CN101684123B - Preparation method of 2-formamido thienopyridine derivatives and medical uses - Google Patents

Abstract

The invention discloses a preparation method of 2-formamido thienopyridine derivatives and uses in preparing antineoplastic medicine compositions. The experiment results show that the 2-formamido thienopyridine derivatives have antitumor action, which can be used as main active components to prepare new antineoplastic medicine and provide new selection for treatment of tumour.

Description

2-formamido thienopyridine derivatives preparation method and medicinal use

Technical field

The invention belongs to field of medicaments, be specifically related to the purposes of 2-formamido thienopyridine derivatives in preparing antineoplastic pharmaceutical compositions.

Background technology

Malignant tumour has become the important killer that harm humans is healthy and survive, and particularly in recent years, along with the deterioration of environment, obvious ascendant trend has appearred in the sickness rate of malignant tumour.Although, along with the application that chemotherapeutics of new generation is defended etc. as taxol, gemcitabine, Gree, patient's existence obtains certain benefit, most of cancer patientss' prognosis is still poor, and mortality ratio is high, typical in liver cancer and prostate cancer etc.Therefore, research and develop novel particularly there is the antitumor drug of brand-new precursor structure or from existing compound the antitumor drug that makes new advances of screening become the task of top priority.

The contriver finds that through research the 2-formamido thienopyridine derivatives has anti-tumor activity.Its structural formula is as follows:

In formula, R ₁for hydrogen, amino or hydroxyl; R ₂for hydrogen, bromine, chlorine, fluorine, methoxyl group or trifluoromethoxy; R ₃for hydrogen, bromine, chlorine, fluorine, methoxyl group or trifluoromethoxy.

And the contriver further finds compound 3-amino-N-(4-chlorobenzyl)-7-(3-methoxyphenyl)-4H-thiopyrano[2 under study for action, 3-b] pyridine-2-carboxamide, it is the compound that a kind of 2-of take formamido thienopyridine is precursor structure, be one of 2-formamido thienopyridine derivatives, this compound has obvious anti-tumor activity.Its relative molecular mass is 423.915, is yellow powder, water-soluble poor, dissolves in dimethyl sulfoxide (DMSO) (DMSO) and ethanol equal solvent.Its structural formula is as follows:

Also about the medicinal use of this compound and other 2-formamido thienopyridine derivatives, do not report at present.

Summary of the invention

The contriver finds that through research the 2-formamido thienopyridine derivatives has anti-tumor activity, 3-amino-N-(4-chlorobenzyl)-7-(3-methoxyphenyl)-4H-thiopyrano[2 particularly, 3-b] pyridine-2-carboxamide, can be used for the pharmaceutical composition that tumor disease is treated in preparation.The 2-formamido thienopyridine derivatives has structure shown in formula I:

Its reaction formula is as follows:

Wherein, R ₁for hydrogen, amino or hydroxyl; R ₂for hydrogen, bromine, chlorine, fluorine, methoxyl group or trifluoromethoxy; R ₃for hydrogen, bromine, chlorine, fluorine, methoxyl group or trifluoromethoxy.

Further, R ₁for hydrogen or amino; R ₂for hydrogen or methoxyl group; R ₃for hydrogen or chlorine.

Preferably, R ₁for amino; R ₂for methoxyl group; R ₃for chlorine.

The contriver finds to have anti-tumor activity preferably as shown in the formula the II compound in the 2-formamido thienopyridine derivatives especially, relative molecular mass 423.915, and structural formula is as follows:

Formula II is yellow powder, is insoluble in water, dissolves in dimethyl sulfoxide (DMSO) (DMSO) and ethanol equal solvent.Its chemistry is by name: English name: 3-amino-N-(4-chlorobenzyl)-7-(3-methoxyphenyl)-4H-thiopyrano[2,3-b] pyridine-2-carboxamide; Chinese named: 3-amino-N-(4-chlorobenzyl)-7-(3-p-methoxy-phenyl)-4H-thiophene [2,3-b] pyridine-2-carboxamide.The 2-formamido thienopyridine derivatives that used in the following embodiments all refers to this compound.

