CN101098686B - Glycogen synthase kinase-3 inhibitors - Google Patents

Abstract

Novel compounds designed to allow interactions with binding sites of GSK-3 and hence are capable of inhibiting GSK-3 activity, via inhibition of substrate binding are disclosed. Further disclosed are pharmaceutical compositions including same and methods of using same in the treatment of GSK-3 mediated conditions.

Description

Glycogen synthase kinase-3 inhibitors

Invention field and background

The present invention relates to suppress the new chemical compound of glycogen synthase kinase-3 (GSK-3); And they in the biology of regulating the active mediation of GSK-3 the purposes in the situation; More specifically, relate to these chemical compounds in treatment such as the purposes in situation biology such as type ii diabetes, neurodegenerative disorders and disease and affective disorder.

Protein kinase---enzyme of phosphorylating protein substrate---is the key enzyme of extracellular incident in Cytoplasm and the transmission of nucleus signal; Life and dead relevant almost any incident that it is participated in cell comprise mitosis, differentiation and programmed cell death.Equally, protein kinase is useful drug targets for a long time.Yet, because the activity of protein kinase is crucial for the health of cell, their inhibition being caused cell death often, they are restricted as the purposes of drug targets.Although cell death is the purpose effect of cancer therapy drug, this treats for most of other is major defect.

Glycogen synthase kinase-3 (GSK-3) is a member of protein kinase family; It is the directed serine-threonine kinase of Cytoplasm proline; It is participated in insulin signaling transmission and regulates with metabolism, and the design of pair cell destiny during transmission of Wnt signal and the fetal development.Identify two kinds of similar isotypes of this enzyme, be called GSK-3 α and GSK-3 β.

GSK-3 has been used for being considered to drug targets useful in the protein kinase family for a long time; Because unlike other protein kinase usually by the signal pathway activation; GSK-3 is activated in resting cell usually; And through some signal pathway of activation, combine the signal pathway that produces with its cell surface receptor, can weaken the activity of GSK-3 like insulin.The activation of Insulin receptor INSR causes the activation of protein kinase B (PKB is also referred to as Akt), itself and then phosphorylation GSK-3, thus make its inactivation.Inhibition to GSK-3 possibly cause the synthetic activation of glycogen.This complicated insulin signaling approach is regulated further complicated to the negative feedback of insulin signaling by GSK-3 self, GSK-3 is phosphorylation insulin-receptor substrate-1 people such as (, 1997) Eldar-Finkelman on serine residue.

Therefore, synthetic GSK-3 inhibitor can be simulated some hormone that utilizes the GSK-3 approach and the effect of somatomedin (like insulin).Under some pathological conditions; This scheme can allow to walk around defective receptor, and perhaps signal transmits another imperfect component of machine, thereby even some upper reaches compositions of signal cascade have fault; As in the non-insulin-dependent type ii diabetes, bio signal also will play a role.

The metabolic adjusting of glycogenolysis is crucial biological function in the pair cell, and it relates to a complex set of signal transmitting element, comprises the hormone insulin.Through crossing multiple amboceptor, insulin is through increasing synthetic bring into play its regulating action of Glycogensynthase (GS) to glycogen.A critical events in the insulin action is IRS (this causes multiple signal is transmitted component for IRS-1, the IRS-2) phosphorylation on a plurality of tyrosine residues, comprises the kinase whose while activation of PI3 people such as (, 1992) Myers).Similarly, the activity of Glycogensynthase receives the inhibition of its phosphorylation.Active and the glycogen levels of Glycogensynthase has remarkable decline people such as (, 1990) Shulman in type ii diabetes patient muscle.

One of variation the earliest relevant with II type (non-insulin-dependent) diabetes is insulin resistance.The characteristic of insulin resistance is hyperinsulinemia and hyperglycemia.Although the accurate molecule mechanism of insulin resistance is also unknown, the defective in the downstream component of insulin signaling approach is considered to the cause of disease.

Glycogen synthase kinase-3 (GSK-3) is one of downstream component of insulin signaling transmission.Find that the high activity of GSK-3 diminishes the effect of insulin in the intact cell; This is through phosphorylation IRS-1 (IRS-1) serine residue (people such as Eldar-Finkelman; 1997) realize; And likewise, the active raising of the GSK-3 that expresses in the cell possibly cause the active inhibition of Glycogensynthase (people such as Eldar-Finkelman, 1996).GSK-3 is active significantly raises for further discovering in the epididymal adipose of diabetic mice of carrying out in this respect people such as (, 1999) Eldar-Finkelman.Afterwards, the GSK-3 that in type ii diabetes patient's skeletal muscle, detects rising active people such as (, 2000) Nickoulina.Extra recent research has confirmed that further (summary is seen Eldar-Finkelman in the effect of GSK-3 in glycogen metabolism and insulin signaling transmission; 2002Woodgett; 2001), thus put forward a kind of approach to showing that the active inhibition of GSK-3 possibly represented in the body increases insulin active.

It is also believed that GSK-3 is the key factor in the morbidity of Alzheimer.GSK-3 is accredited as one of kinases of phosphorylation tau, and tau is a MAP, and it acts on conjugate spirals silk (PHF) and forms---the early sign of Alzheimer.Significantly, the unusual peroxophosphoric acidization of tau is that microtubule goes the stable reason that forms with PHF.Although the multiple proteins kinases shows the phosphorylation can promote tau, find that GSK-3 phosphorylation only directly influences the ability of tau promotion microtubule self-assembly (people such as Mandelkow, 1992; People such as Mulot, 1995).About GSK-3 further evidence in this respect to the cell of overexpression GSK-3 with to the research of the transgenic mice of specific expressed GSK-3 in brain.In both of these case, GSK-3 causes producing PHF appearance epi-position tau (people such as Lucas, 2001).

GSK-3 is further related with Alzheimer through its effect in apoptosis.Insulin is neuronic survival factors, and it starts anti-apoptotic effect through activating PI3 kinases and PKB, and this true hint is transmitted the negative GSK-3 that regulates of component by these signals and promoted Neuron Apoptosis.In fact some researchs have confirmed this viewpoint, and show that GSK-3 is extremely important in life and death determine.In addition, the apoptosis function that has shown it does not rely on the PI3 kinases.GSK-3 cross expressing in the PC12 cell causes apoptosis people such as (, 1998) Pap.The activation of GSK-3 mediation migration (migration) and cell death in cerebellar granule neuron people such as (, 2001) Tong.In people's neuroblastoma SH-SY5Y cell, cross the expressing of GSK-3 promoted the inductive apoptosis of D-82041 DEISENHOFEN people such as (, 2000) Bijur.

Further illustrated the relation between the prevention that GSK-3 suppresses and pair cell is dead through some researchs; These researchs show that the expression of Frat1 (a kind of GSK-3 beta inhibitor) is enough to save neuron and avoids the inductive death of the kinase whose inhibition of PI3 (people such as Crowder, 2000).

Also detect GSK-3 and involve, be i.e. bipolar disorder and manic depression in affective disorder.This contact is based on following discovery: the main mood stabilizer lithium that is usually used in the two-phase property mental sickness is strong and special inhibitor (people such as Klein, 1996 of GSK-3 being used under the clinical treatment concentration range; People such as Stambolic, 1996; People such as Phiel, 2001).This discovery has caused carrying out a series of researchs and has confirmed the GSK-3 active forfeiture of lithium in whether can the analog cell process.In fact, lithium demonstrates and can cause the synthetic activation of glycogen (people such as Cheng, 1983); Stable and the accumulation of beta-catenin (people such as Stambolic; 1996), Xenopus laevis (Xenopus) embryo axis duplicates induces (people such as Klein, 1996); Protection (people such as Bijur, 2000) with neuronal death.Find that also another kind of mood stabilizer valproic acid commonly used is effective GSK-3 inhibitor people such as (, 1999) Chen.These researchs show that together GSK-3 is the interior target of main body of lithium and valproic acid, thereby and in the new therapeutic treatment of affective disorder, have important hint.

Play a role a kind of mechanism with the treatment bipolar disorder of lithium and other GSK-3 inhibitor is to increase the neuronic survival that receives the inductive unusual high-caliber excitement of neurotransmitter glutamate people such as (, 1998) Nonaka.People believe that also the neuronal excitability of glutamate induction is poisoned and is and acute injury the neurodegenerative main cause relevant like cerebral ischemia, traumatic brain injury and bacterial infection.In addition; Think that excessive glutamic acid signal transmission is a factor (Thomas, 1995) of the chronic nerve injury in such as Alzheimer, Huntington Chorea, parkinson disease, AIDS relevant dementia, amyotrophic lateral sclerosis (AML) and multiple sclerosis diseases such as (MS), seen.

Therefore, it is believed that the GSK-3 inhibitor be these with other neurodegenerative disorders in useful treatment.In fact, the active imbalance of recent findings GSK-3 and number of C NS disease and neurodegenerative disease comprise schizophrenia (people such as Beasley, 2001), apoplexy and Alzheimer (AD) (Bhat and Budd, 2002; People such as Lucas, 2001; People such as Mandelkow, 1992) relevant.

In view of GSK-3 involves in multiple signal pathway, think that the special inhibitor of exploitation GSK-3 is likely and important for multiple treatment intervention and basic research.

As mentioned above, find that some mood stabilizers can suppress GSK-3.Yet, suppressing GSK-3 although reported through lithium chloride (LiCl) (pct international patent application WO97/41854) and purine inhibitors (pct international patent application WO98/16528), these inhibitor are not special for GSK-3.In fact, shown the multiple signal pathway of these drug influences, and suppressed other cell target, like inositol monophosphatase (IMpase) and histone deacetylase (people such as Berridge, 1989).

Similarly, a kind of cAMP response element binding protein (CREB) of through engineering approaches---a kind of known substrate of GSK-3 has been described, and other potential GSK-3 inhibitor peptides (people such as Fiol, 1990).Yet, nominally these substrates only suppress the GSK-3 activity.

Reported other GSK-3 inhibitor recently.The micromolecule SB-216763 and the SB-415286 (Glaxo SmithKlinePharmaceutical) that are correlated with on two kinds of structures of special inhibition GSK-3 have been developed; They demonstrate can regulate glycogen metabolism and genetic transcription; And neuroprotective unit avoid the PI3 kinase activity reduce inductive death (people such as Cross, 2001; People such as Coghlan, 2000).Another research shows that the active component Induribin of the Chinese medicine that is used for chronic myelocytic leukemia is the GSK-3 inhibitor.Yet Induribin also suppresses ring-type deopendent protein kinase-2 (CDK-2) (people such as Damiens, 2001).These GSK-3 inhibitor are that ATP is emulative, and they are identified through the high flux screening to chemical library.Usually accept following viewpoint: the major defect of ATP competitive inhibitor is their limited specificity people such as (, 2000) Davies.

The general strategy of exploitation specific peptide and other GSK-3 inhibitor is reported the complete by reference this paper that incorporates into of said patent in WO01/49709 and U.S. Patent number 6,780,625 by the inventor.This generality strategy is based on the architectural feature of definition GSK-3 substrate, and according to these characteristic exploitations GSK-3 inhibitor.Yet although these publications have been described these architectural features and instructed the active multiple small peptide of effective inhibition GSK-3, they are not instructed the micromolecular design that can be used as the GSK-3 inhibitor and synthetic.With the inventor's also complete incorporating into of WO2004/052414, this patent is open by reference: the end that hydrophobic part is attached to peptide GSK-3 inhibitor has strengthened its inhibition activity.

Yet although peptide is attracting drug targets, their purposes receives biological example unstability, immunogenicity often, the ability of biomembrane such as cell membrane of passing is very low and the restriction of blood brain barrier (BBB) or the like.

Therefore, generally acknowledge that need and highly advantageously obtaining non-peptide compound is used to suppress the GSK-3 activity and does not have above-mentioned limitation.

Summary of the invention

The inventor is surprisingly found out that now the compound exhibits according to the specific characteristic design of the identification motif of the substrate of GSK-3 goes out the substrate competition inhibition of GSK-3 active; Thereby can be effective in the multiple application; During these were used, the activity that reduces GSK-3 was useful.

Thereby; According to an aspect of the present invention; A kind of chemical compound is provided; It comprises electronegative group and contains the group (amino moiety-containinggroup) of amino part with at least one; Covalently bound between them through spacerarm (spacer); This spacerarm has certain-length, structure and flexibility, select said length, structure and flexible with at least a interaction between first binding site in the catalyst structure domain that allows electronegative group and GSK-3 with contain in the catalyst structure domain of group and GSK-3 of amino part at least a interaction between second binding site, thereby this chemical compound can suppress the catalytic activity of GSK-3.

In scope of the present invention, get rid of the chemical compound of among WO2005/000192, having described.Thereby, get rid of chemical compound with general formula I I from scope of the present invention:

Formula II

Wherein:

Each is carbon atom or nitrogen-atoms independently for X, Y, Z and W;

A is J;

B is electronegative group;

D is selected from by hydrogen, alkyl, C ₁To C ₆Substituted alkyl; Tri haloalkyl; Cycloalkyl; Thiazolinyl; Alkynyl; Aryl; Heteroaryl; The heterolipid ring; Halo (halo); Hydroxyl; Alkoxyl; Aryloxy group; Sulfydryl (thiohydroxy); Alkylthio group (thioalkoxy); Arylthio (thioaryloxy); Sulfinyl; Sulfonyl; Cyanic acid; Nitro; Azo group (azo); Sulfonamide; Carbonyl; Ketone ester; Thiocarbonyl; Ester; Ether; Thioether; Thiocarbamate; Urea; Thiourea; The O-carbamyl; The N-carbamyl; The O-thiocarbamoyl; The N-thiocarbamoyl; The C-acylamino-; The N-acylamino-; The C-carboxyl; The O-carboxyl; Sulfonamido; Three halo methanesulfonamidos; Amidino groups; Amidino groups alkyl (guanyloalkyl); Guanidine radicals; Guanidine alkylation (guanidinoalkyl); Amino; Hydrazine; The group that aminoalkyl and hydrophobic part are formed; With

R ₁, R ₂, R ₃And R ₄Be selected from independently of one another by the group that contains amino part; Hydrogen; Lone electron pair; Alkyl; Tri haloalkyl; Cycloalkyl; Thiazolinyl; Alkynyl; Aryl; Heteroaryl; The heterolipid ring; Halo; Hydroxyl; Alkoxyl; Aryloxy group; Sulfydryl; Alkylthio group; Arylthio; Sulfinyl; Sulfonyl; Cyanic acid; Nitro; Azo group; Sulfonamide; Carbonyl; Ketone ester; Thiocarbonyl; Ester; Ether; Thioether; Thiocarbamate; Urea; Thiourea; The O-carbamyl; The N-carbamyl; The O-thiocarbamoyl; The N-thiocarbamoyl; The C-acylamino-; The N-acylamino-; The C-carboxyl; The O-carboxyl; Sulfonamido; Three halo methanesulfonamidos; Amidino groups; The amidino groups alkyl; Guanidine radicals; Guanidine alkylation; Amino; Aminoalkyl; The group that hydrazine and ammonium ion are formed

Perhaps its officinal salt,

And at least one of X, Y, Z and W is nitrogen-atoms and/or R ₁, R ₂, R ₃And R ₄At least one be the group that contains amino part.

According to the further feature in following the preferred embodiments of the invention, the catalytic activity that suppresses GSK-3 comprises and reduces combining of substrate and catalyst structure domain.

According to the further feature in the described preferred embodiment, first binding site comprises at least one amino acid residue of the group that is selected from arginine 180, arginine 96 and lysine 205 formations.

According to the further feature in the described preferred embodiment, second binding site comprises at least one amino acid residue of the group that is selected from aspartic acid 181, glutamic acid 97, aspartic acid 90, aspartic acid 181, glutamic acid 200, glutamine 89, tyrosine 215 and agedoite 95 formations.

According to the further feature in the described preferred embodiment, this chemical compound also comprise can with the 3rd the interactional hydrophobic part of binding site of GSK-3.

According to the further feature in the described preferred embodiment, the 3rd binding site is the part of the catalyst structure domain of GSK-3.

According to the further feature in the described preferred embodiment, the 3rd binding site comprises at least one amino acid residue of the group that is selected from isoleucine 217, phenylalanine 67 and tyrosine 215 formations.

According to the further feature in the described preferred embodiment, hydrophobic part forms the part of spacerarm.

According to the further feature in the described preferred embodiment, the length of spacerarm is that 2 dusts are in the scope of 50 dusts.

According to the further feature in the described preferred embodiment, electronegative group has formula

Wherein L is selected from the group of being made up of phosphorus atoms, sulphur atom, silicon atom, boron atom and carbon atom; Q ^*, G and D ^*Be selected from the group of forming by oxygen and sulfur independently of one another; E is selected from the group of being made up of hydroxyl, alkoxyl, aryloxy group, carbonyl, thiocarbonyl, O-carboxyl, sulfydryl, alkylthio group and arylthio or does not exist.

According to the further feature in the described preferred embodiment, L is a phosphorus, Q ^*, D ^*Each is that oxygen and E are hydroxyls with G.

According to the further feature in the described preferred embodiment, the group that contains amino part comprises at least one positively charged group.

According to the further feature in the described preferred embodiment, at least one positively charged group is selected from the group of ammonium ion and guanidinium ion formation.

According to the further feature in the described preferred embodiment, at least one positively charged group has the chemical constitution from positively charged amino acid whose side chain.

According to the further feature in the described preferred embodiment, positively charged aminoacid is selected from the group of being made up of arginine, lysine, histidine, proline and its any derivant.

According to the further feature in the described preferred embodiment, at least one group that contains amino part is selected from the group that guanidine radicals, guanidine alkylation, amino, aminoalkyl, hydrazine, amidino groups and amidino groups alkyl constitute.

According to the further feature in the described preferred embodiment, said at least one contain in the group of amino part at least one form the part of spacerarm.

According to the further feature in the described preferred embodiment, said spacerarm comprises at least one annulus.

According to the further feature in the described preferred embodiment, at least one annulus is selected from the group that alicyclic ring, aryl, heteroaryl and heterolipid ring constitute.

According to the further feature in the described preferred embodiment, spacerarm comprises at least two annulus.

According to the further feature in the described preferred embodiment, at least two annulus condense (fuse) each other.

According to the further feature in the described preferred embodiment, at least two annulus interconnect through joint (linker).

According to the further feature in the described preferred embodiment, the group that joint is selected from key, hetero atom, hydrocarbon chain and the hydrocarbon chain that interrupted by at least one hetero atom constitutes.

According to the further feature in the described preferred embodiment, chemical compound has general formula I:

B-J-(S) ₁-(S) ₂....(S)n-K-(Q)m

Formula I

Wherein:

N is 0 to 10 integer;

M is 1 to 6 integer;

B is electronegative group;

Q contains at least one in the amino group partly at least one;

J-(S) ₁-(S) ₂... (S) n-K is a spacerarm; And:

K is selected from aryl, heteroaryl, alicyclic ring, perhaps the heterolipid ring;

J and S ₁-Sn is selected from and replaces or replacement or unsubstituted hydrocarbon chain that unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted alicyclic ring, replacement or unsubstituted heterolipid ring, key, hetero atom, replacement or unsubstituted hydrocarbon chain, at least one hetero atom interrupt, does not perhaps exist.

According to the further feature in the described preferred embodiment, m is 1 to 2 integer.

According to the further feature in the described preferred embodiment, n is 0 to 2 integer.

According to the further feature in the described preferred embodiment, n is 2 and S ₁, S ₂Each is independently selected from the group of aryl and heteroaryl formation with K.

According to the further feature in the described preferred embodiment, J is a hydrocarbon chain.

According to the further feature in the described preferred embodiment, J is an alkyl.

According to the further feature in the described preferred embodiment, S ₁Be aryl, S ₂Be that heteroaryl and K are aryl.

According to the further feature in the described preferred embodiment, S ₁Be phenyl, S ₂Be that triazole and K are phenyl.

According to the further feature in the described preferred embodiment, J and S ₁Among-the Sn at least one comprises at least one group that contains amino part.

According to the further feature in the described preferred embodiment, said at least one group that contains amino part forms the part of K.

According to the further feature in the described preferred embodiment, J, (S) ₁-(S) at least one among n and the K comprises the hydrophobic part that is attached to it.

According to the further feature in the described preferred embodiment, said hydrophobic part is selected from fatty acid residue, have the saturated alkylidene chain of 4 to 30 carbon atoms and have the group that undersaturated alkylidene chain, aryl, cycloalkyl and the hydrophobic peptide sequence of 4 to 30 carbon atoms constitute.

According to the further feature in the described preferred embodiment, fatty acid is selected from the group of myristic acid, lauric acid, Palmic acid, stearic acid, oleic acid, arachidonic acid, linoleic acid plus linolenic acid formation.

According to the further feature in the described preferred embodiment, J is that hydrocarbon chain does not perhaps exist and n is 0.

According to a further aspect of the invention, pharmaceutical composition is provided, it comprises any above-claimed cpd and pharmaceutically suitable carrier as active component, and said chemical compound can suppress the activity of GSK-3.

Further feature according to following the preferred embodiments of the invention; Pharmaceutical composition is packaged in the packaging material; And be used to treat active relevant biological situation (biologicalcondition) being designated with mode of printing on the said packaging material or among the said packaging material, detail like hereinafter with GSK-3.

According to other embodiment in the described preferred embodiment, pharmaceutical composition also comprises the active at least a extra active component that can change GSK-3, details like hereinafter.

In accordance with a further aspect of the present invention; The method of the treatment biological situation relevant with the activity of GSK-3 is provided; It is through realizing its chemical compound of experimenter's administering therapeutic effective dose of needs; This chemical compound comprises electronegative group and contains the group of amino part with at least one; Connect through spacerarm between them; Wherein this spacerarm has certain-length, structure and flexibility, selects said length, structure and flexible to be suitable for allowing at least a interaction between first binding site in the catalyst structure domain of electronegative group and GSK-3 and contains in the catalyst structure domain of group and GSK-3 of amino acid moiety at least a interaction between second binding site, and is as indicated above.

According to the further feature of following the preferred embodiments of the invention, also comprise according to the method for this aspect of the present invention and at least aly can regulate the active extra active component of GSK-3 the experimenter is co-administered.

This extra active component can be can suppress the active active component of GSK-3 or can reduce the active component of the expression of GSK-3.

According to a further aspect in the invention, provide chemical compound described herein to be used to treat the purposes of the biological situation relevant with the activity of GSK-3.

According to a further aspect in the invention, provide chemical compound described herein to be used to prepare the purposes of the medicine of treating the biological situation relevant with the activity of GSK-3.

Biological situation according to the present invention is preferably selected from the group that obesity, non-insulin-dependent diabetes mellitus, insulin-dependent situation, affective disorder, neurodegenerative disease or disease and psychosis or disease constitute.

Affective disorder can be single-phase (unipolar) obstacle (for example, depression) or two-phase property (bipolar) mental disorder (for example, manic depression).

Neurodegenerative disorders can be caused by the incident that is selected from the group that following incident constitutes: cerebral ischemia, apoplexy, traumatic brain injury and bacterial infection; Perhaps it can be chronic neurodegenerative disorders, and it is caused by the disease that is selected from the group that following disease constitutes: Alzheimer, HD, parkinson disease, dementia, amyotrophic lateral sclerosis (AML) and multiple sclerosis that AIDS is relevant.

According to additional aspect of the present invention; The active method that suppresses GSK-3 is provided; Said method comprises that the cell with expression GSK-3 contacts with the chemical compound that suppresses effective dose; This chemical compound comprises electronegative group and contains the group of amino part with at least one; Between them, connect through spacerarm, wherein the length that has of this spacerarm, structure and flexible is adapted such that and between first binding site at least a interaction takes place in the catalyst structure domain of electronegative group and GSK-3 and contain in the catalyst structure domain of group and GSK-3 of amino part between second binding site at least a interaction to take place.

According to another additional aspect of the present invention, provide chemical compound described herein to be used to suppress the active purposes of GSK-3.Preferably contact with the chemical compound described herein that suppresses effective dose, realize the active inhibition of GSK-3 through the cell that will express GSK-3.

This activity can be phosphorylation activity and/or autophosphorylation (autophosphorylation) activity.

According to another additional aspect of the present invention; Provide the reinforcement insulin signaling to transmit the method for (signaling); Said method comprises the insulin response cell is contacted with the chemical compound of effective dose; This chemical compound comprises electronegative group and contains the group of amino part with at least one; Between them, connect through spacerarm, wherein the length that has of this spacerarm, structure and flexible is adapted such that and between first binding site at least a interaction takes place in the catalyst structure domain of electronegative group and GSK-3 and contain in the catalyst structure domain of group and GSK-3 of amino part between second binding site at least a interaction to take place.

According to a kind of additional aspect of the present invention, provide chemical compound described herein to be used to strengthen the purposes that insulin signaling transmits.Preferably, through the insulin response cell is contacted with the chemical compound described herein of effective dose, realize the reinforcement that insulin signaling is transmitted.

In each of these methods and/or purposes, can be external or body in realize and the contacting of cell.

According to the further feature in the preferred embodiment of following this paper, also comprise according to every kind of method of these additional aspect of the present invention and/or purposes cell is contacted with a kind of extra active component as indicated above.

The present invention is through providing newly-designed, being used to suppress the shortcoming that the active non-peptide compound of GSK-3 has successfully solved current known structure, and said chemical compound can be effective to treat multiple biological situation.

Only if definition in addition, all technology and the scientific terminology of this paper use has the identical implication with those skilled in the art's common sense according to the invention.Although can be used for practice of the present invention or test with method and material similar or that be equal to described herein, hereinafter is described suitable method and material.Situation for conflict is as the criterion with present patent application (comprising definition).In addition, material, method and embodiment only are illustrative and are not intended to qualification.

Singulative " a ", " an ", " the " that this paper uses comprise plural reference, only if context is obviously pointed out on the contrary.For example, term " chemical compound " perhaps " at least a chemical compound " can comprise multiple chemical compound, comprises its mixture.

In disclosure full text, can represent many aspects of the present invention with range format.Be to be understood that: the description of range format just to convenient with for purpose of brevity, and should not be understood that rigid qualification to the scope of the invention.Therefore, will think that the description of scope specifically discloses the single numerical value among a small circle all possible and this scope.For example, the description of scope should be considered to have among a small circle disclosed especially as 1 to 6, as 1 to 3,1 to 4,1 to 5,2 to 4,2 to 6,3 to 6 or the like, and the individual digit in this scope, as 1,2,3,4,5 and 6.This is applicable to the scope of any width.

No matter when point out digital scope in this article, it all is intended to comprise any numeral of quoting (mark or integer) in this scope of pointing out.Phrase first indicate numeral with second indicate numeral " between scope " and from first indicate numeral " to " second indicate digital " scope " interchangeable in this article use, and be intended to comprise first and second digital and all marks and integer number of pointing out between them.

The accompanying drawing summary

Here only through embodiment, the present invention is described with reference to the drawings.Now especially in detail with reference to accompanying drawing; Details shown in should be emphasized that only as an example; Be used for the illustrative discussion of the preferred embodiments of the invention, and be for provide think for the description of principle of the present invention and notion aspect the most useful with easy to understand and providing.In this respect, do not attempt than the necessary more detailed demonstration of basic comprehension of the present invention CONSTRUCTED SPECIFICATION of the present invention, this description makes with accompanying drawing how those skilled in the art obviously embody several kinds of forms of the present invention in practice.

In the accompanying drawing:

Fig. 1 a-b has provided as passing through 2D ¹The peptide p9CREB that H-NMR research obtains (Fig. 1 a) and the computer picture of the 3D structure of CREB (Fig. 1 b) (do not have the signify hydrogen atom; Carbon backbone chain is a Lycoperdon polymorphum Vitt, and nitrogen-atoms is blue, and oxygen atom is red, and phosphorus atoms is yellow);

Fig. 2 has shown based on passing through 2D ¹The 3D structure of the peptide that H-NMR research obtains, the static of p9CREB peptide distributes;

Fig. 3 a-b provided phenyl phosphate, pyridoxal 5-phosphate (P-5-P), GSC-1, GSC-2, GSC-3 and noval chemical compound GSC-4, GSC-5 and GSC-21 (Fig. 3 a) with the chemical constitution of noval chemical compound GSC-6, GSC-7, GSC-8 and GSC-9 (Fig. 3 b);

Fig. 4 a-b has provided phosphoric acid 3, the ESI-MS of 5-two (2-amino-ethyl) benzene methyl (GSC-21) (Fig. 4 a) with HPLC tomographic map (Fig. 4);

Fig. 5 has provided comparison diagram, and during it had showed that the vitro inhibition of using the PGS-1 peptide substrates is measured, the GSK-3 of phenyl phosphate, GSC-1, GSC-2, GSC-3 and pyridoxal 5-phosphate (P-5-P) suppressed active;

Fig. 6 has provided the explanation comparison diagram; It has showed that the GSK-3 of GSC-1, GSC-2, GSC-3, GSC-4 and GS-21 in vitro inhibition is measured suppresses active (the circular expression of black GSC-2; Red circular expression GSC-1; Green circular expression GSC-3, blue circular expression GSC-4, the circular expression of pink colour GSC-21);

Fig. 7 a-b has provided comparison diagram; It has been showed in the vitro inhibition of using the p9CREB peptide substrates is measured; (Fig. 7 a, the circular expression of black GSC-2, the circular expression of white GSC-1, black triangle are represented GSC-3 for GSC-1, GSC-2, GSC-3 and GSC-4; The black rectangle is represented GSC-4) and the GSK-3 of GSC-5, GSC-6 and GSC-7 (Fig. 7 b, rectangle represent that GSC-5, triangle represent GSC-6, circular expression GSC-7) suppress active;

Fig. 8 has provided Lineweaver-Burk figure; Its shown shown under the concentration GSC-7 to the inhibition of GSK-3; This is through the mix expression of phosphoric acid to p9CREB peptide substrates (CPM), and this figure confirms that GSC-7 is that (result has shown the representative experiment of of three experiments to competitive special inhibitor; Each point is the average of duplicate sample);

Fig. 9 is what to exist as substrate ³²The gel electrophoresis of the CDK-2 kinase activity of measuring when P [γ-ATP] and histone h1 is measured, and shows that GSC-4, GSC-5 and GSC-7 suppress active and do not exist;

Figure 10 is a curve chart, has showed GSC-5 (hollow circle) and GSC-7 (solid circles) to the active effect of Glycogensynthase in the C2C12 cell, and this is shown as the stimulation multiple with respect to the cell of handling with carrier (vehicle) 0.1%HCL;

Figure 11 a-b is a bar diagram, showed GSC-21 (Figure 11 b) and GSC-4 (influence that Figure 11 a) takes in the glucose of mice adipose cell, this through in the cell with GSC-4 and GSC-21 processing [ ³H] the activation multiple that mixes the cell of handling as peptide contrast (normalizing to 1 unit) of 2-deoxyglucose representes;

Figure 12 has provided the interactional computer simulation to GSC-4 and GSK-3;

Figure 13 has provided the interactional computer simulation to GSC-5 and GSK-3;

Figure 14 has provided the interactional computer simulation to GSC-6 and GSK-3;

Figure 15 has provided the interactional computer simulation to GSC-7 and GSK-3;

Figure 16 has provided the interactional computer simulation to GSC-8 and GSK-3;

Figure 17 has provided the interactional computer simulation to GSC-9 and GSK-3;

Figure 18 has provided the chemical constitution of newly-designed GSK-3 inhibitor MP-1, MP-2, MP-3, MP-4, MP-5 and MP-6; With

Figure 19 has provided comparison diagram, has showed that the GSK-3 of MP-1, MP-2, MP-3, MP-4, MP-5 and MP-6 in the vitro inhibition of using the p9CREB peptide substrates is measured suppresses active.

