CN106916101B - NAMPT/HDAC dual-target inhibitor and preparation method thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种NAMPT/HDAC双靶点抑制剂及其制备方法；该双靶点抑制剂为酰胺类衍生物及其药学上可接受的盐，其结构通式如式(I)所示：

(I)。药理实验表明，本发明所述的衍生物或盐，对烟酰胺磷酸核糖转移酶和组蛋白去乙酰化酶均具有很强的抑制活性，而且具有较强的体外抗肿瘤活性和优秀的体内抑瘤效果。本发明还提供了上述衍生物及其药学上可接受的盐的制备方法，以及在制备烟酰胺磷酸核糖转移酶抑制剂、组蛋白去乙酰化酶抑制剂和抗肿瘤药物中的应用。

The invention discloses a NAMPT/HDAC dual-target inhibitor and a preparation method thereof; the dual-target inhibitor is an amide derivative and a pharmaceutically acceptable salt thereof, and its general structural formula is shown in formula (I) :

(I). Pharmacological experiments show that the derivatives or salts of the present invention have strong inhibitory activity on both nicotinamide phosphoribosyltransferase and histone deacetylase, and have strong in vitro antitumor activity and excellent in vivo inhibitory activity. tumor effect. The present invention also provides a preparation method of the above derivatives and pharmaceutically acceptable salts thereof, as well as applications in the preparation of nicotinamide phosphoribosyltransferase inhibitors, histone deacetylase inhibitors and antitumor drugs.

Description

NAMPT/HDAC double-target inhibitor and preparation method thereof

Technical Field

The invention belongs to the technical field of medicine; in particular to an application of NAMPT/HDAC double-target inhibitor with anti-tumor activity and a medicinal preparation thereof in the aspect of medicaments for treating malignant tumors and diseases related to differentiation and proliferation.

Background

The multi-target medicine can simultaneously regulate a plurality of links in a network system of tumor diseases, is not easy to generate drug resistance, has a total effect on each target larger than the sum of single effects, and achieves the optimal treatment effect. Furthermore, a single molecule possesses relatively simple absorption, distribution, metabolism and excretion processes, greatly reducing drug-drug interactions (Peters, j., et. j. med. chem.2013, 56, 8955-8971). The FDA successively approves a plurality of multi-target tyrosine kinase inhibitors to be marketed, including sorafenib (sorafenib), dasatinib (dasatinib) and lapatinib (lapatinib), and the like, and marks that multi-target drugs become a new direction for tumor treatment and drug development.

Acetylation/deacetylation of histones is an important regulation mode of chromosome structure change and gene expression, and plays an important role in the life processes of apoptosis, energy metabolism, transcription, translation and the like. The acetylation and deacetylation modification process of histone is mainly completed by co-catalysis of Histone Acetylases (HATs) and Histone Deacetylases (HDACs). In tumor cells such as prostate cancer, gastric cancer, breast cancer, colon cancer and leukemia, the expression of HDACs is abnormally increased, and the development of small molecule drugs aiming at HDACs becomes a hot point of the current anti-tumor targeted therapy. HDAC inhibitors by structural class fall into two main classes: hydroxamic acids and amides. Representative compounds are vorinostat (SAHA) and CI-994, respectively. SAHA is approved by the FDA in the united states for clinical use in the treatment of hematological cancers; CI-994 is currently undergoing phase II clinical laboratory studies. In addition, a number of HDAC inhibitors are undergoing clinical trials. However, single HDAC-targeted drugs lack effective treatment for solid tumors. HDAC has synergistic anti-tumor efficacy with multiple tumor targets, such as microprotein (Tubulin) and heat shock protein 90(Hsp90), and the development of HDAC-based multi-target drugs is an effective means for improving anti-tumor efficacy and reducing tumor resistance.

In recent years, the development of drug research and development aiming at metabolic targets has become an important means and an effective strategy for the discovery of anti-tumor drugs. Nicotinamide phosphoribosyltransferase (NAMPT) is an extremely representative metabolic target. NAMPT catalyzes Nicotinamide (NAM) to generate Nicotinamide Mononucleotide (NMN), regulates the level of an essential energy substance NAD of a mammalian cell, is a rate-limiting enzyme of an NAD generation pathway, and plays a vital role in cell physiological activities. Research shows that NAMPT is closely related to the generation and development of tumors and becomes a very important new target in the research of anti-tumor drugs: 1) the tumor cells have high NAD consumption and metabolic rate, and are more dependent on NAD than normal cells and are more easily influenced by NAMPT inhibitors; 2) NAD in tumor cells is used as an essential coenzyme to participate in the synthesis of various tumor essential substances, and can significantly reduce the level of Reactive Oxygen Species (ROS) in the environment and protect the tumor cells; 3) NAMPT plays an important role in angiogenesis and the induction of the generation of vascular endothelial growth factor. Currently, the NAMPT inhibitors reported in the research include FK866 and CHS-828, both of which have been introduced into clinical studies. Clinical data show that both are dose-dependent thrombocytopenia and gastrointestinal side effects, and further improvement is needed.

Disclosure of Invention

The invention aims to provide an amide HDAC/NAMPT double-target inhibitor with high efficiency and low toxicity and pharmaceutically acceptable salt thereof; the invention also aims to provide a preparation method of the HDAC/NAMPT double-target inhibitor and pharmaceutically acceptable salts thereof; the third purpose of the invention is to provide the application of the HDAC/NAMPT double-target inhibitor and the pharmaceutically acceptable salt thereof in preparing NAMPT inhibitors, HDAC inhibitors and antitumor drugs.

The purpose of the invention is realized by the following technical scheme:

the first aspect of the invention provides an amide derivative and a pharmaceutically acceptable salt thereof, wherein the structural general formula of the compound is shown as the formula (I):

wherein, A group is selected from any one of (1) to (4):

(1) phenyl or substituted phenyl

The substituent on the substituted phenyl benzene ring can be positioned at the ortho position, the meta position and the para position of the benzene ring, and can be mono-substituted or multi-substituted, and the substituent is selected from any one or more of the following groups:

a. halogen atoms (including F, Cl, Br, I),

b. a straight chain alkyl group of 1 to 6 carbon atoms or a branched chain alkyl group of 3 to 6 carbon atoms, (preferably, the straight chain alkyl group of 1 to 6 carbon atoms includes methyl, ethyl, propyl, n-butyl, n-pentyl, n-hexyl; the branched chain alkyl group of 3 to 6 carbon atoms includes isopropyl, isobutyl, tert-butyl, isopentyl, tert-pentyl),

c. hydroxy, cyano, cyanomethyl, amino, nitro, trifluoromethyl, trifluoromethoxy, methoxy, ethoxy, propoxy, isopropoxy, butoxy, acetyl;

