An irreversible HDAC6 subtype selective inhibitor, its preparation method and application

Abstract

The present invention provides an irreversible HDAC6 subtype selective inhibitor, a preparation method thereof and an application thereof. The irreversible HDAC6 subtype selective inhibitor of the present invention has a structure shown in the following formula (I). The inhibitor of the present invention has good selectivity and inhibitory activity for HDAC6, can effectively achieve selective inhibition of the HDAC6 subtype at a relatively low concentration, and at the same time can avoid the toxic and side effects caused by inhibiting other HDAC subtypes, and has high safety. The inhibitor of the present invention has an irreversible inhibitory effect on HDAC6 and has a long-lasting drug effect.

Description

Technical Field

[0001] The present invention relates to an irreversible HDAC6 subtype selective inhibitor, a preparation method thereof and an application thereof, and belongs to the technical fields of organic compound synthesis and pharmaceutical application. Background Art

[0002] Irreversible inhibitors such as penicillin, aspirin and omeprazole have made great contributions to human life and health. Compared with reversible inhibitors, irreversible inhibitors often have the following advantages: 1) stronger target affinity; 2) more complete target inhibition; 3) longer-lasting drug efficacy. Therefore, irreversible inhibitors are an important direction in the current field of drug research and development (Lonsdale, R. etc. Chem. Soc. Rev., 2018, 47, 3816 - 3830; Gehringer, M. etc. J. Med. Chem. 2019, 62, 5673 - 5724). It is worth pointing out that in recent years, 7 irreversible kinase inhibitors (afatinib, ibrutinib, osimertinib, acalabrutinib, neratinib, dacomitinib, zanubrutinib) and 1 irreversible KRASG12C inhibitor (sotorasib) have been approved for marketing for the treatment of cancer (Singh, J. etc. Nat. Rev. Drug Discovery, 2011, 10, 307 - 317; Gehringer, M. etc. J. Med. Chem. 2019, 62, 5673 - 5724).

[0003] Histone deacetylase (HDAC) is a class of hydrolases with epigenetic and post-translational modification regulatory functions. It contains 18 family members, among which HDAC1-11 belong to zinc-dependent HDAC. Different from other zinc-dependent HDAC subtype members, HDAC6 is the only HDAC subtype containing two catalytic domains (CD1 and CD2). HDAC6 is mainly distributed in the cytoplasm and mainly catalyzes the deacetylation of non-histone proteins such as α-tubulin, cortactin, heat shock protein 90 (HSP-90), and peroxiredoxins I / II. Another remarkable feature of HDAC6 is the presence of a zinc finger ubiquitin-binding domain and a dynein motor-binding domain, which endow HDAC6 with the important function of recruiting and transporting polyubiquitinated proteins for autophagic degradation. Given the important functions of HDAC6 in protein post-translational modification and quality control, HDAC6 is regarded as a potential therapeutic target for various cancers, autoimmune diseases, and neurodegenerative diseases (Pulya, S. etc. Pharmacological Research 2021, 163, 105274; Zhang, X. etc. J. Med. Chem. 2021, 64, 1362). Currently, several HDAC6 inhibitors such as ACY-241 and ACY-1215 have entered the clinical research stage for the treatment of diseases such as multiple myeloma, melanoma, chronic lymphocytic leukemia, non-small cell lung cancer, breast cancer, cholangiocarcinoma, Huntington's disease, rheumatoid arthritis, progressive neuropathic peroneal muscular atrophy, and painful diabetic peripheral neuropathy (Zhang, X. etc. J. Med. Chem. 2021, 64, 1362-1391). However, the HDAC6 subtype selectivity of ACY-241 and ACY-1215 is not high (only 10-20 times compared with HDAC1, HDAC2, and HDAC3, Santo, L. etc. Blood, 2012, 16, 2579-2589; Huang, P. etc. Oncotarget, 2017, 8, 2694-2707), and it is still impossible to completely avoid the side effects caused by inhibiting other HDAC subtypes. Therefore, there is an urgent need to develop HDAC6 inhibitors with higher subtype selectivity. In addition, most current HDAC6 inhibitors, including ACY-241 and ACY-1215, are hydroxamic acid compounds. Due to the problem of poor metabolic stability, such compounds often require high-dose multiple administrations to maintain effective blood drug concentrations, and the long-lasting efficacy of irreversible HDAC6 inhibitors is expected to overcome the above defects of existing inhibitors.

