CN108690004A - A kind of antitumor drug acting on tubulin - Google Patents

Abstract

The invention discloses a kind of antitumor drug acting on tubulin, structural formula isWherein, R₁,R₂,R₃,R₄,R₅It is independently selected from H or CF₃.Find that there is good tubulin inhibitory activity by the test of tubulin polymerization assays kit, it can be obtained by Bel-7402 transplanted human hepatocellular carcinomas Experimental model of small mice, the compounds of this invention has anti-tumor drug activity, can be used for antitumor drug and carries out more deep research and development.

Description

A kind of antitumor drug acting on tubulin

Technical field

The invention belongs to drug design and synthesis fields, are related to a kind of antitumor drug acting on tubulin.

Technical background

Cancer cell is maximum frequent and uncontrolled difference lies in the Mitotic abnomality of cancer cell with normal cell.Cancer Dynamic circulation (polymerize and depolymerization) between the schizogamy process of cell Showed Very Brisk and tubulin/micro-pipe in the cell cycle There is close contact.Therefore, by tubulin polymerization during inhibiting tumour cell division at micro-pipe, or inhibit micro-pipe Depolymerization is tubulin, will cause mitosis that can not carry out or stagnate, and the interruption of cell mitogen process will be to swollen Oncocyte generates the influence of bigger, and growth is made to be suppressed and induce the apoptosis of tumour cell, what final inducing cell tune was died Occur, to achieve the purpose that inhibit tumour cell increment growth.

Having now been found that a large amount of natural, synthesis and semi-synthetic compound have influences microtubule system, interferes it normal The effect of function because of most of its energy and tubulin binding, therefore is referred to as Antitubulin.These tubulins inhibit Agent has had a wide range of applications in terms of medicine, such as antitumor, antimycotic, expelling parasite, and tubulin has become research and opens The important target spot of new type antineoplastic medicine is sent out, Antitubulin, which also has become, clinically uses effective important antineoplastic Object.The mechanism of action of such inhibitor is, by being acted on micro-pipe, to inhibit tubulin poly- in the tumour cell quickly divided Conjunction prevents spindle from being formed, or promotes tubulin polymerization, and spindle is prevented to be interfered thin from reverting to micro-pipe net again The positive eumitosis process of born of the same parents makes cell mitogen process interrupt, and is stagnated in the phase, is adjusted so as to cause tumour cell It dies, plays antitumor action.

Colchicin is a kind of Tropolones biology extracted from the seed and its bulb of liliaceous plant colchicum Alkali.Colchicin can be incorporated into the colchicin site on tubulin, and α tubulins is made to deform with beta tubulin, Make tubulin be assembled into micro-pipe this process to be blocked, to make the formation of spindle be obstructed, makes Mitosis arrest And causes cell to generate tune and die.Colchicin may also interfere with the protein metabolism process of tumour cell, inhibit the work of RNA polymerases Power, and the transport process of the synthesis and amino acid of cell membrane lipoids on cell membrane is blocked, to induce a variety of entity tumors Natural death of cerebral cells.Experiment finds that colchicin is significant in efficacy to breast cancer, leukaemia, cutaneum carcinoma, to cervix cancer, the cancer of the esophagus, lung Cancer also has certain curative effect.Research recently finds that colchicin has apparent inhibition and induction to adjust growth of glioma cells in vitro The effect of dying.Clinically colchicin is usually used in treating acute gout, arthralgia, familial Mediterranean fever, hepatic sclerosis etc., but because Toxicity is too big and less for treating tumour.

Invention content

One of the objects of the present invention is to provide a kind of compound, structural formula I is as follows:

Wherein, R₁,R₂,R₃,R₄,R₅It is independently selected from H or CF₃。

Further, Formula I is selected from:

Further, above compound or its pharmaceutically acceptable salt as Antitubulin answering in drug With.

Further, the purposes of above-mentioned compound or its pharmaceutically acceptable salt in the preparation of antitumor drugs.

