WO2022121228A1 - Preparation and application of oxazole and thiazole histone deacetylase inhibitor - Google Patents

Abstract

Disclosed are a preparation and an application of an oxazole and thiazole histone deacetylase (HDAC) inhibitor. The chemical structure of the inhibitor is represented by formula (I). The inhibitor has relatively strong inhibitory activity on HDACs and cancer cells, and has significantly increased activity compared to that of marketed drug, SAHA.

Description

Preparation and application of an oxazole and thiazole histone deacetylase inhibitor technical field

The invention relates to the technical field of new drug compounds, in particular to a preparation method of an oxazole and thiazole type histone deacetylase inhibitor and its application in anticancer drugs.

technical background

Histone deacetylases (HDACs) can inhibit the formation of acetyl groups in histones (including some non-histone proteins) and affect the normal expression of related proteins. The mutation and abnormal expression of HDACs are usually closely related to the occurrence of tumors. HDACs inhibitors can induce a series of cell-level anti-tumor effects such as cell cycle arrest, differentiation and apoptosis by inhibiting the deacetylation of histones and non-histone proteins at the molecular level. .

At present, 5 HDACs inhibitors have been approved for marketing, namely vorinostat (SAHA), romidepsin (FK228), belistat (PXD101), panobinostat (LBH589) and chidamide (CS005). However, HDACs inhibitors generally have problems such as poor inhibitory effect on solid tumors. With the continuous in-depth study of HDACs inhibitors, the development and synthesis of HDACs inhibitors has also entered a new stage.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an oxazole and thiazole type histone deacetylase inhibitor.

Another object of the present invention is to provide a preparation method of oxazole and thiazole type histone deacetylase inhibitors.

Another object of the present invention is to provide an application of oxazole and thiazole type histone deacetylase inhibitors in anticancer drugs.

Above-mentioned purpose of the present invention is achieved through the following scheme:

The present invention relates to an oxazole and thiazole class histone deacetylase inhibitor, the chemical structure of which is shown in formula (I):

In the formula, R=H, F, Cl, CH ₃ , CF ₃ or OMe; X=O or S.

The above compound is one of the following:

The present invention relates to a preparation method of oxazole and thiazole type histone deacetylase inhibitors. The method includes the following steps:

Place 15 mmol of hydroxylamine hydrochloride and 15 mmol of KOH in a round-bottomed flask, add 25 mL of anhydrous methanol to dissolve, stir in an ice bath for 30 min, and filter to obtain potassium hydroxylamine (NH ₂ OK) solution, which is sealed for later use. Dissolve 1 mmol of compound 1A with 25 mL of NH ₂ OK solution, add 20 mmol of hydroxylamine, stir at room temperature for 1 h, check the reaction end point by TLC, remove the solvent under reduced pressure, add an appropriate amount of 2 mol/L hydrochloric acid solution to acidify to pH=3-4, di Extracted with methyl chloride (3×20 mL), dried over anhydrous MgSO ₄ and evaporated to dryness to obtain the crude product. The crude product was separated and purified by silica gel column chromatography (the eluent volume ratio was chloroform:methanol=10:1), recrystallized from ethanol, and vacuum-dried to obtain the target product 2A.

The reaction formula is as follows:

In the formula, R=H, F, Cl, CH ₃ , CF ₃ or OMe; X=O or S.

The oxazole and thiazole type histone deacetylase inhibitors and their biological activities involved in the present invention have been confirmed by tests.

The oxazole and thiazole type histone deacetylase inhibitors involved in the present invention can be prepared into tablets, powder injections, emulsifiers, capsules and the like under the combination of preparations acceptable to pharmaceutical standards.

The application of the oxazole and thiazole compounds of the present invention in histone deacetylase inhibitors and anticancer drugs and the like.

Compared with the prior art, the present invention has the following beneficial effects:

1. The oxazole and thiazole type histone deacetylase inhibitors provided by the present invention have strong inhibitory effects on three kinds of HDACs and cancer cells, and have the potential to be developed as a new generation of HDACs inhibitors and anticancer drugs.

2. Compared with the positive control SAHA, the oxazole and thiazole type histone deacetylase inhibitors provided by the present invention have significantly improved inhibitory activity on HDACs, have better effects on solid tumors such as lung cancer, and can overcome existing histone deacetylases. Sirtuin inhibitors have low activity and poor efficacy in solid tumors.