(2) pyridyl or substituted pyridyl

The substituent on the substituted pyridine can be positioned at any substitutable position on the pyridine ring, and can be monosubstituted or polysubstituted, and the substituent is selected from any one or more of the following:

a. halogen atoms (including F, Cl, Br, I),

b. a straight chain alkyl group of 1 to 6 carbon atoms or a branched chain alkyl group of 3 to 6 carbon atoms, (preferably, the straight chain alkyl group of 1 to 6 carbon atoms includes methyl, ethyl, propyl, n-butyl, n-pentyl, n-hexyl; the branched chain alkyl group of 3 to 6 carbon atoms includes isopropyl, isobutyl, tert-butyl, isopentyl, tert-pentyl),

c. hydroxy, cyano, cyanomethyl, amino, nitro, trifluoromethyl, trifluoromethoxy, methoxy, ethoxy, propoxy, isopropoxy, butoxy, acetyl;

(3) five-membered heterocycle or substituted five-membered heterocycle (five-membered heterocycle refers to furan, thiophene, pyrrole gene)

The substituent on the five-membered heterocyclic ring can be positioned at any substitutable position, and can be monosubstituted or polysubstituted, and the substituent is selected from any one or more of the following:

a. halogen atoms (including F, Cl, Br, I),

b. a straight chain alkyl group of 1 to 6 carbon atoms or a branched chain alkyl group of 3 to 6 carbon atoms, (preferably, the straight chain alkyl group of 1 to 6 carbon atoms includes methyl, ethyl, propyl, n-butyl, n-pentyl, n-hexyl; the branched chain alkyl group of 3 to 6 carbon atoms includes isopropyl, isobutyl, tert-butyl, isopentyl, tert-pentyl),

c. hydroxy, cyano, cyanomethyl, amino, nitro, trifluoromethyl, trifluoromethoxy, methoxy, ethoxy, propoxy, isopropoxy, butoxy, acetyl;

(4) parallel group and bicyclic heterocyclic group composed of five-membered or six-membered ring;

the E group is selected from one of the following groups:

the X group is selected from one of the following groups:

wherein, R1 refers to mono-substituted or poly-substituted, and is selected from any one or more of the following:

a.H，

b. halogen atoms (F, Cl, Br, I),

g.a straight chain alkyl group of 1 to 6 carbon atoms or a branched alkyl group of 3 to 6 carbon atoms, (preferably, the straight chain alkyl group of 1 to 6 carbon atoms includes methyl, ethyl, propyl, n-butyl, n-pentyl, n-hexyl; the branched alkyl group of 3 to 6 carbon atoms includes isopropyl, isobutyl, tert-butyl, isopentyl, tert-pentyl),

h. hydroxy, cyano, cyanomethyl, amino, nitro, trifluoromethyl, trifluoromethoxy, methoxy, ethoxy, propoxy, isopropoxy, butoxy, acetyl;

y is a carbon atom or a nitrogen atom;

the Z group is selected from any one of a to d:

a.H

b. halogen atom (F, Cl, Br, I)

c. Benzene ring or six-membered heterocyclic ring

d. Five-membered heterocycle (including pyrrole, furan, thiophene group)

Preferably, the parallel group and bicyclic heterocyclic group consisting of five-membered or six-membered rings include:

preferably, the pharmaceutically acceptable salt is preferably: hydrochloride, hydrobromide, sulphate, acetate, lactate, tartrate, tannate, citrate, trifluoroacetate, malate, maleate, succinate, p-toluenesulphonate or methanesulphonate;

preferably, the pharmaceutically acceptable salt does not contain water of crystallization, or contains one or more molecules of water of crystallization.

More preferably, the pharmaceutically acceptable salt contains 0.5 to 3.0 molecules of water of crystallization.

HDAC inhibitors and HDAC/NAMPT dual-target inhibitors having differentiation and anti-proliferative activity and pharmaceutical formulations thereof, some preferred compounds of the present invention include, but are not limited to, the following:

n- (2-aminophenyl) -4- (3- (6-fluoropyridin-3-yl) methyl) thioureido) benzamide

N- (2-aminophenyl) -4- (3- (pyridin-3-ylmethyl) ureido) benzamide

N- (2-aminophenyl) -4- (3- (pyridin-3-yl) acrylamido) benzamide

N- (2-aminophenyl) -4- (3- (6-chloropyridin-3-yl) methyl) thioureido) benzamide

N- (2-aminophenyl) -4- (3- (6-chloropyridin-3-yl) methyl) ureido) benzamide

N- (2-aminophenyl) -4- (3- (6-fluoropyridin-3-yl) methyl) ureido) benzamide

N- (2-aminophenyl) -4- (3- (6-aminopyridin-3-yl) methyl) thioureido) benzamide

N- (2-aminophenyl) -4- (3- (6-aminopyridin-3-yl) methyl) ureido) benzamide

Tert-butyl (5- ((3- (4- ((2-aminophenyl) carbamoyl) phenyl) ureido) methyl) pyridin-2-yl) carbamate

N- (2-aminophenyl) -4- (3- (5-fluoropyridin-3-yl) methyl) ureido) benzamide

N- (2-aminophenyl) -4- (3- (6-methylpyridin-3-yl) methyl) ureido) benzamide

N- (2-aminophenyl) -4- (3- (4-fluorobenzyl) thioureido) benzamide

N- (2-aminophenyl) -4- (3- (3-fluorobenzyl) thioureido) benzamide

N- (2-aminophenyl) -4- (3- (2-fluorobenzyl) thioureido) benzamide

N- (2-aminophenyl) -4- (3- (3-chlorobenzyl) thioureido) benzamide

N- (2-aminophenyl) -4- (3- (3-bromobenzyl) thiourea) benzamide

N- (2-aminophenyl) -4- (3- (benzo [ d ] [1, 3] dioxol-5-ylmethyl) thioureido) benzamide

N- (2-aminophenyl) -4- (3- (3-aminobenzyl) thioureido) benzamide

4- (3- ((1H-benzo [ d ] imidazol-2-yl) methyl) thioureido) -N- (2-aminophenyl) benzamide

N- (2-aminophenyl) -4- (2, 3-dichloro-3- (pyridin-3-yl) acrylamido) benzamide

N- (4- ((2-aminophenyl) carbamoyl) benzyl) thieno [3, 2-c ] pyridine-2-carboxamide

N- (4- ((2-aminophenyl) carbamoyl) benzyl) -1H-pyrrolo [2, 3-c ] pyridine-2-carboxamide

N- (4- ((2-aminophenyl) carbamoyl) benzyl) -1H-pyrrolo [3, 2-c ] pyridine-2-carboxamide

N- (4- ((2-aminophenyl) carbamoyl) benzyl) -1H-pyrrolo [2, 3-b ] pyridine-2-carboxamide

N- (4- ((2-aminophenyl) carbamoyl) benzyl) imidazo [1, 5-a ] pyridine-6-carboxamide

N- (4- ((2-aminophenyl) carbamoyl) benzyl) -1H-pyrazolo [4, 3-b ] pyridine-6-carboxamide

N- (4- ((2-aminophenyl) carbamoyl) benzyl) imidazo [1, 2-a ] pyridine-7-carboxamide

N- (2-aminophenyl) -4- ((4- (pyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide

