CN115043824A - Compound with SIRT6 deacetylation inhibiting activity, SIRT6 inhibitor and application thereof - Google Patents

Abstract

The examples of the present application disclose a compound having the activity of inhibiting SIRT6 deacetylation, and the compound is the compound represented by formula (1) or a pharmaceutically acceptable salt thereof. The invention also provides the SIRT6 allosteric inhibitor obtained by using the compound and the application of the SIRT6 allosteric inhibitor in the preparation of cancer drugs.

Description

Compounds with SIRT6 deacetylation inhibitory activity, SIRT6 inhibitors and the same application

technical field

The present application relates to the technical field of medicinal chemistry, in particular to a compound having the activity of inhibiting SIRT6 deacetylation, a SIRT6 allosteric inhibitor obtained by using the compound, and its application in the preparation of cancer drugs.

Background technique

SIRT6, as an important member of family III (Sirtuin 1-7) of histone deacetylases (HDACs), was first reported in 2000 and has been extensively studied in recent years. With the deepening of research, many physiological functions of SIRT6 have been discovered. In addition to its NAD ⁺ -dependent histone deacetylase activity, it also has the functions of single ADP ribosyltransferase and de-fatty acylation.

Human full-length SIRT6 consists of 355 amino acids and contains a catalytic center shared by a putative sirtuins family and poorly conserved N- and C-termini. The crystal structure of the truncated SIRT (3-318) has been solved, which shows that SIRT6 contains a two-domain structure consisting of a 'Rossmann fold' domain and a small stretch The domain of the open Zn ²⁺ binding site is composed of domains, and there is no helical structure at the junction of these two domains; in addition, the NAD ⁺ binding domain of SIRT6 is composed of a firm helical structure. These structural features are different from other members of the sirtuins family.

Studies have shown that SIRT6 activity is regulated by a variety of factors, mainly by catalyzing the deacetylation of related proteins in cells, mainly involved in life activities such as DNA repair, inflammation, and glucose and lipid metabolism, and is closely related to diabetes, cancer, aging and other diseases. closely related. Among them, the mechanism of SIRT6 in the occurrence and development of cancer is complicated.

SIRT6 may play a dual role of tumor suppressor or carcinogenesis in different tumor backgrounds. In most cases, SIRT6 acts as a tumor suppressor gene, such as non-small cell lung cancer, ovarian cancer, etc. In contrast, numerous studies have shown that SIRT6 plays an oncogenic role in various cancer types. Studies have shown that the expression of SIRT6 is significantly up-regulated in breast cancer cells, retinoblastoma cells, squamous epithelial cancer cells, pancreatic cancer cells, ovarian cancer cells, and leukemia cells. Exploring its function and mechanism in the process of tumorigenesis and development by inhibiting SIRT6 activity not only provides confirmation for SIRT6 as a new target for tumor intervention, but also provides new ideas for the treatment of related diseases by studying the function of SIRT6.

At present, there are no reports of highly efficient and highly selective inhibitors of SIRT6, which restricts the functional study of SIRT6 as a target through chemical intervention. It is difficult to inhibit SIRT6 with high selectivity through substrate competition. Allosteric modulators target non-conserved allosteric sites, which are suitable for distinguishing subtype members of protein families, and are expected to achieve high efficiency and high selection of SIRT6. Sexual inhibition.

SUMMARY OF THE INVENTION

The present application provides a compound with the activity of inhibiting SIRT6 deacetylation, a SIRT6 allosteric inhibitor obtained by using the compound, and its application in the preparation of cancer drugs, in order to carry out SIRT6-related chemical and biological research and explore the role of SIRT6 in diseases the basis for the therapeutic effect.

The application provides a compound or a pharmaceutically acceptable salt thereof, the compound has the structure shown in formula (1):

Wherein, R is a C6-C20 aromatic group or a C6-C20 aromatic group containing a substituent, and its structural features include that 1,2,5-oxadiazole (furazan ring) substituted by an amino group is connected to triazole , the triazole and the Ar ring are connected by ethylene hydrazide hydrazide structure.

Optionally, in some embodiments of the present application, the aromatic group is phenyl, pyridyl, furyl, thienyl, pyrrolyl, imidazolyl, thiazolyl, naphthyl, oxazolyl, isoxazole group, triazolyl, tetrazolyl, pyranyl, pyrimidinyl, or isothiazolyl.

Optionally, in some embodiments of the present application, a hydrogen atom at at least one site in the aromatic group is substituted with a substituent.

Optionally, in some embodiments of the present application, the substituent is halogen, hydroxyl, nitro, amino, carboxyl, acid ester, sulfonamide, mercapto, methoxy, ethoxy, benzyloxy, One or more of methyl, tert-butyl, cyano, or dimethoxyethanol. Optionally, in some embodiments of the present application, in the compound of formula (1), R is selected from one of the following groups:

Optionally, in some embodiments of the present application, the halogen includes fluorine, chlorine, bromine or iodine.

Optionally, in some embodiments of the present application, the pharmaceutically acceptable salt is hydrochloride, sulfate, oxalate, acetate, trifluoroacetate of the compound represented by formula (1). , or citrate.

Correspondingly, the present application also provides a use of the compound or a pharmaceutically acceptable salt thereof in the preparation of a SIRT6 allosteric inhibitor.

Optionally, in some embodiments of the present application, the compound has a binding effect on SIRT6.

In addition, the present application also provides a pharmaceutical composition containing the compound or a pharmaceutically acceptable salt thereof. .

The present application also provides an application of the compound or a pharmaceutically acceptable salt thereof in the preparation of a cancer drug.

The compounds with the activity of inhibiting SIRT6 deacetylation described in this application or their pharmaceutically acceptable salts can significantly inhibit the deacetylation activity of SIRT6 in in vitro experiments, which is of great significance to the development of drugs for SIRT6-mediated diseases .

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present application more clearly, the following briefly introduces the drawings that are used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those skilled in the art, other drawings can also be obtained from these drawings without creative effort.