Preferred embodiment is described

The present invention relates to new non-peptide compound, the biological situation that it can suppress the GSK-3 activity and therefore can be used to treat the GSK-3 mediation.Particularly, the present invention relates to (i) chemical compound according to the pharmacophore coordinate design of GSK-3 substrate, it can be chosen wantonly has the hydrophobic part that connects it; The pharmaceutical composition that (ii) contains this chemical compound; (iii) use this chemical compound to be used to suppress the active method of transmitting with the reinforcement insulin signaling of GSK-3; (iv) use the method for the biological situation of this compounds for treating, said biological situation such as but not limited to, obesity, non-insulin-dependent diabetes mellitus, insulin-dependent situation, affective disorder, neurodegenerative disease and disease and psychosis or disease.

Can understand principle of the present invention and operation better with reference to accompanying drawing and appended description.

Before illustrated in detail at least one embodiment of the present invention, should be appreciated that application of the present invention is not limited to the details of illustration among following description and the embodiment.The present invention can comprise other embodiment of being put into practice or can carry out in many ways.And, should be appreciated that wording and term that this paper uses are used for explaining and should not being considered limiting.

One of parameter that acts on the identification of substrate kinases is the element that is positioned at substrate, its so-called " identification motif ".As discussed above, unlike other kinases, GSK-3 has unique identification motif, and it comprises the aminoacid sequence SX that provides among the SEQ ID NO:1 ₁X ₂X ₃S (p), wherein S is serine or threonine, X ₁, X ₂And X ₃Each is arbitrary aminoacid, and S (p) is the serine of phosphorylation or the threonine of phosphorylation.

Like generally instruction in complete introducing this paper WO01/49709 and U.S. Patent number 6,780,625 as a reference, to designing based on this identification motif and synthetic one group of small peptide has been tested their activity as substrate or inhibitor.Measure based on these, confirmed to make the many characteristics of these peptides as active substrate or the inhibitor of GSK-3.One of most important characteristic is that the serine or the threonine of phosphorylation in the motif is that substrate and inhibitor combine GSK-3 necessary.These mensuration are further illustrated: some of these peptides are the effective and special inhibitor of the height of GSK-3.With these defined polypeptides is substrate competition property inhibitor.

Only discern this discovery of substrate (promptly having the serine of phosphorylation or the substrate of threonine residues) of preparatory phosphorylation based on GSK-3, suppose that the GSK-3 of these preparatory phosphorylations has particular structure, its allow they and catalytic core interact.Also definite this unique texture of hypothesis can be developed the micromolecule of the substrate competition property inhibitor that can be used as GSK-3.

Thereby; To simulate in the active micromolecular process of inhibition of the above-mentioned little GSK-3 inhibitor peptides of this paper in searching; Along with making the present invention gradually near practice; Confirmed three dimensional structure and the particular structure characteristic of short Phosphorylated Peptide substrate, and selected the chemical compound lot that is characterized as characteristic with these, tested their activity as the GSK-3 inhibitor.

As the representative example of GSK-3 substrate, select the peptide p9CREB (ILSRRPS (p) YR, SEQ ID NO:2) of short phosphorylation in advance.Confirm corresponding non-phosphorylating peptide CREB (ILSRRPSYR, SEQ ID NO:3) through 2D NMR, as (seeing embodiment 1) is detailed in the embodiment chapters and sections below.

Shown in Fig. 1 a and 1b, the p9CREB substrate of phosphorylation in solution, have definite structure (Fig. 1 a), and corresponding to non-phosphorylating peptide CREB do not demonstrate any unique texture (Fig. 1 b).

Consider these results; Propose: the phosphate group in the Phosphorylated Peptide interacts through the cation between tyrosine (Y8) and the arginine (R4)-pi and has caused " ring-type " structure (seeing table 2 and 3); As a result, the serine of phosphorylation is positioned at the outside of ring on the identification motif.This " bending " structure of substrate makes that the serine of phosphorylation is easy to interact with the substrate binding pocket of enzyme.

Really in the crystallization data of delivering recently of the GSK-3 that people such as Dajani (2001) describe, found support to this proposal.People's such as Dajani crystallization data show that the substrate-binding site of GSK-3 comprises three positively charged residue A rg96, Arg180 and Lys205, and they and phosphate anion interact.

" surface " that Fig. 2 has provided based on the p9CREB peptide of these discoveries distributes.

Along with continuing design the present invention, release from above-mentioned discovery: the structure that will simulate the GSK-3 substrate makes that it brings into play the active micromolecule of substrate competition property inhibition will be according to following characteristic Design:

This molecule will comprise electronegative group, preferably phosphoric acid foundation group;

Electronegative group should not receive sterically hindered; And

Electronegative group will be one or two positively charged group at its both sides flank perhaps at least on one side preferably.

Based on mentioned above, designed the general formula (seeing embodiment 2) that is used to suppress the active potential chemical compound of GSK-3.Like what describe in the following embodiment chapters and sections (seeing embodiment 2); With " first generation " chemical compound; Promptly having the preliminary experiment that the chemical compound of the simple structure of this general formula carries out shows: these chemical compounds can suppress the GSK-3 activity, thereby the preliminary indication about the inhibition potentiality of chemical compound with this general formula is provided.

Also the design and based on mentioned above having synthesized more advancing monobasic chemical compound, it comprises new chemical compound (seeing for example embodiment 2).Like what describe in the following embodiment chapters and sections (seeing embodiment 3); The experiment of carrying out with these chemical compounds shows that further they can suppress the GSK-3 activity and can also strengthen the glucose uptake in the mice adipose cell, thereby shows through having brought into play inhibition likely and therapeutic effect according to the chemical compound of this general formula design.

" first generation " chemical compound family is based on single aryl or heteroaryl core (having an aryl or heteroaryl ring), the group of said core connecting band negative charge and one or two positively charged group.Also close among this paper of this family and be called the GSC molecule.

Carry out extra research to the pharmacophore binding site of the catalyst structure domain of GSK-3 with of the interaction of assessment designed molecules, thereby and will interact and will be with these binding sites are strong through combines to compete the active micromolecule of inhibition GSK-3 with substrate with further design with these binding sites.In these researchs, the protein crystallography data of the GSK-3 of people (2001) such as Ter Haar instruction are used as model.

As (the seeing embodiment 4) described in the embodiment chapters and sections below; Infer that from these researchs the catalyst structure domain of GSK-3 comprises multiple binding site; They can interact with micromolecular multiple functional group (for example, group, the hydrophobic part of electronegative and/or positive charge) simultaneously.Thereby release:, will design micromolecule and make that most (if not all) these multiple degrees of functionality will be approaching and directed suitably with binding site in order to realize and interactional maximum number of these binding sites and intensity.Thereby for example, release: will consider followingly to micromolecular design, the distance between wherein electronegative group and the one or more positively charged group is greater than the distance that obtains with single aromatic rings.

For this reason, designed and the successfully preparation (seeing embodiment 5) and the second filial generation chemical compound of having put into practice (seeing embodiment 6).Design these chemical compounds to comprise spacerarm; The group of its connecting band negative charge and one or more positively charged group; Therefore this spacerarm will have suitable length, structure and flexibility, and will allow the strong interaction of different binding sites in the catalytic core with GSK-3.The representative example of this " second filial generation " chemical compound is following such chemical compound, and wherein spacerarm is made up of three annulus, and wherein electronegative and positively charged group is placed with certain orientation each other.Like (the seeing embodiment 6) described in the following embodiment chapters and sections, the experiment of using these chemical compounds to carry out has shown the active ability of inhibition GSK-3 that they are very strong and has showed the influence (seeing for example Figure 19) of relative orientation between the functional group.

Therefore, according to an aspect of the present invention, a kind of chemical compound is provided, it comprises electronegative group and contains the group of amino part with at least one, and is covalently bound through spacerarm between them.This spacerarm is designed with certain length, structure and flexibility; Select said length, structure and flexible with at least a interaction between first binding site in the catalyst structure domain that allows electronegative group and GSK-3 with contain in the catalyst structure domain of group and GSK-3 of amino part at least a interaction between second binding site, thereby this chemical compound can suppress the catalytic activity of GSK-3.Get rid of like disclosed chemical compound the above-mentioned WO2005/000192 from this aspect of the present invention.

A zone of enzyme described in the phrase " catalyst structure domain " that this paper uses, and in this zone, catalytic reaction takes place.Therefore such part of enzyme described in this phrase, wherein participates in substrate and/or other component and the enzyme interacting of catalytic reaction.In context of the present invention, this phrase is particularly useful for being described in this part of the bonded enzyme of substrate (GSK-3) during the catalytic reaction (for example phosphorylation).This phrase therefore in this article with refer to " substrate binding pocket ", " catalytic site ", " avtive spot " or the like in this area interchangeably.

The specific site in the catalyst structure domain described in the phrase " binding site " (binding site) that this paper uses, and it comprises one or more reactive groups, can realization and the interaction of substrate and/or other component through this reactive group.Usually, binding site is made up of one or two amino acid residue, is usually included in the reactive group on these amino acid whose side chains thereby interact.As will be detailed later, during substrate combined, the conformational change of catalyst structure domain that enzyme takes place was so that the base that induces reaction is in suitable proximity and orientation, and allowed the interaction of the functional group of they and substrate.Therefore, the phrase " binding site " that uses of this paper comprises with such proximity and orientation positions to allow this interactional those amino acid residues.

The interaction of a plurality of functional groups of chemical compound and a plurality of binding sites of enzyme can for example be electrostatic interaction, interaction of hydrogen bond, hydrophobic interaction, fragrance interaction, π-accumulative facies mutual effect or the like, and this depends on participates in interactional reactive group and their property closer to each other and orientation.

Exemplary electrostatic interaction comprises that anionic-cationic interacts and soda acid interacts, like the interaction of ammonium cation and carboxylate anion.

Exemplary interaction of hydrogen bond comprises the interaction between for example oxygen, nitrogen and the sulphur atom of hydrogen and other component of amine, hydroxide or sulfydryl of one or more components.

Exemplary hydrophobic interaction comprises that two or more hydrocarbon parts are like the interaction between alkyl, cycloalkyl and the aryl.

Exemplary fragrance interacts and comprises two or more aromatics parts like the interaction between aryl and the heteroaryl, its overlapping based in the aromatization molecular orbit partly.

Exemplary pi-interacting comprises the interaction between the two or more parts (for example, unsaturated part) that contain π-electronics, its based on the part π-track in overlapping.

As preceding text simple discuss with as known in the art, play a role thereby the catalyst structure domain of substrate competition property enzyme inhibitor through desmoenzyme reduces in catalytic process, to be attached to the ratio of the enzyme molecule of enzyme.When enzyme-to-substrate or inhibitor interaction, the conformational change in the initial interaction rapid induction enzyme, its reinforcement finishes merging and makes the functional group in catalytic site and substrate or the inhibitor approaching.The reactive group that exists in enzyme-substrate/inhibitor interaction pacemaker enzyme and the substrate/inhibitor also makes them approaching each other.Substrate/inhibitor is aimed at reactive group with the combination of enzyme and is made that relevant molecular orbit is overlapping.

Therefore; Should design effective substrate competition property inhibitor; Make the reactive group of inhibitor locate near reactive group corresponding in the enzyme catalysis domain (side chain of amino acid residue usually), so that allow in catalyst structure domain, to exist the inhibitor of valid density, in addition with enough; The reactive group of inhibitor should be located with suitable direction, and is overlapping to allow.In addition, inhibitor should have restriction to its conformation motility, so that avoid influence is perhaps weakened interactional conformational change and will comprise the known interactional structural detail that relates to.

Further discuss like preceding text, the initial GSK-3 substrate of discovering has electronegative group and one or more positively charged group, and wherein electronegative group is with specific spatial orientation location.Therefore, substrate competition property GSK-3 inhibitor comprises these groups and its relative orientation.So,, should the design with suppressed agent make these functional groups will have best proximity and orientation for the reactive group of enzyme for the strong interaction between the reactive group in the catalyst structure domain that allows these functional groups and GSK-3.According to an embodiment of the present invention; Through selecting to connect the spacerarm of these functional groups; Make when with catalyst structure domain in reactive group when interacting, each functional group will have the proximity and the orientation of the best to the one or more compatible reactive group in the enzyme binding site.Therefore the suitable interval arm will have suitable length, flexibility and structure, make to allow between each functional group and the one or more compatible reactive group effective interaction aspect proximity and orientation.

As discussed above; Crystallographic research showed to GSK-3 in the past; The catalyst structure domain of GSK-3 substrate and enzyme interacts; Make one or more interactions in electronegative group (for example, phosphate radical) and arginine 180, arginine 96 and the lysine 205 (seeing people 2001 such as people such as Dajani 2001 and Ter Haar).Therefore, according to a preferred embodiment of the invention a, first binding site (it can interact with electronegative group, as above-mentioned) comprises one or more in these amino acid residues.Thereby the design chemical compound make to allow to interact between one or more in electronegative group and these amino acid residues.These preferably electrostatic interactions that interact.Yet, in the catalyst structure domain of GSK-3 with any other chemically compatible binding site of the interactional proximity of electronegative group that allows inhibitor and orientations also within the scope of the invention.

As (the seeing embodiment 4) that detail in the embodiment chapters and sections below; The interactional molecule Modeling Research of substrate competition property inhibitor shown depended on mutual proximity and orientation, contained the group of amino part and the one or more interactions in aspartic acid 181, glutamic acid 97, aspartic acid 90, aspartic acid 181, glutamic acid 200, glutamine 89, tyrosine 215 and the agedoite 95.Therefore, according to the preferred embodiments of the invention, second binding site (contain amino group can with its interaction, as above-mentioned) comprise one or more in these amino acid residues.Thereby the design chemical compound makes the interaction between one or more in the group allow to contain amino part and these amino acid residues.These interactions can be for example electrostatic interaction and/or interaction of hydrogen bond.Yet, in the catalyst structure domain of GSK-3 with allow with any other chemically compatible binding site of the interactional proximity of group that contains amino part of inhibitor and orientations also within the scope of the invention.

Although chemical compound described herein preferably comprises an electronegative group, they can comprise the group that one or more (for example two, three) are contained amino part.So second binding site described herein comprises the interactional all that reactive group of group that contains amino part with all.

Chemical compound described herein can also comprise the interactional hydrophobic part that possibly participate in chemical compound and enzyme.Hydrophobic part can be attached to spacerarm makes the length, structure and flexible of spacerarm allow it and another (the 3rd) binding site interaction of enzyme.Randomly or extraly, through selecting hydrophobic spacerarm, hydrophobic part can form the part of spacerarm self.

The interaction of hydrophobic part can be in the catalyst structure domain of enzyme and/or in another zone.Thereby, for example, in the hydrophobic band that these interactions can exist in enzyme, as reporting by people such as Dajani (2001) in the past.In another example, these interactions can be one or more interactions of amino acid residue isoleucine 217, phenylalanine 67 and tyrosine 215 in the catalyst structure domain with GSK-3.Yet, in the catalyst structure domain of GSK-3 with allow with any other hydrophobic binding site of the interactional proximity of hydrophobic part of inhibitor and orientations also within the scope of the invention.These interactions can be hydrophobic interaction, fragrance interaction and/or π-accumulative facies mutual effect or the like, and this depends on the chemical constitution and the binding site of part.Additional description to hydrophobic part provides hereinafter.

The inventor has been found that: electronegative group and the optimum distance that contains between the amino group partly are the scopes of about 2 dusts to about 50 dusts.So according to the preferred embodiments of the invention, the length of spacerarm is the scopes of about 2 dusts to about 50 dusts, more preferably about 2 dusts are to about 20 dusts, and more preferably from about 2 dusts are to about 10 dusts.Exemplary spacerarm will have the length of about 7-8 dust.

The inventor also finds the flexibility with preferred limit interval arm.So spacerarm preferably contains one or more annulus.Depend on that the functional moiety (for example in characteristic and the chemical compound of GSK-3; Electronegative group, contain group, the hydrophobic part of amino part) character; Comprise the situation of two or more annulus for spacerarm, said part can condense and/or non-condensed.When non-condensed, each of two annulus can interconnect through joint, joint as but be not limited to, key, hetero atom (for example, oxygen, nitrogen, sulfur) or hydrocarbon chain, these terms define hereinafter.Hydrocarbon chain can be chosen wantonly by one or more hetero atoms (for example, oxygen, nitrogen, sulfur or the like) and interrupt.

The having (for the situation of annulus non-condensed) and do not have (for the condensed situation of annulus) and its character depends on and allows the required flexible degree of above-mentioned interaction of joint.Thereby by the situation of limitation in height, it is preferred comprising two or more rigidity spacerarms that condense annulus for flexibility.Alternatively, the rigidity spacerarm that comprises two or more aromatics parts (aryl and/or heteroaryl) is preferred.For only need flexibility being limited to situation to a certain degree, the semi-rigid spacerarm that allows one or more keys freely to rotate is preferred.In most cases, need following spacerarm, it can interact to allow the reactive group in functional group and the binding site for chemical compound provides certain at least rotatability easily.

In a kind of preferred embodiment of the present invention, spacerarm comprises two or more, preferred three annulus, and it is covalently bound each other through key.

In each of these embodiments, each annulus can for example be isocyclic part or heterocyclic moiety.

" isocyclic part " that this paper uses described the saturated or undersaturated annulus of whole carbon, thereby " heterocyclic moiety " described saturated or undersaturated annulus, and it comprises one or more hetero atoms such as nitrogen, oxygen, sulfur, phosphorus, silicon or the like.

Isocyclic part comprises cycloalkyl and aryl, and these terms define hereinafter.Heterocyclic moiety comprises heterolipid ring and heteroaryl, and these terms define hereinafter.

Aromatic ring-shaped part (aryl and heteroaryl) is owing to have more rigidity but preferred than non-aromatic ring shape part (cycloalkyl and ring grease ring), because existence is to the restriction of conformational change in their structure.Yet the combination of fragrance and non-aromatic ring part also can allow required interaction.

Comprise the situation of the heterocyclic moiety that contains one or more nitrogen-atoms for spacerarm, this part can be used as the group that contains amino part, and it participates in the interaction with binding site.

Comprise the situation of isocyclic part and/or hydrocarbon chain for spacerarm, these parts can be used as participates in and the interactional hydrophobic and/or aromatic portion of binding site, like the preceding text discussion.

Thereby, can further select spacerarm to have permission or to limit it and the interactional structure of binding site of enzyme.Have the exemplary spacerarm that allows with the interactional structure of binding site and include but not limited to, contain hydrophobic part spacerarm, contain the spacerarm of aromatics part and contain the spacerarm of amino part (for example, heterocycle).

Yet the structure of spacerarm can further influence its flexibility, stability and possibly relate to the interactional further feature of binding site in the catalyst structure domain of chemical compound and GSK-3.

Therefore, the preferred compound of this embodiment comprises rigidity or semi-rigid spacerarm, the group that it can the connecting band negative charge.Because this structure is through providing electronegative group (it does not receive sterically hindered and has the geometry similar or identical with phosphate); And further group through allowing this negative electricity and the binding site interaction of the group that contains amino part with GSK-3; Simulate the unique texture of GSK-3 substrate; Therefore these chemical compounds can suppress the GSK-3 activity, preferably combine to suppress the GSK-3 activity through what suppress substrate and enzyme.

Phrase " electronegative group " and " positively charged group " that this paper uses refer to ionogenic group, and it has at least one negative charge or positive charge respectively when ionization (usually in aqueous medium).Charged group can perhaps be present in the chemical compound described herein as form before the ionization with their ionization form.

According to the preferred embodiments of the invention, electronegative group has formula

Wherein L is selected from phosphorus atoms, sulphur atom, silicon atom, boron atom and carbon atom; Q ^*, G and D ^*Be selected from the group of forming by oxygen and sulfur independently of one another; E is selected from the group of being made up of hydroxyl, alkoxyl, aryloxy group, carbonyl, thiocarbonyl, O-carboxyl, sulfydryl, alkylthio group and arylthio or does not exist, and these terms define hereinafter.

Preferably, electronegative group is a phosphate groups, thereby in following formula, L is a phosphorus atoms, and Q ^*, G and D ^*Each is an oxygen.Further preferably, E is a hydroxyl.Alternatively, hydroxyl also can ionization to have another minus electrostatic charge.

Alternatively, according to following formula, electronegative group can be D2EHDTPA foundation group, sulfate radical or sulfonate group, borate or boron root (boronate) group, or the like.

Electronegative group is preferably through alkyl, preferred unsubstituted alkyl and more preferably methyl be attached to spacerarm.

Electronegative group through alkyl to making that this electronegative group is the group that can rotate freely adhering to of ring, otherwise its rotatability is restricted when directly being attached to spacerarm.The rotating freely property of the group that this is electronegative is favourable, because it allows electronegative group to be easy to interact with the binding site of enzyme.

Positively charged group is preferably from the group that contains amine moiety, and it can be present in the chemical compound with ionization form (as as ammonium or guanidinium ion) or as unhindered amina (form before the ionization).

The phrase that this paper uses " group that contains amino part " refers to contain the group of one or more amino parts, perhaps refers to amine itself like this term in this paper definition.

The representative example that contains the group of amino part includes but not limited to, amine, aminoalkyl, hydrazine, urea, thiourea, amidino groups, acylamino-, carbamyl, guanidine radicals, guanidine alkylation and amidino groups alkyl, and these terms define in this article.

As as known in the art, free amino is alkaline under neutrallty condition usually, and therefore under physiological environment, it tends to protonated to produce as positively charged-NH ₃ ⁺Base.As indicated above, electronegative group flank with the binding site of relative enzyme stark right distance and orientation to have the chemical compound of one or two this type of positively charged group be preferred.

Thereby, amino part preferred in this group as protonated part easily, promptly wherein the part of amino nitrogen with part negative charge of essence exists.

Therefore, the preferred example that contains the group of amino part includes but not limited to amine, aminoalkyl, amidino groups, amidino groups alkyl, guanidine radicals, guanidine alkylation and amidino groups alkyl, and these terms define in this article.

The group that contains amino part can former state exist or exist as positively charged group in chemical compound of the present invention, and at least one in the wherein amino part is ionized.

As stated; Positively charged group according to the present invention comprises ammonium ion; Thereby the representative example of positively charged group includes, but not limited to ammonium ion itself (protonated amino) and has any group of ammonium ion; Like preceding text definition, as by the substituted alkyl of ammonium ion, guanidine radicals, amidino groups, hydrazine or the like, cycloalkyl or aryl.

Especially preferred is the positively charged group that has from the chemical constitution of positively charged amino acid side chain, like lysine, arginine, histidine, proline and its derivant, preceding two most preferably.

" from the chemical constitution of positively charged amino acid side chain " refers to have the positively charged group of or identical chemical constitution similar with this side chain.

Representative example comprises guanidine and guanidine alkylation (from arginine), amine and aminoalkyl (from lysine) and imidazoles (from histidine), and preceding two is currently most preferred.

Preferred compound can totally be represented through general formula I according to embodiments of the present invention:

B-J-(S) ₁-(S) ₂....(S)n-K-(Q)m

Formula I

Wherein:

N is 0 to 10 integer;

M is 1 to 6 integer;

B is electronegative group, as above-mentioned;

Q is as above-mentioned one of the amino group partly that contains;

J-(S) ₁-(S) ₂... (S) n-K is as above-mentioned spacerarm; And:

K is selected from aryl, heteroaryl, alicyclic ring, perhaps the group of heterolipid ring formation;

J and S ₁-Sn is independently selected from and replaces or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted alicyclic ring, replacement or unsubstituted heterolipid ring, key, hetero atom, replacement or unsubstituted hydrocarbon chain, the replacement of at least one hetero atom interruption or the group that unsubstituted hydrocarbon chain constitutes; Perhaps do not exist, these terms such as this paper define.When aryl, heteroaryl, cycloalkyl, heterolipid ring and hydrocarbon chain are substituted, substituent group can be following those substituent any.

Thereby the spacerarm in these chemical compounds comprises at least one annulus (K among the top formula I), and it adheres to 1-6 group that contains amino part.Preferably, one or two group that contains amino part is attached to annulus K, thereby the m among the following formula I is 1 to 2.Randomly, one of group that contains amino part that is expressed as the Q among the formula I above forms the part of annulus K, details like preceding text.In these situation, K is heterolipid ring or heteroaryl, preferred heteroaryl.

As known in this field, what fragrant intra-annular nitrogen-atoms will be normally alkaline under neutrallty condition, therefore under biotic environment, it tends to protonated so that produce positively charged=NH ⁺-Ji.

The extra group that contains amino part also may reside in the chemical compound.Thereby, J and S ₁In-Sn the part at least one can comprise one or more groups that contain amino part.The group that contains amino part can be attached to these parts or form its part, and is as indicated above.

Electronegative group (B among the following formula I) can directly be attached to annulus K when not being present among the following formula I (when n=0 and J).Randomly and preferably, the group B of longer spacerarm connecting band negative charge and the group Q that contains amino part, thereby J and S ₁Among-the Sn at least one is present in the spacerarm.

As discussed above, (go up J, S among the facial I through the component of selecting to form spacerarm ₁-Sn and K) number and structure, can confirm the length, structure of spacerarm and flexible so that allow the interaction of above-mentioned hope.

Thereby, for example, through selecting wherein J, S ₁Each is the spacerarm of annulus for-Sn and K, obtains the rigid structure of spacerarm.

Annulus is this spacerarm of aromatics part through selecting wherein, obtains having more rigid structural.Through selecting the condensed this spacerarm of wherein two or more annulus, further had more rigid structural, or the like.

On the other hand, through selecting wherein J and S ₁At least one of-Sn is heteroatomic spacerarm, obtains having more flexible spacerarm.Through selecting wherein J and S ₁At least one of-Sn is the spacerarm of hydrocarbon chain, further had more flexible spacerarm.Through selecting wherein J and S ₁At least one of-Sn is the spacerarm of hetero atom or hydrocarbon chain, further had more flexible spacerarm.

In another example, the number and the big or small length that has determined spacerarm of forming the part of spacerarm.For needing long spacer arm to allow required interactional situation, n is greater than 5.For the situation of the shorter spacerarm of needs, n is less than 5 and can be 0.

Like what describe in the following embodiment chapters and sections; In the molecular studies that the crystallographic data to GSK-3 based on people such as TerHaar (2001) report of incorporating this paper by reference into carries out, find that the preferred substrate binding inhibitors of GSK-3 has the spacerarm of the about 7-8 dust of length.

Based on these discoveries with to the additional result (seeing embodiment 3 and 6) that the activity of multiple chemical compound obtains, derive: preferably chemical compound comprise length be 2 dusts to 50 dusts, preferred 2 dusts are to 20 dusts, more preferably 2 dusts are to the spacerarm of 10 dusts.

Consider the average length of the 2-2.5 dust of annulus, so preferred inhibitors comprises 1-3 annulus.

Thereby in a kind of preferred embodiment of the present invention, n is 0 to 2 integer.

In another preferred embodiment, n is 2 and S ₁, S ₂Be independently selected from aryl and heteroaryl with each of K.

As discussed above, electronegative group can directly or indirectly be attached to spacerarm, the preferred latter.Thereby the J among the following formula I can not exist (when electronegative group directly is attached to spacerarm) or flexible group, and it will allow this group to be easy to interact with above-mentioned first binding site.

Thereby preferably, J is a hydrocarbon chain, like alkyl.Preferably, J is short alkyl (C ₁-C ₆Alkyl), more preferably it is a methyl.

Based on top preferable feature, designed the representative family of chemical compound and successfully prepared the representative compound of this family with following formula I.Like (the seeing embodiment 6) explained in the following embodiment chapters and sections, find that these chemical compounds (being called MP molecule or polyaryl/heteroaryl micromolecule in this article interchangeably) have high activity as the substrate competition property inhibitor of GSK-3.

So, can totally represent according to the preferred chemical compound of these embodiments of the present invention through general formula III:

B-J-(S) ₁-(S) ₂-K-Q

Formula III

Wherein B and Q such as above-mentioned, J is alkyl and S ₁, Sn and K each be aryl or heteroaryl.

Exemplary compounds in this classification is the chemical compound with top general formula, and wherein B is a phosphate radical, and J is alkyl (for example, methyl), S ₁Be aryl (for example, phenyl), S ₂Be heteroaryl (for example, triazole), K is aryl (for example a, phenyl).

The chemical constitution of the representative example of these chemical compounds provides in Figure 18.

Disclosed chemical compound among the WO2005/000192 is described below, and it is got rid of outside scope of the present invention.

These chemical compounds are totally represented through general formula I I:

Formula II

Wherein:

Each is carbon atom or nitrogen-atoms independently for X, Y, Z and W;

A is carbochain or does not have (corresponding to top J); B is like above-mentioned electronegative group;

D is selected from by hydrogen; Alkyl; Tri haloalkyl; Cycloalkyl; Thiazolinyl; Alkynyl; Aryl; Heteroaryl; The heterolipid ring; Halo; Hydroxyl; Alkoxyl; Aryloxy group; Sulfydryl; Alkylthio group; Arylthio; Sulfinyl; Sulfonyl; Cyanic acid; Nitro; Azo group; Sulfonamide; Carbonyl; Ketone ester; Thiocarbonyl; Ester; Ether; Thioether; Thiocarbamate; Urea; Thiourea; The O-carbamyl; The N-carbamyl; The O-thiocarbamoyl; The N-thiocarbamoyl; The C-acylamino-; The N-acylamino-; The C-carboxyl; The O-carboxyl; Sulfonamido; Three halo methanesulfonamidos; Amidino groups; Guanidine radicals; Guanidine alkylation; Amino; The group that aminoalkyl and hydrophobic part are formed; And

R ₁, R ₂, R ₃And R ₄Be selected from the group of forming by hydrogen, lone electron pair, alkyl, tri haloalkyl, cycloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, heterolipid ring, halo, hydroxyl, alkoxyl, aryloxy group, sulfydryl, alkylthio group, arylthio, sulfinyl, sulfonyl, cyanic acid, nitro, azo group, sulfonamide, carbonyl, ketone ester, thiocarbonyl, ester, ether, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino-, N-acylamino-, C-carboxyl, O-carboxyl, sulfonamido, three halo methanesulfonamidos, amidino groups, amidino groups alkyl, guanidine radicals, guanidine alkylation, amino, aminoalkyl, hydrazine and ammonium ion independently of one another, perhaps its officinal salt.

It will be appreciated by one of skill in the art that each substituent group (for example, D, G, E and R ₁-R ₄) be positioned at shown in locational feasibility depend on substituent quantivalence and chemical compatibility, the substituted position of institute and other substituent group.So the present invention is intended to comprise all possible substituent group of arbitrary position.

As noted above, the spacerarm in these family compounds comprises annulus, and preferably, aromatic ring (aryl) or heteroaromatic rings (heteroaryl).

In one embodiment, spacerarm is a heteroaryl, makes that at least one among X, Y, Z and the W is nitrogen-atoms in the superincumbent formula III.As discussed above, the intra-annular nitrogen-atoms of aromatics alkalescence normally under neutrallty condition, therefore under biotic environment, it tend to protonated positively charged to produce=NH ⁺-Ji.So in this spacerarm, the positively charged group that contains amino part forms the part of spacerarm.Should be preferably separated by a distance because find to contain amino group partly with electronegative group, so preferably, Z or W are nitrogen-atoms.

In another preferred embodiment, at least two among X, Y, Z and the W is nitrogen-atoms, and more preferably, one of X and Y are that nitrogen-atoms or X and W are nitrogen-atoms, even more preferably, Z and W are nitrogen-atoms.

Alternative or additional as nitrogen-atoms positively charged in the spacerarm, preferably, R ₁, R ₂, R ₃And R ₄In at least one be the group that contains amino part, as above-mentioned.

Preferably, R ₁And R ₂, perhaps R ₃And R ₄Be the group that contains amino part (for example, positively charged group).

So preferred chemical compound according to the present invention is to have following general formula I IA and those chemical compounds of IIB:

Formula IIA formula IIB

Wherein m is 1 to 6 integer; Q ₁And Q ₂Each is carbon atom or nitrogen-atoms independently; Each is the group that contains amino part (for example, positively charged group) for G and/or K.

