CN1938013A - Methods and agents for inhibiting dynamin-dependent endocytosis - Google Patents

Abstract

There are disclosed methods for inhibiting dynamin-dependent endocytosis in cells comprising treating the cells with an effective amount of a compound of formula (I), or a dimeric tyrphostin, physiologically acceptable salt, or prodrug thereof. Compounds useful in the methods described are also provided. The inhibition of dynamin-dependent endocytosis of cells is applicable to the treatment of epilepsy and neurological disorders and conditions.

Description

The method and the medicament that suppress the dependent endocytosis of dynamin

Invention field

The present invention relates to be used to suppress the medicament of the dependent endocytosis of dynamin, and prevention or treatment are by the disease of dynamin dependent cell encytosis mediation or the method for disease.

Background of invention

Mammiferous cell absorbs nutrient from the extracellular, and makes the cell membrane recirculation by endocytosis, comprising form a large amount of vesicles in the endochylema film.Vesicles is not of uniform size, arrives big phagosome greatly, secondly is the vesicle of clarhrin parcel, and is little of small synaptic vesicle (SV).The mechanism of taking the photograph in the cell is helpful to the performance of many cell functions, comprises the picked-up of the outer nutrient substance of pair cell, the adjusting that cell surface receptor is expressed and signal sends, and what antigen presentation and synapse were transmitted keeps.

The approach of taking the photograph in the cell has two to have significant biochemical characteristics.The firstth, quick synaptic vesicle encytosis (SVE), its be at vesicle after the cell of teleneuron effluxes.SVE interrelates with receptor active specifically, and it acts on the recovery that recharges after the empty SVs, and needs the activity of the enzyme of dynamin I.The secondth, receptor-mediated endocytosis (RME) originates in the part that combines with cell surface receptor, and takes place through the concave point of the clarhrin of all cells parcel, comprises teleneuron.RME provides and has been mainly for the activity that endochylema film component (for example receptor-ligand complex and cell membrane lipid plastid) or extracellular liquid enter the inlet of cell and relate to dynamin II.RME acts on identical neuron with SVE, but the mechanism of action is different.

Though they have similar basic protein structure, RME and SVE symbolic animal of the birth year are with proteic different isomerization.There are a plurality of hypotypes in RME and SVE.For example, the cell internalizing effect of EGF-R ELISA (EGFR) and TfR mediates by RME, and depend on the activity of dynamin, but having only the former is to tyrosine kinase (TK) inhibitor sensitivity, and the different biochemical demand to the RME of two kinds of active acceptors has been described.Endocytosis plays multiple action in the mankind comprise the pathologic condition of sacred disease, understand better how to control endocytosis be very important clinically.

Dynamin is the mediated cell encytosis key enzyme in whole latter stage people such as (, 2000) Brodin.Except that dynamin, the molecules mechanism of endocytosis comprises numerous protein and lipid cofactor, and it causes the gathering and the activation (Cousin and Robinson., 2001) of dynamin.In the protein taken the photograph in endocytosis, play a role continuously, at least 4 morphologys are arranged and biochemically go up classification by stages, although some albumen relate to a plurality of phases in pipeline.These morphologys and biochemical classification comprise:

1. nucleation period: the presynaptic ending synaptotagmin performance on SV as cell efflux with cell in the linking function taken the photograph, mode is to replenish on the nucleation site that AP-2 adaptin complex effluxes.AP-2 replenishes clarhrin to form vesicle coating and amphiphysin subsequently.

2. cave in the phase: Amphiphysin is a kind of butt joint molecule, and it can replenish takes the photograph albumen (dynamin, interior tough albumen and synaptojanin) in the remaining great majority, and it is needed to be that vesicle caves in.

3. division stage: dynamin, amphiphysin and/or interior tough albumen assembling ring formation form as the eckband of the spiral helicine vesicle cervical region that caves in.All these three albumen can both be at external oneself's assembling ring formation.The division of vesicle cervical region causes the release of vesicle, needs the activity of the GTP enzyme of dynamin.The GTP hydrolysis causes the unexpected expansion of helical structure, causes the extruding of vesicle of endochylema film, perhaps causes the ring-type constriction.In fruit bat race shibire, the sudden change in dynamin GTP enzyme zone (not blocking the combination of GTP but the hydrolysis of blocking-up GTP) make the dynamin helical structure assembling and form the division (Koenig and Ikeda., 1989) of back blocking-up SV at it.This can differentiate the GTP combination of dynamin ring reaction and the step of GTP hydrolysis, and the hydrolysis of explanation expression GTP, and promptly the GTP enzymatic activity is the last step before the vesicle division.The overexpression of the damaged mutant of dynamin GTP enzyme has suppressed RME and SVE (people such as Brodin, 2000).

4. shelling: the SV shelling was also filled neurotransmitter before effluxing.

Therefore, dynamin is a kind of GTP enzyme of replying synaptic vesicle after effluxing, and has by stimulating function in the assembling of the helical structure of synaptic vesicle depression cervical region (people such as Brodin, 2000; Cousin and Robinson., 2001).Dynamin still is a kind of phosphoprotein, can be by Protein kinase C (PKC) in external phosphorylation with by cyclin dependent kinases (Cdk5) phosphorylation in vivo.It is taken the photograph and dephosphorylation in the irritation cell by stream in unpolarizing and the calcium by neurocalcin soon, and the blocking-up dephosphorylation can prevent to take the photograph in the cell of teleneuron.Keeping dephosphorization acid effect during in the cell of most vesicles, taking the photograph and in cell, take the photograph finish after phosphorylation again.Therefore, the dephosphorylation of dynamin does not work in not taking the photograph in cell, but may be the setting up procedure before taking the photograph in the cell.

Dynamin I has three dynamin genes to express in neuron, and dynamin II then expresses in all positions.Dynamin III expresses in neuron and is present in the testis in a large number.All dynamins all have four main zones, are called GTP enzyme zone, pleckstrin (pleckstrin) homology (PH) zone, GTP enzyme effect device zone (GED) and proline rich zone (PRD).

There is the significantly low affinity to GTP (10-25 μ m) in GTP enzyme zone and than other GTP enzyme very high renewal rate is arranged.It is that the vesicle division is needed.Crystalline structure that should the zone from the dynamin of Dictyostelium gene is found (people such as Niemann, 2001) recently.It is folding that this chondritic comprises the G-protein core, and its normal six line lamellas extend to eight line structures by inserting 55 independent aminoacid.

Pleckstrin homology (PH) zone is a target area, also is the factor of possible GTP enzyme inhibition, is that encytosis is needed.Dynamin and lipid is through this regional interaction, and with the phosphatidylinositols that contains diphosphate (PtdIns (4,5) P ₂) the bonded dynamin of nanotubules stimulated the activity (people such as Stowell, 1999) of GTP enzyme significantly.The PH zone does not need self-assembly or has the GTP enzymatic activity, and removing its (delta-PH zone) can increase intrinsic GTP enzymatic activity largely.

Interaction between GTP enzyme effect device zone (GED) the control dynamin also is assembled into tetramer configuration to dynamin.Approximately 28-32 tetramer associating oneself is assembled into monocycle or contains PtdIns (4,5) P ₂The helical structure of lipid mixtures periphery.GED has illustrated the tetramer self association in conjunction with GTP enzyme zone.The variation of GED influences endocytosis, and part reduces, and part (astoundingly) has increased encytosis.The effect of GED resembles the GTP enzyme effect albumen that stimulates the GTP enzymatic activity.

At the proline rich zone of dynamin C-terminal (PRD) and most SH3 regional interaction that contains albumen and neurocalcin, it is the position of dynamin phosphorylation in the body.

The inhibition method of taking the photograph inhibitor and endocytosis in the various kinds of cell all exists, as cationically ampholytic medicine (for example chlorpromazine), and concanavalin A, the oxidation arsenobenzene, potassium deficiency in the red sulphonyl pentanediamine, cell, acidify and medium temperature are reduced to 4 ℃ in the cell.Every species specificity all a little less than, act on limited.Even so, they still are to take the photograph some effect in the pair cell.Part proved endocytosis capable of blocking be used for human clinically (Atwood, 2001).

Summary of the invention

One or more specific embodiment of the present invention relates to the chemical compound of the GTP enzymatic activity that can suppress dynamin, and the purposes of these chemical compounds in suppressing dynamin dependent cell encytosis.Especially, discovery has the tyrphostin (tyrphostin) of some dimerization can suppress the endocytosis that is mediated by dynamin at least.

Therefore, one aspect of the present invention provides a kind of method that suppresses dynamin dependent cell encytosis in the cell, and this method comprises the chemical compound with the general formula I of effective dose, or its physiologically acceptable salt processing cell, and wherein general formula I is

M-Sp-M ' general formula I

Wherein M and M ' are independent respectively is the structure division shown in the general formula I I, can be identical or different, and Sp is a spacer;

Wherein V is C or CH;

W is a CH or a linking group; And

Y is hydrogen, cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, sulfur (sulfur), unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group, described substituent group is selected from least a of cyano group, nitro, NH, amino, oxo base, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; Or

W, V and Y and Z group condense to form and replace or unsubstituted 5 Yuans or 6 element heterocycles, or carbocyclic ring, wherein heterocycle comprises 1 to 3 hetero atom that is selected from O, N and S, if heterocycle or carbocyclic ring have substituent group, substituent group is to be selected from cyano group, nitro, NH, amino, oxo base, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, sulfur or unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group at least a, substituent group wherein is selected from least a of cyano group, nitro, NH, amino, oxo base, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; With

R is CH ₂R ', CXR ' or CHX ' R ';

X is O or S;

X ' is cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group or unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group, described substituent group is selected from least a of cyano group, nitro, NH, amino, oxo base, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur;

R ' is NH, O or the S that is connected on the spacer; With

Z is selected from

(a) non-replacement contain one or two independently heterocycle of 5 Yuans or 6 Yuans rings, have to be no more than 3 the hetero atom that is selected from O, N and S;

(b) non-replacement contains one or two independently carbocyclic ring of 5 Yuans or 6 Yuans rings;

(c) contain one or two independently heterocycle of 5 Yuans or 6 Yuans rings, have to be no more than 3 the hetero atom that is selected from O, N and S, wherein heterocycle has one or more substituent groups, and described substituent group independently is selected from:

(i) nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo base, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; With

(ii) C ₁-C ₂Alkyl or C ₁-C ₂Alkenyl group, be selected from nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C by at least one ₁-C ₂Alkoxyl and C ₁-C ₂The group of acyl group replace; With

(d) contain one or two independently carbocyclic ring of 5 Yuans or 6 Yuans rings, when W is a CH or a linking group, or W, V and Y have at least two substituent groups when forming the carbocyclic ring of non-replacement, perhaps when W, V and Y form heterocycle, at least one substituent group is arranged, and described substituent group independently is selected from:

(i) nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; With

(ii) C ₁-C ₂Alkyl or C ₁-C ₂Alkenyl group, have at least one to be selected from nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo base, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂The substituent group of acyl group; Wherein when a Z among M or the M ' is selected from (b), and the Z among another M or the M ' is selected from (a), (c) and (d).

Preferably, the chemical compound of general formula I is the tyrphostin of dimerization.

The endocytosis that the invention still further relates to by suppressing the dynamin dependence prevents or treats the relevant disease or the method for disease.Therefore, another aspect of the present invention provides the disease of the endocytosis mediation that is relied on by dynamin in a kind of prevention or the treatment mammal or the method for disease, this method comprises the chemical compound of the general formula I that gives the mammal effective dose, or its physiologically acceptable salt or prodrug.

Another aspect of the present invention provides disease or the disease that is mediated by the endocytosis of dynamin protein dependent in a kind of prevention or the treatment mammal, this method comprise give the mammal effective dose can be in conjunction with dynamin and therefore suppress tyrphostin or its physiologically acceptable salt of dimerization of the GTP enzymatic activity of dynamin, or its analog, or its prodrug.

Chemical compound with general formula I, or the tyrphostin of dimerization or its analog processing cell or mammal, be meant the chemical compound that gives in vivo can dimerization to produce general formula I, or the tyrphostin of dimerization or its analog, they in vivo can be in conjunction with dynamin, the activity that suppresses its Protein G TP enzyme, can generate in vivo and have, suppress chemical compound or the tyrphostin of dimerization or the prodrug of its analog of the active general formula I of its Protein G TP enzyme in conjunction with dynamin.

Providing the chemical compound of general formula I or its physiologically acceptable salt aspect further in the present invention is used to prevent or treats purposes by the medicine of the mammiferous disease of the dependent endocytosis mediation of dynamin or disease in preparation.

Another aspect of the present invention, then provide the tyrphostin of dimerization, its physiologically acceptable salt, or its analog, or its prodrug is used to prevent or treats purposes by the medicine of the mammiferous disease of the dependent endocytosis mediation of dynamin or disease in preparation, and the tyrphostin of described dimerization or analog can and suppress the GTP enzymatic activity of dynamin in conjunction with dynamin.

Another aspect of the present invention provides as the chemical compound of general formula III or its physiologically acceptable salt, wherein:

M-Sp-M ' general formula III

Wherein M and M ' are independent respectively is the structure division shown in the general formula I V, can be identical or different, and Sp is a spacer;

Wherein V is C or CH;

W is a CH or a linking group; And

Y is hydrogen, cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, sulfur, unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group, described substituent group is selected from least a of cyano group, nitro, NH, amino, oxo base, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; Or

W, V and Y and Z group condense to form and replace or unsubstituted 5 Yuans or 6 element heterocycles, or carbocyclic ring, wherein heterocycle comprises 1 to 3 hetero atom that is selected from O, N and S, if heterocycle or carbocyclic ring have substituent group, substituent group is to be selected from cyano group, NH, nitro, amino, oxo base, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, sulfur or unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group at least a, substituent group wherein is selected from least a of cyano group, nitro, NH, amino, oxo base, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; With

R is CH ₂R ', CXR ' or CHX ' R ';

X is O or S;

X ' is cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group or unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group, described substituent group is selected from least a of cyano group, nitro, NH, amino, oxo base, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur;

R ' is NH, O or the S that is connected on the spacer; With

Z is selected from

(a) non-replacement contain one or two independently heterocycle of 5 Yuans or 6 Yuans rings, have to be no more than 3 the hetero atom that is selected from O, N and S;

(b) non-replacement contains one or two independently carbocyclic ring of 5 Yuans or 6 Yuans rings;

(c) contain one or two independently heterocycle of 5 Yuans or 6 Yuans rings, have to be no more than 3 the hetero atom that is selected from O, N and S, wherein heterocycle has one or more substituent groups, and described substituent group independently is selected from:

(i) nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; With

(ii) C ₁-C ₂Alkyl or C ₁-C ₂Alkenyl group, be selected from nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo base, sulfur, sulfydryl, C by at least one ₁-C ₂Alkoxyl and C ₁-C ₂The group of acyl group replace; With

(d) contain one or two independently carbocyclic ring of 5 Yuans or 6 Yuans rings, when W is a CH or a linking group, or W, V and Y have at least two substituent groups when forming the carbocyclic ring of non-replacement, perhaps when W, V and Y form heterocycle, at least one substituent group is arranged, and described substituent group independently is selected from:

(i) nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; With

(ii) C ₁-C ₂Alkyl or C ₁-C ₂Alkenyl group, have at least one to be selected from nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂The substituent group of acyl group; Wherein when a Z among M or the M ' is selected from (b), and the Z among another M or the M ' is selected from (a), (c) and (d), and condition be when R be CXR ', X is O, and R ' is the NH that is bonded on the spacer, and V is C, W is CH, when Y was cyano group, the Z of at least one was not the benzyl group shown in general formula I Va among M and the M '

R ₁, R ₂And R ₅Be H, R ₃And R ₄It is hydroxyl; Perhaps

When Sp is C ₂-C ₄Alkyl at interval the time, R ₁And R ₅Be H, R ₂To R ₄It is hydroxyl; Wherein Z ' is the carbon atom that is connected with the W group.