Fig. 1 is the inhibitory concentration curve of compound No. 42 provided by the invention to SIRT6 deacetylase activity;

Figure 2 is a schematic diagram showing the changes of intracellular SIRT6 deacetylation active substrates H3 K9Ac, H3 K18Ac and H3 K56Ac after treating pancreatic cancer cell line BXPC-3 with different concentrations of compound 42 for 24 hours;

Figure 3 is a schematic diagram showing the changes of intracellular SIRT6 deacetylation active substrates H3 K9Ac, H3 K18Ac and H3 K56Ac after treating pancreatic cancer cell line Mia-paca-2 with different concentrations of compound 42 for 24 hours;

Fig. 4 is a schematic diagram showing the changes of intracellular SIRT6 deacetylation active substrates H3 K9Ac, H3 K18Ac and H3 K56Ac after treating pancreatic cancer cell line Capan-2 with different concentrations of compound 42 for 24 hours;

Figure 5 is the treatment of leukemia cell lines U937 and HDLM2 with different concentrations of compound No. 42, and the growth curve of the compound inhibiting cell proliferation and the half effective concentration IC ₅₀ are measured;

Fig. 6 is to treat skin cancer cell line A375 with compound No. 42 at different concentrations, and the growth curve of compound inhibiting cell proliferation is measured and the half effective concentration IC ₅₀ is measured;

Figure 7 shows the skin cancer cell line SK-MEL-2 treated with compound No. 42 at different concentrations, and the growth curve of the compound inhibiting cell proliferation and the half effective concentration IC ₅₀ were measured;

Figure 8 is the treatment of cervical cancer cell line Hela S3 with different concentrations of compound No. 42, the growth curve of the compound inhibiting cell proliferation is measured and the half effective concentration IC ₅₀ (24h) is measured;

Figure 9 is the treatment of cervical cancer cell line Hela S3 with different concentrations of compound No. 42, and the growth curve of the compound inhibiting cell proliferation and the half effective concentration IC ₅₀ (48h) are measured;

Figure 10 is the treatment of pancreatic cancer cell line BXPC-3 with different concentrations of compound No. 42, and the growth curve of the compound to inhibit cell proliferation and the half effective concentration IC ₅₀ are measured;

Figure 11 is to treat the pancreatic cancer cell line Mia-paca-2 with different concentrations of compound No. 42, and the growth curve of the compound to inhibit cell proliferation and the half effective concentration IC ₅₀ are measured;

Figure 12 is to treat pancreatic cancer cell line Capan-2 with different concentrations of compound No. 42, and the growth curve of the compound to inhibit cell proliferation and the half effective concentration IC ₅₀ are measured;

Figure 13 is treating pancreatic cancer cell line BXPC-3 with different concentrations of compound No. 42, and detecting the inhibitory effect of the compound on cell migration by real-time label-free cell analysis technology (RTCA);

Figure 14 is the treatment of pancreatic cancer cell line Mia-paca-2 with different concentrations of compound No. 42, and the inhibitory effect of the compound on cell migration was detected by real-time label-free cell analysis (RTCA);

Figure 15 shows that the cervical cancer cell line Hela S3 was treated with different concentrations of compound No. 42, and the inhibitory effect of the compound on cell migration was detected by real-time label-free cell analysis (RTCA);

Figure 16 shows that the skin cancer cell line A375 was treated with compound No. 42 at different concentrations, and the inhibitory effect of the compound on cell migration was detected by real-time label-free cell assay (RTCA).

Detailed ways

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

The present application provides a compound with the activity of inhibiting SIRT6 deacetylation, a SIRT6 allosteric inhibitor obtained by using the compound, and its application in the preparation of cancer drugs. Each of them will be described in detail below.

The "aromatic group" in the present invention includes: phenyl, pyridyl, furyl, thienyl, pyrrolyl, imidazolyl, thiazolyl, naphthyl, oxazolyl, isoxazolyl, triazolyl, Aromatic chemical groups such as tetrazolyl, pyranyl, pyrimidinyl, or isothiazolyl.

"SIRT6" of the present invention refers to one of the seven proteins of histone deacetylase family III. Histone deacetylase can deacetylate histones, bind tightly with negatively charged DNA, and chromatin is dense and coiled, thereby inhibiting gene transcription

SIRT6 allosteric inhibitor means that the inhibitor molecule binds to a site other than the active site or orthosite of SIRT6 protein, and induces a conformational change of SIRT6 protein to achieve functional inhibition of SIRT6 protein.

Deacetylation activity refers to the reactivity of deacetylation _CH3CO- in organic compounds.

The "cancer" of the present invention includes malignant tumors such as pancreatic cancer, leukemia, skin cancer, and cervical cancer.

The compounds of the present invention can be used in mammals, including human or non-human mammals, eg, mice, monkeys, and the like.

Example 1. Synthesis of compounds with inhibitory SIRT6 deacetylation activity

(1) Synthesis of compound 2

In a 100 mL conical flask, compound 1 (1 g) was slowly added in batches to the stirring concentrated sulfuric acid (5 mL) at 0 °C, and then slowly added dropwise to the concentrated sulfuric acid (2 mL) at the same temperature after it was completely dissolved. ) in sodium nitrite (723 mg) and the reaction was continued at 0°C for 1 hour. The reaction system was warmed to 4°C, glacial acetic acid (7 mL) was slowly added to dilute, and 1.3 g of sodium azide was added. The reaction was continued at 4°C for 1 hour. The reaction solution was poured into 30 g of ice and continued to stir, and then 40 mL of ethyl acetate was added for extraction. The organic phase was washed with saturated sodium bicarbonate and saturated brine, and dried with anhydrous sodium sulfate. Compound 2 (344 mg) was obtained by column chromatography. , 28%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.19 (s, 2H). MS (ESI+) m/z: 127.0 [M+H] ⁺

