, J=7.8Hz, 1H ), 7.67 (s, 1H), 5.21 (s, 1H), 4.44 (t, J = 6.2Hz, 2H), 3.41 (t, J = 6.1Hz, 2H), 2.98 (s, 3H), 1.70 (s, 6H).

[0030] Test Example 1: IRAK-4 kinase inhibitory activity test

[0031] Prepare 1× modified TR-FRET assay buffer. The test compound concentration is 1000nM, 3-fold dilution, 10 concentration points, and single-well detection. Dilute the solution with DMSO to 100 times the final concentration in a 96-well plate, then dilute it with 1× buffer to 20 times the final concentration, and transfer 1uL to a 384-well reaction plate for use. Add 1uL of 20% DMSO to the negative control well and the positive control well respectively. Prepare a 5-fold final concentration of IRAK-4-Biotin solution with 1× modified TR-FRET assay buffer. Add 4μL of 5-fold final concentration of IRAK-4-Biotin solution to the compound well and the positive control well respectively; add 4μL of 1× modified TR-FRET assay buffer to the negative control well. Centrifuge at 1000rpm for 30 seconds, shake and mix, and incubate at room temperature for 15 minutes. Prepare a mixed solution of ATP-Eu at 4 times the final concentration and Dyelabeled acceptor at 2 times the final concentration with 1× modified TR-FRET assay buffer. Add 15 μL of PD-1-Eu and Dye labeled acceptor mixed solution. Read using a microplate reader, Em665 / 620, and calculate IC 50 value.

[0032] Compound <![CDATA[IC 50 (nM)]]> Compound 1 0.4 Compound 2 0.3 BAY-839 2.8

[0033] As can be seen from the above table, Example compounds 1 and 2 have excellent IRAK-4 inhibitory activity. Compared with the positive drug BAY-839, the inhibitory activity of Example compounds 1 and 2 is significantly better than that of the positive drug.

[0034] Experimental Example 2: Tumor Cell Proliferation Detection of Compounds

[0035] The OCI-LY10 (human B-cell lymphoma) cell line was selected and cultured in a 37°C, 5% CO2 culture system. Cells growing in the exponential growth phase were collected and counted for plate inoculation. Cells were counted using a Trypan-Blue hemocytometer, and the cell concentration was adjusted to an appropriate density and inoculated in a 96-well plate, with 90 μL of cell suspension per well, and wells with only culture medium added were set as blank controls. The plate was placed in a 37°C, 5% CO2 culture system overnight as a cell test plate. Compounds were diluted with DMSO in a 96-well plate until different gradient concentrations.

[0036] Prepare a V-Base Cell culture plate, add 198μL test medium and 2μL diluted compound solution to each well, add 2μL DMSO to the control well, mix by pipetting up and down, and use this V-base culture plate as a 10× concentration compound plate. From each single well of the compound plate, take 10μL of the compound-medium mixture or the mixture in the control well and transfer it to the cell test plate. The final concentration of DMSO is 0.1%. Put the cell test plate back into the culture system and let it stand for 3 days for cell growth.

[0037] The cell samples were treated with Promega's CellTiter-Glo kit (Promega-G7573) and the luminescent signals were recorded on a 2104EnVision plate reader. The inhibition rate (IR) of the test compound at different doses was calculated as follows:

[0038] IR (%) = (1-(RLU compound-RLU blank) / (RLU control-RLU blank)) × 100%, calculate IC 50 value.

[0039] Compound <![CDATA[IC 50 (nM)]]> Compound 1 0.012 Compound 2 0.035 BAY-839 0.179

[0040] It can be seen from the test results in the above table that both compounds 1 and 2 of the present invention exhibit strong inhibitory activity on the proliferation of OCI-LY10 cells. Compared with the positive drug BAY-839, the inhibitory activity of Example compounds 1 and 2 is significantly better than that of the positive drug.

[0041] Test Example 3: Pharmacokinetic Experiment

[0042] 1. Experimental instruments and materials

[0043] 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, had free access to water, and had free access to water and food during administration.

[0044] (II) Experimental steps

[0045] 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.

[0046] (III) Experimental results

[0047] Table 1 Pharmacokinetic parameters

[0048]

[0049] Compared with the positive drug BAY-839, 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.

[0050] 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 pyridine amide IRAK-4 inhibitor compound represented by formula I and a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from the following structures: 。 2. The deuterated pyridine amide 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 pyridine amide compound and a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an IRAK-4 inhibitor.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition is composed of the deuterated pyridine amide 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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