N- (2-aminophenyl) -4- (2- (4- (pyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide

N- (2-aminophenyl) -4- ((4- (pyridin-4-yl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide

N- (2-aminophenyl) -4- ((4- (6-chloropyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide

N- (2-aminophenyl) -4- ((4- (3-aminophenyl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide

N- (2-aminophenyl) -4- ((4- (m-tolyl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide

4- ((4- (4-acetylphenyl) -1H-1, 2, 3-triazol-1-yl) methyl) -N- (2-aminophenyl) benzamide

N- (2-aminophenyl) -4- ((4- (thien-2-yl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide

N- (4-amino- [1, 1' -biphenyl ] -3-yl) -4- ((5- (pyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide

N- (4-amino- [1, 1' -biphenyl ] -3-yl) -4- ((5- (3-aminophenyl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide

N- (4-amino- [1, 1' -biphenyl ] -3-yl) -4- ((5- (3-hydroxyphenyl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide

4- ((5- (4-acetylphenyl) -1H-1, 2, 3-triazol-1-yl) methyl) -N- (4-amino- [1, 1' -biphenyl ] -3-yl) benzamide

N- (2-aminophenyl) -4- ((4- (4- (3- (pyridin-3-ylmethyl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide

N- (2-aminophenyl) -4- ((4- (4- ((pyridin-3-ylmethyl) carbamoyl) phenyl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide

N- (2-aminophenyl) -8- (4- (pyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) octanoyl amide

N- (2-aminophenyl) -8- (4- (3-aminophenyl) -1H-1, 2, 3-triazol-1-yl) octanoyl amide

N- (4-amino- [1, 1' -biphenyl ] -3-yl) -8- (4- (pyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) octanamide

N- (4-amino- [1, 1' -biphenyl ] -3-yl) -8- (4- (3-aminophenyl) -1H-1, 2, 3-triazol-1-yl) octanoyl amide

N- (4-amino- [1, 1' -biphenyl ] -3-yl) -8- (4- (3-hydroxyphenyl) -1H-1, 2, 3-triazol-1-yl) octanamide

The structural formula and nuclear magnetic mass spectrometry data of the above preferred compounds of the present invention are shown in table 1:

TABLE 1 structural formulas and NMR Mass Spectroscopy data for preferred Compounds of the invention

In a second aspect of the present invention, there are provided processes for the preparation of the above amide derivatives and pharmaceutically acceptable salts thereof;

reaction scheme I (Synthesis of Compound A1-20:)

When the general formula is

And X₂Is O or S, R is-NH-CH₂-a, -CH ═ CH-a or

A is phenyl, amino-substituted phenyl, halogen-substituted phenyl, pyridyl, straight-chain alkyl-substituted pyridyl with 1-6 carbon atoms, branched-chain alkyl-substituted pyridyl with 3-6 carbon atoms, halogen-substituted pyridyl, amino-substituted pyridyl,

The method comprises the following steps:

(wherein, R2 represents hydrogen, halogen, amino or tert-butoxycarbonylamino)

Reagents and conditions: (a) raney nickel, hydrogen, ammonia water methanol solution, room temperature, 12 hours, and the yield is 21-89%; (b) CDIor TCDI, dichloromethane, room temperature, 2 hours, yield 85-95%; (c) tetrahydrofuran, room temperature, 12 hours, yield 62-81%; (d) dichloromethane, room temperature or 70 ℃, 2 hours, yield 60%; (e) tetrahydrofuran, room temperature, 12 hours, yield 70%; (f) o-phenylenediamine, HBTU, triethylamine, DMF, room temperature, 3 hours, yield 43-75%.

The cyano compound 1 is catalyzed by Raney nickel, and is reduced by hydrogen in ammonia methanol solution to obtain an amino compound 2 a. Reaction of p-aminobenzoic acid 3 with CDI or TCDI at room temperature gives intermediate 4. Compounds 2a or 2c and 4 were reacted in tetrahydrofuran at room temperature for 12 hours to give intermediate 5. Condensation of compound 2b with 3 in dichloromethane gave intermediate 5. And reacting the intermediate 5 with o-phenylenediamine in DMF at room temperature for 4 hours, and performing column chromatography to obtain the target compound A1-20.

Reaction scheme two (synthesis of compound B1-7):

when the general formula is

And A is

The method comprises the following steps:

reagents and conditions: (a) R-COOH, EDC, DMAP, DMF, room temperature, 3 hours, 31-69% yield; (b) lithium hydroxide, tetrahydrofuran/methanol/water, room temperature, 24 hours, yield 82-97%; (c) o-phenylenediamine, HBTU, triethylamine, DMF, room temperature, 4 hours, yield 37-75%.

Methyl 4-aminomethylbenzoate 7 and various carboxylic acids were subjected to EDC/DMAP catalyzed condensation to give intermediate 8. In a tetrahydrofuran/methanol/water mixed solvent system, the intermediate 8 is hydrolyzed by lithium hydroxide to obtain the corresponding carboxylic acid compound 9. And reacting the compound 9 with o-phenylenediamine in DMF at room temperature for 3 hours, and performing column chromatography to obtain a target compound B1-7.

Reaction scheme three (Synthesis of Compound C1-14):

when the general formula is

And n is 1 or 2, A is pyridyl, halogen substituted pyridyl, amino substituted pyridyl, straight chain alkyl substituted phenyl of 1-6 carbon atoms, acetylPhenyl, thienyl, substituted phenyl,

When Z is a hydrogen atom or a phenyl group, the method is as follows:

reagents and conditions: (a) trimethylsilyl acetylene, palladium triphenylphosphine, copper iodide and triethylamine are added at room temperature for 1 hour; (b) potassium carbonate, methanol, room temperature, 0.5 hour, two-step yield 32-81%; (c) sodium azide at room temperature for 8 hours, and the yield is 64-85 percent; (d) intermediate 13 or RCN, copper sulfate, sodium ascorbate and nitrogen protection, room temperature, overnight, yield 64-87%; (e) lithium hydroxide, tetrahydrofuran/methanol/water, room temperature, 24 hours, yield 71-92%; (f) o-phenylenediamine or [1, 1' -biphenyl ] -3, 4-diamine, HBTU, triethylamine, at room temperature, 3 hours, yield 27-72%.

The compound 11 reacts with trimethylsilyl acetylene to obtain a compound 12, and trimethylsilyl is removed at room temperature under an alkaline condition to obtain a compound 13. Substitution of bromide 14 with sodium azide provided intermediate 15. The intermediate 15 is subjected to click reaction with 13 or other alkynyl compounds to obtain a product 16. The intermediate 16 is hydrolyzed under alkaline condition and condensed with o-phenylenediamine or 4-phenyl o-phenylenediamine to finally obtain the target compound C1-14.