(2) Preparation of compound 3

In a 50 mL conical flask, compound 2 (1 g) was dissolved in 15 mL of acetonitrile, and then ethyl propiolate (1.56 g), copper sulfate pentahydrate (198 mg) and sodium ascorbate (16 mg) were added in sequence at room temperature, The reaction was continued to stir at the same temperature for 20 hours. After TLC detection was completed, 60 mL of ethyl acetate was added, washed with water, saturated sodium bicarbonate solution and saturated brine respectively, the organic phase was dried with anhydrous sodium sulfate, and the compound 3 (1.40 g, 80%) was obtained by column chromatography. . ¹ H NMR(400MHz,DMSO-d6)δ9.44(s,1H),6.71(s,2H),4.39(m,2H),1.34(t,J=7.1Hz,3H).MS(ESI+)m /z:225.1[M+H]+

(3) Preparation of compound 4

In a 25 mL Erlenmeyer flask, compound 3 (1 g) was placed in 15 mL of ethanol, and then 3 mL of 64% hydrazine hydrate was added. The reaction was heated to 60°C under nitrogen protection and reacted for 4 hours, then the reaction system was moved to room temperature and suction filtered to obtain a white solid, which was washed twice with absolute ethanol to obtain compound 4 (858 mg, 85%). ¹ H NMR (400MHz, DMSO-d ₆ )δ10.02(s,1H), 9.19(s,1H), 6.69(s,2H), 4.60(s,2H).MS(ESI ⁺ )m/z: 211.1[M+H] ⁺ .

(4) Preparation of compounds with inhibitory SIRT6 deacetylation activity

In a Erlenmeyer flask, compound 4 (100 mg) and different aldehydes (1.2 equiv.) were added to a catalytic equivalent of glacial acetic acid in 5 mL of ethanol, and the reaction was heated to 60°C and reacted for 4 hours. The reaction was moved to room temperature, and after the solids were fully separated out, the solids were obtained by suction filtration, washed twice with absolute ethanol, and dried to obtain different derivatives containing formula (1). The structures of the synthesized compounds are characterized as follows:

Example 1(E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(2hydroxybenzylidene)-1hydro-1,2,3-triazole- 4-Carbohydrazide

¹ H NMR (500MHz, DMSO-d ₆ )δ12.61(s,1H), 11.16(s,1H), 9.42(s,1H), 8.79(s,1H), 7.59-7.53(m,1H), 7.36-7.29(m,1H),6.99-6.91(m,2H),6.73(s,2H).[MH] ^- :313.1

Example 2(E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(4-hydroxybenzylidene)-1hydro-1,2,3-triazole -4-Carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J＝8.0Hz,1H),7.47(d,J＝8.0Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :315.1.

Example 3 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(3-nitrobenzylidene)-1hydro-1,2,3-tris oxazol-4-carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J=8.0Hz,1H),7.47(d,J=8.0Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :344.1.

Example 4(E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(2-chlorobenzylidene)-1hydro-1,2,3-triazole -4-Carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J=8.0Hz,1H),7.47(d,J=8.0Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :333.1.

Example 5(E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(4-fluorobenzylidene)-1hydro-1,2,3-triazole -4-Carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J=8.0Hz,1H),7.47(d,J=8.0Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :317.1.

Example 6(E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(furan-2-ylmethylene)-1hydro-1,2,3 -Triazole-4-carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J＝8.0Hz,1H),7.47(d,J＝8.0Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :289.1.

Example 7 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(thiophen-2-ylmethylene)-1hydro-1,2,3 -Triazole-4-carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J=8.0Hz,1H),7.47(d,J=8.0Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :305.1.

Example 8(E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(4-nitrobenzylidene)-1hydro-1,2,3-tris oxazol-4-carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J＝8.0Hz,1H),7.47(d,J＝8.0Hz,1H),6.74(s,2H).(ESI,m/z):[MH] ^- :342.0.

Example 9 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-benzyl-1hydro-1,2,3-triazole-4-carbonyl Hydrazine.

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J＝8.0Hz,1H),7.47(d,J＝8.0Hz,1H),6.74(s,2H).[MH] ^- :297.0.

Example 10(E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(pyridin-3-ylmethylene)-1hydro-1,2,3 -Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.49(s, 1H), 9.42(s, 1H), 8.87(s, 1H), 8.64(s, 2H), 8.17(d, J=8.0Hz, 1H), 7.51(dd,J=7.8,4.8Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :300.1.

Example 11 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(2-(trifluoromethoxy)benzyl)-1hydro-1, 2,3-Triazole-4-carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.75(s,1H),9.43(s,1H),9.01(s,1H),8.26(d,J=7.8Hz,1H),7.76(m,3H), 6.74(s,2H).(ESI,m/z):[M+H] ⁺ :383.1.

Example 12(E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(4-methoxybenzylidene)-1hydro-1,2,3- Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.17(s, 1H), 9.37(s, 1H), 8.52(s, 1H), 7.69(d, J=8.7Hz, 2H), 7.04(d, J=8.7 Hz,2H),6.73(s,2H),3.83(s,3H).(ESI,m/z):[M+H] ⁺ :329.1.

Example 13 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(4-(hydroxymethyl)benzylidene)-1H-1,2,3 -Triazole-4-carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.28(s,1H),9.39(s,1H),8.57(s,1H),7.71(d,J=8.1Hz,2H),7.43(d,J=8.0 Hz,2H),6.73(s,2H),5.30(t,J＝5.7Hz,1H),4.56(d,J＝5.7Hz,2H).(ESI,m/z):[M+H] ⁺ :329.1.

Example 14 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(3-chlorobenzylidene)-1H-1,2,3-triazole- 4-Carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.46(s, 1H), 9.42(s, 1H), 8.57(s, 1H), 7.80(s, 1H), 7.71(d, J=5.0Hz, 1H), 7.54–7.49(m,2H),6.73(s,2H).(ESI,m/z):[M+H] ⁺ :333.1.

Example 15 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(2-(trifluoromethyl)benzyl)-1H-1,2, 3-Triazole-4-carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J＝8.0Hz,1H),7.47(d,J＝8.0Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :367.1.

Example 16(E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(4-ethoxybenzylidene)-1H-1,2,3-tris oxazol-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.17(s, 1H), 9.37(s, 1H), 8.52(s, 1H), 7.68(d, J=8.8Hz, 2H), 7.02(d, J=8.8 Hz, 2H), 6.73(s, 2H), 4.09(q, J=7.0Hz, 2H), 1.36(t, J=7.0Hz, 3H).