Preferably, when the Y substituent group of the M of the chemical compound of administration or M ' was hydrogen as the present invention or according to the present invention, another substituent group of M or M ' then was not a hydrogen.Be typically, the Z of at least one of M and M ' is not 2, the dibasic carboxylic acid cyclic group of 3-.Preferably, the Z of at least one of M and M ' comprises:

When Z is selected from (d) and W is CH or C ₁-C ₃Linking group the time, have two ortho-substituents at least; Or

When Z is when being selected from the heterocycle of (c), one of this substituent group or substituent group are on the carbon atom adjacent with one of this hetero atom or hetero atom; Or

When W, V and Y formation and Z condensed heterocycle, one of this substituent group on carbon atom or substituent group have a bond distance at least with heterocycle.

Another aspect of the present invention provides the prodrug of the chemical compound of general formula I or general formula III.

Another aspect of the present invention provides and contains compound of formula III, or its physiological acceptable salt, or the pharmaceutical composition of its prodrug and the acceptable excipient of physiology, carrier or diluent.

Another aspect of the present invention provides screening to have in conjunction with dynamin and suppresses the tyrphostin of dimerization of dynamin GTP enzymatic activity ability or the method for its analog, and this method comprises:

Dynamin or material with dynamin GTP enzymatic activity are cultivated so that the detection data to be provided with tyrphostin or its analog of dimerization; And

Whether tyrphostin or its analog of determining these dimerization on the basis of these data have the dynamin of inhibition GTP enzymatic activity.

Molecule with dynamin GTP enzymatic activity can be a fragment of dynamin, and this fragment can keep the activity of GTP enzyme, or, for example homologue of dynamin, derivant or analog, they in detection as the substitute of dynamin.

The publication that in this manual all are mentioned all is incorporated in this as a reference.In description of the present invention, any discussion to document, used material, equipment, article etc. all only is for the explanation to purpose of the present invention.Basis or common practise related to the present invention that all these have formed the part prior art promptly existed in Australia or other region before the application's priority date as them, did not need permission.

In this manual, term " comprises " that should be understood to is to certain key element, integral body or step, or the comprising of the combination of key element, integral body or step, but does not get rid of other key element, integral body or step, or the combination of key element, integral body or step.

The features and advantages of the present invention will be illustrated in following preferred implementation of the present invention and accompanying drawing in more detail.

Brief Description Of Drawings

Accompanying drawing 1: show two-tyrphostin and tyrphostin A47 (a, b) the active chart of the GTP enzyme of inhibition dynamin I and dynamin II.0.2 (c, d) (e, the measurement of GTP enzymatic activity f) is to use 1.3Ci to μ g dynamin I with dynamin II

Exist or do not exist two-tyrphostin (c, e) or tyrphostin A47 (d carries out under situation f).Primary activity (open loop) and phospholipid-stress active (Gu ring) compare;

Accompanying drawing 2: the automatic radiogram of NC Nitroncellulose film, description taken in conjunction on dynamin I and dynamin II [α- ³²P]-two-tyrphostin or tyrphostin A47 (a, influence b) that GTP is not added.Data upward show at table (c) with (d);

Accompanying drawing 3:(a) pictorialization two-tyrphostin does not play a role in the PH zone of dynamin I, because chemical compound still suppresses to lack the reorganization dynamin mutant GTP enzymatic activity in this zone (" dynamin I-Delta PH "); (b) the sds gel photo of Coomassie blue stain, it shows that two-tyrphostin do not block dynamin and be attached to lipid, dynamin is retained in the pill (P) rather than in supernatant (S);

Accompanying drawing 4: take the photograph in the extracellular that isolating teleneuron (synaptosome) carries out that (a takes the photograph c) He in the cell that (b, d) fluoroscopic examination show that two-tyrphostin rather than tyrphostin A47 can reduce endocytosis especially.Reclaim usefulness and be the more measure of precision of taking the photograph in the outer discharge capacity cell with respect to the front, two-tyrphostin has tangible blocking effect to reclaiming usefulness (e).

Accompanying drawing 5: after the electron micrograph of isolating rat brain teleneuron (synaptosome) is presented at and adds two-tyrphostin, by depolarization stimulate (a, b) and the curl accumulation of depression (c-h) of vesicle, synaptic vesicle is in the disappearance of tip; With

Accompanying drawing 6: the internalization (e-h) that the siderophillin of pictorialization Texas red marker enters Swiss 3T3 cell (a-d) or HER14 cell is suppressed after handling 15 minutes with the preincubation of 100 μ M, two-tyrphostin.The painted part of DAPI (indigo plant) is a nucleus.

The detailed description of the preferred embodiment for the present invention

Term " alkyl " comprises straight or branched saturated fat group.Term " C ₁-C ₂Alkyl " meaning be the length of alkyl chain.Such alkyl group comprises methyl, ethyl, 1-methyl-ethyl and 1,1-dimethyl-ethyl group.

Term " C ₁-C ₂Group " or " C ₁-C ₃Group " comprise saturated or undersaturated aliphatic group with some carbon atoms, side chain can be arranged, can not have side chain yet.Such group comprises alkyl and alkenyl group.Alkenyl group comprises at least one two key.C ₁-C ₃The example of group comprise methyl, ethyl, propyl group, isopropyl, 1,3-dimethyl propyl, 1-methyl-3-ethyl propyl, vinyl, 1-acrylic, 2-acrylic, 1-methyl-2-acrylic and 2-methyl isophthalic acid-acrylic.

Term " C ₁-C ₂" alkenyl group comprises the C that is connected with heterocycle or the carbon ring group of Z by two keys ₁Group.

Term " C ₁-C ₂Alkoxyl " comprise the alkoxy base of the certain-length of forming by some carbon atoms.Alkoxy base can contain a carbon-carbon double bond.

Term " carbon ring group " comprises one or more cyclic groups of being made up of carbon atom.In carbocyclic ring or the ring at least one contains one or more multiple bonds.In general formula I or general formula III during ring formation, the carbocyclic ring of formation preferably contains one or more pairs of keys at W, V and Y.

Term " heterocyclic group " comprises the group that contains one or more rings, and wherein there is hetero atom at least one ring, is selected from O, N and S.At least one ring can contain one or more multiple bonds.

Term " tyrphostin of dimerization " expression comprises the chemical compound of two tyrphostin parts, and these two parts are connected by a spacer, and two tyrphostin parts can be identical or different.Be typically, tyrphostin partly is identical.Most preferably, tyrphostin partly is the benzylidenemalonitrile group.Two-tyrphostin is a kind of tyrphostin of dimerization, is surprisingly found out that it can and suppress the activity of this proteic GTP enzyme in conjunction with dynamin.

Preferably, the independent respectively group of representing for general formula V of M in the chemical compound of general formula I or general formula III and M '.

Wherein V is C;

W is CH;

Y is hydrogen, cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group or unsubstituted C ₁-C ₂Group or the C of replacement ₁-C ₂Group, substituent group wherein is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; Or

W, V and Y and Z condense and form 5 Yuans or 6 Yuans the replacement or the heterocycle of non-replacement, or carbocyclic ring, wherein heterocycle comprises 1 to 3 hetero atom that is selected from O, N and S, if and carbocyclic ring or heterocycle have replacement, have a substituent group at least, be selected from cyano group, nitro, NH, amino, oxo base, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur or unsubstituted C ₁-C ₂Group or the C of replacement ₁-C ₂Group, substituent group wherein is selected from least a of cyano group, nitro, NH, amino, oxo base, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; With

R is CH ₂R ', CXR ' or CHX ' R ';

X is O or S; With

X ' is cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group or unsubstituted C ₁-C ₂Group or the C of replacement ₁-C ₂Group, substituent group wherein independently is selected from cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; And

R ' is NH, O or the S that is bonded to spacer; And

Z is the group that illustrates as among the general formula I I.

Preferably, when W, V and not cyclization of Y, Y is cyano group, nitro, amino, hydroxyl, carboxyl or thiocarboxyl group.Most preferably, Y is a cyano group.

Preferably, R is CXR ', and wherein X is O or S, and R ' is NH, O or S.More preferably, X is O or S, and R ' is NH.

When Z was carbon ring group, it can contain one or more pairs of keys.Carbon ring group for example can be, group of naphthene base, aromatic yl group such as phenyl or naphthyl, or polyphenyl group such as xenyl.When carbon ring group comprised two rings, the ring that directly is connected on the W preferably contained all substituent groups, or when W is CH or linking group, at least two substituent groups is arranged, or when W, V and Y ring formation, at least one substituent group is arranged.Preferably, Z is selected from following group:

(i) containing one or two independently is 5 Yuans or 6 s' heterocycle, comprises no more than 3 the hetero atom that independently is selected from O, N and S;

(ii) containing one or two independently is 5 Yuans or 6 s' heterocycle, comprise no more than 3 the hetero atom that independently is selected from O, N and S, wherein heterocycle contains one or more substituent groups, and described substituent group independently is selected from nitro, NH, halogen, cyano group, amino, hydroxyl, carboxyl, oxo, sulfur and C ₁-C ₂Alkoxyl; With

(iii) containing one or two is the carbocyclic ring of 5 Yuans or 6 Yuans rings independently, contains at least two substituent groups, independently is selected from nitro, NH, amino, halogen, cyano group, hydroxyl, carboxyl, oxo, sulfur and C ₁-C ₂Alkoxyl.

Preferably, be carbocyclic ring and halogen, cyano group, C are arranged when the Z group ₁-C ₂Alkoxyl or C ₁-C ₂Acyl substituent the time, the Z group also contains at least two other substituent groups usually, preferably independently is selected from nitro, NH, amino, hydroxyl, carboxyl, oxo and sulfur, most preferably is nitro, NH, amino, hydroxyl and carboxyl.Preferably, carbon ring group is an aromatic yl group, most preferably, and the benzyl group of replacement.

Preferably, when the Z group is heterocycle, it has one or two independently is 5 Yuans or 6 s' ring, the hetero atom number is no more than 3, is selected from O and N, and wherein heterocycle has one or more substituent groups, independently be selected from nitro, NH, amino, halogen, hydroxyl, carboxyl and oxo, or an aromatic yl group, have one 5 or 6 Yuans monocycle, and at least two substituent groups independently are selected from nitro, amino, halogen, hydroxyl and carboxyl.Preferably, aromatic yl group is the phenyl group that replaces.

Preferably, heterocycle is to replace or unsubstituted imadazolyl, pyranose, different benzyl furyl, furyl, benzopyranyl, pyrrole radicals, 2H-pyrrole radicals, pyrazolyl, pyridine radicals, pyrazinyl, pyrimidine radicals, pyridazinyl, indolizine base, isoindolyl, 3H-indyl, indyl, indazolyl, purine radicals, quinolizinyl, isoquinolyl, quinolyl, pthalazinyl, naphthyridinyl, quinoxalinyl, quinazolyl, cinnolines base, pteridyl, thienyl or benzothienyl.Most preferably, heterocyclic group is the above-mentioned group that replaces.

Under W, V and Y formed situation with Z condensed 5 or 6 element heterocycles, the bonded group of gained and Z replaced or the heterocyclic group of unsubstituted two rings typically.The gained group can be for example to replace or unsubstituted heterocyclic group, be selected from imadazolyl, benzopyranyl, indolizine base, isoindolyl, indyl, indazolyl, purine radicals, quinolizinyl, isoquinolyl, quinolyl, pthalazinyl, naphthyridinyl, quinoxalinyl, quinazolyl, cinnolines base, pteridyl, benzothienyl, and isobenzofuran-base.Preferably, the gained group is to replace or unsubstituted benzopyranyl, indyl or isoquinolyl.Moreover, the group that the heterocyclic group that is formed by W, V and Y cyclization preferably replaces.

Most preferably, it is following chemical compound that chemical compound of the present invention or the method according to this invention give mammiferous chemical compound, wherein

M and M ' they independently are respectively the described chemical compounds of general formula VI, and they can be identical or different, and

X is O or S;

Y is cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl or thiocarboxyl group; Or

R ₁Form 5 or 6 Yuans replacement or unsubstituted heterocycle or carbocyclic ring with Y, wherein heterocycle contains 1 or 2 hetero atom that is selected from O, N and S, and no matter be heterocycle or carbocyclic ring, at least one substituent group is to be selected from cyano group, nitro, NH, amino, oxo base, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; And

R ₂To R ₅Independent is hydrogen or certain substituent group, and described substituent group independently is selected from nitro, amino, halogen, hydroxyl, carboxyl, sulfydryl, thiocarboxyl group, C ₁-C ₂Alkoxyl or C ₁-C ₂Acyl group; Or

R ₁To R ₅Independent is hydrogen or certain substituent group, and described substituent group independently is selected from nitro, amino, halogen, hydroxyl, carboxyl, sulfydryl, thiocarboxyl group, C ₁-C ₂Alkoxyl or C ₁-C ₂Acyl group; Or

R is NH, O and the S that is connected with the Sp spacer; And

Wherein a feature among M and the M ' is as follows at least: R ₁To R ₅At least two be not hydrogen, and work as R ₁To R ₂When being not hydrogen, R ₃To R ₅Neither hydrogen, perhaps work as R ₁When forming unsubstituted carbocyclic ring with Y, R ₂To R ₅At least two be not hydrogen, as Y and R ₁When forming heterocycle, R ₂To R ₅At least one be not hydrogen.

Most preferably, as Y and R ₁Not cyclization and R ₁And R ₂When being not hydrogen, R ₃Neither hydrogen.Be typically R ₁-R ₅At least two be that the ortho position replaces.When chemical compound had three substituent groups, preferred substituents was adjacent one another are.Preferably, in this case, R ₁-R ₃All be not hydrogen, or R ₂-R ₅All not hydrogen.

Work as R ₁-R ₅Or R ₂-R ₅In at least one be halogen, C ₁-C ₂Alkoxyl or C ₁-C ₂Acyl group, in addition substituent at least one be selected from nitro, amino, hydroxyl, carboxyl and thiocarboxyl group, R ₁Form heterocycle with Y, perhaps at least two other substituent groups are selected from nitro, amino, hydroxyl, carboxyl and thiocarboxyl group and R ₁With Y ring formation or form unsubstituted carbocyclic ring not.

The replacement of halogen is typically and is selected from fluorine, chlorine, bromine and iodine.Preferably, the replacement of halogen is selected from fluorine and chlorine.

Preferably, the linking group of general formula I or general formula III comprises single atom or is no more than the chain of 3 atomic lengths, and wherein one or more atoms are other atoms except that carbon atom, as N, O or S.Preferably, linking group is C ₁-C ₃Group.Linking group can be replacement or unsubstituted, can comprise one or more pairs of keys.Substituent group can be selected from hydroxyl, amino, halogen, nitro or other can not influence the group of compound activity.Most preferably, linking group is unsubstituted.

Preferably, the present invention or according to the present invention the spacer part Sp to the chemical compound of mammal administration allows the compound formation hairpin structure.Most preferably, spacer partly is the chain of a replacement or a unsubstituted 1-7 atom, and it can comprise one or more non-carbon atoms, as N, O or S, and one or more pairs of keys.Yet any proper spacing district all should guarantee the rejection of the dynamin dependent cell encytosis of chemical compound.Spacer can be replaced by one or more groups, and described substituent group is selected from hydroxyl, amino, halogen and nitro, or other does not influence the conformation and the elastic group of strand.Most preferably, spacer partly is to replace or unsubstituted alkane chain.Be typically, this spacer is the non-alkane substitute chain with following structure:

-CH ₂(CH ₂) _nCH ₂-

Wherein n is the integer of 1-5, normally 2 or 3.

The acceptable salt of suitable pharmacy comprises acid-addition salts and amino acid addition salt, and ester and amide in rational income/risk ratio scope, have effect pharmaceutically, contact with animal tissue not have toxicity, zest, anaphylactic reaction.Representational salt comprises hydrochlorate, sulfate, bisulphate, maleate, fumarate, succinate, tartrate, toluene fulfonate, citrate, lactate, phosphate, oxalates and borate.These salt can be by for example being the chemical compound with corresponding acid and general formula I, or the tyrphostin of dimerization or its analog are mixed and prepare.This salt can comprise alkali metal or alkaline earth metal cation for example sodium, calcium, magnesium and potassium, and ammonium salt and amine cation.The example of the suitable acceptable salt of pharmacy is on the books in following document: people's such as S.M Berge J.PharmaceuticalSciences (1997), and 66:1-19, its content quotes in full in this as a reference.Representational ester comprises C ₁-C ₇Arrcostab, phenylester and phenyl (C _1-6) Arrcostab.Preferred ester comprises methyl ester.