Another object of the present invention is to provide a kind of synthetic routes of the compound to be:

Further, specific synthesis step is:

1) 6- (1H- pyrazol-1-yls) niacin (compound 1) undergoes coupling reaction to produce 6- (1H- pyrazoles -1- with benzyl bromide Base) nicotinic acid benzyl ester (compound 2);

2) with nitrating agent nitration reaction occurs for compound 2, generates 2- nitros -6- (1H- pyrazol-1-yls) nicotinic acid benzyl ester (compound 3);

3) compound 3 issues raw reduction reaction in suitable reducing condition, generates 2- amino -6- (1H- pyrazol-1-yls) cigarette Acid benzyl ester (compound 4);

4) compound 4 generates corresponding amides product with corresponding acyl chloride reaction.

Further, the nitrating agent in the step 2) has a concentrated nitric acid, fuming nitric aicd, the mixed acid of nitric acid and sulfuric acid with And dinitrogen pentoxide etc., the preferred mixed acid of nitric acid and sulfuric acid.

Further, the nitro restoring method in the step 3) has ferrous acid reduction method, catalytic hydrogen reduction and hydration Hydrazine reduction method, preferably ferrous acid reduction method.

Obviously, the above according to the present invention is not departing from this hair according to the ordinary technical knowledge and means of this field Under the premise of bright above-mentioned basic fundamental thought, the modification, replacement or change of other diversified forms can also be made.

Specific implementation mode

Embodiment 1:The synthesis of 2- (benzofuran -2- formamido groups) -6- (1H- pyrazol-1-yls) nicotinic acid benzyl ester

1, the synthesis of 6- (1H- pyrazol-1-yls) nicotinic acid benzyl ester

6- (1H- pyrazol-1-yls) niacin (compound 1) (4.86g, 25.7mmol) is dissolved in anhydrous DMF (100mL), Under argon atmosphere, potassium carbonate (10.66g, 77.1mmol) and benzyl bromide (13.19g, 77.1mmol) are added to above-mentioned solution In, it then stirs mixture 15 hours and is transferred in distilled water (150mL), mixture is extracted three times with diethyl ether (100mL). Combined extract is washed, magnesium sulfate drying with distilled water (100mL), and be concentrated under vacuum using rotary evaporator, obtained To crude product 6- (1H- pyrazol-1-yls) nicotinic acid benzyl ester (compound 2), the recrystallization purifying in hexane of crude Compound 2 obtains Pure compound 2, white solid, 6.39g, yield 89%.¹H-NMR(400MHz,CDCl₃)δ:5.22(s,2H),6.53(t, 1H),7.30-7.36(m,6H),7.77(d,1H),8.47(d,1H),8.57(d,1H),9.19(s,1H).¹³C-NMR (125MHz,CDCl₃)δ:67.02,107.94,110.56,127.13,128.16,128.19,129.01,130.95, 137.56,139.64,141.01,150.63,153.33,163.97.LC-MS(ESI,pos,ion)m/z:280[M+H].

2, the synthesis of 2- nitros -6- (1H- pyrazol-1-yls) nicotinic acid benzyl ester

Compound 2 (6.39g, 22.88mmol) is dissolved in chloroform (100mL), under condition of ice bath (10 DEG C or so), by It is added dropwise to the mixed acid solution that 65% concentrated nitric acid (2.5mL, 32.03mmol) and 98% concentrated sulfuric acid (2.5mL, 38.90mmol) are made into. It after being added dropwise, is stirred to react at 10 DEG C about 4 hours, TLC monitors reaction end.After completion of the reaction, reaction solution is concentrated under reduced pressure It to doing, is gradually poured into ice water, it is neutral to be adjusted to pH with sodium carbonate, filters, obtains light yellow product 2- nitros -6- (1H- pyrroles Azoles -1- bases) nicotinic acid benzyl ester (compound 3), 6.68g, yield 90%.¹H-NMR(400MHz,CDCl₃)δ:5.22(s,2H),6.56 (t,1H),7.30-7.36(m,6H),7.88(d,1H),9.21(d,1H),9.24(d,1H).¹³C-NMR(125MHz,CDCl₃) δ:67.02,107.94,116.41,127.13,128.16,128.19,129.01,132.84,137.56,141.01, 147.15,150.20,153.00,168.45.LC-MS(ESI,pos,ion)m/z:325[M+H].