Example 17(E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(pyridin-2-ylmethylene)-1H-1,2,3- Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO)δ12.60(s,1H),9.44(s,1H),8.69-8.59(m,2H),8.02(d,J=7.9Hz,1H),7.91(t,J =7.6Hz,1H),7.51–7.38(m,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :300.1.

Example 18(E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(pyridin-4-ylmethylene)-1H-1,2,3- Triazole-4-carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J＝8.0Hz,1H),7.47(d,J＝8.0Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :300.1.

Example 19 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(3-methylbenzylidene)-1H-1,2,3-triazole -4-Carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J＝8.0Hz,1H),7.47(d,J＝8.0Hz,1H),6.74(s,2H).(ESI,m/z):[MH] ^- :311.1.

Example 20(E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(2-methylbenzylidene)-1H-1,2,3-triazole -4-Carbohydrazide

¹ H NMR (500MHz, DMSO)δ12.29(s,1H),9.40(s,1H),8.93(s,1H),7.88(d,J=7.5Hz,1H),7.32(dt,J=17.8 ,7.3Hz,3H),6.74(s,2H),2.47(s,3H).(ESI,m/z):[M+H] ⁺ :313.1.

Example 21 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(2-fluorobenzylidene)-1H-1,2,3-triazole- 4-Carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J=8.0Hz,1H),7.47(d,J=8.0Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :317.1.

Example 22 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(3-fluorobenzylidene)-1H-1,2,3-triazole- 4-Carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.40(s,1H),9.40(s,1H),8.60(s,1H),7.55(dt,J=14.1,7.6Hz,3H),7.31(t,J =7.7Hz,1H),6.71(s,2H).(ESI,m/z):[M+H] ⁺ :317.1.

Example 23 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(4-methylbenzylidene)-1H-1,2,3-triazole -4-Carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J＝8.0Hz,1H),7.47(d,J＝8.0Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :313.1.

Example 24 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(4-chlorobenzylidene)-1H-1,2,3-triazole- 4-Carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.63(s,1H),7.78(d,J=7.6Hz,1H),7.72(s,1H), 7.63(t,J=8.0Hz,1H),7.47(d,J=8.0Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :333.1.

Example 25 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(3-(trifluoromethoxy)benzyl)-1H-1,2 ,3-triazole-4-carbohydrazide.

¹ H NMR (500MHz, DMSO-d ₆ )δ ¹ H NMR (500MHz, DMSO-d ₆ )δ12.31(s,1H),9.35(s,1H),8.68(s,1H),8.05-8.03( m,2H),7.84–7.63(m,2H),6.59(s,2H).[MH] ^- :365.1

Example 26 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(4-bromophenylidene)-1H-1,2,3-triazole -4-Carbohydrazide

¹ H NMR (500MHz, DMSO)δ12.40(s,1H), 9.41(s,1H), 8.56(s,1H), 7.74–7.66(m,4H), 6.73(s,2H).(ESI, m/z):[MH] ^- :375.0.

Example 27 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(2-nitrobenzylidene)-1H-1,2,3-triazole -4-Carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.71(s,1H),9.43(s,1H),9.01(s,1H),8.13(dd,J=19.1,8.0Hz,2H),7.85(t,J =7.6Hz,1H),7.72(t,J=7.7Hz,1H),6.73(s,2H).(ESI,m/z):[M+H] ⁺ :344.1.

Example 28 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(2-bromophenylidene)-1H-1,2,3-triazole -4-Carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.65(s, 1H), 9.42(s, 1H), 8.97(s, 1H), 8.07-8.00(m, 1H), 7.72(d, J=7.9Hz, 1H) ), 7.50(t, J＝7.5Hz, 1H), 7.43–7.36(m, 1H), 6.74(s, 2H).(ESI, m/z): [M+H] ⁺ : 377.0.

Example 29 (E)-N'-([1,1'-biphenyl]-2-ylmethylene)-1-(4-amino-1,2,5-oxadiazol-3-yl)- 1H-1,2,3-Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO)δ12.35(s,1H), 9.36(s,1H), 8.55(s,1H), 8.16–8.10(m,1H), 7.56–7.45(m,5H), 7.39 (d, J=7.2Hz, 3H), 6.71 (s, 2H). (ESI, m/z): [M+H] ⁺ : 375.1.

Example 30 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(thiophen-3-ylmethylene)-1H-1,2,3- Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.21 (s, 1H), 9.38 (s, 1H), 8.61 (s, 1H), 7.97 (d, J=2.5Hz, 1H), 7.66 (dd, J=4.7 ,3.1Hz,1H),7.52(d,J＝5.0Hz,1H),6.73(s,2H).(ESI,m/z):[M+H] ⁺ :305.1.

Example 31 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(3-hydroxyphenylidene)-1H-1,2,3-triazole -4-Carbohydrazide

¹ H NMR (500MHz, DMSO-d ₆ )δ12.00(s,1H), 9.44(s,1H), 9.30(s,1H), 8.50(s,1H), 7.25(m,2H), 7.12( d,J=7.5Hz,1H),6.86(d,J=6.5Hz,1H),6.56(s,2H).[M+Na] ⁺ :337.1.

Example 32 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(3-bromophenylidene)-1H-1,2,3-triazole -4-Carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.46(s,1H),9.42(s,1H),8.55(s,1H),7.94(s,1H),7.74(d,J=7.7Hz,1H), 7.66(d,J=8.0Hz,1H),7.45(t,J=7.8Hz,1H),6.73(s,2H).(ESI,m/z):[M+H] ⁺ :377.0.

