CN1838954B

CN1838954B - Glycogen synthase kinase-3 inhibitors

Info

Publication number: CN1838954B
Application number: CN2004800240175A
Authority: CN
Inventors: H·埃尔达尔-芬克尔曼
Original assignee: Tel Aviv University Future Technology Development LP
Current assignee: Tel Aviv University Future Technology Development LP
Priority date: 2003-06-27
Filing date: 2004-06-27
Publication date: 2013-01-16
Anticipated expiration: 2024-06-27
Also published as: CA2530111A1; EP1638557A2; EP1638557A4; CN1838954A; WO2005000192A3; WO2005000192A2; KR20060018902A; JP2007527859A

Abstract

Novel conjugates that are capable of inhibiting GSK-3 activity, a process of producing same, pharmaceutical compositions including same and methods of using same in the treatment of GSK-3 mediated conditions are disclosed. Methods of treating affective disorders using GSK-3 inhibitors are further disclosed.

Description

Glycogen synthase kinase-3 inhibitors

Invention field and background of invention

The present invention relates to for the new compound that suppresses glycogen synthase kinase-3 (GSK-3) and in the biological disease purposes of regulating by the active mediation of GSK-3, and in more detail, relate to these chemical compounds and treating biological disease for example type ii diabetes, neurodegenerative disorders and disease and affective disorder.

The protein kinase of phosphorylated protein substrate reaches Cytoplasm and the nuclear crucial material that participates in for the signal with the extracellular event, and participation relates to cells survival and dead nearly all event comprises mitosis, differentiation and apoptosis.Thus, protein kinase is good medicine target always.Yet, because protein kinase activity determines the kilter of cell, suppress it and often cause cell death, so limited its purposes as the medicine target.Although cell death to the effect of anticarcinogen for needing, is major defect to most other treatments.

For protein kinase family member's glycogen synthase kinase-3 (GSK-3) is the serine-threonine kinase of Cytoplasm proline orientation, relate to insulin signaling conduction and regulate with metabolism, also relate to the pattern of cell fate during the conduction of Wnt signal and the fetal development.Two kinds of similar isoforms having differentiated this enzyme are called GSK-3 α and GSK-3 β.

Because be different from the protein kinase that other typically activates by signal pathway, think that always GSK-3 is medicine target good in the protein kinase, GSK-3 activates in akinete usually, and for example is bonded to the signal pathway that its cell surface receptor produces by insulin and weakens the GSK-3 activity by activating certain signal pathway.The activation of Insulin receptor INSR causes the activation of protein kinase B (PKB is also referred to as Akt), thereby causes successively the GSK-3 phosphorylation to make its inactivation.The inhibition of GSK-3 may cause the synthetic activation of glycogen.The insulin signaling approach of this complexity is by regulating the insulin signaling conduction and further complicated (Eldar-Finkelman etc., 1997) through GSK-3 from phosphorylation IRS-1 negative feedback on serine residue.

Therefore, synthetic GSK-3 inhibitor can be simulated some hormone and for example effect of the use GSK-3 approach of insulin of somatomedin.Under some pathological conditions, this pattern can allow to walk around the other defective component of deficiency receptor (defective receptor) or signal transduction mechanism, thus make bio signal in addition as noninsulin dependent type ii diabetes in the signal cascade upstream of amplifying come into force when participating in the material defectiveness.

The former catabolic adjusting of endocellular sugar is a kind of key biological function, and the signal element that relates to series of complex comprises the hormone insulin.Through many kinds of media, insulin is brought into play its adjusting effect by increasing Glycogensynthase (GS) glycogen biosynthesis.Main matter is the phosphorylation of IRS (IRS-1, IRS-2) on a plurality of tyrosine residues in the insulin action, and it causes activating several signal components simultaneously, comprises PI3 kinases (Myers etc., 1992)).Similar, the activity of Glycogensynthase is by its phosphorylation and suppressed.Glycogensynthase activity and glycogen levels significantly reduce (Damsbo etc., 1991 in type ii diabetes patient muscle; Nikoulina etc., 1997; Shulman etc., 1990).

A kind of II of following type (non-insulin dependence) onset diabetes begins changes into insulin resistance the earliest.Insulin resistance be characterized as hyperinsulinemia (hyperinsulemia) and hyperglycemia.Although do not know the molecular mechanism that insulin resistance is clear and definite, think due to the defective of insulin signaling pathway downstream component.

