CN102212613A - Application of (E,E)-2-(phenylmethyl aminocarbonyl)-3-styrene-acrylonitrile and derivatives thereof - Google Patents

Application of (E,E)-2-(phenylmethyl aminocarbonyl)-3-styrene-acrylonitrile and derivatives thereof Download PDF

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CN102212613A
CN102212613A CN2011100775482A CN201110077548A CN102212613A CN 102212613 A CN102212613 A CN 102212613A CN 2011100775482 A CN2011100775482 A CN 2011100775482A CN 201110077548 A CN201110077548 A CN 201110077548A CN 102212613 A CN102212613 A CN 102212613A
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bcl
nhc
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aminocarboxyl
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张志超
谢飞博
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Dalian University of Technology
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Dalian University of Technology
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Abstract

The invention provides application of (E,E)-2-(phenylmethyl aminocarbonyl)-3-styrene-acrylonitrile and derivatives thereof. The compounds serve as Bcl-2 family protein inhibitors, and play a role of killing tumor cells by directly bonding Bcl-2 and/or Mcl-1 and/or Bcl-xl proteins. The compounds can be used for screening the tumor cells which can be induced for apoptosis, and can be also used for preparing anti-tumor drugs such as the Bcl-2 family protein inhibitors or Bcl-2/Bax and/or Bcl-xL/Bak and/or Mcl-1/Bak protein dimer regulators.

Description

(E, E)-application of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivative thereof
Technical field
The present invention relates to the application of a compounds in preparation Bcl-2 family protein inhibitor series antineoplastic medicament.
Background technology
The Bcl-2 micromolecular inhibitor plays a role by simulation BH3 structural domain, is a class effective antitumour medicine.This compounds is by simulation natural B H3-only peptide, and competition is combined in the BH3 structural domain of Bcl-2 or Mcl-1, Bcl-xL protein surface, impels natural BH3-only peptide to discharge, activate, and performance promotes effect of apoptosis.So this type small molecular inhibitor is applicable to all high expression level Bcl-2, Mcl-1, and/or the proteic tumour of Bcl-xl comprise neoplastic hematologic disorder and solid tumor.
The applicant is engaged in the molecular designing of Bcl-2 inhibitor, synthetic and pharmaceutical research for many years.In PCT application WO2010/054575A1, Bcl-2 family protein (comprising Bcl-2, Bcl-xL and Mcl-1 albumen) inhibitor---acenaphthene and heterocyclic compound with single-minded target were disclosed once.According to the fragment-based principle, the applicant is at small molecules Bcl-2 inhibitors 3-parathiazan base-8-oxygen-8H-acenaphthene of applying for a patent and obtaining the authorization also [1,2-b] on pyrroles's-9-nitrile (be called for short S1) the basis, split and the molecule growth by molecule, obtained (E, E)-2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide.The structure of this compound once was disclosed (the 39th page in specification sheets, embodiment 17) in CN100413845C.But in above-mentioned disclosed patent specification; whole compounds of being protected are regarded the purposes of kinase modulator and are mainly used in the activity of regulating Tyrosylprotein kinase; be preferred for suppressing with the high expression level Tyrosylprotein kinase is the cell proliferation of the neoplastic hematologic disorder of one of principal character; and be used to identify the cell of expressing Tyrosylprotein kinase, study the location of Tyrosylprotein kinase.Yet the inventor through discovering this compound be not as described in the prior art be in suppressing cell proliferation, to play a role as kinase modulator, but as the Bcl-2 family protein inhibitor, in conjunction with Bcl-2 and/or Mcl-1 and/or Bcl-xl albumen, brought into play the effect of kill tumor cell by directly.Based on the result of above-mentioned research, the contriver thinks that being necessary to give this compounds locatees to use more accurately.
Summary of the invention
The contriver finds in up-to-date research, with (E, E)-2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide (hereinafter also abbreviating 3g as) for a compounds of representative by directly in conjunction with Bcl-2 and/or Mcl-1 and/or Bcl-xl albumen, brought into play the effect of kill tumor cell, therefore what this application of compound should be not as described in the prior art, being confined to/being preferred for the neoplastic hematologic disorder is the tumour of kinases high expression level of representative, but can be used for all high expression level Bcl-2 and/or Mcl-1 and/or the proteic malignant tumour of Bcl-xl.Secondly, the effect of this killing tumor cell may cause kinase whose downward modulations such as Tyrosylprotein kinase indirectly, but does not rely on kinase whose downward modulation.Once more, in the tumour that does not have the Tyrosylprotein kinase high expression level,, still can treat with this compounds as long as meet the indication of this compound.The 4th, kinases especially Tyrosylprotein kinase is not a tumor specific expression, so kinase inhibitor class medicine will have corresponding toxic side effect and so and next contraindication.
Therefore, the objective of the invention is at so that (E E)-2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide is that the mechanism of action of a compounds killing tumor cell of representative is studied, thereby instructs for the use of this compounds provides more accurately.
At first, the invention provides a kind of screening can by (E, E)-method of the tumour cell of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivative induced apoptosis thereof, this method may further comprise the steps (a) and/or (b):
(a) be positioned at karyomit(e) 18q21.3 in the detection tumour cell and go up the Bcl-2 gene copy number, is benchmark with gene copy number for 2.8 times greater than contrast, determine can by (E, E)-tumour cell of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivative induced apoptosis thereof;
(b) tumour cell is carried out immunohistochemical staining, detect Mcl-1 and/or Bcl-xL expressing quantity, is benchmark with immunohistochemical staining greater than 10%, determine can by (E, E)-tumour cell of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivative induced apoptosis thereof.
Wherein, described tumour cell can be from the tumor cell in vitro culture, or from tumour patient, i.e. tumour cell sample of Li Tiing.
The present invention also provide a kind of be used for the screening can be by (E, E)-test kit of the tumour cell of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivative induced apoptosis thereof, this test kit comprises the element that is used to detect the Bcl-2 gene copy number, and/or is used to detect the element of Mcl-1 and/or Bcl-xL expressing quantity.The described element that is used to detect the Bcl-2 gene copy number preferably includes the probe of identification karyomit(e) 18q21.3 and the probe of No. 18 kinetochores of identification.More preferably also comprise Trizol, fluorescence dye SYBR Green I, ThermoScript II, 6 base random primers.The described element that is used to detect Mcl-1 and/or Bcl-xL expressing quantity preferably includes anti-people Mcl-1 of animal source and Bcl-xL antibody, and biotin labeling two is anti-, the anti-people Mcl-1 in rabbit source for example, and rabbit source anti-people Bcl-xl antibody, the anti-rabbit of biotin labeling two is anti-.More preferably, describedly be used to detect the element that Mcl-1 and/or Bcl-xL express and also comprise rabbit anteserum, staining fluid and colour developing liquid.Wherein, described staining fluid comprises the antibiotin peroxidase, tires 5; Described colour developing liquid comprises the 10mM diaminobenzidine.