The chemical compound of general formula I and general formula III, or the prodrug of the tyrphostin of dimerization and analog thereof, comprise that those are selected from the group of carbonate, carbamate, amide and Arrcostab, free amino group, acylamino-, hydroxyl or the carboxylic group with tyrphostin chemical compound, dimerization and analog thereof its covalency is connected.Suitable prodrug also comprises phosphate derivative as acid, sour salt or ester, and by the chemical compound of phosphorus oxygen key and free hydroxyl or other general formula I or general formula III, or the tyrphostin of dimerization and analog thereof are connected.Prodrug can be inactive when using, and it becomes and can produce active chemical compound in conjunction with dynamin by modification in vivo, and as the GTP enzymatic activity of Profilin, mode can be in the cracking or the hydrolysis of using the back key, or other mode.Preferably, the prodrug forms of reactive compound has the stronger membrane permeability performance of specific activity chemical compound, usefulness that can the enhanced activity chemical compound.When also may designing hydrolysis in the adult, prodrug avoids the form of too early doing the trick, as the suitable membrane permeability performance of using for the general that reaches optimum cell action effect and chemical compound in outside.

Endocytosis is the direct or chief reason of various crowd's diseases.The specific vesicles of one class transports the property disease and has announced, referring to Aridor and Hannan 2000, and Traffic1:836-851 and Aridor and Hannan 2002,3:781-790, these documents quote in full in this as a reference.Therefore, method of the present invention can be used in prevention or treatment cancer, ophthalmic diseases, immunodeficiency, gastroenteropathy, virus and bacterial infection, other pathogenic infection, neurodegenerative disease, neural disease and nephropathy and disease, and other relates to the disease of dynamin dependent cell encytosis, or to the disease of the inhibitory action sensitivity of dynamin dependent cell encytosis.

For example, as everyone knows, human polyoma virus JCV is the pathogen of progressive multifocal leukoencephalopathy, this disease is a kind of fatal central nervous system (CNS) demyelination, this pathogen is to neuronic invasion, blocked the example of this kind inhibitor such as chlorpromazine (Atwood W., 2001) by the endocytosis inhibitor.Similarly, HIV infects (Wyss S.et al., 2001), and influenza virus (Roy A., et al.2000) and adeno associated virus (Duan D.etal., 1999) be by the endocytosis onset, its pair cell encytosis inhibitor sensitivity.

In addition, growth factor receptors (needs dynamin to carry out cell internalizing and keeps cell growth (Seto E.et al., 2002) by instruction as E (GF-R).Endocytosis by the blocking-up dynamin stops cell proliferation that many examples (Grieb T.etal., 2000) are arranged and evidence is provided, and promptly dynamin II (non-neuron form) inhibitor possesses active anticancer.Dent ' s disease (POLYCYSTIC KIDNEY DISEASE) also relates to the endocytosis of C1C-5 chloride approach, and takes the photograph (Schwake M.etal., 2001) in the endocytosis blocker prevention cell.

Dynamin is all from taking the photograph the core of transportation in cell surface, Golgi body, Inclusion and mitochondrion carry out.Some neurodegenerative diseases are relevant with these transport channels.Two forms that relate to the generation of amyloid beta are called as encytosis and secretory pathway (Aridor﹠amp; Hannan 2000).At brain, disease that endocytosis is played an important role and disease comprise Alzheimer, Huntington's disease (HD), stiff property syndrome, Lewy body disease and Niemann-Pick cell C type disease (Cateldo et al., 2001; Metzler et al, 2001; Ong et al., 2001; Smith et al., 2000).

The precursor protein of amyloid beta (APP) is taken the photograph in the aixs cylinder cell surface to the vesicles of clarhrin parcel and letter sorting in the synapse bubble of recirculation in Alzheimer, be transported to (Marquez-Sterling N.etal., 1997) in Inclusion and the Cytoplasm.Inclusion is to take the photograph first compartment (the Smythies J. that takes the photograph passage in the dynamin dependent cell of back in APP or the ApoE, 2000) and tangible change (Cataldo A.et al., 1997) arranged in the taper neuron in Alzheimer patient's brain.In the presence of APP and amyloid beta take the photograph the significantly active of approach in the cell when forming, and be some patients with Alzheimer disease brain rapid wears zone neuronic early signs (Cataldo A.et al., 2001).

Huntington's disease (HD) is a kind of neurodegenerative disease, mainly influences striatal neuron, though mutant gene product Huntingdon is not a distinctive material in the brain.The Huntingdon strong effect is in the member of the same clan of Huntingdon action protein 1 (HIP1).The interaction of Huntingdon-HIP1 is subjected to the restriction of brain, and the polyglutamic amide length of its influence and Huntingdon is inversely proportional to.The disappearance of normal Huntingdon-HIP1 can cause integrity damaged of theca cell support in the brain.HIP1 is a basic composition (Metzler M.et al., 2001) of taking the photograph device in the cell of dynamin mediation.Therefore, a large amount of reports all are about taking the photograph (the Aridor ﹠amp of unusual HD neurologic defect in the cell; Hannan, 2000; Metzler M.etal., 2001).

Another example is presynaptic cicatrization albumen (synuclein protein), and this albumen is the mat woven of fine bamboo strips one candidate's factor of the sick cause of disease of Lewy body, comprises parkinson, Lewy body dementia and Lewy body AD variation.Exogenous cicatrization is owing to the formation of inclusion in endocytosis and the endochylema causes nerve cell death.It is the direct result (Sung J.et al., 2001) of people's neuroblastoma endocytosis that cell death and α-cicatrix is assembled.Endocytosis also relates to the generation of epilepsy.For example, take the photograph in the targeted disruption bilateral and take the photograph proteic rat in albumen synaptojanin (SJ) and the amphibious plant and reduced SVE and died from induced seizures (DiPaolo et al., 2002), show its effect on neuronal excitability and relevant with epilepsy.

The approach of taking the photograph in the cell also can be utilized to enter cell by virus, toxin and symbiotic Institute of Micro-biology.For example, botulic neurotoxin and methods of preparing tetanus all are bacterioproteins, and the mediator that can suppress specific synapses discharges, and causes two kinds of serious paralysis neuralis diseases, the plain bacillus of tetanus and meat Shen poison.Their behavior depends on the endocytosis (Humeau et al., 2000) of its neurad tip.Therefore, the inhibition of the endocytosis of targeting can be used for clinical.

Therefore, the specified disease of the prevention of method application of the present invention or treatment includes but not limited to disease, Alzheimer, Huntington's disease, stiff syndrome, Lewy body disease, Lewy asthenia (dimentias), parkinson, epilepsy, tetanus, botulism, HIV infection, influenza and the sticking fat disease that multifocal leukoencephalopathy, POLYCYSTIC KIDNEY DISEASE, amyloid-beta are correlated with.

Preferably, the present invention is applied to benzylidenemalonitrile tyrphostin or its prodrug that the chemical compound of mammiferous general formula I is a dimerization.Most preferably, the tyrphostin of dimerization is two-tyrphostin or its analog.The feature of the tyrphostin of known two-tyrphostin or dimerization and/or group can provide in conjunction with the ability of dynamin and suppress the activity of dynamin, its analog more particularly analogies can be designed to the structure difference, and activity can keep.The application of the tyrphostin analog of dimerization and specific two-tyrphostin analog is also included within the middle of the present invention in the methods described herein.

Term " analog " comprises that molecular structure is different from the tyrphostin of dimerization but has kept the biology performance of tyrphostin of one or more dimerization or living features similar.Analog has basically and the on all four architectural feature of the tyrphostin of dimerization, or comprise that one or more dimerization tyrphostins provide the zone of biology performance or living features, or it relates in addition biology performance or activity is provided.The analog of two-tyrphostin can obtain by replace one or two hydroxyl substituent with the perhaps how different proper group of other suitable group on one or two aromatic group of chemical compound.In addition, one or more groups of chemical compound also can be by cancellation, modification or replacement.

The design of analog typically comprises the physical property of determining original chemical, whether keeps ability in conjunction with dynamin as molecular size, CHARGE DISTRIBUTION and tertiary structure and/or authenticating compound.Particularly, primary chemical compound will be considered its stereochemical structure and utilize X-ray diffraction, nuclear magnetic resonance, NMR and be purchased the physical property that design software is determined.Preferably, model should be considered the interaction of chemical compound and dynamin, because any variation of the conformation that interaction causes all should take in when analog designs.Such modelling technology is a technique known, is in the scope that those of ordinary skill can be known.Suitable analogue technique comprises Accelrys Catalyse_Pharmacore Development and AccelrysCerius 4.8 LigandFit_ protocols (Accelrys Inc., San Diego, California, application USA).More suitable analogue technique comprises MacSpartan ProVersion 1.1 protocols (Wave Function Inc, Irvine, California, application USA).

Analog can also be selected or derived from template molecule, contain chemical group on it, can provide molecule required physicochemical properties, or for obtaining required physicochemical properties chemical reaction easily.The selection of template molecule and chemical group is based on the easy degree of chemosynthesis, the potential danger of degradation in vivo, ability stable and maintenance biologic activity after using.The technical staff is understood that easily pharmaceutically acceptable property etc. takes in too in design.

The chemical compound of using according to the present invention can or medicinally together be used with one or more other chemical compounds.For example, chemical compound can or combine with the chemotherapy drugs associating and give mammal jointly, described chemotherapy drugs be routine be used to prevent or treat the specific mammalian diseases or the medicine of disease.It is with approach identical or inequality administration simultaneously, or with approach order administration identical or inequality with same dosage form or two kinds of different dosage forms that term " gives " meaning jointly.Term " order administration " meaning is that chemical compound and other medicines give in proper order in certain time interval, and interval is from the very short time to several hrs or several days.

The appropriate drug compositions comprises the solution that is suitable for injecting.Injectable compositions like this is the fluid with injectability, is typically can store some months at least keep stable after production.Carrier can be solvent or disperse medium, comprises one or more surfactants, normal saline, ethanol, polyhydric alcohol (for example glycerol, propylene glycol, liquid polyethylene glycol or the like), vegetable oil and their mixture.

For oral administration, chemical compound can add and is suitable for the preparation of oral inert diluent, assimilable edible carrier, perhaps for example is encapsulated in the gelatine capsule of hard system or soft system.Selectable, can directly join in the middle of the food.And, chemical compound can give jointly with one or more excipient, disintegrating agent, the example of excipient is a dicalcium phosphate, examples of disintegrants is corn starch, potato starch or alginic acid, and dosage form can be absorbable tablet, buccal tablet, lozenge, capsule, elixir, suspensoid and syrup.

Tablet, pill etc. can also contain one or more binding agents, lubricant, sweeting agent and aromatic, the example of binding agent is tragakanta, arabic gum, corn starch or gelatin, the example of lubricant is a magnesium stearate, and the example of sweeting agent is sucrose, lactose, glucide.When dosage form is capsule, except one or more mentioned components can also contain liquid-carrier.Various other components may reside in the coating.In addition, chemical compound may reside in the middle of the dosage form of any lasting release suitably.

Chemical compound can typically be prepared the pharmaceutical composition that contains pharmaceutically acceptable carrier or excipient that becomes the particular individual administration.Any conventional carrier diluent and excipient of be sure oing can use.Suitable pharmaceutically acceptable carrier and excipient comprise known solvent, disperse medium and isosmotic solution.The medicinal of these compositions and medium is known.Be typically, compositions of the present invention can also be in conjunction with one or more antiseptic, as metagin, chlorobutanol, phenol, sorbic acid and thimersal.The example that is used for the suitable pharmaceutical acceptable carrier of the present composition and preparation all has description in the middle of the handbook of routine or textbook, for example " Remington:The Science and Practice of Pharmacy (MackPublishing Co.; 1995) ", the content of the document quotes in full in this as a reference.

Particularly preferred dosage form is the parenteral route compositions of unit dosage form, and its easy administration and dosage are even.The unit dosage form here refers to the dosage unit that gives patient's appropriateness, and each unit contains the prevention that can produce needs as calculated or the active substance of therapeutic effect and the carrier and/or the excipient of selecting of scheduled volume.

The dosage of chemical compound depends on many factors, comprise the prevention of chemical compound or therapeutic purposes, the condition of administration, the order of severity, patient's age and some other correlative factor of disease, such as body weight and the health status generally determined by common principle by doctor and nursing staff.For example, can give low dosage, then after the reaction of assess patient, increase gradually again at first.Similarly, the frequency of administration can determine that also promptly continuous monitoring reaction between each administration if necessary, can increase the frequency of administration with identical method, or lowers the frequency of administration.

The pharmaceutical composition route of administration also depends on and will give the disease of compositions or the essence of disease.Suitable route of administration includes but not limited in the respiratory system, trachea, nasal cavity, vein, intraperitoneal, subcutaneous, intradermal, intramuscular, infusion, oral, per rectum, part and slow release are implanted.For the intravenously administrable approach, particularly suitable is to be injected in the blood vessel of organ of tumor or particular treatment.Chemical compound can also be released in the body cavity, and for example in pleural space or the peritoneal cavity, or direct injection is to tumor or illing tissue.

In order to understand the present invention better, preferred form of the present invention will be described by the embodiment of following indefiniteness.

Embodiment 1: the inhibiting evaluation of the tyrphostin of dynamin GTP enzymatic activity

1.1 material and method

Phosphatidylserine, 1,2-diolein, calmodulin, ATP, GTP, leupeptin, phenylmethyl sulfonylfluoride, Tween 80, two (sulfosuccinimide base) suberate (BS3) and glutathione agarose derive from Sigma.Papain and antipain dihydrochloride derive from Boehringer Mannheim (the Federal Republic of Germany).Gel electrophoresis reagent and device derive from Bio-Rad.

(3000 Ci/mmol) and (25 μ Ci/mmol) derives from Amersham plc, UK.Molecular weight protein marker device and chromatography resin derive from Pharmacia.All other reagent all are analytical pure levels or better.

1.1.1 proteinic preparation

The plasmid of GST-Amph2-SH3 (muscle Amph2) (Butler et al., 1997) is by Pieto DeCamilli, Yale, and Conneticut, USA provides, and places the pGEX2T carrier.Plasmid is grown in escherichia coli, the GST-Amph2-SH3 fusion rotein carries out purification by eluting in glutathion (GSH)-agarose, eluent is the 20mM Tris-HCl solution of 10mM reduced form GSH, pH7.5, in the same buffer that does not contain GSH, dialyse, and 4 ℃ of preservations.Purification dynamin from the sheep brain, method is to extract (Robinson et al. from the peripheral membrane portions of whole brain, 1993) and as discussed previously carry out purification (Marks and McMahon. at the GST-Amph2-SH3-agarose, 1998), from 250g sheep brain, obtain 8mg protein.Dynamin II is in expressed in insect cells in reorganization, and derive from Dr Sandra Schmid (Scripps, San Diego, CA).The reorganization dynamin I (dynamin PH derives from Robin Scaife) that lacks the PH zone passes through the granule viral infection in expressed in insect cells (Salim et al., 1996).

1.1.2 the detection of GTP enzyme

The activity of dynamin GTP enzyme is by hydrolysis Determine that method is previous described method (Robinson et al., 1993) substantially.Briefly, the dynamin I of purification or dynamin II (0.2 μ g/ pipe) are at GTP enzyme buffer liquid (10mM Tris, 10mM NaCl, 2mM Mg ²⁺, 0.05% Tween 80, pH7.4,1 μ g/ml leupeptin and 0.1mM PMSF) and contain 0.3mM GTP and 1.3 Ci

Cultivated 10 minutes down at 30 ℃ in the tail wine, condition is inhibitor or the DMSO medium that contains or do not contain variable concentrations.Final detection volume is 40 μ l.The dynamin activity stress descend be measured at baseline and the phospholipid that adds the L-Phosphatidylserine of 5 μ g/ml.(pH1.9) termination then adds the charcoal solution (acid solution of 7% charcoal (w/v)) and the 100 μ l BSA (5mg/ml) of 600 μ l pickling for 2% formic acid, 8% acetic acid with the GTP enzyme stop buffer of 100 μ l in reaction.After the centrifugalize 5 minutes, (under the room temperature 13,000rpm), upper strata liquid is got 200 μ l and is counted in enumerator, counting from

Release ³²Pi.