3, the synthesis of 2- amino -6- (1H- pyrazol-1-yls) nicotinic acid benzyl ester

Compound 3 (6.68g, 20.60mmol) and ammonium chloride (0.55g, 10.30mmol) are added to 95% second successively In alcohol (150mL), glacial acetic acid (0.5mL, 2.06mmol) is added, is warming up to 80 DEG C, reaction after ten minutes, is gradually added into also in batches Former iron powder (9.23g, 164.8mmol), reacts after about 3 hours and finishes, and TLC monitors reaction end.It is filtered while hot to remove dereaction Iron cement in liquid.Iron cement filter cake is boiled 1 hour under reflux conditions with 95% ethyl alcohol, then is filtered while hot.3 times repeatedly, merge filter Liquid is concentrated to dryness, and 45 DEG C of vacuum obtains gray solid 2- amino -6- (1H- pyrazol-1-yls) nicotinic acid benzyl ester (compound 4), 5.52g, yield 91%.¹H-NMR(400MHz,CDCl₃)δ:1.28(s,2H),5.22(s,2H),6.53(t,1H),7.30- 7.35(m,6H),7.69(d,1H),7.78(d,1H),8.26(d,1H).¹³C-NMR(125MHz,CDCl₃)δ:67.02, 101.48,107.94,112.66,127.13,128.16,128.19,129.01,137.56,141.01,141.91,152.73, 158.76,171.44.LC-MS(ESI,pos,ion)m/z:295[M+H].

4, the synthesis of 2- (benzofuran -2- formamido groups) -6- (1H- pyrazol-1-yls) nicotinic acid benzyl ester

Compound 4 (5.52g, 18.76mmol) is added in absolute ethyl alcohol (150mL), benzo furan is then gradually added into It mutters -2- formyl chlorides (28.36mmol), stirring is warming up to reaction reflux.It reacts and finishes after about 6 hours, TLC monitors reaction end. It after reaction solution is stood cooling, filters, a small amount of absolute ethyl alcohol washing filter cake is used in combination, obtains white solid product 2- (benzofurans- 2- formamido groups) -6- (1H- pyrazol-1-yls) nicotinic acid benzyl ester, 7.98g, yield 97%.¹H-NMR(400MHz,CDCl₃)δ:5.22 (s,2H),6.53(t,1H),7.18-7.36(m,9H),7.50(d,1H),7.59(d,1H),8.45(d,1H),8.52(d, 1H),8.57(d,1H).¹³C-NMR(125MHz,CDCl₃)δ:67.02,107.94,109.48,112.76,112.84, 118.97,123.11,123.50,125.05,127.13,128.16,128.19,129.01,129.10,137.56,141.01, 142.62,150.05,154.43,155.04,156.54,159.80,169.82.LC-MS(ESI,pos,ion)m/z:439[M+ H].

Embodiment 2:2- (7- trifluoromethyls-benzofuran -2- formamido groups) -6- (1H- pyrazol-1-yls) nicotinic acid benzyl ester Synthesis

Compound 4 (5.52g, 18.76mmol) is added in absolute ethyl alcohol (150mL), 7- trifluoros are then gradually added into Methl-benzofuran -2- formyl chlorides (28.36mmol), stirring are warming up to reaction reflux.It reacts and finishes after about 6 hours, TLC prisons Survey reaction end.It after reaction solution is stood cooling, filters, a small amount of absolute ethyl alcohol washing filter cake is used in combination, obtains white solid product 2- (7- trifluoromethyls-benzofuran -2- formamido groups) -6- (1H- pyrazol-1-yls) nicotinic acid benzyl ester, 8.74g, yield 92%. LC-MS(ESI,pos,ion)m/z:507[M+H].

Embodiment 3:2- (bis- trifluoromethyls of 6,7--benzofuran -2- formamido groups) -6- (1H- pyrazol-1-yls) niacin benzyl The synthesis of ester

Compound 4 (5.52g, 18.76mmol) is added in absolute ethyl alcohol (150mL), 6,7- bis- is then gradually added into Trifluoromethyl-benzofuran -2- formyl chlorides (28.36mmol), stirring are warming up to reaction reflux.It reacts and finishes after about 6 hours, TLC monitors reaction end.It after reaction solution is stood cooling, filters, a small amount of absolute ethyl alcohol washing filter cake is used in combination, obtains white solid Product 2- (6,7- bis- trifluoromethyls-benzofuran -2- formamido groups) -6- (1H- pyrazol-1-yls) nicotinic acid benzyl ester, 10.24g, production Rate 95%.LC-MS(ESI,pos,ion)m/z:575[M+H].