On the other hand, the invention provides a kind of examination be suitable for (E, E)-method of the tumour patient of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivatives for treatment thereof, this method may further comprise the steps:
(a) obtain the tumour cell of tumour patient, preparation becomes stripped tumour cell sample;
(b) detect Bcl-2 gene copy number in the tumour cell, and/or tumour cell is carried out immunohistochemical staining, detect Mcl-1 and/or Bcl-xL expressing quantity;
(c) with gene copy number greater than 2.8 times of contrasts, and/or immunohistochemical staining be benchmark greater than 10%, screening suitable (E, E)-tumour patient of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivatives for treatment thereof.
The present invention also provides a kind of examination to be suitable for (E, E)-test kit of the tumour patient of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivatives for treatment thereof, this test kit comprises the element that is used to detect the Bcl-2 gene copy number, and/or be used to detect the element that Mcl-1 and/or Bcl-xL express, and/or be used to detect the element that short antiapoptotic factors Bax and Bak express.
In the optimal technical scheme, the described element that is used to detect the Bcl-2 gene copy number comprises the probe of identification karyomit(e) 18q21.3 and the probe of No. 18 kinetochores of identification.More preferably, this element also comprises Trizol, fluorescence dye SYBR Green I, ThermoScript II, 6 base random primers.
In another optimized technical scheme, describedly be used to detect the element that Mcl-1 and/or Bcl-xL express and comprise anti-people Mcl-1 of animal source and Bcl-xL antibody, biotin labeling two is anti-, for example the anti-people Mcl-1 in rabbit source, rabbit source anti-people Bcl-xl antibody, anti-rabbit two resists biotin labeling.More preferably, this element also comprises rabbit anteserum, staining fluid and colour developing liquid.Wherein, described staining fluid comprises the antibiotin peroxidase, tires 5; Described colour developing liquid comprises the 10mM diaminobenzidine.
In another technical scheme, above-mentioned suitable (the E of examination that is used for, E)-test kit of the tumour patient of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivatives for treatment thereof comprises that also being used to detect the element that short antiapoptotic factors Bax and Bak express comprises anti-people Bax of animal source and Bak antibody, the anti-people actin of animal source antibody, for example anti-people Bax in rabbit source and Bak antibody, mouse source anti-people actin antibody, rabbit source anti-people actin antibody, the anti-rabbit two of HPR mark is anti-, the anti-rabbit two of FITC mark is anti-, and the anti-mouse two of Cy3 mark is anti-.Preferably, describedly be used to detect the element that short antiapoptotic factors Bak expresses and also comprise lysate, confining liquid, washings, commentaries on classics film damping fluid, colour developing liquid and stationary liquid.Wherein, described confining liquid is 5%BSA; Described washings comprises 10mM Tris-HCl, pH8.0,150mM NaCl and 0.1%Tween-20; Described lysate comprises 62.5mM Tris-HCl, and pH 6.8,2%w/v SDS, 10%glycerol, 50mMDTT and 1mM PMSF; Described commentaries on classics film damping fluid comprises the 39mM glycine, 48mM Tris, 0.0375% (w/v) SDS and 20% (v/v) methyl alcohol; Described stationary liquid is 10% (v/v) formaldehyde.
Again on the one hand, the invention provides a kind of method of inducing apoptosis of tumour cell, comprise give the tumour cell effective dose (E, E)-step of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide or derivatives thereof.Wherein said tumour cell refers to the proteic tumour cell of high expression level Bcl-2 and/or Bcl-xL and/or Mcl-1, especially solid tumor cells such as liver cancer, mammary cancer, lung cancer, prostate cancer also comprise the proteic various leukemia of high expression level Bcl-2 and/or Bcl-xL and/or Mcl-1.
Can amplify out a kind of tumor treatment method thus, comprise (the E that gives from the tumour cell treatment effective dose of tumour patient, E)-2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide or its derivative, detect Bcl-2/Bax, the content of Mcl-1/Bak and Bcl-xl-Bak, contrast is without E, E)-tumour cell that 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide or its derivative are handled, with the release of Bax/Bak as the effective standard of treatment.
The present invention also provides a kind of employing (E, E)-test kit of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide or its derivatives for treatment tumour patient, this test kit comprises the (E of significant quantity, E)-and 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide or its derivative, be used to detect the dimeric element of Bcl-2/Bax and/or Mcl-1/Bak and/or Bcl-xl/Bak.Preferably, the described measuring element that is used for comprises the anti-people Bcl-2 of animal source, Bax, and Mcl-1 and Bak and Bcl-xl antibody, for example anti-people Bcl-2 in rabbit source and Bax antibody, the anti-people Mcl-1 in rabbit source, Bcl-xl and Bak antibody, the anti-rabbit two of HPR mark is anti-.More preferably, the described measuring element that is used for also comprises albumin A/G agar beads, confining liquid, washings, lysate, commentaries on classics film damping fluid, colour developing liquid and stationary liquid.Wherein, described confining liquid is 5%BSA; Described washings comprises 10mM Tris-HCl, pH8.0,150mM NaCl and 0.1%Tween-20; Described lysate comprises 150mM NaCl, 10mM HEPES pH 7.4,2%CHAPS, 50mM DTT and 1mM PMSF; Described commentaries on classics film damping fluid comprises the 39mM glycine, 48mM Tris, 0.0375% (w/v) SDS and 20% (v/v) methyl alcohol; Described stationary liquid is 10% (v/v) formaldehyde.
The present invention also provides a kind of adjusting, preferably suppresses Bcl-2/Bax and/or Bcl-xL/Bak and/or the dimeric method of Mcl-1/Bak albumen in the cell, comprise give the cell effective dose (E, E)-step of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide or derivatives thereof.
Again on the one hand, the invention provides (the E that the tumour patient of this compounds for treating of needs is given significant quantity, E)-and 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide or its derivative disturb the dimeric method of Bcl-2/Bax and/or Mcl-1/Bak and/or Bcl-xl/Bak, and this method may further comprise the steps:
(a) give this compounds that tumour patient is treated significant quantity, described tumour patient is the tumour patient of determining according to above-mentioned method that is suitable for this compounds for treating;
Preferably, aforesaid method is further comprising the steps of:
(a) detect Bcl-2/Bax in the tumour cell of taking from tumour patient, Mcl-1/Bak, the dimeric content of Bcl-xk-Bak;
(b), determine that this compounds for treating tumour patient is effective according to Bcl-2/Bax and/or Mcl-1/Bak and/or Bcl-xl/Bak is dimeric dissociates.
In addition, The compounds of this invention can be used for research tool, is used to study the interaction between the Bcl-2 family protein.