Glycogen synthase kinase-3 (GSK-3) is a kind of downstream component of insulin signaling conduction.Found that highly active GSK-3 is by making IRS-1 (IRS-1) serine residue phosphorylation damage insulin action (Eldar-Finkelman etc. in intact cell, 1997), and same, the GSK-3 increased activity causes the inhibition (Eldar-Finkelman etc., 1996) of Glycogensynthase activity in the cell.The other research of carrying out in this respect discloses active significantly strengthen (Eldar-Finkelman etc., 1999) of GSK-3 in the epididymal adipose of diabetic mice.Measured afterwards GSK-3 increased activity in the type ii diabetes patient skeletal muscle (Nickoulina etc., 2000).Recently other research has determined that further (summary is seen, Eldar-Finkelman, 2002 in the effect of GSK-3 in glycogen metabolism and insulin signaling conduction; Grimes and Jope, 2001; Woodgett, 2001), can represent a kind of method that improves the insulin activity in vivo thereby prompting suppresses the GSK-3 activity.

Considered that also GSK-3 is the important participation material in the Alzheimer's disease pathogenesis.Differentiated GSK-3 as a kind of tyrosine phosphorylation microtubule-associated protein tau, tau is relevant with pairing taenidium (the paired helical filaments) formation (PHF) that is the Alzheimer's disease early sign.Significantly, the unusual hyperphosphorylation of tau is the reason that microtubule destabilization and PHF form.Although in fact several protein kinases all show promotion tau phosphorylation, only find that the GSK-3 phosphorylation directly affects ability (Hanger etc., 1992 that tau promotes the microtubule self-assembly; Mandelkow etc., 1992; Mulot etc., 1994; Mulot etc., 1995).The further evidence of GSK-3 effect is crossed the research of expressing GSK-3 from cell and the transgenic mice of specific expressed GSK-3 in brain in this regard.GSK-3 all causes the generation (Lucas etc., 2001) of PHF sample epi-position tau in two kinds of situations.

GSK-3 acts on further relevant with Alzheimer's disease in apoptosis by it.Insulin is a kind of neuronal survival factor (Barber etc., 2001) and causes its anti-apoptotic function (Barber etc., 2001) by activating PI3 kinases and PKB that the GSK-3 that prompting is reduced by these signal elements promotes Neuron Apoptosis.A plurality of researchs really confirm this viewpoint and are presented at the key important function that determines GSK-3 in the life and death.And, show that its apoptosis function does not rely on the PI3 kinases.GSK-3 crosses to express and causes apoptosis (Pap etc., 1998) in the PC12 cell.In cerebellar granule neuron, activate GSK-3 mediation migration and cell death (Tong etc., 2001).In human neuroblastoma SH-SYSY cell, GSK-3 crosses and expresses (stauroaporine-induced) apoptosis (Bijur etc., 2000) that promotes that D-82041 DEISENHOFEN is induced.

As the GSK-3 beta inhibitor, the expression of Fratl is enough to make neuron to be rescued in the death that the PI3 kinases induces from suppressing, and has further proved by this research and has suppressed GSK-3 and prevent relation (Crowder etc., 2000) between cell death.

Find that GSK-3 is that bipolar disorder and manic depressive illness are relevant with affective disorder also.This contact is based on the potent and specific inhibitor (Klein etc., 1996 that in the treatment concentration range of first-selected mood stabilizer lithium in clinical use of finding through being usually used in bipolar disorder are a kind of GSK-3; Stambolic etc., 1996; Phiel etc., 2001).This discovery causes beginning the forfeiture of a series of researchs to determine that can lithium simulate the GSK-3 activity in cytopathy.In fact; shown that lithium-activated glycogen synthesizes (Cheng etc.; 1983); stable and accumulate beta-catenin (Stambolic etc.; 1996), induce Xenopus (Xenopus) embryo axis to copy (Klein etc., 1996); and neuroprotective unit is in order to avoid dead (Bijur etc., 2000).Have been found that the another kind of mood stabilizer valproic acid that often uses also is a kind of effective GSK-3 inhibitor (Chen etc., 1999).Generally speaking, these studies show that GSK-3 is the main body giving drugs into nose target of lithium and valproic acid and therefore significant in novel affective disorder therapy.

