CN114315670B - SIRT2/HDAC6 double-target inhibitor and application thereof - Google Patents

SIRT2/HDAC6 double-target inhibitor and application thereof Download PDF

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CN114315670B
CN114315670B CN202210057576.6A CN202210057576A CN114315670B CN 114315670 B CN114315670 B CN 114315670B CN 202210057576 A CN202210057576 A CN 202210057576A CN 114315670 B CN114315670 B CN 114315670B
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CN114315670A (en
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何彬
王攀
赵永龙
陈蕾
李燕
李勇军
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Guizhou Medical University
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Abstract

The invention discloses a SIRT2/HDAC6 double-target inhibitor and application thereof. The SIRT2/HDAC6 double-target inhibitor can effectively and selectively inhibit the activity of SIRT2 and HDAC6 in vitro and in vivo, has cheap synthetic raw materials, low cost and obvious anti-tumor activity, and can be used for novel SIRT2/HDAC6 double-target inhibitor anti-tumor drugs with high efficiency and low toxicity.

Description

SIRT2/HDAC6 double-target inhibitor and application thereof
Technical Field
The invention relates to the technical field of chemistry and pharmacy, in particular to a SIRT2/HDAC6 double-target inhibitor and application thereof.
Background
Microtubules (microtubules) are the main component of cytoskeleton, and meanwhile, the dynamic assembly of microtubules has unique effects in the processes of cell mitosis and cell proliferation, and is the action target point of current first-line antitumor drugs such as taxol, vinblastine, colchicine and the like. Microtubules (microtubules) are also the first identified as non-histone acetylation substrates, assembled from dimers formed by tubulin alpha-tubulin and beta-tubulin, where the 40 th lysine (K40) of alpha-tubulin was confirmed as its acetylation site. Research shows that the alpha-tubulin K40 acetylation level can be used as a marker for stabilizing microtubules, regulating microtubule structure and controlling stress and immune response; in addition, the level of K40 acetylation has important potential roles in various other cellular processes, including intracellular trafficking, cilia assembly, cell signaling, cell migration, and the like.
The level of alpha-tubulin K40 acetylation was found to be regulated by the tubulin acetyltransferase (alpha-tubulin acetyltransferase, ATA 1) and histone deacetylase family members HDAC and Sirtuin. Among them, HDAC6 was the first histone deacetylase demonstrated to be able to remove alpha-tubulin K40 acetylation, and SIRT2 was subsequently also found to be able to remove alpha-tubulin K40 acetylation. Although HDAC6 is the predominant α -tubulin K40 deacetylase, SIRT2 compensatory α -tubulin K40 deacetylase under certain conditions, e.g., predominantly SIRT2 down-regulates α -tubulin K40 acetyl levels during the mitotic spindle phase of cells; during the macrophage inflammatory body activation phase, predominantly SIRT2 exhibits alpha-tubulin K40 deacetylase activity. More importantly, the tubulin a-tubulin K40 acetylation levels are mostly shown to be significantly reduced in a variety of pathologies, such as: neurological diseases such as Alzheimer's disease and Parkinson's disease; tumor diseases such as multiple myeloma and cylindrical tumor; heart diseases such as atrial fibrillation; chronic Obstructive Pulmonary Disease (COPD); inflammation and immune diseases such as colitis and transplant rejection, etc. From this, it can be seen that the decrease in the K40 acetylation level of α -tubulin is closely related to diseases such as tumor, neurodegenerative diseases, heart diseases, inflammation, viral infection, etc.