Description of drawings

FIG. 1 is the chemical structural formula of the oxazole and thiazole type histone deacetylase inhibitors according to the present invention.

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings described in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are only For some embodiments of the present invention, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without any creative effort.

Detailed ways

The present invention will be further described below through specific embodiments, but the specific embodiments do not limit the present invention in any way.

Example 1: Synthesis of compound 9g

Place hydroxylamine hydrochloride (1.04g, 15.00mmol, 15.00equiv) and KOH (0.84g, 15.00mmol, 15.00equiv) in a round-bottomed flask, add 25mL of anhydrous methanol to dissolve in an ice bath, stir at room temperature for 30min, and filter to obtain potassium hydroxylamine ( _NH2OK ) solution. Compound 8g (0.36g, 1.00mmol, 1.00equiv) was dissolved in 25mL of NH ₂ OK solution, hydroxylamine (0.66g, 20.00mmol, 20.00equiv) was added, and after stirring at room temperature for 1h, the reaction end was detected by TLC, the solvent was removed by concentration, and 20mL of The 2 mol/L hydrochloric acid solution was acidified to PH=3-4, extracted with dichloromethane (3×20 mL), dried over anhydrous MgSO ₄ and evaporated to dryness to obtain the crude product. The crude product was separated and purified by silica gel chromatography (the eluent volume ratio was chloroform:methanol=10:1), recrystallized from ethanol, and dried in vacuo to obtain 9g (0.15g, 42%) of white solid powder. ¹ H NMR (500MHz, DMSO-d ₆ )δ10.34(s,1H), 8.92(s,1H), 8.67(s,1H), 7.79-7.71(m,3H), 7.08(d, J=8.9 Hz, 2H), 3.81(s, 3H), 3.22(s, 2H), 1.93(s, 2H), 1.53–1.45(m, 4H), 1.31–1.22(m, 4H). ¹³ C NMR (125MHz, DMSO-d ₆ )δ169.19,160.20,154.76,153.86,152.55,126.39,122.07,119.49,114.79,55.46,38.95,32.32,28.96,28.41,26.22,25.19.HRMS(AP- _ESId )m/z calc for C H ₂₃ N ₃ O ₅ [M+H] ⁺ 362.1710, found 362.1713.

Example 2: Synthesis of compound 9o

Place hydroxylamine hydrochloride (1.04g, 15.00mmol, 15.00equiv) and KOH (0.84g, 15.00mmol, 15.00equiv) in a round-bottomed flask, add 25mL of anhydrous methanol to dissolve in an ice bath, stir at room temperature for 30min, and filter to obtain potassium hydroxylamine ( _NH2OK ) solution. Compound 8o (0.34 g, 1.00 mmol, 1.00 equiv) was dissolved in 25 mL of NH ₂ OK solution, hydroxylamine (0.66 g, 20.00 mmol, 20.00 equiv) was added, and after stirring at room temperature for 1 h, TLC detected the end of the reaction, concentrated to remove the solvent, and added 20 mL of The 2 mol/L hydrochloric acid solution was acidified to PH=3-4, extracted with dichloromethane (3×20 mL), dried over anhydrous MgSO ₄ and evaporated to dryness to obtain the crude product. The crude product was separated and purified by silica gel chromatography (the eluent volume ratio was chloroform:methanol=10:1), recrystallized from ethanol, and dried in vacuo to obtain a white solid powder 9o (0.16 g, 47%). ¹ H NMR (500MHz, DMSO-d ₆ )δ10.34(s,1H), 8.89(s,1H), 8.68(s,1H), 8.40(s,1H), 7.78(s,2H), 7.48( s, 2H), 7.42(s, 1H), 3.24(s, 2H), 1.93(s, 2H), 1.50(s, 4H), 1.27(s, 4H). ¹³ C NMR(125MHz, DMSO-d ₆ ) _{δ169.19,162.76,159.03,143.60,139.93,130.40,129.50,129.29,126.91,39.12,32.33,29.03,28.41,26.24,25.20.HRMS} (AP-ESI) _m /z calcd for C ₁₇ H ₂₁ N S[M+H] ⁺ 348.1376, found 348.1374.

