Oxazole histone deacetylase inhibitor and preparation method thereof
阅读说明：本技术 一种噁唑类组蛋白去乙酰化酶抑制剂及其制备方法 (Oxazole histone deacetylase inhibitor and preparation method thereof ) 是由 赵登高 张瑞强 马燕燕 张焜 莫华龙 李冬利 徐学涛 盛钊君 于 2020-12-07 设计创作，主要内容包括：本发明提供一种噁唑类组蛋白去乙酰化酶抑制剂及其制备方法,特征包括用NH_2OK溶液溶解噁唑化合物1A,加入羟胺,室温搅拌后,TLC检测反应终点,减压除去溶剂,加入盐酸溶液酸化,二氯甲烷萃取,蒸干得粗产物；本发明的所提供的噁唑类化合物对组蛋白去乙酰化酶具有很强的抑制作用,对癌细胞具有很强的抑制作用,本发明对比已上市的组蛋白去乙酰化酶抑制剂,本发明提供的噁唑类化合物对组蛋白去乙酰化酶的活性显著提高,对肺癌等实体瘤的效果更好,可克服已有组蛋白去乙酰化酶抑制剂活性较低,对实体瘤疗效较差等问题。(The invention provides an oxazole histone deacetylase inhibitor and a preparation method thereof, which is characterized in that NH is used 2 Dissolving an oxazole compound 1A in OK solution, adding hydroxylamine, stirring at room temperature, detecting the reaction end point by TLC, decompressing to remove the solvent, adding hydrochloric acid solution for acidification, extracting by dichloromethane, and evaporating to dryness to obtain a crude product; compared with the existing histone deacetylase inhibitors, the oxazole compound provided by the invention has the advantages that the activity of the histone deacetylase is obviously improved, the effect on solid tumors such as lung cancer is better, and the problems of low activity, poor curative effect on the solid tumors and the like of the existing histone deacetylase inhibitor can be solved.)
1. An oxazole histone deacetylase inhibitor, which has a chemical structure represented by formula (I):
wherein R is H, Cl, CH3,CF3Or OMe; n is 6.
2. An oxazole histone deacetylase inhibitor according to claim 1, wherein: the chemical structure of the inhibitor is shown as follows:
3. an oxazole histone deacetylase inhibitor according to claim 1, wherein: the chemical structure of the inhibitor is shown as follows:
4. an oxazole histone deacetylase inhibitor according to claim 1, wherein: the chemical structure of the inhibitor is shown as follows:
5. an oxazole histone deacetylase inhibitor according to claim 1, wherein: the chemical structure of the inhibitor is shown as follows:
6. an oxazole histone deacetylase inhibitor according to claim 1, wherein: the chemical structure of the inhibitor is shown as follows:
7. a process for the preparation of an inhibitor of the deacetylases of oxazolines histone deacetylases, for use in the preparation of the inhibitor of any one of claims 1 to 6, characterized in that: with 25mL of hydroxylamine potassium solution NH2Dissolving 1mmol of oxazole compound 1A in OK solution, adding 20mmol of hydroxylamine, stirring at room temperature for 30min-1h, detecting the reaction end point by TLC, and removing the solvent under reduced pressure; then addAdding a proper amount of 2mol/L hydrochloric acid solution to acidify until the PH value is 3-4, extracting with dichloromethane for 1-3 times, and evaporating to dryness to obtain a crude product;
the reaction formula is as follows:
wherein R is H, Cl, CH3,CF3Or OMe; n is 6.
8. The application of an oxazole histone deacetylase inhibitor is characterized in that: the application in histone deacetylase inhibitors, anti-cancer drugs and cell cycle drugs.
Technical Field
The invention relates to the technical field of new medicine compounds, in particular to an oxazole histone deacetylase inhibitor and a preparation method thereof.
Background
Histone Deacetylases (HDACs) are a class of proteases that are widely found in eukaryotic cells and remove acetyl groups from histone lysines, thereby inhibiting gene transcription.
In addition, HDACs can remove acetyl groups from some non-histone proteins, such as transcription factors, structural proteins and inflammatory mediators, affecting their function. Inhibiting the enzyme activity of HDACs not only affects the cellular processes of gene expression, apoptosis, growth arrest, differentiation, etc., but also inhibits angiogenesis. Because HDACs play an extremely important role in gene expression and other cellular activities, HDAC inhibitors have tremendous research and development value in cancer therapy.
Vorinostat is the first HDAC inhibitor approved by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T-cell lymphoma (CTCL), ruminants are FDA-approved for the treatment of CTCL and peripheral T-cell lymphoma (PTCL) in 2009 and 2011, respectively, and belinostat is FDA-approved for the treatment of PTCL in 2014. However, the efficacy of HDAC inhibitors for the treatment of B cell lymphoma is not yet clear.
