CN102670625A

CN102670625A - Application of urea compound

Info

Publication number: CN102670625A
Application number: CN2011104474889A
Authority: CN
Inventors: 缪朝玉; 张偌瑜; 管云枫; 秦烨
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2011-03-18
Filing date: 2011-11-30
Publication date: 2012-09-19
Anticipated expiration: 2031-11-30
Also published as: CN102670625B

Abstract

The invention discloses an application of a urea compound (shown in figure 1) in preparation of a nicotinamide phosphoribosyltransferase inhibitor. The invention also discloses an application of a compound I in preparation of medicines for prevention and/or treatment of cancer. The invention finds a fully new application of the compound I. The compound I shows good inhibiting activity on nicotinamide phosphoribosyltransferase, can be used for preparing medicines for prevention and/or treatment of diseases by inhibiting nicotinamide phosphoribosyltransferase, and also can be used for preventing and treating cancer.

Description

A kind of application of carbamide compounds

Technical field

The present invention relates to a kind of new application of carbamide compounds.

Background technology

Nicotiamide phosphoribosyltransferase (Nampt) has another name called plain visfatin of interior fat or pre B lymphocyte clone enhancer PBEF, the level of essential energy matter NAD in the regulation and control mammalian cell.Cancerous cell has very high NAD and consumes and metabolic rate, thereby the rate-limiting enzyme Nampt of NAD route of synthesis becomes the new target drone of treatment of cancer, its enzyme inhibitor FK866 (Hasmann M, et al., Cancer Research 2003; 63:7436-7442.) and CHS-828 (Hjarnaa PJV, et al., Cancer Research1999; 59:5751-5757.) the present clinical research that has got into treatment of cancer.

Summary of the invention

Technical problem to be solved by this invention has provided the application of a kind of carbamide compounds in preparation nicotiamide phosphoribosyltransferase inhibitor, with and prevent and/or treat the application in the medicine of cancer in preparation.The present invention has found a kind of brand-new purposes of carbamide compounds; This chemical compound shows better inhibited activity for the nicotiamide phosphoribosyltransferase; Can be used for preparing the medicine of the disease that prevents and/or treats through inhibition nicotiamide phosphoribosyltransferase, also can be used for diseases such as anti-curing cancers.

Therefore, the present invention relates to that (chemical name is: N-[4-(piperdine sulfonyl) phenyl]-N '-(3-pyridylmethyl) thiourea promptly: the application in the preparation nicotiamide phosphoribosyltransferase inhibitor thiourea of N-[4-(piperidinosulfonyl) phenyl]-N '-(3-pyridylmethyl)) suc as formula the carbamide compounds shown in the I;

Therefore, compound I of the present invention can be used for preparing the medicine of the disease that prevents and/or treats through inhibition nicotiamide phosphoribosyltransferase.Described disease comprises that this area is through suppressing all diseases that the nicotiamide phosphoribosyltransferase prevents and/or treats, like cancer.

The invention further relates to above-claimed cpd I and prevent and/or treat the application in the medicine of cancer in preparation.

Among the present invention, described cancer can be ovarian cancer, hepatocarcinoma, pulmonary carcinoma, colon cancer or osteosarcoma.

Among the present invention, the tumor cell of described cancer can be Proliferation of Human Ovarian Cell (like human oophoroma cell line A2780), human liver cancer cell (like human hepatoma cell strain HepG 2), the high transfer of people lung carcinoma cell (like the high lung cancer cell line 95-D that shifts of people), human colon cancer cell (like human colon cancer cell strain HCT-116), people's non-small cell lung cancer cell (like people's non-small cell lung cancer cell A549) or human osteosarcoma cell (like human osteosarcoma cell's strain U2OS).

Among the present invention, described medicine can be any suitable regular dosage form, and wherein the consumption of contained compound shown by formula I can be selected for more than the treatment effective dose as the case may be, is generally 0.01～100mg/kg/ days.Described medicine can be only with compound shown by formula I as unique active component, also can also contain other active component except that compound shown by formula I.Described other active component does not have harmful effect can unite other suitable active component for the activity to the inhibition nicotiamide phosphoribosyltransferase of compound shown by formula I, like other active component through the medicine that suppresses the nicotiamide phosphoribosyltransferase and prevent and treat.

On the basis of this area general knowledge, above-mentioned each optimum condition, but combination in any promptly get each preferred embodiments of the present invention.

Agents useful for same of the present invention and raw material are all commercially available to be got, and formula I chemical compound wherein can be buied through Britain maybrige company.

Positive progressive effect of the present invention is: the present invention has found a kind of brand-new purposes of carbamide compounds; This chemical compound shows better inhibited activity for the nicotiamide phosphoribosyltransferase; Can be used for preparing the medicine of the disease that prevents and/or treats through inhibition nicotiamide phosphoribosyltransferase, also can be used for diseases such as anti-curing cancers.