Example 3: Synthesis of compound 9s

Place hydroxylamine hydrochloride (1.04g, 15.00mmol, 15.00equiv) and KOH (0.84g, 15.00mmol, 15.00equiv) in a round-bottomed flask, add 25mL of anhydrous methanol to dissolve in an ice bath, stir at room temperature for 30min, and filter to obtain potassium hydroxylamine ( _NH2OK ) solution. Compound 8s (0.36 g, 1.00 mmol, 1.00 equiv) was dissolved in 25 mL of NH ₂ OK solution, hydroxylamine (0.66 g, 20.00 mmol, 20.00 equiv) was added, and after stirring at room temperature for 1 h, the reaction end was detected by TLC, the solvent was removed by concentration, and 20 mL of The 2 mol/L hydrochloric acid solution was acidified to PH=3-4, extracted with dichloromethane (3×20 mL), dried over anhydrous MgSO ₄ and evaporated to dryness to obtain the crude product. The crude product was separated and purified by silica gel chromatography (the eluent volume ratio was chloroform:methanol=10:1), recrystallized from ethanol, and dried in vacuo to obtain a white solid powder 9s (0.14 g, 38%). ¹ H NMR (500MHz, DMSO-d ₆ )δ10.34(s,1H), 8.89(s,1H), 8.67(s,1H), 8.37(s,1H), 7.86–7.80(m,2H), 7.33(t,J＝8.8Hz,2H),3.25(d,J＝6.9Hz,2H),1.93(t,J＝7.4Hz,2H),1.50(d,J＝19.3Hz,4H),1.31– _The ^_ HRMS(AP-ESI) m/z calcd for C ₁₇ H ₂₀ FN ₃ O ₃ S[M+H] ⁺ 366.1282, found 366.1287.

Example 4: Synthesis of compound 9u

Place hydroxylamine hydrochloride (1.04g, 15.00mmol, 15.00equiv) and KOH (0.84g, 15.00mmol, 15.00equiv) in a round-bottomed flask, add 25mL of anhydrous methanol to dissolve in an ice bath, stir at room temperature for 30min, and filter to obtain potassium hydroxylamine ( _NH2OK ) solution. Compound 8u (0.36 g, 1.00 mmol, 1.00 equiv) was dissolved in 25 mL of NH ₂ OK solution, hydroxylamine (0.66 g, 20.00 mmol, 20.00 equiv) was added, and after stirring at room temperature for 1 h, the reaction end was detected by TLC, the solvent was removed by concentration, and 20 mL of The 2 mol/L hydrochloric acid solution was acidified to PH=3-4, extracted with dichloromethane (3×20 mL), dried over anhydrous MgSO ₄ and evaporated to dryness to obtain the crude product. The crude product was separated and purified by silica gel chromatography (the eluent volume ratio was chloroform:methanol=10:1), recrystallized from ethanol, and dried in vacuo to obtain a white solid powder 9u (0.20 g, 55%). ¹ H NMR (500MHz, DMSO-d ₆ )δ10.34(s, 1H), 8.86(s, 1H), 8.67(s, 1H), 8.34(s, 1H), 7.65(d, J=8.1Hz, 2H), 7.29(d, J＝8.0Hz, 2H), 3.24(d, J＝7.0Hz, 2H), 2.34(s, 3H), 1.93(s, 2H), 1.50(d, J＝18.6Hz, 4H), 1.26(d, J=6.5Hz, 4H). ¹³ C NMR (125MHz, DMSO-d ₆ )δ169.18, 162.20, 159.06, 143.77, 139.40, 139.00, 130.02, 127.60, 126.79, 39.10, 32.32, 29.03, 28.41, 26.23, 25.19, 20.93. HRMS(AP-ESI) m/z calcd for C ₁₈ H ₂₃ N ₃ O ₃ S[M+H] ⁺ 362.1533, found 362.1530.