In addition, the existing marketed HDAC inhibitors generally have the problems of low activity, poor curative effect on solid tumors and the like. Therefore, the development of a novel histone deacetylase inhibitor has wide application prospect.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an oxazole histone deacetylase inhibitor and a preparation method thereof.
The technical scheme of the invention is as follows: an oxazole histone deacetylase inhibitor, which has a chemical structure shown in formula (I):
wherein R is H, Cl, CH3,CF3Or OMe; n is 6.
Further, the chemical structure of the inhibitor is as follows:
further, the chemical structure of the inhibitor is as follows:
further, the chemical structure of the inhibitor is as follows:
further, the chemical structure of the inhibitor is as follows:
further, the chemical structure of the inhibitor is as follows:
the invention also provides a preparation method of the oxazole histone deacetylase inhibitor, which comprises the following steps:
s1), placing 15mmol of hydroxylamine hydrochloride and 15mmol of KOH in a round-bottom flask, adding 25mL of anhydrous methanol for dissolving, stirring in ice bath for 20-40min, and filtering to obtain a hydroxylamine potassium solution NH2OK；
S2), using 25mL of NH2Dissolving 1mmol of oxazole compound 1A in OK solution, adding 20mmol of hydroxylamine, stirring at room temperature for 30min-1h, detecting the reaction end point by TLC, and decompressing and removingRemoving the solvent;
s3), then adding an appropriate amount of 2mol/L hydrochloric acid solution to acidify to PH 3-4, extracting with dichloromethane for 1-3 times, and then using anhydrous MgSO4Drying and evaporating to obtain a crude product;
the reaction formula is as follows:
wherein R is H, Cl, CH3,CF3Or OMe; n is 6.
The invention has the beneficial effects that:
1. the oxazole compound provided by the invention has strong inhibition effect on histone deacetylase and cancer cells, and can form a new generation of anticancer lead compound;
2. compared with the histone deacetylase inhibitor on the market, the oxazole compound provided by the invention has obviously improved activity on the histone deacetylase, has better effect on solid tumors such as lung cancer and the like, and can overcome the problems of lower activity, poorer curative effect on the solid tumors and the like of the existing histone deacetylase inhibitor;
3. the oxazole compound of the invention can be prepared into tablets, powder injection, emulsifying agents, capsules and the like under the preparation combination which can be accepted by pharmaceutical specifications, and the oxazole compound of the invention can be applied to histone deacetylase inhibitors, anti-cancer drugs and the like.
Detailed Description
The following further illustrates embodiments of the invention:
example 1
The embodiment provides a preparation method of an oxazole histone deacetylase inhibitor, which comprises the following steps:
s1), respectively placing 15mmol of hydroxylamine hydrochloride and 15mmol of KOH in a round-bottom flask, adding 25mL of anhydrous methanol for dissolving, stirring in ice bath for 30min, and filtering to obtain hydroxylamine potassium (NH)2OK) solution, sealing for standby;
s2), mixing1mmol of methyl 7- (5-phenyloxazole-2-amide) heptanoate was used with a prepared 25mL NH2Dissolving OK solution, adding 20mmol of hydroxylamine, stirring at room temperature for 1h, detecting the reaction end point by TCL, and removing the solvent under reduced pressure;
s3), acidifying to PH 3-4 by adding appropriate 2mol/L hydrochloric acid solution, extracting with dichloromethane (3 × 20mL), anhydrous MgSO4Drying and evaporating to dryness to obtain a crude product which is recorded as 10 d.
This example prepares the reaction as follows:
the crude product was purified by column chromatography on silica gel (eluent chloroform: methanol 10:1), recrystallized from ethanol and dried in vacuo to give 0.35mmol of the desired product 10d, with a yield of about 35%.
Target product 10 d: white powder solid.1H NMR(500MHz,DMSO-d6)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＝7.6Hz,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)；13C NMR(126MHz,DMSO-d6)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。
Example 2
The embodiment provides a preparation method of an oxazole histone deacetylase inhibitor, which comprises the following steps:
s1) respectively putting 15mmol of hydroxylamine hydrochloride and 15mmol of KOH into a round-bottom flask, adding 25mL of anhydrous methanol for dissolving, stirring in ice bath for 30min, and filtering to obtain hydroxylamine potassium (NH)2OK) solution, sealed for use.
S2), 1mmol of methyl 7- (5- (4-methoxyphenyl oxazole) -2-amide) heptanoate was used with a prepared 25mL of NH2Dissolving OK solution, adding 20mmol of hydroxylamine, stirring at room temperature for 1h, detecting the reaction end point by TCL, and removing the solvent under reduced pressure;
s3), adding proper 2mol/L hydrochloric acid solution to acidify until the pH value is 3-4, and dichloroMethane (3X 20mL) extraction, anhydrous MgSO4Dried and evaporated to dryness to give 9g of crude product.