RAS is a protein which plays a critical role in classical tumor treatment pathway, EGFR regulates the phosphorylation of RAF, PI3K and other proteins at the downstream of the RAS by activating RAS protein, and the expression of various tumor genes is regulated by cascade amplification. K-RAS is a mutant of RAS protein, the mutant occupies a high proportion in various cancers such as pancreatic cancer, non-small cell lung cancer and the like, and the mutant has strong tolerance to EGFR-targeting drugs, so the K-RAS is also an important target for tumor treatment nowadays. In 2013 Yang et al found that SIRT2 could promote tumor proliferation and migration by down-regulating the level of acetylation of lysine 104 in K-RAS and up-regulating the level of phosphorylation of its downstream substrate, and when studying its mechanism, researchers found that this pathway was not inhibited when SIRT2 inhibitors were administered, and subsequently found that when SIRT2 activity was decreased, cells could increase HDAC6 activity by compensation to reduce K-RAS K104 acetylation. Therefore, HDAC6 and SIRT2, which are capable of down-regulating the levels of acetylation of α -tubulin K40 and K-RAS K104, have become hot spot targets for drug development of related diseases, and HDAC6 inhibitors and SIRT2 inhibitors are also the leading edge and hot spots for research in drug development of related diseases. Therefore, the research and development of the HDAC6 and SIRT2 double-target inhibitor have very important theoretical and practical significance. However, only studies on dual inhibitors of HDAC and other targets have been reported, such as dual inhibitors of HDAC and FGFR1, HDAC and PI3K, HDAC and PDE5, HDAC and Ras, HDAC and JAK2, HDAC and topoismerase II, HDAC and LSD1, HDAC and NAMPT. Two target inhibitors of HDAC and Sirtuin, particularly HDAC6 and SIRT2, with high selectivity are reported.
Disclosure of Invention
The purpose of the invention is that: the SIRT2/HDAC6 double-target inhibitor can effectively and selectively inhibit the activity of SIRT2 and HDAC6 in vitro and in vivo, has cheap synthetic raw materials and low cost, and has important application value for the drug screening and pharmaceutical industry.
The invention is realized in the following way: a SIRT2/HDAC6 dual-target inhibitor, which takes lysine as a framework and has a structure shown in one of the following general formulas:
wherein said R is 1 Is methylene or imino; r is R 2 Is tetradecyl, phenyl or adamantyl, polyethoxy; r is R 3 Is alkyl, phenyl, benzyl, cyclohexenyl, polyethoxy, benzothiazolyl, thiazolyl, or piperazinyl; r is R 4 Is hydroxamic acid, mercapto, phthalic diamido, para-fluorophthalic diamido or ethyl acetohydrazino; r is R 5 Is quinolinyl, benzyl, adamantyl, triphenylamine, pyrido [4,3-b]Indolyl, 1,2,3, 4-tetrahydroquinolinyl, indolyl, benzothiazolyl, benzothienyl or 5-phenylisoxazolyl; r is R 6 Is alkyl, phenyl, polyethoxy or piperazinyl.
The synthetic route is one of the following four synthetic routes:
synthetic route 1:
step a: glycine (5 mmol,1 eq) was dissolved in anhydrous methanol and thionyl chloride (5 mmol,1 eq) was slowly added dropwise followed by reflux for 2h and drying under reduced pressure to give product A.
Step b: thiocbz-lys (1 mmol,1 eq) was dissolved in anhydrous tetrahydrofuran, DIEA (3 mmol,3 eq) was then added, HBTU (1.5 mmol,1.5 eq) was added, product A was added after stirring at room temperature for 10min, the solvent was removed under reduced pressure after reacting at room temperature for 4h, the residue was dissolved in dichloromethane, washed three times with saturated sodium chloride, dried over anhydrous sodium sulfate, and PE/EA=2:1 column chromatography gave product B.
Step c: product B (1 mmol,1 eq) was dissolved in anhydrous methanol, then hydroxylamine hydrochloride (20 mmol,20 eq) and then potassium hydroxide (22 mmol,22 eq) were added, the solvent was removed under reduced pressure after reaction for 4h at room temperature, DCM/meoh=20:1 column chromatography to give product C.