The preparation method of formula II compound is: take p-methoxy-acetophenone 1 as raw material, elder generation and N, dinethylformamide dimethylacetal (DMF-DMA) reaction obtains ketones with Enamino-esters 2, close ring with cyano-thioacetamide again and obtain 3, after obtain formamido thienopyridine class target product with acid amides 5 condensations again, 3 steps reactions obtain formula II target product with 32% overall yield.

This preparation method's advantage is: reactions steps is brief, and desired raw material reagent is cheap and easy to get, does not need column chromatography purification, and whole process only needs recrystallization 1 time, can obtain highly purified target product.

Second technical problem to be solved by this invention is to provide a kind of antineoplastic pharmaceutical compositions.This antineoplastic pharmaceutical compositions is to take the 2-formamido thienopyridine derivatives shown in formula I to add pharmaceutically acceptable complementary composition as main active ingredient and be prepared from:

Wherein, R ₁for hydrogen, amino or hydroxyl; R ₂for hydrogen, bromine, chlorine, fluorine, methoxyl group or trifluoromethoxy; R ₃for hydrogen, bromine, chlorine, fluorine, methoxyl group or trifluoromethoxy.

Further, R ₁for hydrogen or amino; R ₂for hydrogen or methoxyl group; R ₃for hydrogen or chlorine.

Preferably, R ₁for amino; R ₂for methoxyl group; R ₃for chlorine.

Beneficial effect of the present invention is, creatively by experiment in vivo and vitro, has proved that the 2-formamido thienopyridine derivatives has good antitumor action, can be for the preparation of antineoplastic pharmaceutical compositions.For the antitumor drug preparation field provides a kind of new selection, there are good market outlook.

The accompanying drawing explanation

The inhibited proliferation of Fig. 1 SKLB-703 to the different people JEG-3

The inhibited proliferation of Fig. 2 SKLB-703 to different mouse cancer cells

Fig. 3 SKLB-703 is to the effect of Mice Bearing Lewis Lung Cancer growth-inhibiting

Fig. 4 SKLB-703 is to the effect of people A549 lung cancer growth-inhibiting

Embodiment

Below in conjunction with embodiment and Figure of description, the present invention is further set forth but be not limitation of the present invention.

The preparation of Preparation Example 3-amino-N-(4-chlorobenzyl)-7-(3-p-methoxy-phenyl)-4H-thiophene [2,3-b] pyridine-2-carboxamide (SKLB-703)

The 2-formamido thienopyridine derivatives used in the present invention is synthetic voluntarily for the contriver, relative molecular mass 423.915, and structural formula is suc as formula shown in II:

The preparation method of formula II compound is: take p-methoxy-acetophenone 1 as raw material, elder generation and N, dinethylformamide dimethylacetal (DMF-DMA) reaction obtains ketones with Enamino-esters 2, close ring with cyano-thioacetamide again and obtain 3, after obtain the Thienopyridines target product with acid amides 5 condensations again, 3 steps reactions obtain formula II target product with 32% overall yield.

Experimental implementation and data division:

1, the preparation of ketones with Enamino-esters:

By p-methoxy-acetophenone 1 (3.19g, 21.2mmol) and N, dinethylformamide dimethylacetal (DMF-DMA) (5.5ml, 32.2mmol) stirring and refluxing reaction 20 hours, TLC shows that raw material reaction is complete, directly casts step reaction (productive rate is in 90%) after reaction solution is chilled to room temperature.

¹H-NMR(400MHz，CDCl3)：δ2.90(s，3H)，3.12(s，3H)，3.80(s，3H)，5.80(d，J＝12.2Hz，1H)，6.95(d，J＝8.6Hz，2H)，7.66(d，J＝12.2Hz，1H)，7.88(d，J＝8.6Hz，1H)ppm；

2, the preparation of intermediate product 3

First to above-mentioned reaction solution, add the 19ml aqueous solution of cyano-thioacetamide (2.20g, 22.0mmol) under room temperature, the potassium hydroxide aqueous solution 15ml of rear dropping 10%, be warming up to 30-35 ℃ of reaction 30 minutes, follow-up continuous 40-45 ℃ of reaction 5 hours that are warming up to.This reaction solution is let cool naturally to room temperature, it is neutral dripping concentrated hydrochloric acid 3.2ml adjusting pH, the red solid of separating out is water successively, normal hexane and ethanol are washed, naturally obtain red solid 3.1 grams after drying, two step productive rates 60% (in p-methoxy-acetophenone), this solid not recrystallization is directly cast the step reaction.