This example prepares the reaction as follows:
the crude product prepared in this example was purified by column chromatography on silica gel (eluent chloroform: methanol 10:1), recrystallized from ethanol and dried in vacuo to give 9g of 0.42mmol of the desired product in about 42% yield.
9g of target product: white powder solid.1H NMR(500MHz,DMSO-d6)δ10.34(s,1H),8.92(s,1H),8.67(s,1H),7.79–7.71(m,3H),7.08(d,J＝8.9Hz,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)；13C NMR(126MHz,DMSO-d6)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。
Example 3
The embodiment provides a preparation method of an oxazole histone deacetylase inhibitor, which comprises the following steps:
s1), respectively placing 15mmol of hydroxylamine hydrochloride and 15mmol of KOH in a round-bottom flask, adding 25mL of anhydrous methanol for dissolving, stirring in ice bath for 30min, and filtering to obtain hydroxylamine potassium (NH)2OK) solution, sealed for use.
S2), 1mmol of methyl 7- (5- (4-chlorophenyloxazole) -2-amide) heptanoate was used with the prepared 25mL NH2Dissolving OK solution, adding 20mmol of hydroxylamine, stirring at room temperature for 1h, detecting the reaction end point by TCL, and removing the solvent under reduced pressure;
s3), acidifying to PH 3-4 by adding appropriate 2mol/L hydrochloric acid solution, extracting with dichloromethane (3 × 20mL), anhydrous MgSO4Drying and evaporating to dryness to obtain a crude product which is recorded as 10 m.
This example prepares the reaction as follows:
the crude product prepared in this example was purified by column chromatography on silica gel (eluent chloroform: methanol 10:1), recrystallized from ethanol and dried in vacuo to give 0.28mmol of the desired product 10m, with a yield of about 28%.
Target product 10 m: white powder solid.1H NMR(500MHz,DMSO-d6)δ10.34(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)；13C NMR(126MHz,DMSO-d6)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。
Example 4
The embodiment provides a preparation method of an oxazole histone deacetylase inhibitor, which comprises the following steps:
s1), respectively placing 15mmol of hydroxylamine hydrochloride and 15mmol of KOH in a round-bottom flask, adding 25mL of anhydrous methanol for dissolving, stirring in ice bath for 30min, and filtering to obtain hydroxylamine potassium (NH)2OK) solution, sealed for use.
S2), 1mmol of methyl 7- (5- (4-methyloxazole) -2-amide) heptanoate was used with the prepared 25mL NH2Dissolving OK solution, adding 20mmol of hydroxylamine, stirring at room temperature for 1h, detecting the reaction end point by TCL, and removing the solvent under reduced pressure;
s3), then acidified to PH 3-4 by adding an appropriate amount of 2mol/L hydrochloric acid solution, extracted with dichloromethane (3 × 20mL), anhydrous MgSO4Drying and evaporating to dryness to obtain a crude product which is recorded as 10 n.
This example prepares the reaction as follows:
the crude product prepared in this example was purified by column chromatography on silica gel (eluent chloroform: methanol 10:1), recrystallized from ethanol and dried in vacuo to give 0.30mmol of the desired product 10n, in about 30% yield.
Target product 10 n: white powder solid.1H NMR(500MHz,DMSO-d6)δ10.34(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)；13C NMR(126MHz,DMSO-d6)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。
Example 5
The embodiment provides a preparation method of an oxazole histone deacetylase inhibitor, which comprises the following steps:
s1), respectively placing 15mmol of hydroxylamine hydrochloride and 15mmol of KOH in a round-bottom flask, adding 25mL of anhydrous methanol for dissolving, stirring in ice bath for 30min, and filtering to obtain hydroxylamine potassium (NH)2OK) solution, sealed for use.
S2), 1mmol of methyl 7- (5- (4-trifluoromethyloxazole) -2-amide) heptanoate was used with a prepared 25mL of NH2Dissolving OK solution, adding 20mmol of hydroxylamine, stirring at room temperature for 1h, detecting the reaction end point by TCL, and removing the solvent under reduced pressure;
s3), acidifying to PH 3-4 by adding appropriate 2mol/L hydrochloric acid solution, extracting with dichloromethane (3 × 20mL), anhydrous MgSO4Drying and evaporating to dryness to obtain a crude product which is recorded as 10 p.
This example prepares the reaction as follows:
the crude product prepared in this example was purified by column chromatography on silica gel (eluent chloroform: methanol 10:1), recrystallized from ethanol and dried in vacuo to give 0.30mmol of the desired product 10p in about 20% yield.