1.1.3[α ^-32P]-the bonded detection of GTP

[α ^-32P]-the bonded detection of GTP finishes on the microplate of 96-hole.Dynamin (O.2 μ g/ hole) joins in the GTP enzyme buffer liquid, cultivates 10 minutes at 4 ℃ of dark down places.[α ^-32P]-GTP (2 μ Ci/ pipe) joins in the reaction system subsequently, further cultivated 10 minutes at 4 ℃ of dark down places.Microplate shone 30 minutes under 315nm with short wavelength's uviol lamp subsequently, and range of exposures is 8cm.The specificity of the sign of taking a picture is by relatively existing or not existing the sign of the cold GTP of 1mM to be determined.Sample is applied on the NC Nitroncellulose film by 24 orifice plates with suction method immediately.NC Nitroncellulose is given a baby a bath on the third day after its birth time with PBS, drying.Associating nucleotide is measured (molecular dynamics) by phosphorometer.

1.1.4 phospholipids incorporate and spiral combination

Purification is in dynamin I of the full brain of sheep (50 μ g/ml) and Phosphatidylserine liposome (80 μ g/ml, sonication in 30mM Tris/HCl pH7.4) together at 100 μ l assembling buffer (1mM EGTA, 30mM Tris, 100mM NaCl, 1mM DTT, 1mMPMSF, with adequate proteins enzyme inhibitor cocktail tablet (Roche)) the middle cultivation, condition is existence or does not have 1mM Mg/GTP, 25 ℃ of temperature, one hour time.Sample is 14, and 000rpm centrifugal treating 15 minutes is to separate lipid material in conjunction with (P) and the dissociate dynamin of (S) and the fraction of analyzing with gel electrophoresis under the 12%SDS polyacrylamide gel.When existing, medicine (10 μ M and 100 μ M) and phospholipid premix before cultivating with dynamin I.

1.1.5 the picked-up of texas Red-siderophillin in cell

The picked-up of siderophillin (Tf) is by being analyzed on Swiss 3T3 and HER14 cell with the method for before having described (van derBliek et al., 1993).Briefly, cell is placed in the DMEM medium and adds in 10% hyclone, and 60% fusion is arranged, and cell is not at first containing the DMEM overnight incubation of hyclone (8-10 hour).(Tf-TxR, Molecular Probes Oregon) add that to obtain ultimate density be 5 μ g/ml to texas Red-siderophillin, and cell was cultivated 10 minutes down at 37 ℃.The dyeing of cell surface is by (0.2 M acetic acid+0.5M NaCl, pH2.8) the middle cultivation after 15 minutes removed at the solution of ice bath pickling with cell.Cell is fixed 10 minutes with 4% paraformaldehyde apace, and it is inferior to give a baby a bath on the third day after its birth with PBS.(Molecular Probes's nucleus Oregon) dyes with DAPI.Load slide with DABCO, detect fluorescence with Leica DMLB bright field microscope and SPOT digital camera.With in the experiment of inhibitor, before 15 or 60 minutes of adding Tf-TxR, replenish two-tyrphostin with DMEM.

1.1.6 endocytosis

Isolating teleneuron (synaptosome) separates the cerebral cortex that (Dunkley et al., 1986) derive from rat by being interrupted Silicon stone colloidal suspension liquid gradient centrifugation.Fraction 3 and 4 mixes and is used for all experiments.Use the method (Cousin and Robinson 2000a) of previously described picked-up fluorescent dye FM2-10 and measure endocytosis.Synaptosome (0.6mg/2ml) adds or does not add Ca under 37 ℃ ²⁺Cultivated under the situation of KrebsShi solution 5 minutes.Added FM2-10 (100M) at preceding 1 minute that stimulates with 30mM KCl (S1).FM2-10 by vesicle when encytosis absorbs in the S1 stimulation period, synaptosome is being cultivated with antagonist during this period.Especially, synaptosome and tyrphostin A47 or two-tyrphostin were cultivated before stimulation 5 minutes together.Stimulated back 2 minutes, synaptosome adds the Ca of the hyclone that contains 1mg/ml ²⁺Solution is washed twice.Washing step has been removed FM2-10 and the tyrphostin that does not have picked-up.Washed synaptosome adds Ca ²⁺Solution is transferred in the fluorescence container and the 30mM KCl (S2) of the standard of using activation at 37 ℃ of following resuspending.The S2 of standard activates and discharges the FM2-10 that all build up, and measures encytosis (excite at 488nm, be transmitted in 540nm) by the minimizing that dyestuff discharges into the FM2-10 fluorescence that solution causes.

The calculating of endocytosis be by with 30mM KCl during S2 fluorescence stress minimizing finish.The labelling that shows is represented that meansigma methods that FM2-10 discharges from synaptosome deducts and is not contained Ca ²⁺The value of synaptosome context marker of FM2-10.Reclaim usefulness and can carry out taking the photograph in the more accurate pair cell and measure owing to consider the effect of effluxing.Reclaim in being calculated as of usefulness and take the photograph/efflux, wherein the definition of encytosis is the same, and the effect of effluxing is stress back 2 minutes Ca ²⁺The release of the glutamic acid that relies on.Reclaim efficiency value 30 mM KCl specifications are turned to ratio 1.0.

1.1.7 discharging, glutamic acid detects

It is to use the enzyme connection fluoroscopic examination that glutamic acid discharges (Cousin andRobinson., 2000a b) finishes that glutamic acid discharge to detect.In brief, synaptosome (0.6mg/2ml) adds (1.2mM CaCl under 37 ℃ ₂) or do not add (1mM EGTA) Ca ²⁺KrebsShi solution (118.5mM NaCl, 4.7mM KCl, 1.18mM MgCl ₂, 0.1mM Na ₂HPO ₄, 20mM Hepes, the 10mM glucose, pH 7.4) situation under carry out resuspending.Experiment is to add 1mM NADP ⁺After begin.Add the 50U glutamte dehydrogenase after 1 minute, the synaptosome suspension activated with 30mM KCl after 4 minutes.Cause the increase of fluorescence under the condition that 340nm activates and 460nm launches, to be measured owing to producing NADPH by Perkin-Elmer LS-50B spectrofluorophotometer.Experiment is by adding the standardization of 4nmol glutamic acid.Data representation Ca ²⁺The release of-dependency glutamic acid is by adding or do not add Ca under identical stressed condition ²⁺The difference of solution is calculated.In the experiment of using inhibitor, stress the prolapse contact with KCl and tyrphostin A47 or the preincubation of two-tyrphostin 5 minutes.

1.1.8 electron micrograph

Synaptosome is comprising 1.2mM Ca ²⁺KrebsShi solution in cultivated 5 minutes, stimulated 2 minutes with 30mM KCl then.Synaptosome is with 100 μ M, two-tyrphostin preincubation 5 minutes, before adding KCl.After the stimulation, synaptosome Pelleting 1 minute in microfungus, condition is in room temperature, resuspending is fixed under the phosphate-buffered salt that contains 5% glutaraldehyde of ice bath then.After 1 hour, at room temperature low speed (2500rpm) made the synaptosome piller loose in centrifugal 5 minutes.Piller washed three times in low speed rotation (2500rpm) mode with the MOPs buffer in 7 minutes altogether, and resuspending in the aqueous solution of 10% bovine serum albumin (BSA) was at room temperature stablized 20 minutes then.Synaptosome and then under low speed (2500rpm) centrifugal 7 minutes covers and 4 ℃ of following overnight incubation with Karnovsky ' s fixative.Piller is rinsing immediately, and fixes 3 hours in Osmic acid. buffer solution.Synaptosome and then rinsing, and dyeing 1 hour in 2% aqueous acetic acid uranyl, continuous washing 10 minutes and dry before, solutions employed is: 50% ethanol adds 0.1%NaCl; 70% ethanol adds 0.1%NaCl; 95% ethanol adds 0.1%NaCl, and 100% ethanol adds twice of 0.1%NaCl and 100% acetone twice.Soaked into 1 hour with the mixed liquid (1: 1) of acetone/resin then, 70 ℃ down with 10 fens clock times with Spur ' s epoxy resin washing 3 times, replant in the vessel of filling Spur ' s epoxy resin 70 ℃ of maintenances 10 hours down.

(Reichert Germany) is used to obtain the epoxy resin of 0.5m to ultramicrotome Ultracut-E.Cutting use diamond cutter (Diatome Switzerland), suspends in water droplet, places ultramicroscope grid and double staining: earlier with the alcoholic solution of 2% uranyl acetate 15 minutes, in Reynold ' s lead citrate 4 minutes again.Grid washes with water, blots with filter paper, and is saved to Electronic Micro-Analysis when beginning.Analysis is that (Einhoven Netherlands) carries out on the ultramicroscope, and picture is printed on the dull and stereotyped film of ultramicroscope (Kodak, 4489,8.3cm * 10.2cm) at Phillips1L-BioTwin.

1.2 result

1.2.1 two-tyrphostin suppresses the activity of the GTP enzyme of dynamin I and dynamin II

Dynamin GTP enzymatic activity plays crucial effect from the process of plasmalemma generation vesicle in endocytosis.For finding nerve-specific dynamin I inhibitor at first, the lipid kinase inhibitors that many kinases inhibitors and some have high atpase activity position rejection all detects.The selection of these chemical compounds is based on a kind of hypothesis, and promptly the atpase activity site is similar to the avtive spot of GTP enzyme, and some atpase inhibitors can also the targeting dynamin.The gained result shows that the inhibitor with lower dynamin I GTP enzymatic activity inhibition ability can use.

A series of tyrphostins are assessed immediately, find that wherein two have the inhibition ability, and one is tyrphostin A47 (IC ₅₀=100 μ M), and the inhibitor of tool potentiality is two-tyrphostins, its IC ₅₀Be 2 μ M (referring to accompanying drawing 1c and 1d).

Tyrphostin A47 and two-tyrphostin are tested, and whether suppress dynamin I especially to assess it, or can also influence ubiquitous dynamin II.Two medicines proofs all to dynamin II more effective force referring to accompanying drawing 1e and 1f).More in particular, tyrphostin A47 shows the IC to dynamin II ₅₀IC for 9M two-tyrphostin ₅₀Only be 0.5 μ M.This explanation medicine acts on the dynamin gene outcome especially, and pharmacology control that can the pair cell encytosis designs.

1.2.2 two-tyrphostin and tyrphostin A47 can not stop dynamin I or dynamin II to combine with GTP

For assessing two-tyrphostin and tyrphostin A47 hydrolysis at the GTP that stops dynamin, medicine is detected, whether can the GTP avtive spot on the dynamin be at war with to see it.[α- ³²P]-the bonded detection of GTP is that its process contains or do not contain two-tyrphostin and tyrphostin A47 or BIM I (accompanying drawing 2a-d) by the finishing with the bonded GTP of dynamin of visible light electronic marker.Matched group (not containing medicine) demonstration [α- ³²P]-GTP is in conjunction with dynamin.Have medicine two-tyrphostin and tyrphostin A47, minimizing is not seen in the combination of GTP, still, when high concentration, then is significantly increased.Tyrphostin A47 particularly, when high concentration to the GTP of dynamin II in conjunction with having greatly increased.TGP enzyme inhibitor BIM I also finds to compete the GTP in conjunction with dynamin, found [α- ³²P]-the bonded minimizing of GTP.

These medicines to GTP 4 small G-proteins (Rab3A, Ras, Arf2, the competition of RalA) avtive spot also detects.Find medicine do not influence [α- ³²P]-GTP is not to proteic combination (having video data).This shows that two-tyrphostin and tyrphostin A47 are active special to dynamin, and not special to other G albumen that is present in equally in teleneuron or the cell.

1.2.3 two-tyrphostin does not work in the PH zone of dynamin I

For confirming that whether two-tyrphostin suppresses dynamin I through the PH zone, does contrast experiment (accompanying drawing 3a) with the reorganization two-tyrphostin (Δ PH, the zone of dynamin I) that lacks the PH zone.The PH zone of dynamin I is equivalent to the negativity control band of GTP enzymatic activity, and the dynamin I in Δ PH zone has basic activity and is not subjected to the influence of phospholipid.The result show two-tyrphostin still can suppress Δ PH zone dynamin I surpass 50% hydrolysis, be approximately 10 μ M.Illustrate that the PH zone is not the avtive spot of two-tyrphostin to dynamin I, illustrate that two-tyrphostin must be that dynamin I molecule allosteric site is played inhibitory action.Yet BIM I has lost the inhibitory action to the GTP enzymatic activity of the dynamin I in disappearance PH zone, illustrates that this medicine stops the GTP hydrolysis by the PH zone.

Dynamin and the interaction of phospholipid have stimulated the activity of GTP enzyme by the assembling of inducing relevant dynamin helical structure.The dynamin that assembles easily uses simple intermediate processing to detect, and this feature can be used for determining whether two-tyrphostin regulates the interaction of assembling of dynamin spiral or phospholipid.Dynamin does not form precipitation in detection separately, is retained in (accompanying drawing 3b, row 1-2) in the supernatant, but then is present in the deposit seed in a large number when existing at PS liposome (row 3-4).The combination of phospholipid is arranged, and the assembling success of dynamin helical structure then is not subjected to 10 or 100 μ M, two-tyrphostin (row 5-12) fully.Adding Mg/GTP detects but can not change the result.This explanation two-tyrphostin can not stop the dynamin relevant with phospholipid.Therefore, two-tyrphostin all suppresses dynamin GTP enzymatic activity to the site of any allosteric, and it is inhibited that it can also form the back in the spiral assembling.

1.2.4 two-tyrphostin, rather than tyrphostin A47, the synaptic vesicle that can suppress dynamin I mediation restores, and forms the ring of dynamin I in this process

Fluorimetry is used for determining that two-tyrphostin and tyrphostin A47 are to the influence (synaptosome, accompanying drawing 4) of OVE at the cranial nerve tip of colony rat.The effect of taking the photograph outward of two-tyrphostin and A47 pair cell is influence (Ca not ²⁺The release of-dependency glutamic acid, accompanying drawing 4a and 4b).Two-tyrphostin (100 μ M, 10 minutes) significantly suppresses SVE, and tyrphostin A47 (100 μ M) then can not (accompanying drawing 4c and d).The value of SVE depends on the degree that had before effluxed in this detection, and the inhibition of taking the photograph in the cell can be in addition quantitative by calculate reclaiming usefulness.This parameter is the value taken the photograph in the cell ratio (Cousin et al., 2001) divided by the value that effluxes.Reclaim in the usefulness 1 expression medicine pair cell and take the photograph not influence.The recovery efficiency value of tyrphostin A47 is 0.95 (± 0.05), and two-tyrphostin is 0.7 (± 0.05, accompanying drawing 4e).This explanation can significantly reduce SVE with two-tyrphostin.

Because two-tyrphostin suppresses the GTP enzymatic activity (accompanying drawing 1) of dynamin I, but do not combine (accompanying drawing 2) with GTP, so study to determine whether two-tyrphostin also can catch dynamin between the given period of SVE, wherein dynamin is in the ring structure that synaptic vesicle generates.The synaptosome of resting state or in 41mM KCl (S1) once unpolarized synaptosome in 10 seconds shown normal morphology (accompanying drawing 5a-b) under electron microscopic (EM).Being characterized as of teleneuron: i) the endochylema film of level and smooth sealing, ii) in the synaptic vesicle that complete filling is little and iii) always comprise one to three NM and contain synaptosome once in a while, and relevant with synaptosome density.After the synaptosome that does not stimulate is handled with two-tyrphostin, do not show morphologic any influence (accompanying drawing 5a).Yet,, the loss (accompanying drawing 5b) of a large amount of vesicles is arranged for unpolarized.Sub-fraction endochylema film depression also detects (accompanying drawing 5e-f), and illustrating does not have endocytosis.Hinder the TGP hydrolysis but do not hinder the prediction of GTP in conjunction with GTP, a certain amount of curling nuclear occurs, the cervical region of vesicle clearly by dense neck ring around (accompanying drawing 5c, d, g and h).