¹H-NMR(400MHz，DMSO-d6)：δ3.86(s，3H)，7.14(d，J＝7.6Hz，1H)，7.22(d，J＝8.0Hz，1H)，7.34(d，J＝8.0Hz，1H)，7.36(s，1H)，7.45(t，J＝8.0Hz，1H)，8.69(d，J＝8.0Hz，1H)，10.39(s，1H)，11.39(s，1H)，13.83(s，1H)ppm；

HRMS(Maldi)Calcd?for?C13H11N20S：243.0593，Found243.0598；

3, the preparation of reactant 5

Under ice-water bath stirs, to to chlorobenzylamine (19.74g, 140mmol) and salt of wormwood (22.8g, methylene dichloride 165mmol) (250ml) hanging drop adds chloroacetyl chloride (18.47g, 165mmol), drip rear temperature rising reflux reaction 4 hours, TLC shows the raw material complete reaction, is poured in frozen water after naturally being chilled to room temperature, methylene dichloride for water (150ml3) extraction, merge organic phase, saturated common salt washing, anhydrous sodium sulfate drying, filter, filtrate being spin-dried for obtains white solid 29 grams, productive rate 95%, and this solid not recrystallization is directly cast the step reaction.

¹H-NMR(400MHz，CDCl3)：δ3.81(s，3H)，4.09(s，2H)，4.43(d，J＝5.6Hz，2H)，6.80(s，1H)，6.88(d，J＝8.8Hz，2H)，6.88(d，J＝8.8Hz，2H)，7.23(d，J＝8.4Hz，2H)ppm；

4, the preparation of target product

Under stirring at room, to the KOH solution 23.4ml that adds 10% in DMF (80ml) suspension of 3 (10.1g, 41.7mmol), after add 5 (10.93g, 50mmol), under room temperature, stirring reaction is 15 minutes, after add again 10% KOH solution 23.4ml, be warming up to 80-85 ℃ of reaction 7 hours, cooling reaction solution, a large amount of solids are separated out, filter, solid is water successively, and second alcohol and water (v:v=1:1) is washed, dry and obtain solid 12.06 grams, the crude product productive rate is 68%.

Get crude product 4 grams, obtain 3.2 grams with ethanol 100ml recrystallization, recrystallization productive rate 80%.

¹H-NMR(400MHz，DMSO-d6)：δ3.84(s，3H)，4.40(d，J＝6.0Hz，2H)，7.07(d，J＝8.8Hz，2H)，7.21(s，broad，2H)，7.37(dd，J＝8.4，9.2Hz，4H)，7.99(d，J＝8.4Hz，1H)，8.14(d，J＝8.8Hz，2H)，8.30(t，J＝5.8Hz，1H)，8.46(d，J＝8.8Hz，1H)ppm；

¹³C-NMR(100MHz，DMSO-d6)：δ42.22，55.74，97.03，114.72(2C)，116.04，124.96，128.62(2C)，128.90(2C)，129.59(2C)，130.83，131.66，132.01，139.64，146.23，156.78，159.21，161.10，165.53ppm；

ESI-MS(m/z，％)422.3(M-H)-；

The tumor cell in vitro proliferation inhibition test of EXPERIMENTAL EXAMPLE 1 2-formamido thienopyridine derivatives

1, experiment material

1.1 main agents

RPMI-1640, DMEM, foetal calf serum, pancreatin etc. are purchased from Gibco BRL company (InvitrogenCorporation, USA), and thiazole bromide blue tetrazolium (MTT), methyl-sulphoxide (DMSO) are Sigma company (USA) product.The 2-formamido thienopyridine derivatives is bought from Specs company (Holland), is the chemical standard product, during experiment in vitro, with DMSO, is mixed with the 20mg/ml storage liquid, puts 4 ℃ of refrigerators and keeps in Dark Place standbyly, faces the used time to be diluted to desired concn with complete culture solution.