Target product 10 p: white powder solid.1H NMR(500MHz,DMSO-d6)δ10.34(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).13C NMR(126MHz,DMSO-d6)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。
Example 6
Study of Activity of oxazoles on human Histone deacetylase 1(HDAC1)
(1) Experimental Material
Target compound and positive control vorinostat (SAHA), human histone deacetylase 1(HDAC1) kit (Abnova, Catalog Number KA1320), microplate reader (Biotek NEO)2) And black 96-well plates.
(2) Experimental methods
The procedure was as provided by HDAC1 kit manufacturing. The black 96-well plate contains 3 systems of blank, negative and sample sets, 3 parallel sets per system. Wherein blank group (Bw): 150 μ L buffer, 10 μ L DMSO, and 10 μ L HDAC1 substrate; negative group (F)0): 140 μ L buffer, 10 μ L HDAC1 enzyme, 10 μ L DMSO, and 10 μ L HDAC1 substrate; sample set (F): 140 μ L buffer, 10 μ L HDAC1 enzyme, 10 μ L of different concentrations of compound, and 10 μ L HDAC1 substrate. Then incubating in a microplate constant temperature oscillator at 37 deg.C for 30min, adding 40 μ L of developer to each well, incubating for 15min, and using Biotek NEO2Measuring fluorescence intensity values of an enzyme-labeling instrument under the conditions that the excitation wavelength is 350nm and the emission wavelength is 450nm, calculating the inhibition rate of compounds with different concentrations on HDAC1 enzyme, and performing nonlinear curve fitting analysis on the inhibition rate of each concentration to obtain half inhibition concentration IC50The value is obtained. Median inhibitory concentration IC50Lower values represent a stronger inhibition of HDAC1 enzyme by the compound.
As shown in table 1, the half inhibitory concentration of the positive control SAHA to human histone deacetylase 1(HDAC1) was 214.8nM, and the half inhibitory concentrations of the compounds 9g, 10d, 10m, 10n and 10p were 40.07nM, 36.42nM, 31.38nM, 44.41nM and 42.82nM, respectively, which were superior to the positive control, showing that the target compound had a strong inhibitory activity to HDAC 1.
TABLE 1 comparison of experimental results for inhibition of histone deacetylase 1(HDAC1) by oxazoles
Example 7
Study on effect of oxazole histone deacetylase inhibitor on cancer cell proliferation
(1) Experimental Material
Target compound and positive control vorinostat (SAHA), pancreatin, cleaning solution PBS, fetal bovine serum, human lung cancer cell A549, double antibody (penicillin and streptomycin), CCK-8 kit (Biyunyan), enzyme labeling instrument (Biotek NEO)2) And 96-well plates.
(2) Experimental methods
Human lung cancer cell a549 was from shanghai institute of biochemistry and cell biology. The cells were cultured in the presence of 5% CO2In the 37 ℃ incubator, DMEM medium containing 10% serum and 1% diabase (penicillin, streptomycin) was used.
When tested by the SRB method, cells in the logarithmic growth phase are first digested and counted. Cells were seeded into 96-well plates overnight. After the cells are attached to the wall, compounds with different concentrations are added, wherein 7 concentrations are set for each compound, and 3-5 concentrations are set for each compound in parallel. The cells and the drug are cultured for 72h and then taken out, 50 mu L of 50% (m/v) trichloroacetic acid is added into each hole to fix the cells, and the cell plate needs to be placed at room temperature for 5min and then placed at 40 ℃ for 1 h. The stationary liquid was discarded, washed with distilled water 5 times, and naturally dried in the air. Then, 100 pieces of 0.4% SRB solution were added to each well, left at room temperature for 30min, washed with 1% acetic acid solution more than 10 times, and air-dried. Finally, 150. mu.L of Tris solution was added using Biotek NEO2The absorbance was measured by a microplate reader, and the absorption wavelength was set at 570 nm. Carrying out nonlinear curve fitting analysis on the inhibition rate of each concentration to obtain half inhibition concentration IC50The value is obtained.
As shown in table 2, the half inhibitory concentration of SAHA of the positive control to a549 in human lung cancer cells was 2.043 μm, and the half inhibitory concentrations of compounds 9g, 10d, 10m, 10n and 10p were 0.6087 μm, 0.4195 μm, 0.5139 μm, 0.6601 μm and 0.6410 μm, respectively, which were superior to the positive control, showing that the target compound had a strong inhibitory activity against cancer cells.
TABLE 2 comparison of experimental results of half inhibitory concentrations of oxazoles on A549 lung cancer cells
It is demonstrated by the above examples that the oxazole compounds provided according to the present invention have strong inhibitory effects on histone deacetylase and cancer cells. Compared with the histone deacetylase inhibitor on the market, the oxazole compound provided by the invention has obviously improved activity and can be developed into a new generation of anticancer drugs.
The foregoing embodiments and description have been presented only to illustrate the principles and preferred embodiments of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention as hereinafter claimed.
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