Moreover, the invention also discloses (E, E)-and 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and the application of derivative in preparation Bcl-2 family protein inhibitor series antineoplastic medicament or albumen dimer conditioning agent thereof, described albumen dimer comprises the dimer of Bcl-2/Bax and/or Bcl-xL/Bak and/or Mcl-1/Bak.Described antitumor drug or albumen dimer conditioning agent be described (E, E)-2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide or derivatives thereof mixes with the conventional medicinal assistant agent of accepting, and is prepared into the pharmaceutical dosage form of routine.Assistant agent commonly used is pharmacy available vehicle, disintegrating agent, tackiness agent, Drug coating etc. for example.Those skilled in the art can use the conventional formulation technology to obtain the said products.
More than in each technical scheme, from the tumour cell of tumour patient, promptly the tissue samples of Li Tiing comprises patient's peripheral blood sample, neoplasmic tissue sample, acupuncture sample, marrow sample, the lymphoglandula sample, urine sample, ascites sample, the gastric lavage sample, oesophagus flushing sample, bladder or lung's flushing sample, the cerebrospinal fluid sample, conduit is drawn sample, galactography sample, the hydrothorax sample, tissue freezing sample, paraffin-embedded tissue sample.Tissue samples is used for detecting after certain processing.For example, from patient's peripheral blood sample, isolate the specific epidermal cells group, be used for detecting.Micro-separation tumor tissues obtains to be rich in the sample of tumour cell, is used for detecting.Preferred tissue samples comprises peripheral blood sample, neoplasmic tissue sample, acupuncture sample, paraffin embedding sample and marrow sample.
The test of protein two dimension nuclear-magnetism, the fluorescence polarization test, enzyme linked immunosorbent assay, (comprise human liver cancer cell SMMC-7721 in a plurality of clones, mouse liver cancer cell H22, human breast cancer cell MCF-7, human lung carcinoma cell H1688, H520, Human Prostate Cancer Cells DU145, with people's acute lymphoblastic leukemia Nalm-6) in Cell Biology Experiment, and the experiment of subcutaneous lotus liver cancer H22 solid tumor mouse all proves (E, E)-2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide is a BH3 analogue, be combined in Bcl-2, Mcl-1, BH3 structural domain with Bcl-xl, and therefore in culturing cell and at somatocyte internal disintegration Bcl-2/Bax, the dimer of Mcl-1/Bak, discharge and activate Bax and Bak, thereby with the mode kill tumor cell of inducing apoptosis of tumour cell.For example at high expression level Bcl-2, Mcl-1, in the cell of Bcl-xl from liver cancer resection organization, 3g can induce Bcl-2/Bax, the dimeric downward modulation of Mcl-1/Bak and Bcl-xl/Bak, therefore and cause apoptosis, but can not be at high expression level Bcl-2 not, apoptosis-induced in the cell of Mcl-1 and Bcl-xl from people's hepatic cyst resection organization.Especially, this apoptosis-induced effect does not rely on kinase whose downward modulation.In the acute lymphoblastic leukemia cell strain, the following mediation apoptosis of 3g inductive Mcl-1/Bak all occurs in before the Brc-ABL kinases downward modulation, prove that the 3g inductive is the apoptosis reduced of dependent kinase not, and its direct target spot is the Bcl-2 family protein.At last, in liver cancer model mouse body, 0.1mg/kg body weight 3g abdominal injection just caused Bcl-2/Bax in 10 days, the dimeric downward modulation of Mcl-1/Bak and Bcl-xl/Bak, and tumor control rate is about 30% under the corresponding dosage.
More than in each method, the tumorous cellular density of carrying out common cultivation is 10 5～10 7/ mL, the concentration of The compounds of this invention is 5～10 μ M, incubation time is 24 hours altogether.
Further two-dimentional nuclear-magnetism and structural simulation show, have and (E, E)-some row compounds in 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide analog structure territory all have similar activity, this compounds all contains α cyano group structure, the cyano group in the structure and with the be separated by R of one or two heavy atom of cyano group 1In N, the O atom together with Mcl-1/ albumen in R263 and N260 form hydrogen bond network, thereby occupy Mcl-1 protein B H3 in conjunction with groove.Bcl-2 and Bcl-xl albumen have R and N amino-acid residue equally in corresponding position, and they also are same molecular basises with these two proteic combinations.Therefore, (the E that above-mentioned any one technical scheme of the present invention is addressed, E)-2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivative thereof also comprise a series of compounds that have the same structure territory with this compound, comprises the compound of general formula I, general formula I I and general formula III, wherein:
The compound of described general formula I has following general structure:
In the general formula I:
R 1, R 2And R 3Be selected from independently of one another: H, OH, C (1-6)Alkyl, (CH 2) nAr, OC (1-6)Alkyl, O (CH 2) nAr, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, SH, SC (1-6)Alkyl, S (CH 2) nAr, NO 2, CF 3, OCF 3And halogen;
R 4Be selected from: C (X) R 5, SO 3Ar, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), NH (CH 2) nAr, PO (OH) 2, PO (OC (1-6)Alkyl) 2And C (NH 2)=C (CN) 2
X is selected from: O, S, NH and NC (1-6)Alkyl;
R 5Be selected from: OH, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, N[(CH 2) nAr] [(CH 2) qAr], NH (CH 2) nOH, (CH 2) nOC (1-6)Alkyl, O (CH 2) nAr, C (1-6)Alkyl, NHNH 2, NHNHC (1-6)Alkyl, NHNH (CH 2) nAr, NHC (O) NH 2, NHC (O) OC (1-6)Alkyl, N-morpholine and N-pyrroles;
Ar does not have to replace or 1～4 fragrance or the assorted aromatic group that replaces, and wherein substituting group is independently selected from: OH, C (1-6)Alkyl, (CH 2) nAr, OC (1-6)Alkyl, O (CH 2) nAr, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), NH (CH 2) nAr, SH, SC (1-6)Alkyl, S (CH 2) nAr, NO 2, CF 3, OCF 3And halogen;
N is 0～4;
The compound of described general formula I I has following general structure:
Figure BDA0000052705140000071
Among the general formula I I:
R 1Be selected from: C (X) R 2, SO 2R 2, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), NH (CH 2) nAr, C (NH 2)=C (CN) 2, PO (OH) 2, PO (OC (1-6)Alkyl) 2And PO[O (CH 2) nAr] 2
X is selected from: O, S, NH, NC 1-6Alkyl and N (CH 2) nAr;
R 2Be selected from: H, OH, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, N[(CH 2) nAr] [(CH 2) qAr], NH (CH 2) nOH, (CH 2) nOC (1-6)Alkyl, O (CH 2) nAr, C (1-6)Alkyl, (CH 2) nAr, NHNH 2, NHNHC (1-6)Alkyl, NHNH (CH 2) nAr, NHC (O) NH 2, NHC (O) OC (1-6)Alkyl, N-morpholine, N-parathiazan, N-piperidines, N-piperazine and N-pyrroles;
Ar is fragrance or the assorted aromatic group that does not have replacement or 1-4 replacement, and wherein substituting group is independently selected from: OH, C (1-6)Alkyl, (CH 2) nAr, OC (1-6)Alkyl, O (CH 2) nAr, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, SH, SC (1-6)Alkyl, S (CH 2) nAr, NO 2, CF 3, OCF 3And halogen;
N is 0～4.