As above-mentioned and as following embodiment in further specify, designed chemical compound lot according to above-mentioned general formula, their are synthetic by successfully, and find that they combine the high inhibition activity of performance to substrate and GSK-3.These chemical compounds also are called GS molecule, GSC molecule or single aryl/hetaryl micromolecule in this article interchangeably.The chemical constitution of these chemical compounds is shown among Fig. 3 a and the 3b.These chemical compounds provide in for example Fig. 5-7 as the effect of GSK-3 inhibitor, and they are illustrated in embodiment 9 to the specificity of GSK-3, and the beneficial effect that they are taken in glucose in the mice adipose cell is showed in Figure 11 a and 11b.

With shown in the following embodiment chapters and sections, some in the chemical compound of test do not have positively charged group (for example, GSC-1 and GSC-2) like Fig. 5-7, yet performance suppresses active to these chemical compounds to GSK-3.Yet shown in further among Fig. 5-7, discovery has nitrogen-atoms in the alkalescence ring chemical compound is more activated inhibitor, thereby has pointed out the beneficial effect of these groups.

Therefore, be chemical compound according to the extra preferred chemical compound of this embodiment according to above-mentioned general formula I I, wherein each of X, Y, Z and W is a carbon atom; R ₃And R ₄In at least one be the group that contains amino part (for example, positively charged group); D is hydrogen or alkyl.Preferred chemical compound is that wherein each of X, Y and Z is that carbon atom and W are those chemical compounds of nitrogen-atoms.

Should be noted that in this article; Although phosphate radical or any other electronegative group of the present invention directly perhaps are attached to indirectly on aromatics or the heteroaromatic rings and realize through simple steps; And produced simple chemical compound on the structure, but synthesized only a limited number of this compounds up to now.These comprise pyridoxin phosphate, phosphoric acid benzene methyl, phenyl phosphate and its a limited number of derivant (for example, substituted pyridoxin phosphate, phosphoric acid benzene methyl and phenyl phosphate).Suppose that compounds is not relevant therewith because have biological activity up to now, but those of ordinary skills there is not motivation that this compounds is provided.However, still get rid of the current compound known that following formula comprises according to the chemical compound of this method of the present invention.

Particularly, the chemical compound that has following general formula A and a B is got rid of outside the scope aspect this of the present invention:

Formula A formula B

Wherein, be that methylene does not perhaps exist for formula A:M; Each is hydrogen for Ra, Rb and T; Rc and Rd each be independently selected from hydrogen ,-CH=N-NH-C (=NH)-NH ₂,-CH ₂-N (NH ₂)-CH ₃With-CH ₂-NH-C (=NH)-group that OH constitutes,

Be methylene or do not exist for formula B:M; Each is nitrogen or carbon independently for Z and W, and at least one among Z and the W is nitrogen; Each is independently selected from hydrogen, nitro, alkyl, halo, hydroxyl and methyl Re, Rf, Rg, Rh and T.

Detail in WO2004/052404 (PCT/IL03/10157) like one of inventor of the present invention, it is active to find that hydrophobic part is attached to the terminal inhibition that strengthens this peptide of the N-of GSK-3 inhibitor peptides.Crystallographic data according to people such as Dajani people such as (2001) report; Can be owing to there be hydrophobic band in this enhanced activity in enzyme, and therefore cause the enhancing of the membrane permeability of inhibitor peptides with the interaction of this hydrophobic band and/or owing to hydrophobic tail owing to inhibitor peptides.

Because the phosphorylation residue is positioned at its C-end in the inhibitor peptides; So it is active to suppose will to bring into play enhanced inhibition according to chemical compound of the present invention; Said chemical compound comprises the hydrophobic part that for electronegative group, is positioned at the far-end distance, like the situation for inhibitor peptides.

Thereby in a kind of preferred embodiment aspect this of the present invention, every kind of chemical compound described herein has the one or more hydrophobic parts that are attached to it.Preferably, hydrophobic part is attached to spacerarm, and preferably, the following rheme of hydrophobic part is put and is attached to spacerarm, and said position makes the interaction between the binding site will allow this part and GSK-3, and is as discussed above.

Thereby, for example, according to this embodiment, in chemical compound with formula I, J, (S) ₁-(S) at least one among n and the K comprises the hydrophobic part that is attached to it.Preferably, K has the hydrophobic part that is attached to it.Alternatively or extraly, Sn has the hydrophobic part that is attached to it.

In the chemical compound of formula II on have, D is a hydrophobic part.

The phrase " hydrophobic part " that uses like this paper refers to that characteristic is hydrophobic any material or its residue.

As recognized in the art, the major part of material described in term " residue ", and it is covalently bound to another material, is the chemical compound that preceding text are described here.

Therefore, according to the hydrophobic part of the present invention residue of hydrophobic substance preferably, and preferably covalently is attached to above-mentioned chemical compound.

Hydrophobic part of the present invention can from the representative example of hydrophobic substance include but not limited to, saturated alkylidene chain, undersaturated alkylidene chain, aryl, cycloalkyl and hydrophobic peptide sequence, these terms define in this article.

Refer to the hydrocarbon linear chain like phrase described herein " alkylidene chain ", it can be saturated or undersaturated.Alkylidene chain can be to be substituted or unsubstituted, as said about alkyl among this paper, and can be further interrupted by one or more hetero atoms such as nitrogen, oxygen, sulfur, phosphorus or the like.Alkylidene chain preferably includes at least 4 carbon atoms, at least 8 carbon atoms more preferably, and more preferably at least 10 carbon atoms, and can have much 20,25 and even 30 carbon atoms.

Therefore hydrophobic part of the present invention can comprise the residue of above-described hydrophobic substance.

A preferred example according to the alkylidene chain of this aspect of the present invention is the alkylidene chain that comprises carboxyl, that is, and and fatty acid residue.

The preferred fatty acid that is suitable for use in the context of the present invention includes but not limited to; Have more than 10 carbon atoms; Saturated or the undersaturated fatty acid of preferred 12 to 24 carbon atoms; Such as but not limited to, myristic acid, lauric acid, Palmic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid or the like.

Alternatively, according to the present invention, hydrophobic part can be the hydrophobic peptide sequence.According to the present invention, the hydrophobic peptide sequence preference comprises 2 to 15 amino acid residues, more preferably 2 to 10 amino acid residues, and more preferably 2 to 5 amino acid residues, wherein at least one amino acid residue is a hydrophobic amino acid residue.

The representative example of hydrophobic amino acid residue comprises; But be not limited to; Alanine residue, cysteine residues, glycine residue, isoleucine residue, leucine residue, valine residue, phenylalanine residue, tyrosine residue, methionine residues, proline residue and trp residue; Perhaps its any modification is as indicated above.

Alternatively, hydrophobic amino acid residue can comprise any other amino acid residue, can modify it through mixing hydrophobic part.

Preferably, the hydrophobic amino acid sequence comprises at least two and at least 5 hydrophobic amino acid residues more preferably, and it more preferably interconnects, so that its continuous sequence is provided in the hydrophobic amino acid sequence.

The hydrophobic amino acid sequence (be called interchangeably in the art " the permeable sequence of film " perhaps the example of " MPS " can for example in Hagiwer (1999), find.

The phrase " amino acid residue " that this paper uses also is called " aminoacid " in this article interchangeably, and it has described the intrachain aminoacid unit of peptide.Amino acid residue in the hydrophobic peptide sequence can be natural or through modified amino acid residue, these phrases define hereinafter.

The phrase " natural amino acid residue " that this paper uses described following amino acid residue, defined at preceding text like this term, and it is included in one of 20 seed amino acids of finding in the nature.

The phrase " through modified amino acid residue " that this paper uses described following amino acid residue, defined at preceding text like this term, and it is included in the natural amino acid that its side chain is modified.This type of modification is as known in the art and for example comprises, in side chain, mixes functional group, such as but not limited to hydroxyl, amino, carboxyl and phosphate.Only if point out in addition; Therefore this phrase comprises the aminoacid of chemical modification; Comprise amino acid analogue (like penicillamine, 2-sulfydryl-D-valine), naturally occurring nonprotein property (proteogenic) aminoacid (like nor-leucine) and have the chemical compound of the chemosynthesis that is known as the aminoacid ins and outs in this area.Term " protein property " expression aminoacid can be incorporated in the protein in the cell through known metabolic pathway.

Therefore, use like this paper, term " aminoacid (or its plural form) " should be understood to include 20 kinds of naturally occurring aminoacid; Those common aminoacid of post translational modification in vivo for example comprise hydroxyproline, phosphoserine and phosphothreonine; With other uncommon aminoacid, include, but not limited to 2-aminoadipic acid, hydroxylysine, isodensmosine, norvaline and ornithine.In addition, term " aminoacid " comprises D-and L-aminoacid, and it is connected at least one additional aminoacid through peptide bond or peptide bond analog, defines in this article like this term.

Because hydrophobic part provides enhanced unpredictable activity, in the scope aspect this of the present invention, also comprise compound known, for example phenyl phosphate and pyridoxal 5-phosphate, and above-described by substituted other compound known of hydrophobic part.

Be definition below to the number of chemical part of quoting among the application.

Singly-bound, two key or triple bond described in term " key " as this paper uses.

The part of mainly being made up of carbon and hydrogen atom described in phrase " hydrocarbon chain " that this paper uses and term " hydrocarbon ".Therefore hydrocarbon chain comprises one or more in alkyl, thiazolinyl, alkynyl, cycloalkyl and the aryl, and these terms are defined in this article, and wherein each can be unsubstituted or substituted, as described herein.Hydrocarbon chain can further be interrupted by one or more hetero atoms like this paper definition.

Having described like the term " hetero atom " of this paper use is not any atom that carbon still can form covalent bonds with one or more carbon atoms.Exemplary hetero atom includes, but not limited to nitrogen, oxygen, sulfur, phosphorus, silicon, boron or the like.

The saturated aliphatic hydrocarbon that comprises straight chain and branched group described in term " alkyl " as this paper uses.Preferably, alkyl has 1 to 20 carbon atom.When digital scope is described in this article; For example when " 1-20 ", it is implied under the situation of alkyl, and this group can contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, or the like, up to and comprise 20 carbon atoms.More preferably, alkyl is the medium sized alkyl with 1 to 10 carbon atom.Most preferably, only if point out in addition, alkyl is the low alkyl group with 1 to 4 carbon atom.Alkyl can be substituted or unsubstituted.When being substituted, substituent group can be for example hydroxy alkyl, tri haloalkyl, cycloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, heterolipid ring, halo, hydroxyl, alkoxyl, aryloxy group, sulfydryl, alkylthio group, arylthio, sulfinyl, sulfonyl, cyanic acid, nitro, azo group, sulfonamide, sulfinyl, sulfonamide, ketone ester, carbonyl, thiocarbonyl, ester, ether, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino-, N-acylamino-, C-carboxyl, O-carboxyl, three halo methanesulfonamidos, amidino groups, amidino groups alkyl, guanidine radicals, guanidine alkylation, amino, aminoalkyl, hydrazine and ammonium ion.

Term " cycloalkyl " or " alicyclic ring " have been described full carbon monocycle or condensed ring (that is, the ring of total a pair of adjacent carbon atom) group, the one or more pi-electron systems that do not have total conjugated in its medium ring.The example of cycloalkyl (not limiting) is cyclopropane, Tetramethylene., Pentamethylene., cyclopentenes, cyclohexane extraction, cyclohexadiene, cycloheptane, cycloheptatriene and diamantane (obsolete).Cycloalkyl can be substituted or unsubstituted.When replacing, substituent group can be for example alkyl, hydroxy alkyl, tri haloalkyl, cycloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, heterolipid ring, halo, hydroxyl, alkoxyl, aryloxy group, sulfydryl, alkylthio group, arylthio, sulfinyl, sulfonyl, cyanic acid, nitro, azo group, sulfonyl, sulfinyl, sulfonamide, ketone ester, carbonyl, thiocarbonyl, ester, ether, carboxyl, thiocarboxyl group, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino-, N-acylamino-, C-carboxyl, O-carboxyl, sulfonamido, three halo methanesulfonamidos, amidino groups, amidino groups alkyl, guanidine radicals, guanidine alkylation, amino, aminoalkyl, hydrazine and ammonium ion.

The alkyl like the preceding text definition described in term " thiazolinyl ", and it is made up of at least two carbon atoms and at least one carbon-to-carbon double bond.

The alkyl like the preceding text definition described in term " alkynyl ", and it is made up of at least two carbon atoms and at least one carbon-to-carbon triple bond.

Term " aryl " has been described full carbon monocycle or fused rings multi-ring (that is, total several rings to adjacent carbon atom) group, and it has the pi-electron system of total conjugated.The example of aryl is (not limiting) phenyl, naphthyl and anthryl.Aryl can be replacement or unsubstituted.When replacing; Substituent group for example can be, alkyl, hydroxy alkyl, tri haloalkyl, cycloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, heterolipid ring, halo, hydroxyl, alkoxyl, aryloxy group, sulfydryl, alkylthio group, arylthio, sulfinyl, sulfonyl, cyanic acid, nitro, azo group, sulfonyl, sulfinyl, sulfonamide, ketone ester, carbonyl, thiocarbonyl, ester, ether, carboxyl, thiocarboxyl group, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino-, N-acylamino-, C-carboxyl, O-carboxyl, sulfonamido, three halo methanesulfonamidos, amidino groups, amidino groups alkyl, guanidine radicals, guanidine alkylation, amino, aminoalkyl, hydrazine and ammonium ion.

Monocycle or fused rings (that is, the ring of total a pair of adjacent atom) group described in term " heteroaryl ", and it has one or more atoms in ring, like nitrogen, oxygen and sulfur, in addition, have the pi-electron system of total conjugated.The example of heteroaryl includes but not limited to, pyrroles, furan, thiophene, imidazoles 、 oxazole, thiazole, pyrazoles, pyridine, pyrimidine, quinoline, isoquinolin and purine.Heteroaryl can be replacement or unsubstituted.When being substituted; Substituent group for example can be, alkyl, hydroxy alkyl, tri haloalkyl, cycloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, heterolipid ring, halo, hydroxyl, alkoxyl, aryloxy group, sulfydryl, alkylthio group, arylthio, sulfinyl, sulfonyl, cyanic acid, nitro, azo group, sulfonyl, sulfinyl, sulfonamide, ketone ester, carbonyl, thiocarbonyl, ester, ether, carboxyl, thiocarboxyl group, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino-, N-acylamino-, C-carboxyl, O-carboxyl, sulfonamido, three halo methanesulfonamidos, amidino groups, amidino groups alkyl, guanidine radicals, guanidine alkylation, amino, aminoalkyl, hydrazine and ammonium ion.

Monocycle or the fused rings group that in ring, has one or more atoms such as nitrogen, oxygen and sulfur described in term " heterolipid ring ".Ring can also have one or more pairs of keys.Yet ring does not have the pi-electron system of total conjugated.Alicyclic ring can be to be substituted or unsubstituted.When being substituted, substituent group can be for example lone electron pair, alkyl, hydroxy alkyl, tri haloalkyl, cycloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, heterolipid ring, halo, hydroxyl, alkoxyl, aryloxy group, sulfydryl, alkylthio group, arylthio, sulfinyl, sulfonyl, cyanic acid, nitro, azo group, sulfonyl, sulfinyl, sulfonamide, ketone ester, carbonyl, thiocarbonyl, ester, ether, carboxyl, thiocarboxyl group, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino-, N-acylamino-, C-carboxyl, O-carboxyl, sulfonamido, three halo methanesulfonamidos, amidino groups, amidino groups alkyl, guanidine radicals, guanidine alkylation, amino, aminoalkyl, hydrazine and ammonium ion.Representative example is piperidines, piperazine, oxolane, Pentamethylene oxide., morpholino (morpholino) or the like.

The electron pair of not participating in into key described in term " lone electron pair ".Only, X, Y, Z or W have lone electron pair when being unsubstituted nitrogen-atoms.

Term " hydroxyl " has been described-the OH group.

Term " hydroxyl " has been described-the N=N group.

Term " alkoxyl " described as this paper definition-O-alkyl and-O-cycloalkyl.

Term " aryloxy group " described as this paper definition-O-aryl and-O-heteroaryl.

Term " sulfydryl " has been described-the SH group.

Term " alkylthio group " described as this paper definition-S-alkyl and-S-cycloalkyl.

Term " arylthio " described as this paper definition-S-aryl and-S-heteroaryl.

Term " carbonyl " described-C (=O)-and R ' group, wherein R ' is hydrogen, alkyl, thiazolinyl, cycloalkyl, aryl, heteroaryl (through ring carbon Cheng Jian) or the heterolipid ring (through ring carbon Cheng Jian) like this paper definition.

Carbonyl described in term " aldehyde ", and wherein R ' is a hydrogen.

Term " thiocarbonyl " described-C (=S)-R ' group, wherein R ' such as this paper to R ' definition.

Term " C-carboxyl " described-C (=O)-O-R ' group, wherein R ' defines like this paper.

Term " O-carboxyl " described R ' C (=O)-the O-group, wherein R ' defines like this paper.

The C-carboxyl described in term " carboxylic acid ", and wherein R is a hydrogen.

Fluorine, chlorine, bromine or iodine described in term " halogen ".

Term " trihalomethyl group " has been described-CX ₃Group, wherein X is the halogen like this paper definition.

X described in term " three halo mesyls " ₃CS (=O) ₂-group, wherein X is the halogen like this paper definition.

Term " sulfinyl " described-S (=O)-and R ' group, wherein R ' defines like this paper.

Term " sulfonyl " described-S (=O) ₂-R ' group, wherein R ' defines like this paper.

Term " S-sulfonamido " described-S (=O) ₂-NR ' R " group, R ' is like this paper definition and R " such as to R ' definition.

Term " N-sulfonamido " described R ' S (=O) ₂-NR ", wherein R ' and R " define like this paper.

X described in term " three halo sulfonamidos " ₃CS (=O) ₂NR '-group, wherein R ' and X such as this paper define.

Term " O-carbamyl " described-OC (=O)-NR ' R " group, wherein R ' and R " define like this paper.

R described in term " N-carbamyl " " OC (=O)-and NR '-group, wherein R ' and R " define like this paper.

Term " O-thiocarbamoyl " described-OC (=S)-NR ' R " group, wherein R ' and R " define like this paper.

R described in term " N-thiocarbamoyl " " and OC (=S) NR '-group, wherein R ' and R " define like this paper.

Term " amino " has been described-NR ' R " group, wherein R ' and R " define like this paper.

Term " aminoalkyl " has been described by amino substituted alkyl like the preceding text definition.Preferably, alkyl finishes with amino.

Term " C-acylamino-" described-C (=O)-NR ' R " group, wherein R ' and R " define like this paper.

Term " N-acylamino-" described R ' C (=O)-NR " group, wherein R ' and R " define like this paper.

Term " urea " described-NR ' C (=O)-and NR " R " ' group, wherein R ' and R " like this paper definition, and R " ' like R ' or R " definition.

Term " guanidine radicals " described-R ' NC (=NR " ")-NR " R " ' group, wherein R ', R " and R " ' like this paper definition and R " " like R ', R " or R " ' definition.

Term " guanidine alkylation " has been described by the substituted alkyl of guanidine radicals, and these terms define in this article.Preferably, alkyl finishes with guanidine radicals.

R ' R described in term " amidino groups " " NC (=NR " ")-group, wherein R ', R ", R " ' and R " " define like this paper.

Term " amidino groups alkyl " has been described by the substituted alkyl of amidino groups, and these terms define in this article.Preferably, alkyl stops with amidino groups.

Term " nitro " has been described-NO ₂Base.

Term " cyanic acid " has been described-C ≡ N base.

Term " ketone ester " described-C (=O)-C (=O)-the O-base.

Term " thiourea " described-NR '-C (=S)-and NR '-Ji, R ' and R " define like preceding text.

NR '-NR described in term " hydrazine " " base, R ' and R " define like preceding text.

Term " ammonium ion " described-and (NR ' R " R " ') ⁺, wherein R ', R " and R " ' define like preceding text.

Term " guanidinium ion " described-(R ' NC (=NR " ")-NR " R " ' R " ") ⁺Base, wherein R ', R ", R " ' and R " " define like this paper.

The present invention also comprises the officinal salt of chemical compound described herein.

Phrase " officinal salt " refers to the charged species and its equilibrium ion of parent compound.The non-limitative example of officinal salt will be to comprise ammonium or guanidine cation and the cationic chemical compound of acid like HCl, trifluoroacetic acid (TFA) etc.Like what in preceding text discussion and embodiment chapters and sections below, further specify, above-mentioned every kind of chemical compound is based on the three dimensional structure design of GSK-3 substrate and is the active potential substrate competition property inhibitor of GSK-3 therefore.

So, according to a further aspect of the invention, inhibition GSK-3 being provided active method, it contacts with any above-claimed cpd that suppresses effective dose through the cell that will express GSK-3 and realizes.

The term " inhibition effective dose " that this paper uses is to consider definite amount through known in the art this type of, and it is enough to suppress the activity of GSK-3.Activity can be that phosphorylation and/or the autophosphorylation of GSK-3 is active.

Method according to this aspect of the present invention can realize through cell is contacted in external and/or body with chemical compound.This method can also realize through cell is contacted with the active extra active component that can change GSK-3, detail like hereinafter.

Active inhibition is a kind of method that increases insulin active in the body to GSK-3.Insulin action in the high activity of GSK-3 infringement intact cell people such as (, 1997) Eldar-Finkelman.This infringement is owing to GSK-3 causes the phosphorylation of IRS-1 (IRS-1) serine residue.Has type ii diabetes (non-insulin-dependent diabetes mellitus; NIDDM) research of carrying out among the patient is illustrated in the active significantly reduction of Glycogensynthase among these patients; And detect activation (people such as Shulman, (1990) of reduction of the upstream regulation agent of protein kinase B (PKB)-GSK-3; People such as Cross, (1995).The mice of inductive diabetes of high fat diet and obesity susceptible had the GSK-3 of obvious rising active people such as (, 1999) Eldar-Finkelman in epididymal adipose.The GSK-3 activity of the increase of in cell, expressing causes the active containment of Glycogensynthase (people such as Eldar-Finkelman, 1996).

Therefore, active inhibition provides the process useful that increases insulin active in the insulin-dependent situation to GSK-3.Thereby, in accordance with a further aspect of the present invention, providing and strengthened the method that insulin signaling transmits, it is realized through the insulin response cell is contacted with any above-claimed cpd of effective dose (like this paper definition).

The phrase that this paper uses " strengthen the insulin signaling transmission " and comprise the Insulin receptor INSR downstream component phosphorylation increase with not subject experimenter or cell in glucose take in the increase of comparing the glucose uptake rate.

Method according to this aspect of the present invention can realize through cell is contacted in external or body with the chemical compound of this embodiment, and can realize through further cell being contacted with insulin.

Use chemical compound described herein and in vivo the insulin signaling transmission is strengthened can be used as the clinical endpoint supervision.The easy method of in principle, keeping watch on that insulin strengthens is to carry out the glucose tolerance test.After the fasting, to the patient give glucose and through algoscopy measurement of glucose well known in the art from the disappear speed of (being that glucose is taken in by cell) of blood flow.(comparing with the health volunteer) LG clearance rate will show insulin resistance.The insulin resistance patient is used inhibitor to be compared with untreated patient and has increased glucose and take in speed.Can for a long time inhibitor be applied to the insulin resistance patient, and can measure the level (very high usually) of insulin in the blood circulation, glucose and leptin.The reduction of glucose level will show that inhibitor strengthened insulin action.Only the reduction of insulin and leptin level not necessarily shows the reinforcement of insulin action, but this will point out the improvement to this disease condition that causes through other mechanism.

Can effectively utilize above-claimed cpd to be used to treat any biological situation relevant with GSK-3.

So,, the method for the treatment biological situation relevant with the GSK-3 activity is provided according to additional aspect of the present invention.This method according to the present invention is through realizing its any above-claimed cpd of experimenter's administering therapeutic effective dose of needs.

The phrase " with the active relevant biological situation of GSK-3 " that uses like this paper comprises any biology or medical condition or disease, identifies that wherein the effective GSK-3 that is in normal or abnormal level is active.It can be characteristic with the GSK-3 activity simply that said situation or disease can cause perhaps by the GSK-3 activity.This situation can track the GSK-3 activity with active relevant certain aspect of this situation that is meant of GSK-3.

Here, term " treatment " comprises elimination, suppress in fact, slow down or the development of reverse situation or disease, and essence alleviates the appearance of clinical symptoms of clinical symptoms or the essence prevention situation or the disease of situation or disease.Reduction or neurodegenerative disorders aspect that these effects can for example show as the glucose absorption of type ii diabetes aspect stop neuronal cell death, detail like hereinafter.

The term administering that this paper uses " described chemical compound of the present invention and the cell that receives the invasion and attack of said situation or disease so that chemical compound can influence the method that the active mode of GSK-3 in these cells gathers together.Can use chemical compound of the present invention through medically acceptable any approach.Route of administration can depend on disease, situation or the damage of being treated.Possible route of administration comprises injection, passes through parenteral route; As in the blood vessel, in intravenous, intra-arterial, subcutaneous, intramuscular, the tumor, in the intraperitoneal, ventricle, in the dura mater, in the cerebrovascular or other approach, and per os, nose, eye, rectum, locally perhaps use through suction.The present invention uses particularly including continuing to discharge, as perhaps being prone to the erosion implant through the depot formulation injection.Using can also be endarterial, internal rectum, intraperitoneal, intramuscular, subcutaneous or through the aerosol inhalant.When treatment is general; Chemical compound can per os or parenteral; Use like (intracisternally) in intravenous, intramuscular, subcutaneous, the eye socket, in the capsule, in intraperitoneal or the brain pond, as long as, detail like hereinafter to be suitable for realizing that the compositions that chemical compound is imported target cell uses.

The individual amount that is applied to described in the phrase " treatment effective dose " that this paper uses; It is enough to eliminate, essence suppresses, slow down or the development of the situation that reverse is relevant with the GSK-3 activity, and essence alleviates the appearance of the clinical symptoms or the clinical symptoms that essence is prevented this situation of this situation.The GSK-3 activity can be the GSK-3 kinase activity.Can directly confirm amount of suppression to the active inhibition of GSK-3 through measuring, perhaps for example, when desirable effect be to the approach of inducing GSK-3 in the active effect in the active downstream of GSK-3, can measure this inhibition through measuring downstream effects so.Thereby; For example; When the inhibition to GSK-3 caused the prevention of phosphorylation of Glycogensynthase, the effect of chemical compound can comprise the effect to the relevant approach of insulin-dependent or insulin, and can compound administration to glucose be taken in the time point that be increased to optimum level.And, when the inhibition to GSK-3 causes the phosphorylation of required protein of other biological activity such as Protein tau not exist, can use this chemical compound so and receive substantive the inhibition up to the polymerization of the Protein tau of phosphorylation.Therefore, active inhibition will partly depend on the character that relates to active downtrod approach of GSK-3 or process to GSK-3, and depend on the active inhibition of GSK-3 the effect in given biological background.

The amount of forming the chemical compound of amount of suppression will become according to multiple parameter; Said parameter for render a service such as chemical compound and its, the development speed of half life, the disease of being treated or the biological situation of chemical compound in health, situation is to the assessment to medical condition of the response of therapeutic dose or mode of administration, preparation, the doctor in charge; The factor relevant with other; General health situation with the patient; Consider that with other like before using of other therapeutic agent, it is active or will influence therapeutic agent co-administered of the active or approach that the GSK-3 activity mediates of GSK-3 perhaps will influence the inhibition of chemical compound.The expection amount of suppression will fall into wide relatively scope, and it can be confirmed through normal experiment.

Like what go through at preceding text, GSK-3 participates in multiple biological approach and therefore, this method according to the present invention can be used to treat multiple biological situation, details like hereinafter.

GSK-3 participates in the insulin signaling pipeline and therefore, in an example, this method according to the present invention can be used to treat any insulin-dependent situation.

Because adipose cell was to the differentiation of adipose cell before known GSK-3 inhibitor suppressed, therefore, in another example, the method for this aspect of the present invention can be used for treatment of obesity.

In a further embodiment, the method according to this aspect of the present invention can be used to treat diabetes, especially non-insulin-dependent diabetes mellitus.

Diabetes are heterogeneous protopathy diseases of carbohydate metabolism, have multiple nosetiology factor, and it is usually directed to insufficient insulin or insulin resistance or both.The insulin dependent diabetes mellitus (IDDM) of I type teenager outbreak is present in and has very little endogenous insulin secretion ability or do not have among the patient of endogenous insulin secretion ability.Serious hyperglycemia takes place and relies on exogenous insulin fully to treat and keep nearest survival in these patients.II type or adult show effect or non-insulin-dependent diabetes mellitus takes place in the certain endogenous insulin secretion capacity patient of maintenance, but their great majority are perhaps insulin resistances of insufficient insulin.In the U.S., in all diabeticss about 95% be the type ii diabetes (NIDDM) of non-insulin-dependent, therefore, this is the diabetes form that accounts for most medical problems.The diabetes toleration is the potential characteristic features of NIDDM, and this metabolic deficiency causes diabetic syndrome.Insulin resistance can be because the insulin binding affinity that insufficient Insulin receptor INSR is expressed, reduced, perhaps any unusual (the seeing U.S. Patent number 5,861,266) of arbitrary step in the insulin signaling pipeline.

Chemical compound of the present invention can be used to treat the patient's with type ii diabetes type ii diabetes, as follows: to this chemical compound of patient's administering therapeutic effective dose, and detect clinical marker, like blood sugar level.Chemical compound of the present invention can also be used for preventing as follows experimenter's type ii diabetes: the patient is used the chemical compound of prevention effective dose, and monitor clinical marker, like the IRS-1 phosphorylation.

Confirm treatment of diabetes through the standard medical method.A target of treating diabetes is as far as possible safely blood glucose to be reduced near normal level.Usually the target of setting is a 80-120 milligram/decilitre (mg/dl) and the preceding 100-140mg/dl that sleeps ante cibum.Concrete doctor can set different targets for the patient, depends on other factors, has the frequency of hypoglycemic reaction like the patient.Useful medical science test comprises to be tested to confirm blood sugar level, test glycolated hemoglobin level (HbA patient's blood and urine _1cThe measurement of average blood sugar level in 2-3 month in the past, be 4-6% normal range), the test of the test of cholesterol and fat level and urine protein level.This class testing is standard testing well known by persons skilled in the art (sees, for example, AmericanDiabetes Association, 1998).Through making, also can confirm successful treatment plan in that to have diabetic oculopathy, nephropathy or neuropathic patient in the works less.

So in a specific embodiments according to the method aspect this of the present invention, the method for treatment non-insulin-dependent diabetes mellitus is provided: the patient is diagnosed at the early stage quilt of non-insulin-dependent diabetes mellitus.Chemical compound of the present invention is formulated as the intestinal capsule.The instruction patient is whenever taking a slice with stimulation insulin signaling pipeline after the meal, and therefore controls glucose level to the level of eliminating the needs of using exogenous insulin.

That further discuss like preceding text and in WO2004/052404, illustrate, that GSK-3 suppresses is relevant with affective disorder.Therefore, in another example, can be used to treat affective disorder according to the method for this aspect of the present invention, like unipolar disorders (for example, depression) and bipolar disorder (for example, manic depression).

Owing to think that also GSK-3 is the key factor in the pathogeny of neurodegenerative disorders and disease, so can also be used to treat multiple this type of disease and disease according to the method for this aspect of the present invention.

In an example, because the inhibition of GSK-3 is caused stopping neuronal cell death, so can be used to treat the neurodegenerative disorders that causes that the dead incident of neuronal cell causes according to the method for this aspect of the present invention.This type of incident can be for example cerebral ischemia, apoplexy, traumatic brain injury or bacterial infection.

In another example; Because GSK-3 is active relevant with multiple central nervous system disorders and neurodegenerative disease; So the method according to this aspect of the present invention can be used to treat multiple chronic neurodegenerative disease; Such as but not limited to, dementia, amyotrophic lateral sclerosis (AML) and multiple sclerosis that Alzheimer, HD, parkinson disease, AIDS are correlated with.