1.2.5 the endocytosis of the receptor-mediated transferrins to Swiss 3T3 and HER14 cell of two-tyrphostin blocking-up dynamin II mediation

Transferrins is transported in the cell by taking the photograph process in the receptor-mediated cell, and this process is mediated by dynamin II.Detect inhibition of two-tyrosine phosphorylation and tyrphostin A47 to taking the photograph influence (accompanying drawing 6) in the transferrins to non-neurocyte.Control cells shows the Cytoplasm dyeing (table a and e) of vast scale, and this shows to take the photograph in the transferrins and has entered cell.Nucleus is dyed blueness with DAPI, has represented the position (table b, d, f and h) of cyton.Add two-tyrphostin, can observe the painted minimizing of a large amount of transferrinss.Tyrphostin A47 also has such effect, though be not to resemble remarkable (not shown) two-tyrphostin.Find that also inhibitory action is a concentration dependent.Medium DMSO is to taking the photograph not effect in the transferrins.

1.3 discuss

Because at first proved fruit bat mutant dyeing shibire, blocking-up dynamin and teleneuron endocytosis have caused the consume of most SVs.Because a large amount of SVs has the morphological feature of teleneuron, its loss is clearly.And the form of endochylema film can provide as everyone knows testifies that significantly the blocking-up of taking the photograph in the clear-cells has taken place really.Two-tyrphostin has reduced the teleneuron of most SVs and has produced the vesicle of the depression on a spot of endochylema film, has the circle or the ring of dynamin clearly.Such result shows that ring is modified and the avtive spot of open loop two-tyrphostin before.But surprisingly, the dynamin neck ring is rarely found.What the situation explanation two-tyrphostin of this complexity was blocked is another site, and there are two remarkable different sites in the activity of prompting dynamin GTP enzyme in the mechanism of SVE.

Three dynamin gene outcomes can mediate the endocytosis of at least three kinds of forms.Dynamin I mediates SVE, and dynamin II mediates RME, takes the photograph (Gray et al., 2003) in the cell at dynamin III mediation post-synapse spinal column place.Further mechanism of action known.The distinguishing ability that this discrepant inhibitory action of dynamin has been given these cell functions.Particularly, selective depressant is the instrument of the dissimilar endocytosis of very important identification, and the pathology targeting based on multi-form endocytosis is had clinical meaning.

This result further specifies the GTP enzymatic activity that two-tyrphostin (BisT or AG537) suppresses dynamin I and II, and the SVE of equal block nerves tip (synaptosome) and the RME that transferrins is taken the photograph in 3T3 or HER14 cell.Its avtive spot is not the GTP binding site, neither the PH zone, so it is the inhibitor of allosteric body.Because it does not influence the combination of GTP, so do not influence dynamin assembling ring formation yet.This has just formed the unique tools of the dynamin of assembling back targeting.The original discovery of two-tyrphostin can suppress EGFR-TK (IC ₅₀=0.4M) breed (IC with the EGF-dependent cell ₅₀=3M) (Gazit etal., 1996).Therefore, can design and keep dynamin to suppress active but lose analog (because the specific determiner of tyrosine kinase has been known, Gazit et al., 1996) the effect of EGFR tyrosine kinase.

Embodiment 2: the development of tyrphostin analog

?

Two-tyrphostin (1) and tyrphostin A47 (2)

2.1 the development of analog

The structure of two-tyrphostin and tyrphostin A47 is as implied above.These 3, the existence of the similarity of the compound structure of 4-dihydroxy benzenes and cyanamide or thioamides structure illustrates that these groups are extremely important for the dynamin inhibitory action.These features can be developed synthetic problem in the settlement procedure storehouse, and having two program libraries to be used for determining the type and the quantity of the active aromatic substituent of decision, the needs of symmetry synthetic (1vs2) and these program libraries of importance that are positioned at the center alkane gap length between two amino parts of two-tyrphostin are called program library 1 (dimeric compounds) and program library 2 (asymmetric single polyacetylene compound).

2.2 the program library analog is synthetic

The simple application of Knoevenagel chemistry and a series of suitable α, w-diamidogen give needed program library (scheme 1) product with good yield apace.

Scheme 1: program library 1 synthetic.R ¹-R ⁵Substituent group and alkyl at interval n in the following table 2 definition

This application can be according to 5 products in alkyl n=1-5 speed-generating program storehouse 1 at interval.Initial screening biology of the GTP enzymatic activity of dynamin I is carried out under 100 μ M.More analog screens under the concentration of certain limit subsequently, to determine their IC ₅₀Value (table 2).In synthetic 80 analog, find IC smaller or equal to 100 μ M ₅₀The material of value has significant inhibitory effect.R ¹-R ⁵Described in substituent definition such as the following table 1.

2.3 the dimerization tyrphostin is synthetic

2.3.1 general the introduction

All raw materials are available from Aldrich Chemical Company and LancasterSynthesis. ¹H and ¹³The C chromatograph on the Bruker Advance AMX 300 MHz spectrometers respectively 300.1315 and 75.4762MHz under record.With respect to the chemical shift of TMS as interior mark.

2.3.2 synthetic method

Chemical compound 5a:2-cyano group-N-[3-(2-cyanoacetamide base)-ethyl]-acetamide

(1.5g, 25mmol) (5g 50mmol) at room temperature stirred two hours ethylenediamine (3a) with the methyl cyano-acetate.The white solid that obtains mixes with 10ml ethanol immediately, filters and collects.Obtain white solid with ethyl alcohol recrystallization, 6.3g (81%).mp?182℃(Lit?183℃) ²⁹。

¹H?NMR(DMSO)：8.25(2H，t，J＝5.5Hz)，3.56(4H，s)，3.13(4H，br?s)。

¹³C?NMR(DMSO)：162.31，115.96，38.41，25.25。

Chemical compound 5b:2-cyano group-N-[3-(2-cyanoacetamide base)-propyl group]-acetamide

(2.2g, 30mmol) (6.4g 65mmol) at room temperature stirred two hours propyl diamine (3b) with the methyl cyano-acetate.The white solid that obtains mixes with 20ml ethanol.Filter and collect.Obtain 4.995g white solid (81%) with ethyl alcohol recrystallization.mp146℃(Lit148℃) ²⁹。

¹H?NMR(DMSO)：8.21(2H，t，J＝5.5Hz)，3.59(4H，s)，3.07(4H，q，J＝6.7Hz)，1.53(2H，quin，＝6.7Hz)。

¹³C?NMR(DMSO)：162.45，116.64，39.28，28.90，25.67。

Chemical compound 5c:2-cyano group-N-[3-(2-cyanoacetamide base)-butyl]-acetamide

1,4-diaminobutane (3c) (3g, 34mmol) and the methyl cyano-acetate (7g 70mmol) at room temperature stirs and obtained white solid in two hours.Solid mixes solid collected by filtration with ethanol (10ml).Obtain white solid with ethyl alcohol recrystallization, 5.995g (78%).mp145℃(Lit?145℃)。

¹H?NMR(DMSO)：8.15(2H，t，J＝5.5Hz)，3.56(4H，s)，3.05(4H，br?s)，1.38(4H，br?s)。

¹³C?NMR(DMSO)：161.84，116.09，38.63，26.07，25.17。

Chemical compound 5d:2-cyano group-N-[3-(2-cyanoacetamide base)-amyl group]-acetamide

1, (2g, 20mmol) (3.9g 40mmol) at room temperature stirred two hours 5-diaminourea pentane (3d), obtained white solid with the methyl cyano-acetate.Solid mixes with ethanol (10ml), filters and collects.Obtain white solid with ethyl alcohol recrystallization, 4.62g (98%).mp?125℃(Lit?125℃)

¹H?NMR(DMSO)：8.14(2H，t，J＝5.4Hz)，3.55(s，4H)，3.03(4H，q，J＝6.4Hz)，1.39(4H，quin，J＝7Hz)，1.23(2H，quin，＝7Hz)。

¹³C?NMR(DMSO)：161.79，116.11，38.84，28.26，25.17，23.43。

Chemical compound 5e:2-cyano group-N-[3-(2-cyanoacetamide base)-hexyl]-acetamide

(3g, 26mmol) (6g 60mmol) at room temperature stirred two hours 1 (3e), obtained white solid with the methyl cyano-acetate.Solid mixes with ethanol (10ml), filters and collects.Obtain white solid with ethyl alcohol recrystallization, 6.2g (95%).mp141℃(Lit?140℃)

¹HNMR(DMSO)：8.15(2H，t，J＝5.5Hz)，3.56(4H，s)，3.04(4H，q，J＝6.1Hz)，1.37(4H，quin，J＝5.9Hz)，1.24(4H，brs)。

¹³C?NMR(DMSO)：161.76，116.12，38.85，28.58，25.?82，25.16。

Chemical compound 9:2-cyano group-N-{3-[2-cyano group-3-(3, the 4-dihydroxy phenyl) acrylamido]-ethyl }-3-(3, the 4-dihydroxy phenyl)-acrylamide

2-cyano group-N-[3-(2-cyano group-acetylamino)-ethyl]-acetamide (5a) (0.3g, 1.5mmol), 3, (0.42g, 3mmol), 3 piperidines and ethanol (10ml) refluxed 2 hours the 4-4-dihydroxy benzaldehyde.Cooling is filtered and is washed with cold ether (10ml), obtains the yellow green solid, 0.54g (81%).mp290℃(Lit?295℃)

¹H?NMR(DMSO)：8.32(2H，t，J＝5.5Hz)，7.92(2H，s)，7.53(2H，d，J＝2.1?Hz)，7.25(2H，dd，J＝8.2，2.1Hz)，6.85(2H，d，J＝2.1Hz)，3.45(4H，brs)。

¹³C?NMR(DMSO)：162.50，151.63，161.61，146.22，125.76，123.45，117.65，116.53，116.31，100.85，39.60。

Chemical compound 10:2-cyano group-N-{3-[2-cyano group-3-(3,4,5-trihydroxy phenyl)-acrylamido]-ethyl]-3-(3 4,5-trihydroxy phenyl)-acrylamide

2-cyano group-N-[3-(2-cyano group-acetylamino)-ethyl]-acetamide (5a) (0.056g, 0.3mmol), 3,4, (0.1g's 5-tri hydroxybenzaldehyde 0.65mmol) refluxed 1 hour with 1 piperidines and ethanol (2mL).Cooling is filtered and is washed with cold ethanol (10mL), obtains the orange colour solid, 0.11g (82%).mp＞300℃

¹H?NMR(DMSO)：8.29(2H，t，J＝5.5?Hz)，7.79(2H，s)，6.99(4H，s)，3.32(4H，brs).

¹³C?NMR(DMSO)：162.15，150.7，145.96，140.24，121.26，117.30，109.97，99.76，39.40.

Chemical compound 11:2-cyano group-N-{3-[2-cyano group-3-(3,4-dihydroxy-4-methoxyphenyl)-acrylamido]-ethyl-3-(3,4 dihydroxy-5-anisyl)-acrylamide

2-cyano group-N-[3-(2-cyano group-acetylamino)-ethyl]-acetamide (5a) (0.06g, 3mmol), 3,4-dihydroxy-5-methoxybenzaldehyde (0.1g, 0.6mmol), 1 piperidines and 2mL alcohol reflux 2 hours.Cooling is filtered and is washed with cold ethanol (5mL), obtains the orange colour solid, 0.101g (66%).mp?274℃

¹H?NMR(DMSO)：8.34(2H，t，J＝5.5Hz)，7.93(1H，s)，7.20(2H，d，J＝1.92Hz)，7.13(2H，d，J＝1.92Hz)，3.77(6H，s)，3.35(4H，br?s).

¹³C?NMR(DMSO)：161.90，150.85，148.03，145.83，139.90，121.76，117.20，111.09，107.20，100.83.

Chemical compound 22:2-cyano group-N-{3-[2-cyano group-3-(3, the 4-dihydroxy phenyl)-acrylamido]-propyl group }-3-(3, the 4-dihydroxy phenyl)-acrylamide

2-cyano group-N-[3-(2-cyanoacetamide base)-propyl group]-acetamide (5b) (0.3g1.4mmol), (0.4g, 2.8mmol) 3,4-4-dihydroxy benzaldehyde, 3 piperidines and 10mL alcohol reflux two hours.Cooling is filtered and is washed with cold ether (10mL), obtains the yellow green solid, 0.55g (85%).mp?274℃(Lit?277℃)

¹H?NMR(DMSO)：8.24(2H，t，J＝5.5Hz)，7.92(s，2H)，7.52(2H，d，J＝2.1Hz)，7.26(2H，dd，J＝8.2，2.1Hz)，6.85(2H，d，J＝8.2Hz)，3.23(4H，q，J＝6Hz)，1.70(2H，quin，J＝6.7Hz).

¹³C?NMR(DMSO)：161.50，150.60，150.50，125.10，123.21，117.10，116.00，115.80，100.50，37.27，28.82.

Chemical compound 23:2-cyano group-N-{3-[2-cyano group-3-(3,4,5-trihydroxy phenyl)-acrylamido]-propyl group }-3-(3,4,5-trihydroxy phenyl)-acrylamide

2-cyano group-N-[3-(2-cyanoacetamide base)-propyl group]-acetamide (5b) (0.06g0.29mmol), 3,4, (0.1g, 0.58mmol), 1 piperidines and ethanol (10mL) refluxed 2 hours the 5-tri hydroxybenzaldehyde.Cooling is filtered and is washed with cold ethanol (10mL), obtains the orange colour solid, 0.097g (70%) .Mp＞300 ℃ (Lit＞300 ℃)

¹H?NMR(DMSO)：8.18(2H，t，J＝5.5Hz)，7.78(2H，s)，6.99(4H，s)，3.21(4H，q，J＝6.8Hz)，1.68(2H，quin，J＝6.8Hz).

¹³C?NMR(DMSO)：161.80，150.70，145.95，140.30，121.22，117.30，109.90，99.50，38.20，28.90.

Chemical compound 24:2-cyano group-N-{3-[2-cyano group-3-(3,4-dihydroxy-5-methoxyphenyl)-acrylamido]-propyl group }-3-(3,4-dihydroxy-5-methoxyphenyl)-acrylamide

2-cyano group-N-[3-(2-cyanoacetamide base)-propyl group]-acetamide (5b) (0.3g 1.4mmol), 0.44g3,4-dihydroxy-4-methoxybenzaldehyde, 3 piperidines and ethanol (10mL) refluxed two hours.Cooling is filtered and is washed with cold ethanol (5mL) and obtains orange colour solid, 0.31g (42%) .mp＞300 ℃

¹H?NMR(DMSO)：8.35(2H，t，J＝5.4Hz)，7.95(2H，s)，7.21(2H，d，J＝1.9Hz)，7.12(2H，d，J＝1.9Hz)，3.21(4H，q，J＝6.8Hz)，1.71(2H，quin，J＝6.8Hz).

¹³C?NMR(DMSO)：161.30，150.61，147.20，145.30，121.04，117.60，110.60，107.65，98.71，38.35，28.88.

Chemical compound 35:2-cyano group-N-{3-[2-cyano group-3-(3, the 4-dihydroxy phenyl)-acrylamido]-butyl }-3-(3, the 4-dihydroxy phenyl)-acrylamide

2-cyano group-N-[3-(2-cyanoacetamide base)-butyl]-acetamide (5c) (0.3g, 1.35mmol), 3, (0.37g, 2.7mmol), 3 piperidines and ethanol (10mL) refluxed two hours the 4-4-dihydroxy benzaldehyde.Cooling is filtered and is washed with cold ether (10mL), obtains yellow solid, 0.61g (97%).mp?281℃(Lit?283℃)

¹H?NMR(DMSO)：8.25(2H，t，J＝5.5Hz)，7.91(2H，s)，7.53(2H，d，J＝1.9Hz)，7.26(2H，dd，J＝8.3，1.9Hz)，6.85(2H，d，J＝8.3Hz)，3.20(4H，br?s)，1.49(4H，br?s).

¹³C?NMR(DMSO)：161.52，150.86，150.42，145.65，125.23，123.09，117.20，115.81，100.51，39.31.