Reaction scheme four (Synthesis of Compound D1-5):

when the general structure is

And R' is amino, hydroxyl or hydrogen atom, X₃When the N or CH and Z are hydrogen atoms or phenyl, the method comprises the following steps:

reagents and conditions: (a) sodium azide, DMF, 85 degrees, 6 hours; (b) o-phenylenediamine or [1, 1' -biphenyl ] -3, 4-diamine, HBTU, triethylamine, at room temperature for 3 hours; (c) copper sulfate, sodium ascorbate, tert-butanol and water 5: 1 at room temperature under nitrogen for 24 hours.

The compound 18 reacts with sodium azide to obtain an intermediate 19, and the intermediate 20 is obtained by condensation reaction of the intermediate 19 with o-phenylenediamine or 4-phenyl o-phenylenediamine under the catalysis of HBTU and triethylamine. The intermediate 20 is subjected to Click reaction by using aryl acetylene containing different substituents to obtain a compound D1-5.

In a third aspect of the present invention, there is provided a use of the above amide derivatives and pharmaceutically acceptable salts thereof in the preparation of nicotinamide phosphoribosyltransferase inhibitors, histone deacetylase inhibitors or HDAC/NAMPT dual-target inhibitors.

Experiments on the inhibition of NAMPT and HDAC1 activity prove that most compounds have better NAMPT and HDAC1 inhibition activity, and A2, A3, C1, C2, C9, C13, C14, D1, D2 and D3 have balanced inhibition activity on NAMPT and HDAC 1.

The invention also provides the application of the amide derivatives and the pharmaceutically acceptable salts thereof in preparing antitumor drugs.

Preferably, the tumor is liver cancer, lung cancer, intestinal cancer, ovarian cancer, prostatic cancer, gastric cancer, lymph cancer, etc.

The anti-tumor activity experiment proves that most of the compounds have better in-vitro anti-tumor activity, and the preferable compounds have excellent in-vivo tumor growth inhibition activity.

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

according to the invention, the amide HDAC/NAMPT double-target inhibitor is reasonably constructed by a pharmacophore fusion strategy, so that high-efficiency small molecules with balanced inhibition activity on NAMPT and HDAC1 are obtained, and the in-vivo drug effect is remarkably improved; opens up a new way for the deep research and development of new structural antitumor drugs and provides a new strategy.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a schematic diagram showing the change of tumor volume of human intestinal cancer HCT116 nude mice; wherein, (a) a tumor volume line graph; (B) tumor anatomy after dosing was completed.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention. The percentages stated in the present invention are by weight unless otherwise specified.

EXAMPLE 1 preparation of N- (2-aminophenyl) -4- (3- (6-fluoropyridin-3-yl) methyl) thioureido) benzamide(A1)

1.1 preparation of intermediate 2: 6-fluoro-3-aminomethylpyridines

6-fluoro-3-cyanopyridine (300mg, 2.46mmol) was dissolved in 2mol/L ammonia/methanol solution (30mL) and Raney nickel (0.5g), H, was added₂The reaction was carried out at room temperature under an atmosphere for 12 hours. After completion of the reaction, the reaction mixture was filtered through celite, washed with methanol (5mL × 2), the filtrate was evaporated to dryness, and the residue was purified by silica gel column chromatography (dichloromethane: methanol 100: 4) to give 262mg of a yellow oily solid in a yield of 85%.¹H-NMR(DMSO-d₆，300MHz)δ：8.11-6.84(m，3H)，3.86(s，2H)，1.47(s，2H).

1.2 preparation of intermediate 4: 4-isothiocyanatobenzoic acid

Triethylamine (1.4mL, 194mmol) was dissolved in dichloromethane (12mL), thiocarbonyldiimidazole (2.11g, 11.85mmol) and 4-aminobenzoic acid (1.25g, 11.57mmol) were added under ice-bath, and stirring was carried out for 1 hour under ice-bath. Concentrated hydrochloric acid (3mL, 33.60mmol) was added dropwise to n-hexane (12mL) to prepare a mixed solution, and the mixed solution was slowly added dropwise to the reaction mixture and stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was placed in an ice bath, filtered, and the filter cake was washed with water (5 mL. times.2) to give 1.93g of an off-white solid in a yield of 93%.¹H-NMR(DMSO-d₆，300MHz)δ：13.20(br s，1H)，7.52(d，J＝8.7Hz，2H)，7.97(d，J＝8.7Hz，2H).

1.3 preparation of intermediate 5: 4- (3- ((6-Fluoropyridin-3-yl) methyl) thioureido) benzoic acid

6-fluoro-3-aminomethylpyridine (133mg, 1.06mmol) and 4-isothiocyanatobenzoic acid (189mg, 1.06mmol) were dissolved in dry THF (10mL) and reacted at room temperature for 12 hours, after which the solvent was evaporated to dryness to give 0.26g of a yellow solid in 81% yield.¹H-NMR(DMSO-d₆，300MHz)δ：10.24(s，1H)，8.75(s，1H)，8.36-8.34(m，2H)，7.87(d，J＝8.5Hz，2H)，7.63-7.58(m，3H)，4.80(d，J＝5.4Hz，2H).ESI-MS(m/s)：287.12[M+1].

1.4 preparation of the desired product A1

4- (3- ((6-Fluoropyridin-3-yl) methyl) thioureido) benzoic acid (90mg, 0.30mmol) was dissolved in dry DMF (5mL), and o-phenylenediamine (32mg, 0.30mmol), HBTU (114mg, 0.30mmol) and triethylamine (41. mu.L, 0.30mmol) were added and reacted at room temperature for 4 hours. After the reaction, the reaction mixture was poured into water (40mL) and filtered to obtain a crude product. Column chromatography of the crude product (dichloromethane: methanol 100: 3) gave 59mg of a white powder in 51% yield.¹H-NMR(DMSO-d₆，300MHz)δ：9.95(s，1H)，9.56(s，1H)，8.46(s，1H)，8.22(s，1H)，8.04-7.88(m，3H)，7.58(d，J＝8.3Hz，2H)，7.24-7.10(m，2H)，6.96(t，J＝7.1Hz，1H)，6.85-6.72(m，1H)，6.58(t，J＝7.3Hz，1H)，4.99-4.72(m，4H).ESI-MS(m/s)：396.38[M+1]，394.17[M-1].

Other preparation methods of A series of compounds A2-20 refer to example 1.

Example 2N- (4- ((2-aminophenyl) carbamoyl) phenyl) chlorazol [1, 2-a]Preparation of pyridine-7-carboxamidePrepare (B7)

2.1 preparation of intermediate 8: 4- ((imidazo [1, 2-a ] pyridine-7-carboxamide) methyl) benzoic acid methyl ester

Imidazo [1, 2-a)]Pyridine-6-carboxylic acid (200mg, 1.00mmol), EDC (230mg, 1.20mmol) and DMAP (146mg, 1.20mmol) were dissolved in dry DMF (5mL) and stirred at room temperature for 0.5 h. Additional methyl 4-aminomethylbenzoate (165mg, 1.00mmol) was added and stirring was continued for 2 hours. After the reaction is finished, pouring the reaction liquid into ten times of water,solid precipitated. The solid was filtered, the filter cake dried and column chromatographed to yield 120mg of intermediate, 37%.¹H-NMR(DMSO-d₆，300MHz)δ：9.21(t，J＝5.7Hz，1H)，9.16(s，1H)，8.08(s，1H)，7.90(d，J＝8.3Hz，2H)，7.73-7.60(m，3H)，7.44(d，J＝8.4Hz，2H)，4.57(d，J＝5.8Hz，2H)，3.84(s，3H).ESI-MS(m/s)：310.51[M+1]，300.23[M-1].