As discussed above, the GSK-3 activity is especially relevant with the pathogeny of Alzheimer (Alzheimer ' sdisease).So; In a representative embodiment according to the method aspect this of the present invention; Provide treatment to suffer from the patient's of Alzheimer method: the patient who is diagnosed as Alzheimer is used chemical compound of the present invention; It suppresses the tau hyperphosphorylation of GSK-3 mediation, to pass the formulation preparation of blood brain barrier (BBB).Through regularly on the SDS-PAGE gel, analyzing the existence of the tau of phosphorylation form from the isolating protein of patient's brain cell, the patient is detected the polymer of tau phosphorylation, the tau that suppresses said phosphorylation form characterizes the existence and the progress of said disease.The dosage that can regulate chemical compound as required exists with the Protein tau matter that reduces phosphorylation form.

GSK-3 is also relevant with mental disorder such as schizophrenia, and therefore the method according to this aspect of the present invention can be further used for treating psychosis or disease, like schizophrenia.

Can also be according to the method for this aspect of the present invention through co-administered one or more are extra, can regulate the active active component of GSK-3 realizes to the experimenter.

" co-administered " used herein (co-administering) described with extra active component (also being called activating agent or therapeutic agent in this article) combined administration according to chemical compound of the present invention.Said extra activating agent can be any therapeutic agent that is used to treat patient's situation.Co-administered can be simultaneously, for example, through using the mixture of this chemical compound and therapeutic agent, perhaps can pass through separate administration, in during the short time, uses this chemical compound and activating agent.Co-administered this chemical compound of sequential application and one or more another kind of therapeutic agents of also comprising.One or more extra therapeutic agents can be used before or after said chemical compound.Dosage treatment can be single agent scheme or multi-agent scheme.

Extra active component can be an insulin.

Preferably; Extra active component can suppress the activity of GSK-3; Thereby extra active component according to the present invention can be any GSK-3 inhibitor except chemical compound of the present invention; Like the small peptide GSK-3 inhibitor of describing in WO01/49709, WO2004/052404 and the U.S. Patent number 6,780,625.Alternatively, the GSK-3 inhibitor can be for example lithium, valproic acid and/or lithium ion.

Alternatively, extra active component can be the active component that can reduce the expression of GSK-3.

The activating agent that downward modulation GSK-3 expresses refers in the mRNA level or on protein level, influences perhaps any activating agent of degraded (acceleration) of GSK-3 synthetic (deceleration).For example, being designed for the little interference polynucleotide molecule of the expression of downward modulation GSK-3 can be as according to the extra active component of this embodiment of the present invention.

The example of little interference polynucleotide molecule that can reduce the expression of GSK-3 is siRNA or siRNA, like people such as Munshi (Munshi CB, Graeff R, Lee HC, JBiol Chem 2002 Dec 20; 277 (51): the morpholino antisense oligonucleotide of describing 49453-8); It comprises the duplex oligonucleotide, and it disturbs (RNAi) mechanism to instruct the sequence-specific degraded (Hutvagner and Zamore (2002) Curr.Opin.Genetics and Development12:225-232) of mRNA through the RNA that described in the past.

The phrase " duplex oligonucleotide " that uses like this paper refers to oligonucleotide structure or its analogies, and it forms through self complementary nucleic acid chains or through at least two complementary nucleic acid chains." duplex oligonucleotide " of the present invention can be made up of the RNA that double-stranded RNA (dsRNA), DNA RNA hybrid, single stranded RNA (ssRNA), isolating RNA (that is, partially purified RNA, pure basically RNA), synthetic RNA and reorganization produce.

Preferably, the little interference duplex of specificity of the present invention oligonucleotide is the oligonucleotide of mainly being made up of ribonucleic acid.

Generation can the interferential duplex oligonucleotide of mediate rna be taught in Www.amhion.comIn provide

So little interference polynucleotide molecule according to the present invention can be RNAi molecule (rnai molecule).

Alternatively, little interference polynucleotide molecule can be an oligonucleotide, and like GSK-3 specific antisense molecule or ribozyme molecule, it is further definition hereinafter.

Antisense molecule is an oligonucleotide, and it contains two or more chemically different zones, and each zone is made up of at least one nucleotide.These oligonucleotide contain at least one following zone usually, wherein this oligonucleotide through modifying so that giving the cell of the resistance of oligonucleotide to the increase of nuclease degradation, increase takes in, and/or to the binding affinity of the increase of target polynucleotide.The extra zone of oligonucleotide can be used as the substrate of the enzyme that can cut RNA:DNA or RNA:RNA crossbred.This example comprises RNA enzyme H (RNase H), and it is the cell endonuclease, the RNA chain of cutting RNA:DNA duplex.Therefore, the activation of RNA enzyme H is caused the cutting to the RNA target, thereby the oligonucleotide that greatly increases gene expression suppresses efficient.Therefore, when using chimeric oligonucleotide, compare, obtain suitable result usually with short oligonucleotide with deoxy-oligonucleotide thiophosphate to identical target region.Cutting to the RNA target can detect through gel electrophoresis routinely, if necessary, and in conjunction with nucleic acid hybridization technique as known in the art.

Antisense molecule of the present invention can form the composite construction of the oligonucleotide of two or more oligonucleotide, warp modification, as above-mentioned.Instruction includes but not limited to the representative United States Patent (USP) of the preparation of this type of hybrid structure, U.S. Patent number 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; With 5,700,922, each complete introducing this paper as a reference with them.

Ribozyme (rybozyme) molecule is used for through cutting mRNA sequence-specific being carried out in gene expression just gradually and suppresses.Some ribozyme sequences can merge with oligonucleotide of the present invention.These sequences include but not limited to that the ANGIOZYME specificity of VEGF-R (Vascular Endothelial Growth Factor (VEGF) receptor) (a kind of key component in the blood vessel generation approach) suppresses to form; And HEPTAZYME; It is that design is used for the ribozyme (Rybozyme Pharmaceuticals, Incorporated-WEB homepage) of selective destruction hepatitis C virus (HCV) RNA.

Also alternatively, according to the present invention, little interference polynucleotide molecule can be DNA enzyme (DNAzymes).

The DNA enzyme is a strand catalytic RNA molecule.The universal model (＂ 10-23 ＂ model) of DNA enzyme has been proposed.＂ 10-23 ＂ DNA enzyme has the catalyst structure domain of 15 deoxyribonucleotides, and its flank has two substrate recognition structure territories, and each domain is 7 to 9 deoxyribonucleotides.The DNA enzyme of the type can be at purine: its substrate RNA (Santoro, S.W.& Joyce, G.F.Proc.Natl, Acad.Sci.USA199 effectively cut at pyrimidine contact place; About DNA enzyme summary, see Khachigian, LM Curr Opin Mol Ther2002; 4:119-21).

The example of the synthetic through engineering approaches DNA of the DNA enzyme of structure and amplification identification strand and double-stranded target cleavage site is open in people's such as Joyce U.S. Patent number 6,326,174.Observe DNA enzyme to the similar design of human urokinase receptor recently and suppress urokinase receptor and express, and suppress colon cancer cell in the successful terrain and shift (people such as Itoh, 20002, Abstract409, Ann Meeting Am Soc Gen Ther Www.asgt.org).In Another Application, the complementary DNA enzyme of bcr-ab1 oncogene successfully suppresses the oncogene expression among the leukaemia, and has reduced the relapse rate in the autologous bons marrow transplantation plant for the situation of CML and ALL.

Can produce the oligonucleotide of instructing design according to the present invention like enzymatic synthesis or solid phase synthesis according to any oligonucleotide synthesis method as known in the art.Equipment and the reagent of carrying out solid phase synthesis can obtain from for example Applied Biosystems through crossing commercial sources.Can also use this type of synthetic any other method; The reality of oligonucleotide is synthesized within those skilled in the art's ability.

Although be the efficacious therapy agent; And the individual need of treatment is used effective amount of actives because treatment is used usually; So chemical compound described herein preferably is included in the pharmaceutical composition as active component, said pharmaceutical composition also comprises pharmaceutically suitable carrier to assist chemical compound using and assist perhaps cell of active component target approach tissue possibly to the individuality of being treated.

So, according to a further aspect in the invention, pharmaceutical composition being provided, it comprises any chemical compound described herein and pharmaceutically suitable carrier as active component.

Hereinafter, phrase " pharmaceutically suitable carrier " and " physiologically acceptable carrier " refer to following carrier or diluent, and it does not cause the serious stimulation to the experimenter, does not eliminate the biological activity and the character of the chemical compound of being used.The non-limitative example of carrier is the mixture of propylene glycol, saline, Emulsion and organic solvent and water.

Among this paper, term " excipient " refers to join in the pharmaceutical composition inert substance of using with further assistance chemical compound.The non-limitative example of excipient comprises calcium carbonate, calcium phosphate, multiple sugar and starch type, cellulose derivative, gelatin, vegetable oil and Polyethylene Glycol.

Pharmaceutically suitable carrier can also comprise other reagent; Such as but not limited to, absorption delay agent, antibacterial agent, antifungal, antioxidant, adhesive, buffer agent, filler, cationic liquid agent, coloring agent, diluent, disintegrating agent, dispersant, emulsifying agent, excipient, fumet, fluidizer, isotonic agent, liposome, microcapsule, solvent, sweetener, viscosity dressing agent, humidizer and skin penetration promoter (skin penetration enhancers).

The preparation and the technology of drug administration can be seen " Remington ' s PharmaceuticalSciences, " Mack Publishing Co., Easton, and PA, latest edition is incorporated it into this paper by reference.Suitable route of administration can for example comprise that per os, rectum, through mucous membrane, percutaneous, intestinal or rectum send, comprise in intramuscular, subcutaneous and intramedullary injection and the sheath, directly in the ventricle, intravenous, intraperitoneal, intranasal or intraocular injection.

Pharmaceutical composition of the present invention can be produced by means commonly known in the art, as through routine mixing, dissolving, granulation, system ingot, levigate, emulsifying, encapsulate, hold back or freeze drying process is produced.

Thereby can use one or more pharmaceutically suitable carrier to prepare according to pharmaceutical composition of the present invention with the mode of routine, said carrier comprises excipient and accessory drugs, and it is convenient to chemical compound is processed into the preparation that can pharmaceutically use.Can be with the delivery form compositions formulated, said delivery form is implant, gel, gel capsule, pellet, injection, ingestible solution, the solution that can suck, lotion, oil solution, pill, suppository, ointment, suspensoid, lasting release, syrup, tablet, tincture, local cream, dermal delivery form such as aerosol delivery form, aqueous solution, bolus injection, capsule, colloid, extended release, depot formulation, soluble powder, drop, Emulsion, easy erosion.Suitable dosage form depends on selected route of administration.

For injection, chemical compound of the present invention can be prepared with aqueous solution, preferably uses buffer such as Hank buffer, Ringer's solution or the normal saline buffer solution (contain or do not contain organic solvent like propylene glycol, Polyethylene Glycol) of physical compatibility to prepare.For mucosal administration, in preparation, use penetrating agent.This type of penetrating agent is well known in the art.

For oral administration, through chemical compound and pharmaceutically suitable carrier well known in the art combination can easily be prepared chemical compound.Examples of such carriers make chemical compound of the present invention can be formulated as tablet, pill, lozenge, capsule, liquid, gel, syrup, unguentum, suspensoid, or the like, be used for oral absorption by the patient.Being used for the pharmaceutical formulations that per os uses can prepare with solid excipients, the optional gained mixture of will milling, and processing granular mixture, if desired, add suitable accessory drugs after, obtain tablet or lozenge core.Suitable excipient is a filler especially, like sugar, comprises lactose, sucrose, mannitol or sorbitol; Cellulose preparation; Like corn starch, wheaten starch, rice starch, potato starch, gelatin, tragakanta, methylcellulose, hydroxypropyl emthylcellulose, sodium carboxymethyl cellulose and/or physiologically acceptable polymer, like polyvinylpyrrolidone (PVP).If desired, can add disintegrating agent, like crosslinked polyvinylpyrrolidone, agar, perhaps alginic acid or its salt are like sodium alginate.

For the lozenge core provides suitable coating (coating).For this purpose, can use spissated sugar juice, it can be chosen wantonly and contain arabic gum, Talcum, polyvinylpyrrolidone, carbopol gel, Polyethylene Glycol, titanium dioxide, lacquer solution and appropriate organic solvent or solvent mixture.Can in tablet or lozenge coating, add dyestuff or pigment, become the various combination of divided dose with sign or identified activity.

The pharmaceutical composition that can orally use comprises lightpressure fit formula (push-fit) capsule of being made by gelatin, and by the soft seal capsule of gelatin with plasticizer such as glycerol or sorbitol manufacturing.Lightpressure fit formula capsule can contain active component and filler, like lactose, and adhesive such as starch, lubricant such as Talcum or magnesium stearate and the mixture of stabilizing agent randomly.In soft capsule, active component can dissolve or be suspended in suitable liquid, in fatty oil, liquid paraffin or liquid macrogol.In addition, can add stabilizing agent.All preparations that are used for oral administration will be in the dosage that is suitable for selected route of administration.

Use for sucking, compositions can adopt the tablet of preparation in a usual manner or the form of lozenge.

For using through suction, chemical compound of the present invention can be sent with the form from the spray of the packing of supercharging or aerosol apparatus, uses suitable propellant, like dichlorodifluoromethane, Arcton 11, dichlorotetra-fluoroethane or carbon dioxide.For the aerocolloidal form of supercharging,, the valve of sending measured quantity can confirm dosage unit through being provided.The for example gelatine capsule and the cartridge case that are used for inhaler or insufflator can be mixed with the mixture of powders that contains said composition and suitable powder substrate (like lactose or starch).

Can prepare chemical compound described herein and be used for parenteral administration, for example, through bolus injection or continuous infusion.Injection preparation can exist with unit dosage forms, for example, is present in ampoule or the multi-dose container its optional antiseptic with interpolation.Compositions can be suspensoid, solution or the Emulsion that is in oiliness or the aqueous carrier, and can contain preparation and use reagent, as suspending, stablizing and/or dispersant.

The pharmaceutical composition that is used for parenteral administration comprises the aqueous solution of the water-soluble form of chemical compound.In addition, can the suspension of chemical compound be mixed with suitable oily injection suspensoid.Suitable lipophilic solvent or carrier comprise fatty oil, and like Oleum sesami, perhaps synthetic fatty acid ester is like ethyl oleate, triglyceride or liposome.The aqueous injection suspension can contain the material of the viscosity that increases suspensoid, like sodium carboxymethyl cellulose, sorbitol or glucosan.Randomly, suspensoid can also contain suitable stabilizers or increase the material of the dissolubility of active component with permission compounding high concentration solution.

Alternatively, chemical compound can be powder form, uses suitable carriers before using, for example, and aseptic, no pyrogen water preparation.

Chemical compound of the present invention can also be formulated as rectal compositions, like suppository or enema,retention, for example uses, and conventional suppository base is like cocoa butter or other glyceride.

Pharmaceutical composition described herein can also comprise the suitable solid of gel phase carrier or excipient.The example of examples of such carriers or excipient includes, but not limited to calcium carbonate, calcium phosphate, multiple sugar, starch, cellulose derivative, gelatin and polymer, like Polyethylene Glycol.

The pharmaceutical composition that is suitable for use in the context of the present invention comprises such compositions, and the amount of wherein contained chemical compound can effectively realize intended purposes.More specifically, the treatment effective dose refers to effectively to influence the amount of chemical compound of the symptom of situation or the experimenter's that prolongation is treated survival.

The treatment effective dose is fixed within those skilled in the art's limit of power really, especially when provide according to this paper disclose in detail the time.

For any active component that is used for the inventive method, can treat effective dose or dosage according to a preliminary estimate from the activation measurement cell culture and/or the animal.This type of information can be used for confirming more accurately at the useful dosage of human body.

Dosage can become according to used dosage form and used route of administration.Can by each doctor according to patient's situation select definite dosage form, route of administration and dosage (for example see that Fingl waits the people, 1975, in ＂ The Pharmacological Basis of Therapeutics ＂, Ch.1p.1).

If desired, compositions of the present invention may reside in packing or the dispenser device, and in the test kit like the FDA approval, it can contain the one or more unit dosage forms that comprise said chemical compound.Packing can for example comprise metal or plastic foil, like blister package (blisterpack).Packing or dispenser device can also attach uses description.Packing or allotter can also attach the bulletin of following container, and it is the form of government organs' regulation of management production of medicine, use or sale, and this this mechanism of bulletin reflection has ratified said composition form or people or veterinary and used.This type of bulletin for example can be that food and drug administration's approval is used for the label of prescription drugs or the product inset of approval.Can also prepare the compound compositions of the present invention that comprises with compatible pharmaceutical carrier preparation, be placed in the suitable containers, and labelling be used to treat pointed situation.The suitable situation of on label, pointing out for example comprises, above listed and GSK-3 active relevant any biology of situation.

So pharmaceutical composition of the present invention can be packaged in the packaging material, and with printing means be identified on the packaging material or within, be used to treat or situation biology that prevention is relevant with GSK-3.

Pharmaceutical composition of the present invention also can comprise can regulate the active extra active component of GSK-3, as indicated above.

When the embodiment below the inspection, additional object of the present invention, advantage and novel feature will will become apparent to those skilled in the art that these embodiment are not intended to qualification.In addition, describe like preceding text with embodiment part in require each of of the present invention a plurality of embodiments and aspect of protection all to find the experiment support among below the embodiment.

Embodiment

With reference now to following embodiment,, they illustrate the present invention with top description with non-limiting mode.

Material

By Genemed Synthesis Inc. (San Francisco, CA) synthetic peptide.

Buy active material from Amersham Ltd.

Obtain phenyl phosphate and pyridoxal 5-phosphate (being also referred to as P-5-P in this article) from Sigma (Israel).

Only if point out in addition, all other reagent and dissolving all from commercial source (for example, Sigma, Acros Aldrich) obtains, and uses by that kind that is provided.

According to method as known in the art synthetic GSC-1, GSC-2 and GSC-3, detail like hereinafter.

It is said to press hereinafter, and design is also implemented synthesizing noval chemical compound GSC-4, GSC-5, GSC-6, GSC-7 and GSC-21.

It is said to press hereinafter, and design is also implemented synthesizing noval chemical compound MP1-MP6.

Embodiment 1

Confirm the 3D structure of GSK-3 and based on its Structure Calculation through the NMR spectrographic method

The peptide of test:

Confirm graphic little Phosphorylated Peptide (being called p9CREB) and two kinds of extra peptides behind the known GSK-3 substrate CREB: a kind of unphosphorylated peptide 9CREB and variant (S wherein ¹With glutamic acid (thinking that it simulates charged group) alicyclic ring) 9ECREB, be used for listing in these researchs and the table 1 below.

Table 1

Peptide	Sequence	SEQ?ID?NO：
			p9CREB	ILSRRPS(p)YR	2
9CREB	ILSRRPSYR					3
			9ECREB	ILSRRPEYR
	4

Time course analysis to the peptide phosphorylation of GSK-3 confirms: the peptide p9CREB that has only phosphorylation is the substrate of GSK-3, and 9CREB and 9ECREB fully can not be by the GSK-3 phosphorylations, thereby the serine of pointing out phosphorylation once more is the GSK-3 absolute demand.

Measure the 3D structure of the peptide of test:

Step below using is passed through 2D ¹H NMR spectrographic method carry out to p9CREB (Fig. 1 a), the mensuration of the 3D structure of 9CREB (Fig. 1 b) and 9ECREB (not shown):

Through freeze dried powder dissolution is being contained 10%D ₂The solution for preparing every kind of peptide in the water of O.On Bruker Advance DMX spectrogrph with 600.13MHz's ¹The H proton frequency obtains 2D-NMR spectrum.Carrier frequencies is arranged on the water signal and it suppresses through the low-power radiation through the WATERGATE method or at relaxation period.Optimization experiment temperature (280 ° of K) because quick exchange causes under the temperature of more multi-ring border colony is average, and keeps most probable spectral resolution to reduce.Carry out all experiments and with the actual t of spectrum width, 4K of 12ppm with phase sensitive mode (TPPI or States-TPPI) ₂Data point and 512t ₁-incremental record.The two dimension of collecting comprises TOCSY that the incorporation time of use MLEV pulse train and 150msec is carried out and the NOESY experiment of carrying out with 100 to 750msecs incorporation time with the check figure certificate.Usually, relaxation delay was respectively 1.5 and 2.0 seconds in TOCSY and NOESY experiment.In ROESY measured, the persistent period of spin-lock (spin-lock) was set to 400msec, and power is 3.4KHz.All spectrum are all calibrated tetramethylsilane.

Use Bruker XWINNMR software (Bruker Analytische Messtechnik, GmbH, version 2 .7) process data.All processed, calculating and analysis are all carried out on SiliconGraphics work station (INDY R4000 and INDIGO2 R10000).Use the apodization of the free induction decay of skew squared sinusoidal window function on zero padding and two dimensions of indirect dimension, carry out Fourier transformation then with enhanced spectrum resolution.Automatic multinomial baseline correction through Application of B ruker exploitation is carried out further phasing to spectrum.

According to the order distribution method of W ü thrich exploitation,, be based on the distribution of resonating of the OCSY that measures under the same experimental conditions and NOESY spectrum with Bruker software program AURELIA (Bruker Analytic GmbH, version 2 .7).

The NOE distance limit obtains from the NOESY spectrum at the 450msec record.Through comparing the NOE signal intensity and the incorporation time that from 100msec to 750msec, changes in the serial experiment, for p9CREB peptide sample is confirmed this best mixing time.Selected incorporation time provides maximum NOE accumulation, does not spread the remarkable contribution of (spin diffusion) from spin.This value is used for unphosphorylated analog experiment, so that keep identical experiment condition.The peak volume of integration is used r ^-6Dependency changes into distance limit, and with between two adjacent protons of tyrosine aromatic rings 2.47

Known distance be used for the calibration.Will limit classified as strong (1.8-2.5 ), moderate (1.8-3.5

) and weak (1.8-5.0

).With 0.5

empirical calibration join the restriction upper limit that relates to methyl.

Use XPLOR (version 3 .856) to come computation structure through the annealing of hybridization distance geometry-dynamics simulation.Introduce the NOE energy as square-well potential, the constant force constant is 50Kcal/mol ²Simulated annealing by following 1500 the 3fsec steps of 1000K be cooled among the 300K 3000 1fsec steps and form.At last, use the conjugate gradient energy minimization method of 4000 iteration to minimize structure.(Molecular Modeling System version97.0, Molecular Simulations Inc.) are used for showing and analyzing the deutero-structure of NMR to INSIGHTII.Use their quality of PROCHECK assessment.

The result:

Following table 2 and 3 has provided the structure coordinate data, and they are used for the input structure analysis software to show the 3D structure.

The gained 3D structure that from Fig. 1 a and 1b, provides, only observing, the peptide of phosphorylation has definite structure conformation.Shown in Fig. 1 a, for p9CREB, phosphorylation has caused remarkable " corner " of peptide main chain, makes that Tyr8 and Arg4 are approaching, and forms " circulus ", thereby the residue of phosphorylation stretches out from ring.This conformation makes the minimize interference of positively charged residue (Arg4 and Arg5) Yu catalyzed combination pocket of enzyme on the one hand, makes the serine of phosphorylation easily near enzyme on the other hand.This structural analysis provides the explanation to unique substrate identification of GSK-3.Therefore, to simulating the micromolecular design of this structure that provides here, the method for the selective depressant that obtains GSK-3 is provided.

Embodiment 2

The chemosynthesis of single aryl/hetaryl GSK-3 inhibitor

(GSC micromolecule)

Instrument data:

Proton, carbon, fluorine and phosphorus NMR spectrum obtain from Bruker AMX500 spectrogrph or Brucker AV300 spectrogrph, and with ppm (δ) report.Tetramethylsilane (TMS) is as the spectrographic interior mark of proton, and phosphoric acid is as the interior mark of phosphorescence spectrum, and solvent peak is as carbon and the spectrographic reference peak of fluorine.

On Finnigan LCQ Duo LC-MS ion trap electrospray ionisation (ESI) mass spectrograph, obtain mass spectrum.

Use the Analtech silica gel plate to carry out thin layer chromatography (TLC), perhaps show through flat board being dyeed with the 0.2 wt% 1,2,3-indantrione monohydrate in the butanols through ultraviolet (UV) line.

By Quantitative Technologies, (Whitehouse NJ) carries out elementary analysis to Inc..

Use Hypersil BDS C18 post, 4.6 * 150mm, 5 μ m at room temperature also use the detector of operation under 220nm to obtain the HPLC analysis, use standard solvent gradient program as mobile phase, as follows:

Time (minute)	Flow velocity (ml/min)	％A	％B
				0.0	1.0	100	0.0
4.0	1.0	100	0.0
				20.0	1.0	92.0	8.0
21.0	1.0	100	0.0
				22.0	1.0	100	0.0

0.1%TFA in the A=water

0.1%TFA in the B=acetonitrile

The preparation of GSK-3 inhibitor:

Based on 3D structure determination mentioned above, designed and successfully prepared a series of micromolecule, as follows as the potential selective depressant of GSK.

A. phosphoric acid right-methylbenzene methyl ester (GSC-2) synthetic:

To describing in the synthetic general way of the GSC-2 scheme 1 below.

Scheme 1

Di(2-ethylhexyl)phosphate-tert-butyl group. to the preparation of methylbenzene methyl ester: with 1H-tetrazolium solution (0.45M in the acetonitrile, 20ml, 9mmol; 3 equivalents) join (0.4 gram of 4-methylbenzyl alcohol among the anhydrous THF (3ml) with portion; 3.3mmol, 1.1 equivalent) and phosphoramidite. di-t-butyl diisopropyl ester (0.95ml, 0.83 gram; In the solution of stirring 3mmol, 1 equivalent).Mixture was stirred 15 minutes at 20 ℃.Cooling mixture adds 85% m-chloro benzoic acid (mCPBA) (0.81g in the 1ml dichloromethane, 4mmol, the 1.3 equivalents) solution in the dichloromethane (4ml) fast to-40 ℃ (through dry ice/acetonitrile), and reaction temperature remains on below 0 ℃ simultaneously.Let solution be warmed up to room temperature, after 5 minutes, add 10%NaHSO 20 ℃ of stirrings ₃Aqueous solution (10ml) carries out stirring in 10 minutes to mixture again.Extract mixture and abandon water with ether (70ml).Use 10%NaHSO ₃Aqueous solution (2x20ml), 5% saturated NaHCO ₃Aqueous solution (2x20ml) washing ether phase, dry and filtration on sodium sulfate.Evaporate organic filtrating and, use EtOAc/ hexane 1:15 as eluent through purification residue on silicagel column, obtain product (the di(2-ethylhexyl)phosphate tert-butyl group. to the methylbenzene methyl ester) with the mixture of raw material, it is not further purified and directly use.

¹H?NMR(200MHz，CDCl ₃)：δ＝7.22(m，4H，Ar)，4.93(d，J＝7.22Hz，2H，CH ₂O)，2.33(s，3H，CH ₃)，1.46(s，18H，OtBu).

¹³C?NMR(50.4MHz，CDCl ₃)：δ

137.7(Ar)，137.0(Ar)，129.0(Ar)，127.7

82.3(c)，68.3(CH ₂O)，29.8(OtBu)，21.1(CH ₃).

³¹P?NMR(81.3MHz，CDCl ₃)：δ -9.4ppm.

Phosphoric acid is to the preparation of methylbenzene methyl ester: at 20 ℃ to the gained di(2-ethylhexyl)phosphate tert-butyl group. the methylbenzene methyl ester adds 4M in the HCl diox, 2ml, 8mmol, 2.6 equivalents) with the solution of diox (6ml), and keep watch on through TLC and to react.In case hydrolysis is accomplished, with regard to the reduction vaporization diox, and residue is dissolved in the water (15ml), wash with ether (2x15ml) and remove excessive benzyl alcohol raw material.Solvent evaporated under reduced pressure then, under the long-time high vacuum dry, the oil that gained is transparent slowly becomes colorless solid, obtains 0.18g (30%) end-product.

¹H?NMR(200MHz，D ₂O)：δ＝7.27(d，J＝8.1Hz，2H，Ar)，7.19(d，J＝8.1Hz，2H，Ar)，4.82(d，J＝7.0Hz，2H，CH ₂O)，2.26(s，3H，CH ₃).

¹³C?NMR(50.4MHz，D ₂O)：

＝138.7(Ar)，

129.2? 128.0 68.0

20.1

³¹P?NMR(81.3MHz，D ₂O)：δ 0.6ppm.

B. phosphoric acid benzene methyl (GSC-1) is synthetic:

Describe in the general way scheme 2 below of synthetic GSC-1.

Describe in the general synthetic scheme 2 below of phosphoric acid benzene methyl:

Scheme 2

The di(2-ethylhexyl)phosphate tert-butyl group. the preparation of benzene methyl: with 1H-tetrazolium solution (0.45M in the acetonitrile, 20ml, 9mmol; 3 equivalents) join benzyl alcohol (34ml among the anhydrous THF (3ml) with portion; 3.3mmol, 1.1 equivalent) and phosphoramidite. di-t-butyl diisopropyl ester (0.95ml, 0.83 gram; In the solution of stirring 3mmol, 1 equivalent).Mixture was stirred 15 minutes at 20 ℃, be cooled to-40 ℃ (through dry ice/acetonitrile) afterwards.Add fast 85%mCPBA (in 1ml dichloromethane 0.81g, 4mmol, the 1.3 equivalents) solution of dichloromethane (DCM) in (4ml), reaction temperature remains on below 0 ℃ simultaneously.Let solution be warmed up to room temperature and 20 ℃ stir 5 minutes after, add 10%NaHSO ₃Aqueous solution (10ml) continued to stir the mixture 10 minutes.Extract mixture and abandon water with ether (70ml).Use 10%NaHSO ₃Aqueous solution (2x20ml), 5% saturated NaHCO ₃Aqueous solution (2x20ml) washing ether phase, dry and filtration on sodium sulfate.Evaporation organic layer, and come the purification residue through on silicagel column, carrying out chromatography wherein uses EtOAc/ hexane 1:15 as eluent, obtain product (the di(2-ethylhexyl)phosphate tert-butyl group. benzene methyl) with the mixture of raw material, it is not further purified and directly use.

¹H?NMR(200MHz，CDCl ₃)：δ＝7.36(m，5H，Ar)，4.99(d，J＝7.33Hz，2H，CH ₂O)，1.46(s，18H，OtBu).

³¹P?NMR(81.3MHz，CDCl ₃)：δ -9.3ppm.

The preparation of phosphoric acid benzene methyl: at 20 ℃ to the gained di(2-ethylhexyl)phosphate tert-butyl group. benzene methyl adds 4M in the HCl diox, 2ml, 8mmol, 2.6 equivalents) with the solution of diox (6ml), and keep watch on through TLC and to react.In case hydrolysis is accomplished, with regard to the reduction vaporization diox, and residue is dissolved in the water (15ml), wash with ether (2x15ml), remove excessive benzyl alcohol raw material.Solvent evaporated under reduced pressure then, under long-time high vacuum dry, the oil that gained is transparent slowly becomes colorless solid, obtains 0.17g (30%) end-product.

¹H?NMR(200MHz，D ₂O)：δ＝7.34(m，5H，Ar)，4.82(d，J＝7.09Hz，2H，CH ₂O).

³¹P?NMR(81.3MHz，D ₂O)： ＝0.7ppm.

