CN103724251A - STAT3 (Signal Transducer and Activator of Transcription 3)-targeting small molecular compound as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses an STAT3 (Signal Transducer and Activator of Transcription 3)-targeting small molecular compound as well as a preparation method and application thereof. The STAT3 inhibitor is a compound shown as a general formula I in the specification, wherein R<1> is 5-chloro-2-hydroxyphenyl, (E)-3-(6-bromo-2-pyridine)-2-cyan or 4-(2-phenylquinoline); R<2> is hydrogen or C1-C6 linear alkyl or branched alkyl. The compound has the advantages that the proliferation and the survival of various tumor cells of breast cancer, lung cancer, liver cancer, cervical cancer, prostate cancer and the like can be remarkably inhibited; the STAT3 signal channel of the tumor cells is inhibited; therefore, the tumor cell apoptosis is induced and the effect of treating the tumor diseases is achieved.

Description

Micromolecular compound of a kind of target STAT3 and its preparation method and application

Technical field

The invention belongs to pharmaceutical chemistry field, relate to STAT3 inhibitor, be specifically related to micromolecular compound of a kind of target STAT3 and its preparation method and application.

Background technology

Signal transduction and activating transcription factor (Signal Transducers and Activators of Transcription, STATs) family is that molecule amount is the plasmosin of 79-113kDa, wide expression is in the dissimilar cell of body and tissue, participate in the regulation and control of the different physiological roles such as Growth of Cells, differentiation, apoptosis, and close with inflammation, tumour and immune response pathologic, physiologic functional relationship.This class STATs family protein had not only been had a signal conduction function but also have transcription activating function as difunctional plasmosin, can from different cytokine receptor combinations, extracellular signal is passed in nucleus, thereby and in core, is combined with specific DNA promoter sequence and causes the transcribing of corresponding target genes.Up to the present, the STATs member who has found has STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6.

As one of important member of STATs family, STAT3 plays an important role in promotion tumor cell proliferation, inhibition tumor cell apoptosis, promotion Invasion and Metastasis and immunologic escape, thereby enjoys researchist to pay close attention to.Increasing result of study shows, the activation of STAT3 albumen is strictly controlled under normal physiological conditions, but all can find this albumen excessive activation and high level expression in the cell of nearly all solid tumor and blood tumour (as mammary cancer, ovarian cancer, prostate cancer, malignant melanoma, multiple myeloma, lymphoma, squamous cell carcinoma of the head and neck, brain tumor, nonsmall-cell lung cancer and various leukemia etc.).Therefore, STAT3 signalling channel becomes the focus of research in recent years as one of developing anti-tumor medicaments new action target spot.

From 1994 discovery STAT3 by now, existing in a large number about the inhibitor of STAT3 and the research of anti-tumor activity report, comprising polypeptide or class peptide compounds and small molecules non-peptide compound, only have at present only a few small molecules non-peptide compound to enter I phase/II clinical trial phase, also do not ratify so far the medicine of listing.Wherein hinder effective STAT3 micromolecular inhibitor and enter a clinical principal element to be into the property of medicine poor, physicochemical property poor (as poorly water-soluble), the biochemical property (for example anti-tumor activity is not high) of for example polypeptide or class peptide compounds are undesirable; The pharmacokinetic property of small molecules non-peptide compound is bad, easily produce untoward reaction or toxic side effect is large; A few micromolecular non-peptide compounds cLogP value and the molecular weight as higher in OPB-31121 has that enters I phase/II clinical trial phase, affects its further clinical development.Therefore, actively find and there is high anti-cancer activity, low toxicity and become the good New type of S TAT3 inhibitor of the property of medicine to have very important meaning for cancer therapy.The outstanding advantages that the present invention has comprises: STAT3 inhibitor of the present invention is small molecule selective depressant, by measuring it to its active result of the evaluation of effect of cancer cells, can show that small molecules STAT3 selective depressant of the present invention can be used for the exploitation of relevant cancer treatment drugs, has very wide purposes.Small molecules STAT3 selective depressant of the present invention, preparation method is simple, and kind is many, and product purity is high, and yield is high, practical.