Description of drawings

Fig. 1 is among the effect embodiment 1, and formula I chemical compound is to the inhibition curve chart of nicotiamide phosphoribosyltransferase.

Fig. 2 is among the effect embodiment 2, and compound I is to the amount effect curve of HepG 2 HCCs NAD level inhibition in 24 hours.

The specific embodiment

Mode through embodiment further specifies the present invention below, but does not therefore limit the present invention among the described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example according to conventional method and condition, or is selected according to catalogue.

Effect embodiment 1 compound I suppresses the activity of nicotiamide phosphoribosyltransferase

Following enzyme is meant the nicotiamide phosphoribosyltransferase, can also can prepare voluntarily available from health peptide bio tech ltd.

1, the preparation of enzyme: BL21 (DE3) the plysS cell inoculation that conversion is had recombiant plasmid (Nampt-pET28a+) is in 2 * YT culture medium (37ug/ml chloromycetin and 75ug/ml kanamycin); 37 ℃ of joltings are spent the night; Resuspended behind the collection thalline with 20 times of fresh cultures to original volume; 37 ℃ to be cultured to OD600 about 0.6, under 0.3mM IPTG, 28 ℃ of conditions, induces 5h.Centrifugal collection thalline, and be resuspended among the lysis buffer (20mM Tris-HCl pH 8.0,300mM NaCl), 200W ultrasonic degradation cell, ultrasonic 1s gap 9s carries out 30min altogether.Lysate is drawn supernatant in 12500rpm, 4 ℃ of centrifugal 50min.This supernatant and Ni-NTA post (available from QIAGEN company) on ice jolting hatch 1h, use binding buffer (5mM imidazole, 0.5M NaCl, 20mMTris-Hcl more successively; PH=7.5), wash buffer (40mM imidazole, 0.5M NaCl, 20mM Tris-HCl; PH=7.5) flush away foreign protein is successively used Elution buffer (200mM imidazole, 0.5MNaCl at last; 20mM Tris-Hcl, pH=7.5) eluting destination protein, and carry out SDS-PAGE and detect.The destination protein of eluting is transferred in the bag filter, and the Tris-HCl (20mM pH=7.5) with sterilization in 4 ℃ of refrigerators dialyses 4～5 times, after 20%PEG20000 concentrates, measures protein concentration with the Bradford method.

2, enzyme reaction system is 25 μ l, and wherein various component concentrations are: the chemical compound of 50mM Tris-HCl (pH7.5), 0.02%BSA, 12mM MgCl2,2mM ATP, 0.4mM PRPP, 2mM DTT, 2 μ g/mlNampt, 0.2 μ M NAM, 2%DMSO and doubling dilution.Elder generation is added on 96 orifice plates with the DMSO solution of the variable concentrations of 0.5 μ l chemical compound; Add 20 μ l enzyme reaction mixed solutions (the enzyme reaction component except that substrate) again; Behind 37 ℃ of preincubate 5min; Add 4.5 μ l substrate NAM solution to start reaction, stop enzyme reaction in 95 ℃ of heating 1min behind 37 ℃ of reaction 15min.

3, treat enzyme reaction solution after cooled on ice, add 10 μ l, 20% 1-Phenylethanone. and 2M KOH successively, on the vortex mixed appearance behind the mixing in 0 ℃ the effect 2min, add 45 μ l, 88% formic acid, 70 ℃ the heating 5min, cooled on ice.

4, use ELIASA to measure the fluorescent value at excitation wavelength 382nm, emission wavelength 445nm place.

5, according to formula: E=R/ (1+ (C/IC ₅₀) ^S(wherein E is an enzymatic activity to)+B, and C is a compound concentration, R, IC ₅₀, S, B be the parameter of treating match), in origin software, enzymatic activity is carried out the match (see figure 1) to the curve of compound concentration, obtain the IC of chemical compound ₅₀, the result sees table 1:

Table 1

Effect embodiment 2 compound I reduce the experiment of the level of NAD in the cell

Tumor cell has high-caliber NAD to generate and wear rate; For satisfying its quick value-added needs; The dependence of NAD level is stronger than normal cell in the tumor cell pair cell; And Nampt is the synthetic rate-limiting enzyme of NAD in the mammalian body, so tumor cell receives the influence of Nampt inhibitor more easily.We are according to the influence of NAD level in the following method detection compound pair cell:

(1) collect the exponential phase cell, after the adjustment cell suspension density, at 96 porocyte culture plate middle berth system cells, every hole 200 μ l cell suspension, about 5000 cells, density is about 2.5 * 10 ⁴/ ml.