1.2 clone and cultivation

This tests human hepatoma cell strain used (HepG2), human prostate cancer cell line (PC-3), human breast cancer cell strain (MCF-7), human lung carcinoma cell line (A549), human colon cancer cell strain (HCT-116), people's squamous cell cancer (A431), mouse colonic cell strain (CT26), Lewis lung cancer cells strain (LL2) and mouse mastopathy cell strain (4T1) all are purchased from U.S. ATCC company, by this laboratory, are preserved.

Human liver cancer cell (HepG2) and Lewis lung cancer cells (LL2) are with containing 10% foetal calf serum, 100U/mL penicillin, the DMEM perfect medium of 100 μ g/mL Streptomycin sulphates, 5%CO2,37 ℃ of cultivations.Human Prostate Cancer Cells (PC-3), human breast cancer cell (MCF-7), human colon cancer cell (HCT-116), mouse colonic cell (CT26), mouse mastopathy cell (4T1) and human lung carcinoma cell (A549) are with containing 10% foetal calf serum, 100U/mL penicillin, the RPMI-1640 perfect medium of 100 μ g/mL Streptomycin sulphates, 5%CO2,37 ℃ of cultivations.

2 experimental techniques and result

2.1 experimental technique (mtt assay)

Adjusting cell concn with complete culture solution is 1-2 * 104/mL, is inoculated in 96 orifice plates, every hole 200 μ L, overnight incubation, use respectively next day various dose the 2-formamido thienopyridine derivatives (final concentration is respectively 20,5,1.25,0.31g/ml) the processing cell, establish not containing negative control group and isopyknic solvent control group of medicine, DMSO concentration is 0.1% simultaneously, and each dosage group is established 5 multiple holes, 37 ℃, 5%CO2 cultivates.Cultivate after 48 hours, every hole adds 5mg/mL MTT reagent 20 μ L, continues to cultivate 2-4h, abandon supernatant, then add DMSO150 μ L, vibration mixes 15min, measure absorbancy (A) value (the A value is directly proportional to viable count) by microplate reader (λ=570nm), get its mean value.Relative cell proliferation inhibition rate (%)=(control group A 570-experimental group A570)/control group A 570 * 100%.Experiment at least repeats 3 times.Experimental data means by mean ± standard deviation, adopts the SPSS13.0 statistical software to process.Measurement data adopts the t check, and there is statistical significance P<0.05 for difference.(the control group here refers to the solvent control group, and the acellular inhibited proliferation of solvent control group, therefore do not list especially)

2.2 experimental result

After the different cells of different concns SKLB-703 effect, with drug level, increase, cell inhibitory effect is more obvious, and each dosage group is compared with control group, and cell proliferation inhibition rate difference all has statistical significance (P<0.05) (referring to Fig. 1 and Fig. 2).The half-inhibition concentration (IC50) that SKLB-703 processes MCF-7, A549, HepG2, PC-3 cell, A431 cell and HCT116 cell 48h is respectively 9.89 μ g/ml, 13.38 μ g/ml, 2.67 μ g/ml, 7.42 μ g/ml, 38.3 μ g/ml and 35.42 μ g/ml.The half-inhibition concentration (IC50) that SKLB-703 processes LL2 cell, CT26 cell and 4T1 cell 48h is respectively 5.49 μ g/ml, 4.1 μ g/ml and 9.18 μ g/ml (referring to table 1).

Table 1SKLB-703 is to different tumor cell line inhibited proliferations (inhibiting rate %)

The anti-tumor in vivo experiment of EXPERIMENTAL EXAMPLE 2 2-formamido thienopyridine derivatives

1, experiment material

Referring to EXPERIMENTAL EXAMPLE 1.