A kind of mechanism that lithium and other GSK-3 inhibitor can be treated bipolar disorder has improved the neuronic survival rate (Nonaka etc., 1998) that stands after the unusual high-level excitement that neurotransmitter glutamate induces for it.The neuronal excitability poisoning of also be sure oing glutamate induction is the main cause that neural degeneration follows for example cerebral ischemia of acute injury, traumatic brain injury and antibacterial to infect.And be sure of the factor (Thomas, 1995) that chronic neuron that the conduction of excessive glutamic acid signal is for example seen in relevant dull-witted, the amyotrophic lateral sclerosis (AML) of Alzheimer's disease, Huntington Chorea, parkinson disease, AIDS and the multiple sclerosis (MS) for disease injures.

Therefore be sure of that the GSK-3 inhibitor is conducive to treat these neurodegenerative diseases and other neurodegenerative disease.In fact, show that recently the active imbalance of GSK-3 is disorderly relevant with neurodegenerative disease with several CNS, comprise schizophrenia (Beasley etc., 2001; Kozlovsky etc., 2002), apoplexy and Alzheimer's disease (AD) (Bhat and Budd, 2002; Hernandez etc., 2002; Lucas etc., 2001; Mandelkow etc., 1992).

Recently research proves that further GSK-3 relates to other cell processes and comprises that growth (He etc., 1995), tumor generate (Rubinfeld etc., 1996) and protein synthesis (Welsh etc., 1993).Important, GSK-3 plays negative effects in these approach.This just further points out GSK-3 is the cytostatics of signal transduction path.

In view of GSK-3 relates generally to various signal transduction paths, exploitation specificity GSK-3 inhibitor gets involved (therapeutic interventions) for various treatments and basic research all is likely and is important.

As previously mentioned, found that some mood stabilizers suppress GSK-3.But when report suppressed GSK-3 by lithium chloride (LiCI) (pct international patent application WO97/41854) with by purine inhibitors (pct international patent application WO98/16528), these inhibitor were not that specificity is for GSK-3.In fact, can find out these drug influence multiple signal approach and suppress other cellular targets such as inositol monophosphatase (IMpase) and histone deacetylase (histone deacetylases) (Berridge etc., 1989; Phiel and Klein, 2001).

Similar, having described a kind of known GSK-3 substrate is genetic engineering cAMP response element binding protein (CREB) (Fiol etc., 1994), and other potential GSK-3 inhibitor peptides (Fiol etc., 1990).Nominally but also only (nominally) inhibition GSK-3 is active for these substrates.

Recently reported other GSK-3 inhibitor.Developed relevant micromolecule SB-216763 and SB-415286 (Glaxo SmithKline Pharmaceutical) on two kinds of structures, its specificity suppresses GSK-3 and demonstrates glycogen metabolism and the genetic transcription effect regulated, also demonstrate the effect of neuroprotective unit in case because the PI3 kinase activity lowers neuronal death (Cross etc., 2001 of inducing; Coghlan etc., 2000).Another studies show that the active ingredient of Chinese herbs for chronic myelocytic leukemia is that Induribin is a kind of GSK-3 inhibitor.But Indirubin also suppresses cell cycle deopendent protein kinase (cyclic-dependent proteinkinase-2) (CDK-2) (Damiens etc., 2001).These GSK-3 inhibitor are that ATP is emulative and differentiate by chemicals library high flux screening.The major defect that it has been generally acknowledged that the ATP-competitive inhibitor is its limited specificity (Davies etc., 2000).

In the general policies that the inventor's WO01/49709 and U.S. Patent Application Publication No. 20020147146 have been reported exploitation specific peptide and other GSK-3 inhibitor, its disclosed full content is incorporated herein by reference.This general policies is based on the definition of the architectural feature of GSK-3 substrate, and according to these features exploitations GSK-3 inhibitor.But, although its multiple small peptide of openly having described these architectural features and having provided establishment GSK-3 activity, these open design and synthetic micromolecular methods that can be used as the GSK-3 inhibitor of not providing.The end that the inventor's PCT/IL03/01057 discloses a kind of peptide GSK-3 inhibitor adheres to a hydrophobic part and improves its inhibition activity.

Although peptide is interesting drug target, its purposes often is subject to biological example and learns unstability, immunogenicity, passes through such as cell membrane and blood brain barrier (BBB) ability etc. of biomembrane.

Therefore extensively thinking does not have the non-peptide compound of above-mentioned circumscribed inhibition GSK-3 activity to need, and is very useful.

Summary of the invention

At present surprised discovery of the inventor is active and can therefore to be effective to reduce the GSK-3 activity be in the favourable various application for the substrate competition inhibition of GSK-3 according to the compound exhibits of the characteristic feature design of GSK-3 substrate identification motif (recognitionmotif).