Chemical compound 36:2-cyano group-N{3-[2-cyano group-3-(3,4,5-trihydroxy phenyl)-acrylamido]-butyl }-3-(3,4,5-trihydroxy phenyl)-acrylamide

2-cyano group-N-[3-(2-cyanoacetamide base)-butyl]-acetamide (5c) (0.065g, 0.3mmol), 3,4, (0.1g, 0.6mmol), 1 piperidines and ethanol (2mL) refluxed 1 hour the 5-tri hydroxybenzaldehyde.Cooling is filtered and is washed with cold ether (5mL), obtains yellow solid, 0.121g (82%).mp＞300℃(Lit＞310℃) ²⁹

¹H?NMR(DMSO)：8.16(2H，t，J＝5.5Hz)，7.78(2H，s)，6.98(4H，s)，3.19(4H，br?s)，1.48(4H，br?s).

¹³C?NMR(DMSO)：161.70，150.56，145.90，140.20，121.30，117.30，109.90，99.80，39.26，26.37.

Chemical compound 37:2-cyano group-N{3-[2-cyano group-3-(3,4-dihydroxy-5-anisyl)-acrylamido]-butyl }-3-(3,4-dihydroxy-5-anisyl)-acrylamide

2-cyano group-N-[3-(2-cyanoacetamide base)-butyl]-acetamide (5c) (0.065g, 0.3mmol), 3, (0.1g, 0.6mmol), 1 piperidines and ethanol (2mL) refluxed 1 hour 4-dihydroxy-5-methoxybenzaldehyde.Cooling is filtered and is washed with cold ether (5mL), obtains yellow solid, 0.110g (70%).mp＞300℃

¹H?NMR(DMSO)：8.09(2H，t，J＝5.5Hz)，7.86(2H，s)，7.18(2H，d，J＝1.9Hz)，7.10(2H，d，J＝1.9Hz)，3.75(6H，s)，3.19(4H，br?s)，1.48(4H，br?s)。

¹³C?NMR(DMSO)：161.71，150.23，148.70，146.24，120.25，117.51，109.50，106.80，98.81，55.76，39.31，26.64.

Chemical compound 48:2-cyano group-N{3-[2-cyano group-3-(3, the 4-dihydroxy phenyl)-acrylamido]-butyl }-3-(3, the 4-dihydroxy phenyl)-acrylamide

2-cyano group-N-[3-(2-cyanoacetamide base)-amyl group]-acetamide (5d) (0.2g, 0.85mmol), 3, the 4-4-dihydroxy benzaldehyde (0.23g, 1.7mmol), 3 piperidines and 7mL alcohol reflux two hours.Cooling is filtered and is washed with cold ether (10mL), obtains yellow solid, 0.36g (90%).mp?252℃(Lit?248℃) ²⁹

¹H?NMR(DMSO)：8.15(2H，t，J＝5.5Hz)，7.85(2H，s)，7.50(2H，d，J＝2.1Hz)，7.20(2H，dd，J＝8.5Hz，2Hz)，6.75(2H，d，J＝8.5Hz)，3.16(4H，q，J＝6.2Hz)，1.50(4H，quin，J＝7.1Hz)，1.28(2H，quin，J＝6.9Hz)。

¹³C?NMR(DMSO)：161.85，153.88，150.34，146.28，126.16，121.47，117.70，115.71，114.65，98.40，39.46，28.63，23.73.

Chemical compound 49:2-cyano group-N{3-[2-cyano group-3-(3,4,5-trihydroxy phenyl)-acrylamido]-butyl }-3-(3,4,5-trihydroxy phenyl)-acrylamide

2-cyano group-N-[3-(2-cyanoacetamide base)-amyl group]-acetamide (5d) (0.068g, 0.29mmol), (0.1g, 058mmol) 3,4, the 5-tri hydroxybenzaldehyde (0.1g, 0.58mmol), 1 piperidines and 2mL alcohol reflux 1 hour.Cooling is filtered and is washed with cold ether (5mL), obtains yellow solid, 0.123g (83%).mp＞300℃32

¹H?NMR(DMSO)：8.12(2H，t，J＝5.5Hz)，7.76(2H，s)，6.98(4H，s)，3.16(4H，br?s)，1.50(4H，quin，J＝6.8Hz)，1.28(2H，quin，J＝6.7Hz).

¹³C?NMR(DMSO)：161.80，150.49，146.11，146.01，141.25，120.69，117.53，109.90，99.12，39.47，28.62，22.30.

Chemical compound 50:2-cyano group-N-{3-[2-cyano group-3-(3,4-dihydroxy-5-methoxyphenyl)-acrylamido]-amyl group }-3-(3,4-dihydroxy-5-methoxyphenyl)-acrylamide

2-cyano group-N-[3-(2-cyanoacetamide base)-amyl group]-acetamide (5d) (0.069g, 0.29mmol), 3, (0.1g, 0.58mmol), 1 piperidines and ethanol (2mL) refluxed one hour 4-dihydroxy-5-methoxybenzaldehyde.Cooling is filtered and is washed with cold ether (5mL), obtains yellow solid, 0.126g (81%).mp256℃

¹H?NMR(DMSO)：8.09(2H，t，J＝5.5Hz)，7.86(2H，s)，7.18(2H，d，J＝2Hz)，7.10(2H，d，J＝2Hz)3.75(6H，s)，3.17(4H，br?s)，1.50(4H，quin，J＝6.8Hz)，1.28(4H，quin，J＝6.9Hz)。

¹³C?NMR(DMSO)：161.81，150.50，148.00，146.20，120.05，117.81，110.50，107.80，98.71，55.71，39.41，28.64，22.47。

Chemical compound 61:2-cyano group-N-{3-[2-cyano group-3-(3, the 4-dihydroxy phenyl)-acrylamido]-hexyl }-3-(3, the 4-dihydroxy phenyl)-acrylamide

2-cyano group-N-[3-(2-cyanoacetamide base)-hexyl]-acetamide (5e) (0.3g, 1.2mmol), 3, the 4-4-dihydroxy benzaldehyde (0.33g, 2.4mmol), 3 piperidines and 10mL alcohol reflux two hours.Cooling is filtered and is washed with cold ether (10mL), obtains yellow solid, 0.52g (89%).mp?263℃(Lit?260℃)

¹H?NMR(DMSO)：8.18(2H，t，J＝5.5Hz)，7.89(2H，s)，7.51(2H，d，J＝2Hz)，7.24(2H，dd，J＝2Hz，8.3Hz)，6.83(2H，d，J＝8.3Hz)，3.17(4H，q，J＝6.1Hz)，1.47(4H，quin，J＝6.1Hz)，1.28(4H，br?s).

¹³C?NMR(DMSO)：161.52，151.18，150.30，145.72，125.22，122.91，117.24，115.80，115.72，100.38，39.48，28.78，25.98.

Chemical compound 62:2-cyano group-N-{3-[2-cyano group-3-(3,4,5-trihydroxy phenyl)-acrylamido]-hexyl }-3-(3,4,5-trihydroxy phenyl)-acrylamide

2-cyano group-N-[3-(2-cyanoacetamide base)-hexyl]-acetamide (5e) (0.073g, 0.29mmol), 3,4, (0.1g, 0.58mmol), 1 piperidines and ethanol (2mL) refluxed 1 hour the 5-tri hydroxybenzaldehyde together.Cooling is filtered and is washed with cold ether (5mL), obtains yellow solid, 0.1g (67%).mp＞300℃

¹H?NMR(DMSO)：8.11(2H，t，J＝5.5Hz)，7.76(2H，s)，6.98(4H，s)，3.16(4H，br?s)，1.47(4H，quin，J＝6.1Hz)，1.28(4H，br?s).

¹³C?NMR(DMSO)：161.80，150.46，145.99，141.19，120.71，117.53，109.89，99.15，39.68，28.84，26.01.

Chemical compound 63:2-cyano group-N-{3-[2-cyano group-3-(3,4-dihydroxy-5-methoxyphenyl)-acrylamido]-hexyl }-3-(3,4-dihydroxy-5-methoxyphenyl)-acrylamide

2-cyano group-N-[3-(2-cyanoacetamide base)-hexyl]-acetamide (5e) (0.069g, 0.28mmol), 3, (0.1g, 0.56mmol), 1 piperidines and ethanol (2mL) refluxed one hour 4-dihydroxy-5-methoxybenzaldehyde.Cooling is filtered and is washed with cold ether (5mL), obtains yellow solid.0.132g(86％)。mp?243℃

¹H?NMR(DMSO)：8.17(2H，t，J＝5.5Hz)，7.89(2H，s)，7.19(2H，d，J＝1.6Hz)，7.13(2H，d，J＝1.6Hz)，3.77(6H，s)，3.17(4H，br?s)，1.48(4H，quin，J＝6.1Hz)，1.29(4H，br?s).

¹³C?NMR(DMSO)：161.80，150.49，146.11，146.01，141.25，120.69，117.53，109.90，99.12，39.47，28.62，22.30。

2.3.2 the activity of dimerization tyrphostin

Table 1: the chemical compound of program library 1 (dimer) is to the influence of the GTP enzymatic activity of dynamin I

Mono-substituted aromatic does not contain substituent group, or only contains single substituent group, as list-OH (for example, R ₁Or R ₂Be OH) ,-Cl (R ₂Or R ₄Be Cl) ,-OMe (R ₂Or R ₃Be OMe), or-COOH (R ₃Be COOH), such chemical compound does not show that dynamin suppresses ability.And just possessed significant effect after having introduced second substituent group that antioxidant capacity arranged.3, and 4-two-OH (11, IC ₅₀=5.1 ± 0.6 μ M) thing has just shown the ability of similar compound 1, and (2,3-two-OH) to be known as two-tyrphostin.3,4,5-three-substituted aromatic (10) also has the ability that is equal to chemical compound 1.Same situation also exists in the chemical compound of different chain length.

The extending in n＞3 o'clock and can some influence be arranged of alkyl spacer chain to usefulness.For example, chain elongation analog 9 (n=0), 22 (n=1), 35 (n=2), the IC of 48 (n=3) and 61 (n=4) ₅₀It is not 5.1 ± 0.6,1.7 ± 0.2,3.2 ± 1,5 ± 1.4 and 26 ± 1.5 μ M that value is divided in addition.Say that in essence originally then there was opposite report tyrosine kinase inhibition aspect.The detection compound of the poly-GAT zymolyte of antagonism EGF receptor tyrosine kinase people such as () Gazit is inhibited, and does not rely on length people such as (, 1996) Gazit of chain.

R ₁The analog that forms with the position cyclization that is occupied by cyano group (CN) also can use.For example, work as R ₁When being hydroxyl, oh group energy and cyano group reaction form imminochromene, shown in following scheme 2.

Synthesizing of the imminochromene analog of 2: two-tyrphostin of scheme

For explaining the similarity of analog 9 on inhibiting value of chain elongation, the model of all 5 alkane spacer analog is analyzed and is shown the result of the following low-yield conformer of MacSpartanPro.As our finding, the low-yield conformer of all 5 analog all has the conformation of hair clip, reaches the interactional maximum of pi-pi of terminal phenyl ring.Therefore, the length of increase spacer can restriction effect begin to be formed at metastable hairpin structure (n 〉=5) up to encytosis.The tyrphostin of this and dimerization compares the effect of the tyrosine kinase that kept the configuration that prolongs, and therefore makes them be suitable for the dimerization intermediate of EGFR tyrosine kinase.

In table 2, two-tyrphostin such as chemical compound 9 samples identify that it is synthetic according to flow process 1.Therefore chemical compound 1 is identical with 9.

For studying the bonded and 1 correlation technique effect of potential H-, chemical compound 71 is developed.This chemical compound have firm relatively and 1 size similarly piperazine be connected.But it is presented at≤lack the dynamin inhibitory action during 100 μ M.Similarly, obtain also not observing inhibitory action behind the methylated analog 72 of N-at methylate 1 alkyl spacer of N-.These observations show that the hairpin structure of the tyrphostin of dimerization is desirable to inhibitory action, and support model is observed (hairpin structure rather than prolongation chain structure), and the amide groups substituent group is also played an important role in the dynamin combination.

Successfully developed the effective symmetrical analog of some μ M based on two-tyrphostin, the modification of having studied an aromatic proton is used for measuring it in the effect that suppresses dynamin.Therefore, another compound library based on tyrphostin A47 (2) is developed, and shown in scheme 3, and has checked that this analog suppresses the ability of the GTP enzymatic activity of dynamin I.

Synthesizing of scheme 3. storehouses 2

Surprisingly, the screening of storehouse 2 chemical compounds does not show that any dynamin suppresses≤100 μ M.That more surprised is the dynamin IC that the initial data show tyrphostin A47 (2) that screens shows ₅₀=70 μ M.

Further the detection of tyrphostin A47 has been provided the chemical compound that does not detect storehouse 2 and suppressed an active possible explanation, promptly list-S may effectively be used for solution and is oxidized to corresponding dimeric structure.Simple tautomer produces corresponding disulphide (121) (referring to scheme 4) after the oxidation.2 freshly prepd solution does not demonstrate the inhibition ability, and at room temperature preserving then has weak inhibition ability after 24 hours.When reducing agent dithiothreitol (2mM) is present in the dynamin detection medium, 121 IC ₅₀Value reduces to＞300 μ M.When using dithiothreitol, DTT separately, then lack dynamin GTP enzymatic activity (data not shown).121 in position generations of dimerization give the major function group that similar low-yield conformation is possessed needed suitable layout, guarantee dynamin inhibitory action preferably.Some similar results also are observed for the sulfo-indole, and they are EGFR tyrosine kinase inhibitors, show increase activity to oxidation people such as (, 1993) Thompson.

Scheme 4

2.4 discuss

The structure-activity relation of the dimerization tyrphostin of antagonism GTP enzyme dynamin is by being assessed through synthetic and screening storehouse chemical compound based on lead compound two-tyrphostin and tyrphostin A47.Find out that from the result of gained containing at 3,4 has the dimerization tyrphostin chemical compound of two aromatic rings of hydroxyl replacement to have strong inhibitory activity.Can be used to form spacer and finish easily by changing functional group the modification of these chemical compounds.

Embodiment 3: the exploitation of prodrug

The prodrug of two-tyrphostin and analog thereof are developed with the transparent performance that increases cell membrane and therefore increase the usefulness of medicine in cell.Provide in the suitable scheme that is reflected at 5 of dimerization tyrphostin chemical compound prodrug explanation is arranged.Two-tyrphostin is the example of initial action agent.Dimerization tyrphostin chemical compound and suitable anhydride or acyl chlorides (molal quantity is excessive) stir together in dinethylformamide (DMF) solution, and have suitable catalyst such as dimethyl aminopyridine (DMAP) at pyridine/N.In some cases, solution need reflux so that react completely.After reacting completely, esterification products carries out purification by recrystallization or chromatograph.The example of the prodrug of exploitation is as shown in table 2 and table 3.

Synthesizing of scheme 5. prodrugs

Table 2: the prodrug of two-tyrphostin

Table 3: the prodrug forms of dimerization tyrphostin

Prodrug Pro-BisT has 4 acetyl group ester groups to replace 4 oh groups of two-tyrphostin, and estimates its ability by the outer cell membrane of cell.Pro-BisT is transformed into active substance two-tyrphostin in cell, therefore it can also suppress endocytosis in conjunction with dynamin then.Find that Pro-BisT suppresses receptor-mediated cell line Hela, HER14, COS7, Swiss 3T3, A431, the transferrins of B104 and B35 or the endocytosis of EGF (RME).Pro-BisT has more significant usefulness (30x) than two-tyrphostin, can block the RME of the cell line of testing effectively, concentration is that 10-20 μ M detects down, shows the ability that the penetration cell of mutually big improvement is arranged than two-tyrphostin.

Prodrug 80-1 suppresses RME being similar to Pro-BisT concentration (10-20 μ M), is developed to be used for reducing the too early hydrolysis before extracellular environment enters cell of this prodrug.When storing with powder type, compares this prodrug the shelf life that Pro-BisT has improvement.

Those of ordinary skills should be able to understand various to variation of the present invention and/or modification, and do not deviate from the spirit or scope of the present invention.The specific embodiment of the present invention is to be used for explanation rather than restriction the present invention.