The compound of described general formula III has following general structure:
Figure BDA0000052705140000081
In the general formula III:
R 1, R 2, R 3, R 4And R 5Be independently selected from: H, OH, C (1-6)Alkyl, (CH 2) nAr, OC (1-6)Alkyl, O (CH 2) nAr, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, SH, S C (1-6)Alkyl, S (CH 2) nAr, NO 2, CF 3, OCF 3And halogen;
R 6Be selected from: C (X) R 7, SO 2R 7, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), NH (CH 2) nAr, C (NH 2)=C (CN) 2, PO (OH) 2, PO (OC (1-6)Alkyl) 2And PO[O (CH 2) nAr] 2
X is selected from: O, S, NH, NC 1-6Alkyl and N (CH 2) nAr;
R 7Be selected from: H, OH, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, N[(CH 2) nAr] [(CH 2) qAr], NH (CH 2) nOH, (CH 2) nOC (1-6)Alkyl, O (CH 2) nAr, C (1-6)Alkyl, (CH 2) nAr, NHNH 2, NHNHC (1-6)Alkyl, NHNH (CH 2) nAr, NHC (O) NH 2, NHC (O) OC (1-6)Alkyl, N-morpholine, N-parathiazan, N-piperidines, N-piperazine and N-pyrroles;
Ar is fragrance or the assorted aromatic group that does not have replacement or 1-4 replacement, and wherein substituting group is independently selected from: OH, C (1-6)Alkyl, (CH 2) nAr, OC ( 1-6)Alkyl, O (CH 2) nAr, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, SH, SC (1-6)Alkyl, S (CH 2) nAr, NO 2, CF 3, OCF 3And halogen;
N is 0～4.
Technical scheme of the present invention, defined with (E, E)-2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide is that a compounds of representative is as the correct pharmacological Mechanism of anticarcinogen with go for all high expression level Bcl-2 and/or Mcl-1 and/or the proteic malignant tumour of Bcl-xl, the application prospect that comprises solid tumor and hematopoietic tumor, this compounds clinical application and exploitation are of great immediate significance, clear and definite with (E, E)-and 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide is the proteic inhibitor of a compounds of representative as the tumour-specific high expression level, the contraindication that is not subjected to and kinase inhibitor is general limited.
Description of drawings
Below, describe embodiment of the present invention in conjunction with the accompanying drawings in detail, wherein:
Fig. 1 shows 3g and Bcl-2 (A), Mcl-1 (B), and the protein two-dimensional nucleus magnetic knot fruit of Bcl-xl (C), 3g is combined in three proteic BH3 grooves respectively, causes the tangible chemical shift of amino acid in the BH3 groove, and other position amino acid does not have obvious chemical shift.
Fig. 2 shows 3g and Bcl-2, Mcl-1, and the competition binding curve of Bcl-xl shows that 3g can be combined in three proteic BH3 grooves.
Accompanying drawing 3 shows that the apoptosis-induced immediate cause of 3g is Bcl-2/Bax, the dimeric downward modulation of Mcl-1/Bak and Bcl-xl/Bak, rather than kinases downward modulation.The kinases downward modulation is the downstream events of apoptosis.Wherein: Fig. 3 A: flow cytometer detects 3g inductive Nalm-6 apoptosis.3 hours time points for the apoptosis startup.Fig. 3 B: co-immunoprecipitation detects 3g inductive Bcl-2/Bax, the dimeric downward modulation of Mcl-1/Bak and Bcl-xl/Bak.The release of Bax and Bak occurs in 2.5 hours, early than the apoptosis starting point.Fig. 3 C:western detects the BRC-ABL downward modulation.Downward modulation occurs in 5 hours, is later than the apoptosis starting point.
Accompanying drawing 4 shows that 3g relies on Bax and Bak cell death inducing fully.Utilize siRNA, delete intracellular Bax and Bak.Negative control product gossypol and 3g act on wild-type cell and transfectional cell respectively, and flow cytometer detects finds that the apoptosis-induced ability of 3g completely loses in transfectional cell.
Accompanying drawing 5 shows 3g at high expression level Bcl-2 and Mcl-1, and is apoptosis-induced in the cell of Bcl-xl.Wherein: Fig. 5 A:H1688, H520, NCI-H345, SKOV3, healthy human peripheral blood white corpuscle and normal cell are the Bcl-2 gene quantification among the HEK293.Wherein, H1688, H520, the gene expression amount of NCI-H345, SKOV3 are higher than more than 2.8 times of HEK293, and the healthy human peripheral blood cell is high expression level not.Fig. 5 B: immunohistochemical staining detects H1688, and H520, NCI-H345, SKOV3, healthy human peripheral blood white corpuscle and normal cell are that Mcl-1 and the Bxl-xl rate of dyeing among the HEK293 is higher than 10%.Fig. 5 C: flow cytometer detects the effect of 3g to above-mentioned clone.3g induces H1688 in dosage dependence mode, H520, and NCI-H345 and SKOV3 apoptosis, can not induce healthy human peripheral blood white corpuscle and normal cell is the apoptosis of HEK293 cell.
Accompanying drawing 6 shows that 3g can kill liver cancer cell, can not kill the hepatic cyst cell.Fig. 6 A:Bcl-2 gene quantification shows liver cancer cell high expression level Bcl-2 gene, and hepatic cyst does not have high expression level.Fig. 6 B: immunohistochemical staining detects and shows that liver cancer tissue section Mcl-1 and Bxl-xl rate of dyeing are higher than 10%, and the hepatic cyst section is negative for dyeing.Fig. 6 C: flow cytometer detects the effect of 3g to two routine cells.3g can not kill the hepatic cyst cell with the apoptosis-induced liver cancer cell that kills.Fig. 6 D: co-immunoprecipitation detects 3g and induces Bcl-2/Bax in the liver cancer cell, and the dimeric downward modulation of Mcl-1/Bak and Bcl-xl/Bak can not be induced above-mentioned dimeric downward modulation in the hepatic cyst cell.
The test-results that accompanying drawing 7 shows behind lotus H22 mouse vivo medicine-feeding 3g, show 3g by reducing Bcl-2/Bax, Mcl-1/Bak and Bcl-xl/Bak dimer suppress the intravital tumor growth of lotus H22 mouse, and with after the ineffective dose effect in the body, above-mentioned dimer is downward modulation not.Wherein: Fig. 7 A: the tumor tissues that strips.0.02mg/kg 3g does not have tumor-inhibiting action, 0.1mg/kg 3g tumour inhibiting rate is about 30%.Fig. 7 B, Fig. 7 C and Fig. 7 D show respectively: 0.1mg/kg 3g reduces Bcl-2/Bax, Mcl-1/Bak and Bcl-xl/Bak dimer, not downward modulation effect of 0.02mg/kg 3g.