Synthetic route 2:
step d: thiocbz-lys (1 mmol,1 eq) was dissolved in anhydrous tetrahydrofuran, DIEA (3 mmol,3 eq) was then added, HBTU (1.5 mmol,1.5 eq) was added, after stirring at room temperature for 10min 5-phenylisoxazolamine (1.2 mmol,1.2 eq) was added, the solvent was removed under reduced pressure after reaction at room temperature for 4h, the residue was dissolved in dichloromethane, washed three times with saturated sodium chloride, dried over anhydrous sodium sulfate, and PE: EA=1:1 column chromatography gave product D.
Step e: product D (1 mmol,1 eq) was dissolved in THF/H 2 O=1: 1, lithium hydroxide (3 mmol,3 eq) was then added, after overnight reaction, the pH was adjusted to 3, the EA was extracted, concentrated and dried and dissolved in anhydrous THF, then DIEA (3 mmol,3 eq) was added, then HBTU (1.5 mmol,1.5 eq) was added, after stirring for 10min at room temperature, 7-aminoheptanoic acid methyl ester (1.2 mmol,1.2 eq) was added, after reaction at room temperature for 4h the solvent was removed under reduced pressure, the residue was dissolved in dichloromethane, washed three times with saturated sodium chloride, dried over anhydrous sodium sulfate and PE: EA=1:2 column chromatography gave product E.
Step f: product E (1 mmol,1 eq) was dissolved in anhydrous methanol, then hydroxylamine hydrochloride (20 mmol,20 eq) and then potassium hydroxide (22 mmol,22 eq) were added, the solvent was removed under reduced pressure after reaction for 4h at room temperature, DCM/meoh=20:1 column chromatography to give product F.
Synthetic route 3:
step g: thiocbz-lys (1 mmol,1 eq) was dissolved in anhydrous tetrahydrofuran, DIEA (3 mmol,3 eq) was then added, HBTU (1.5 mmol,1.5 eq) was added, after stirring at room temperature for 10min, 4-azidoaniline was added, the solvent was removed under reduced pressure after reaction at room temperature for 4h, the residue was dissolved in dichloromethane, washed three times with saturated sodium chloride, dried over anhydrous sodium sulfate, and PE/EA=2:1 column chromatography gave product G.
Step h: product G (0.1 mmol,1 eq) was dissolved in anhydrous DMF, then N-hydroxy-4- ((prop-2-yn-1-yl (quinolin-8-yl) amino) methyl) benzamide (0.1 mmol,1 eq) was added, then anhydrous copper sulfate (0.02 mmol,0.2 eq) was added, then TBTA (0.01 mmol,0.1 eq) was added, then sodium ascorbate (0.05 mmol,0.5 eq) was dissolved in 200 μl of water, reacted for 16H at room temperature, EA was added, saturated sodium chloride was washed three times, anhydrous sodium sulfate was dried, DCM/meoh=10:1 column chromatography to give product H.
Synthetic route 4:
step g: thiocbz-lys (1 mmol,1 eq) was dissolved in anhydrous tetrahydrofuran, DIEA (3 mmol,3 eq) was then added, HBTU (1.5 mmol,1.5 eq) was added, after stirring at room temperature for 10min, 4-azidoaniline was added, the solvent was removed under reduced pressure after reaction at room temperature for 4h, the residue was dissolved in dichloromethane, washed three times with saturated sodium chloride, dried over anhydrous sodium sulfate, and PE/EA=2:1 column chromatography gave product G.
Step h: product G (0.1 mmol,1 eq) was dissolved in anhydrous DMF, then (E) -N-hydroxy-4- (((2 ' -oxo-1' - (prop-2-yn-1-yl) - [2,3' -biindolin ] -3-yleidene) amino) oxide) bunamide (0.1 mmol,1 eq) was added, then anhydrous copper sulfate (0.02 mmol,0.2 eq) was added, then TBTA (0.01 mmol,0.1 eq) was added, then sodium ascorbate (0.05 mmol,0.5 eq) was dissolved in 200 μl water, after 16h reaction at room temperature EA was added, saturated sodium chloride was washed three times, anhydrous sodium sulfate was dried, DCM/meoh=10:1 column chromatography to give product I.