Test example 1:Tubulin inhibitory activity

With reference to the Tubulin PolymerizationAssay Kit (tubulin polymerizations point of Cyroskeleton companies Analyse kit) specification, it is operated and is measured.With 80mM PIPES pH 6.9,0.5mM EGTA, 2mM MgCl₂, 1mM The glycerol of Mg GTP and 10% at 3mg/ml cow brain tissue tubulin solution.The tubulin solution 100 μ L are taken, it is fast Speed is added in 96 orifice plates containing various concentration untested compound and colchicin (colchicine), and control wells are added identical The DMSO (s &lt of final concentration;0.5%).37 DEG C are set, 5%CO₂Lower incubation, initiated polymerization.With multi-function microplate reader measure its Wavelength is the absorption value of 355nm, primary per 3min readings, 45min is surveyed altogether, with prism Software on Drawing tubulin polymerization power Curve is learned, and acquires IC₅₀(when a concentration of its original concentration half of tubulin, the concentration for the compound being added).

IC of 1 compound of table to tubulin₅₀

Compound	IC50(nM)
		colchicine	1782
COLC-101	163
		COLC-102	159
COLC-103	177
		COLC-104	169
COLC-105	173
		COLC-106	178
COLC-107	161

It can be obtained by upper table, listed compound tubulin inhibitory activity IC in table₅₀Value is respectively less than It is lower to illustrate that compound listed in table reaches the concentration needed when tubulin half inhibits by colchicine.Confirm this The prepared compound of invention has tubulin inhibitory activity, can carry out antitumor drug as Antitubulin Exploitation.

Embodiment 2:Bel-7402 transplanted human hepatocellular carcinoma mouse models are studied

One, human liver cancer nude mouse xenotransplant cancer model is established

SPF grades of Balb/c nude mices, male, 4 week old, people's Bel-7402 liver cancer cells nude mice model tumor formations of learning from else's experience, then through naked 2 more than generation tumor mass of mouse passage, is cut into about 2*2*2mm, and it is subcutaneous to be inoculated in nude mice armpit using trochar, establishes human liver cancer nude mice Heteroplastic transplantation cancer model.

Two, dosage regimen

With the length and width of each animal tumor of vernier caliper measurement, by formula:V=1/2*ab²Knurl product is calculated, waits for that knurl is accumulated It grows to about 100mm³Start to be administered, all animals are randomly divided into model group, positive drug capecitabine group (400mg/ by knurl product Kg), medicine group (100mg/kg) to be measured, every group 8, each group gastric infusion, dosage is l0ml/kg, 1 time/d, successive administration 14d。

Three, knurl weight and tumour inhibiting rate

Strip tumor mass within 16th day, assay balance claims knurl weight, calculates tumour inhibiting rate as follows.

Tumour inhibiting rate %=(model group average knurl weight-administration group average knurl weight)/model group average knurl weight * 100%.

Data analysis is carried out to testing result using 5 data processing softwares of GraphPadPrism.

Four, knurl weight and tumour inhibiting rate

The different group Bel-7402 transplanted human hepatocellular carcinoma mouse knurl weights of table 2 and tumour inhibiting rate (N=8)

Note:* the P&lt compared with model group;0.05

As can be seen from the above table, compared with model group, the equal conspicuousness of other each group knurl weights reduces, the listed present inventionization in table It closes object (100mg/kg) tumour inhibiting rate and is higher than capecitabine (400mg/kg), illustrate that the compounds of this invention dosage has in 100mg/kg There is good tumors inhibition activity.It can be obtained by Bel-7402 transplanted human hepatocellular carcinomas Experimental model of small mice, the compounds of this invention tool There is anti-tumor drug active, can be used for antitumor drug and carry out more deep research and development.

The above is only a preferred embodiment of the present invention, it is noted that come for those of ordinary skill in the art It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as The content that the present invention is covered.

Claims (4)

1. a kind of compound, which is characterized in that its structural formula I is as follows:

Wherein, R₁,R₂,R₃,R₄,R₅It is independently selected from H or CF₃。

2. compound as described in claim 1, characterized in that be further selected from:

3. compound as described in claim 1 or its pharmaceutically acceptable salt are as Antitubulin in drug Using.

4. the purposes of compound as described in claim 1 or its pharmaceutically acceptable salt in treatment is antitumor.