C. phosphoric acid 3-pyridyl methyl ester (GSC-3) is synthetic:

Describe in the general way scheme 3 below of synthetic phosphoric acid 3-pyridyl methyl ester:

Scheme 3

The preparation of di(2-ethylhexyl)phosphate-tert-butyl group .3-pyridyl methyl ester: with 1H-tetrazolium solution (0.45M in the acetonitrile, 20ml, 9mmol; 3 equivalents) join 3-pyridine radicals methanol (0.31ml, 3.3mmol, 1.1 equivalent) and phosphoramidite among the anhydrous THF (3ml) with portion. di-t-butyl diisopropyl ester (0.95ml; 0.83 gram; In the solution of stirring 3mmol, 1 equivalent), and mixture stirred 15 minutes at 20 ℃ afterwards.Cooling mixture adds 85%mCPBA (0.81g among the 1ml DCM, 4mmol, the 1.3 equivalents) solution among the DCM (4ml) fast to-40 ℃ (through dry ice/acetonitrile), and reaction temperature remains on below 0 ℃ simultaneously.Let solution be warmed up to room temperature and 20 ℃ stir 5 minutes after, add 10%NaHSO ₃Aqueous solution (10ml), restir mixture 10 minutes.Extract mixture and abandon water with ether (70ml).Use 10%NaHSO ₃Aqueous solution (2x20ml), 5% saturated NaHCO ₃Aqueous solution (2x20ml) washing ether phase, dry and filtration on sodium sulfate.Evaporate organic filtrating and come the purification residue, use CHCl through on silicagel column, carrying out chromatography ₃/ hexane 1:1 obtains the mixture of product di(2-ethylhexyl)phosphate tert-butyl group .3-pyridyl methyl ester and raw material as eluent, and it is not further purified and directly uses.

¹H?NMR(200MHz，CDCl ₃)：δ＝8.52(d，J＝1.56Hz，1H，Ar)，8.51(dd，J＝4.83Hz，J＝1.53Hz，1H，Ar)，7.80(m，1H，Ar)，7.33(dd，J＝7.44Hz，J＝4.62Hz，1H，Ar)，4.94(d，J＝6.83Hz，2H，CH ₂O)，1.46(s，18H，OtBu).

³¹P?NMR(81.3MHz，CDCl ₃)：

＝-9.4ppm.

A preparation of phosphoric acid 3-pyridyl methyl ester: add HCl to gained di(2-ethylhexyl)phosphate tert-butyl group .3-pyridyl methyl ester at 20 ℃! (6ml) is with the solution of diox (6ml), and keeps watch on reaction through TLC with diox for 4M in the diox, 2ml, 8mmol, 2.6 equivalent).In case hydrolysis is accomplished, with regard to the reduction vaporization diox, and residue is dissolved in the water (15ml), wash with ether (2x15ml).Solvent evaporated under reduced pressure obtains 0.19g (30%) end-product then.

1H?NMR(400MHz，D ₂O)：δ＝8.72(t，J＝0.81Hz，1H，Ar)，8.62(d，J＝5.71Hz，1H，Ar)，8.51(d，J＝8.27Hz，1H，Ar)，7.96(dd，J＝8.15Hz，J＝5.94Hz，Ar)，5.04(d，J＝8.23Hz，2H，CH ₂O).

³¹P?NMR(162MHz，D ₂O)：

＝0.76ppm.

D. phosphoric acid 3,5-two (2-amino-ethyl) benzene methyl (GSC-21) synthetic:

The general strategy (describing in the scheme 4 below) of design and the synthetic GSC-21 of practice and the purification schemes of end-product.Obtain phosphoric acid 3,5-two (2-amino-ethyl) benzene methyl (GSC-21) through synthetic gross production rate of eight steps with 5%.Also prepared corresponding trifluoroacetate.

Scheme 4

Benzyl alcohol intermedium (square case 4) is accredited as: from the raw material 1,3 of cheapness, 5-benzenetricarboxylic acid trimethyl is with available key intermediate species of four steps, as describing in hereinafter detailed description and the scheme 5.

Scheme 5

According to Johns (Tetrahedron Lett.1988,29, method 2369-2372) is in the presence of tetrazolium, through with the alcohol and the phosphoramidite tert-butyl group. the diisopropyl ester reaction imports the phosphoric acid part.Do not separate the gained phosphite ester through right-chloro benzylhydroperoxide (mCPBA) oxidation immediately and obtain corresponding phosphate ester.Use trifluoroacetic acid under controlled condition, to realize the whole removing protection of amine and phosphate ester.Material obtains with its trifluoroacetate then.Spent ion exchange resin processing behind its recrystallization is obtained the purpose product, and it has the enough purity of about 90% (AUC of HPLC) usually, as describing in the following scheme 6.

Scheme 6

Synthetic detailed description when following:

1,3, the preparation of 5-three (hydroxymethyl) benzene: under nitrogen atmosphere, to be equipped with the suspention agitator, application of sample funnel and reflux condenser 3 liters of round-bottomed flasks pack into lithium aluminium hydride (49.7 the gram, 1.31mol) with anhydrous THF (500ml).The gained suspension slowly is heated to backflow, and is dissolved in 1,3 of anhydrous THF (1.0 liters) to wherein dripping, (100.0 grams, solution 0.40mol) keeps gentle reflux (3 hours) to 5-benzenetricarboxylic acid trimethyl simultaneously.Under refluxing, the gained grey suspension was stirred extra 7 hours, in inner ice-water bath, cool off then.Through dripping water (50ml, 45 minutes),, add 15%NaOH (50ml, slowly liquid stream) then, and finally add more water (150ml, slow-flowing stream) and come the excessive lithium aluminium hydride of hydrolysis.With the gained suspension stirring at room 14 hours.The filtering solid also will be filtrated at the concentrated water white oil that obtains of fine vacuum, and its slow curing obtains 1,3, and 5-three (hydroxymethyl) benzene (62.3 grams, 94%) is white solid. ¹H NMR with ¹³C NMR is consistent with the structure of distribution.

¹H?NMR(500MHz，d ⁶-DMSO)：δ＝4.48(d，J＝5.5Hz，6H)，5.14(t，J＝6Hz，3H)，7.14(s，3H).

¹³C?NMR(500MHz，d ⁶-DMSO)：δ＝62.97(CH ₂O)，122.92(Ar)，141.96(Ar).

The preparation of 3,5 two (bromomethyl) benzyl alcohol: 2 liters of round-bottomed flasks to being equipped with magnetic stirring bar and application of sample funnel pack 1,3 into, and 5-three (hydroxymethyl) benzene [33.7 grams, 0.20mol) and anhydrous acetonitrile (750ml).Add bromo trimethyl silyl (TMSBr) 979.0ml to the gained suspension with slow liquid stream under stirring, 0.60mol).White serosity overstrike and heavy-gravity.Then reactant mixture is heated to 40 ℃, 25 minutes, obtains settled solution.(the 90:10 methylene chloride is through UV colour developing, initial substance R through the TLC analysis _f0.07, product R _f0.77) judge to react and accomplish.Decompression removes to desolvate down and obtains the brown pastel.Through column chromatography purification (silica gel, 0-5%MeOH/CH ₂Cl ₂) crude material.Obtain 3,5-two (bromomethyl) benzyl alcohol (33.5 grams, 57%) is white solid. ¹H NMR with ¹³C NMR spectrum is consistent with the structure of distribution.

¹H?NMR(500MHz，d ⁶-DMSO)：δ＝4.49(s，2H，CH ₂O)，4.69(s，4H，CH ₂Br)，7.34(s，2H，Ar)，7.38(s，1H，Ar).

¹³C?NMR(500MHz，d ⁶-DMSO)：δ＝62.19(CH ₂O)，127.06(CH ₂Br)，128.18(CH ₂Br)，138.14(Ar)，143.63(Ar).

3, the preparation of 5-two (cyano methyl) benzyl alcohol:

1 liter of round-bottomed flask to being equipped with suspention agitator, application of sample funnel and reflux condenser packs 3 into, and 5-two (bromomethyl) benzyl alcohol (33.1 grams, 0.11mol) and methanol (400ml).The settled solution that obtains is heated to backflow.The Cyanogran. (16.2 grams, the 0.33mol) solution that slowly add water-soluble (25ml).Continue heating 6 hours under refluxing, (the 95:5 methylene chloride is through UV colour developing, initial substance R through TLC analytical judgment reaction completion then _f0.62, product R _f0.38).Reaction mixture is to room temperature, and removal of solvent under reduced pressure obtains the brown pastel.(6 * 100ml) grind with MTBE with it.The MTBE extract is merged and removal of solvent under reduced pressure.Through column chromatography purification (silica gel, 0-5%MeOH/CH ₂Cl ₂) yellow oil of gained.Obtain 3,5-two (cyano methyl) benzyl alcohol (18.7 grams, 89%) is light yellow oil, and it slowly becomes wax shape white solid.Take out the 1g sample and obtain the sample of purification through column chromatography purification. ¹H NMR with ¹³C NMR spectrum is consistent with the structure of being distributed.

¹H?NMR(500MHz，d ⁶-DMSO)：δ＝4.06(s，4H，CH ₂CN)，4.52(s，2H，CH ₂O)，7.20(s，1H，Ar)，7.27(s，2H，Ar).

¹3C?NMR(500MHz，d ⁶-DMSO)：δ＝62.20(CH ₂O)，127.06(CH ₂CN)，125.14(CH ₂CN)125.86(CH ₂CN)，131.79(Ar)，144.24(Ar).

3; The preparation of 5-two (amino-ethyl) benzyl alcohol: with 3,5-two (cyano methyl) benzyl alcohol (8.0 grams, sample 0.04mol) is divided into three parts; In packing 500-ml Parr bottle into independent by every part of 2.5-3.0g, add ethanol (100ml) and NaOH aqueous solution (1.2g in the 5ml water) then.In gained solution, add Raney Ni (suspension in 50% water, 1.2 grams).Mixture on the Parr agitator with 30psi hydrogenation.Through ¹H NMR keeps watch on reaction and after 3 hours, judges and accomplish.Filtering catalyst is also with ethanol (200ml) washing diatomite layer on diatomite layer.To merge from the filtrating of all third-order reactions, solvent evaporated under reduced pressure obtains 3,5-two (amino-ethyl) benzyl alcohol, and it is brown pastel (14.16g).Product ¹H NMR spectrum shows the ethanol that has 25% (w/w).When sample is dry for a long time in fine vacuum, do not observe the remarkable change of ethanol content.This material is not further purified and is used for directly that next step is synthetic.

3; The preparation of 5-two (tert-butoxycarbonyl amino-ethyl) benzyl alcohol: in the 3l that is equipped with magnetic stirring bar, thermometer and gas inlet attack three neck round-bottomed flasks, packing into is dissolved in 3 of HF (590ml), 5-two (amino-ethyl)-1-hydroxymethyl benzyl alcohol (29.4 gram) and 2N NaOH aqueous solution (590ml).(59.4 restrain, 272mmol) to the disposable adding bicarbonate of stirred mixture di tert butyl carbonate.Heating blends to 45 ℃, 4 hours.Remove volatile organic matter with gained solution cool to room temperature and through vacuum.Add methanol (600ml) to the gained aqueous mixtures.The solution that stirs is heated to 45 ℃, 2 days, so that selective hydrolysis carbonic acid uncle fourth oxygen ester moiety and expose carbamate.Behind the cool to room temperature, the volatile matter of removing solution is also with chloroform (3 * 600ml) aqueous phase extracted mixture.Merge organic layer, with saline (600ml) washing and concentrated.After the high vacuum dry, the productive rate with 67% obtains bullion 3,5-two (tert-butoxycarbonyl amino-ethyl) benzyl alcohol (24.88 gram). ¹H NMR spectrum is consistent with the structure of being distributed.Thick material is not further purified and directly uses.

Shielded phosphoric acid 3; The preparation of 5-two (2-amino-ethyl) benzene methyl: the 500ml round-bottomed flask to being equipped with magnetic stirring bar and application of sample funnel packs 3 into; 5-two (tert-butoxycarbonyl amino-ethyl) benzyl alcohol (2.3 grams, 0.0058mol) and anhydrous methylene chloride (45ml).Cooling gained solution is to about 5 ℃ in ice-water-bath.Be dissolved in the phosphoramidite di-t-butyl the anhydrous acetonitrile (45ml) from the application of sample funnel with slow liquid stream adding. the solution of diisopropyl ester.Slowly add tetrazolium (1.0 grams, 0.0144mol) solution in the 1:1 mixture (90ml) of anhydrous acetonitrile/anhydrous methylene chloride then.Stirred the gained white suspension 1 hour at about 5 ℃, (95:5 chloroform/isopropyl alcohol is through the colour developing of dyeing with ninhydrin, initial substance R through TLC analytical judgment reaction completion then _f0.23, product R _f0.30).Removal of solvent under reduced pressure obtains pastel, is dissolved in the anhydrous chloroform (75ml) it and cooling in dry ice/acetonitrile is bathed.Disposable adding CPBA (1.3 grams, the 0.0144mol) solution in anhydrous methylene chloride.Stirred the gained mixture 1 hour, and be allowed to warm to room temperature (1 hour) and continue and stirred 30 hours.Use 1.0M sodium thiosulfate solution (100ml) and saturated sodium bicarbonate (2 * 100ml) continuous washing reactant mixtures then.Organic extract is also filtered with anhydrous sodium sulfate drying.Removal of solvent under reduced pressure obtains raw phosphoric acid salt, and it is a yellow oil, then it is passed through column chromatography purification (silica gel, 0-5%MeOH/CH ₂Cl ₂).Obtain shielded phosphoric acid 3,5-two (2-amino-ethyl) benzene methyl (2.1 grams, 61%) is heavy-gravity water white oil.Product ¹HNMR, ¹³C NMR with ³¹P NMR spectrum is consistent with the structure of being distributed.

¹H?NMR(500MHz，CDCl ₃)：δ＝1.43(s，18H，NH-t-Boc)，1.48(s，18H，OP(＝O)O-t-Boc)，2.77(t，4H，Ar-CH ₂CH ₂)，3.36(m，4H，CH ₂NH)，4.59(s，2H，CH ₂O)，4.95(d，2H，NH)，7.20(s，1H，Ar)，7.04(d，1H，Ar)，7.27(s，2H，Ar).

³¹P?NMR(500MHz，CDCl ₃)：δ＝-9.69ppm.

Phosphoric acid 3, the preparation of 5-two (2-amino-ethyl) benzene methyl tfa salt: to the 250ml round-bottomed flask that is equipped with magnetic stirring bar pack into shielded phosphate ester (2.9 grams, 0.0049mol), anhydrous methylene chloride (30ml) and trifluoroacetic acid (30ml).The gained settled solution was stirring at room 3 hours.Through ¹H NMR with ³¹The reaction of P NMR analytical judgment finishes.Removal of solvent under reduced pressure obtains heavy-gravity orange oil, it is dissolved in the methane (7.5ml) and stirs down add diethyl ether (500ml), causes the deposition of product.The gained serosity stirring at room 1 hour, is allowed solid precipitation then.Pour out settled solution from the top also with ether (2 * 100ml) grinding products.Let solid sedimentation and pour out settled solution at every turn.Product finally in vacuum drying oven 55 ℃ of dryings 108 hours, extra 192 hours of 65 ℃ of dryings, obtain phosphoric acid 3 then, 5-two (2-amino-ethyl) benzene methyl tfa salt (1.78 grams, 71%) is white solid. ¹H NMR, ¹³C NMR with ³¹P NMR spectrum is consistent with the structure of being distributed. ¹H NMR spectrum is illustrated in and has 8.5% (w/w) ether in the product.Prove that this ether is very difficult to remove and is accredited as this material hygroscopic.

³¹P?NMR(500MHz，CDCl ₃)：δ＝1.14ppm.

MS：m/z(％)＝275.22(100，[C ₁₁H ₁₉N ₂O ₄P+H] ⁺)，549.43(80)，276.23(60)，408.24(40).

Phosphoric acid 3; The preparation of 5-Bis (2-amino-ethyl) benzene methyl (GSC-21): under the nitrogen atmosphere; Add 3 in the anhydrous methylene chlorides (1 liter) to being equipped with the boost 5l three neck round-bottomed flasks of isostatic application of sample funnel of power and gas inlet attack of suspension mechanical agitator, thermometer, 1; 5-two (tert-butoxycarbonyl amino-ethyl) benzyl alcohol (24.88 grams, 63.15mmol) solution.With ice/brine bath reaction mixture.Through the application of sample funnel of isostasy, with reaction temperature remain on 6 ℃ speed adds and is dissolved in the phosphoramidite di-t-butyl in the anhydrous acetonitrile (1 liter). diisopropyl ester (49.8ml, 157.9mmol).(the 0.45M solution in the 351ml acetonitrile 157.9mmol), and adds with the speed that temperature remains on 6 ℃ of substrates through the application of sample funnel of isostasy with anhydrous acetonitrile (150ml) and anhydrous methylene chloride (500ml) dilution tetrazolium.After add accomplishing, be placed in the cryostat flask and stirred reaction mixture 1 hour.Bathe the cooling flask to-35 ℃ through dry ice/acetonitrile then.3-chloro-peroxy benzoic acid in the disposable adding anhydrous methylene chloride (500ml) (18.4 grams, 82.1mmol).Let mixture be warming up to room temperature, stirred afterwards 2 hours.Pour solution into Na ₂S ₂O ₃(20 gram) and K ₂CO ₃In the aqueous solution of (50 gram).The pH of gained two-phase mixture is 11.Stir after 15 minutes, vacuum is removed volatile organic matter also with chloroform extraction water layer (4 * 750ml).With the organic layer that dried over mgso merges, filter and the concentrated yellow oil (69 gram) that obtains.Through column chromatography (silica gel, MTBE/ heptane, 6:4) the thick material of purification.Merge the mixing fraction that contains product, concentrate and obtain light yellow oil (32.6 gram).28.6g oil is dissolved in the dichloromethane (287ml, 10 volumes), in 1 liter of round-bottomed flask of pack into the application of sample funnel that is equipped with the 500ml isostasy and magnetic stirring bar.Application of sample funnel through isostasy adds trifluoroacetic acid (287ml, 10 volumes) fast.Stirred gained solution 5 hours.Concentrate and after the fine vacuum dried overnight, obtain condensed orange oil (37.88 gram).In the methanol (90 volume) that residue is dissolved in the water (57ml, 1.5 volumes) and dropwise adding is stirred, obtain precipitate.Stir after 30 minutes, allow solid sedimentation 1 hour, pour out liquid.Vacuum is removed remaining liquid, obtains the 13.72g solid.With substance dissolves in the water (68ml, 5 volumes) and be loaded in the Dowex 50WX8-200 ion exchange resin (137 gram).Water (550ml, 40 volumes) washing pillar.Use 3:1MeOH/NH ₄OH aqueous solution (2 liters, 145 volumes) eluted product.Concentrating under reduced pressure ethanol fraction obtains pale solid.Solid is dissolved in the minimum water and joins in the methanol (40 volume) of stirring.Through filtering collecting precipitation thing and vacuum dried overnight.Water (7 volume) grinds the gained powder.After filtration and the high vacuum dry, obtain end product (2.0 gram) and be white powder.Concentrated filtrate and water grind residue (5 volume).Filter and after fine vacuum concentrates, obtain after-crop product [0.9 gram).Merge two batches of products, mixed 10 minutes.Obtain phosphoric acid 3,5-two (2-amino-ethyl) benzene methyl (GSC-21) is a white powder.Product ¹H NMR, ³¹P NMR with ¹³C NMR spectrum is consistent with the structure of being distributed.

¹H?NMR(500MHz，D ₂O)：δ＝2.93(t，J＝7Hz，Ar-CH ₂CH ₂)，3.28(m，CH ₂NH)，4.66(s，CH ₂O)，4.75(t，J＝8.5Hz，NH)，7.09(s，1H，Ar)，7.24(s，2H，Ar).

¹³C?NMR(500MHz，D ₂O)：δ＝32.56，40.43，65.6，126.74，128.47，137.48，140.54.

³¹P?NMR(500MHz，D ₂O)：δ＝3.97ppm.

The molecule peak value that the mass spectrum of the end-product that in Fig. 4 a, provides is illustrated in m/z is 275 [C ₁₁H ₁₉N ₂O ₄P+H] ⁺The HPLC tomographic map that in Fig. 4 b, provides (using said method A to obtain) has shown 96.7% product purity.End-product is characterized by non-hygroscopic.

Use identical strategy, synthetic as follows new chemical compound GSC-4 and GSC-5:

E. phosphoric acid 3-(guanidine radicals methyl) benzene methyl (GSC-5) is synthetic:

Describe in the general synthetic scheme 7 below of GSC-5 as its trifluoroacetate:

Scheme 7

GSC-5 (tfa salt)

The preparation of 3-(amino methyl) benzyl alcohol: the THF solution with 3-(hydroxymethyl) benzonitrile under the vigorous stirring slowly adds LiAlH ₄In the reflux solution of THF, remain under the nitrogen.Solution in the heating of spending the night down that refluxes, is slowly dripped water afterwards and reacts (up to H with cancellation ₂Further emit not obvious).Decompression is evaporated THF down and is added ether/acidifying water.Abandon the ether phase.Wash water and abandon organic facies with ether.Add NaOH and reach pH7 up to the pH of water.With THF extraction solution 3 times, use MgSO ₄Dry and reduction vaporization obtains light yellow residue, through chromatography on silicagel column it is further purified, and uses from ethyl acetate to begin the gradient eluent with the mixture end of 1:1 ethyl acetate MeOH, and the productive rate with 40% obtains intermediate product.

1H?NMR(200MHz，d ⁶-DMSO)：δ＝7.16-7.27(m，4H，Ar)，4.47(s，2H，CH ₂O)，3.94(bs，2H，NH ₂)，3.72(s，2H，CH ₂NH ₂).

¹³C?NMR(50.4MHz，CDCl ₃)δ＝143.1，142.8，128.2，125.9，?125.7，124.9，63.3，45.6.

The preparation of 3-(N, N '-two-BOC-guanidine radicals methyl) benzyl alcohol: at room temperature (N, N '-two (tert-butoxycarbonyl)-S-methyl isothiourea and the solution stirring of triethylamine in dry DMF are spent the night to 3-(amino methyl) benzyl alcohol, 3-.Add ether/aqueous mixtures then and separate organic layer, use the extracted with diethyl ether water layer simultaneously.With the organic extract that water washing merges, use MgSO ₄Dry also reduction vaporization.Through flash chromatography purification crude product, wherein use from hexane to begin the ethyl acetate with 1:5: the gradient eluent that the mixture of hexane finishes, productive rate is 85%.

¹H?NMR(200MHz，CDCl ₃)：δ＝11.51(bs，1H)，8.56(bs，1H)，7.22-7.38(m，4H)，4.70(s，2H)，4.65(d，J＝5.1Hz，2H)，1.52(s，9H)，1.48(s，9H).

¹³C?NMR(50MHz，CDCl ₃)：δ＝155.9，153.1，141.5，137.7，128.9，127.0，126.4，126.2，65.0，45.0，28.2，27.9.

The preparation of di(2-ethylhexyl)phosphate tert-butyl group .3-(N, N '-two-BOC-guanidine radicals methyl) benzene methyl: the 3-in anhydrous THF (3ml) (N, N '-two-BOC-guanidine radicals methyl) benzyl alcohol (1 equivalent) and phosphoramidite di-t-butyl. isopropyl ester (1.42ml; 1.24 gram; 4.5mmol, 1.5 equivalents) through the disposable adding 1H-of agitating solution tetrazolium solution (0.45M in the acetonitrile, 20ml; 9mmol, 3 equivalents).Stirred the mixture 30 minutes at 20 ℃, be cooled to-40 ℃ (through dry ice/acetonitrile) then.Add 85%mCPBA (1.25 grams are dissolved in 1.5ml DCM, 6.15mmol, the 2.0 equivalents) solution among the DCM (4ml) fast, keep reaction temperature to be lower than 0 ℃ simultaneously.Let solution be warmed up to room temperature, stirred afterwards 20 minutes, add 10%NaHSO ₃Aqueous solution (10ml) also carries out 5 minutes extra stirring to mixture.Extract mixture and abandon water with ether (70ml).Ether is used 10%NaHSO mutually ₃Aqueous solution (2x20ml) and saturated NaHCO ₃Aqueous solution (2x20ml) washing is with dried over sodium sulfate and filtration.Evaporate organic filtrating and on silicagel column, passed through the chromatography purification residue, use the gradient eluent of ethyl acetate/hexane 1:9 to 1:5, obtain the mixture of phosphate product and benzyl alcohol raw material, it is further purified through chromatography on silicagel column, uses CHCl ₃: MeOH30:1 obtains pure di(2-ethylhexyl)phosphate tert-butyl group .3-(N, N '-two-BOC-guanidine radicals methyl) benzene methyl to the gradient eluent of 20:1, and productive rate is 70%.

¹H?NMR(200MHz，CDCl ₃)：δ＝11.52(bs，1H)，8.53(bs，1H)，7.25-7.35(m，4H)，4.99(d，J＝7.2Hz，2H)，4.63(d，J＝5.1Hz，2H)，1.51(s，9H)，1.47(s，27).

³¹P?NMR(162MHz，CDCl ₃)：δ＝-9.3.

The preparation of the trifluoroacetate of phosphoric acid 3 (guanidine radicals methyl) benzene methyl: 25% trifluoroacetic acid (TFA) solution among the DCM is added 20 ℃ di(2-ethylhexyl)phosphate tert-butyl group .3-(N, N '-two-BOC-guanidine radicals methyl) benzene methyl, and stirred reaction mixture 18 hours.Solvent evaporated under reduced pressure and TFA afterwards are dissolved in residue in the water and with ether and wash.Solvent evaporated under reduced pressure obtains pure product then, and productive rate is 60%.

¹H?NMR(200MHz，D ₂O)：δ＝7.25-7.35(m，4H)，4.84(d，J＝7.2Hz，2H)，4.37

(s，1H).

³¹P?NMR(162MHz，CDCl ₃)：δ＝0.85.

¹⁹F?NMR：δ＝-76.6.

Use method well known in the art, remove or, obtain free guanidine for example through HCl displacement trifluoroacetic acid, perhaps for example, the hydrochlorate of this chemical compound.

F. phosphoric acid 3-guanidine radicals benzene methyl (GSC-4) is synthetic:

Described generality synthetic in the following scheme 8 as the GSC-4 of its trifluoroacetate:

Scheme 8

GSC-4 (tfa salt)

The preparation of 3-(N, N '-two-BOC-guanidine radicals) benzyl alcohol: with N, N '-two (tert-butoxycarbonyl)-S-methyl isothiourea (1.32 grams, 4.4mmol; 1.1 equivalent), mercuric chloride (1.22 grams, 4.4mmol; 1.1 equivalent) and the solution of triethylamine (1.72ml, 12mmol, 3 equivalents) add 3-aminobenzene methanol (0.5 gram in the anhydrous dimethyl formamide (DMF); 4mmol, 1.0 equivalent) in, stirring at room reactant mixture 5 hours.Afterwards with ether/water extraction mixture, and with saturated NH4Cl aqueous solution and brine wash organic layer.Use the extracted with diethyl ether water layer.With the dry ethereal solution that merges of MgSO4, and reduction vaporization.On silica gel, through hurried (flash) chromatography purification crude product, use hexane to the 40:60 ethyl acetate: the gradient eluent of hexane, obtain said intermediate product, productive rate is 60%.

¹H?NMR(200MHz，CDCl ₃)：δ＝11.60(brs，1H)，10.30(brs，1H)，?7.11-7.55(m，4H)，4.65(s，2H)，1.49(s，9H)，1.48(s，9H).

¹³C?NMR(400MHz，CDCl ₃)：δ＝171.4，163.4，153.7，142.0，136.7，129.0，123.4，121.4，120.8，65.8，64.7，28.1，27.9.

The preparation of di(2-ethylhexyl)phosphate tert-butyl group .3-(N, N '-two-BOC-guanidine radicals) benzene methyl: 3-in anhydrous THF (3ml) (N, N '-two-BOC guanidine radicals) benzyl alcohol (1 gram; 2.8mmol, 1 equivalent) and the phosphoramidite di-t-butyl. diisopropyl ester (1.13ml, 3.6mmol; 1.3 disposable adding 1-H-tetrazolium solution (0.45M in acetonitrile equivalent); 18.4ml, 8.3mmol, 3 equivalents).Mixture was stirred 30 minutes at 20 ℃, be cooled to-40 ℃ (through dry ice/acetonitrile) then.Add 85%mCPBA (0.85 gram is dissolved in 1.5ml DCM, 4.20mmol, the 1.5 equivalents) solution among the DCM (4ml) fast, keep reaction temperature to be lower than 0 ℃ simultaneously.Let reactant be warmed up to room temperature, stirred afterwards 20 minutes, add 10%NaHSO ₃Aqueous solution (10ml) stirred extra 10 minutes mixture.With ether (50ml) extraction mixture, and abandon water.Use 10%NaHSO ₃Aqueous solution (2x20ml) and saturated NaHCO ₃Aqueous solution (2x20ml) washing ether phase is used MgSO ₄Dry also filtration.Evaporating solvent also passed through the chromatography purification residue on silicagel column, use the gradient eluent of ethyl acetate/hexane 10:90 to 30:70, obtained shielded product, and productive rate is 60%.

¹H?NMR(200MHz，CDCl ₃)：δ＝11.65(brs，1H)，10.40(brs，1H)，7.10-7.64(m，4H)，4.97(d，J＝7.0Hz，2H)，1.49(s，18H)，1.45(s，18H).

¹³C?NMR(400MHz，CDCl ₃)：δ＝153.3，136.6，129.2，124.3，122.1，121.3，67.9，δ29.8，28.0.

³¹P?NMR(200MHz，CDCl ₃)：δ＝-9.3.

The preparation of phosphoric acid 3-guanidine radicals benzene methyl: under 20 ℃, 25%TFA (1.5ml) solution among the DCM (4.5ml) is added the di(2-ethylhexyl)phosphate tert-butyl group-3--(N, N '-two-BOC guanidine radicals) benzene methyl (0.3 gram, 0.54mmol, 1 equivalent), and stirred reaction mixture 18 hours.Solvent evaporated under reduced pressure and TFA afterwards, and residue is dissolved in the water, wash with ether.Decompression (freeze dryer) evaporating solvent obtains pure product, and productive rate is 40% (C ₁₀H ₁₃F ₃N ₃O ₆P; The Mw=359.2 gram/mol).

¹H?NMR(200MHz，CDCl ₃)：δ＝7.13-7.38(m，4H)，4.83(d，J＝7.6Hz，2H)

¹³C?NMR(400MHz，CDCl ₃)：δ＝156.3，139.5，134.3，130.1，126.8，125.3，124.6，66.4.

³¹P?NMR(200MHz，CDCl ₃)：δ＝0.8.

Use method well known in the art, remove or, obtain free guanidine for example through HCl displacement trifluoroacetic acid, perhaps for example, the hydrochlorate of this chemical compound.

G. phosphoric acid 3-(GE) benzene methyl (GSC-6) is synthetic:

To as describing in the synthetic scheme 9 below of the generality of the GSC-6 of its trifluoroacetate:

Scheme 9

The preparation of 3-(cyano methyl) essence of Niobe: vigorous stirring 3 (bromomethyl) essence of Niobe under the room temperature (0.6 gram, 2.6mmol), (0.35 gram is 5.2mmol) with the mixture of 60mg18-crown-6 in the 6ml acetonitrile 24 hours for potassium cyanide.Filtering mixt, filtrating is concentrated to half volume, adds entry and extracts with dichloromethane to remaining mixture.With the dry organic facies of MgSO4, and evaporating solvent, obtain yellow liquid product, its not purified and directly use.

TLC(30％EtOAc/Hexanes)；R _f＝0.45.

¹H?NMR(200MHz，DMSO-d ₆)：δ＝7.96(m，1H)，7.93(m，1H)，7.62(m，1H)，7.55(m，1H)，3.88(s，3H)，3.82(s，2H)；

¹3C?NMR(50MHz，DMSO-d ₆)：δ＝166.4，133.5，132.8，130.8，130.1，129.2，129.0，119.6，52.8，22.7.

The preparation of 3-(2-amino-ethyl) benzyl alcohol: (4 grams of 3-(cyano methyl) essence of Niobe under nitrogen atmosphere in the anhydrous THF of 5ml; 0.023mol, 1 molar equivalent) and solution, add THF (60ml; 5 molar equivalents) and (MeO) 2MLiBH among the 3B (3ml, 1 molar equivalent) ₄Stirred reaction mixture and 70-75 ℃ the heating 16-18 hour.After the cooling, evaporation THF, and with 2N H ₂SO ₄Add residue (different souring soln is to pH1-2).Wash gained solution with ether, and add Powdered K to water ₂CO ₃Reach 7 up to pH.Add NaCl then, and use the THF extraction solution.Use MgSO ₄Dry organic facies, and evaporating solvent obtain 2.3g (67%) pure products, its not purified direct use.

