CN104434922A - Application of benzamide compound with fluorophores - Google Patents

Abstract

The invention relates to an application of a benzamide compound with fluorophores as shown in the formula I. The formula 1 is as shown in the specification. The compound shows relatively good inhibitory activity on nicotinamide phosphoribosyltransferase, and can be used for preparing medicines for preventing and/or treating diseases by inhibiting the nicotinamide phosphoribosyltransferase. A cell experiment proves that proliferation of a human hepatocellular carcinoma cell HepG2 can be inhibited; the benzamide compound can be used for developing the medicines for preventing and treating the liver cancer; and in addition, the compound has the fluorophores, and can also be applied to imaging diagnosis of the liver cancer.

Description

A kind of application with the benzamide compound of fluorophor

Technical field

The present invention relates to the novelty teabag of known compound, specifically, relate to a kind of application with the benzamide compound of fluorophor.

Background technology

Hepatocarcinoma is one of significant tumor disease of China, and sickness rate is high, and mortality rate is high, develops new protective agents very important.At present, the level of required energy matter NAD in Nampt (Nampt) controllable mammalian cell has been found.And hepatoma carcinoma cell has very high NAD consumption and metabolic rate, comparatively responsive for Nampt inhibitor.Thus the rate-limiting enzyme Nampt of NAD route of synthesis becomes the new target drone for the treatment of of cancer, its enzyme inhibitor FK 866(Hasmann M, et al., Cancer Research 2003; 63:7436-7442.) with CHS-828(Hjarnaa PJV, et al., Cancer Research 1999; 59:5751-5757.) the current clinical research having entered treatment of cancer.

Compound 4-(3-oxo-4-propyl group-3,4-dihydro-quinoxaline-2-base)-N-(pyridin-3-yl methyl) Benzoylamide is a kind of benzamide compound, its English 4-(3-oxo-4-propyl-3 by name, 4-dihydroquinoxalin-2-yl)-N-(pyridin-3-ylmethyl) benzamide, structural formula, such as formula shown in I, have not been reported about the application that this compound is preventing and/or treating liver cancer at present.

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Summary of the invention

The object of the invention is for deficiency of the prior art, provide such as formula the novelty teabag with the benzamide compound of fluorophor shown in I,

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For achieving the above object, the technical scheme that the present invention takes is:

In a first aspect of the present invention, such as formula the benzamide compound with fluorophor shown in I for the preparation of the medicine preventing and/or treating hepatocarcinoma.

In a second aspect of the present invention, such as formula the benzamide compound with fluorophor shown in I for the preparation of reagent, described reagent is for suppressing the propagation of human hepatoma cell strain HepG 2.

In a third aspect of the present invention, such as formula the benzamide compound with fluorophor shown in I for the preparation of reagent, described reagent is for suppressing the activity of Nampt.

In a fourth aspect of the present invention, such as formula the benzamide compound with fluorophor shown in I for the preparation of reagent, described reagent is used for the imaging diagnosis of hepatocarcinoma.

Described " medicine " can be any applicable regular dosage form, and wherein the contained consumption such as formula the compound shown in I is for more than treatment effective dose, can select as the case may be, is generally 0.01 ~ 100mg/kg/ days.Described medicine can only using such as formula the compound shown in I as sole active agent, also can also containing except such as formula other active component except the compound shown in I.Other described active component for not having harmful effect can other active component of conbined usage to the activity of the suppression Nampt such as formula the compound shown in I, as other active component of medicine by suppressing Nampt and preventing and treating.

Described " reagent " refers to biological chemical reagent or reagent, and its concept comprises the medicine etc. of diagnostic reagent, prevention or disease therapy.

The invention has the advantages that:

Present invention finds a kind of brand-new purposes with the benzamide compound of fluorophor, this compound shows good inhibit activities for Nampt, can be used for preparing the medicine by the disease suppressing Nampt to prevent and/or treat, can be used for preventing and treating hepatocarcinoma; This compound also has fluorophor simultaneously, therefore also can be used for diagnosing liver cancer.

Accompanying drawing explanation

Fig. 1 is in embodiment 1, and Compound I is to the suppression curve chart of Nampt.

Fig. 2 is in embodiment 2, the amount effect curve figure that Compound I suppresses human liver cancer cell HepG 2 multiplication capacity.

Fig. 3 is in embodiment 3, the fluorescence imaging figure of Compound I in human liver cancer cell HepG 2.

Detailed description of the invention

Below in conjunction with accompanying drawing, detailed description of the invention provided by the invention is elaborated.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.

embodiment 1 Compound I suppresses the activity of Nampt

Following enzyme refers to Nampt, purchased from Kang Tai bio tech ltd (003-82) or ChemDiv company of the U.S. (M049-0244), also can prepare voluntarily.