Example 5: Synthesis of compound 9w

Place hydroxylamine hydrochloride (1.04g, 15.00mmol, 15.00equiv) and KOH (0.84g, 15.00mmol, 15.00equiv) in a round-bottomed flask, add 25mL of anhydrous methanol to dissolve in an ice bath, stir at room temperature for 30min, and filter to obtain potassium hydroxylamine ( _NH2OK ) solution. Compound 8w (0.41 g, 1.00 mmol, 1.00 equiv) was dissolved in 25 mL of NH ₂ OK solution, hydroxylamine (0.66 g, 20.00 mmol, 20.00 equiv) was added, and after stirring at room temperature for 1 h, the reaction end was detected by TLC, the solvent was removed by concentration, and 20 mL of The 2 mol/L hydrochloric acid solution was acidified to PH=3-4, extracted with dichloromethane (3×20 mL), dried over anhydrous MgSO ₄ and evaporated to dryness to obtain the crude product. The crude product was separated and purified by silica gel chromatography (the eluent volume ratio was chloroform:methanol=10:1), recrystallized from ethanol, and dried in vacuo to obtain a white solid powder 9w (0.19 g, 48%). ¹ H NMR (500MHz, DMSO-d ₆ )δ10.34(s, 1H), 8.97(s, 1H), 8.67(s, 1H), 8.56(s, 1H), 8.01(d, J=8.1Hz, 2H), 7.84(d, J＝8.3Hz, 2H), 3.25(s, 2H), 1.93(t, J＝7.4Hz, 2H), 1.50(dd, J＝14.7, 7.2Hz, 4H), 1.32– 1.20(m, 4H). ¹³ C NMR (125MHz, DMSO-d ₆ )δ169.19, 164.15, 158.85, 141.71, 141.59, 134.44, 127.61, 126.37, 126.34, 126.31, 39.01, 32.32, 28.9, 9, 2.28.40, HRMS(AP-ESI) m/z calcd for C ₁₈ H ₂₀ F ₃ N ₃ O ₃ S[M+H] ⁺ 416.1250, found 416.1246.

Example 6: Synthesis of compound 9y

Place hydroxylamine hydrochloride (1.04g, 15.00mmol, 15.00equiv) and KOH (0.84g, 15.00mmol, 15.00equiv) in a round-bottomed flask, add 25mL of anhydrous methanol to dissolve in an ice bath, stir at room temperature for 30min, and filter to obtain potassium hydroxylamine ( _NH2OK ) solution. Compound 8y (0.38 g, 1.00 mmol, 1.00 equiv) was dissolved in 25 mL of NH ₂ OK solution, hydroxylamine (0.66 g, 20.00 mmol, 20.00 equiv) was added, and after stirring at room temperature for 1 h, the reaction end was detected by TLC, the solvent was removed by concentration, and 20 mL of The 2 mol/L hydrochloric acid solution was acidified to PH=3-4, extracted with dichloromethane (3×20 mL), dried over anhydrous MgSO ₄ and evaporated to dryness to obtain the crude product. The crude product was separated and purified by silica gel chromatography (the eluent volume ratio was chloroform:methanol=10:1), recrystallized from ethanol, and dried in vacuo to obtain a white solid powder 9y (0.18 g, 50%). ¹ H NMR (500MHz, DMSO-d ₆ )δ10.34(s, 1H), 8.83(s, 1H), 8.68(s, 1H), 8.47(s, 1H), 7.92(d, J=9.3Hz, 1H), 7.42(t, J＝8.6Hz, 1H), 7.21(d, J＝8.0Hz, 1H), 7.07(t, J＝7.6Hz, 1H), 3.95(s, 3H), 3.25(d, J=7.0Hz, 2H), 1.93 (t, J=7.4Hz, 2H), 1.48 (s, 4H), 1.27 (s, 4H). ¹³ C NMR (125MHz, DMSO-d ₆ ) δ 169.20, 162.88, 159.61 ,155.04,141.08,137.94,130.52,128.03,121.21,119.04,112.36,55.97,39.02,32.34,29.09,28.43,26.25,25.21.HRMS(AP-ESI)m/z calcd for C ₁₈ H ₂₃ N ₃ O ₄ S[M+H] ⁺ 378.1482, found 378.1483.