Example 33 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(2-methoxybenzylidene)-1H-1,2,3-tris oxazol-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.31 (s, 1H), 9.39 (s, 1H), 8.93 (s, 1H), 7.90 (dd, J=7.7, 1.3 Hz, 1H), 7.48–7.42 (m ,1H),7.13(d,J=8.3Hz,1H),7.05(t,J=7.5Hz,1H),6.73(s,2H),3.88(s,3H).(ESI,m/z): [M+H] ⁺ :329.1

Example 34 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(3-methoxybenzylidene)-1H-1,2,3-tris oxazol-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.33(s, 1H), 9.40(s, 1H), 8.56(s, 1H), 7.40(t, J=7.8Hz, 1H), 7.31(d, J=7.6 Hz,2H),7.07–7.02(m,1H),6.74(s,2H),3.83(d,J＝6.0Hz,3H).(ESI,m/z):[M+H] ⁺ :329.1

Example 35 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-nitrogen'-(2-hydroxy-4-(trifluoromethyl)benzylidene)-1H -1,2,3-Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.72(s, 1H), 11.43(s, 1H), 9.44(s, 1H), 8.86(s, 1H), 7.87(d, J=8.0Hz, 1H), 7.31–7.20(m,2H),6.73(s,2H).(ESI,m/z):[M+H] ⁺ :383.1.

Example 36 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(3-(trifluoromethoxy)benzyl)-1H-1,2 ,3-triazole-4-carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.50(s,1H),9.43(s,1H),8.62(s,1H),7.78(d,J=7.8Hz,1H),7.72(s,1H), 7.63(t,J=8.0Hz,1H),7.47(d,J=8.3Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :383.1.

Example 37 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(quinolin-4-ylmethylene)-1H-1,2,3 -Triazole-4-carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.53(s,1H),9.42(s,1H),9.31(s,1H),9.00(s,1H),8.72(d,J=6.0Hz,1H), 8.12(d,J=8.2Hz,1H),7.91–7.80(m,2H),7.75(s,1H),6.65(s,2H).(ESI,m/z):[M+H] ⁺ : 350.1.

Example 38 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(2-chloro-4-(trifluoromethyl)benzyl)-1H- 1,2,3-Triazole-4-carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.79(s,1H),9.45(d,J=4.0Hz,1H),9.05(d,J=3.3Hz,1H),8.30-8.20(m,1H), 7.98(s,1H),7.83(d,J＝6.9Hz,1H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :401.0.

Example 39 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(2-hydroxy-5-nitrobenzylidene)-1H-1,2, 3-Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.74(s, 1H), 12.18(s, 1H), 9.44(s, 1H), 8.87(s, 1H), 8.59(d, J=2.8Hz, 1H), 8.19(dd,J=9.1,2.9Hz,1H),7.13(d,J=9.1Hz,1H),6.74(s,2H).(ESI,m/z):[M+Na] ⁺ :382.1.

Example 40(E)-3-((2-(1-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4-carbonyl) Hydrazino)methyl)benzoic acid

¹ H NMR (500MHz, DMSO) δ 12.45(s, 1H), 9.43(s, 1H), 8.66(s, 1H), 8.34(s, 1H), 8.00(dd, J=26.5, 7.8Hz, 2H ),7.62(t,J=7.7Hz,1H),6.74(s,2H).(ESI,m/z):[M+Na] ⁺ :365.1.

Example 41 (E)-Methyl 4-((2-(1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4- Carbonyl)hydrazonyl)-3-nitrobenzoic acid methyl ester

¹ H NMR(500MHz, DMSO)δ12.87(s,1H),9.45(s,1H),9.06(s,1H),8.53(d,J=1.4Hz,1H),8.32(t,J=9.7 Hz,2H),6.73(s,2H),3.94(s,3H).(ESI,m/z):[M+H] ⁺ :402.1.

Example 42 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(3-chloro-5-(trifluoromethyl)benzylidene)-1H -1,2,3-Triazole-4-carbohydrazide

¹ H NMR (400MHz, DMSO) δ 12.67(s, 1H), 9.44(s, 1H), 8.63(s, 1H), 8.10(s, 1H), 8.05(s, 1H), 7.94(s, 1H) ),6.73(s,2H).(ESI,m/z):[M+H] ⁺ :401.0.

Example 43 (E)-Methyl 5-((2-(1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4- Carbonyl)hydrazonyl)-2-hydroxybenzoic acid methyl ester

¹ H NMR (500MHz, DMSO) δ 12.28(s, 1H), 10.79(s, 1H), 9.39(s, 1H), 8.52(s, 1H), 8.15(d, J=2.1Hz, 1H), 7.88(dd,J=8.7,2.1Hz,1H),7.10(d,J=8.6Hz,1H),6.73(s,2H),3.94(s,3H).(ESI,m/z):[M +H] ⁺ :373.1.

Example 44 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(4-(dimethylamino)benzylidene)-1H-1,2, 3-Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 11.96(s, 1H), 9.33(s, 1H), 8.41(s, 1H), 7.55(d, J=7.8Hz, 2H), 6.85-6.61(m, 4H) ),2.99(s,6H).(ESI,m/z):[M+H] ⁺ :342.1.

Example 45 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(quinolin-5-ylmethylene)-1H-1,2,3 -Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.46(s, 1H), 9.45(s, 1H), 9.35(d, J=8.5Hz, 1H), 9.23(s, 1H), 9.01(dd, J=4.1 ,1.5Hz,1H),8.14(d,J＝8.4Hz,1H),8.02(d,J＝6.6Hz,1H),7.91–7.85(m,1H),7.72(dd,J＝8.7,4.1Hz ,1H),6.76(s,2H).(ESI,m/z):[M+H] ⁺ :350.1.

Example 46 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-((5-fluoronaphthalen-1-yl)methylene)-1H-1 ,2,3-Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.46(s, 1H), 9.45(s, 1H), 9.33(s, 1H), 8.63(d, J=8.6Hz, 1H), 8.21(d, J=8.4 Hz,1H),8.10(d,J=7.2Hz,1H),7.79–7.67(m,2H),7.47(dd,J=10.5,7.9Hz,1H),6.76(s,2H).(ESI, m/z):[M+H] ⁺ :367.1.