[0004] Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides an irreversible HDAC6 subtype selective inhibitor, its preparation method and application. The irreversible HDAC6 subtype selective inhibitor of the present invention has good selectivity and inhibitory activity for HDAC6, can effectively achieve the selective inhibition of HDAC6 subtype at a relatively low concentration, and at the same time can avoid the toxic and side effects caused by inhibiting other HDAC subtypes, with high safety. The inhibitor of the present invention has an irreversible inhibitory effect on HDAC6 and has a long-lasting drug effect.

[0006] The technical solution of the present invention is as follows:

[0007] I. An irreversible HDAC6 subtype selective inhibitor

[0008] An irreversible HDAC6 subtype selective inhibitor containing a benzosulfonyl oxidofurazan structure has the structure shown in the following formula (I):

[0009]

[0010] II. A preparation method of an irreversible HDAC6 subtype selective inhibitor

[0011] The preparation method of the irreversible HDAC6 subtype selective inhibitor of the present invention includes the steps:

[0012] Compound 1 reacts with terephthalyl alcohol to form compound 2; compound 2 is then oxidized to obtain carboxylic acid compound 3; compound 3 is condensed with hydroxylamine hydrochloride to obtain the HDAC6 subtype selective inhibitor (I).

[0013]

[0014] Preferably according to the present invention, the preparation method of compound 2 includes the steps: under stirring conditions, add terephthalyl alcohol to the THF solution of compound 1, and then dropwise add an aqueous NaOH solution; after stirring reaction, concentrate the reaction solution, extract the residue with ethyl acetate, wash the extract with saturated brine, dry it with anhydrous magnesium sulfate, filter, and concentrate to obtain a crude product, and then obtain compound 2 through silica gel column chromatography.

[0015] Preferably, the molar ratio of compound 1 to the volume of THF is 0.1 - 1 mol / L.

[0016] Preferably, the molar ratio of compound 1 to NaOH is 1∶1 - 3.

[0017] Preferably, the molar ratio of compound 1 to terephthalyl alcohol is 1∶3.5 - 4.

[0018] Preferably, the stirring reaction temperature is 3 - 8°C, and the stirring reaction time is 20 - 40 minutes.

[0019] According to the present invention, preferably, the preparation method of compound 3 includes the steps of: adding Jones reagent to the acetone solution of compound 2 at (-5) - 5°C, stirring the reaction at room temperature; filtering off the precipitate, evaporating the acetone, and then adding ethyl acetate; washing the organic phase with saturated brine, drying with anhydrous magnesium sulfate, filtering, and concentrating to obtain the crude product, and then obtaining compound 3 through silica gel column chromatography.

[0020] Preferably, the molar amount of compound 2 and the volume ratio of acetone are 0.1 - 1 mol / L.

[0021] Preferably, the molar amount of compound 2 and the volume ratio of Jones reagent are 2 - 3 mol / L.

[0022] Preferably, the stirring reaction time is 5 - 15 hours.