Embodiment
Below in conjunction with embodiment the present invention is further described in detail, the embodiment that provides is only in order to illustrate the present invention, rather than in order to limit the scope of the invention.
Below among each embodiment, employed various test materialss and reagent source are as follows:
Tumor cell line DU145, K562, MKN-28, HCT116, SMMC-7721, H22, HL-60, Nalm-6 is available from Chinese Academy of Sciences's cell bank.
Bak antibody (sc-7873), actin antibody (sc-130656), Bcl-2 antibody (sc-783), Bax antibody (sc-8265) is available from U.S. Santa Cruz company; Mcl-1 antibody (BS1220), Bxl-xl antibody is available from U.S. Bioworld company.Anexin V apoptosis detection reagent (C1063), ProteinA/G-agarose co-immunoprecipitation test kit (P2012) is available from China green skies Bioisystech Co., Ltd.Lipofectamine 2000 is available from American I nvitrogen company.
BALB/c mouse is available from Dalian Medical Univ's Experimental Animal Center.
Conventional culture of tumor cell method is with reference to " mammalian cell is cultivated handbook.
Embodiment 1:3g directly is combined in Bcl-2, the proteic BH3 structural domain of Mcl-1 and Bcl-xL
At first make up Bcl-2, Mcl-1 and Bcl-xL expression plasmid.Extract the total RNA of human leukemia HL-60 cell, carry out reverse transcription PCR.Utilize primer 5 '-GGATCCGAGGACGAGTTGTACCGGC-3 ' and 5 '-AAGCTTCTAGCCACCTTCTAGGTC CTCT-3 ', obtain the Mcl-1 target gene sequences.Extension increasing sequence is connected on the expression vector pET28a (+), thereby obtains the Mcl-1 protein expressing plasmid.Make up the Bcl-xL expression plasmid: reverse transcription PCR amplification total length Bcl-xL cDNA, utilize PCR method to reject amino acid whose coding region of 45-85 and the amino acid whose coding region of C end 212-233.By restriction enzyme site, be connected on the pHis-TEV carrier; Make up the Bcl-2 expression plasmid: reverse transcription PCR amplification Bcl-2 target gene sequences (coded amino acid 1-218).Extension increasing sequence is connected on the expression vector pET28a (+), thereby obtains the Bcl-2 protein expressing plasmid.Three kinds of target proteins are expressed in E.coli BL21 (DE3) bacterium, are used for the NMR experiment.Thalli growth is adding 15The M9 of N mark ammonium chloride cultivates and concentrates.0.4mM behind the IPTG abduction delivering 4 hours, collect thalline, the cracking fragmentation utilizes the Ni post to carry out the target protein purifying.
1H/ 15N albumen hsqc spectrum figure is at 0.1mM 15Measure under the N labelled protein concentration. 1H/ 15N compound/albumen hsqc spectrum figure is at the 0.1mM protein concentration, and compound mixed under is measured at 1: 1.Experiment is carried out on Bruker DRX600MHz two dimension nuclear magnetic resonance spectrometer.Data processing is utilized NMRPipe software.Chemical displacement value formula ((Δ 1H ppm) 2+ (0.2 * Δ 15N ppm) 2) 0.5) the calculating acquisition.
Compound respectively with Bcl-2, Mcl-1 and the combination of Bcl-xL hydrophobic pocket are induced the chemistry of amino acids displacement result as shown in Figure 1.The chemistry of amino acids shift value is defined as with compound above 0.1ppm interaction.Compound is induced the displacement of Mcl-1 chemistry of amino acids, and 78% site is positioned at the BH3 structural domain; Compound is induced the displacement of Bcl-xL chemistry of amino acids, and 63% site is positioned at the BH3 structural domain; Compound is induced the displacement of Bcl-2 chemistry of amino acids, and 69% site is positioned at the BH3 structural domain.
Embodiment 2: compete in conjunction with Bcl-2 and/or Bcl-xL and/or the proteic ability of Mcl-1 by fluorescence polarization assay method detection 3g and other testing compound
Synthetic one have 21 amino acid whose Bid BH3 peptide sections (amino acid: 79-99:QEDIIRNIARHLAQVGDSMDR), and on N end mark 6-Fluoresceincarboxylic acid succinimide ester (FAM) as fluorescence labels (FAM-Bid).Competition is that GST-Bcl-2 albumen (40nM) or Mcl-1 albumen or Bxl-xl albumen and FAM-Bid polypeptide (5nM) are dissolved in (100mM K in the reaction buffer in conjunction with used reaction system in the experiment 3PO 4, pH 7.5; 100 μ g/mL ox γ albumin; 0.02% sodiumazide).In 96 orifice plates, every hole adds 100 μ L reaction systems, add then 1 μ L different concns the compound to be detected that is dissolved in DMSO (E, E)-mother liquor of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide is to the required final concentration of experimental design.Set up two control groups simultaneously, a control group is only to contain Bcl-2 or Mcl-1 or Bcl-xL and FAM-Bid (being equivalent to 0% inhibiting rate) in the reaction system, and the reaction system in another control group only contains FAM-Bid peptide section.96 orifice plates carry out detecting on the microplate reader after hatching through 4 hours lucifuge.Fluorescence polarization value (mP) is measured under the 485nm emission wavelength that is excited generation by the 530nm wavelength.Ki value root calculation formula is derived and is drawn.Test-results as shown in Figure 2.The proteic competition binding constant of this compound and Bcl-2 is 25nM, is 5nM to the competition binding constant of Mcl-1, is 35nM to Bcl-xl.
Detect other 7 compounds and Bcl-2 and the proteic binding constant of Mcl-1, Bcl-xl (being called for short binding constant in the table 1) also in the nM level according to above-mentioned identical test method, concrete outcome is as shown in table 1.
Table 1
Figure BDA0000052705140000121
Embodiment 3:3g cell death inducing Mechanism Study
Human liver cancer cell SMMC7721, lung carcinoma cell H1688 and leukemia cell NALM-6 cell are carried out routine cultivate, to density be 106/mL.Add DMSO and 10 μ M 3g respectively and handled 24 hours, collecting cell, 2%CHAPS lysate, 4 ℃ of cracking 10 minutes.The quantitative albumen of Xylene Brilliant Cyanine G, each sample is got 500 μ g, use the anti-Mcl-1 antibody of 2 μ g or 4 ℃ of overnight incubation of anti-Bcl-2 antibody respectively, add proteinA/G-agarose co-precipitation test kit subsequently, hatched 1 hour centrifugal collection immunoprecipitation complex for 4 ℃, electrophoresis, change film, use corresponding protein in the anti-Bak antibody test Mcl-1 mixture respectively, with corresponding protein in the anti-Bax antibody test Bcl-2 mixture.