Example 7: Synthesis of compound 10d

Place hydroxylamine hydrochloride (1.04g, 15.00mmol, 15.00equiv) and KOH (0.84g, 15.00mmol, 15.00equiv) in a round-bottomed flask, add 25mL of anhydrous methanol to dissolve in an ice bath, stir at room temperature for 30min, and filter to obtain potassium hydroxylamine ( _NH2OK ) solution. Compound 9d (0.33 g, 1.00 mmol, 1.00 equiv) was dissolved in 25 mL of NH ₂ OK solution, hydroxylamine (0.66 g, 20.00 mmol, 20.00 equiv) was added, and after stirring at room temperature for 1 h, the reaction end was detected by TLC, the solvent was removed by concentration, and 20 mL of The 2 mol/L hydrochloric acid solution was acidified to PH=3-4, extracted with dichloromethane (3×20 mL), dried over anhydrous MgSO ₄ and evaporated to dryness to obtain the crude product. The crude product was separated and purified by silica gel column chromatography (the eluent volume ratio was chloroform:methanol=10:1), recrystallized from ethanol, and vacuum-dried to obtain white solid powder 10d (0.11 g, 35%). ¹ H NMR (500MHz, DMSO-d ₆ )δ10.34(s, 1H), 8.98(s, 1H), 8.67(s, 1H), 7.90(s, 1H), 7.83(d, J=7.2Hz, 2H), 7.52(t, J＝7.6 Hz, 2H), 7.44(t, J＝7.4Hz, 1H), 3.24(q, J＝6.7Hz, 2H), 1.93(t, J＝7.4Hz, 2H) , 1.53–1.47 (m, 4H), 1.30–1.24 (m, 4H); ¹³ C NMR (125MHz, DMSO-d ₆ ) δ169.18, 154.68, 154.45, 152.36, 129.51, 129.32, 126.87, 124.69, 123.70, 38.98, 32.32, 28.93, 28.40, 26.21, 25.18. HRMS(AP-ESI) m/z calcd for C ₁₇ H ₂₁ N ₃ O ₄ [M+H] ⁺ 332.1605, found 332.1604.

Example 8: Synthesis of Compound 10m

Place hydroxylamine hydrochloride (1.04g, 15.00mmol, 15.00equiv) and KOH (0.84g, 15.00mmol, 15.00equiv) in a round-bottomed flask, add 25mL of anhydrous methanol to dissolve in an ice bath, stir at room temperature for 30min, and filter to obtain potassium hydroxylamine ( _NH2OK ) solution. Compound 9m (0.37 g, 1.00 mmol, 1.00 equiv) was dissolved in 25 mL of NH ₂ OK solution, hydroxylamine (0.66 g, 20.00 mmol, 20.00 equiv) was added, and after stirring at room temperature for 1 h, the reaction end was detected by TLC, the solvent was removed by concentration, and 20 mL of The 2 mol/L hydrochloric acid solution was acidified to PH=3-4, extracted with dichloromethane (3×20 mL), dried over anhydrous MgSO ₄ and evaporated to dryness to obtain the crude product. The crude product was separated and purified by silica gel chromatography (the eluent volume ratio was chloroform:methanol=10:1), recrystallized from ethanol, and dried in vacuo to obtain 10m of white solid powder (0.24g, 28%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 10.34(s, 1H), 9.00(s, 1H), 8.67(s, 1H), 7.94(s, 1H), 7.85(d, J=8.6Hz, 2H), 7.60(d, J=8.6Hz, 2H), 3.25(s, 2H), 1.93(s, 2H), 1.51(s, 4H), 1.27(s, 4H); ¹³ C NMR (125MHz, DMSO) -d ₆ ) _{δ169.18,154.60,154.57,151.32,133.96,129.43,126.46,125.77,124.34,39.00,32.32,28.92,28.40,26.21,25.18.HRMS} (AP-ESI)m/z calcd for C ₁₇ H ₃ O ₄ [M+H] ⁺ 366.1215, found 366.1212.

Example 9: Synthesis of Compound 10n

Place hydroxylamine hydrochloride (1.04g, 15.00mmol, 15.00equiv) and KOH (0.84g, 15.00mmol, 15.00equiv) in a round-bottomed flask, add 25mL of anhydrous methanol to dissolve in an ice bath, stir at room temperature for 30min, and filter to obtain potassium hydroxylamine ( _NH2OK ) solution. Compound 9n (0.34 g, 1.00 mmol, 1.00 equiv) was dissolved in 25 mL of NH ₂ OK solution, and hydroxylamine (0.66 g, 20.00 mmol, 20.00 equiv) was added, and after stirring at room temperature for 1 h, the reaction end was detected by TLC, the solvent was removed by concentration, and 20 mL of The 2 mol/L hydrochloric acid solution was acidified to PH=3-4, extracted with dichloromethane (3×20 mL), dried over anhydrous MgSO ₄ and evaporated to dryness to obtain the crude product. The crude product was separated and purified by silica gel chromatography (the eluent volume ratio was chloroform:methanol=10:1), recrystallized from ethanol, and dried in vacuo to obtain a white solid powder 10n (0.10 g, 30%). ¹ H NMR (500MHz, DMSO-d ₆ )δ 10.34(s, 1H), 8.95(s, 1H), 8.67(s, 1H), 7.82(s, 1H), 7.71(d, J=8.1Hz, 2H), 7.33(d, J=8.0Hz, 2H), 3.23(s, 2H), 2.35(s, 3H), 1.93(s, 2H), 1.51(s, 4H), 1.26(s, 4H); ¹³ C NMR (125MHz, DMSO-d ₆ )δ169.21,154.73,154.16,152.58,139.26,129.87,124.68,124.17,123.02,38.98,32.33,28.95,28.41,26.23,25.20,21.07.HRMS(AP) /z calcd for C ₁₈ H ₂₃ N ₃ O ₄ [M+H] ⁺ 346.1761, found 346.1768.