[0023] According to the present invention, preferably, the method for preparing the HDAC6 subtype selective inhibitor (I) by condensing compound 3 with hydroxylamine hydrochloride includes the steps of: dropping isobutyl chloroformate into the anhydrous THF solution of compound 3 at (-5) - 5°C under stirring conditions, stirring for 0.3 - 1 hour, then dropping triethylamine, continuing to stir for 0.5 - 2 hours, and then filtering off the precipitate to obtain solution A; dissolving potassium hydroxide and hydroxylamine hydrochloride in anhydrous methanol to obtain solution B; adding solution A to solution B, and carrying out a stirring reaction at room temperature; then evaporating the solvent, adding hydrochloric acid to the residue and extracting with ethyl acetate, washing the obtained organic phase with saturated brine, drying with anhydrous magnesium sulfate, filtering, and concentrating to obtain the crude product, and then obtaining the HDAC6 subtype selective inhibitor (I) through silica gel column chromatography.

[0024] Preferably, the molar amount of compound 3 and the volume ratio of THF are 0.1 - 1 mol / L.

[0025] Preferably, the molar ratio of compound 3, isobutyl chloroformate, and triethylamine is 1∶1 - 1.1∶0.5 - 0.8.

[0026] Preferably, the molar ratio of potassium hydroxide and hydroxylamine hydrochloride is 1∶1; the molar amount of hydroxylamine hydrochloride and the volume ratio of anhydrous methanol are 0.03 - 1 mol / L.

[0027] Preferably, the molar ratio of compound 3 and hydroxylamine hydrochloride is 1∶1 - 2, preferably 1∶1.5.

[0028] Preferably, after adding solution A to solution B, the stirring reaction time at room temperature is 3 - 5 hours.

[0029] The synthesis route of the inhibitor of the present invention is as follows:

[0030]

[0031] III. Application of an irreversible HDAC6 subtype selective inhibitor

[0032] The present invention also provides an application of an irreversible HDAC6 subtype selective inhibitor containing a benzenesulfonyl oxidized furazan structure in the preparation of a drug for preventing or treating a disease associated with abnormal HDAC6 activity or expression.

[0033] Preferably according to the present invention, the disease associated with abnormal HDAC6 activity or expression is a tumor disease or a non-tumor disease.

[0034] Preferably, the tumor disease is multiple myeloma, melanoma, chronic lymphocytic leukemia, non-small cell lung cancer, breast cancer or cholangiocarcinoma; the non-tumor disease is Huntington's chorea, rheumatoid arthritis, Charcot-Marie-Tooth disease or painful diabetic peripheral neuropathy.

[0035] IV. A pharmaceutical composition

[0036] The present invention also provides a pharmaceutical composition suitable for oral or parenteral administration, comprising the HDAC6 subtype selective inhibitor of the present invention or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers or excipients.

[0037] The technical features and beneficial effects of the present invention are as follows:

[0038] The irreversible HDAC6 subtype selective inhibitor of the present invention has good selectivity and inhibitory activity for HDAC6, can effectively achieve selective inhibition of the HDAC6 subtype at a relatively low concentration, and at the same time can avoid the toxic and side effects caused by inhibiting other HDAC subtypes, with high safety. The inhibitor of the present invention has an irreversible inhibitory effect on HDAC6 and has a long-lasting drug effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Effects of the inhibitor (I) prepared in Example 1 and Tubastatin A (Tub A) on the levels of ac-tub and ac-HH4 in A549 cells at different concentrations;

[0040] Figure 2 Effects of the inhibitor (I) prepared in Example 1 and Tubastatin A (Tub A) on the levels of ac-tub and ac-HH4 in A549 cells after elution;

[0041] Figure 3 Results of apoptosis induction of RPMI8266 cells by the inhibitor (I) prepared in Example 1 and ACY-241 at different concentrations. Detailed implementation manners

[0042] The present invention will be further described below in conjunction with embodiments, but not limited thereto.

[0043] Meanwhile, the reagents used in the embodiments can be obtained commercially without special instructions; the methods and equipment used can be according to the existing technologies without special instructions.