Summary of the invention

The object of the present invention is to provide micromolecular compound of a kind of target STAT3 and its preparation method and application, this compound can significantly suppress propagation and the survival of the kinds of tumor cells such as mammary cancer, lung cancer, liver cancer, cervical cancer and prostate cancer, inhibition tumor cell STAT3 signal path, thereby inducing apoptosis of tumour cell, reaches the effect for the treatment of tumor disease.

For achieving the above object, the present invention adopts following technical scheme:

A kind of STAT3 inhibitor is to have the compound shown in general formula I

, wherein R ¹for 5-chlorine-2-hydroxyl phenyl, (E)-3-(6-bromo-2-pyridyl)-2-cyano group or 4-(2-phenylquinoline); R ²for the straight or branched alkyl of hydrogen, C1-C6.

A kind of method of preparing STAT3 inhibitor as above comprises the following steps:

(1) 6-nitroindoline and iodo-alkyl reaction obtain

, R ¹for the straight or branched alkyl of hydrogen, C1-C6, then obtain through reduction reaction

, R ¹for the straight or branched alkyl of hydrogen, C1-C6;

(2) will

, R ¹for the straight or branched alkyl of hydrogen, C1-C6, and acid-respons obtains

,

or

, R ¹for the straight or branched alkyl of hydrogen, C1-C6;

(3) will

, R ¹for the straight or branched alkyl of hydrogen, C1-C6, obtain under the catalysis of ammonium acetate with 6-bromopyridine 2-formaldehyde

, R ¹for the straight or branched alkyl of hydrogen, C1-C6.

Described STAT3 inhibitor is for the preparation of the medicine for the treatment of mammary cancer, lung cancer, liver cancer, cervical cancer and prostate cancer.

Remarkable advantage of the present invention is: the micromolecular compound of target STAT3 of the present invention can significantly suppress propagation and the survival of the kinds of tumor cells such as mammary cancer, lung cancer, liver cancer, cervical cancer and prostate cancer, inhibition tumor cell STAT3 signal path, thereby inducing apoptosis of tumour cell, reaches the effect for the treatment of tumor disease.

Embodiment

Compound shown in formula I is as STAT3 inhibitor, and its structural formula is as follows:

Wherein R ¹for 5-chlorine-2-hydroxyl phenyl, (E)-3-(6-bromo-2-pyridyl)-2-cyano group, 4-(2-phenylquinoline); R ²for the straight or branched alkyl of hydrogen, C1-C6.

Concrete compound structure is as follows:

Wherein R ²for the straight or branched alkyl of hydrogen, C1-C6.

Preparation method's concrete reaction formula is as follows:

Wherein: NaH is sodium hydride, DMF is dimethyl formamide, NH ₄cl is ammonium chloride, and MeOH is methyl alcohol, and 5-chlorosalicylic acid is 5-chloro-salicylic acid, and HBTU is benzotriazole-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester; NH ₄oAc is ammonium acetate, and EtOH is ethanol.

6-nitroindoline and different iodo-alkyl reactions obtain intermediate 2, and intermediate 2 obtains the amino intermediate 3 of 6-by reduction.Intermediate 3 and different acid-responss obtain formula II, intermediate 4 and IV.The compound of formula III can be obtained by intermediate 4 and 6-bromopyridine 2-formaldehyde under the effect of ammonium acetate.

Shown in formula I, II, III and IV, the structure of compound and compound are to inhibiting tumour cells activity research.Through applicant's further investigation discovery, compound shown in formula I, II, III and IV is mainly by suppressing STAT3 signal path, and then the growth of inhibition tumor cell.In addition, after the MDA-MB-231 of compound treatment breast carcinoma cell strain shown in formula I, II, III and IV, extract total protein of cell through sds gel electrophoresis analysis, after the anti-processing of primary antibodie two, result shows that it can suppress STAT3 tyrosine 705 phosphorylations, suppress the expression of downstream albumen Bcl-xl and c-Myc, and activate Caspase-3, thereby suppress STAT3 signal path.

The inhibitor being formed by compound shown in formula I, II, III and the IV of significant quantity and pharmaceutically acceptable auxiliary material.The described auxiliary material of accepting auxiliary material and can adopt this area routine, according to conventional preparation method's preparation, the Optimization of Adjuvant of accepting of the present invention is from sorbyl alcohol, N.F,USP MANNITOL, Xylitol, cyclodextrin, sucrose.Wherein, described STAT3 inhibitor is preferably oral preparations, and described oral preparations is preferably tablet.