(2) it is about 60～70% to treat that cell grows to, and changes and does pastille culture medium incubated cell 24 hours.

(3) use perchloric acid method's cell lysis: inhale and abandon the pastille culture medium, wash cell 3 times, clap and do with 4 ℃ of pre-cooling 1 * D-Hanks liquid.Add 50 μ l 1M HClO ₄Liquid reacts 30min on ice.Get 40 μ l reactant liquors, add 16 μ l1M K ₂CO ₃, react 20min on ice.Use 13000g, 4 ℃ of centrifugal 2min.

(4) NAD measures: the cell pyrolysis liquid 10 μ l that take perchloric acid method's processing are added in the 96 hole lamella lucidas various components below the adding successively: 50mM Tris-HCl (pH 7.5), 3% dehydrated alcohol, 1.66mMPES, 0.42mM MTT, 90 μ g/ml ADH.The reaction system cumulative volume is 100 μ l, with behind each component mix homogeneously in 37 ℃ of reaction 40min, measure the ultraviolet absorption value of the 570nm product MTT-formazan of place with ELIASA, the concentration of NAD and UV signal are linearity.

As shown in Figure 2, along with increasing of compound I concentration, the NAD level progressively reduces in the cell, when the 0.8 μ M left and right sides, the synthetic inhibitory action of NAD is reached platform, and maximal percentage inhibition is about 70%.The half-inhibition concentration IC of compound I ₅₀Value is 93.7 ± 3.4nM.

Effect embodiment 3 compound I suppress the experiment of the in-vitro multiplication of tumor cell

We have investigated the inhibitory action of compound I to tumor cell in-vitro multiplication ability.In the cell strain of exponential phase 3 days, utilize srb assay to detect with the compound effects of gradient concentration:

(1) behind the cell dissociation with exponential phase, blows and beats into single cell suspension, be inoculated in 96 well culture plates; 5 * 10 ³Cells/well, every hole culture medium 100 μ l, 37 ℃, 5%CO ₂Overnight incubation in the incubator.

(2) treat cell attachment after, the test-compound that adds gradient concentration was cultivated in incubator 3 days again.

(3) 10% trichloroacetic acid are fixed, 4mg/ml SRB (the sulphonyl rhodamine B is available from sigma company) dyeing, and 1% acetic acid is cleaned SRB.

(4) add 10mM Tris (100 μ l/ hole), culture plate is placed the 10min that vibrates on the microwell plate agitator, make the crystal dissolving.

(5) ELIASA detects each hole OD value and (detects wavelength: 515nm), write down the result, calculate suppression ratio by following formula: suppression ratio (%)=(OD contrasts an OD administration)/OD contrast * 100%.

The result shows, compound I shows inhibitory action in various degree to the propagation of several kinds of cell strains being tested, and table 2 has shown the IC of compound I ₅₀Value.

Table 2

Cell strain	IC ₅₀(μM)
		Human oophoroma cell line A2780	4.61±2.37
Human hepatoma cell strain HepG 2	2.43±0.47
		The high lung cancer cell line 95-D that shifts of people	4.25±1.15
Human colon cancer cell strain HCT-116	41.76±3.08
		People's non-small cell lung cancer cell A549	2.00±0.50
Human osteosarcoma cell's strain U2OS	8.69±3.84

Claims

1. one kind suc as formula the application of the carbamide compounds shown in the I in preparation nicotiamide phosphoribosyltransferase inhibitor;

2. compound I as claimed in claim 1 prevents and/or treats the application in the medicine of cancer in preparation.

3. application as claimed in claim 2 is characterized in that: described cancer is ovarian cancer, hepatocarcinoma, pulmonary carcinoma, colon cancer or osteosarcoma.

4. application as claimed in claim 2 is characterized in that: the tumor cell of described cancer is Proliferation of Human Ovarian Cell, human liver cancer cell, the high transfer of people lung carcinoma cell, human colon cancer cell, people's non-small cell lung cancer cell or human osteosarcoma cell.

5. application as claimed in claim 4 is characterized in that: described Proliferation of Human Ovarian Cell is human oophoroma cell line A2780.

6. application as claimed in claim 4 is characterized in that: described human liver cancer cell is human hepatoma cell strain HepG 2.

7. application as claimed in claim 4 is characterized in that: the high high lung cancer cell line 95-D that shifts of lung carcinoma cell behaviour that shifts of described people.

8. application as claimed in claim 4 is characterized in that: described human colon cancer cell behaviour colon cancer cell line HCT-116.

9. application as claimed in claim 4 is characterized in that: described people's non-small cell lung cancer cell behaviour non-small cell lung cancer cell A549.

10. application as claimed in claim 4 is characterized in that: the described human osteosarcoma cell osteosarcoma cell line U2OS that behaves.