[0044] Example 1. Preparation of HDAC6 subtype selective inhibitor (I)

[0045]

[0046] The specific synthesis steps are as follows:

[0047] (1) Synthesis of compound 2:

[0048] Under the conditions of 5 °C and stirring, p - xylene glycol (1.4 g, 10 mmol) was added to a solution of compound 1 (1.0 g, 2.7 mmol) in THF (10 mL), and then 25 wt% aqueous NaOH solution (1 mL) was added dropwise. After stirring the reaction at 5 °C for 30 minutes, the reaction solution was concentrated, and the residue was extracted with ethyl acetate. The extract was washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain a crude product, which was then purified by silica gel column chromatography to obtain compound 2 (white solid, 0.39 g, 40% yield).

[0049] The NMR data of the product are as follows:

[0050] 1 H NMR (400 MHz, DMSO - d6) δ 8.00 - 7.95 (m, 2H), 7.90 (t, J = 7.5 Hz, 1H), 7.73 (t, J = 7.9 Hz, 2H), 7.40 (q, J = 8.1 Hz, 4H), 5.46 (s, 2H), 5.27 (s, 1H), 4.53 (s, 2H).

[0051] (2) Synthesis of compound 3:

[0052] At 0 °C, Jones reagent (1 mL) was added to a solution of compound 2 (0.80 g, 2.2 mmol) in acetone (10 mL). After stirring the reaction at room temperature for 10 hours, the precipitate was filtered off, the acetone was evaporated, and ethyl acetate was added. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain a crude product, which was then purified by silica gel column chromatography to obtain compound 3 (white solid, 0.65 g, 78% yield).

[0053] The NMR data of the product are as follows:

[0054] 11H NMR (400 MHz, DMSO-d6) δ 13.09 (s, 1H), 8.03 - 7.89 (m, 5H), 7.75 (t, J = 7.8 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 5.57 (s, 2H).

[0055] (3) Synthesis of HDAC6 subtype selective inhibitor (I):

[0056] Under stirring at 0 °C, isobutyl chloroformate (0.9 mL, 3.9 mmol) was added dropwise to a solution of compound 3 (1.48 g, 3.8 mmol) in anhydrous THF (10 mL). After 0.5 h, triethylamine (0.9 mL, 2.5 mmol) was added dropwise, and stirring was continued for 1 h. The precipitate was filtered off to obtain filtrate A for later use. Potassium hydroxide (0.32 g, 5.7 mmol) and hydroxylamine hydrochloride (0.40 g, 5.7 mmol) were added to anhydrous methanol (10 mL) and fully dissolved. The precipitate was filtered off to obtain filtrate B. Filtrate A was added to filtrate B, and the mixture was stirred at room temperature for 4 h. Then the solvent was evaporated. 1 mol / L hydrochloric acid was added to the residue, and the mixture was extracted with ethyl acetate. The obtained organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain the crude product, which was then purified by silica gel column chromatography to obtain the target compound (I) (white solid, 0.62 g, 42% yield).

[0057] The NMR data of the product are as follows:

[0058] 1 1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 9.12 (s, 1H), 8.02 - 8.00 (m, 2H), 7.91 (t, J = 7.5 Hz, 1H), 7.82 (d, J = 8.0 Hz, 2H), 7.75 (t, J = 7.8 Hz, 2H), 7.53 (d, J = 7.9 Hz, 2H), 5.54 (s, 2H). 13 13C NMR (100 MHz, DMSO-d6) δ 163.81, 158.65, 137.30, 137.12, 136.20, 133.11, 130.07, 128.34, 127.96, 127.14, 110.60, 71.80. HRMS (AP-ESI) m / z calcd for C 16 H 14 N3O7S [M + H] + 392.0552 found 392.0544.

[0059] Experimental Example 1. In vitro HDAC6 inhibitory activity and selectivity evaluation experiment of the target compound

[0060] Using the known HDAC6 subtype-selective inhibitor Tubastatin A as a positive control, the inhibitory activities and selectivities of the inhibitor (I) prepared in Example 1 against HDAC6 and HDAC2 were tested.