Therefore, provide according to an aspect of the present invention a kind of chemical compound or its pharmaceutically acceptable salt with following general formula:

Wherein:

X, Y, Z and W independently are carbon atom or nitrogen-atoms separately;

A is alkyl or does not exist;

B is for having formula Electronegative group, wherein L is selected from phosphorus atoms, sulphur atom, silicon atom, boron atom and carbon atom; Q, G and D independently are selected from oxygen and sulfur separately; And E is selected from hydroxyl, alkoxyl, aryloxy group, carbonyl, thiocarbonyl, O-carboxyl, sulfydryl, alkylthio group and arylthio or does not exist.

D is selected from hydrogen, alkyl, tri haloalkyl, cycloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, the heterolipid ring, halogen, hydroxyl, alkoxyl, aryloxy group, sulfydryl, alkylthio group, arylthio, sulfinyl, sulfonyl, cyano group, nitro, azo, amino-sulfonyl, carbonyl, ketone ester, thiocarbonyl, ester, ether, thioether, thiocarbamate, urea, thiourea, the O-carbamoyl, the N-carbamoyl, the O-thiocarbamoyl, the N-thiocarbamoyl, the C-acylamino-, the N-acylamino-, the C-carboxyl, the O-carboxyl, sulfonamido, the trihalomethyl group sulfonamido, amidino groups, amidino groups alkyl (guanylinoalkyl), guanidine radicals, guanidine alkylation (guanidinoalkyl), amino, aminoalkyl, hydrazine and hydrophobic part; And

R ₁, R ₂, R ₃And R ₄Independently be selected from separately hydrogen; lone pair electrons; alkyl; tri haloalkyl; cycloalkyl; thiazolinyl; alkynyl; aryl; heteroaryl; the heterolipid ring; halogen; hydroxyl; alkoxyl; aryloxy group; sulfydryl; alkylthio group; arylthio; sulfinyl; sulfonyl; cyano group; nitro; azo; amino-sulfonyl; carbonyl; ketone ester; thiocarbonyl; ester; ether; thioether; thiocarbamate; urea; thiourea; the O-carbamoyl; the N-carbamoyl; the O-thiocarbamoyl; the N-thiocarbamoyl; the C-acylamino-; the N-acylamino-; the C-carboxyl; the O-carboxyl; sulfonamido; the trihalomethyl group sulfonamido; amidino groups; the amidino groups alkyl; guanidine radicals; guanidine alkylation; amino; aminoalkyl; hydrazine and ammonium ion

Condition is that at least one is nitrogen-atoms and/or R among X, Y, Z and the W ₁, R ₂, R ₃And R ₄For containing group of at least one amino part, and precondition is that described chemical compound is not pyridoxal 5-phosphate.

The further feature in the preferred embodiment of following description according to the present invention, D is hydrophobic part in following formula, and therefore provides according to another aspect of the present invention the chemical compound with above-mentioned formula, wherein D is hydrophobic part.The compounds of this invention also comprises the not chemical compound that is replaced by described hydrophobic part in above-mentioned scope according to this aspect of the invention.

The chemical compound of above describing can suppress the activity of GSK-3.

According in the described preferred embodiment further feature A be alkyl.

According in the described preferred embodiment further feature L be phosphorus atoms.

According to respectively do for oneself oxygen and the preferred hydroxyl of E of feature Q, G and D further in the described preferred embodiment.

According to further at least a among feature X, Y, Z and the W in the described preferred embodiment be nitrogen-atoms.

According in the described preferred embodiment further among feature X, Y, Z and the W at least two kinds be nitrogen-atoms.Preferred X and Y nitrogen-atoms or Z and the W nitrogen-atoms of respectively doing for oneself of respectively doing for oneself.

According to feature R further in the described preferred embodiment ₁, R ₂, R ₃And R ₄In at least a for containing group of at least one amino part.

According to feature R further in the described preferred embodiment ₁, R ₂, R ₃And R ₄In at least two kinds for containing group of at least one amino part.Preferred R ₁And R ₂Respectively do for oneself and contain group or the R of at least one amino part ₃And R ₄Respectively do for oneself and contain the group of at least one amino part.

The example that contains the group of at least one amino part includes but not limited to guanidine radicals, guanidine alkylation, aminoalkyl, its analog, its derivant and any combination thereof.

According in the described preferred embodiment further feature group of containing at least one amino part comprise at least a positively charged group.