¹H NMR (200MHz, methanol-d ₄): δ=7.46-7.32 (m, 4H), 4.60 (s, 2H), 3.18-3.07 (m, 2H), 2.98-2.90 (m, 2H);

¹³C NMR (50MHz, methanol-d ₄): δ=141.9,136.8,128.2,127.2,126.8,125.3,63.5,40.7,33.7.

The preparation of 3-(N, N '-two-Boc-guanidine radicals methyl) benzyl alcohol: with 3-(amino-ethyl) benzyl alcohol (2.3 grams, 0.015mol, 1 molar equivalent); N, N '-two (tert-butoxycarbonyl)-S-methyl isothiourea (4.8 grams, 0.016mol, 1.1 molar equivalents); Mercuric chloride (4.5 grams, 0.016mol, 1.1 molar equivalents); And mixture under room temperature nitrogen the stirred overnight of triethylamine (7ml, 0.048mol, 3 molar equivalents) in dry DMF (25ml).Add ethyl acetate (100ml) then, and use the diatomite filtration suspension.Water, saturated NH ₄Cl aqueous solution and brine wash filtrating, dry and solvent evaporated under reduced pressure on MgSO4.Through flash chromatography purification crude product (use EtOAc: the 10:90 of hexane is to the gradient eluent of 30:70 mixture), obtain 1.9 gram (32%) pure products.

¹H?NMR(200MHz，CDCl ₃)：δ＝11.4(bs，1H)，8.28(bs，1H)，7.23-7.09(m，4H)，4.59(s，2H)，3.61(q，J＝6.9Hz，2H)，2.80(t，J＝6.9Hz，2H)，1.45(s，9H)，1.37(s，9H).

The preparation of di(2-ethylhexyl)phosphate tert-butyl group .3-(N, N '-two-Boc-GE) benzene methyl: under the nitrogen atmosphere, to 3-(N, N '-two-Boc-GE) benzyl alcohol (1.8 grams; 4.6mmol, 1 molar equivalent) and the phosphoramidite di-t-butyl. diisopropyl ester (1.9 grams, 2.2ml; 7mmol, 1.5 molar equivalents) the solution in the anhydrous THF of 8ml through stirring, disposable adding 1H-triazole solution (0.45M in the acetonitrile; 30ml, 14mmol, 3 molar equivalents).Stirred the mixture under the room temperature 50 minutes, and be cooled to-40 ℃ of solution that also add the 77%m-CPBA (1.8 grams, 9.2mol, 2 molar equivalents) in the 10ml anhydrous methylene chloride fast then, keep reaction temperature to be lower than 0 ℃ simultaneously.Let solution be warmed up to room temperature, and after stirring 30 minutes, add 20ml10%NaHSO ₃Aqueous solution also stirred the mixture 10-15 minute.Use 10%NaHSO afterwards ₃, 5%NaHCO ₃And brine wash and use MgSO ₄Drying composite.The evaporation organic layer also passes through flash chromatography purification residue (use EtOAc: the 10:90 of hexane is to the gradient eluent of 30:70 mixture), obtains impure product.Use CHCl such as degree such as grade ₃, obtaining pure product through chromatography purification, productive rate is 35%.

¹H?NMR(200MHz，CDCl ₃)：δ＝11.36(bs，1H)，8.29(bs，1H)，7.19-7.06(m，4H，Ar)，4.89(d，J＝7.1Hz，2H)，3.56(q，J＝7.0Hz，2H)，2.78(t，J＝7.1Hz，2H)，1.40(s，9H)，1.32(s，27H)；

¹³C?NMR(100MHz，CDCl ₃)：δ＝163.5，156.05，153.1，138.6，137.4，128.6，128.4，127.9，125.8，82.9，82.3，79.2，68.2，42.1，35.1，29.8，28.3；

³¹P?NMR(81MHz，CDCl ₃)：δ＝-9.2.

The preparation of phosphoric acid 3-(GE) benzene methyl hydrochlorate: di(2-ethylhexyl)phosphate tert-butyl group .3-(N, N '-two-Boc-GE) benzene methyl (100mg) is dissolved in the 1M solution of the HCl in the ether (5ml), and stirring at room solution 5-6 hour.Evaporate ether then, add entry and wash gained solution with ether to residue.The lyophilizing aqueous solution obtains 40mg (72%) product then.

¹H?NMR(400MHz，D ₂O)：δ＝7.12-7.02(m，4H)，4.67(d，J＝6.5Hz，2H)，3.21(t，J＝6.0Hz，2H)，2.66(t，J＝6.0Hz，2H)；

¹³C?NMR(100MHz，D ₂O)：δ＝156.6，138.7，137.4，129.0，128.6，128.1，125.9，67.3，42.1，34.1；

³¹P?NMR(00MHz，D ₂O)：δ＝0.7.

H. phosphoric acid 3,5-two guanidine radicals benzene methyls (GSC-7) synthetic:

Be prepared as follows GSC-7:

3, the preparation of 5-two (N, N '-two-Boc-guanidine radicals) benzyl alcohol: to 3,5-diaminobenzene methanol dichloride (2.2 grams, 0.01mol; 1.0 molar equivalent) in the solution in dry DMF (30ml), add N, N '-two (tert-butoxycarbonyl)-S-methyl isothiourea (6.6 grams, 0.023mol; 2.3 molar equivalent), mercuric chloride (6.2 grams, 0.023mol, 2.3 molar equivalents); With triethylamine (10ml, 0.07mol, 3 molar equivalents), and in ambient temperature overnight stirred reaction mixture (about 18 hours).Add ethyl acetate then, and with diatomite filtration gained suspension.Water, saturated NH4Cl solution and brine wash organic layer are at MgSO ₄Last dry, and solvent evaporated under reduced pressure.Through flash chromatography purification crude product on silica gel (using the gradient eluent of 20% mixture of EtOAc/ hexane), obtain 6.0 gram (96%) products to 30% mixture of EtOAc/ hexane.

¹H?NMR(200MHz，CDCl ₃)：δ＝11.50(bs，2H)，10.36(bs，2H)，7.70(t，J＝3.0Hz，1H)，7.42(d，J＝3.0Hz，2H)，4.59(s，2H)，1.42(s，18H)，1.36(s，18H)；

¹³C?NMR(50MHz，CDCl ₃)：δ＝163.2，153.4，153.1，142.7，137.3，116.8，114.6，83.6，79.6，64.7，28.1，28.0.

The di(2-ethylhexyl)phosphate tert-butyl group-3, the preparation of 5-(N, N '-two-Boc-guanidine radicals) benzene methyl: under the nitrogen atmosphere, to 3; 5-two (N, N '-two-Boc-guanidine radicals) benzyl alcohol (2 grams, 0.003mol, 1 molar equivalent) and phosphoramidite di-t-butyl. diisopropyl ester (1.15ml; 0.0036mol, 1.2 molar equivalents) and solution in the anhydrous THF of 5ml through stirring, disposable adding 1H-triazole solution (0.45M in the acetonitrile; 20ml, 0.009mol, 3 molar equivalents).Stir the gained mixture 50-60 minute under the room temperature, be cooled to-40 ℃ (through dry ice/acetonitrile) then, and add the solution of the 77%m-CPBA (0.8 gram, 0.0045mol, 1.5 molar equivalents) in the 5ml dichloromethane fast, keep reaction temperature to be lower than 0 ℃ simultaneously.Let reactant mixture be warmed up to room temperature afterwards, and after stirring 30 minutes, add 10%NaHSO ₃Aqueous solution (10ml) also carries out extra 10-15 minute stirring to mixture.With ether (20-80ml) extraction mixture, and use 10%NaHSO ₃Aqueous solution and brine wash, and use MgSO ₄Dry also filtration.The evaporation organic layer also passes through the flash chromatography purification residue (use EtOAc: the 10:90 of hexane is to the gradient eluent of 30:70 mixture) on silicagel column, obtains impure product.Use CHCl such as degree such as grade ₃, obtaining pure product through extra chromatography purification, productive rate is 40%.

¹H?NMR(200MHz，CDCl ₃)：δ＝11.5(bs，2H)，10.40(bs，2H)，7.81(s，1H)，7.38(s，2H)，4.91(d，J＝7.0Hz，2H)，1.41(s，18H)，1.40(s，18H)；

¹³C?NMR(50MHz，CDCl ₃)：δ＝163.1，153.2，153.1，138.1(d，J＝8.3Hz)，137.3，83.6，82.2(d，J＝6.8Hz)，79.4，67.8(d，J＝4.9Hz)，29.7，27.9；

³¹P?NMR(81MHz，CDCl ₃)：δ＝-9.4.

Phosphoric acid 3; The preparation of 5-two guanidine radicals benzene methyl dihydrochlorides: the 1M solution of HCl in ether (35ml) is added the di(2-ethylhexyl)phosphate tert-butyl group-3; 5-two (N, N '-two-Boc-guanidine radicals) benzene methyl (0.45 the gram, 0.57mmol) in; And at room temperature stirred the gained mixture 22-24 hour, during form white precipitate.Pour out ether afterwards, and with absolute ether washing precipitation 3-4 time, evaporation also obtains 0.13 gram (65%) end-product at the high vacuum dry residue.

¹H?NMR(400MHz，D ₂O)：δ＝7.16(s，2H)，7.11(s，1H)，4.8(d，J＝8.2Hz，2H)；

¹³C?NMR(100MHz，D ₂O)：δ＝156.10，141.2，135.8，123.3，121.9，66.0；

³¹P?NMR(162MHz，D ₂O)：δ＝0.7.

Embodiment 3

Activation measurement

Method:

Vitro inhibition algoscopy: with purified recombinant rabbit GSK-3 β people such as (, 1996) Eldar-Finkelman and peptide substrates PGS-1 (YRRAAVPPSPSLSRHSSPSQS (p) EDEEE) (SEQ ID NO:1) or peptide substrates p9CREB (SEQ ID NO:2) and phenyl phosphate, pyridoxal 5-phosphate (P-5-P), GSC-1, GSC-2, GSC-3, GSC-4, GSC-5, GSC-6, GSC-7 or GSC-21 (structural formula is described in Fig. 3) incubation under pointed concentration.Reactant mixture comprises Tris50mM (pH=7.3), 10mM MgAc, ³²P [γ-ATP] (100 μ M), 0.01% beta-mercaptoethanol was 30 ℃ of incubations 10 minutes.The reactant point sample on cellulose phosphate paper (p81), is used the 100mM phosphoric acid washing, and radioactivity is counted (like people such as Eldar-Finkelman, described in 1996).

With CDK-2 (1 unit) be similar to mentioned above and contain the reactant mixture incubation of histone h1 substrate (5 μ g), test GSC-4, GSC-5 and GSC-7 (100 and 200 μ M) influence to other protein kinase.Reactant is boiled with the SDS sample buffer, on gel electrophoresis, separate and autoradiography.

Through GSK-3 inhibitor activation Glycogensynthase: the activation of Glycogensynthase can be used as the excellent marker that GSK-3 suppresses.Our data in the past show GSK-3 inhibitor peptides activation Glycogensynthase.Thereby, in the C2C12 myotube, tested GSC-5 and GSC-7 activation to Glycogensynthase.The C2C12 cell was handled 2.5 hours under pointed concentration with GSC-5 and GSC-7, active to lysate supernatant test Glycogensynthase afterwards.Activity with Glycogensynthase in the cell of carrier DMSO (0.1%DMSO) processing is normalized to 1 unit, and the active value representation of observed Glycogensynthase is the stimulation multiple with respect to the cell of handling with carrier 0.1%HCL in the cell that uses the GSK-3 inhibitor to handle.

Glucose is taken in the isolating adipose cell: like former description people such as (, 1977) Lawrence through with 0.8mg/ml collagenase (Worthington Biochemical) digestion, from epididymal adipose tissues pad separation mice adipose cell.The fat pad of order digestion is through the nylon drainage screen; And with contain 1% bovine serum albumin (Fraction V, Boehringer Mannheim, Germany); 10mMHEPES (pH=7.3), Krebs-bicarbonate buffer (pH=7.4) washed cell of 5mM glucose and 200nM adenosine three times.GSC-4 under cell and the pointed concentration and GSC-21 incubation 2.5 hours, add then the 2-deoxidation [ ³H] glucose (0.5 μ ci/ bottle) 10 minutes.(ICN, USA) centrifuge cell stops measuring through passing dinonyl phthalate.Quantitative through liquid scintillation analyser (Packard) afterwards ³H.Before adding radioactive substance, through add cytochalasin B (50 μ M) measured in 30 minutes the 2-deoxidation [ ³H] the non-special absorption of glucose.The result:

The vitro inhibition algoscopy: in preliminary inhibition was measured, it is active that the GSK-3 of test compound known phenyl phosphate described above, pyridoxal 5-phosphate, GSC-1, GSC-2 and GSC-3 suppresses.The result who in Fig. 5, provides shows: all performance suppresses active to the chemical compound of all tests to GSK-3, the phosphate derivative of pyridine, and promptly pyridoxal 5-phosphate and GSC-3 are higher than phosphate derivative (phenyl phosphate, GSC-1 and the GSC-2) activity of phenyl.

In an extra inhibition was measured, the GSK-3 that has tested GSC-1, GSC-2, GSC-3, GSC-4 and GSC-21 suppressed active.In the presence of these chemical compounds of pointed concentration, measure the ability of GSK-3 phosphorylation PGS-1 peptide substrates.Result's representative that in table 6, provides and the contrast incubation that does not have inhibitor be the active percentage ratio of GSK-3 Comparatively speaking, and this result is the meansigma methods ± SEM of two independent experiments, and wherein each point is with triplicate mensuration.

As shown in Figure 6, find that the chemical compound of all tests all has high activity (the IC50 value is 1-5mM) in suppressing the GSK-3 activity, GSC-3 and GSC-4 are the highest active chemical compounds.To have one or more nitrogen-atoms (for example, being directly connected to annular atoms) be can influence (enhancing) newly-designed micromolecular GSK-3 to suppress active a kind of characteristic to these results suggest in ring or its adjacent position.

In mensuration again, the GSK-3 that has tested GSC-1, GSC-2, GSC-3, GSC-4, GSC-5, GSC-6 and GSC-7 suppresses active.In the presence of these chemical compounds of pointed concentration, measure the ability of GSK-3 phosphorylation p9CREB peptide substrates.Result's representative that in table 7a and 7b, provides and the contrast incubation that does not have inhibitor be the active percentage ratio of GSK-3 Comparatively speaking, and these results are meansigma methods ± SEM of two independent experiments, and wherein each point is with triplicate mensuration.

Shown in Fig. 7 a-b, find that the chemical compound of all tests all has high activity (the IC50 value is 1-5mM) in suppressing the GSK-3 activity, GSC-4 and GSC-7 are the highest active chemical compounds (IC50=0.5Mm).These results support following prompting: in ring or to have one or more nitrogen-atoms (for example, being directly connected to annular atoms) be can influence (enhancing) newly-designed micromolecular GSK-3 to suppress active a kind of characteristic in its adjacent position.These results also support following prompting: the existence of guanidine part is can influence (enhancing) newly-designed micromolecular GSK-3 to suppress active a kind of characteristic.

Study the kinetic property of inhibitor through measuring initial velocity (it is as the function of concentration of substrate under several kinds of inhibitor concentration).These inhibitor have confirmed that to the Lineweaver-Burk figure that GSK-3 suppresses these are hypothesis (data not shown) of substrate competition property inhibitor.Fig. 8 has provided the Lineweaver-Burk figure that the gsc-7 of property inhibitor as an example suppresses GSK-3, and this is through the mix expression of phosphoric acid to p9CREB peptide substrates (CPM).The result has shown the once representative experiment in 3 experiments.Terminal point is the meansigma methods of duplicate sample.

Measure the selectivity of noval chemical compound through estimating to GSK-3 with the inhibition of the closely-related a kind of kinase c DK-2 of GSK-3.Thereby, as substrate ³²P [γ-ATP] and histone h1 exist down, and it is active to measure CDK-2.Add GSC-4, GSC-5 and GSC-7 with final concentration 2mM.The result provides in Fig. 9 and clearly shows the inhibition of not observing the histone h1 phosphorylation, thereby shows the high selectivity of chemical compound to GSK-3.

The GSK-3 inhibitor is to the activation of Glycogensynthase: the activation of Glycogensynthase in the C2C12 cell that mensuration as indicated above is handled with GSC-5 and GSC-7.The activity of Glycogensynthase is normalized to 1 unit in the cell of handling with carrier DMSO (0.1%DMSO), in that (observed Glycogensynthase activity value is expressed as with respect to the stimulation multiple with the cell of carrier 0.1%HCL processing in the cell that GS4 (hollow circular) GS6 (solid circles) handles with GSK-3.

The result provides in Figure 10 and clearly illustrates that GSC-5 (hollow circular) and GSC-7 (solid circles) activate Glycogensynthase and be respectively 1.5 times and 1.8 times.

Glucose is taken in: by mentioned above, newly-designed chemical compound GSC-4 of test and GSC-21 promote the ability that glucose is taken in the former fat subsitutes cell of mice.In untreated adipose cell observed relatively [ ³H] the 2-deoxyglucose takes in and to be normalized to 1 unit, in the adipose cell that uses GSC-4 or GSC-21 to handle be [ ³H] value representation that obtains of 2-deoxyglucose is for respect to the activation multiple with the cell of peptide control treatment, and it is the meansigma methods ± SEM of 6 independent experiments, and wherein each data point is with triplicate mensuration.

The result who in Figure 11 a (GSC-4) and Figure 11 b (GSC-21), provides shows: GSC-21 increases glucose respectively and takes in 2.5 times and 1.7 times under the concentration of 5 μ M and 0.5 μ M.In the presence of GSC-4, observe the effect that reduces to a certain extent, GSC-4 strengthens glucose and takes in about 2 times under the concentration of 10 μ M.Like what further show among Figure 11 a and the 11b, the activation that the glucose that GSC-4 and GSC-21 realize is taken in is suitable with the activation that in the presence of the 100nM insulin, realizes.These results show that further these newly-designed chemical compounds can strengthen the disease of insulin signaling transmission and treatment GSK-3 mediation as insulin-mimickers, like diabetes.

Embodiment 4

Interactional stimulation to GSC inhibitor and GSK-3 catalyst structure domain

Simulated annealing is the molecule modeling method that is used to find proteinic stable conformation.The interaction of above-mentioned newly-designed GSK-3 inhibitor (being also referred to as the GSC molecule in this article) and GSK-3 catalyst structure domain has been checked in this research, and it is based on the protein crystallography of the GSK-3 that instructs like people (2001) such as Ter Haar.

The repetition heating and cooling of simulated annealing using system are with the energy minimum of the best of discovery system.In this research, extra parameter " protein-ligand combination energy " (" Protein-Ligand Binding Energy ") is joined in each heating at interval, selecting in the consideration of new starting point.

Experimental technique:

The preparation of complex: the GSK subunit B structure as people (2001) such as Ter Haar describe is used the initial model that acts on calculating.Add in the structure hydrogen atom and fixing non-complete residue.Because according to the structure of describing among the Ter Haar (preceding text), phosphoric acid is between Arg96 and Arg180, with manual its position between Arg96 and Arg180 that is inserted into of the phosphoric acid of little (GSC) molecule of testing.Four conformers that manual generation has each micromolecular different orientation keep the phosphoric acid position as common basis as starting point simultaneously.Calculate independently of each other.Afterwards through minimizing the lax of realization system, wherein with the phosphoric acid atom 15

radius is released with interior part, hydrogen atom, side chain and hydrone.

Mimic annealing: mimic annealing comprises following step:

1000 step balance to 1000 ° K;

1000 ° of 7500 steps of K simulation, thereby per 500 steps collection coordinate produces structure diversity, totally 15 times;

Interaction between the enzyme of computational minimization and the top ligand structure;

Be used for the dynamics simulation above the repetition from 15 kinds of best interaction structures of minimized structure choice; And

Repeat dynamics simulation, minimize, calculate and select 15 times

Minimize with kinetics during, with the phosphoric acid atom 15

hydrogen atom, substrate analogue and side chain and hydrone in the radius be released.

The distal portions of immobilized enzyme (greater than 15

) and protein main chain.

Burble point (cut off): dependent 9.5A distance; Per step is 1fs.

Calculate through program discover3/InsightII of Accelrys inc.

In order to minimize and kinetics service routine Discover3.The field of force that is used for the energy of computing system is CVFF.

Select three kinds of parallel methods to produce interactional good repertoire between GSK-3 and its part.The known method that molecular simulation under multiple starting point conformation and the high temperature is to use; In addition, the simulated annealing method of enzyme-part binding energy during use is stressed to simulate.

This combined method produces many conformations.For each starting point, be evaluated at the conformation that has minimum binding energy in last (the 15th) simulation loop.Relatively one of binding energy and selection the best.In some cases, to more than one conformation of every kind of ligand analysis.

During simulating, part moves freely.In all simulations, the interaction of phosphoric acid and arginine residues 180 and 96 does not rub.In the majority simulation, lysine 205 does not keep producing salt bridge with the interaction of phosphoric acid and with glutamic acid 211.

The result:

In Figure 12-17, illustrated the data that obtain in this research, it has shown the interaction between the aminoacid in the catalyst structure domain of chemical compound of being analyzed (inhibitor) and GSK-3, as determined through above-mentioned simulated annealing.Amine or guanidine part and aminoacid that main interaction between chemical compound of being analyzed and the catalyst structure domain comprises inhibitor are (for example; Glutamic acid and aspartic acid) side chain on the hydroxyl of acidic moiety or tyrosine between static and/or interaction of hydrogen bond; And the fragrance of the aromatic portion of inhibitor and aminoacid (for example, phenylalanine, tyrosine and isoleucine) or fragrance and/or the hydrophobic interaction between the hydrocarbon side chain.Consider the insertion of the part of phosphoric acid between Arg96 and the Arg180, find that these interactional majorities are the interactions with Ile217, Tyr215, Phe67, Asp181, Glu97, Asp90, Asp181 and Glu200.Phe67 be bound fraction important in the binding site of enzyme and, in addition, it is as the door of closing on the binding site.

Below table 4 provided the inhibitor of being analyzed a plurality of atoms and and a plurality of amino acid residues of the enzyme of these atomic interactions between distance.Numeral (providing with

) is the short distance between inhibitor and the residue.

The interaction of GSC-4 and GSK-3; As shown in Figure 12, the best conformation of GSC-4 (phosphoric acid 3-guanidine radicals benzene methyl) shown with the hydrophobic interaction of Ile217 and Tyr216 and with the electrostatic interaction of Asp181 and randomly with the hydrogen bond of the hydroxyl of Tyr215.

The interaction of GSC-5 and GSK-3: as shown in Figure 13, the best conformation of this chemical compound and acidic-group Glu97Asp90 or Asp181 have low the interaction.Observe suitable interaction with Phe67.

The interaction of GSC-7 and GSK-3; As shown in Figure 14; Because it is too short to be attached to the arm of guanidine radicals of aromatic core; It is that fragrance with Phe67 interacts that the majority of this chemical compound interacts; Thereby with acidic-group (for example; Asp90 and Glu97) interaction very a little less than, particularly in having the conformation of minimum binding energy (with Asp903.48

and with Glu974.92

).

The interaction of GSC-6 and GSK-3: as shown in Figure 15; Compare with GSC-4 (wherein guanidine radicals directly is attached to aromatic rings); (for example, Asp181) interaction is closely for guanidine radicals among the GSC-6 (wherein guanidine radicals is attached to ring through the ethylidene spacerarm) and acidic-group.Also observe fragrance interaction with Phe67 and Tyr216.

The interaction of GSC-8 and GSK-3: as shown in Figure 16, GSC-8 is fit to this binding site fully.Best conformation comprises points to Asp90 (1.71

) guanidine radicals arm with point to Glu97 (1.88 ) another guanidine radicals arm and with Phe67 (3.6), Arg96 (2.06

) and Arg180 (2.63

) extra interaction.This complicacy interacts and has the good binding ability.Because two extra guanidine radicals are attached to aromatic rings through the methylene spacerarm, so these groups can interact with Asp90 and Glu97 simultaneously.Obviously, GsC-8 has the better interaction than GSC-7, and wherein guanidine radicals directly is attached to ring.

The interaction of GSC-9 and GSK-3: as shown in Figure 17, GSC-9 is fit to this binding site fully, has the interaction that increases number.Optimal conformation including links to Asp90 (3.43?

) of a guanidine arm and pointing Glu97 (2.38?

) guanidine another arm.Although this interaction is not closely, guanidine radicals and extra residue such as Glu200, Gln89 and main chain o Leu88 interact, thereby combine to be enhanced.Although Phe67 does not interact strongly, as if near binding site.

Other conformation (Asp90 Glu97) that should be noted that the type have good energy and with carboxyl in addition interact more closely.

Other interaction with the GSK-3 binding site: except the interaction at the catalyzed combination position of GSC molecule and the GSK-3 of design, it is required to the best combination in this site that this research also is used for definite which interaction.In a word, find that best inhibitor will interact with Phe67, Glu97, Gln89 and Asn95.These find further to support the specific characteristic of GSK-3.Although Glu97 is conservative and be important in catalytic activity in protein kinase, the role of Phe67, Gln89 and Asn95 is novel and is that GSK-3 is unique.

Therefore carried out extra research, thereby so that design will can produce the new chemical compound of the special inhibition of GSK-3 with these residues interactions.

Embodiment 5

The chemosynthesis of polyaryl/heteroaryl GSK-3 inhibitor

(MP micromolecule)

Based on above-mentioned molecule Modeling Research, the inventor has designed and the new family that has successfully prepared the GSK-3 inhibitor now, and the skeleton that wherein contains polyaryl and/or heteroaryl is as the spacerarm between phosphoric acid part and the part (for example, guanidine) that contains amine.

Provide the general step and the data in each step in the route of synthesis of representative compound of this family below.In Figure 18, provide the chemical constitution of chemical compound.

Experimental data:

Proton, carbon and phosphorus nuclear magnetic resonance spectrum obtain on Bruker AVANCE200 or 400MHz spectrogrph, and with ppm (δ) report.Unless otherwise indicated, tetramethylsilane (TMS) is as the interior mark of proton spectra, and phosphoric acid is as the interior mark of phosphorescence spectrum, and solvent peak is as carbon and the spectrographic reference peak of fluorine.

On Micromass VG autospec M250, obtain mass spectrum.

N-(iodine substituted phenyl), N ', N " preparation of two (tert-butoxycarbonyl) guanidine:

N-(iodine substituted phenyl), N ' describes in the general preparation process scheme 10 below of N ＂-two (tert-butoxycarbonyl) guanidine.

Scheme 10

With N, N '-two (tert-butoxycarbonyl)-S-methyl isothiourea (1.32 grams, 4.4mmol; 1.1 molar equivalent), mercuric chloride (1.22 grams, 4.4mmol; 1.1 molar equivalent) and the solution of triethylamine (1.72ml, 12.0mmol, 3.0 molar equivalents) add iodo aniline (0.88 gram in the dry DMF (15ml); 4.0mmol, 1.0 molar equivalents) in, and at room temperature stirred the gained mixture 5 hours.Then mixture is imported ethyl acetate (60ml) and passes through diatomite filtration.Water (50ml), saturated NH ₄Cl aqueous solution (50ml) and brine wash organic layer are also used MgSO ₄Dry.Solvent evaporated under reduced pressure.The bullion chemical compound is pure basically and is not further purified and directly use.

Use this general step, the chemical compound below having prepared:

N-(3-iodine substituted phenyl), N ', N " two (tert-butoxycarbonyl) guanidine:

Productive rate: 85%

¹H?NMR(200MHz，CDCl ₃)：δ＝11.60(s，1H)，10.33(s，1H)，7.89(s，1H)，7.71(d，J＝8.3Hz，lH)，7.45(d，J＝7.8Hz，1H)，7.05(t，J＝8.1Hz，1H)，1.53(bs，18H).

¹³C?NMR(100MHz，CDCl ₃)：δ＝162.5，153.2，137.7，133.9，130.6，130.4，121.5，93.7，84.3，80.5，27.9，27.3.

N-(2-iodine substituted phenyl), N ', N " two (tert-butoxycarbonyl) guanidine:

By this chemical compound that obtains mentioned above,, but difference was to stir lasting 4 days.

Productive rate: 85%.

¹H?NMR(200MHz，CDCl ₃)：δ＝11.64(s，1H)，10.17(s，1H)，7.91(d，J＝8.1Hz)，7.64(d，J＝7.9Hz，1H)，7.18(t，J＝7.7Hz，1H)，6.70(t，J＝7.6Hz，1H)，1.42(bs，9H)，1.36(bs，9H).

¹³C?NMR(50MHz，CDCl ₃)：δ＝163.2，153.8，152.8，138.8，137.9，128.4，126.6，125.9，92.4，83.4，79.3，27.9

N-(4-iodine substituted phenyl), N ', N " two (tert-butoxycarbonyl) guanidine:

Productive rate: 85%

¹H?NMR(200MHz，CDCl ₃)：δ＝11.63(s，1H)，10.44(s，1H)，7.62(d，J＝8.7Hz，2H)，7.38(d，J＝8.7Hz，2H)，1.52(bs，9H)，1.50(bs，9H).

¹³C?NMR(50MHz，CDCl ₃)：δ＝163.1，153.2，137.7，136.6，123.9，88.2，83.3，79.8，28.0

N-(TMS-ethynyl phenyl), N ', the preparation of N '-two (tert-butoxycarbonyl) guanidine:

Preparation N-(TMS-ethynyl phenyl), N ' describes in the general step scheme 11 below of N ＂-two (tert-butoxycarbonyl) guanidine.

Scheme 11

With Pd (PPh ₃) ₂Cl ₂(16mg, 0.02mmol, 0.02 molar equivalent), CuI (8mg; 0.05mmol, 0.04 molar equivalent), trimethyl silyl acetylene (0.20ml, 1.2mmol; 1.1 molar equivalent) solution in diethylamine (1.8ml, 14.8mmol, 16 equivalents) and dimethyl formamide (0.6ml) adds N-(iodine substituted phenyl), N '; In N ＂-two (tert-butoxycarbonyl) guanidine (0.50 gram, 1.1mmol, 1 molar equivalent), and at nitrogen chamber relaxing the bowels with purgatives of warm nature stirred overnight gained mixture.Afterwards reactant mixture is poured in the water (8ml), and with diethyl ether (3x8ml) extraction 3 times.The organic layer that water and saline (8ml) washing merges, and with diethyl ether (2x8ml) aqueous phase extracted twice once more, and the organic layer of merging used MgSO ₄Dry also concentrating under reduced pressure.On silica gel, pass through flash chromatography purification residue (use hexane and 20:80 ethyl acetate: the gradient eluent of hexanes mixtures).

Use this general step, the chemical compound that preparation is following:

N-(3-TMS-ethynyl phenyl), N ', N '-two (tert-butoxycarbonyl) guanidine:

Productive rate: 80%

¹H?NMR(200MHz，CDCl ₃)：δ＝11.61(s，1H)，10.36(s，1H)，7.77(d，J＝8.0Hz，1H)，7.58(bs，1H)，7.26(t，J＝7.7Hz，1H)，7.19(d，J＝7.7Hz，1H)，1.51(bs，9H)，1.45(bs，9H)，0.24(s，9H).

¹³C?NMR(100MHz，CDCl ₃)：δ＝163.1，154.4，137.9，129.9，126.1，124.7，123.4，105.7，95.4，85.1，81.0，29.1，0.9

N-(2-TMS-ethynyl phenyl), N ', N '-two (tert-butoxycarbonyl) guanidine:

Obtain this chemical compound by mentioned above, but difference was to stir lasting 7 days.

Productive rate: 80%.