In the above route, the R 1 Is methylene or imino; r is R 2 Is tetradecyl, phenyl orAdamantyl, polyethoxy; r is R 3 Is alkyl, phenyl, benzyl, cyclohexenyl, polyethoxy, benzothiazolyl, thiazolyl, or piperazinyl; r is R 4 Is hydroxamic acid, mercapto, phthalic diamido, para-fluorophthalic diamido or ethyl acetohydrazino; r is R 5 Is quinolinyl, benzyl, adamantyl, triphenylamine, pyrido [4,3-b]Indolyl, 1,2,3, 4-tetrahydroquinolinyl, indolyl, benzothiazolyl, benzothienyl or 5-phenylisoxazolyl; r is R 6 Is alkyl, phenyl, polyethoxy or piperazinyl.
Application of SIRT2/HDAC6 double-target inhibitor in preparing solid tumor and hematological tumor medicines.
By adopting the technical scheme, the SIRT2/HDAC6 double-target inhibitor can effectively and selectively inhibit the activity of SIRT2 and HDAC6 in vivo and in vitro, has cheap synthetic raw materials, low cost and obvious anti-tumor activity, and can be used for high-efficiency and low-toxicity novel SIRT2/HDAC6 double-target inhibitor anti-tumor medicines.
Detailed Description
Embodiments of the invention: the following (1) synthetic route for SIRT2/HDAC6 dual target inhibitors was employed:
through this synthetic route, compounds 1-3 were obtained, the structures of compounds 1-3 were as follows:
embodiments of the invention: the following (2) synthetic route for SIRT2/HDAC6 dual target inhibitors was employed:
by this synthetic route, compound 4 was obtained, the structure of compound 4 being as follows:
embodiments of the invention: the following (3) synthetic route for SIRT2/HDAC6 dual target inhibitors was employed:
through this synthetic route, compound 5 was obtained, the structure of compound 5 being as follows:
embodiments of the invention: the following (4) synthetic route for SIRT2/HDAC6 dual target inhibitors was employed:
by this synthetic route, compound 6 was obtained, the structure of compound 6 being as follows:
compounds 1 to 6 1 H-NMR， 13 C-NMR and HRMS data
Compound 1: 1 H NMR(400MHz,DMSO-d6)δ(ppm):10.57(t,J＝6.5Hz,2H),8.41(t,J＝5.6Hz,2H),7.83(d,J＝8.8Hz,2H),7.66(d,J＝8.6Hz,1H),7.45(d,J＝2.3Hz,1H),7.31(dd,J＝8.7,5.6Hz,1H),4.32(tt,J＝5.8Hz,1H),3.31–3.25(m,2H),2.98(dd,J＝12.7,6.7Hz,2H),2.58(dd,J＝6.7Hz,2H),2.24(d,J＝5.7Hz,2H),1.78(s,2H),1.72–1.63(m,2H),1.41(dt,J＝15.0,7.4Hz,4H),1.30–1.19(m,22H),0.94(t,J＝7.4Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ204.51，171.75,169.56,169.48,138.12，127.09,126.19,124.32,118.37,117.75,43.37,42.67,40.73,31.15,30.58,30.49,29.26,23.16,23.10,18.94,14.06.HRMS(ESI)for C 30 H 50 N 4 O 4 S(M+H + ):calcd,563.36255.,found,563.36227.