Test-results is seen accompanying drawing 3, with acute lymphoblastic leukemia cell Nalm-6 is example, 5 μ M3g function cells are after 2.5 hours, discovery is combined in the Bax on the Bcl-2, the Bak that is combined on Mcl-1 and the Bcl-xl is released, the apoptosis of cell (11%) beginning subsequently, and continue to increase, to 7 hours, cell survival rate only had 10%.In this process, detected the BRC-ABL kinase activity, the kinases horizontal down-regulation appears at 5 hours, obviously is later than the time of origin of apoptosis.
The above results shows: 3g is by inducing Mcl-1/Bak, and Bcl-xl/Bak and Bcl-2/Bax are dimeric to be dissociated, and performance is the effect of the killing tumor cell of mechanism with the apoptosis.
Embodiment 4: the cytotoxicity experiment of compound dependence BAX/BAK is verified the characteristic of its BH3 analogue
Coprecipitation of calcium phosphate transfection 3 μ g BAX/BAK interference plasmids are to human breast cancer cell MCF-7 cell, after the transfection 24 hours, collecting cell, Western detects RNA and disturbs back BAX and BAK protein expression situation, and same treatment plasmid-free cells transfected group is made as control group.Cell inoculation after the transfection is in 96 orifice plates (1 * 10 5Individual/hole), the parallel control experiment of carrying out untransfected plasmid cell group, add compound (E to be detected by the experimental design concentration gradient, E)-2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide, act on after 48 hours, MTT detects cell viability, and the result as shown in Figure 4, Gossypol is as non-specific BH3 analogue and compound of the present invention contrast parallel processing, and visible 3g has the cytotoxicity of absolute dependence BAX/BAK.
Detect other 5 compounds (compound as described below 1.～8.) according to above-mentioned identical test method, the result shows that institute's detection compound also all has the effect characteristics of absolute dependence BAX/BAK.These compounds comprise:
1. (E, E)-the 2-[(3-hydrocinnamyl) aminocarboxyl]-3-styryl vinyl cyanide;
2. (E, E)-2-aminocarboxyl-3-styryl vinyl cyanide;
3. 2-cyano group-N-phenmethyl ethanamide
4. (E)-2-aminocarboxyl-3-phenyl vinyl cyanide;
5. (E)-2-B aminocarbonyl-3-phenyl vinyl cyanide.
This absolute test-results that relies on Bax/Bak shows that further 3g and derivative thereof are only apoptosis-induced by Bcl-2 family protein path, do not have other any target spot.The kinases downward modulation that occurs in the apoptotic process is the downstream molecules incident of plastosome apoptosis pathway, does not play decisive role.3g and the derivative that the present invention relates to not are kinase inhibitor.
Embodiment 5: screening can be by the method for the apoptosis-induced tumour cell of 3g
Present embodiment is with human lung cancer cell line H1688, H520, and NCI-H345, Proliferation of Human Ovarian Cell are that SKOV3 and healthy human peripheral blood white corpuscle are example, screening can be by the apoptosis-induced tumour cell of 3g.Healthy human peripheral blood is from the healthy blood donor, and vein extracts the postheparin anti-freezing, handles the white corpuscle in the separation and Extraction whole blood through lymphocyte separation medium.Specifically may further comprise the steps:
A. detect the gene copy number of Bcl-2 in the cell.Concrete operations are as follows:
1) collects 10 6-10 7Individual cell adds 1mL Trizol, and room temperature was placed 5 minutes.2) add 0.2mL chloroform, concuss 15 seconds.3) get the upper strata water in a new pipe, add the 0.5mL Virahol, room temperature was placed 10 minutes, centrifugal 10 minutes of 12000g.4) abandon supernatant, drying at room temperature 10 minutes is dissolved in RNA 50 μ L water then.5) mix 0.5 μ L, 6 primers at random, the RNA that 0.5 μ L ThermoScript II and 3 μ L extract carries out reverse transcription.Reaction conditions is 37 ℃, 15 minutes, 85 ℃, 5 seconds, circulates 3 times.6) parallelly carry out the reaction of two real-time quantitative PCRs.Use 18q21.3 to go up the Bcl-2 gene primer in the reaction, as experimental group.Use No. 18 kinetochore gene primers in another reaction, in contrast group.Mix 12 μ L SYBR fluorescence dyes, 1 μ L upstream primer, 1 μ L downstream primer, 2 μ L template cDNA, 0.5 μ L transcriptase is quantitatively transcribed.Reaction conditions is 60 ℃, 30 seconds, 95 ℃, 5 seconds, circulates 40 times.7) transcription product is embedded fluorescence dye intensity 30 and be set at threshold value, the reference control group reaches the cycle index of threshold value, represents single copy, quantitative experiment group Bcl-2 gene copy number.To be higher than contrast is screening criteria for 2.8 times.
B. immunohistochemical methods detects the expressing quantity of Mcl-1 and Bcl-xl.
1) tumour cell smear at first carries out pre-treatment, is summarized as follows: roasting sheet, dimethylbenzene dewaxing, gradient ethanol aquation, 3%H 2O 2Handled 20 minutes.2) the normal rabbit serum sealing is 20 minutes.3) adding dilution in 1: 800 rabbit anti-people Mcl-1 in source or Bcl-xL antibody, 4 ℃ of overnight incubation.The parallel laboratory test group adds and does not contain an anti-diluent, as negative control group.4) add 1: 500 anti-rabbit two of dilution biotin labeling and resist incubated at room 30 minutes.5) add antibiotin peroxidase stain liquid, room temperature dyeing 10 minutes.6) add 10mM diaminobenzidine colour developing liquid, color development at room temperature 10 minutes.7) opticmicroscope is observed coloration result down.Reference negative control group staining power, statistics tumour cell stained positive ratio.To be higher than 10% is screening criteria.
C. will cultivate altogether by tumour cell and the 3g that above-mentioned screening obtains.
Cultivate and reach 10 in routine 6In the above-mentioned cell of/mL, add DMSO (control group) and 10 μ M 3g respectively and handled 24 hours.Centrifugal collecting cell.PBS washes twice, and the Annexin V that is connected with FITC-was hatched 10 minutes jointly with 1: 40 ratio.Painted apoptotic cell is added up via flow cytometer.
The result as shown in Figure 5, as seen H1688, H520, the Bcl-2 gene of NCI-H345, SKOV3 cell all surpasses and all contrasts more than 2.8 times, the immunohistochemical staining positive rate is also all more than 10%, and they can both be killed with apoptosis-induced form by 3g, and healthy human peripheral blood cell and normal cell are that the HEK293 cell does not have high expression level said gene and albumen, (＜2%) can not be apoptosis-induced by 3g.
Embodiment 6: examination is suitable for the method for the tumour patient of 3g treatment
This enforcement is with the patient and a routine multiple hepatic cyst patient of a routine primary hepatocarcinoma, and examination is suitable for the patient's of 3g treatment method.Specifically may further comprise the steps:
A. obtain the liver cancer tissue and the benign tumor tissue of excision, grind the back and obtain single cell suspension and the section of preparation paraffin organization.
B. detect the Bcl-2 gene copy number of two routine single cell suspensions and/or the expression amount of Mcl-1 and/or Bcl-xl.