2.2 preparation of intermediate 9: 4- ((imidazo [1, 2-a ] pyridine-7-carboxamide) methyl) benzoic acid

4- ((imidazo [1, 2-a)]Pyridine-7-carboxamide) methyl) benzoic acid methyl ester (120mg.0.387mmol) in THF: MeOH: H₂O is 3: 2: 1(6 mL). LiOH (28mg, 1.16mmol) was added and the reaction was carried out at room temperature for 24 hours. After the reaction, the solvent was evaporated to dryness, water (10mL) was added to the residue, the pH was adjusted to 3-4 with dilute hydrochloric acid, a white solid precipitated, and the filter cake was dried after filtration to give 111mg of the product as a white solid in 97% yield.¹H-NMR(DMSO-d₆，300Hz)δ：12.92(sbr，1H)，9.22(t，J＝5.7Hz，1H)，9.17(s，1H)，8.08(s，1H)，7.91(d，J＝8.3Hz，2H)，7.72-7.60(m，3H)，7.45(d，J＝8.3Hz，2H)，4.57(d，J＝5.7Hz，2H).ESI-MS(m/s)：296.42[M+1]，294.19[M-1].

2.3 preparation of the desired product B7

4- ((imidazo [1, 2-a)]Pyridine-7-carboxamide) methyl) benzoic acid (111mg, 0.38mmol), o-phenylenediamine (45mg, 0.41mmol), HBTU (157mg, 0.41mmol) were dissolved in dry DMF (3mL), triethylamine (60. mu.L, 0.41mmol) was added dropwise, and the reaction was stirred at room temperature for 4 hours. After completion of the reaction, the reaction mixture was poured into water (30mL), extracted with ethyl acetate (30 mL. times.3), and dried over anhydrous sodium sulfate. The extract was evaporated to dryness, and the residue was subjected to column chromatography (dichloromethane: methanol 100: 3) to give 56mg of a white powdery solid in 39% yield.¹H-NMR(DMSO-d₆，300MHz)δ：9.62(s，1H)，9.22(t，J＝6.1Hz，1H)，9.17(s，1H)，8.08(s，1H)，7.94(d，J＝8.4Hz，2H)，7.77-7.57(m，3H)，7.45(d，J＝8.2Hz，2H)，7.15(d，J＝7.6Hz，1H)，6.95(t，J＝6.9Hz，1H)，6.76(d，J＝7.1Hz，1H)，6.58(t，J＝7.4Hz，1H)，4.57(d，J＝4.6Hz，2H).¹³C-NMR(DMSO-d₆，150MHz)δ：165.57，164.76，144.84，143.55，143.38，143.26，134.37，133.68，129.15，128.27，127.80，127.63，127.15，126.94，124.97，123.79，123.66，120.24，119.59，116.74，116.60，116.40，114.92，110.07，49.06，42.96.ESI-MS(m/s)：386.38[M+1]，384.30[M-1].

Other preparation methods of B series compound B1-6 refer to example 2.

Example 3N- (2-aminophenyl) -4- ((4- (pyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) methyl) benzylAmide preparation (C1)

3.1 preparation of intermediate 15: 4-Azomethylbenzoic acid methyl ester

Methyl 4-bromomethylbenzoate (342mg, 1.5mmol) was dissolved in dry DMF (5mL) and NaN3(195mg, 3mmol) was added slowly with stirring at room temperature. Then, the reaction solution was put in an oil bath at 80 ℃ to react for 8 hours. After completion of the reaction, the reaction mixture was poured into water (50mL), extracted with ethyl acetate (50 mL. times.3), and dried over anhydrous magnesium sulfate. The solvent was evaporated to dryness to obtain 310mg of intermediate methyl 4-azidobenzoate with a yield of 85%.¹H-NMR(DMSO-d₆，300MHz)δ：7.90(d，J＝8.3Hz，2H)，7.43(d，J＝8.3Hz，2H)，3.83(s，3H)，3.77(t，J＝7.0Hz，2H)，3.20(t，J＝7.0Hz，2H).ESI-MS(m/s)：206.35[M+1].

3.2 preparation of intermediate 16: 4- ((4- (pyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) methyl) benzoic acid methyl ester

3-ethynylpyridine (154mg, 1.5mmol), methyl 4-azidomethylbenzoate (287mg, 1.5mmol), Vc Na (30mg, 0.15mmol) and CuSO₄.5H₂O (3.8mg, 0.015mmol) in a mixed solvent of tert-butanol/water (1: 1, 6mL), N₂The reaction is carried out for 12 hours at room temperature under the protection condition. After the reaction was completed, the reaction mixture was poured into water (30mL) to precipitate a white solid. And filtering the mixed solution to obtain a filter cake. Chromatography of the filter cake by column (1: 2. petroleum ether: ethyl acetate) gave 360mg of a white powdery solid in 82% yield.¹H-NMR(DMSO-d₆，300Hz)δ：9.04(d，J＝1.7Hz，1H)，8.79(s，1H)，8.53(dd，J＝1.5，4.7Hz，1H)，8.21(tt，J＝1.8，7.9Hz，1H)，7.97(d，J＝8.2Hz，2H)，7.49-7.43(m，3H)，5.78(s，2H)，3.83(s，3H).ESI-MS(m/s)：295.35[M+1]，589.39[2M+1].

3.3 preparation of intermediate 17: 4- ((4- (pyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) methyl) benzoic acid

Intermediate 16(303mg, 1.03mmol) was dissolved in THF: MeOH: H₂O in a 3: 2: 1 mixed solvent (12 mL). LiOH (74mg, 3.09mmol) was added thereto, and the reaction was carried out at room temperature for 24 hours. After the reaction, the solvent is evaporated to dryness, 15mL of aqueous solution is added, the pH value is adjusted to 3-4 by using dilute hydrochloric acid, white solid is separated out, the filter cake is dried after filtration, and the product of 260mg is obtained, wherein the yield is 90%.¹H-NMR(DMSO-d6，300MHz)δ：13.05(s，1H)，9.06(s，1H)，8.80(s，1H)，8.55-8.53(m，1H)，8.23(d，J＝8.0Hz，1H)，7.96(d，J＝8.4Hz，2H)，7.50-7.43(m，3H)，5.79(s，2H).ESI-MS(m/s)：295.35[M+1].