Example 47 (E)-4-((2-(1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4-carbonyl) Hydrazone)methyl)benzoic acid

¹ H NMR (500MHz, DMSO) δ 13.13(s, 1H), 12.49(s, 1H), 9.43(s, 1H), 8.65(s, 1H), 8.04(d, J=8.3Hz, 2H), 7.87(d,J=8.3Hz,2H),6.74(s,2H).(ESI,m/z):[M+H] ⁺ :343.1.

Example 48 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(3-hydroxy-4-nitrobenzylidene)-1H-1,2 ,3-triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.57(s, 1H), 11.22(s, 1H), 9.44(s, 1H), 8.56(s, 1H), 7.98(d, J=8.5Hz, 1H), 7.54(d,J=1.1Hz,1H),7.32(dd,J=8.6,1.2Hz,1H),6.74(s,2H).(ESI,m/z):[M+Na] ⁺ :382.1.

Example 49 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(4-(methylthio)benzyl)-1H-1,2,3 -Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.27(s, 1H), 9.39(s, 1H), 8.53(s, 1H), 7.68(d, J=8.4Hz, 2H), 7.35(d, J=8.4 Hz,2H),6.73(s,2H),2.53(s,3H).(ESI,m/z):[M+H] ⁺ :345.1.

Example 50 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(3,5-bis(trifluoromethyl)benzylidene)-1H-1 ,2,3-Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.94(s, 1H), 9.47(s, 1H), 9.06(s, 1H), 8.48(d, J=8.3Hz, 1H), 8.19(d, J=8.4 Hz,1H),8.11(s,1H),6.75(s,2H).(ESI,m/z):[M+Na] ⁺ :457.1.

Example 51 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(2-hydroxy-4-nitrobenzylidene)-1H-1,2 ,3-triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.74(s, 1H), 11.52(s, 1H), 9.44(s, 1H), 8.88(s, 1H), 7.96(d, J=8.6Hz, 1H), 7.78–7.71(m,2H),6.73(s,2H).(ESI,m/z):[M+H] ⁺ :360.1.

Example 52 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(2-hydroxy-3-methoxybenzylidene)-1H-1, 2,3-Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.58(s, 1H), 10.82(s, 1H), 9.42(s, 1H), 8.79(s, 1H), 7.17(dd, J=7.9, 1.1Hz, 1H ),7.08–7.04(m,1H),6.88(t,J=7.9Hz,1H),6.73(s,2H),3.83(s,3H).(ESI,m/z):[M+H] ⁺ :345.1.

Example 53 (E)-Methyl 3-((2-(1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4- Carbonyl)hydrazino)methyl)-4-hydroxybenzoate

¹ H NMR (500MHz, DMSO) δ12.65(s, 1H), 11.70(s, 1H), 9.43(s, 1H), 8.85(s, 1H), 8.30(d, J=2.2Hz, 1H), 7.90(dd,J=8.6,2.2Hz,1H),7.05(d,J=8.6Hz,1H),6.74(s,2H),3.86(s,3H).(ESI,m/z):[M +H] ⁺ :373.1.

Example 54 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(3,4,5-trihydroxybenzylidene)-1H-1,2, 3-Triazole-4-carbohydrazide

¹ H NMR(500MHz, DMSO)δ12.49(s,1H), 11.42(s,1H), 9.54(s,1H), 9.39(s,1H), 8.60(s,1H), 8.53(s,1H) ),6.80(d,J＝8.5Hz,1H),6.73(s,2H),6.42(d,J＝8.4Hz,1H).(ESI,m/z):[M+H] ⁺ :373.1.

Example 55 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(2-nitro-4-(trifluoromethyl)benzyl)-1H -1,2,3-Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ12.90(s, 1H), 9.46(s, 1H), 9.04(s, 1H), 8.45(s, 1H), 8.37(d, J=8.2Hz, 1H), 8.21(d,J=8.2Hz,1H),6.74(s,2H).(ESI,m/z):[M+Na] ⁺ :434.1.

Example 56 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-((6-(trifluoromethyl)pyridin-3-yl)methylene )-1H-1,2,3-triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.20(s, 1H), 9.27(s, 1H), 9.04(s, 1H), 8.64(s, 1H), 8.37(d, J=7.8Hz, 1H), 7.92(d,J=8.2Hz,1H),6.42(s,2H).(ESI,m/z):[M+H] ⁺ :368.1.

Example 57 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(2-hydroxy-4-nitrobenzyl)-1H-1,2, 3-Triazole-4-carbohydrazide

¹ H NMR (500MHz, DMSO) δ 12.97(s, 1H), 12.71(s, 1H), 9.47(s, 1H), 8.86(s, 1H), 8.03(dd, J=8.2, 1.5Hz, 1H ),7.94(dd,J=7.7,1.3Hz,1H),7.14(t,J=7.9Hz,1H),6.74(s,2H).(ESI,m/z):[M+Na] ⁺ : 382.1.

Example 58 (E)-1-(4-Amino-1,2,5-oxadiazol-3-yl)-N'-(2,3-dihydroxybenzylidene)-1H-1,2,3 -Triazole-4-carbohydrazide

¹ H NMR(500MHz,DMSO)δ12.62(s,1H), 11.02(s,1H), 9.42(s,1H), 9.24(s,1H), 8.74(s,1H), 6.98(d,J =7.7Hz,1H),6.91–6.87(m,1H),6.76(m,3H).(ESI,m/z):[M+H] ⁺ :333.1.

Example 59 (E)-2-((2-(1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4-carbonyl) Hydrazone)methyl)benzoic acid

¹ H NMR (500MHz, DMSO) δ13.39(s, 1H), 12.53(s, 1H), 9.42(s, 1H), 9.29(s, 1H), 8.08(d, J=7.7Hz, 1H), 7.93(dd,J=7.8,0.8Hz,1H),7.68(t,J=7.4Hz,1H),7.56(td,J=7.8,1.0Hz,1H),6.74(s,2H).(ESI, m/z):[M+H] ⁺ :343.1.