According in the described preferred embodiment further this positively charged group of feature comprise ammonium ion.Selectable, this positively charged group has the chemical constitution derived from positively charged amino acid whose side chain, such as but not limited to arginine, lysine, histidine, proline and any derivant thereof.

If D is hydrophobic part, this hydrophobic part is preferably selected from fatty acid residue, has the saturated alkylene chain (alkylene chain) of 4-30 carbon atom, has unsaturated alkylene chain, aryl, cycloalkyl and the hydrophobic peptide sequence of 4-30 carbon atom.

Fatty acid can be for example myristic acid, lauric acid, Palmic acid, stearic acid, oleic acid, arachidonic acid, linoleic acid plus linolenic acid.

Preferred compound of the present invention further comprises wherein X, Y, Z and the W carbon atom of respectively doing for oneself; And R ₁, R ₂, R ₃And R ₄In at least a for containing as mentioned above group of at least one amino part.

Further respectively do for oneself carbon atom and W of X, Y and Z is nitrogen-atoms in the preferred chemical compound.

Another aspect of the present invention provides a kind of Pharmaceutical composition that can suppress the GSK-3 activity, comprises chemical compound as noted before and pharmaceutically acceptable carrier as active component.

According to further feature in the preferred embodiments of the invention what follows, Pharmaceutical composition be packaged in the packaging material and on packaging material or in stamp sign, be used for the treatment of the active relevant biological disease with GSK-3, such as detailed description hereinafter.

According to feature further in the described preferred embodiment, this Pharmaceutical composition further comprises at least a other active component that can change the GSK-3 activity, such as detailed description hereinafter.

Another aspect of the present invention provides the method for the treatment biological disease relevant with the GSK-3 activity, and it comprises that the curee who needs it treats the chemical compound that can suppress the GSK-3 activity of effective dose, and is as indicated above.

According to further feature in the preferred embodiments of the invention what follows, the method for this aspect of the present invention further comprises and jointly gives the curee at least a other active component that can change the GSK-3 activity.

This other active component can be the active component that can suppress the GSK-3 activity, maybe can reduce the active component that GSK-3 expresses.

Biological disease of the present invention is preferably selected from obesity, non-insulin-dependent diabetes mellitus, insulin dependency disease, affective disorder, neurodegenerative disease or obstacle and psychosis or obstacle.

Affective disorder can be unipolar disorders (for example depression) or bipolar disorder (for example manic depressive illness).

This neurodegenerative disease can be by the chronic neurodegenerative disease that is selected from due to the disease that cerebral ischemia, apoplexy, traumatic brain injury and antibacterial infect, perhaps can serves as reasons due to the disease that is selected from relevant dull-witted, the amyotrophic lateral sclerosis (AML) of Alzheimer's disease, Huntington Chorea, parkinson disease, AIDS and multiple sclerosis.

Another aspect of the present invention provides a kind of method of the GSK-3 of inhibition activity, and it comprises the cell of expressing GSK-3 with the compounds of this invention contact that suppresses effective dose.

This activity is that phosphorylation activity and/or autophosphorylation are active.

Another aspect of the present invention provides a kind of method that strengthens the insulin signaling conduction, and it comprises the above described chemical compound contact insulin sensitivity cell with effective dose.

In each method, this exposing cell can carry out or carry out in vivo external.

According to further feature in the preferred embodiments of the invention what follows, each method further comprises with at least a other active component exposing cell in these other aspects of the present invention, and is as indicated above.

The proposition of the newly-designed non-peptide compound success of the present invention by being provided for suppressing the GSK-3 activity defective of present configuration known, the compounds of this invention can be effective to treat various biological diseases.

Unless otherwise defined, all technical terms used herein all have the implication identical with the common understanding of those of ordinary skills with scientific terminology.Although in practice of the present invention or test, can use and herein described similar or suitable method and material following suitable method and the material described.If conflict is arranged, will contrast patent specification and comprise definition.In addition, material, method and embodiment only are descriptive, have no intention to limit the present invention.

The accompanying drawing summary

This paper only describes the present invention by example and mode with reference to the accompanying drawings.With detailed specifically with reference to the accompanying drawings together, only emphasize to show details by the mode of example and the purpose only discussed for the illustrative of the preferred embodiment of the invention, and to present details be to be that the principle of the invention and concept aspect are the most useful and hold intelligible description most because be sure of it.Because of this consideration, do not attempt to show that employing figure and description make and those skilled in the art know that how imbody is in putting into practice for several forms of the present invention than understanding the substantially required more detailed detailed construction of the present invention of the present invention.