3.4 preparation of the desired product C1

4- ((4- (pyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) methyl) benzoic acid (250mg, 0.89mmol), o-phenylenediamine (106mg, 0.98mmol), HBTU (372mg, 0.98mmol) were dissolved in DMF (5mL), triethylamine (136. mu.L, 0.98mmol) was added dropwise, and the reaction was stirred at room temperature for 4 hours. After completion of the reaction, the reaction solution was poured into water (40mL), extracted with ethyl acetate (40mL × 3), the organic layers were combined, dried over anhydrous sodium sulfate, the solution was evaporated under reduced pressure, and the residue was subjected to column chromatography (dichloromethane: methanol ═ 100: 2) to give 88mg of a pale yellow powder solid in 27% yield.¹H-NMR(DMSO-d₆，300Hz)δ：9.64(s，1H)，9.06-9.05(d，J＝1.9Hz，1H)，8.79(s，1H)，8.53(dd，J＝1.4，4.7Hz，1H)，8.21(dt，J＝1.7，7.9Hz，1H)，7.98(d，J＝8.1Hz，2H)，7.49-7.45(m，3H)，7.14(d，J＝7.8Hz，1H)，6.98-6.92(m，1H)，6.77-6.74(m，1H)，6.58(t，J＝7.6Hz，1H)，5.77(s，2H)，4.88(s，2H).ESI-MS(m/s)：371.29[M+1].

Other C series compounds C2-14 were prepared according to example 3.

Example 4N- (2-aminophenyl) -8- (4- (pyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) octanoyl amide (D1)

4.1 preparation of intermediate 20: n- (2-aminophenyl) -8-azidooctanoyl amide

8-azidooctanoic acid (0.43g, 2.32mmol) and o-phenylenediamine (0.28g, 2.55mmol) were dissolved in 5mL of DMF, HBTU (1.06g, 2.79mmol) and triethylamine (1.30 mL) were added in this order, and the mixture was stirred at room temperature for 30 minutes, after completion of the reaction, the reaction mixture was poured into water (50mL), extracted with ethyl acetate (50 mL. times.3), the organic layers were combined, dried, and spin-dried under reduced pressure, and the residue was subjected to column chromatography (dichloromethane: methanol ═ 100: 1) to give 0.07g of a white solid in 92.1% yield.¹H-NMR(DMSO-d₆，600MHz)δ：1.29-1.33(m，6H)，1.50-1.55(m，2H)，1.55-1.59(m，2H)，2.29(t，J＝7.78Hz，2H)，3.31(t，J＝6.91Hz，2H)，4.80(s，2H)，6.50-6.55(m，1H)，6.70(dd，J＝7.96Hz，1.38Hz，1H)，6.85-6.89(m，1H)，7.13(dd，J＝7.83Hz，1.51Hz，1H)，9.08(s，1H).MS(ESI，positive)m/z：276.17[M+H].

4.2 preparation of the desired product D1

Compound 20(0.2g, 0.726mmol) was dissolved in a mixed solvent of tert-butyl alcohol and water (5: 1) (10mL), N₂Protection, sequentially injecting CuSO₄Aqueous solution (1.45mL) and aqueous VcNa solution (7.26mL) were stirred at room temperature overnight. After completion of the reaction, the reaction solution was spin-dried under reduced pressure, the remaining aqueous layer was extracted with ethyl acetate (50mL × 3), the organic layers were combined, the solution was evaporated to dryness, and the residue was subjected to column chromatography (dichloromethane: methanol ═ 100: 2) to give 0.16g of a white solid in 62% yield.¹H-NMR(DMSO-d₆，600MHz)δ：1.28-1.35(m，6H)，1.55-1.59(m，2H)，1.85-1.88(m，2H)，2.29(t，J＝7.78Hz，2H)，4.41(t，J＝7.08Hz，2H)，4.78(s，2H)，6.49-6.53(m，1H)，6.69(dd，J＝7.91Hz，1.23Hz，1H)，6.85-6.88(m，1H)，7.12(d，J＝6.74Hz，1H)，7.46(dd，J＝7.97Hz，4.81Hz，1H)，8.18-8.20(m，1H)，8.52(dd，J＝4.91Hz，1.35Hz，1H)，8.70(s，1H)，9.03(d，J＝1.44Hz，1H)，9.06(s，1H).MS(ESI，positive)m/z：379.22[M+H].

Other preparation methods of D series compound D2-5 refer to example 4.

Example 5N- (2-aminophenyl) - (4- (4- (4- ((pyridin-3-ylidenemethyl) carbamoyl) phenyl) -1H-1,preparation of 2, 3-triazol-1-yl) methyl) benzamide hydrochloride (Compound C14 hydrochloride)

0.1g of N- (2-aminophenyl) - (4- (4- (4- ((pyridin-3-ylidenemethyl) carbamoyl) phenyl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide is dissolved in 5mL of hot ethanol and a continuous flow of dry hydrogen chloride gas is passed through. After the reaction was stirred for 2 hours, a white solid precipitated, which was cooled, filtered, washed and dried to obtain 0.05g of N- (2-aminophenyl) - (4- (4- (4- ((pyridine-3-trimethylene) carbamoyl) phenyl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide hydrochloride.

EXAMPLE 6 preparation of N- (2-aminophenyl) -4- (3- (pyridin-3-ylmethylene) ureido) benzamide succinate(Compound A2 succinate)

0.5g of N- (2-aminophenyl) -4- (3- (pyridine-3-methylene) ureido) benzamide succinate is dissolved in 10mL of hot ethanol, a hot ethanol solution of succinic acid is added, the mixture is reacted for 3 hours at room temperature, and after the reaction is finished, the mixture is filtered, washed and dried to obtain 0.1g of N- (2-aminophenyl) -4- (3- (pyridine-3-methylene) ureido) benzamide succinate.

Example 7, N- (2-aminophenyl) - (4- (4- (4- ((pyridin-3-ylidenemethyl) carbamoyl) phenyl) -1H-1,preparation of 2, 3-triazol-1-yl) methyl) benzamide maleate (Compound C14 maleate)

In a 50mL round-bottom flask, 0.3g of N- (2-aminophenyl) - (4- (4- (4- ((pyridine-3-methylene) carbamoyl) phenyl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide was added, the mixture was dissolved in ethanol, a hot ethanol solution of maleic acid was added, the mixture was reacted at room temperature for 3 hours, and after the completion of the reaction, the mixture was filtered with suction, washed, and dried to obtain 0.15g of N- (2-aminophenyl) - (4- (4- (4- ((pyridine-3-methylene) carbamoyl) phenyl) -1H-1, 2, 3-triazol-1-yl) methyl) benzamide maleate.

Example 8N- (2-aminophenyl) -4- (2- (4- (pyridin-3-yl)) -1H-1, 2, 3-triazol-1-yl) benzoylAmine malate preparation (Compound C1 malate)

In a 50mL round-bottom flask, 0.5g of N- (2-aminophenyl) -4- (2- (4- (pyridine-3-yl)) -1H-1, 2, 3-triazole-1-yl) benzamide was added, the mixture was dissolved in ethanol, a hot ethanol solution of malic acid was added, the mixture was reacted at room temperature for 3 hours, and after the reaction was completed, the mixture was filtered, washed and dried to obtain 0.09g of N- (2-aminophenyl) -4- (2- (4- (pyridine-3-yl)) -1H-1, 2, 3-triazole-1-yl) benzamide malate.