Compound shown in formula I, II, III and IV can significantly suppress propagation and the survival of the kinds of tumor cells such as mammary cancer, lung cancer, prostate cancer and cervical cancer, inhibition tumor cell STAT3 signal path, thereby inducing apoptosis of tumour cell, reaches the effect for the treatment of tumor disease.

Described STAT3 inhibitor can be applicable to treat tumor disease, and described tumor disease is preferably mammary cancer, lung cancer, liver cancer, cervical cancer and prostate cancer.

Below in conjunction with following examples, the present invention is further elaborated, but these embodiment do not limit the scope of the invention.

Preparation Example:

Embodiment 1:5-chlorine-2-hydroxyl- n-(1-methyl-6-indoles) benzamide (preparation of Compound I Ia)

Step 1:1-methyl-6-nitroindoline (intermediate 2): add the 60%NaH of 800mg in the DMF of 20mL solution, add the 6-nitroindoline of 4g at 0 ℃ in batches.Mixed solution stirs after 10 minutes and is added dropwise to 1.86mL methyl iodide at 0 ℃.Mixed solution at room temperature stirred after 24 hours, slowly joined and in 100mL frozen water, then added 100mL ethyl acetate.After the organic phase saturated sodium-chloride washing that extraction obtains, organic phase obtains 4.50g intermediate 2 with after being spin-dried for after anhydrous sodium sulfate drying. ¹H?NMR?(400?MHz,?CDCl ₃)? δ?8.27?(d,?1H),?7.97?(d,?1H),?7.61?(d,?1H),?7.33?(d,?1H),?6.56?(d,?1H),?3.87?(s,?3H)。

Step 2: 1g intermediate 2 is dissolved in 20mL methyl alcohol, adds 100mg zinc powder.At 0 ℃, add 20mL saturated aqueous ammonium chloride, stirred overnight at room temperature.Vacuum rotating is removed methanol solution, water arrives neutral with the sodium hydrogen carbonate solution adjust pH of 20mL10%, then with the extraction of 50mL ethyl acetate, after the organic phase saturated sodium-chloride washing that extraction obtains, organic phase obtains 950mg intermediate 3 with after being spin-dried for after anhydrous sodium sulfate drying.Do not need purifying to be directly used in next step reaction.

Step 3:146mg intermediate 3 is dissolved in the DMF of 10mL, add the 5-chloro-salicylic acid of 200mg, add HBTU(benzotriazole-N of 500mg, N, N', N'-tetramethyl-urea phosphofluoric acid ester) and the DIPEA(diisopropylethylamine of 0.5mL), after stirring at room 24 hours, add 10mL water and 30mL ethyl acetate, the organic phase that extraction obtains is again with the extraction of 10mL water, after the organic phase saturated sodium-chloride washing that extraction obtains, organic phase is with after being spin-dried for after anhydrous sodium sulfate drying, and crude product is crossed silicagel column and obtained 100mg final product IIa. ¹H?NMR?(400?MHz,?DMSO- d ₆)? δ?12.01?(s,?1H),?10.28?(s,?1H),?7.89?(s,?1H),?7.81?(d,?1H),?7.78?(d,?1H),?7.48-7.59?(m,?4H),?6.58?(d,?1H),?3.82?(s,?3H)。

Embodiment 2:(E)-3-(6-bromopyridine-2)-2-cyano group- n-(1-methyl-6-indoles) acrylamide (preparation of compound III a)

Step 1 is identical with synthetic compound IIa with step 2, and 5 chloro-salicylic acids of step 3 change 2-cyanoacetic acid into.Being specially 146mg intermediate 3 is dissolved in 10mLDMF, add 100mg2-cyanoacetic acid, add 500mgHBTU and 0.5mLDIPEA, after stirring at room 24 hours, add 10mL water and 30mL ethyl acetate, the organic phase that extraction obtains is again with the extraction of 10mL water, after the organic phase saturated sodium-chloride washing that extraction obtains, organic phase is with after being spin-dried for after anhydrous sodium sulfate drying, and crude product is crossed silicagel column and obtained 120mg intermediate 4. ¹H?NMR?(400?MHz,?DMSO- d ₆)? δ?10.83?(s,?1H),?8.26?(d,?1H),?7.95?(d,?1H),?7.60?(d,?1H),?7.32?(d,?1H),?6.55(d,1H),?3.97?(s,?2H),?3.82?(s,?3H)。