1, the preparation of enzyme: conversion is had the BL21(DE3 of recombiant plasmid (Nampt-pET 28a+)) plysS cell is inoculated in 2 × YT culture medium (37 μ g/ml chloromycetin and 75 μ g/ml kanamycin), 37 DEG C of joltings are spent the night, resuspended with 20 times of fresh cultures to original volume after collecting thalline, 37 DEG C are cultured to OD ₆₀₀about 0.6, induce 5 hours under 0.5 mM IPTG, 28 DEG C of conditions.Collected by centrifugation thalline, and be resuspended in lysis buffer(20 mM Tris-HCl, 300 mM NaCl, pH=7.5) in, 200 W ultrasonic degradation cells, ultrasonic 1 second interval 9 seconds, carries out 30 minutes altogether.By lysate in 12500 rpm, 4 DEG C of Aspirate supernatant after centrifugal 50 minutes.This supernatant and Ni-NTA post (purchased from QIAGEN company) on ice jolting hatch 2 hours, use binding buffer(5 mM imidazole more successively, 0.5 M NaCl, 20 mM Tris-HCl, pH=7.5), wash buffer 1(20 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH=7.5), wash buffer 2(40 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH=7.5), wash buffer 3(60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH=7.5) foreign protein is washed away successively, finally use Elution buffer(200 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH=7.5) eluting collects destination protein, and carry out SDS-PAGE detection.The destination protein collected is mixed with 1:1 ratio with sterile glycerol, for subsequent use with being stored in-20 DEG C after Bradford method mensuration protein concentration.

2, enzyme reaction system is 25 μ l, and wherein the concentration of various component is: 50mM Tris-HCl(pH=7.5), 0.02 % BSA, 12 mM MgCl ₂, 2 mM ATP, 0.4 mM PRPP, 2 mM DTT, 2 μ g/ml Nampt, 0.2 μM of NAM, 2% DMSO and doubling dilution Compound I.First the Compound I of 0.5 μ l variable concentrations is added on 96 orifice plates, add 20 μ l enzyme reaction mixed solutions (the enzyme reaction component except substrate) again, incubated at room is after 5 minutes, add 4.5 μ l substrate NAM solution to start reaction, 37 DEG C of reactions stopped enzyme reaction in 1 minute in 95 DEG C of heating after 15 minutes.

3, until enzyme reaction solution after cooled on ice, add 10 μ l 20% 1-Phenylethanone .s and 10 μ l 2M KOH successively, on vortex mixed instrument mixing after in frozen water act on 2 minutes, add 45 μ l 88% formic acid, 37 DEG C heating 10 minutes, cooled on ice.

4, microplate reader is used to measure the fluorescent value at excitation wavelength 382 nm, emission wavelength 445 nm place.

5, according to formula: E=R/(1+ (C/IC ₅₀) ^s)+B(wherein E be enzymatic activity, C is compound concentration, R, IC ₅₀, S, B be the parameter treating matching), in origin 7.0 software, enzyme relative activity is carried out matching (see figure 1) to the curve of Compound I concentration, obtains the IC of Compound I ₅₀, the results are shown in Table 1:

Table 1

embodiment 2 Compound I suppresses the in-vitro multiplication of human liver cancer cell

We have investigated the inhibitory action of Compound I to human liver cancer cell in-vitro multiplication ability.Act on the cell strain 2 days of exponential phase by the Compound I of gradient concentration, utilize cell viability method to detect:

1, by after HepG 2 cell dissociation of exponential phase, blow and beat into single cell suspension, be inoculated in 96 well culture plates, 1 × 10 ⁴cells/well, every hole culture medium 100 μ l, 37 DEG C, 5% CO ₂overnight incubation in incubator.

2, after cell attachment, the test-compound I adding gradient concentration cultivates 2 days again in incubator.

3, cell viability measures: get cell viability reagent (CCK-8 reagent) 10 μ l and be added in 96 porocyte culture plates successively, in 37 DEG C of reactions 30 minutes after mix homogeneously, microplate reader detects each hole OD value (determined wavelength: 450 nm), record result, by following formulae discovery suppression ratio: suppression ratio (%)=(OD contrasts-OD administration)/OD contrasts × 100%.

Result display (see figure 2), the propagation of Compound I to human hepatoma cell strain HepG 2 shows inhibitory action in various degree, the IC that compound I on cell vigor suppresses ₅₀value is 3050 ± 950 nM.

the fluorescence imaging of embodiment 3 Compound I in human liver cancer cell

We have observed the fluorescence imaging of Compound I in human liver cancer cell HepG 2 by laser confocal microscope.Concrete grammar is:

1, by after the cell dissociation of exponential phase, blow and beat into single cell suspension, be inoculated in culture dish, 37 DEG C, 5% CO ₂overnight incubation in incubator.

2, after cell attachment, add the Compound I that final concentration is 3 μMs, solvent control then adds the solvent DMSO of equal volume, i.e. dimethyl sulfoxide.

3, after 30 minutes, under laser confocal microscope, compound fluorescence is observed.

4, after observation, remove rapidly the culture fluid containing compound, and under laser confocal microscope, after cleaning 3 times with phosphate buffer, again observe the fluorescence in cell.

5, after observation, be changed at once and continue to hatch 4 hours without compound culture fluid.

6, after 4 hours, the fluorescence in cell observed again by taking-up culture dish.

The fluorescence imaging figure of Compound I in human liver cancer cell HepG 2 is shown in Fig. 3, in vitro under condition of culture, finds that it can be enriched in hepatoma carcinoma cell, therefore can be used for the diagnostic imaging of hepatocarcinoma.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.

Claims (4)

1. preparing such as formula the benzamide compound with fluorophor shown in I the application prevented and/or treated in the medicine of hepatocarcinoma,

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2. such as formula the benzamide compound with fluorophor shown in I preparing the application in reagent, it is characterized in that, described reagent for suppressing the propagation of human hepatoma cell strain HepG 2,

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3. such as formula the benzamide compound with fluorophor shown in I preparing the application in reagent, it is characterized in that, described reagent for suppressing the activity of Nampt,

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4. such as formula the benzamide compound with fluorophor shown in I preparing the application in reagent, it is characterized in that, described reagent is used for the imaging diagnosis of hepatocarcinoma,

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