Example 10: Synthesis of Compound 10p

Place hydroxylamine hydrochloride (1.04g, 15.00mmol, 15.00equiv) and KOH (0.84g, 15.00mmol, 15.00equiv) in a round-bottomed flask, add 25mL of anhydrous methanol to dissolve in an ice bath, stir at room temperature for 30min, and filter to obtain potassium hydroxylamine ( _NH2OK ) solution. Compound 9p (0.39 g, 1.00 mmol, 1.00 equiv) was dissolved in 25 mL of NH ₂ OK solution, hydroxylamine (0.66 g, 20.00 mmol, 20.00 equiv) was added, and after stirring at room temperature for 1 h, the reaction end was detected by TLC, the solvent was removed by concentration, and 20 mL of The 2 mol/L hydrochloric acid solution was acidified to PH=3-4, extracted with dichloromethane (3×20 mL), dried over anhydrous MgSO ₄ and evaporated to dryness to obtain the crude product. The crude product was separated and purified by silica gel chromatography (the eluent volume ratio was chloroform:methanol=10:1), recrystallized from ethanol, and dried in vacuo to obtain a white solid powder 10p (0.078g, 20%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ 10.34(s, 1H), 9.06(s, 1H), 8.67(s, 1H), 8.10(s, 1H), 8.05(d, J=8.2Hz, 2H), 7.89(d, J＝8.3Hz, 2H), 3.25(q, J＝6.7Hz, 2H), 1.93(t, J＝7.4Hz, 2H), 1.57–1.43(m, 4H), 1.27( s, 4H). ¹³ C NMR (125MHz, DMSO-d ₆ )δ169.18,155.13,154.51,150.88,130.64,126.32,126.29,125.80,125.31,39.04,32.32,28.91,28.40,26.21,25.18.HRMS(AP) ESI) m/z calcd for C ₁₈ H ₂₀ F ₃ N ₃ O ₄ [M+H] ⁺ 400.1479, found 400.1474.

Biological activity evaluation:

Experiment 1: Inhibitory activity test of oxazole and thiazole histone deacetylase inhibitors on HDAC1, 3 and 6.

(1) Experimental materials

HDAC1 kit (KA1320) was purchased from Abcam company, HDAC3 kit (K363-100) was purchased from BioVision company, HDAC6 (ab42632) was purchased from Abcam company; trypsin (T8003-1g), bovine serum albumin (BSA), Tri -HCl, NaCl, KCl and _MgCl2 were purchased from Sigma-Aldrich Company, microplate reader (Biotek NEO2) was purchased from Biotek Company of the United States, and black 96-well plate was purchased from Corning Company of the United States.

(2) Material handling

HDACs Buffer: 25 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl ₂ , and 0.1 mg/mL BSA, pH=8.

HDACs solution: Dilute different kinds of HDACs enzymes to appropriate concentrations with HDACs buffer.

Developer: 6 mg/mL Trypsin, 33 μM TSA for HDAC1, HDAC3 and HDAC6.

HDACs substrate (Substrate): The HDACs fluorescent substrate was prepared into a 2mM stock solution with DMSO, and then diluted with HDACs buffer to obtain a solution of appropriate concentration.

Test compound (Inhibitor): The test compounds were sequentially diluted with HDACs buffer (10×, containing 10% DMSO) to prepare 5-7 compound solutions of different test concentrations.