After extracting the mRNA of two routine cells respectively, utilize real-time quantitative PCR to detect 18q21.3 and go up the Bcl-2 gene copy number.Specific embodiments is with embodiment 6.Carry out the positive rate that immunohistochemical staining detects Mcl-1 and Bcl-xL at tissue slice simultaneously.Concrete grammar is with embodiment 5.
C. two routine cells and 3g are cultivated altogether.
Above-mentioned patient's single cell suspension, density are 10 5～10 7/ mL adds DMSO (control group) and 1 μ M 3g respectively and handled 24 hours.Centrifugal collecting cell.PBS washes twice, and the Annexin V that is connected with FITC-was hatched 10 minutes jointly with 1: 40 ratio.Painted apoptotic cell is added up via flow cytometer.
D. co-immunoprecipitation detects without in the cell that 3g handles and process 3g handles, Mcl-1/Bak, the content of Bcl-xl-Bak and Bcl-2/Bax.The co-immunoprecipitation method is with embodiment 3.
The result as shown in Figure 6, liver cancer patient Bcl-2 gene copy number is higher than control group more than 2.8 times, Mcl-1 and/or Bcl-xl protein immunization group positive rate are more than 10%.There are not high expression level said gene and albumen in hepatic cyst patient's the sample.3g kills liver cancer cell with apoptosis-induced form, can not kill the hepatic cyst cell.In the tumour cell of apoptosis, Mcl-1/Bak, the content downward modulation of Bcl-xl-Bak and Bcl-2/Bax, Bax and Bak are released, and do not have in the apoptotic cells, do not have the release of the above-mentioned dimeric Bax/Bak of mediation down.
Embodiment 7: the method that adopts 3g treatment tumour patient
Present embodiment provides the method that adopts 3g treatment tumour patient, specifically may further comprise the steps:
A. before giving tumour patient 3g treatment, detect Bcl-2 gene copy number and/or Mcl-1 and/or the proteic content of Bcl-xl in the tumor sample.Leave and take sample segment back to be treated simultaneously and detect validity.
(1) gathers the patient tumors sample, comprise patient's peripheral blood sample, neoplasmic tissue sample, the acupuncture sample, marrow sample, lymphoglandula sample, urine sample, ascites sample, gastric lavage sample, oesophagus flushing sample, bladder or lung's flushing sample, cerebrospinal fluid sample, conduit is drawn sample, galactography sample, hydrothorax sample, the tissue freezing sample, the paraffin-embedded tissue sample.Tissue samples is used for detecting after certain processing.For example, from patient's peripheral blood sample, isolate the specific epidermal cells group, be used for detecting.Micro-separation tumor tissues obtains to be rich in the sample of tumour cell, is used for detecting.The preferential tissue samples of selecting comprises peripheral blood, tumor tissues sample, acupuncture sample, paraffin embedding sample and marrow sample.(2) real-time quantitative PCR detects 18q21.3 and goes up the Bcl-2 gene copy number.To be higher than contrast is screening criteria for 2.8 times.Concrete grammar is with embodiment 6.(3) expression amount of Mcl-1 and Bcl-xl in the immunohistochemical methods method test sample.Concrete grammar is with embodiment 6.Being higher than 10% with positive rate is screening criteria.
B. give the 3g of tumour patient treatment significant quantity, at 1 day, 3 days, 5 days, after 10 days, the content of Mcl-1/Bak and Bcl-2/Bax in the sample of leaving and taking before the co-immunoprecipitation method detection tumor sample while contrast therapy, method is with embodiment 5.
C. the content of Mcl-1/Bak and Bcl-2/Bax before and after the contrast therapy is determined the tumour patient treatment effectively according to discharging from Bal-2 and/or above the Mcl-1 of Bax and/or Bak.Method is with embodiment 3.
By the Bcl-xl/Bak that dissociates, Mcl-1/Bak and Bcl-2/Bax realize suppressing tumor growth and prolong lifetime embodiment 8:3g in the tumor model animal body.
To BALB/c mouse shoulder, visible knurl piece forms after 14 days with rat liver cancer H22 injection cell.Random packet is three groups, one group 10, and the beginning administration.Wherein, two groups of subcutaneous injection administration 3g, dosage are respectively 0.02 and 0.1mg/kg mouse body weight, and administration time is 10 days, the next day once.Another group injection DMSO organizes in contrast.Administration is measured and is respectively organized the mouse tumor volume simultaneously.Put to death mouse on the 11st day, and peeled off tumour and observe.After each is organized mouse tumor and peels off, grind broken back cracking, each sample protein of detection by quantitative is got 500 μ g sample Bcl-2, and Mcl-1 and Bcl-xl antibody precipitation target protein detect the variation of measuring with its bonded Bax and Bak.
Test-results is seen Fig. 7, and wherein, 0.02mg/kg dosage 3g can not effectively suppress mouse tumor, fails the Bcl-2/Bax that dissociates on molecular level, and Mcl-1/Bak and Bcl-xl/Bak interact; 0.1mg/kg dosage 3g can effectively suppress mouse tumor, above-mentioned dimer can dissociate.Above-mentioned test-results proves, Bcl-2/Bax is effective in Mcl-1/Bak and the Bcl-xl/Bak downward modulation decision 3g body.

Claims (10)

  1. A screening can by (E, E)-method of the tumour cell of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivative induced apoptosis thereof, this method may further comprise the steps (a) and/or (b):
    (a) be positioned at karyomit(e) 18q21.3 in the detection tumour cell and go up the Bcl-2 gene copy number, is benchmark with gene copy number for 2.8 times greater than contrast, determine can by (E, E)-tumour cell of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivative induced apoptosis thereof;
    (b) tumour cell is carried out immunohistochemical staining, detect Mcl-1 and/or Bcl-xL expressing quantity, is benchmark with immunohistochemical staining greater than 10%, determine can by (E, E)-tumour cell of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivative induced apoptosis thereof.
  2. 2. the described method of claim 1 is characterized in that, described tumour cell is from tumor cell in vitro culture or tumour patient.