2.1 experimental technique

Set up mouse tumor model: the Mice Bearing Lewis Lung Cancer model, animal model adopts the female C57BL/6 mouse of 6～8 week age, the about 18-20g of body weight left and right.Purchased from experimentation on animals center, Sichuan University West China.In this laboratory animal room, raise, animal freely advances diet, and alternately, start experiment daytime at night after animal conforms.After the LL/2 cell is inoculated into to mouse, flank is subcutaneous, and about 5 * 105 cells (0.1ml) of every inoculation are treated that tumour grows to can lay one's hand in time, mouse are divided into to two groups at random: solvent control group, 60mg/kg SKLB-703 treatment group, 10 every group.SKLB-703 is dissolved in 20%PEG and 10% ethanolic soln, every day intraperitoneal administration, successive administration 15 days.Within every three days, measure gross tumor volume and Mouse Weight.

A549 people's lung cancer model: animal model adopts the female BALB/C nude mice of 6～8 week age, the about 18-20g of body weight left and right, purchased from experimentation on animals center, Sichuan University West China.In this laboratory animal room, raise, animal freely advances diet, and alternately, start experiment daytime at night after animal conforms.After the A549 cell is inoculated into to mouse, flank is subcutaneous, inoculates approximately 5 * 106 cells (0.1ml) for every, treats that tumour grows to 50-100mm3, mouse is divided into to two groups at random: solvent control group, 60mg/kg SKLB-703 treatment group, 5 every group.Every day intraperitoneal administration, successive administration 30 days.Observation index is the same.

2.2 experimental result

We find that the SKLB-703 processing can obviously suppress Mice Bearing Lewis Lung Cancer tumour and people A549 lung cancer tumor growth.Compare with the solvent control group, after the SKLB-703 administration, the inhibiting rate of Mice Bearing Lewis Lung Cancer tumour and people A549 lung cancer tumour reached respectively to 50% and 67%, compared significant difference (P<0.05) (referring to Fig. 3 and Fig. 4) with the solvent control group.During administration, the physiological saline control group, obvious untoward reaction does not all appear in solvent control group and SKLB-703 treatment group mouse, the change relevant with medication all do not appear in the generalized cases such as the fur of mouse, diet, behavior.

The preparation of example of formulations 1 coating tablet

The label formula:

The compounds of this invention 10.0g

Lactose 50.0g

Starch 40.0g

Hydroxypropylcellulose 4.0g

10% pregnant dimension ketone is appropriate

Magnesium Stearate 0.5g

Get mentioned component and mix, the whole grain that sieves after granulation, drying, compressing tablet is made 100

The coating liquid formula:

Opadry (Opadry) 5g, 80% appropriate amount of ethanol dressing.

The preparation of example of formulations 2 capsules:

Formula:

The compounds of this invention 100g

Starch 10g

Carboxymethyl starch is received 20g

The low replacement through the third Mierocrystalline cellulose 10g

Tween 80 is appropriate

Polyvinylpyrrolidone 5% ethanol is appropriate

Sodium lauryl sulfate 8g

1000 of No. 0 gastric-dissolved capsules

Make 1000 capsules.

The preparation method:

Get the formula ratio supplementary material, sieve respectively, add 5% polyvinylpyrrolidone liquid and tween 80 softwood processed, get 20 mesh sieves and granulate, dry under 15 ° of C of room temperature, add sodium lauryl sulphate, mix, by the 0.27g/S gastric-dissolved capsule of packing into No. 0, sample examination, the stripping limit is Q=80%, and content should be the 90-110% of labelled amount.

Claims (3)

1. suc as formula the preparation method of the 2-formamido thienopyridine derivatives shown in I, its step is as follows:

Wherein, R ₁for hydrogen, amino or hydroxyl; R ₂for hydrogen, bromine, chlorine, fluorine, methoxyl group or trifluoromethoxy; R ₃for hydrogen, bromine, chlorine, fluorine, methoxyl group or trifluoromethoxy.

2. preparation method as claimed in claim 1, is characterized in that R ₁for hydrogen or amino; R ₂for hydrogen or methoxyl group; R ₃for hydrogen or chlorine.

3. preparation method as claimed in claim 1, is characterized in that R ₁for amino; R ₂for methoxyl group; R ₃for chlorine.

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