Step 4: 213mg intermediate 4 is dissolved in 5mL ethanolic soln, adds 39mg ammonium acetate, add 6-bromopyridine 2-formaldehyde, room temperature reaction spends the night, and obtains solid.Solid filtering is removed ethanol, water and washing with alcohol solid, the dry 350mg final product IIIa that obtains. ¹H?NMR?(400?MHz,?DMSO- d ₆)? δ?10.97?(s,?1H),?8.29?(s,?1H),?8.15?(s,?1H),?7.96?(d,?1H),?7.91-7.93?(m,?2H),?7.85?(d,?1H),?7.34?(d,?1H),?6.55(d,?1H),?3.89?(s,?3H)。

Embodiment 3: n-(1-methyl-6-indoles)-2-phenylquinoline-4-methane amide (preparation of compound IV a)

Step 1 is identical with synthetic compound IIa with step 2, and in the preparation of Compound I Ia, 5 chloro-salicylic acids of step 3 change 2-phenylquinoline-4-acetic acid into.Being specially 146mg intermediate 3 is dissolved in 10mLDMF, add 300mg2-phenylquinoline-4-acetic acid, add 500mgHBTU and 0.5mLDIPEA, after stirring at room 24 hours, add 210mL water and 50mL ethyl acetate, the organic phase that extraction obtains is again with the extraction of 20mL water, after the organic phase saturated sodium-chloride washing that extraction obtains, organic phase is with after being spin-dried for after anhydrous sodium sulfate drying, and crude product is crossed silicagel column and obtained 220mg final product IVa. ¹H?NMR?(400?MHz,?DMSO- d ₆)? δ?11.31?(s,?1H),?8.45?(s,?1H),?8.39?(d,?2H),?8.35?(s,?1H),?8.21?(d,?2H),?7.92?(d,?1H),?7.89?(t,?1H),?7.55-7.75?(m,?6H),?6.54?(d,?1H),?3.89?(s,?3H)。

Embodiment 4:5-chlorine-2-hydroxyl- n-(1-sec.-propyl-6-indoles) benzamide (preparation of Compound I Ib)

The preparation of the preparation of Compound I Ib and Compound I Ia is similar, and 2-iodopropane is replaced to methyl iodide. ¹H?NMR?(400?MHz,?DMSO- d ₆)? δ?12.00?(s,?1H),?10.27?(s,?1H),?7.88?(s,?1H),?7.85?(d,?1H),?7.75?(d,?1H),?7.46-7.55?(m,?4H),?6.59?(d,?1H),?4.05-4.09?(m,?3H),?1.60?(d,?6H)。

Embodiment 5:(E)-3-(6-bromopyridine-2)-2-cyano group- nthe preparation of-(1-sec.-propyl-6-indoles) acrylamide (preparation of compound III b) compound III b and the preparation of compound III b are similar, and 2-iodopropane is replaced to methyl iodide. ¹H?NMR?(400?MHz,?DMSO- d ₆)? δ?10.91?(s,?1H),?8.49?(s,?1H),?8.25?(s,?1H),?7.90?(d,?1H),?7.90-7.93?(m,?2H),?7.86?(d,?1H),?7.39?(d,?1H),?6.54?(d,1H),?4.04-4.09?(m,?3H),?1.61?(d,?6H)。

Embodiment 6: nthe preparation of-(1-sec.-propyl-6-indoles)-2-phenylquinoline-4-methane amide (preparation of compound IV b) compound IV b and the preparation of compound IV a are similar, and 2-iodopropane is replaced to methyl iodide. ¹H?NMR?(400?MHz,?DMSO- d ₆)? δ?11.38?(s,?1H),?8.46?(s,?1H),?8.39?(d,?2H),?8.35?(s,?1H),?8.21?(d,?2H),?7.91?(d,?1H),?7.88?(t,?1H),?7.56-7.78?(m,?6H),?6.55?(d,?1H),?4.08-4.09?(m,?3H),?1.65?(d,?6H)。

Application Example:

Embodiment 1: the restraining effect of compound to tumor cell proliferation and survival

Material: breast cancer cell MDA-MB-231, lung cancer cell line Α 549, hepatoma cell strain HepG2, cervical cancer cell strain HeLa and prostate cancer PC3 cell strain.