(3) Experimental method

Compounds of different test concentrations were incubated with different kinds of HDACs enzymes in a microplate constant temperature shaker at 37°C. After 10 minutes of action, the incubation was stopped, the substrate solution was quickly added, and then incubated for 30-90 minutes. Finally, a high concentration of developer was added to stop the reaction between the HDACs enzyme and the compound. After 10-15min incubation at 37°C, the fluorescence intensity values were detected at specific excitation and emission wavelengths. The inhibition rates of different concentrations of compounds were calculated, and after nonlinear curve fitting analysis was performed on the inhibition rates of different concentrations, the IC ₅₀ value of the half-inhibitory concentration was obtained. The lower the IC ₅₀ value, the stronger the ability of the test compound to inhibit the activity of HDACs. The specific detection method is as follows:

HDAC1 kit detection:

a). Blank group (B _w ): 150 μL Buffer, 10 μL DMSO and 10 μL HDAC1 substrate.

b). Negative group (F ₀ ): 140 μL Buffer, 10 μL HDAC1 enzyme, 10 μL DMSO and 10 μL HDAC1 substrate.

c). Sample group (F): 140 μL Buffer, 10 μL HDAC1 enzyme, 10 μL compounds of different concentrations and 10 μL HDAC1 substrate.

All substances were co-incubated at 37°C for 0.5h, then 40μL of Developer was added to each well, and the incubation was continued for 15min. The inhibition of HDAC1 by the compounds was determined at excitation wavelength (Ex )/emission wavelength (E _m ) ₌ 350/450 nm detection wavelength Effect.

HDAC3 kit detection:

a). Blank group (B _w ): 75 μL Buffer.

b). Negative group (F ₀ ): 48 μL Buffer, 2 μL HDAC3 enzyme, 25 μL buffer.

c). Sample group (F): 48 μL Buffer, 2 μL HDAC3 enzyme, 25 μL compounds of different concentrations.

Incubate the diluted HDAC3 and compounds at 37°C for 0.5h. After adding 25μL of HDAC3 substrate to each well, continue to incubate for 60min. After adding 10μL of Developer to all wells, incubate for another 5min. At E _x /E _m =380/ Under the detection wavelength of 500nm, the inhibitory effect of the compounds on HDAC3 was determined.

HDAC6 model test:

a). Blank group (B _w ): 40 μL Buffer.

b). Negative group (F ₀ ): 29 μL Buffer, 1 μL HDAC6 enzyme (50 μg/mL), 10 μL buffer.

c). Sample group (F): 29 μL Buffer, 1 μL HDAC6 enzyme (50 μg/mL), 10 μL compound solution.

Incubate the diluted _HDAC6 and compounds at 37°C for 10 min, add 25 μL of _HDAC6 substrate (50 μM) to each well, and continue to incubate for 60 min. After adding 50 μL of Developer to each well, incubate for another 30 min. At the detection wavelength of /450nm, the inhibitory effect of the compound on HDAC6 was determined.

As shown in Table 1 and Table 2, in the HDAC1 activity test, the IC ₅₀ value of SAHA was 214.80 nM, and the IC ₅₀ values of 10 compounds such as compounds 9g, 9o, 9s, 9u, and 10p ranged from 24.71 to 102.70 nM, wherein , the IC ₅₀ values of compounds 9s and 10d are 8.6 times and 5.9 times that of SAHA; in the HDAC3 activity test, the IC ₅₀ value of SAHA is 1559 nM, and the IC ₅₀ values of 5 compounds such as compounds 9g, 9o, 9s, 9u, and 9y are The range is 140.70-2061 nM, wherein the IC ₅₀ value of compound 9y is 11.1 times that of SAHA; in the HDAC6 activity test, the IC 50 value of SAHA is 21.08 nM, and the IC ₅₀ value of compound 9g, 9o, 9s, 9u, and 10p and other 10 compounds. The _IC50 values ranged from 2.56 to 14.77 nM, wherein the _IC50 values of compounds 10d and 9y were 8.2-fold and 5.8-fold higher than SAHA.

Table 1 Inhibitory activity of oxazole histone deacetylase inhibitors on HDAC1 and 6

Table 2 Inhibitory activity of thiazole histone deacetylase inhibitors on HDAC1, 3 and 6

The above results show that the test compounds have strong inhibitory effects on the three HDACs.

Experiment 2: Cytotoxicity test of oxazole and thiazole histone deacetylase inhibitors on human lung cancer cell A549.

(1) Experimental materials

Target compound and positive control vorinostat (SAHA), trypsin, washing solution PBS, fetal bovine serum, human lung cancer cell A549, double antibody (penicillin, streptomycin), CCK-8 kit (Biyuntian), enzyme A standard marker (Biotek NEO ₂ ) and a transparent 96-well plate.