[0061] Table 1 The experimental results showed that the half-maximal inhibitory concentration (IC 50 ) of compound (I) against HDAC6 was 0.033 μM, far lower than its IC 50 against HDAC2 (1.5 μM), and the selectivity index (SI) was 45.5, higher than those of the HDAC6 inhibitors ACY-241 (SI = 17.3) and ACY-1215 (SI = 10.2) currently in clinical research stage.

[0062] Table 1. Evaluation results of in vitro HDAC6 inhibitory activity and selectivity

[0063]

[0064] a Performed under repeated conditions (n≥2), the SD value of IC 50 was less than 20% of the mean value. b SI = HDAC2 IC 50 / HDAC6 IC 50

[0065] Test Example 2. Immunoblot analysis experiment of the target compound

[0066] The effects of the inhibitor (I) prepared in Example 1 and Tubastatin A (Tub A) on the levels of ac-tub and ac-HH4 in A549 cells at different concentrations were tested, as Figure 1 shown. The DMSO group was used as the negative control group.

[0067] From Figure 1 the results, it can be seen that at a concentration of 0.4 μM, both compound (I) and the positive control Tubastatin A (Tub A) could significantly increase the level of the HDAC6 substrate acetyl-α-tubulin (ac-tub) in A549 cells without affecting the level of the class I HDACs (HDAC1 / 2 / 3) substrate acetyl-histone H4 (ac-HH4), indicating that compound (I) could selectively inhibit intracellular HDAC6. It is worth noting that at a concentration of 80 nM, the effect of compound (I) on increasing the level of ac-tub was significantly better than that of Tubastatin A (Tub A), indicating that at a lower concentration, compound (I) had a stronger inhibitory effect on intracellular HDAC6 than Tubastatin A (Tub A).

[0068] To verify the selective irreversible inhibitory effect of compound (I) on intracellular HDAC6, the present invention used an elution experiment to investigate the effects of compound (I) and Tubastatin A (TubA) on the levels of acetyl-α-tubulin (ac-tub) and acetyl-histone H4 (ac-HH4) in A549 cells, respectively. Figure 2 The results showed that the elevation effect of Tubastatin A (TubA) on the intracellular acetyl-α-tubulin (ac-tub) level was significantly reduced 1 hour after elution, indicating that Tubastatin A (TubA) is a reversible HDAC6 inhibitor. In sharp contrast to Tubastatin A (TubA), compound (I) still had a highly significant elevation effect on acetyl-α-tubulin (ac-tub) even 24 hours after elution, which confirmed its irreversible inhibitory effect on intracellular HDAC6. Consistent with Figure 1 the results, Tubastatin A (TubA) and compound (I) had no significant effect on the intracellular acetyl-histone H4 (ac-HH4) level, which further confirmed their HDAC6 selectivity.

[0069] Test Example 3. In vitro anti-proliferation of the target compound against human multiple myeloma cells and cytotoxicity to normal cells

[0070] Using the HDAC6 inhibitor ACY-241 in the clinical research stage as a positive control, the present invention evaluated the in vitro anti-proliferation activity of the inhibitor (I) prepared in Example 1 against human multiple myeloma cells by a CCK-8 assay. The results in Table 2 showed that the inhibitory activity of compound (I) against each strain of human multiple myeloma cells was significantly better than that of the positive control ACY-241. In addition, compound (I) had a higher selectivity index (SI) than ACY-241, and thus was expected to have better safety than ACY-241.