compound 2: 1 H NMR(400MHz,DMSO-d6)δ(ppm):10.52(t,J＝6.2Hz,2H),8.31(t,J＝5.9Hz,2H),7.73(d,J＝6.8Hz,2H),7.60(d,J＝6.6Hz,1H),7.42(d,J＝2.5Hz,1H),7.29(dd,J＝6.7,5.6Hz,1H),4.45(t,J＝5.5Hz,1H),3.31–3.22(m,2H),2.87(dd,J＝12.7,6.7Hz,2H),2.68(dd,J＝5.9Hz,4H),2.34(d,J＝5.4Hz,4H),1.68(t,4H),1.72–1.63(m,4H),1.39(dt,J＝15.0,7.4Hz,4H),1.30–1.09(m,22H),0.92(t,J＝7.0Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ205.21，174.25,171.56,170.48,141.12，140.09,140.02,139.02,122.19,120.32,119.27,113.15,43.37,42.67,40.73,31.15,30.58,30.49,29.26,23.16,23.10,18.94,14.26.HRMS(ESI)for C 32 H 54 N 4 O 4 S(M+H + ):calcd,591.39385.,found,591.39302.
compound 3: 1 H NMR(400MHz,DMSO-d6)δ(ppm):10.59(d,J＝5.2Hz,2H),8.41(t,J＝4.9Hz,2H),7.53(d,J＝6.2Hz,2H),7.53(d,J＝6.3Hz,1H),7.41(d,J＝2.4Hz,1H),7.25(dd,J＝5.6Hz,1H),4.25(t,J＝5.5Hz,1H),3.22(t,J＝6.3Hz,2H),2.87(dd,J＝11.7,6.7Hz,4H),2.48(dd,J＝5.2Hz,4H),2.34(d,J＝5.4Hz,2H),1.68(t,8H),1.72–1.63(m,4H),1.49(dt,J＝15.0,7.4Hz,4H),1.40–1.09(m,22H),0.92(t,J＝7.0Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ205.51，172.25,171.56,170.48,141.12，140.09,140.02,139.02,122.19,120.32,119.27,113.15,43.37,42.67,40.73,37.15,35.58,35.49,34.2631.15,30.58,30.49,29.26,23.16,23.10,18.94,14.26.HRMS(ESI)for C 34 H 58 N 4 O 4 S(M+H + ):calcd,619.42515.,found,619.42527.
compound 4: 1 H NMR(400MHz,DMSO-d6)δ(ppm):10.53(d,J＝5.2Hz,2H),8.41(t,J＝4.9Hz,2H),7.83(d,J＝6.1Hz,4H),7.43(d,J＝6.2Hz,2H),7.41(d,J＝2.4Hz,2H),7.25(dd,J＝5.3Hz,2H),5.05(t,J＝5.2Hz,2H),4.22(t,J＝3.5Hz,1H),3.25(t,J＝5.3Hz,2H),3.05(t,J＝6.1Hz,2H),2.42(dd,J＝5.2Hz,2H),2.35(d,J＝5.4Hz,2H),1.68(t,6H),1.72–1.63(m,2H),1.49(dt,J＝15.0,7.4Hz,6H),1.40–1.09(m,22H),0.90(t,J＝7.0Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ204.51,171.95,171.26,170.38,160.18,159.02,158.22,150.09,141.22，140.29,132.02,131.92,130.19,130.02,129.27,123.15,123.02,120.09,100.97,65.97,60.50,45.27,43.37,42.67,40.73,38.15,36.58,35.19,34.26,31.15,30.58,30.49,29.26,23.16,23.10,18.93,14.22.HRMS(ESI)for C 45 H 66 N 4 O 7 S(M+H + ):calcd,835.47865.,found,835.47802.