¹H?NMR(200MHz，CDCl ₃)：δ＝11.69(s，1H)，10.80(s，1H)，8.53(d，J＝8.4Hz，1H)，7.41(dt，J＝9.1Hz，1.4Hz，1H)，7.32(dd，J＝8.3，1.2Hz，1H)，7.02(dt，J＝7.6Hz，1.0Hz，1H)，1.53(bs，18H)，0.28(s，9H).

¹³CNMR(100MHz，CDCl ₃)：δ＝164.5，154.5，153.5，140.1，133.3，130.4，124.7，123.4，115.1，102.9，101.1，84.3，80.7，29.2，0.9.

N-(4-TMS-ethynyl phenyl), N ', N '-two (tert-butoxycarbonyl) guanidine:

Productive rate: 80%

¹H?NMR(200MHz，CDCl ₃)：δ＝11.61(s，1H)，10.42(s，1H)，7.58(d，J＝8.7Hz，2H)，7.41(d，J＝8.7Hz，2H)，1.51(bs，9H)，1.49(bs，9H)，0.24(s，9H).

¹³C?NMR(100MHz，CDCl ₃)：δ＝164.7，164.4，154.3，137.8，134.2，133.2，122.6，120.4，118.6，84.9，83.8，80.7，80.1，29.3，0.9

N-(ethynyl phenyl), N ', the preparation of N '-two (tert-butoxycarbonyl) guanidine:

Preparation N-(ethynyl phenyl), N ' describes in the general step scheme 12 below of N ＂-two (tert-butoxycarbonyl) guanidine.

Scheme 12

(use ice: salt bath), under nitrogen, stir with N-(TMS-ethynyl phenyl) N ', the solution of N '-two (tert-butoxycarbonyl) guanidine (97mg, 0.24mmol, 1 molar equivalent) in THF (3ml) at-5 ℃.In a few minutes, drip TBAF (1.0M among the THF, 0.4ml, 0.4mmol, 1.6 molar equivalents), and let mixture be warmed up to room temperature, and stirred 3 hours.Solvent evaporated under reduced pressure afterwards, and with ethyl acetate (6ml) extraction product.Water (3x15ml) and brine wash organic facies three times are used MgSO ₄Dry also reduction vaporization.The bullion chemical compound is enough pure and be not further purified and directly use.

Use this general step, the chemical compound that preparation is following:

N-(3-ethynyl phenyl), N ', N '-(tert-butoxycarbonyl) guanidine:

Productive rate: 85%

¹HNMR(200MHz，CDCl ₃)：δ＝11.62(s，1H)，10.36(s，1H)，7.75(d.J＝7.4Hz，1H)，7.63(bs，1H)，7.21-7.33(m，2H)，3.07(s，1H)，1.52(bs，18H).

¹³C?NMR(100MHz，CDCl ₃)：δ＝162.1，153.4，136.8，128.9，128.4，125.3，125.2，122.5，83.8，83.1，79.8，28.0.

N-(2-ethynyl phenyl), N ', N '-two (tert-butoxycarbonyl) guanidine:

Productive rate: 85%

¹H?NMR(200MHz，CDCl ₃)：δ＝11.64(s，1H)，10.88(s，1H)，8.49(d，J＝8.4Hz，1H)，7.33-7.47(m，2H)，7.05(dt，J＝7.7Hz，2.1Hz，1H)，2.97(s，1H)，1.53(bs，18H).

¹³C?NMR(50MHz，CDCl ₃)：δ＝163.3，153.4，152.6，139.4，132.1，129.7，123.6，122.4，113.2，84.4，83.4，79.7，79.1，28.0.

N-(4-ethynyl phenyl), N ', N '-two (tert-butoxycarbonyl) guanidine:

Productive rate: 85%

¹H?NMR(200MHz，CDCl ₃)：δ＝11.61(s，1H)，10.44(s，1H)，7.62(d，?J＝10.8Hz，2H)，7.45(d，J＝8.7Hz，2H)，3.07(s，1H)，1.53(bs，18H).

¹³C?NMR(100MHz，CDCl ₃)：δ＝164.4，154.3，138.4，133.8，122.6，119.0，85.0，84.5，80.9，29.2.

The preparation of triazobenzene methanol:

Described the general step of preparation triazobenzene methanol in the scheme 13 below.

Scheme 13

To the 50ml round-bottomed flask stirring rod of packing into, 25ml2N HCl and aminobenzene methanol (2.15 grams, 17.5mmol, 1 molar equivalent).In salt-ice bath, solution is cooled to-5 ℃.The solution that in 5 minutes, slowly adds the ice pre-cooling of the sodium nitrite (1.45 grams, 21mmol, 1.2 molar equivalents) in the 5ml water, thus the temperature of reactant is not elevated to and is higher than-3 ℃.After 5 minutes, add 125mg carbamide and destroy excessive nitrous acid.Then the solution of gained diazol was joined in 5 minutes in the solution of the Hydrazoic acid,sodium salt (2.28 grams, 35mmol, 2 molar equivalents) and the ice pre-cooling of the stirring of sodium acetate (4.20 restrain 51mmol, 3 molar equivalents) in 25ml water.Stirred the mixture 2 hours at 0 ℃, and with diethyl ether (2x50ml) extraction black oily product.Ethereal solution with 1N NaOH (2x50ml) and water (2x50ml) washing, is used MgSO ₄Drying, and be evaporated to dried.The gained chemical compound is enough pure and be not further purified and directly use.

Use this general step, the chemical compound that preparation is following:

3-triazobenzene methanol:

Productive rate: 83%

¹H?NMR(200MHz，CDCl ₃)：δ＝7.29(t，J＝7.6Hz，1H)，7.04(bd，J＝7.8Hz，1H)，6.95(s，1H)，6.91(d，J＝8Hz，1H)，4.55(s，2H).

¹³C?NMR(100MHz，CDCl ³)：δ＝142.8，140.1，129.8，122.5，118.0，117.1，64.4.

4-triazobenzene methanol:

Productive rate: 90%

¹H?NMR(200MHz，CDCl ₃)：δ＝7.28(d，J＝8.4Hz，2H)，6.97(d，J?＝8.4Hz，2H)，4.55(s，2H)，3.00(s，1H).

¹³C?NMR(100MHz，CDCl ₃)：δ＝139.1，137.5，128.4，118.9，64.2

The di(2-ethylhexyl)phosphate tert-butyl group. the preparation of triazobenzene methyl ester:

The preparation di(2-ethylhexyl)phosphate tert-butyl group. the general step of triazobenzene methyl ester is described in the scheme 14 below.

Scheme 14

With 1-H-tetrazolium solution (0.45M in the acetonitrile; 44.7ml, 20.2mmol, 3 molar equivalents) and disposable adding triazobenzene methanol (1.0 grams; 6.7mmol; 1 molar equivalent) and the phosphoramidite di-t-butyl. the solution of diisopropyl ester (3.17ml, 10.1mmol, 1.3 molar equivalents) in anhydrous THF (7ml).Stirred the gained mixture 30 minutes at 20 ℃, be cooled to-40 ℃ (using dry ice/acetonitrile to bathe) afterwards.Add 85%mCPBA (1.35 grams among the 2.4ml DCM, 10.05mmol, the 1.5 molar equivalents) solution among the DCM (9ml) fast and keep reaction temperature to be lower than 0 ℃.After letting reactant mixture reach room temperature and stirring 20 minutes, add 10%NaHSO3 aqueous solution (24ml) and also continued to stir the mixture 10 minutes.Use ether (120ml) extraction mixture then, and abandon water.Use 10%NaHSO ₃Aqueous solution (2x48ml) and saturated NaHCO ₃Aqueous solution (2x48ml) washing ether phase is used MgSO ₄Dry also filtration.Evaporating solvent and on silicagel column (using the gradient eluent of hexane) to the 40:60 mixture of ethyl acetate/hexane the chromatography residue obtain product.

Use this general step, the chemical compound that preparation is following:

Di(2-ethylhexyl)phosphate tert-butyl group .3-triazobenzene methyl ester:

Productive rate: 10%

¹H?NMR(200MHz，CDCl ₃)：δ＝7.32(t，J＝7.7Hz，1H)，7.12(d，J＝7.6Hz，1H)，7.05(bs，1H)，6.95(d，J＝6.6Hz，1H)，4.97(d，J＝7.4Hz，2H)，1.47(bs，18H). ¹³C?NMR(100MHz，CDCl ₃)：δ＝140.1，138.6，?129.6，123.6，118.4，117.7，82.5，67.4，29.7.

³¹P?NMR(81MHz，CDCl ₃)：δ＝-9.3

Di(2-ethylhexyl)phosphate tert-butyl group .4-triazobenzene methyl ester:

Productive rate: 12%

¹H?NMR(200MHz，CDCl ₃)：δ＝7.33(d，J＝8.4Hz，2H)，6.98(d，J＝8.4Hz，2H)，4.93(d，J＝7.8Hz，2H)，1.40(bs，18H).

¹³C?NMR(100MHz，CDCl ₃)：δ＝139.7，133.4，129.2，118.9，82.5，67.6，29.8.

³¹P?NMR(81MHz，CDCl ₃)：δ＝-9.3

Phosphoric acid triazobenzene methyl ester and N-(ethynyl phenyl), N ', N " the dipole cycloaddition of two (tert-butoxycarbonyl) guanidine:

Describe in the general step of the dipole cycloaddition of above-mentioned triazo-compound and the acetylene scheme 15 below.

Scheme 15

With CuSO ₄(0.02 gram, 0.125mmol, 0.5 molar equivalent) and copper cash add N-(ethynyl phenyl); N ', N '-two (tert-butoxycarbonyl) guanidine (0.07 gram, 0.212mmol; 1 molar equivalent) and the di(2-ethylhexyl)phosphate tert-butyl group. triazobenzene methyl ester (0.07 gram; 0.205mmol, 1 molar equivalent) solution in DMF (5ml), and under nitrogen stirred overnight gained mixture.Afterwards with solution with ethyl acetate (10ml) extraction, and with saline to organic layer washing three times, and with ethyl acetate (2x10ml) to water layer extracted twice again.Under reduced pressure concentrate the organic layer that merges.Obtain shielded addition compound product through going up the chromatography purification residue at silicagel column (using the gradient eluent of ethane) to 60:40 ethyl acetate/hexane mixture.

Use this general step, the chemical compound that preparation is following:

Shielded 3-(4-(3-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate (shielded 2-Gua-3-Phos):

Productive rate: 85%

¹H?NMR(400MHz，CDCl ₃)：δ＝11.61(s，1H)，10.42(s，1H)，8.26(s，1H)，8.01(s，1H)，7.81(s，1H)，7.69-7.74(m，2H)，7.61(d，J＝7.6Hz，1H)，7.48(t，J＝8Hz，1H)，7.41(d，J＝7.6Hz，1H)，7.35(t，J＝7.9Hz，1H)，5.04(d，J＝8Hz，2H)，1.51(bs，18H).

¹³C?NMR(100MHz，CDCl ₃)：δ＝164.4，154.7，154.3，148.9，140.0，138.4，138.1，131.8，130.8，130.5，128.5，123.4，123.1，120.8，120.4，120.3，119.0，84.8，83.8，80.6，68.4，30.9，30.3.

³¹P?NMR(81MHz，CDCl ₃)：δ＝-9.3

Shielded 3-(4-(2-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate (shielded 2-Gua-3-Phos):

By this chemical compound that obtains mentioned above, difference was stirred reaction mixture 4 days.

Productive rate: 50%

¹H?NMR(400MHz，CDCl ₃)：δ＝8.33(bs，1H)，7.98-8.00(m，2H)，7.88(s，1H)，7.72(m，1H)，7.47(bs，2H)，7.36(t，J＝7.2Hz，1H)，7.23(t，J＝7.6Hz，1H)，5.07(d，J＝8.4Hz，2H)，1.51(bs，18H).

Part ¹³C NMR (100MHz, CDCl ₃): δ=155.1,146.1,140.0,138.2,131.4,129.9,128.5,124.7,120.9,120.5,83.8,68.5,30.9,29.1.

³¹P?NMR(81MHz，CDCl ₃)：δ＝-9.3

MS (FAB): C ₃₄H ₄₉N ₆O ₈P (MH ⁺) m/z theoretical value=701.3; Experiment value=701.2.

Shielded 3-(4-(4-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate (shielded 4-Gua-3-Phos):

Productive rate: 73%

¹H?NMR(400MHz，CDCl ₃)：δ＝11.67(s，1H)，10.48(s，1H)，8.20(s，1H)，7.86-7.90(m，3H)，7.71-7.79(m，3H)，7.44-7.58(m，2H)，5.10(d，J＝7.7Hz，2H)，1.50(m，18H).

Part ¹³C NMR (100MHz, CDCl ₃): δ=163.2,153.3,147.9,138.8,137.0,129.8,127.4,126.3,122.3,119.8,119.1,117.2,83.8,82.7,79.7,67.3,29.8,28.0.

³¹P?NMR(81MHz，CDCl ₃)：δ＝-9.3

Shielded 4-(4-(3-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate (shielded 3-Gua-4-Phos):

Productive rate: 90%

¹H?NMR(200MHz，CDCl ₃)：δ＝11.63(s，1H)，10.44(s，1H)，8.26(s，1H)，8.07(s，1H)，7.64-7.80(m，4H)，7.52-7.56(bd，2H)，7.38(m，1H)，5.05(d，J＝7.4Hz，2H)，1.51(bs，9H)，1.24(bs，9H).

¹³C?NMR(100MHz，CDCl ₃)：δ＝163.1，153.4，153.1，147.8，137.2，136.5，130.6，129.4，128.7，127.8，122.1，121.9，120.1，119.2，117.8，83.7，82.5，79.5，67.2，29.6，27.9.

³¹P?NMR(81MHz，CDCl ₃)：δ＝-9.3

Shielded 4-(4-(2-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate (shielded 2-Gua-4-Phos):

Like above-mentioned this chemical compound that obtains, difference was stirred reaction mixture 48 hours, and should reaction still not accomplish.

Productive rate: 43%

¹H?NMR(400MHz，CDCl ₃)：δ＝10.53(bs，1H)，8.34(bs，1H)，7.81-7.85(bd，3H)，7.50-7.55(m，2H)，7.38(t，J＝7.4Hz，1H)，7.21-7.29(m，2H)，5.06(d，J＝7.6Hz，2H)，1.49(bs，9H)，1.25(bs，9H).

Part ¹³C NMR (100MHz, CDCl ₃): δ=153.9,144.8,137.3,136.6,128.8,128.6,127.9,127.8,125.6,124.5,123.7,120.3,82.7,82.5,67.3,29.5,28.0.

³¹P?NMR(81MHz，CDCl ₃)：δ＝-9.3

Shielded 4-(4-(4-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate (shielded 4-Gua-4-Phos):

Productive rate: 75%

¹H?NMR(200MHz，CDCl ₃)：δ＝11.64(s，1H)，10.45(s，1H)，8.19(s，1H)，7.69-7.88(m，6H)，7.56(d，J＝8.4Hz，2H)，5.06(d，J＝7.6Hz，2H)，1.52(bs，18H).

¹³C?NMR(100MHz，CDCl ₃)：δ＝163.3，153.4，137.5，137.4，136.9，136.6，128.8，127.9，126.5，126.3，122.4，120.3，83.8，82.7，79.7，67.4，29.8，28.1

³¹P?NMR(81MHz，CDCl ₃)：δ＝-9.3

The preparation of MP-1-MP-6 (t-Bu and BOC group go protection):

Thereby described in the scheme 15 below to go to protect t-Bu and BOC protection base to obtain the general step of end-product.

Scheme 15

Add HCl to shielded chemical compound in the time of 20 ℃! 4M in the diox, 2ml, 8mmol, 2.6 molar equivalent) are with the solution of diox (6ml) and stirred overnight gained mixture.The reduction vaporization diox obtains end-product afterwards.

Use this general step, the chemical compound (, seeing Figure 18) that preparation is following about the chemical constitution of chemical compound:

3-(4-(3-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate (3-Gua-3-Phos, MP1):

Productive rate: 70%

¹H?NMR(400MHz，DMSO-d ₆)：δ＝10.02(s，1H)，9.39(s，1H)，7.96(s，1H)，7.85(m，2H)，7.79(s，1H)，7.60(t，J＝7.6Hz，1H)，7.54(bs，2H)，7.49(t，J＝7.2Hz，1H)，7.21(d，J＝7.2Hz，1H)，4.98(d，J＝7.2Hz，2H)，

¹³C?NMR(100MHz，DMSO-d ₆)：δ＝157.5，148.1，141.3，138.0，137.5，133.1，131.9，131.5，128.9，125.8，124.8，122.8，121.7，120.7，120.0，67.5.

³¹P-NMR(81MHz，DMSO-d ₆)：δ＝-0.6

MS (FAB): C ₁₆H ₁₈N ₆O ₄P (MH ⁺) m/z theoretical value=389.1; Experiment value=389.0.

3-(4-(2-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate (2-Gua-3-Phos, MP3):

Productive rate: 72%

¹H?NMR(400MHz，DMSO-d ₆)：δ＝10.01(s，1H)，9.34(s，1H)，7.98-8.07(m，2H)，7.87(s，1H)，7.37-7.56(m，6H)，4.89(bs，2H).

¹³C?NMR(100MHz，DMSO-d ₆)：δ＝158.1，155.4，152.9，145.3，141.2，137.9，132.7，131.5，131.2，130.7，130.4，129.2，，123.0，120.8，120.2，67.8.

³¹P?NMR(81MHz，DMSO-d ₆)：δ＝-0.6

MS (FAB): C ₁₆H ₁₈N ₆O ₄P (MH ⁺) m/z theoretical value=389.1; Experiment value=389.0.

3-(4-(4-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate (4-Gua-3-Phos, MP4):

Productive rate: 72%

¹H?NMR(400MHz，DMSO-d ₆)：δ＝10.21(s，1H)，9.37(s，1H)，7.97-?8.13(m，3H)，7.61(bs，3H)，7.33(bs，3H)，4.84(bs，2H).

¹³C?NMR(50MHz，DMSO-d ₆)：δ＝156.4，147.0，140.1，136.9，135.5，130.4，129.0，127.7，126.9，125.1，120.2，119.6，118.9，66.7.

³¹P?NMR(81MHz，DMSO-d ₆)：δ＝-0.6

MS (FAB): C ₁₆H ₁₈N ₆O ₄P (MH ⁺) m/z theoretical value=389.1; Experiment value=389.0.

4-(4-(3-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate (3-Gua-4-Phos, MP5):

Productive rate: 75%

¹H?NMR(200MHz，DMSO-d ₆)：δ＝10.20(s，1H)，9.38(s，1H)，7.18-7.90(m，9H)，4.69(bs，2H).

¹³C?NMR(100MHz，DMSO-d ₆)：δ＝156.4，146.8，138.4，136.2，131.9，130.7，129.0，127.9，124.4，123.6，121.4，120.5，120.1，66.6.

³¹P?NMR(81MHz，DMSO-d ₆)：δ＝-0.6

MS (FAB): C ₁₆H ₁₆N ₆O ₄P{ (M-H) ^-M/z theoretical value=387.1; Experiment value=387.1

4-(4-(2-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate (2-Gua-4-Phos, MP6):

Like above-mentioned this chemical compound that obtains, difference is to mix and continues 22 hours.

Productive rate: 76%

¹H?NMR(400MHz，DMSO-d ₆)：δ＝9.91(s，1H)，8.47(s，1H)，7.87(bs，2H)，7.07-7.49(m，7H)，4.46(bs，2H).

¹³C?NMR(100MHz，DMSO-d ₆)：δ＝156.8，144.0，137.9，136.3，136.0，131.9，131.4，130.8，130.0，129.2，127.9，121.7，120.5，66.6.

³¹P?NMR(81MHz，DMSO-d ₆)：δ＝-0.6

MS (FAB): C ₁₆H ₁₂D ₄N ₆O ₄P{ (M-H) ^-M/z theoretical value=391.1; Experiment value=391.4.

4-(4-(4-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate (4-Gua-4-Phos, MP2):

Productive rate: 75%

¹H?NMR(400MHz，DMSO-d ₆)：δ＝10.31(s，1H)，9.35(s，1H)，7.94-8.02(m，3H)，7.59-7.68(m，3H)，7.33-7.37(m，3H)，4.98(d，J＝7.6Hz，?2H).

³¹P?NMR(81MHz，DMSO-d ₆)：δ＝-0.6

Embodiment 6

The MP molecule is to the inhibition of GSK-3

In the presence of the MP of pointed concentration molecule, measure the ability of GSK-3 phosphorylation p9CREB peptide substrates with the scheme of describing among the preceding text embodiment 3.The result who in Figure 19, provides represents the active percent of GSK-3 in the contrast incubation, wherein omits inhibitor.The result is the meansigma methods ± SEM of 2 independent experiments, and wherein each data point is with duplicate mensuration.

As shown in Figure 19, it is active to find that MP-1 and MP-4 demonstrate best inhibition, and the IC50 value is about 1mM.