  3. 3. the described method of claim 1 is characterized in that, described (E, E)-derivative of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide comprises the compound of general formula I, general formula I I and general formula III, wherein:
    The compound of described general formula I has following general structure:
    Figure FDA0000052705130000011
    In the general formula I:
    R 1, R 2And R 3Be selected from independently of one another: H, OH, C (1-6)Alkyl, (CH 2) nAr, OC (1-6)Alkyl, O (CH 2) nAr, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, SH, SC (1-6)Alkyl, S (CH 2) nAr, NO 2, CF 3, OCF 3And halogen;
    R 4Be selected from: C (X) R 5, SO 3Ar, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), NH (CH 2) nAr, PO (OH) 2, PO (OC (1-6)Alkyl) 2And C (NH 2)=C (CN) 2
    X is selected from: O, S, NH and NC (1-6)Alkyl;
    R 5Be selected from: OH, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, N[(CH 2) nAr] [(CH 2) qAr], NH (CH 2) nOH, (CH 2) nOC (1-6)Alkyl, O (CH 2) nAr, C (1-6)Alkyl, NHNH 2, NHNHC (1-6)Alkyl, NHNH (CH 2) nAr, NHC (O) NH 2, NHC (O) OC (1-6)Alkyl, N-morpholine and N-pyrroles;
    Ar does not have to replace or 1～4 fragrance or the assorted aromatic group that replaces, and wherein substituting group is independently selected from: OH, C (1-6)Alkyl, (CH 2) nAr, OC (1-6)Alkyl, O (CH 2) nAr, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), NH (CH 2) nAr, SH, SC (1-6)Alkyl, S (CH 2) nAr, NO 2, CF 3, OCF 3And halogen;
    N is 0～4;
    The compound of described general formula I I has following general structure:
    Figure FDA0000052705130000021
    Among the general formula I I:
    R 1Be selected from: C (X) R 2, SO 2R 2, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), NH (CH 2) nAr, C (NH 2)=C (CN) 2, PO (OH) 2, PO (OC (1-6)Alkyl) 2And PO[O (CH 2) nAr] 2
    X is selected from: O, S, NH, NC 1-6Alkyl and N (CH 2) nAr;
    R 2Be selected from: H, OH, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, N[(CH 2) nAr] [(CH 2) qAr], NH (CH 2) nOH, (CH 2) nOC (1-6)Alkyl, O (CH 2) nAr, C (1-6)Alkyl, (CH 2) nAr, NHNH 2, NHNHC (1-6)Alkyl, NHNH (CH 2) nAr, NHC (O) NH 2, NHC (O) OC (1-6)Alkyl, N-morpholine, N-parathiazan, N-piperidines, N-piperazine and N-pyrroles;
    Ar is fragrance or the assorted aromatic group that does not have replacement or 1-4 replacement, and wherein substituting group is independently selected from: OH, C (1-6)Alkyl, (CH 2) nAr, OC (1-6)Alkyl, O (CH 2) nAr, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, SH, SC (1-6)Alkyl, S (CH 2) nAr, NO 2, CF 3, OCF 3And halogen;
    N is 0～4.
    The compound of described general formula III has following general structure:
    Figure FDA0000052705130000031
    In the general formula III:
    R 1, R 2, R 3, R 4And R 5Be independently selected from: H, OH, C (1-6)Alkyl, (CH 2) nAr, OC (1-6)Alkyl, O (CH 2) nAr, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, SH, S C (1-6)Alkyl, S (CH 2) nAr, NO 2, CF 3, OCF 3And halogen;
    R 6Be selected from: C (X) R 7, SO 2R 7, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), NH (CH 2) nAr, C (NH 2)=C (CN) 2, PO (OH) 2, PO (OC (1-6)Alkyl) 2And PO[O (CH 2) nAr] 2
    X is selected from: O, S, NH, NC 1-6Alkyl and N (CH 2) nAr;
    R 7Be selected from: H, OH, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, N[(CH 2) nAr] [(CH 2) qAr], NH (CH 2) nOH, (CH 2) nOC (1-6)Alkyl, O (CH 2) nAr, C (1-6)Alkyl, (CH 2) nAr, NHNH 2, NHNHC (1-6)Alkyl, NHNH (CH 2) nAr, NHC (O) NH 2, NHC (O) OC (1-6)Alkyl, N-morpholine, N-parathiazan, N-piperidines, N-piperazine and N-pyrroles;
    Ar is fragrance or the assorted aromatic group that does not have replacement or 1-4 replacement, and wherein substituting group is independently selected from: OH, C (1-6)Alkyl, (CH 2) nAr, OC (1-6)Alkyl, O (CH 2) nAr, NH 2, NHC (1-6)Alkyl, N (C (1-6)Alkyl) (C (1-6)Alkyl), N (C (1-6)Alkyl) [(CH 2) nAr], NH (CH 2) nAr, SH, SC (1-6)Alkyl, S (CH 2) nAr, NO 2, CF 3, OCF 3And halogen;
    N is 0～4.
  4. One kind be used for the screening can be by (E, E)-test kit of the tumour cell of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivative induced apoptosis thereof, this test kit comprises the element that is used to detect the Bcl-2 gene copy number, and/or is used to detect the element of Mcl-1 and/or Bcl-xL expressing quantity.
  5. An examination be suitable for (E, E)-method of the tumour patient of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivatives for treatment thereof, this method may further comprise the steps:
    (a) the stripped tumour cell sample of preparation;
    (b) detect Bcl-2 gene copy number in the tumour cell, and/or tumour cell is carried out immunohistochemical staining, detect Mcl-1 and/or Bcl-xL expressing quantity;
    (c) with gene copy number greater than 2.8 times of contrasts, and/or immunohistochemical staining be benchmark greater than 10%, screening suitable (E, E)-tumour patient of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivatives for treatment thereof.
  6. 6. an examination is suitable for (E, E)-test kit of the tumour patient of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and derivatives for treatment thereof, this test kit comprises the element that is used to detect the Bcl-2 gene copy number, and/or be used to detect the element that Mcl-1 and/or Bcl-xL express, and/or be used to detect the element that short antiapoptotic factors Bax and Bak express.
  7. 7. the method for inducing apoptosis of tumour cell, comprise give the tumour cell effective dose (E, E)-step of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide or derivatives thereof.
  8. 8. (E, E)-2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and the application of derivative in preparation Bcl-2 family protein inhibitor series antineoplastic medicament thereof.
  9. 9. Bcl-2/Bax and/or Bcl-xL/Bak and/or the dimeric method of Mcl-1/Bak albumen in the adjusting cell, comprise give the cell effective dose (E, E)-step of 2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide or derivatives thereof.
  10. 10. (E, E)-2-(phenmethyl aminocarboxyl)-3-styryl vinyl cyanide and the application of derivative in preparation Bcl-2/Bax and/or Bcl-xL/Bak and/or Mcl-1/Bak albumen dimer conditioning agent thereof.
CN2011100775482A 2011-03-29 2011-03-29 Application of (E,E)-2-(phenylmethyl aminocarbonyl)-3-styrene-acrylonitrile and derivatives thereof Pending CN102212613A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1138458A (en) * 1995-03-21 1996-12-25 伊莱利利公司 Treatment and prevention of prostatic cancer

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Publication number Priority date Publication date Assignee Title
CN1138458A (en) * 1995-03-21 1996-12-25 伊莱利利公司 Treatment and prevention of prostatic cancer

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《中国药理学通报》 20091031 张志超 一个新的BH3模拟分子--广谱Bcl-2蛋白抑制剂 第182页 1-10 , *
张志超: "一个新的BH3模拟分子——广谱Bcl-2蛋白抑制剂", 《中国药理学通报》 *
金礼吉等: "一个全新的有机小分子Bcl-2蛋白抑制剂", 《中国科学 C辑：生命科学》 *

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