compound 5: 1 H NMR(400MHz,DMSO-d6)δ(ppm):10.51(d,J＝5.2Hz,2H),8.40(t,J＝4.9Hz,2H),7.81(d,J＝6.2Hz,4H),7.72(d,J＝6.1Hz,4H),7.61(d,J＝2.4Hz,4H),7.52(d,J＝4.1Hz,4H),7.32(dd,J＝5.2Hz,4H),7.02(d,J＝2.2Hz,1H),5.62(d,J＝5.2Hz,2H),5.02(t,J＝4.2Hz,2H),4.22(t,J＝5.5Hz,1H),3.92(t,J＝6.1Hz,2H),3.80(t,J＝4.3Hz,2H),3.52(t,J＝2.3Hz,4H),3.22(t,J＝6.3Hz,2H),2.97(d,J＝11.7,6.7Hz,6H),2.48(dd,J＝4.2Hz,2H),1.72(t,6H),1.65–1.58(m,4H),1.49(dt,J＝15.0,7.4Hz,4H),1.40–1.09(m,22H),0.96(t,J＝7.0Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ204.21，173.25,171.06,162.48,157.82,142.32,142.12，141.09,140.42,139.52,130.09,129.87,129.02,128.81,127.69,122.19,120.32,101.27,100.15,67.28,65.42,64.29,60.09,58.29,56.72,54.29,53.19,50.09,43.17,42.07,41.73,37.05,35.28,35.09,33.26,31.05,30.18,30.09,29.23,23.12,23.19,18.24,14.37.HRMS(ESI)for C 61 H 80 N 10 O 7 S(M+H + ):calcd,1097.60049.,found,1097.60012.
compound 6: 1 H NMR(400MHz,DMSO-d6)δ(ppm):10.57(d,J＝5.2Hz,2H),8.50(t,J＝4.9Hz,2H),7.83(d,J＝6.2Hz,4H),7.62(d,J＝6.1Hz,4H),7.54(d,J＝2.6Hz,4H),7.52(d,J＝4.1Hz,2H),7.02(d,J＝4.2Hz,2H),6.92(d,J＝2.1Hz,2H),5.05(t,J＝4.1Hz,2H),4.85(t,J＝6.1Hz,2H),4.29(t,J＝5.1Hz,1H),3.91(t,J＝6.1Hz,2H),3.86(t,J＝4.1Hz,2H),3.42(t,J＝5.3Hz,4H),3.22(t,J＝6.3Hz,2H),3.02(d,J＝11.2,6.7Hz,2H),2.49(dd,J＝4.1Hz,2H),2.29(d,J＝6.1Hz,2H),1.71(t,6H),1.62–1.59(m,4H),1.44(dt,J＝13.0,7.1Hz,4H),1.40–1.09(m,22H),0.91(t,J＝6.2Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ204.41，173.15,172.06,171.09,170.92,169.99,155.48,147.82,142.12,141.12，131.29,130.82,130.52,130.29,129.77,129.02,128.91,127.69,122.19,120.22,111.28,110.25,109.87,71.09,70.97,67.28,61.42,64.29,60.09,52.12,50.09,44.27,42.42,41.33,36.15,35.08,35.29,33.16,31.23,30.28,30.02,29.23,23.22,23.13,18.24,14.58.HRMS(ESI)for C 62 H 79 N11O 9 S(M+H + ):calcd,1154.58557.,found,1154.58237.
example 2:
in vitro SIRT2 and HDAC6 inhibition activity screening:
SIRT2 inhibitory activity screening:
the specific operation steps are as follows:
1) An in vitro inhibitory enzyme reaction mixture (ddwater, reaction volume: 38.4. Mu.L) was prepared in accordance with Table 1, and the reaction solution of the desired measurement sample was placed in a 1.5mL centrifuge tube;
2) The enzymatic reaction mixture was dispensed into labeled 1.5mL centrifuge tubes (52. Mu.L/each), 2. Mu.L of 6. Mu. Mol/L of the target compound (final concentration 0.2. Mu. Mol/L) was added, and the mixture was placed in an ice box;
3) Vortex, centrifuge, put all samples together in incubator, pre-incubate for 30min at 37 ℃;
4) Taking out the sample, placing in an ice box, cooling, and sequentially adding 6 mu L of substrate (final concentration: 200 mu M), wherein the total reaction volume is 60 mu L;
5) Vortex, centrifuge, add a sample at intervals of 30s, pre-incubate for 60min at 37 ℃, quench with the queue buffer quenching liquid in turn;
6) Centrifugation was carried out at 10000rpm for 10min at high speed, 100. Mu.L of sample was taken in 96-well plates and detected at UV 280nm wavelength of HPLC, and the sample volume was 40. Mu.L.