Example 9N- (2-aminophenyl) -8- (4- (pyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) octanamide tartratePreparation of acid salt (Compound D1 tartrate)

A50 mL round-bottomed flask was charged with 0.5g of N- (2-aminophenyl) -8- (4- (pyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) octanamide, dissolved in ethanol, and reacted at room temperature for 3 hours by adding a hot ethanol tartaric acid solution, followed by suction filtration, washing and drying after completion of the reaction to give 0.23g of N- (2-aminophenyl) -8- (4- (pyridin-3-yl) -1H-1, 2, 3-triazol-1-yl) octanamide tartrate.

Example 10 enzyme inhibitory Activity and in vitro antitumor Activity of the Compound of interest

(1) Target compound NAMPT enzyme inhibition assay

The following enzymes are nicotinamide phosphoribosyltransferase available from KANGPEITH Biotech, Inc. or prepared by itself.

1) Preparation of enzyme:

will be transformed with recombinant plasmid NAMPT-pET28a⁺BL21(DE3) plysS cells were inoculated in 2 XYT medium (37. mu.g/mL chloramphenicol and 100. mu.g/mL kanamycin), shaken overnight at 37 ℃, collected, resuspended in 20-fold the original volume of fresh medium, cultured at 37 ℃ to OD600 of about 0.6-0.8, and induced at 28 ℃ for 8 hours with 0.5mM IPTG. The cells were collected by centrifugation, resuspended in lysis buffer (20mM Tris-HCl pH 8.0, 300mM NaCl), and 0.1% Triton X-100, 1% PMSF, 200W sonicated cells were added for 30min in 1s gap with sonication for 9 s. The lysate was centrifuged at 12000rpm at 4 ℃ for 50min to aspirate the supernatant. The supernatant was incubated with a Ni-NTA column (from QIAGEN) for 2 hours on ice with shaking, followed by washing sequentially with binding buffer (5mM imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.5), buffer (10mM/20mM/40mM/60mM imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.5) to remove albumin, and finally eluting sequentially with elution buffer (200mM imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.5) to elute the objectiveProteins were detected by SDS-PAGE and protein concentration was determined by BCA method.

2) The enzyme reaction system was 25 μ L, with final concentrations of the various components: 50mM Tris-HCl (pH 7.5), 0.02% BSA, 12mM MgCl₂2mM ATP, 0.4mM PRPP, 2mM DTT, 2. mu.g/mL NAMPT, 0.2. mu.M NAM, DMSO and a dilution by fold of a compound of the invention. First, 0.5. mu.L of a solution of the compound of the present invention with different concentrations was added to a 96-well plate, 20. mu.L of an enzyme reaction mixture solution (enzyme reaction components except for the substrate) was added, and after incubation at room temperature for 5 minutes, 4.5. mu.L of a substrate NAM solution was added to initiate the reaction, and after reaction at 37 ℃ for 15 minutes, the enzyme reaction was terminated by heating at 95 ℃ for 1 minute.

3) After cooling the reaction solution on ice, 10. mu.L of 20% acetophenone and 2M KOH were added in sequence, mixed uniformly on a vortex mixer, acted at 0 ℃ for 2 minutes, added with 45. mu.L of 88% formic acid, and incubated at 37 ℃ for 10 minutes.

4) Fluorescence values at an excitation wavelength of 382nm and an emission wavelength of 445nm were measured using a microplate reader.

5) According to the formula: e ═ R/(1+ (C/IC)₅₀)^s) + B (where E is the enzyme activity and C is the compound concentration, R, IC₅₀S, B are parameters to be fitted), the curve of enzyme activity versus compound concentration was fitted in origin software to determine the IC of the compound₅₀。

(2) Target compound HDAC1 enzyme inhibition assay

1) Experimental materials:

HDAC1 enzyme, buffer (137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride, 0.1mg/mL BSA, Tris-HCl 25mM at PH 8), HDAC substrate 3, trypsin, 96-well black plate.

2) The experimental method comprises the following steps:

(a) the 96-well black plate was equilibrated to room temperature.

(b) The test compound was diluted with a buffer containing 10% DMSO at a concentration of 100. mu.M, 30. mu.M, 10. mu.M, 3. mu.M, 1. mu.M, 0.3. mu.M, 0.1. mu.M, 0.03. mu.M, 0.01. mu.M, 0.003. mu.M, in that order.

(c) Add 11. mu.L of HDAC1 to 400. mu.L of buffer and shake;

(d) to the 2 nd to 11 th wells of the 96-well plate, 35. mu.L of the freshly prepared buffer containing HDAC1 enzyme was added, and 5. mu.L of the diluted compounds at different concentrations were sequentially added to the corresponding reaction wells, and to the negative control (first well) and the blank control well (eleventh well), 40. mu.L and 5. mu.L of the buffer were added, respectively.

(e) To all reaction wells, 35. mu.L of 100. mu.M HDAC substrate and 5. mu.L of 0.5mg/mL trypsin were added, and the reading was taken after incubation at 37 ℃ for 30 min.

(f) The inhibition rate was calculated according to the formula: inhibition rate (100% active wells-sample wells)/100% active wells 100, and IC of compound was determined by fitting a curve of enzyme activity versus compound concentration in orgin software₅₀The value is obtained.

(3) In vitro antitumor Activity test of target Compounds

1) Sample preparation: after dissolution in DMSO (Merck), 1000. mu.M solution or homogeneous suspension was prepared by adding PBS (-) and then diluted with DMSO-containing PBS (-). The final concentration of the sample was 100, 10, 1, 0.1, 0.01, 0.001. mu.M.

2) Cell line

HCT116 (human intestinal cancer cells), MDA-MB-231 (human breast cancer cells), and HepG2 (human liver cancer cells), were frozen and passaged in this laboratory.

3) Culture solution

DMEM + 10% FBS + double antibody

4) Test method

MTT method. The concentration of the added solution in each hole of a 96-hole plate is 4-5 multiplied by 10⁴Cell suspension 100. mu.l/mL, at 37 ℃ in 5% CO₂In the incubator. After 24 hours, the sample solution was added at 10. mu.L/well in triplicate wells at 37 ℃ with 5% CO₂The reaction was carried out for 72 hours. Adding 20 μ L of 5mg/mL MTT solution into each well, reacting for 4 hr, adding 100 μ L/well of dissolving solution, placing in incubator, dissolving, and measuring OD value at 570nm with full-wavelength multifunctional microplate reader

(4) Results of enzyme inhibitory Activity and in vitro antitumor Activity test for the object Compound (Table 2)

TABLE 2 enzyme inhibitory Activity and in vitro Activity results (μ M) for the Compounds of interest

Pharmacological experiments show that the compound or the salt thereof has strong inhibitory activity on histone deacetylase 1 subtype (HDAC1) closely related to tumors.