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Claims (80)

1, a kind of method that suppresses the dependent endocytosis of dynamin in the cell, this method comprises the chemical compound with the general formula I of effective dose, or its physiologically acceptable salt handles described cell, wherein

M-Sp-M ' general formula I

Wherein M and M ' are independent respectively is the structure division shown in the general formula I I, can be identical or different, and Sp is a spacer;

Wherein V is C or CH;

W is a CH or a linking group; And

Y is hydrogen, cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, sulfur, or unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group, described substituent group is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; Or

W, V and Y form and the condensed replacement of Z group or unsubstituted 5 Yuans or 6 element heterocycles or carbocyclic ring, wherein heterocycle comprises 1 to 3 hetero atom that is selected from O, N and S, if heterocycle or carbocyclic ring have substituent group, substituent group is to be selected from cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, sulfur, or unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group at least a, replacement C wherein ₁-C ₃Substituent group be selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; With

R is CH ₂R ', CXR ' or CHX ' R ';

X is O or S;

X ' is cyano group, nitro, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, or unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group, described substituent group is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur;

R ' is NH, O or the S that is connected on the spacer; With

Z is selected from

(a) non-replacement by one or two heterocycles of forming of 5 Yuans or 6 Yuans ring independently, have to be no more than 3 the hetero atom that is selected from O, N and S;

(b) non-replacement by one or two carbocyclic rings of forming of 5 Yuans or 6 Yuans ring independently;

(c) by one or two heterocycles of forming of 5 Yuans or 6 Yuans ring independently, have to be no more than 3 the hetero atom that is selected from O, N and S, wherein heterocycle has one or more substituent groups, and described substituent group independently is selected from:

(i) nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; With

(ii) C ₁-C ₂Alkyl or C ₁-C ₂Alkenyl group, be selected from nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C by at least one ₁-C ₂Alkoxyl and C ₁-C ₂The group of acyl group replace; With

(d) by one or two carbocyclic rings of forming of 5 Yuans or 6 Yuans ring independently, when W is a CH or a linking group, or W, V and Y at least two substituent groups when forming the carbocyclic ring of non-replacement, perhaps when W, V and Y formation heterocycle, at least one substituent group independently is selected from:

(i) nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; With

(ii) C ₁-C ₂Alkyl or C ₁-C ₂Alkenyl group, have at least one to be selected from nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂The substituent group of acyl group;

Wherein when one Z among M or the M ' is selected from (b), and other Z is selected from (a), (c) or (d) among M or the M '.

2. the method for claim 1, wherein:

V is C;

W is CH;

Y is hydrogen, cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, or unsubstituted C ₁-C ₂Group or the C of replacement ₁-C ₂Group, substituent group wherein independently is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; Or

W, V and Y form 5 Yuans or 6 s' replacement or non-replacement with Z condensed heterocycle or carbocyclic ring, wherein heterocycle comprises 1 to 3 hetero atom that is selected from O, N and S, if and carbocyclic ring or heterocycle have replacement, has a substituent group at least, be selected from cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur, or unsubstituted C ₁-C ₂Group or the C of replacement ₁-C ₂Group, replacement C wherein ₁-C ₂The substituent group of group independently is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; With

R is CH ₂R ', CXR ' or CHX ' R ';

X is O or S; With

X ' is cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, or unsubstituted C ₁-C ₂Group or the C of replacement ₁-C ₂Group, replacement C wherein ₁-C ₂The substituent group of group independently is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur.

3. method as claimed in claim 2, wherein:

Y is the C of cyano group, nitro, amino, carboxyl, hydroxyl, sulfydryl, thiocarboxyl group, replacement ₁-C ₂Group, substituent group wherein independently is selected from cyano group, nitro, amino, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group;

W, V and Y form 5 Yuans or 6 s' replacement or non-replacement with Z condensed heterocycle or carbocyclic ring, wherein heterocycle contains 1 to 3 hetero atom that is selected from O, N and S, if and heterocycle or carbocyclic ring have replacement, contain at least one substituent group, described substituent group is selected from cyano group, nitro, amino, hydroxyl, sulfydryl, carboxyl and thiocarboxyl group, or the C that replaces ₁-C ₂Group replaces C ₁-C ₂The substituent group of group is selected from cyano group, nitro, amino, hydroxyl, sulfydryl, carboxyl and thiocarboxyl group; And

R is CXR '.

4. each described method of claim 1-3, wherein Z is selected from:

(i) independently be 5 Yuans or 6 Yuans heterocycles that ring is formed by one or two, comprise no more than 3 the hetero atom that independently is selected from O, N and S;

Independently be 5 Yuans or 6 Yuans heterocycles that ring is formed (ii) by one or two, comprise no more than 3 the hetero atom that independently is selected from O, N and S, wherein heterocycle contains one or more substituent groups, and described substituent group independently is selected from nitro, NH, halogen, cyano group, amino, hydroxyl, carboxyl, oxo, sulfur and C ₁-C ₂Alkoxyl; With

Independently be 5 Yuans or 6 Yuans carbocyclic rings that ring is formed (iii), contain at least two substituent groups, be selected from nitro, NH, amino, halogen, cyano group, hydroxyl, carboxyl, oxo, sulfur and C by one or two ₁-C ₂Alkoxyl.

5. as each method of claim 1-4, wherein the Z of at least one is not 2 among M and the M ', the dibasic carbon ring group of 3-.

6. as each method of claim 1-5, wherein the Z of at least one comprises among M and the M ':

When Z is selected from (d) and W is CH or C ₁-C ₃Linking group the time, at least two mutual ortho positions of substituent group or at adjacent the position of substitution; Or

When Z is when being selected from the heterocycle of (c), the above substituent group of carbon atom or one of adjacent with one of hetero atom or its; Or

When W, V and Y cyclisation formed with the Z condensed heterocycle, one of the substituent group on carbon atom or its had at least a bond distance at interval with heterocycle.

7. as each method of claim 1-6, wherein one Y substituent group is a hydrogen among M and the M ', and the Y substituent group of the another one of M and M ' then is not a hydrogen.

8. as each method of claim 1-7, wherein W, V and Y form 5 Yuans or 6 Yuans and Z condensed heterocycle.

9. method as claimed in claim 8 wherein forms two ring heterocyclic groups with the Z condensed heterocycle.

10. as each method of claim 1-8, comprising aromatic yl group, to form by one or two ring, described ring independently is 5 or 6 Yuans rings, at least two substituent groups independently are selected from nitro, NH, amino, halogen, cyano group, hydroxyl, carboxyl, oxo, sulfur and C ₁-C ₂Alkoxyl.

11. method as claimed in claim 10, wherein Z comprises aromatic yl group and at least two substituent groups of being made up of one 6 Yuans rings, and described substituent group independently is selected from nitro, amino, halogen, cyano group, hydroxyl, carboxyl and C ₁-C ₂Alkoxyl.

12. method as claimed in claim 11, wherein aromatic yl group has two substituent groups at least, independently is selected from nitro, amino and hydroxyl.

13. as each method of claim 1-8, wherein Z contains heterocyclic group, described heterocycle comprises one or two independently 5 or 6 Yuans rings, contain and be no more than 3 the hetero atom that is selected from O, N and S, wherein said heterocycle contains one or more substituent groups, independently is selected from nitro, NH, halogen, cyano group, amino, hydroxyl, carboxyl, oxo, sulfur and C ₁-C ₂Alkoxyl.

14. method as claimed in claim 13, wherein heterocyclic group contains one or more substituent groups, independently is selected from nitro, amino and hydroxyl.

15. as each method of claim 1-5, wherein M and M ' difference are independent is the following radicals part:

Wherein:

X is O or S;

Y is cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl or thiocarboxyl group; Or

R ₁Form 5 or 6 Yuans replacement or unsubstituted heterocycle or carbocyclic ring with the Y cyclisation, wherein heterocycle contains 1 or 2 hetero atom that is selected from O, N and S, and heterocycle or carbocyclic ring have the substituent group that at least one is selected from cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur when replacing; And

R ₂To R ₅Independent is hydrogen or certain substituent group, and described substituent group independently is selected from nitro, amino, halogen, hydroxyl, carboxyl, sulfydryl, thiocarboxyl group, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; Or

R ₁To R ₅Independent is hydrogen or certain substituent group, and described substituent group independently is selected from nitro, amino, halogen, hydroxyl, carboxyl, sulfydryl, thiocarboxyl group, halogen, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; With

R is NH, O or the S that links on the basic Sp in interval; And

Wherein at least one among M and the M ' is characterised in that: R ₁To R ₅At least two be not hydrogen, and work as R ₁To R ₂When being not hydrogen, R ₃To R ₅In at least one neither hydrogen, perhaps work as R ₁When cyclisation forms unsubstituted carbocyclic ring with Y, R ₂To R ₅At least two be not hydrogen, perhaps work as R ₁When forming heterocycle with the Y cyclisation, R ₂To R ₅At least one be not hydrogen.

16. method as claimed in claim 15, wherein R ₁-R ₃Not hydrogen.

17. method as claimed in claim 15, wherein R ₁-R ₅At least two at the ortho position.

18. method as claimed in claim 15, wherein at least one has three substituent groups among M and the M ', and substituent group is adjacent.

19. method as claimed in claim 18, wherein R ₁-R ₃Not hydrogen, or R ₂-R ₅Not hydrogen.

20. method as claimed in claim 15 is wherein worked as R ₁-R ₅Or R ₂-R ₅In at least one be halogen, C ₁-C ₂Alkoxyl or C ₁-C ₂Acyl group the time, at least one other substituent group is selected from nitro, amino, hydroxyl, carboxyl and thiocarboxyl group, works as R ₁With the Y cyclisation and when forming heterocycle, perhaps at least two other substituent groups are selected from nitro, amino, hydroxyl, carboxyl and thiocarboxyl group, work as R ₁With Y ring formation or form unsubstituted carbocyclic ring not.

21. as each described method of claim 15-20, wherein Y is a cyano group, X is that O and R are NH.

22. as each described method of claim 1-21, wherein M is identical with M '.

23. as each described method of claim 1-22, wherein to allow be the chemical compound of hairpin structure to spacer Sp.

24. as each described method of claim 1-23, wherein spacer Sp comprises unsubstituted alkane chain, structure is as follows:

-CH ₂(CH ₂) _nCH ₂-

Wherein n is the integer of 1-5.

25. as each described method of claim 1-24, the chemical compound of wherein said general formula I is the tyrphostin of dimerization.

26. the disease of an endocytosis mediation that prevents or treat to rely on by dynamin in the mammal or the method for disease, this method comprises the chemical compound of the general formula I that gives the mammal effective dose, or its physiologically acceptable salt or prodrug, the structure of general formula I is:

M-Sp-M ' general formula I

Wherein M and M ' are independent respectively is the structure division shown in the general formula I I, can be identical or different, and Sp is a spacer;

Wherein V is C or CH;

W is a CH or a linking group; And

Y is hydrogen, cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, sulfur, or unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group, described substituent group is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; Or

W, V and Y form and the condensed replacement of Z group or unsubstituted 5 Yuans or 6 element heterocycles or carbocyclic ring, wherein heterocycle comprises 1 to 3 hetero atom that is selected from O, N and S, if heterocycle or carbocyclic ring have substituent group, substituent group is to be selected from cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, sulfur, or unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group, substituent group wherein is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; With

R is CH ₂R ', CXR ' or CHX ' R ';

X is O or S;

X ' is cyano group, nitro, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, or unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group, described substituent group is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur;

R ' is NH, O or the S that is connected on the spacer; With

Z is selected from

(a) non-replacement by one or two heterocycles of forming of 5 Yuans or 6 Yuans ring independently, have to be no more than 3 the hetero atom that is selected from O, N and S;

(b) non-replacement by one or two carbocyclic rings of forming of 5 Yuans or 6 Yuans ring independently;

(c) by one or two heterocycles of forming of 5 Yuans or 6 Yuans ring independently, have to be no more than 3 the hetero atom that is selected from O, N and S, wherein heterocycle has one or more substituent groups, and described substituent group independently is selected from:

(i) nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; With

(ii) C ₁-C ₂Alkyl or C ₁-C ₂Alkenyl group, be selected from nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C by at least one ₁-C ₂Alkoxyl and C ₁-C ₂The group of acyl group replace; With

(d) by one or two carbocyclic rings of forming of 5 Yuans or 6 Yuans ring independently, when W is a CH or a linking group, or W, V and Y at least two substituent groups when forming the carbocyclic ring of non-replacement, perhaps when W, V and Y formation heterocycle, at least one substituent group independently is selected from:

(i) nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; With

(ii) C ₁-C ₂Alkyl or C ₁-C ₂Alkenyl group, have at least one to be selected from nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂The substituent group of acyl group;

Wherein when one Z among M or the M ' is selected from (b), and other Z is selected from (a), (c) or (d) among M or the M '.

27. method as claimed in claim 26, wherein:

V is C;

W is CH;

Y is hydrogen, cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, or unsubstituted C ₁-C ₂Group or the C of replacement ₁-C ₂Group, substituent group wherein independently is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; Or

W, V and Y form 5 Yuans or 6 s' replacement or non-replacement with Z condensed heterocycle or carbocyclic ring, wherein heterocycle comprises 1 to 3 hetero atom that is selected from O, N and S, if and carbocyclic ring or heterocycle have replacement, has a substituent group at least, be selected from cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur, or unsubstituted C ₁-C ₂Group or the C of replacement ₁-C ₂Group, substituent group wherein independently is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; With

R is CH ₂R ', CXR ' or CHX ' R ';

X is O or S; With

X ' is cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, or unsubstituted C ₁-C ₂Group or the C of replacement ₁-C ₂Group, substituent group wherein independently is selected from cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; And

R ' is NH, O or the S that is bonded on the spacer.

28. method as claimed in claim 27, wherein:

Y is the C of cyano group, nitro, amino, carboxyl, hydroxyl, sulfydryl, thiocarboxyl group, replacement ₁-C ₂Group, substituent group wherein independently is selected from cyano group, nitro, amino, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group;

W, V and Y form 5 Yuans or 6 s' replacement or non-replacement with Z condensed heterocycle or carbocyclic ring, wherein heterocycle contains 1 to 3 hetero atom that is selected from O, N and S, if and heterocycle or carbocyclic ring have replacement, contain at least one substituent group, described substituent group is selected from cyano group, nitro, amino, hydroxyl, sulfydryl, carboxyl and thiocarboxyl group, or the C that replaces ₁-C ₂Group, substituent group are selected from cyano group, nitro, amino, hydroxyl, sulfydryl, carboxyl and thiocarboxyl group; And

R is CXR '.

29. as each described method of claim 26-28, wherein the Z of at least one is not 2 among M and the M ', the dibasic carbon ring group of 3-.

30. as each described method of claim 26-29, wherein the Z of at least one comprises among M and the M ':

When Z is selected from (d) and W is CH or C ₁-C ₃Linking group the time, have two ortho-substituents at least; Or

When Z is when being selected from the heterocycle of (c), one of this substituent group or substituent group are on the carbon atom adjacent with one of this hetero atom or hetero atom; Or

When W, V and Y cyclisation formation and Z annelated heterocycles, one of this substituent group on the carbon atom of Z or substituent group have a bond distance at least with heterocycle.

31. as each described method of claim 26-30, the Y substituent group of one of them M or M ' is a hydrogen, and the Y substituent group of other M or M ' is not a hydrogen.

32. as each described method of claim 26-31, wherein M and M ' independently are selected from following group part:

Wherein:

X is O or S;

Y is cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl or thiocarboxyl group; Or

R ₁Form 5 or 6 Yuans replacement or unsubstituted heterocycle or carbocyclic ring with the Y cyclisation, wherein heterocycle contains 1 or 2 hetero atom that is selected from O, N and S, and heterocycle or carbocyclic ring have the substituent group that at least one is selected from cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur when replacing; And

R ₂To R ₅Independent is hydrogen or certain substituent group, and described substituent group independently is selected from nitro, amino, halogen, hydroxyl, carboxyl, sulfydryl, thiocarboxyl group, C ₁-C ₂Alkoxyl or C ₁-C ₂Acyl group; Or

R ₁To R ₅Independent is hydrogen or certain substituent group, and described substituent group independently is selected from nitro, amino, halogen, hydroxyl, carboxyl, sulfydryl, thiocarboxyl group, halogen, C ₁-C ₂Alkoxyl or C ₁-C ₂Acyl group; With

R is NH, O and the S that links on the basic Sp in interval; And

Wherein at least one among M and the M ' is characterised in that: R ₁To R ₅At least two be not hydrogen, and work as R ₁To R ₂When being not hydrogen, R ₃To R ₅In at least one neither hydrogen, perhaps work as R ₁When cyclisation forms unsubstituted carbocyclic ring with Y, R ₂To R ₅At least two be not hydrogen, perhaps work as R ₁When forming heterocycle with Y, R ₂To R ₅At least one be not hydrogen.