(2) Experimental method

Cancer cells were cultured in DMEM medium in a CO ₂ constant temperature incubator (5%, 37°C). First, the A549 growth phase cells were digested, and the number of A549 cells was counted with a microscope, and then the A549 cells were seeded into 96-well plates and waited for adherence. In the sample group, the compounds to be tested were dissolved with DMSO and diluted to 5-7 gradient concentrations, each with 3 replicate wells; the blank group was replaced with DMSO. After the cells adhered, incubate the diluted compound and cells together in a CO ₂ constant temperature incubator. After the compound and cells were fully interacted, add 10 μL of CCK-8 solution to all wells, and continue to incubate in a CO ₂ constant temperature incubator. Incubate for 2 h in 2 h, use a microplate reader (Biotek NEO2) to detect the OD value of different gradient concentrations on human lung cancer cells A549, and calculate the inhibition rate of the compound. The calculation method of IC ₅₀ value is consistent with the HDACs test. The lower the IC ₅₀ value, the test compound The stronger the anti-proliferative activity of human lung cancer cell A549.

As shown in Table 3, in the cytotoxic activity test of oxazole and thiazole-type histone deacetylase inhibitors on human lung cancer cell A549, the _IC50 value of the positive control SAHA was 2.47 μM, the compounds 9g, 9o, 9s, 9u The IC ₅₀ values of 10 compounds such as , and 10p ranged from 0.42 to 1.73 μM, and their inhibitory activity on human lung cancer cell A549 was better than SAHA. Among them, the _IC50 values of compounds 10d and 9y were 5.8 times and 4.8 times higher than SAHA. The test results show that the test compounds have strong inhibitory activity on human lung cancer cell A549.

Table 3 Inhibitory activity of oxazole and thiazole histone deacetylase inhibitors on human lung cancer cell A549

Compound number _IC50 (μM±SD) ^a SAHA 2.47±0.32 9g 0.61±0.07 9o 1.47±0.20 9s 1.46±0.31 9u 1.72±0.24 9w 1.73±0.25 9y 0.52±0.09 10d 0.42±0.19 10m 0.51±0.15 10n 0.66±0.08 10p 0.64±0.20

^a Data are expressed as mean±SD(standard deviations) from at least three independent experiments.

The above examples prove that the oxazole and thiazole histone deacetylase inhibitors provided according to the present invention have strong inhibitory effects on HDACs and cancer cells. Compared with the positive control SAHA, the activity of the oxazole and thiazole type histone deacetylase inhibitors provided by the present invention is significantly improved, and has the potential to be developed as a new generation of HDACs inhibitors.

The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the points that are different from other embodiments, and the same and similar parts between the various embodiments can be referred to each other.

The above description of the disclosed embodiments enables those skilled in the art to practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (3)

An oxazole and thiazole class histone deacetylase inhibitor, characterized in that the chemical structure of the compound is shown in formula (I):

In the formula, R=H, F, Cl, CH ₃ , CF ₃ or OMe; X=O or S. The preparation method of oxazole and thiazole class histone deacetylase inhibitor according to claim 1, is characterized in that, comprises the following steps: Place 15mmol of hydroxylamine hydrochloride and 15mmol of KOH in a round-bottomed flask, add 25mL of anhydrous methanol to dissolve, stir in an ice bath for 30min and filter to obtain potassium hydroxylamine (NH ₂ OK) solution, which is sealed for later use; 25mL of NH ₂ OK solution is dissolved in the middle of 1mmol. Product 1A was added with 20 mmol of hydroxylamine, stirred at room temperature for 1 h, TLC detected the reaction end point, removed the solvent under reduced pressure, added an appropriate amount of 2 mol/L hydrochloric acid solution to acidify to pH=3-4, extracted with dichloromethane three times, anhydrous MgSO ₄ Dry and evaporate to dryness to obtain the crude product; the crude product is separated and purified by silica gel column chromatography, and the eluent volume ratio is chloroform:methanol=10:1, recrystallized from ethanol, and vacuum-dried to obtain the target product 2A; The reaction formula is as follows:

In the formula, R=H, F, Cl, CH ₃ , CF ₃ or OMe; X=O or S. The application of the oxazole and thiazole type histone deacetylase inhibitor according to claim 1 in the aspect of histone deacetylase inhibitor and anticancer drug.

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