Pharmacological experiments show that the compound or the salt thereof has strong inhibitory activity on nicotinamide phosphoribosyltransferase (NAMPT) related to tumors.

Pharmacological experiments show that part of the compounds or salts thereof disclosed by the invention have better inhibitory activity on HDAC1 and NAMPT. Particularly, the inhibition activity of the compound C14 on HDAC1 is 55nM, the inhibition activity on NAMPT is 31nM, and the inhibition activity on double targets reaches better balance; the inhibition activity of compound D1 on HDAC1 was 15nM, and the inhibition activity on NAMPT was 18nM, showing better in vitro activity. The in vitro inhibitory activity of the compound D2 on HDAC1 and NAMPT is 31nM and 36nM respectively, and the in vitro activity is better. The compound D3 had in vitro inhibitory activities of 40nM and 41nM on HDACl and NAMPT, respectively, and the in vitro activity was better.

In vitro anti-tumor activity test adopts MTT method, most compounds show stronger growth inhibition activity to intestinal cancer (HCT116), breast cancer (MDA-MB-231) and liver cancer (HepG2) tumor strains. A2, A4, A5, A8, A13-18, A20, B1-3, B5, B7, C1-5, C7-10, C14 and D3 have better activity on intestinal cancer than positive medicaments CI-994. A1-3, A5, A8, A12-18, A20, B1-5, B7, C1-10, C12-14, D1-3 and D5 have better activity on liver cancer than that of positive medicine CI-994.

Example 11 HDAC Selective assay of target Compounds

Pharmacological experimental results (table 3) indicate that compound C14 has selective inhibitory effects on HDAC1 and HDAC 2.

TABLE 3 inhibition of HDAC isoforms by Compound C14

Example 12 antitumor Effect of the object Compounds in vivo

According to the results of in vitro anti-tumor experiments and the structural characteristics of the compounds, human colon cancer HCT116 is selected as a nude mouse transplanted tumor model, a compound C14 is used as a research object, and SAHA and FK866 are used as positive control drugs.

The administration dose of the designed animal experiment is 25mg/kg twice a day, and the intraperitoneal injection is carried out continuously for 21 days. The result shows that the survival state of the nude mice is good, and no obvious weight change of the mice is observed. The inhibition rate of the compound on the tumor growth is 68.88% (figure 1, table 4), which is obviously higher than that of positive drugs SAHA (33.05%) and FK866 (39.35%). Therefore, compared with a single-target inhibitor, the HDAC/NAMPT double-target inhibitor has obvious advantages, shows the advantages of high efficiency and low toxicity, and is worthy of further research.

TABLE 4 therapeutic effect of target compounds on human intestinal cancer HCT116 nude mouse transplantation tumor

Comparison with Control: p < 0.05, P < 0.01.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims (6)

1. An amide derivative and pharmaceutically acceptable salt thereof, wherein the amide derivative has a general structural formula

Wherein, in the step (A),

X₂is S, R is-NH-CH₂-A, A isHalogen substituted pyridyl, methyl alkyl substituted pyridyl, halogen substituted phenyl, amino substituted phenyl, methyl alkyl substituted phenyl, methyl,

Or

；

Or, X₂Is O, R is-NH-CH₂-A, A is a halogen substituted pyridyl group.

2. The amide-based derivative and the pharmaceutically acceptable salt thereof according to claim 1, wherein the pharmaceutically acceptable salt is hydrochloride, hydrobromide, sulfate, acetate, lactate, tartrate, tannate, citrate, trifluoroacetate, malate, maleate, succinate, p-toluenesulfonate or methanesulfonate.

3. An amide derivative and a pharmaceutically acceptable salt thereof, wherein the amide derivative is:

N- (2-aminophenyl) -4- (3- (6-fluoropyridin-3-yl) methyl) thioureido) benzamide,

N- (2-aminophenyl) -4- (3- (pyridin-3-ylmethyl) ureido) benzamide,

N- (2-aminophenyl) -4- (3- (6-chloropyridin-3-yl) methyl) thioureido) benzamide,

N- (2-aminophenyl) -4- (3- (6-chloropyridin-3-yl) methyl) ureido) benzamide,

N- (2-aminophenyl) -4- (3- (6-fluoropyridin-3-yl) methyl) ureido) benzamide,

N- (2-aminophenyl) -4- (3- (6-aminopyridin-3-yl) methyl) thioureido) benzamide,

N- (2-aminophenyl) -4- (3- (6-aminopyridin-3-yl) methyl) ureido) benzamide,

N- (2-aminophenyl) -4- (3- (5-fluoropyridin-3-yl) methyl) ureido) benzamide,

N- (2-aminophenyl) -4- (3- (6-methylpyridin-3-yl) methyl) ureido) benzamide,

N- (2-aminophenyl) -4- (3- (4-fluorobenzyl) thioureido) benzamide,

N- (2-aminophenyl) -4- (3- (3-fluorobenzyl) thioureido) benzamide,

N- (2-aminophenyl) -4- (3- (2-fluorobenzyl) thioureido) benzamide,

N- (2-aminophenyl) -4- (3- (3-chlorobenzyl) thioureido) benzamide,

N- (2-aminophenyl) -4- (3- (3-bromobenzyl) thioureido) benzamide,

N- (2-aminophenyl) -4- (3- (3-aminobenzyl) thioureido) benzamide,

Or 4- (3- ((1)H-benzo [2 ]d]Imidazol-2-yl) methyl) thioureido) -N- (2-aminophenyl) benzamide.

4. A preparation method of amide derivatives and pharmaceutically acceptable salts thereof is characterized in that: the amide derivative has a structural general formula of

Wherein, in the step (A),

X₂is S, R is-NH-CH₂-A, A is halogen substituted pyridyl, methyl alkyl substituted pyridyl, halogen substituted phenyl, amino substituted phenyl or

；

Or, X₂Is O, R is-NH-CH₂-A, A is a halogen-substituted pyridyl group;

the method comprises the following steps:

a1, cyano Compound 1

The amino compound 2a is obtained by hydrogen reduction in ammonia water methanol solution under the catalysis of Raney nickel

，X₁Is N or CH, R2 is halogen, amino or methyl;

a2 reaction of p-aminobenzoic acid with CDI or TCDI at room temperature to obtain intermediate 4

；

A3, Compound 2a or Compound 2c

Reacting the intermediate 4 in tetrahydrofuran at room temperature to obtain an intermediate 5

；

A4 and the intermediate 5 react with o-phenylenediamine in DMF at room temperature, and a target compound is obtained by column chromatography

。

5. Use of the amide derivatives according to claim 1 and pharmaceutically acceptable salts thereof for the preparation of nicotinamide phosphoribosyltransferase inhibitors, histone deacetylase inhibitors or HDAC/NAMPT dual-target inhibitors.

6. The amide derivatives and the pharmaceutically acceptable salts thereof according to claim 1 are used for preparing antitumor drugs, wherein the tumors are liver cancer, lung cancer, intestinal cancer, ovarian cancer, prostate cancer, stomach cancer or lymph cancer.

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