Tested medicine: 0, the compound of 7 concentration of 0.1 μ Μ, 0.5 μ Μ, 1.0 μ Μ, 5 μ Μ, 10 μ Μ, 50 μ Μ.

The restraining effect of Compound I Ia to breast cancer cell MDA-MB-231 propagation and survival: after MDA-MB-231 cell cultures, add respectively compound to hatch 72h, then microscopic examination and MTS method analytical results show the decline of MDA-MB-231 cell proliferation capacity, cell density significance reduces, and cell presents apoptosis performance.In MTS analytical results, find that Compound I Ia is consistent with microscopic examination result to the viability of MDA-MB-231 cell, the survival rate of cell is on a declining curve along with IIa concentration increase (0,0.1 μ Μ, 0.5 μ Μ, 1.0 μ Μ, 5 μ Μ, 10 μ Μ, 50 μ Μ), at 1.0 μ M, is down to nearly 70%.

Embodiment 2: the impact of compound on signal path

Material: breast cancer cell MDA-MB-231;

Tested medicine: 0, the compound of 7 concentration of 0.1 μ Μ, 0.5 μ Μ, 1.0 μ Μ, 5 μ Μ, 10 μ Μ, 50 μ Μ.

The impact of STAT3 signal path: IIa was processed respectively to MDA-MB-231 cell after 48 hours, extract total protein of cell, through sds gel electrophoresis, separate, immunoblotting analyzing total STAT3, the STAT3 (p-STAT3) of phosphorylation and the expression level of downstream albumen Bcl-xl and c-Myc, result shows can find IIa dose-dependent inhibition STAT3 tyrosine 705 phosphorylations, suppress the expression of downstream albumen Bcl-xl and c-Myc, and activate Caspase-3, thereby suppress STAT3 signal path.

Embodiment 3: the preparation of lyophilized injectable powder

Get IIa(100 mg) be dissolved in ethanol (1 mL), add beta-cyclodextrin (1g is dissolved in 10 mL water), stir after 1 hour, vacuum rotating is removed after ethanol, and freeze-drying obtains powder injection.

Embodiment 4: the preparation of tablet

Get IIa, sucrose and the cyclodextrin ratio by weight 1:2:2, make according to a conventional method tablet.

The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.

Claims (3)

1. a STAT3 inhibitor, is characterized in that: described STAT3 inhibitor is to have the compound shown in general formula I

, wherein R ¹for 5-chlorine-2-hydroxyl phenyl, (E)-3-(6-bromo-2-pyridyl)-2-cyano group or 4-(2-phenylquinoline); R ²for the straight or branched alkyl of hydrogen, C1-C6.

2. a method of preparing STAT3 inhibitor as claimed in claim 1, is characterized in that: comprise the following steps:

(1) 6-nitroindoline and iodo-alkyl reaction obtain

, R ¹for the straight or branched alkyl of hydrogen, C1-C6, then obtain through reduction reaction

, R ¹for the straight or branched alkyl of hydrogen, C1-C6;

(2) will

, R ¹for the straight or branched alkyl of hydrogen, C1-C6, and acid-respons obtains

,

or

, R ¹for the straight or branched alkyl of hydrogen, C1-C6;

(3) will

, R ¹for the straight or branched alkyl of hydrogen, C1-C6, obtain under the catalysis of ammonium acetate with 6-bromopyridine 2-formaldehyde

, R ¹for the straight or branched alkyl of hydrogen, C1-C6.

3. an application for STAT3 inhibitor as claimed in claim 1, is characterized in that: described STAT3 inhibitor is for the preparation of the medicine for the treatment of mammary cancer, lung cancer, liver cancer, cervical cancer and prostate cancer.