[0071] Table 2. In vitro anti-proliferation of the compound against human multiple myeloma cells and cytotoxicity to normal cells

[0072]

[0073] a Performed under repeated conditions (n≥3), values are shown as mean ± SD values

[0074] b Selectivity index = IC of LO2 cells 50 / Average of IC of three strains of MM cells 50

[0075] Test Example 4. Experiment on apoptosis induction of the target compound in RPMI8266 cells ​

[0076] Using the HDAC6 inhibitor ACY-241 in the clinical research stage as a positive control, the present invention analyzed the activity of the inhibitor (I) prepared in Example 1 in inducing apoptosis of multiple myeloma cells RPMI8266 by flow cytometry. Figure 3 The results show that compound (I) can significantly induce apoptosis of RPMI8266 cells at both 0.1 μM and 0.5 μM concentrations, and has a dose-dependence. In sharp contrast, the apoptosis-inducing activity of ACY-241 is not obvious.

Claims

1. An irreversible HDAC6 subtype selective inhibitor, characterized in that, It has the structure shown in the following formula (I):

2. The preparation method of the irreversible HDAC6 subtype selective inhibitor according to claim 1, comprising the steps of: Compound 1 reacts with terephthalyl alcohol to form Compound 2; Compound 2 is then oxidized to obtain carboxylic acid Compound 3; Compound 3 is condensed with hydroxylamine hydrochloride to obtain the HDAC6 subtype selective inhibitor (I); 3. The preparation method of the irreversible HDAC6 subtype selective inhibitor according to claim 2, wherein, The preparation method of Compound 2 comprises the steps of: under stirring conditions, add terephthalyl alcohol to the THF solution of Compound 1, and then dropwise add an aqueous NaOH solution; after stirring reaction, concentrate the reaction solution, extract the residue with ethyl acetate, wash the extract with saturated brine, dry with anhydrous magnesium sulfate, filter, concentrate to obtain the crude product, and then obtain Compound 2 by silica gel column chromatography.

4. The preparation method of the irreversible HDAC6 subtype selective inhibitor according to claim 2, wherein, The preparation method of Compound 3 comprises the steps of: at (-5)-5 °C, add Jones reagent to the acetone solution of Compound 2, and stir at room temperature; filter off the precipitate, evaporate the acetone and then add ethyl acetate; after washing the organic phase with saturated brine, dry with anhydrous magnesium sulfate, filter, concentrate to obtain the crude product, and then obtain Compound 3 by silica gel column chromatography.

5. The preparation method of the irreversible HDAC6 subtype selective inhibitor according to claim 2, wherein, The method for condensing Compound 3 with hydroxylamine hydrochloride to prepare the HDAC6 subtype selective inhibitor (I) comprises the steps of: at (-5)-5 °C and under stirring conditions, dropwise add isobutyl chloroformate to the anhydrous THF solution of Compound 3, stir for 0.3-1 hour, then dropwise add triethylamine, continue to stir for 0.5-2 hours and then filter off the precipitate to obtain solution A; dissolve potassium hydroxide and hydroxylamine hydrochloride in anhydrous methanol to obtain solution B; add solution A to solution B and carry out stirring reaction at room temperature; then distill off the solvent, add hydrochloric acid to the residue and extract with ethyl acetate, wash the obtained organic phase with saturated brine, dry with anhydrous magnesium sulfate, filter, concentrate to obtain the crude product, and then obtain the HDAC6 subtype selective inhibitor (I) by silica gel column chromatography.

6. The use of the irreversible HDAC6 subtype selective inhibitor according to claim 1 in the preparation of a drug for preventing or treating a disease associated with abnormal HDAC6 activity or expression.

7. The application according to claim 6, wherein The disease associated with abnormal HDAC6 activity or expression is a tumor disease or a non-tumor disease.

8. The application according to claim 7, wherein The tumor disease is multiple myeloma, melanoma, chronic lymphocytic leukemia, non-small cell lung cancer, breast cancer or cholangiocarcinoma; the non-tumor disease is Huntington's chorea, rheumatoid arthritis, Charcot-Marie-Tooth disease or painful diabetic peripheral neuropathy.

9. A pharmaceutical composition suitable for oral or parenteral administration, comprising the HDAC6 subtype selective inhibitor according to claim 1 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers or excipients.

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