1 parallel control group, one negative control (DMSO), one positive control (TM) (final concentration 0.2. Mu.M) was set up in the experiment.
TABLE 1 enzymatic reaction mixture composition of Sirtuin in vitro inhibitory Activity
The HPLC liquid chromatography detection conditions were as follows:
1) Chromatographic column: inertSustatin AQ-C18X 46mm,5 μm;
2) Mobile phase: phase A, 0.1% TFA/acetonitrile (v/v); phase B, 0.1% TFA/ddwater (v/v);
3) Detection wavelength: 254nm and 284nm;
4) Column temperature: 25 DEG C
5) The elution conditions were as follows:
TABLE 2 gradient conditions for HPLC elution
The conversion of the substrate is calculated as 1.1:
convergence% = A product/(A product+A substrate) ×100% (1.1)
The inhibition rate calculation formula of the target compound is shown in 1.2:
inhibition% = (1-C target compound/C negative control) 100% (1.2)
SIRT2 inhibitory activity results for compounds 1-3 are shown in table 3:
TABLE 3 SIRT2 inhibitory Activity of Compounds 1-6
HDAC6 inhibitory activity screening:
all HDAC kits were purchased from BPS Bioscience. Enzymatic HDAC assays were performed following the manufacturer's protocol. Take HDAC6 as an example. (fluorescent HDAC6 assay kit, catalog number: 50076;BPS Bioscience).
The specific operation steps are as follows:
1) HDAC assay buffer (35 μl) was mixed with BSA (1 mg/mL,5 μl) and HDAC substrate (200 μl,5 μl) on a 96-well black plate;
2) HDAC6 (7 ng/μl,5 μl) and various concentrations of compound (5 μl) or TSA (5 μl) (as positive control) were added to the wells;
3) Incubating the resulting mixture at 37 ℃ for 30 minutes;
4) After incubation, 50 μl of 2xHDAC development was added to each well;
5) After incubating the mixture at room temperature for 15 minutes, the fluorescence intensity was measured at 360nm excitation and 460nm emission wavelength using a microplate reader.
The HDAC6 inhibitory activity results for compounds 1-6 are shown in table 4:
TABLE 4 HDAC6 inhibitory Activity of Compounds 1-6
Example 3:
anti-tumor activity of SIRT2/HDAC6 dual target inhibitors against different tumor cells:
candidate compounds were evaluated for antiproliferative activity on 5 human cancer cells using the accepted CCK8 method, which can be used for large-scale antitumor drug screening and cytotoxicity assay. The test compound is compound 3, and the negative control group is a group without adding medicine; the positive control groups were TM and ACY-1215.
Cell lines: human breast cancer cells MCF-7, human liver cancer cells HepG2, human colorectal cancer cells HCT-116, human acute leukemia cells MV411 and human myelogenous leukemia cells K562.
Cell proliferation inhibition = (negative control OD-drug OD) ×100%/negative control OD. The IC50 values (μM) were calculated by inhibition of the compound series concentrations and the results are shown in Table 5.
TABLE 5 determination of IC of Compound 3 for different tumor cells by CCK8 method 50 Value (mu M)
It is noted that the above examples and test examples are only limited to further explanation and understanding of the technical solutions of the present invention, and should not be construed as further limiting the technical solutions of the present invention, and the invention without significant essential features and significant improvements made by those skilled in the art still falls within the scope of protection of the present invention.

Claims (2)

1. A SIRT2/HDAC6 dual target inhibitor, characterized by: the compound takes lysine as a framework, and specifically adopts one of the following structural formulas:
2. use of the SIRT2/HDAC6 dual target inhibitor of claim 1 for the preparation of a medicament for solid and hematological tumors.
CN202210057576.6A 2022-01-19 2022-01-19 SIRT2/HDAC6 double-target inhibitor and application thereof Active CN114315670B (en)

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