Table 1

NA: No activity A: IC50>20μM B: IC50<20μM

Embodiment 2. Validation experiment of the inhibitory effect of SIRT6 allosteric inhibitor on SIRT6

In this example, the inhibitory effect of the SIRT6 allosteric inhibitor of the present invention on the deacetylation activity of SIRT6 was verified. The SIRT6 allosteric inhibitor uses the compounds listed in the above examples 1 to 59 as active ingredients. In this example, the deacetylation activity of SIRT6 was detected by the method of fluorescence quantification. After SIRT6 deacetylates the ε-acetyl on the lysine of the polypeptide, trypsin is used to cleave to generate free AMC, and then the reaction is quantified by fluorescence to determine the amount of the product.

The specific experimental method is as follows: take 50 μL of the reaction solution and react at 37°C for 2 hours, then add nicotinamide with a final concentration of 20 mM to terminate the reaction; then add trypsin with a final concentration of 3 mg/mL to carry out the cleavage reaction for 30 minutes; Fluorescence quantitative detection of the reaction was carried out with an instrument (Synergy H4 Hybrid Reader, BioTek), the excitation wavelength was 360 nm, and the emission wavelength was 460 nm. The blank group reaction solution contained 2.5 mM NAD+, 75 μM substrate polypeptide RHKK-Ac-AMC, 5 μM SIRT6, and the reaction buffer consisted of 50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl ₂ , the pH value is 8.0. The reaction solution of the experimental group was the reaction solution of the blank group, which further contained 100 μM of inhibitor, and the inhibitor was dissolved in DMSO, and the SIRT6 deacetylation activity of the blank group and the experimental group was compared. The SIRT6 allosteric inhibitor used the compounds listed in the above examples 1 to 59 as active ingredients, and the inhibition rate of the compounds on SIRT6 enzyme activity at a concentration of 100 μM was measured. The results are shown in Table 1.

In this example, the SIRT6 allosteric inhibitor using the compounds listed in Example 1-Example 59 as the active ingredient was further determined to the half-inhibitory concentration IC ₅₀ of SIRT6 deacetylase activity. The SIRT6 deacetylation activity was measured by the above method, and the measured data were obtained by GraphPad Prism 6 software to obtain the IC ₅₀ of the inhibitor on SIRT6 inhibitory effect under various concentration conditions. The results are shown in Table 1. The results show that the SIRT6 allosteric inhibitors using the compounds listed in the above examples 1 to 59 as active ingredients have an inhibitory effect on the activity of SIRT6 deacetylase.

The deacetylation activity of SIRT6 was measured at different concentrations of the inhibitor, and the results are shown in Figure 1. Figure 1 shows that the inhibitor using the compound of Example 42 as an active ingredient inhibited the deacetylation activity of SIRT6 in a concentration-dependent manner.

Example 3. Cell detection

This example verifies that the compounds of the present invention inhibit the deacetylation activity of SIRT6 in cells. The cell lines used in this example were selected from tumor cell lines THP1, K562, U937, HDLM-2, Hela, Hela S3, BXPC-3, Mia-paca-2, Panc-1, A375, SK-MEL-2, Capan- 2 is the test object, these cell lines include leukemia cell lines, cervical cancer cell lines, pancreatic cancer cell lines and skin cancer cell lines. The series of cell lines were cultured according to the cell culture conditions recommended by ATCC to select appropriate cell culture medium. The above cell lines were all identified and analyzed by Short Tanden Repeat (STR).

Cell detection is divided into three ways: 1. Western blot to detect the effect of compounds on the activity of intracellular SIRT6 deacetylase; 2. Cell growth experiments to detect the growth inhibition of compounds on tumor cells; 3. RTCA experiments to detect compounds on tumor cells. Cell migration inhibition. The specific method is as follows:

1. Western blot detection: BXPC-3, Mia-paca-2 and Capan-2 and other tumor cell lines were cultured in the corresponding cell culture medium containing double antibody. A gradient concentration of inhibitor stock solutions (dissolved in DMSO) was prepared and cells were treated at final concentrations of 0 μM, 10 μM, 20 μM and 40 μM. The well-conditioned cells grown in the corresponding medium were seeded into 6-well plates, and 3*10 ⁵ cells per well were cultured at 37°C and 5% CO ₂ . If the cells are in good condition at 24h, change to fresh cell culture medium (2ml), then add the above-prepared inhibitor to the cell culture medium of the experimental group at a ratio of 1:1000, and add the same volume of DMSO (2μL) to the control group. The culture was continued for a certain period of time (12 hours, 24 hours, 48 hours). The inhibitory activity of the inhibitor on histone H3 deacetylase activity of SIRT6 was verified by Western blot.

The intracellular SIRT6 deacetylation was detected by Western blot after treating the pancreatic cancer cell line BXPC-3, the pancreatic cancer cell line Mia-paca-2, and the pancreatic cancer cell line Capan-2 with different concentrations of the compound of Example 42 for 24 hours. The active substrates H3 K9Ac, H3 K18Ac and H3 K56Ac (H3 K9Ac, H3 K18Ac and H3 K56Ac refer to the acetylation of the 9th, 18th, and 56th lysine residues of histone H3, respectively) are shown in Figure 2- Figure 4 shows that the compound of Example 42 can concentration-dependently up-regulate H3 K9Ac, H3 K18Ac and H3 K56Ac, thus indicating that the compound of Example 42 can inhibit SIRT6 depletion in BXPC-3, Mia-paca-2, Capan-2 cells Acetylation activity.

2. Functional detection of tumor cell proliferation and death: IncuCyte ZOOM (Essen, USA) live cell real-time dynamic imaging system was used to detect the effect of compound 42 on cell proliferation and death. Cells grown in the corresponding medium in good condition were seeded into 96-well cell culture plates with 8000 cells per well, and cultured at 37°C and 5% CO ₂ . Inhibitors (0 μM, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, 250 μM) and 0.1 μM fluorescent dye YOYO-1 Iodide were added. After that, the IncuCyte ZOOM system was used to measure the degree of cell confluency and the number of fluorescently stained cells every 2 hours. The half effective concentration _IC50 of the compound to inhibit cell proliferation was then calculated from the cell confluency data.