Table 2

REMARK?FILENAME＝＂refine_1_4.pdb＂

REMARK

＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝

REMARK?overall，bonds，angles，improper，vdw，noe，cdih

REMARK?energies：49.6206，2.76302，18.089，2.9318，0.894357，24.9424，

SCDIH

REMARK

＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝

REMARK?bonds，angles，impropers，noe，cdih

REMARK?rms-d：4.019702E-03，0.622994，0.465061，9.195132E-02，0

REMARK

＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝

REMARK?noe，cdih

REMARK?violations.：0，0

REMARK

＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝

REMARK?DATE：27-Apr-00?08:12:42 created?by?user：orish

ATOM 1 CA ?ILE 1 11.861 ?-0.265 ?-1.755 1.00 0.00

ATOM 2 HA ?ILE 1 11.773 ?0.710 -1.305 1.00 0.00

ATOM 3 CB ?ILE 1 11.788 ?-0.143 ?-3.278 1.00 0.00

ATOM 4 HB ?ILE 1 10.810 ?0.216 -3.564 1.00 0.00

ATOM 5 CG1 ILE 1 12.034 ?-1.514 ?-3.911 1.00 0.00

ATOM 6 HG11?ILE 1 12.789 ?-2.041 ?-3.347 1.00 0.00

ATOM 7 HG12?ILE 1 12.368 ?-1.385 ?-4.930 1.00 0.00

ATOM 8 CG2 ILE 1 12.852 ?0.841 -3.766 1.00 0.00

ATOM 9 HG21?ILE 1 13.794 ?0.326 -3.880 1.00 0.00

ATOM 10?HG22?ILE 1 12.963 ?1.638 -3.045 1.00 0.00

ATOM 11?HG23?ILE 1 12.551 ?1.256 -4.717 1.00 0.00

ATOM 12?CD1 ILE 1 10.735 ?-2.319 ?-3.895 1.00 0.00

ATOM 13?HD11?ILE 1 10.871 ?-3.209 ?-3.300 1.00 0.00

ATOM 14?HD12?ILE 1 10.472 ?-2.596 ?-4.905 1.00 0.00

ATOM 15?HD13?ILE 1 9.945 -1.718 ?-3.469 1.00 0.00

ATOM 16?C ILE 1 10.762 ?-1.195 ?-1.232 1.00 0.00

ATOM 17?O ILE 1 10.856 ?-2.402 ?-1.334 1.00 0.00

ATOM 18?N ILE 1 13.205 ?-0.849 ?-1.475 1.00 0.00

ATOM 19?HT1 ILE 1 13.115 ?-1.875 ?-1.335 1.00 0.00

ATOM 20?HT2 ILE 1 13.599 ?-0.414 ?-0.615 1.00 0.00

ATOM 21?HT3 ILE 1 13.839 ?-0.665 ?-2.278 1.00 0.00

ATOM 22?N LEU 2 9.722 -0.643 ?-0.667 1.00 0.00

ATOM 23 HN ?LEU 2 ?9.666 ?0.337 -0.591 1.00 0.00

ATOM 24 CA ?LEU 2 ?8.619 ?-1.500 ?-0.136 1.00 0.00

ATOM 25 HA ?LEU 2 ?8.809 ?-2.544 ?-0.349 1.00 0.00

ATOM 26 CB ?LEU 2 ?8.63D ?-1.265 ?1.375 ?1.00 0.00

ATOM 27 HB1 LEU 2 ?7.626 ?-1.058 ?1.714 ?1.00 0.00

ATOM 28 HB2 LEU 2 ?9.269 ?-0.424 ?1.604 ?1.00 0.00

ATOM 29 CG ?LEU 2 ?9.156 ?-2.514 ?2.083 ?1.00 0.00

ATOM 30 HG ?LEU 2 ?9.725 ?-3.111 ?1.384 ?1.00 0.00

ATOM 31 CD1 LEU 2 ?10.056 -2.100 ?3.249 ?1.00 0.00

ATOM 32 HD11?LEU 2 ?11.090 -2.148 ?2.940 ?1.00 0.00

ATOM 33 HD12?LEU 2 ?9.897 ?-2.770 ?4.081 ?1.00 0.00

ATOM 34 HD13?LEU 2 ?9.816 ?-1.091 ?3.548 ?1.00 0.00

ATOM 35 CD2 LEU 2 ?7.977 ?-3.332 ?2.615 ?1.00 0.00

ATOM 36 HD21?LEU 2 ?7.730 ?-3.000 ?3.613 ?1.00 0.00

ATOM 37 HD22?LEU 2 ?8.246 ?-4.378 ?2.640 ?1.00 0.00

ATOM 38 HD23?LEU 2 ?7.123 ?-3.195 ?1.968 ?1.00 0.00

ATOM 39 C LEU 2 ?7.279 ?-1.067 ?-0.736 1.00 0.00

ATOM 40 O LEU 2 ?7.213 ?-0.161 ?-1.544 1.00 0.00

ATOM 41 N SER 3 ?6.209 ?-1.707 ?-0.349 1.00 0.00

ATOM 42 HN ?SER 3 ?6.283 ?-2.435 ?0.304 ?1.00 0.00

ATOM 43 CA ?SER 3 ?4.875 ?-1.331 ?-0.898 1.00 0.00

ATOM 44 HA ?SER 3 ?4.861 ?-0.288 ?-1.173 1.00 0.00

ATOM 45 CB ?SER 3 ?4.700 ?-2.201 ?-2.142 1.00 0.00

ATOM 46 HB1 SER 3 ?5.077 ?-1.671 ?-3.007 1.00 0.00

ATOM 47 HB2 SER 3 ?3.655 ?-2.421 ?-2.286 1.00 0.00

ATOM 48 OG ?SER 3 ?5.414 ?-3.418 ?-1.967 1.00 0.00

ATOM 49 HG ?SER 3 ?4.796 ?-4.082 ?-1.654 1.00 0.00

ATOM 50C SER 3 ?3.777 ?-1.630 ?0.126 ?1.00 0.00

ATOM 51 O SER 3 ?3.504 ?-2.771 ?0.442 ?1.00 0.00

ATOM 52 N ARG 4 ?3.145 ?-0.613 ?0.646 ?1.00 0.00

ATOM 53 HN ?ARG 4 ?3.380 ?0.300 0.375 ?1.00 0.00

ATOM 54 CA ?ARG 4 ?2.063 ?-0.839 ?1.649 ?1.00 0.00

ATOM 55 HA ?ARG 4 ?1.545 ?-1.766 ?1.443 ?1.00 0.00

ATOM 56 CB ?ARG 4 ?2.784 ?-0.921 ?3.001 ?1.00 0.00

ATOM 57 HB1 ARG 4 ?2.113 ?-0.610 ?3.789 ?1.00 0.00

ATOM 58 HB2 ARG 4 ?3.648 ?-0.270 ?2.990 ?1.00 0.00

ATOM 59 CG ?ARG 4 ?3.234 ?-2.362 ?3.257 ?1.00 0.00

ATOM 60 HG1 ARG 4 ?4.312 ?-2.395 ?3.326 ?1.00 0.00

ATOM 61 HG2 ARG 4 ?2.906 ?-2.991 ?2.444 ?1.00 0.00

ATOM 62 CD ?ARG 4 ?2.627 ?-2.865 ?4.571 ?1.00 0.00

ATOM 63 HD1 ARG ?4 1.720 ?-2.327 ?4.798 1.00 0.00

ATOM 64 HD2 ARG ?4 3.341 ?-2.763 ?5.378 1.00 0.00

ATOM 65 NE ?ARG ?4 2.321 ?-4.302 ?4.326 1.00 0.00

ATOM 66 HE ?ARG ?4 1.389 ?-4.605 ?4.292 1.00 0.00

ATOM 67 CZ ?ARG ?4 3.292 ?-5.157 ?4.155 1.00 0.00

ATOM 68 NH1 ARG ?4 4.516 ?-4.825 ?4.463 1.00 0.00

ATOM 69 HH11?ARG ?4 4.710 ?-3.915 ?4.830 1.00 0.00

ATOM 70 HH12?ARG ?4 5.259 ?-5.481 ?4.333 1.00 0.00

ATOM 71 NH2 ARG ?4 3.038 ?-6.344 ?3.677 1.00 0.00

ATOM 72 HH21?ARG ?4 2.100 ?-6.599 ?3.441 1.00 0.00

ATOM 73 HH22?ARG ?4 3.782 ?-7.000 ?3.546 1.00 0.00

ATOM 74 C ARG ?4 1.081 ?0.336 1.636 1.00 0.00

ATOM 75 O ARG ?4 1.468 ?1.482 1.752 1.00 0.00

ATOM 76 N ARG ?5 -0.187 0.062 1.495 1.00 0.00

ATOM 77 HN ?ARG ?5 -0.480 -0.873 ?1.402 1.00 0.00

ATOM 78 CA ?ARG ?5 -1.192 1.168 1.475 1.00 0.00

ATOM 79 HA ?ARG ?5 -0.705 2.127 1.595 1.00 0.00

ATOM 80 CB ?ARG ?5 -1.844 1.083 0.094 1.00 0.00

ATOM 81 HB1 ARG ?5 -2.917 1.041 0.204 1.00 0.00

ATOM 82 HB2 ARG ?5 -1.499 0.192 -0.412?1.00 0.00

ATOM 83 CG ?ARG ?5 -1.465 2.316 -0.727?1.00 0.00

ATOM 84 HG1 ARG ?5 -0.748 2.908 -0.179?1.00 0.00

ATOM 85 HG2 ARG ?5 -2.350 2.907 -0.918?1.00 0.00

ATOM 86 CD ?ARG ?5 -0.848 1.876 -2.057?1.00 0.00

ATOM 87 HD1 ARG ?5 -0.300 0.955 -1.931?1.00 0.00

ATOM 88 HD2 ARG ?5 -0.202 2.651 -2.445?1.00 0.00

ATOM 89 NE ?ARG ?5 -2.008 1.659 -2.965?1.00 0.00

ATOM 90 HE ?ARG ?5 -2.795 2.241 -2.903?1.00 0.00

ATOM 91 CZ ?ARG ?5 -1.977 0.695 -3.845?1.00 0.00

ATOM 92 NH1 ARG ?5 -0.857 0.392 -4.441?1.00 0.00

ATOM 93 HH11?ARG ?5 -0.022 0.898 -4.225?1.00 0.00

ATOM 94 HH12?ARG ?5 -0.834 -0.347 ?-5.115?1.00 0.00

ATOM 95 NH2 ARG ?5 -3.067 0.035 -4.127?1.00 0.00

ATOM 96 HH21?ARG ?5 -3.925 0.267 -3.670?1.00 0.00

ATOM 97 HH22?ARG ?5 -3.043 -0.704 ?-4.801?1.00 0.00

ATOM 98 C ARG ?5 -2.236 0.954 2.575 1.00 0.00

ATOM 99 O ARG ?5 -2.260 -0.078 ?3.214 1.00 0.00

ATOM 100?N PRO ?6 -3.068 1.946 2.758 1.00 0.00

ATOM 101?CA ?PRO ?6 -4.149 1.876 3.807 1.00 0.00

ATOM 102?HA ?PRO ?6 -3.740 1.628 4.790 1.00 0.00

ATOM 103 CB PRO 6 ?-4.710 3.304 ?3.802 1.00 ?0.00

ATOM 104 HB1?PRO 6 ?-4.213 3.918 ?4.537 1.00 ?0.00

ATOM 105 HB2?PRO 6 ?-5.780 3.296 ?3.976 1.00 ?0.00

ATOM 106 CG PRO 6 ?-4.411 3.814 ?2.426 1.00 ?0.00

ATOM 107 HG1?PRO 6 ?-4.357 4.887 ?2.428 1.00 ?0.00

ATOM 108 HG2?PRO 6 ?-5.177 3.477 ?1.740 1.00 ?0.00

ATOM 109 CD PRO 6 ?-3.087 3.236 ?2.027 1.00 ?0.00

ATOM 110 HD2?PRO 6 ?-3.044 3.093 ?0.948 1.00 ?0.00

ATOM 111 HD1?PRO 6 ?-2.275 3.877 ?2.353 1.00 ?0.00

ATOM 112 C ?PRO 6 ?-5.282 0.893 ?3.432 1.00 ?0.00

ATOM 113 O ?PRO 6 ?-6.393 1.035 ?3.902 1.00 ?0.00

ATOM 114 N ?SER 7 ?-5.030 -0.093 2.607 1.00 ?0.00

ATOM 115 HN SER 7 ?-4.145 -0.207 2.235 1.00 ?0.00

ATOM 116 CA SER 7 ?-6.110 -1.051 2.233 1.00 ?0.00

ATOM 117 HA SER 7 ?-5.833 -1.603 1.348 1.00 ?0.00

ATOM 118 CB SER 7 ?-6.238 -2.001 3.415 1.00 ?0.00

ATOM 119 HB1?SER 7 ?-6.552 -2.972 3.057 1.00 ?0.00

ATOM 120 HB2?SER 7 ?-6.974 -1.619 4.102 1.00 ?0.00

ATOM 121 OG SER 7 ?-4.984 -2.104 4.077 1.00 ?0.00

ATOM 122 HG SER 7 ?-5.045 -2.814 4.720 1.00 ?0.00

ATOM 123 C ?SER 7 ?-7.430 -0.316 2.010 1.00 ?0.00

ATOM 124 O ?SER 7 ?-8.251 -0.211 2.899 1.00 ?0.00

ATOM 125 N ?TYR 8 ?-7.643 0.184 ?0.831 1.00 ?0.00

ATOM 126 HN TYR 8 ?-6.966 0.078 ?0.127 1.00 ?0.00

ATOM 127 CA TYR 8 ?-8.925 0.904 ?0.559 1.00 ?0.00

ATOM 128 HA TYR 8 ?-9.535 0.924 ?1.451 1.00 ?0.00

ATOM 129 CB TYR 8 ?-8.533 2.329 ?0.179 1.00 ?0.00

ATOM 130 HB1?TYR 8 ?-9.278 2.738 ?-0.498?1.00 ?0.00

ATOM 131 HB2?TYR 8 ?-7.570 2.317 ?-0.324?1.00 ?0.00

ATOM 132 CG TYR 8 ?-8.466 3.172 ?1.458 1.00 ?0.00

ATOM 133 CD1?TYR 8 ?-7.422 4.091 ?1.648 1.00 ?0.00

ATOM 134 HD1?TYR 8 ?-6.664 4.205 ?0.901 1.00 ?0.00

ATOM 135 CD2?TYR 8 ?-9.451 3.035 ?2.465 1.00 ?0.00

ATOM 136 HD2?TYR 8 ?-10.261?2.331 ?2.352 1.00 ?0.00

ATOM 137 CE1?TYR 8 ?-7.360 4.861 ?2.815 1.00 ?0.00

ATOM 138 HE1?TYR 8 ?-6.553 5.566 ?2.952 1.00 ?0.00

ATOM 139C?E2 TYR 8 ?-9.379 3.809 ?3.629 1.00 ?0.00

ATOM 140 HE2?TYR 8 ?-10.134?3.702 ?4.394 1.00 ?0.00

ATOM 141 CZ TYR 8 ?-8.336 4.721 ?3.803 1.00 ?0.00

ATOM 142 OH TYR 8 ?-8.270 5.483 ?4.951 1.00 ?0.00

ATOM 143 HH TYR 8 -7.345 ?5.662 ?5.136 ?1.00 ?0.00

ATOM 144 C ?TYR 8 -9.680 ?0.230 ?-0.587 1.00 ?0.00

ATOM 145 O ?TYR 8 -9.400 ?0.456 ?-1.747 1.00 ?0.00

ATOM 146 N ?ARG 9 -10.639 -0.592 -0.266 1.00 ?0.00

ATOM 147 HN ARG 9 -10.848 -0.751 0.681 ?1.00 ?0.00

ATOM 148 CA ARG 9 -11.423 -1.283 -1.334 1.00 ?0.00

ATOM 149 HA ARG 9 -11.870 -0.561 -1.999 1.00 ?0.00

ATOM 150C?B ?ARG 9 -10.408 -2.140 -2.099 1.00 ?0.00

ATOM 151 HB1?ARG 9 -9.528 ?-1.554 -2.314 1.00 ?0.00

ATOM 152 HB2?ARG 9 -10.849 -2.477 -3.027 1.00 ?0.00

ATOM 153 CG ARG 9 -10.015 -3.354 -1.253 1.00 ?0.00

ATOM 154 HG1?ARG 9 -10.141 -3.120 -0.206 1.00 ?0.00

ATOM 155 HG2?ARG 9 -8.982 ?-3.606 -1.444 1.00 ?0.00

ATOM 156 CD ARG 9 -10.907 -4.543 -1.618 1.00 ?0.00

ATOM 157 HD1?ARG 9 -11.932 -4.342 -1.351 1.00 ?0.00

ATOM 158 HD2?ARG 9 -10.556 -5.439 -1.125 1.00 ?0.00

ATOM 159 NE ARG 9 -10.781 -4.677 -3.096 1.00 ?0.00

ATOM 160 HE ARG 9 -11.439 -4.252 -3.684 1.00 ?0.00

ATOM 161 CZ ARG 9 -9.794 ?-5.361 -3.607 1.00 ?0.00

ATOM 162 NH1?ARG 9 -8.770 ?-5.684 -2.865 1.00 ?0.00

ATOM 163 HH11ARG 9 -8.742 ?-5.408 -1.904 1.00 ?0.00

ATOM 164 HH12ARG 9 -8.014 ?-6.208 -3.256 1.00 ?0.00

ATOM 165 NH2?ARG 9 -9.831 ?-5.723 -4.860 1.00 ?0.00

ATOM 166 HH21ARG 9 -10.615 -5.476 -5.429 1.00 ?0.00

ATOM 167 HH22ARG 9 -9.074 ?-6.247 -5.252 1.00 ?0.00

ATOM 168 C ?ARG 9 -12.504 -2.167 -0.705 1.00 ?0.00

ATOM 169 OT1?ARG 9 -13.492 -2.425 -1.372 1.00 ?0.00

ATOM 170 OT2?ARG 9 -12.324 -2.570 0.433 ?1.00 ?0.00

END

Table 3

REMARK?FILENAME＝＂refine_1_20.pdb＂

REMARK

＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝

REMARK?overall，bonds，angles，improper，vdw，noe，cdih

REMARK?energies：104.733，3.47295，70.7767，3.51384，6.64866，

20.3204，SCDIH

REMARK

＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝

REMARK?bonds，angles，impropers，noe，cdih

REMARK?rms-d：4.442149E-03，1.21652，0.496579，7.90724E-02，0

REMARK

＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝

REMARK noe，cdih

REMARK?violations.：0，0

REMARK

＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝

REMARK?DATE：03-Apr-00?08:41:00 created?by?user：orish

ATOM 1 CA ILE 1B ?-9.783 -1.457 ?-0.558 1.00 ?0.00

ATOM 2 HA ILE 1B ?-9.677 -0.665 ?-1.298 1.00 ?0.00

ATOM 3 CB ILE 1B ?-11.259 ?-1.637 ?-0.199 1.00 ?0.00

ATOM 4 HB ILE 1B ?-11.578 ?-0.796 ?0.417 ?1.00 ?0.00

ATOM 5 CG1 ?ILE 1B ?-11.441 ?-2.945 ?0.598 ?1.00 ?0.00

ATOM 6 HG11 ILE 1B ?-12.251 ?-2.816 ?1.316 ?1.00 ?0.00

ATOM 7 HG12 ILE 1B ?-10.519 ?-3.167 ?1.135 ?1.00 ?0.00

ATOM 8 CG2 ?ILE 1B ?-12.101 ?-1.660 ?-1.481 1.00 ?0.00

ATOM 9 HG21 ILE 1B ?-12.492 ?-0.662 ?-1.677 1.00 ?0.00

ATOM 10?HG22 ILE 1B ?-12.930 ?-2.357 ?-1.35B 1.00 ?0.00

ATOM 11?HG23 ILE 1B ?-11.480 ?-1.978 ?-2.318 1.00 ?0.00

ATOM 12?CD1 ?ILE 1B ?-11.776 ?-4.119 ?-0.334 1.00 ?0.00

ATOM 13?HD11 ILE 1B ?-11.998 ?-5.004 ?0.263 ?1.00 ?0.00

ATOM 14?HD12 ILE 1B ?-10.926 ?-4.325 ?-0.983 1.00 ?0.00

ATOM 15?HD13 ILE 1B ?-12.644 ?-3.866 ?-0.941 1.00 ?0.00

ATOM 16?C ?ILE 1B ?-8.973 -1.137 ?0.677 ?1.00 ?0.00

ATOM 17?O ?ILE 1B ?-9.510 -0.787 ?1.709 ?1.00 ?0.00

ATOM 18?N ?ILE 1B ?-9.351 -2.764 ?-1.130 1.00 ?0.00

ATOM 19?HT1 ?ILE 1B ?-8.379 -2.681 ?-1.489 1.00 ?0.00

ATOM 20?HT2 ?ILE 1B ?-9.987 -3.028 ?-1.910 1.00 ?0.00

ATOM 21?HT3 ?ILE 1B ?-9.383 -3.494 ?-0.391 1.00 ?0.00

ATOM 22 N LEU 2 ?-7.676 ?-1.250 ?0.593 ?1.00 ?0.00

ATOM 23 HN ?LEU 2 ?-7.221 ?-1.531 ?-0.230 1.00 ?0.00

ATOM 24 CA ?LEU 2 ?-6.745 ?-0.970 ?1.725 ?1.00 ?0.00

ATOM 25 HA ?LEU 2 ?-7.286 ?-0.659 ?2.617 ?1.00 ?0.00

ATOM 26 CB ?LEU 2 ?-6.051 ?-2.305 ?1.992 ?1.00 ?0.00

ATOM 27 HB1 LEU 2 ?-5.606 ?-2.675 ?1.069 ?1.00 ?0.00

ATOM 28 HB2 LEU 2 ?-6.782 ?-3.027 ?2.359 ?1.00 ?0.00

ATOM 29 CG ?LEU 2 ?-4.955 ?-2.110 ?3.041 ?1.00 ?0.00

ATOM 30 HG ?LEU 2 ?-5.142 ?-1.190 ?3.595 ?1.00 ?0.00

ATOM 31 CD1 LEU 2 ?-4.955 ?-3.296 ?4.007 ?1.00 ?0.00

ATOM 32 HD11?LEU 2 ?-5.261 ?-2.958 ?4.997 ?1.00 ?0.00

ATOM 33 HD12?LEU 2 ?-3.952 ?-3.720 ?4.062 ?1.00 ?0.00

ATOM 34 HD13?LEU 2 ?-5.651 ?-4.055 ?3.651 ?1.00 ?0.00

ATOM 35 CD2 LEU 2 ?-3.595 ?-2.020 ?2.345 ?1.00 ?0.00

ATOM 36 HD21?LEU 2 ?-3.732 ?-1.671 ?1.322 ?1.00 ?0.00

ATOM 37 HD22?LEU 2 ?-3.127 ?-3.004 ?2.334 ?1.00 ?0.00

ATOM 38 HD23?LEU 2 ?-2.956 ?-1.320 ?2.884 ?1.00 ?0.00

ATOM 39 C LEU 2 ?-5.725 ?0.080 1.347 ?1.00 ?0.00

ATOM 40 O LEU 2 ?-4.915 ?0.491 2.155 ?1.00 ?0.00

ATOM 41 N SER 3 ?-5.747 ?0.528 0.122 ?1.00 ?0.00

ATOM 42 HN ?SER 3 ?-6.390 ?0.212 -0.547 1.00 ?0.00

ATOM 43 CA ?SER 3 ?-4.806 ?1.562 -0.398 1.00 ?0.00

ATOM 44 HA ?SER 3 ?-4.771 ?1.557 -1.486 1.00 ?0.00

ATOM 45 CB ?SER 3 ?-5.388 ?2.889 0.083 ?1.00 ?0.00

ATOM 46 HB1 SER 3 ?-6.272 ?3.133 -0.510 1.00 ?0.00

ATOM 47 HB2 SER 3 ?-4.648 ?3.677 -0.034 1.00 ?0.00

ATOM 48 OG ?SER 3 ?-5.738 ?2.778 1.457 ?1.00 ?0.00

ATOM 49 HG ?SER 3 ?-6.250 ?3.554 1.696 ?1.00 ?0.00

ATOM 50 C SER 3 ?-3.416 ?1.369 0.164 ?1.00 ?0.00

ATOM 51 O SER 3 ?-3.131 ?1.747 1.283 ?1.00 ?0.00

ATOM 52 N ARG 4 ?-2.534 ?0.782 -0.597 1.00 ?0.00

ATOM 53 HN ?ARG 4 ?-2.747 ?0.466 -1.515 1.00 ?0.00

ATOM 54 CA ?ARG 4 ?-1.116 ?0.522 -0.175 1.00 ?0.00

ATOM 55 HA ?ARG 4 ?-0.840 ?1.104 0.716 ?1.00 ?0.00

ATOM 56 CB ?ARG 4 ?-1.095 ?-0.975 ?0.176 ?1.00 ?0.00

ATOM 57 HB1 ARG 4 ?-1.739 ?-1.526 ?-0.506 1.00 ?0.00

ATOM 58 HB2 ARG 4 ?-1.453 ?-1.114 ?1.200 ?1.00 ?0.00

ATOM 59 CG ?ARG 4 ?0.323 -1.521 ?0.077 ?1.00 ?0.00

ATOM 60 HG1 ARG 4 ?1.018 -0.714 ?-0.142 1.00 ?0.00

ATOM 61 HG2 ARG 4 ?0.369 -2.273 ?-0.711 1.00 ?0.00

ATOM 62 CD ARG 4 0.681 -2.146 1.415 1.00 0.00

ATOM 63 HD1 ?ARG 4 -0.203 -2.604 1.849 1.00 0.00

ATOM 64 HD2 ?ARG 4 1.067 -1.373 2.079 1.00 0.00

ATOM 65 NE ARG 4 1.715 -3.169 1.096 1.00 0.00

ATOM 66 HE ARG 4 2.519 -3.100 1.652 1.00 0.00

ATOM 67 CZ ARG 4 1.576 -4.075 0.168 1.00 0.00

ATOM 68 NH1 ?ARG 4 1.048 -5.233 0.460 1.00 0.00

ATOM 69 HH11 ARG 4 0.750 -5.424 1.395 1.00 0.00

ATOM 70 HH12 ARG 4 0.942 -5.927 -0.252 1.00 0.00

ATOM 71 NH2 ?ARG 4 1.965 -3.825 -1.052 1.00 0.00

ATOM 72 HH21 ARG 4 2.370 -2.938 -1.276 1.00 0.00

ATOM 73 HH22 ARG 4 1.859 -4.520 -1.764 1.00 0.00

ATOM 74 C ARG 4 -0.156 0.835 ?-1.306 1.00 0.00

ATOM 75 O ARG 4 0.292 -0.044 -2.015 1.00 0.00

ATOM 76 N ARG 5 0.150 2.088 ?-1.507 1.00 0.00

ATOM 77 HN ARG 5 -0.225 2.805 ?-0.970 1.00 0.00

ATOM 78 CA ARG 5 1.062 2.546 ?-2.605 1.00 0.00

ATOM 79 HA ARG 5 1.447 1.693 ?-3.157 1.00 0.00

ATOM 80 CB ARG 5 0.167 3.349 ?-3.540 1.00 0.00

ATOM 81 HB1 ?ARG 5 0.684 3.496 ?-4.489 1.00 0.00

ATOM 82 HB2 ?ARG 5 -0.044 4.319 ?-3.089 1.00 0.00

ATOM 83 CG ARG 5 -1.142 2.596 ?-3.784 1.00 0.00

ATOM 84 HG1 ?ARG 5 -1.832 3.235 ?-4.334 1.00 0.00

ATOM 85 HG2 ?ARG 5 -1.587 2.319 ?-2.828 1.00 0.00

ATOM 86 CD ARG 5 -0.861 1.333 ?-4.602 1.00 0.00

ATOM 87 HD1 ?ARG 5 -1.790 0.801 ?-4.79B 1.00 0.00

ATOM 88 HD2 ?ARG 5 -0.168 0.687 ?-4.058 1.00 0.00

ATOM 89 NE ARG 5 -0.259 1.834 ?-5.868 1.00 0.00

ATOM 90 HE ARG 5 0.592 1.404 ?-6.094 1.00 0.00

ATOM 91 CZ ARG 5 -0.804 2.756 ?-6.613 1.00 0.00

ATOM 92 NH1 ?ARG 5 -2.099 2.919 ?-6.607 1.00 0.00

ATOM 93 HH11 ARG 5 -2.673 2.338 ?-6.031 1.00 0.00

ATOM 94 HH12 ARG 5 -2.516 3.626 ?-7.178 1.00 0.00

ATOM 95 NH2 ?ARG 5 -0.054 3.515 ?-7.365 1.00 0.00

ATOM 96 HH21 ARG 5 0.938 3.389 ?-7.370 1.00 0.00

ATOM 97 HH22 ARG 5 -0.472 4.221 ?-7.936 1.00 0.00

ATOM 98 C ARG 5 2.235 3.432 ?-2.176 1.00 0.00

ATOM 99 O ARG 5 3.149 3.598 ?-2.959 1.00 0.00

ATOM 100?N PRO 6 2.225 4.009 ?-0.990 1.00 0.00

ATOM 101?CA PRO 6 3.362 4.885 ?-0.604 1.00 0.00

ATOM 102 HA ?PRO 6 ?3.562 ?5.623 ?-1.380 1.00 ?0.00

ATOM 103 CB ?PRO 6 ?2.877 ?5.579 ?0.665 ?1.00 ?0.00

ATOM 104 HB1 PRO 6 ?2.405 ?6.534 ?0.423 ?1.00 ?0.00

ATOM 105 HB2 PRO 6 ?3.704 ?5.730 ?1.362 ?1.00 ?0.00

ATOM 106 CG ?PRO 6 ?1.867 ?4.642 ?1.236 ?1.00 ?0.00

ATOM 107 HG1 PRO 6 ?1.119 ?5.192 ?1.780 ?1.00 ?0.00

ATOM 108 HG2 PRO 6 ?2.357 ?3.932 ?1.887 ?1.00 ?0.00

ATOM 109 CD ?PRO 6 ?1.228 ?3.922 ?0.080 ?1.00 ?0.00

ATOM 110 HD2 PRO 6 ?1.038 ?2.890 ?0.343 ?1.00 ?0.00

ATOM 111 HD1 PRO 6 ?0.316 ?4.415 ?-0.219 1.00 ?0.00

ATOM 112 C PRO 6 ?4.587 ?4.040 ?-0.334 1.00 ?0.00

ATOM 113 O PRO 6 ?5.195 ?4.120 ?0.714 ?1.00 ?0.00

ATOM 114 N SRP 7 ?4.973 ?3.235 ?-1.287 1.00 ?0.00

ATOM 115 HN ?SRP 7 ?4.501 ?3.180 ?-2.153 1.00 ?0.00

ATOM 116 CA ?SRP 7 ?6.178 ?2.345 ?-1.198 1.00 ?0·00

ATOM 117 C SRP 7 ?5.986 ?1.217 ?-0.187 1.00 ?0.00

ATOM 118 O SRP 7 ?6.890 ?0.401 ?0.000 ?1.00 ?0.00

ATOM 119 CB ?SRP 7 ?7.409 ?3.196 ?-0.806 1.00 ?0.00

ATOM 120 OG1 SRP 7 ?7.614 ?4.247 ?-1.816 1.00 ?0.00

ATOM 121 PG2 SRP 7 ?9.111 ?4.826 ?-1.692 1.00 ?0.00

ATOM 122 OG3 SRP 7 ?9.841 ?4.741 ?-3.126 1.00 ?0.00

ATOM 123 OG2 SRP 7 ?9.883 ?4.016 ?-0.692 1.00 ?0.00

ATOM 124 OG4 SRP 7 ?9.056 ?6.362 ?-1.210 1.00 ?0.00

ATOM 125 HA ?SRP 7 ?6.354 ?1.890 ?-2.170 1.00 ?0.00

ATOM 126 HG3 SRP 7 ?10.770 4.312 ?-3.012 1.00 ?0.00

ATOM 127 HG4 SRP 7 ?8.124 ?6.751 ?-1.413 1.00 ?0.00

ATOM 128 HB1 SRP 7 ?8.290 ?2.553 ?-0.751 1.00 ?0.00

ATOM 129 HB2 SRP 7 ?7.240 ?3.659 ?0.163 ?1.00 ?0.00

ATOM 130 N TYR 8 ?4.845 ?1.147 ?0.452 ?1.00 ?0.00

ATOM 131 HN ?TYR 8 ?4.121 ?1.772 ?0.312 ?1.00 ?0.00

ATOM 132 CA ?TYR 8 ?4.521 ?0.098 ?1.463 ?1.00 ?0.00

ATOM 133 HA ?TYR 8 ?4.788 ?0.421 ?2.466 ?1.00 ?0.00

ATOM 134 CB ?TYR 8 ?3.001 ?-0.061 1.371 ?1.00 ?0.00

ATOM 135 HB1 TYR 8 ?2.757 ?-1.115 1.255 ?1.00 ?0.00

ATOM 136 HB2 TYR 8 ?2.631 ?0.495 ?0.510 ?1.00 ?0.00

ATOM 137 CG ?TYR 8 ?2.351 ?0.471 ?2.630 ?1.00 ?0.00

ATOM 138 CD1 TYR 8 ?2.895 ?0.164 ?3.884 ?1.00 ?0.00

ATOM 139 HD1 TYR 8 ?3.776 ?-0.453 3.953 ?1.00 ?0.00

ATOM 140C?D2 ?TYR 8 ?1.202 ?1.269 ?2.544 ?1.00 ?0.00

ATOM 141 HD2 TYR 8 ?0.776 ?1.504 ?1.579 ?1.00 ?0.00

ATOM 142 CE1 TYR 8 ?2.293 ?0.655 ?5.048 ?1.00 0.00

ATOM 143 HE1 TYR 8 ?2.713 ?0.417 ?6.014 ?1.00 0.00

ATOM 144 CE2 TYR 8 ?0.600 ?1.759 ?3.710 ?1.00 0.00

ATOM 145 HE2 TYR 8 ?-0.284 2.374 ?3.643 ?1.00 0.00

ATOM 146 CZ ?TYR 8 ?1.146 ?1.452 ?4.961 ?1.00 0.00

ATOM 147 OH ?TYR 8 ?0.553 ?1.936 ?6.110 ?1.00 0.00

ATOM 148 HH ?TYR 8 ?1.222 ?2.407 ?6.613 ?1.00 0.00

ATOM 149 C TYR 8 ?5.198 ?-1.217 1.126 ?1.00 0.00

ATOM 150 O TYR 8 ?5.057 ?-1.728 0.033 ?1.00 0.00

ATOM 151 N ARG 9 ?5.936 ?-1.788 2.046 ?1.00 0.00

ATOM 152 HN ?ARG 9 ?6.065 ?-1.397 2.951 ?1.00 0.00

ATOM 153 CA ?ARG 9 ?6.655 ?-3.095 1.832 ?1.00 0.00

ATOM 154 HA ?ARG 9 ?5.958 ?-3.901 1.569 ?1.00 0.00

ATOM 155 CB ?ARG 9 ?7.597 ?-2.847 0.639 ?1.00 0.00

ATOM 156 HB1 ARG 9 ?7.078 ?-2.256 -0.115 1.00 0.00

ATOM 157 HB2 ARG 9 ?7.886 ?-3.805 0.204 ?1.00 0.00

ATOM 158 CG ?ARG 9 ?8.858 ?-2.097 1.089 ?1.00 0.00

ATOM 159 HG1 ARG 9 ?8.772 ?-1.825 2.139 ?1.00 0.00

ATOM 160 HG2 ARG 9 ?8.975 ?-1.193 0.489 ?1.00 0.00

ATOM 161 CD ?ARG 9 ?10.085 -2.996 0.895 ?1.00 0.00

ATOM 162 HD1 ARG 9 ?10.070 -3.810 1.617 ?1.00 0.00

ATOM 163 HD2 ARG 9 ?10.998 -2.408 1.013 ?1.00 0.00

ATOM 164 NE ?ARG 9 ?9.950 ?-3.518 -0.493 1.00 0.00

ATOM 165 HE ?ARG 9 ?9.658 ?-4.453 -0.534 1.00 0.00

ATOM 166 CZ ?ARG 9 ?10.193 -2.808 -1.561 1.00 0.00

ATOM 167 NH1 ARG 9 ?11.414 -2.436 -1.833 1.00 0.00

ATOM 168 HH11 ?ARG 9 ?12.163 -2.695 -1.223 1.00 0.00

ATOM 169 HH12 ?ARG 9 ?11.600 -1.892 -2.651 1.00 0.00

ATOM 170 NH2 ARG 9 ?9.215 ?-2.471 -2.357 1.00 0.00

ATOM 171 HH21 ?ARG 9 ?8.280 ?-2.756 -2.149 1.00 0.00

ATOM 172 HH22 ?ARG 9 ?9.402 ?-1.927 -3.175 1.00 0.00

ATOM 173 C ARG 9 ?7.449 ?-3.492 3.057 ?1.00 0.00

ATOM 174 OT1 ARG 9 ?7.344 ?-2.801 4.057 ?1.00 0.00

ATOM 175 OT2 ARG 9 ?8.155 ?-4.485 2.985 ?1.00 0.00

END

Will be appreciated that characteristics more of the present invention of in the context of independent embodiment, describing in order to know also can provide in the combination in single embodiment.On the contrary, also can provide separately or with any suitable time combination for the various features of succinctly in the context of single embodiment, describing of the present invention.

Although combined specific embodiments of the present invention to describe the present invention, obvious many alternatives, modification and work-around solution are that those skilled in the art are conspicuous.Therefore, be intended to comprise spirit and interior all these type of alternatives, modification and the work-around solution of wide region that falls into accompanying claims.Incorporate in this description all publications, patent and the patent application mentioned in this description are all complete by reference, this is special with each independent publication, patent or patent application and individually to incorporate this paper by reference into the same.In addition, quoting or identifying and not to be understood that to admit that this list of references is as prior art of the present invention in this application to any list of references.

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Sequence table

<110>Eldar-Finkelman，Hagit

 

< 120>glycogen synthase kinase-3 inhibitors

 

<130>28881

 

<160>4

 

< 170>PatentIn version 3 .2

 

<210>1

<211>5

<212>PRT

< 213>artificial sequence

 

<220>

< 223>GSK-3 identification motif consensus sequence

 

<220>

<221>misc_feature

<222>(1)..(1)

<223>Ser?or?Thr

 

<220>

<221>misc_feature

<222>(2)..(4)

< 223>Xaa can be any naturally occurring aminoacid

<220>

<221>misc_feature

<222>(5)..(5)

< 223>Ser of phosphorylation or Thr

 

<400>1

 

Xaa?Xaa?Xaa?Xaa?Xaa

1 5

 

<210>2

<211>9

<212>PRT

< 213>artificial sequence

 

<220>

< 223>synthetic peptide

 

<220>

<221>MOD_RES

<222>(7)..(7)

< 223>phosphorylation

 

<400>2

 

Ile?Leu?Ser?Arg?Arg?Pro?Ser?Tyr?Arg

1 5

<210>3

<211>9

<212>PRT

< 213>artificial sequence

 

<220>

< 223>synthetic peptide

 

<400>3

 

Ile?Leu?Ser?Arg?Arg?Pro?Ser?Tyr?Arg

1 5

 

<210>4

<211>9

<212>PRT

< 213>artificial sequence

 

<220>

< 223>synthetic peptide

 

<400>4

 

Ile?Leu?Ser?Arg?Arg?Pro?Glu?Tyr?Arg

1 5

Claims (16)

1. chemical compound; It comprises electronegative phosphate groups and contains the group of guanidine radicals part with at least one; Covalently bound between them through spacerarm; This spacerarm has certain-length, structure and flexibility; Select said length, structure and flexible to contain in the catalyst structure domain of group and said GSK-3 of guanidine radicals part at least a interaction between second binding site with at least a interaction between first binding site in the catalyst structure domain that allows said phosphate groups and GSK-3 and said, thereby this chemical compound can suppress the catalytic activity of GSK-3, said chemical compound has following formula

Wherein said phosphate groups and the said group that contains guanidine radicals part are connected in any position of the phenyl ring that links to each other separately with them.

2. the chemical compound of claim 1, the catalytic activity that wherein suppresses said GSK-3 comprise and reduce the combination of substrate to said catalyst structure domain.

3. the chemical compound of claim 1, wherein said first binding site comprises at least one amino acid residue that is selected from the group of being made up of arginine 180, arginine 96 and lysine 205.

4. the chemical compound of claim 1, wherein said second binding site comprises at least one amino acid residue that is selected from the group of being made up of aspartic acid 181, glutamic acid 97, aspartic acid 90, aspartic acid 181, glutamic acid 200, glutamine 89, tyrosine 215 and agedoite 95.

5. the chemical compound of claim 1, it is selected from 3-(4-(3-guanidino phenyl)-1,2; The 3-triazol-1-yl) benzyl phosphate hydrochlorate, 4-(4-(4-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate, 3-(4-(2-guanidino phenyl)-1; 2,3-triazol-1-yl) benzyl phosphate hydrochlorate, 3-(4-(4-guanidino phenyl)-1,2; The 3-triazol-1-yl) benzyl phosphate hydrochlorate, 4-(4-(3-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate and 4-(4-(2-guanidino phenyl)-1; 2, the 3-triazol-1-yl) benzyl phosphate hydrochlorate.

6. the chemical compound of claim 1, it is 3-(4-(3-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate.

7. the chemical compound of claim 1, it is 3-(4-(4-guanidino phenyl)-1,2,3-triazoles-1-yl) benzyl phosphate hydrochlorate.

8. pharmaceutical composition, it comprises as each chemical compound and pharmaceutically suitable carrier among the claim 1-7 of active component.

9. the pharmaceutical composition of claim 8, it is packaged in the packaging material, and is used to treat the active relevant biological situation with GSK-3 being designated with mode of printing on the said packaging material or among the said packaging material.

10. the pharmaceutical composition of claim 8, it also comprises can regulate the active at least a extra active component of GSK-3.

11. the active method of vitro inhibition GSK-3, said method comprise each external contact of chemical compound among the claim 1-7 of the cell of expressing GSK-3 and inhibition effective dose.

12. the method for claim 11, wherein said activity are that phosphorylation activity and/or autophosphorylation are active.

13. the method for claim 11, it also comprises and said cell is contacted the activity that said extra active component can be regulated GSK-3 with at least a extra active component.

14. the method that external reinforcement insulin signaling transmits, it comprises each external contact of chemical compound among the claim 1-7 of insulin response cell and effective dose.

15. the method for claim 14 also comprises said cell is contacted with insulin.

16. each chemical compound is used to prepare the purposes that treatment is selected from the medicine of following biological situation among the claim 1-7: obesity, non-insulin-dependent diabetes mellitus, insulin-dependent situation, affective disorder, neurodegenerative disease or disease and psychosis or disease.

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