33. method as claimed in claim 32, wherein R ₁-R ₃Not hydrogen.

34. as claim 26-31 each described method, wherein R ₁-R ₅At least two be that the ortho position replaces.

35. as each described method of claim 26-31, wherein M and M ' at least one three substituent groups are arranged, and substituent group is adjacent.

36. method as claimed in claim 33, wherein R ₁-R ₃All be not hydrogen, or R ₂-R ₅All not hydrogen.

37., wherein work as R as each described method of claim 26-32 ₁-R ₅Or R ₂-R ₅In at least one be halogen, C ₁-C ₂Alkoxyl or C ₁-C ₂Acyl group the time, at least one other substituent group is selected from nitro, amino, hydroxyl, carboxyl and thiocarboxyl group, works as R ₁With the Y cyclisation and when forming heterocycle, perhaps at least two other substituent groups are selected from nitro, amino, hydroxyl, carboxyl and thiocarboxyl group, work as R ₁With Y ring formation or form unsubstituted carbocyclic ring not.

38. as each described method of claim 26-37, wherein Y is a cyano group, X is that O and R are NH.

39. as each described method of claim 26-38, wherein M is identical with M '.

40. as each described method of claim 26-39, wherein to allow be the chemical compound of hairpin structure to spacer Sp.

41. as each described method of claim 26-40, wherein the chemical compound of general formula I is the tyrphostin of dimerization.

42. as each described method of claim 26-41, wherein said disease or disease are selected from cancer, ophthalmic diseases, immunodeficiency, gastroenteropathy, pathogenicity infection, nephropathy, epilepsy and sacred disease, neurodegenerative disease and neural disease and disease.

43. method as claimed in claim 42, wherein sacred disease, neurodegenerative disease and nervous system disease and disease are selected from demyelination, Alzheimer, Huntington's disease, parkinson and Lewy body disease.

44. method as claimed in claim 42, wherein disease or disease are epilepsies.

45. one kind is prevented or treats mammal by the disease of dynamin dependent cell encytosis mediation or the method for disease, this method comprises the dimerization tyrphostin of the inhibition dynamin GTP enzymatic activity that gives the mammal effective dose, or the analog of this dimerization tyrphostin, physiologically acceptable salt, or prodrug.

46. method as claimed in claim 45, wherein the dimerization tyrphostin comprises the structure division of two tyrphostins, it links to each other by a compartment, and wherein at least one tyrphostin partly is a benzal Cyanoacetyl-Cyacetazid part.

47. method as claimed in claim 46, wherein two tyrphostin parts all are benzal Cyanoacetyl-Cyacetazid parts.

48. claim 46 or 47 described methods, wherein tyrphostin partly is identical.

49. as each described method of claim 45-48, wherein the dimerization tyrphostin comprises two-tyrphostin.

50. as each described method of claim 45-49, wherein said disease or disease are selected from the disease and the disease of cancer, ophthalmic diseases, immunodeficiency, gastroenteropathy, pathogenicity infection, nephropathy, epilepsy and sacred disease, neurodegenerative disease and nervous system disease.

51. method as claimed in claim 50, wherein sacred disease, neurodegenerative disease and nervous system disease and disease are selected from demyelination, Alzheimer, Huntington's disease, parkinson and Lewy body disease.

52. method as claimed in claim 50, wherein said disease or disease are epilepsies.

53. differentiate that dimerization tyrphostin or its analog have the method that suppresses dynamin GTP enzymatic activity ability for one kind, this method comprises: dimerization tyrphostin or its analog cultivated with dynamin or molecule with dynamin GTP enzymatic activity obtain detecting data; And

Detect on the basis of data at this, determine whether dimerization tyrphostin or its analog have the effect of the GTP enzymatic activity that suppresses dynamin.

54. dimerization tyrphostin or the application in the dependent endocytosis of dynamin in suppressing cell of its analog by the described method evaluation of claim 53.

55. the described chemical compound of following general formula III or its physiologically acceptable salt, or prodrug, wherein:

M-Sp-M ' general formula III

Wherein M and M ' are independent respectively is the structure division shown in the general formula I V, can be identical or different,

Sp is a spacer;

Wherein V is C or CH;

W is a CH or a linking group; And

Y is hydrogen, cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, sulfur, or unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group, described substituent group independently is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; Or

W, V and Y form and the condensed replacement of Z group or unsubstituted 5 Yuans or 6 element heterocycles or carbocyclic ring, wherein heterocycle comprises 1 to 3 hetero atom that is selected from O, N and S, if heterocycle or carbocyclic ring have substituent group, substituent group is to be selected from cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, sulfur, or unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group, wherein replace C ₁-C ₃The substituent group of group is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; With

R is CH ₂R ', CXR ' or CHX ' R ';

X is O or S;

X ' is cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, or unsubstituted C ₁-C ₃Group or the C of replacement ₁-C ₃Group, described substituent group independently is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur;

R ' is NH, O or the S that is connected on the spacer; With

Z is selected from

(a) non-replacement by one or two heterocycles of forming of 5 Yuans or 6 Yuans ring independently, have to be no more than 3 the hetero atom that is selected from O, N and S;

(b) non-replacement by one or two carbocyclic rings of forming of 5 Yuans or 6 Yuans ring independently;

(c) by one or two heterocycles of forming of 5 Yuans or 6 Yuans ring independently, have to be no more than 3 the hetero atom that is selected from O, N and S, wherein heterocycle has one or more substituent groups, and described substituent group independently is selected from:

(i) nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; With

(ii) C ₁-C ₂Alkyl or C ₁-C ₂Alkenyl group, be selected from nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C by at least one ₁-C ₂Alkoxyl and C ₁-C ₂The group of acyl group replace; With

(d) by one or two carbocyclic rings of forming of 5 Yuans or 6 Yuans ring independently, when W is a CH or a linking group, or W, V and Y at least two substituent groups when forming the carbocyclic ring of non-replacement, perhaps when W, V and Y formation heterocycle, at least one substituent group independently is selected from:

(i) nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; With

(ii) C ₁-C ₂Alkyl or C ₁-C ₂Alkenyl group, have at least one to be selected from nitro, NH, amino, cyano group, halogen, hydroxyl, carboxyl, oxo, sulfur, sulfydryl, C ₁-C ₂Alkoxyl and C ₁-C ₂The substituent group of acyl group;

Wherein when one Z among M or the M ' is selected from (b), and other Z is selected from (a), (c) or (d) among M or the M '.And condition be when R be CXR ', X is O, R ' is the NH on being bonded at interval, V is C, W is CH, Y is cyano group and R ₁, R ₂And R ₅Be H, R ₃And R ₄When being hydroxyl, one Z is not a benzyl group shown in general formula I Va among M and the M ' at least,

Perhaps

When Sp is C ₂-C ₄Alkyl at interval the time, R ₁And R ₅Be H, R ₂To R ₄It is hydroxyl; And wherein Z ' is the carbon atom that is connected with the W group.

56. as the chemical compound of claim 55, wherein:

V is C;

W is CH;

Y is hydrogen, cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, or unsubstituted C ₁-C ₂Group or the C of replacement ₁-C ₂Group, substituent group wherein independently is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; Or

W, V and Y form 5 Yuans or 6 s' replacement or non-replacement with Z condensed heterocycle or carbocyclic ring, wherein heterocycle comprises 1 to 3 hetero atom that is selected from O, N and S, if and carbocyclic ring or heterocycle have replacement, has a substituent group at least, be selected from cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur, or unsubstituted C ₁-C ₂Group or the C of replacement ₁-C ₂Group, wherein replace C ₁-C ₂The substituent group of group independently is selected from least a of cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur; With

R is CH ₂R ', CXR ' or CHX ' R ';

X is O or S; With

X ' is cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group, or unsubstituted C ₁-C ₂Group or the C of replacement ₁-C ₂Group, wherein replace C ₁-C ₂The substituent group of group independently is selected from cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur.

57. as the chemical compound of claim 56, wherein:

Y is the C of cyano group, nitro, amino, carboxyl, hydroxyl, sulfydryl, thiocarboxyl group, replacement ₁-C ₂Group, substituent group wherein independently is selected from cyano group, nitro, amino, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group;

W, V and Y form 5 Yuans or 6 s' replacement or non-replacement with Z condensed heterocycle or carbocyclic ring, wherein heterocycle contains 1 to 3 hetero atom that is selected from O, N and S, if and heterocycle or carbocyclic ring have replacement, contain at least one substituent group, described substituent group is selected from cyano group, nitro, amino, hydroxyl, sulfydryl, carboxyl and thiocarboxyl group, or the C that replaces ₁-C ₂Group replaces C ₁-C ₂The group substituent group is selected from cyano group, nitro, amino, hydroxyl, sulfydryl, carboxyl and thiocarboxyl group; And

R is CXR '.

58. as each described chemical compound of claim 55-57, wherein Z is selected from:

(i) independently be 5 Yuans or 6 Yuans heterocycles that ring is formed by one or two, comprise no more than 3 hetero atom that is selected from O, N and S;

Independently be 5 Yuans or 6 Yuans heterocycles that ring is formed (ii) by one or two, comprise no more than 3 hetero atom that is selected from O, N and S, wherein heterocycle contains one or more substituent groups, and described substituent group independently is selected from nitro, NH, halogen, cyano group, amino, hydroxyl, carboxyl, oxo, sulfur and C ₁-C ₂Alkoxyl; With

Independently be 5 Yuans or 6 Yuans carbocyclic rings that ring is formed (iii), contain at least two substituent groups, be selected from nitro, NH, amino, halogen, cyano group, hydroxyl, carboxyl, oxo, sulfur and C by one or two ₁-C ₂Alkoxyl.

59. as each described chemical compound of claim 55-58, wherein the Z of at least one is not 2 among M and the M ', the dibasic carbon ring group of 3-.

60. as each chemical compound of claim 55-59, wherein the Z of at least one comprises among M and the M ':

When Z is selected from (d) and W is CH or C ₁-C ₃Linking group the time, have two ortho-substituents at least; Or

When Z is when being selected from the heterocycle of (c), one of this substituent group or substituent group are on the carbon atom adjacent with one of this hetero atom or hetero atom; Or

When W, V and Y cyclisation formation and Z annelated heterocycles, one of this substituent group on carbon atom or substituent group have a bond distance at least with heterocycle.

61. as each chemical compound of claim 55-60, wherein one Y substituent group is a hydrogen among M and the M ', the Y substituent group of another one then is not a hydrogen.

62. as each chemical compound of claim 55-61, wherein W, V and Y form the heterocycle with condensed 5 Yuans or 6 Yuans of Z.

63. chemical compound as claimed in claim 62 wherein forms the heterocyclic group of two rings with the Z-shaped annelated heterocycles that becomes.

64. as each chemical compound of claim 55-63, wherein Z comprises aromatic yl group, and described aryl is made up of one or two ring, and described ring independently is 5 or 6 Yuans rings, with have two substituent groups at least, independently be selected from nitro, NH, amino, halogen, cyano group, hydroxyl, carboxyl, oxo, sulfur and C ₁-C ₂Alkoxyl.

65. as the described chemical compound of claim 64, wherein Z comprises 6 Yuans aromatic yl group and has two substituent groups at least, independently is selected from nitro, amino, halogen, cyano group, hydroxyl, carboxyl and C ₁-C ₂Alkoxyl.

66. as the described chemical compound of claim 65, wherein aromatic yl group has two substituent groups at least, independently is selected from nitro, amino and hydroxyl.

67. as each chemical compound of claim 55-62, wherein Z contains heterocyclic group, described heterocycle comprises one or two independently 5 or 6 Yuans rings, contain and be no more than 3 the hetero atom that is selected from O, N and S, wherein said heterocycle contains one or more substituent groups, independently is selected from nitro, NH, halogen, cyano group, amino, hydroxyl, carboxyl, oxo, sulfur and C ₁-C ₂Alkoxyl.

68. as the described chemical compound of claim 67, wherein heterocyclic group contains one or more substituent groups, independently is selected from nitro, amino and hydroxyl.

69. as each chemical compound of claim 55-59, wherein M and M ' difference are independent is the following radicals part:

Wherein:

X is O or S;

Y is cyano group, nitro, amino, halogen, hydroxyl, sulfydryl, carboxyl or thiocarboxyl group; Or

R ₁Form 5 or 6 Yuans replacement or unsubstituted heterocycle or carbocyclic ring with the Y cyclisation, wherein heterocycle contains 1 or 2 hetero atom that is selected from O, N and S, and heterocycle or carbocyclic ring have the substituent group that at least one is selected from cyano group, nitro, NH, amino, oxo, halogen, hydroxyl, sulfydryl, carboxyl, thiocarboxyl group and sulfur when replacing; And

R ₂To R ₅Independent is hydrogen or certain substituent group, and described substituent group independently is selected from nitro, amino, halogen, hydroxyl, carboxyl, sulfydryl, thiocarboxyl group, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; Or

R ₁To R ₅Independent is hydrogen or certain substituent group, and described substituent group independently is selected from nitro, amino, halogen, hydroxyl, carboxyl, sulfydryl, thiocarboxyl group, halogen, C ₁-C ₂Alkoxyl and C ₁-C ₂Acyl group; With

R is NH, O or the S that links on the basic Sp in interval; And

Wherein at least one among M and the M ' is characterised in that: R ₁To R ₅At least two be not hydrogen, and work as R ₁To R ₂When being not hydrogen, R ₃To R ₅In at least one neither hydrogen, perhaps work as R ₁When cyclisation forms unsubstituted carbocyclic ring with Y, R ₂To R ₅At least two be not hydrogen, perhaps work as R ₁When forming heterocycle with the Y cyclisation, R ₂To R ₅At least one be not hydrogen.

70. as the described chemical compound of claim 69, wherein R ₁-R ₃Not hydrogen.

71. as the described chemical compound of claim 69, wherein R ₁-R ₅At least two mutually at the ortho position.

72. as the described chemical compound of claim 69, wherein at least one has three substituent groups among M and the M ', and substituent group is adjacent.

73. as the described chemical compound of claim 72, wherein R ₁-R ₃Not hydrogen, or R ₂-R ₅Not hydrogen.

74., wherein work as R as the described chemical compound of claim 69 ₁-R ₅Or R ₂-R ₅In at least one be halogen, C ₁-C ₂Alkoxyl or C ₁-C ₂Acyl group the time, at least one other substituent group is selected from nitro, amino, hydroxyl, carboxyl and thiocarboxyl group, works as R ₁When forming heterocycle with the Y cyclisation, perhaps at least two other substituent groups are selected from nitro, amino, hydroxyl, carboxyl and thiocarboxyl group, work as R ₁With Y ring formation or when forming unsubstituted carbocyclic ring not.

75. as each described chemical compound of claim 69-74, wherein Y is a cyano group, X is that O and R are NH.

76. as each described chemical compound of claim 55-75, wherein M is identical with M '.

77. as each described chemical compound of claim 55-76, wherein to allow be the chemical compound of hairpin structure to spacer Sp.

78. as each described chemical compound of claim 55-77, wherein spacer Sp comprises unsubstituted alkane chain, structure is as follows:

-CH ₂(CH ₂) _nCH ₂-

Wherein n is the integer of 1-5.

79. according to the arbitrary chemical compound among the claim 1-78, the chemical compound of wherein said general formula III is the tyrphostin of dimerization.

80. a pharmaceutical composition, said composition comprise as the defined chemical compound of each claim of claim 55-58, and physiologically acceptable excipient, carrier or diluent.

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