The leukemia cell line U937 and HDLM2 were treated with the compound of Example 42 at different concentrations (0 μM, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, 250 μM), and the growth curve of the compound inhibiting cell proliferation and the compound’s inhibition of cell proliferation were examined. The half effective concentration _IC50 is shown in Figure 5, indicating that the compound of Example 42 can inhibit the cell proliferation of leukemia cells U937 and HDLM2 in a concentration-dependent manner.

The skin cancer cell line A375 was treated with the compound of Example 42 at different concentrations for 24 hours, and the growth curve of the compound to inhibit cell proliferation and the half effective concentration IC50 of the compound to inhibit cell proliferation were shown in Figure 6, indicating that the compound of Example 42 can be concentration-dependently Inhibits cell proliferation of skin cancer cells A375.

The skin cancer cell line SK-MEL-2 was treated with different concentrations of the compound of Example 42 for 48 hours, and the growth curve of the compound to inhibit cell proliferation and the IC50 of the half effective concentration of the compound to inhibit cell proliferation are shown in Figure 7, indicating that the compound of Example 42 can inhibit cell proliferation. Concentration-dependent inhibition of cell proliferation of skin cancer cells SK-MEL-2.

The cervical cancer cell line Hela S3 was treated with different concentrations of the compound of Example 42 for 24 hours, and the growth curve of the inhibition of cell proliferation by the test compound is shown in Figure 8, indicating that the compound of Example 42 can inhibit the cell proliferation of cervical cancer cell Hela S3 in a concentration-dependent manner .

The cervical cancer cell line Hela S3 was treated with different concentrations of the compound of Example 42 for 48 hours, and the growth curve of the compound to inhibit cell proliferation and the half effective concentration IC50 of the compound to inhibit cell proliferation were shown in Figure 9, indicating that the compound of Example 42 can be concentration-dependent. inhibited the proliferation of cervical cancer cells Hela S3.

The pancreatic cancer cell line BXPC-3 was treated with the compound of Example 42 at different concentrations, and the growth curve of the compound to inhibit cell proliferation and the IC50 of the half effective concentration of the compound to inhibit cell proliferation are shown in Figure 10, indicating that the compound of Example 42 can be concentration-dependently Inhibits cell proliferation of pancreatic cancer cells BXPC-3.

The pancreatic cancer cell line Mia-paca-2 was treated with the compound of Example 42 at different concentrations, and the growth curve of the obtained compound to inhibit cell proliferation and the half effective concentration IC50 of the compound to inhibit cell proliferation are shown in Figure 11, indicating that the compound of Example 42 can inhibit cell proliferation. Concentration-dependent inhibition of cell proliferation of pancreatic cancer cell Mia-paca-2.

The pancreatic cancer cell line Capan-2 was treated with the compound of Example 42 at different concentrations, and the growth curve of the compound to inhibit cell proliferation and the half-effective concentration IC50 of the compound to inhibit cell proliferation were shown in Figure 12, indicating that the compound of Example 42 can be concentration-dependently Inhibits cell proliferation of pancreatic cancer cells Capan-2.

3. Functional detection of tumor cell migration: Real-time label-free cell analysis (RTCA) detection of cell migration: Resuspend 6*10 ⁴ cells in serum-free culture with good growth status in 100 μL serum-free medium, and in the medium. Contains 30 ng/mL PMA and a range of concentrations (0 μM, 5 μM, 10 μM, 20 μM, 40 μM) of inhibitor. The cell resuspension was added to the upper chamber, and the lower chamber was added with complete medium containing 10 ng/mL IL8, 30 ng/mL PMA and inhibitors at the same concentrations (0 μM, 5 μM, 10 μM, 20 μM, 40 μM) as in the upper chamber. The cell index was detected and recorded every 15 min, and the migration curve was analyzed with RTCAsoftware v1.2 (Roche Applied Science).

Pancreatic cancer cell line BXPC-3, pancreatic cancer cell line Mia-paca-2, cervical cancer cell line Hela S3 and skin cancer cell line A375 were treated with different concentrations of the compound of Example 42, and the inhibitory effect of the compound on cell migration was detected by RTCA method , the results are shown in Figures 13-16, indicating that: with the increase of the concentration of the compound of Example 42, the cell index decreased and the cell migration rate decreased. It is illustrated that the compound of Example 42 can inhibit cell migration in BXPC-3, Mia-paca-2, Hela S3, A375 cells.

A compound with the activity of inhibiting SIRT6 deacetylation provided in the examples of this application, the SIRT6 allosteric inhibitor obtained by using the compound and its application in the preparation of cancer drugs have been described in detail above. The principles and implementations of the present application are described in a single example, and the descriptions of the above embodiments are only used to help understand the method and the core idea of the present application; There will be changes in the method and application scope. To sum up, the content of this specification should not be construed as a limitation on the application.

Claims (9)

1. A compound represented by formula (1) or a pharmaceutically acceptable salt thereof:

wherein R is an aromatic group of C6-C20 or a C6-C20 aromatic group containing a substituent.

2. The compound of claim 1, wherein said aromatic group is phenyl, pyridyl, furyl, thienyl, pyrrolyl, imidazolyl, thiazolyl, naphthyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyranyl, pyrimidinyl, or isothiazolyl.

3. The compound of claim 2, wherein a hydrogen atom of at least one site in said aromatic group is substituted with a substituent.

4. A compound according to claim 3, wherein the substituents are one or more of halogen, hydroxy, nitro, amino, carboxy, ester, sulfonamide, mercapto, methoxy, ethoxy, benzyloxy, methyl, tert-butyl, cyano, or dimethoxyethanol.

5. The compound of claim 1, wherein the compound is selected from the group consisting ofIn the compound of formula (1), R is selected from one of the following groups:

6. the compound of claim 1, wherein the pharmaceutically acceptable salt is a hydrochloride, sulfate, oxalate, acetate, trifluoroacetate or citrate of the compound of formula (1).

7. Use of a compound of any one of claims 1-6 in the preparation of a SIRT6 allosteric inhibitor.

8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6.

9. Use of a compound according to any one of claims 1 to 6 for the manufacture of a medicament for the treatment of cancer.

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