Among the figure:

Fig. 1 a-b represents that (Fig. 1 a) and the computer image of the 3D structure of CREB (Fig. 1 b) passes through 2D to peptide class p9CREB ¹H-NMR research obtains (signify hydrogen atom not; Carbon backbone chain is Lycoperdon polymorphum Vitt, and nitrogen-atoms is blue, and oxygen atom is that redness and phosphorus atoms are yellow);

The image that Fig. 2 distributes for the static that shows the p9CREB peptide is based on deriving from 2D ¹The peptide 3D structure of H-NMR research;

Fig. 3 represents the chemical constitution of phenyl phosphate, pyridoxal 5-phosphate (P-5-P), GS-1, GS-2, GS-3 and the chemical constitution of new compound GS-4, GS-5 and GS-21;

Fig. 4 a-b represents i.e. 1,3,5-three (hydroxymethyl) benzene of a kind of GS-21 synthetic intermediate ¹HNMR spectrum (Fig. 4 a) and ¹³C NMR composes (Fig. 4 b);

Fig. 5 a-b represents i.e. 3,5-two (bromomethyl) benzyl alcohol of a kind of GS-21 synthetic intermediate ¹HNMR spectrum (Fig. 5 a) and ¹³C NMR composes (Fig. 5 b);

Fig. 6 a-b represents i.e. 3,5-two (cyano methyl) benzyl alcohol of a kind of GS-21 synthetic intermediate ¹H NMR spectrum (Fig. 6 a) and ¹³C NMR composes (Fig. 6 b);

Fig. 7 represents i.e. 3,5-two (amino-ethyl) benzyl alcohol of a kind of GS-21 synthetic intermediate ¹The HNMR spectrum;

Fig. 8 represents i.e. 3,5-two (uncle-butoxy carbonyl amino-ethyl) benzyl alcohol of a kind of GS-21 synthetic intermediate ¹H NMR spectrum;

Fig. 9 a-c represents the phosphoric acid 3 that a kind of GS-21 synthetic intermediate is namely protected, 5-two (2-amino-ethyl) benzyl esters ¹H NMR spectrum (Fig. 9 a), ¹³C NMR spectrum (Fig. 9 b) and ³¹P NMR composes (Fig. 9 c);

Figure 10 a-d represents phosphoric acid 3,5-two (2-amino-ethyl) benzyl esters tfa salt (GS-21TFA salt) ¹H NMR spectrum (Figure 10 a), ¹³C NMR spectrum (Figure 10 b), ³¹P NMR composes (Figure 10 c) and ESI-MS (Figure 10 d);

Figure 11 a-e represents phosphoric acid 3,5-two (2-amino-ethyl) benzyl esters (GS-21) ¹HNMR spectrum (Figure 11 a), ¹³C NMR spectrum (Figure 11 b), ³¹P NMR composes (Figure 11 c), ESI-MS (Figure 11 d) and HPLC chromatogram (Figure 11 e);

Figure 12 is illustrated in proves in the body outer suppressioning test that phenyl phosphate, GS-1, GS-2, GS-3 and pyridoxal 5-phosphate (P-5-P) GSK-3 suppresses active comparison chart (comparative plots);

Figure 13 is illustrated in proves in the body outer suppressioning test that GS-1, GS-2, GS-3, GS-5 and GS-21GSK-3 suppress active comparison chart (black circle indication GS-1, red circle indication GS-2, green circle indication GS-3, blue circle indication GS-5 and pink circle indication GS-21); With

Figure 14 a-b for proof GS-21 (Figure 14 b) and GS-5 (Figure 14 is a) on the bar chart of the impact of the glucose uptake of mice adipose cell, be incorporated in the cell of processing with usefulness peptide reference substance in the cell of processing with GS-5 and GS-21 [ ³H] multiple of 1,5-anhydroglucitol activates expression (being normalized into 1 unit).

The description of preferred embodiment

The present invention includes novel non-peptide compound, it can suppress the active biological disease that also can therefore be used for the treatment of the GSK-3 mediation of GSK-3.Specifically, the present invention includes (i) according to the chemical compound of stearic coordination (stearic coordinates) design of GSK-3 substrate, it can be chosen wantonly has a hydrophobic part that is attached to this; (ii) contain the Pharmaceutical composition of same compound; (iii) use the identical method that suppresses the GSK-3 reactive compound and strengthen the insulin signaling conduction; (iv) the same compound of use is used for the treatment of the method such as but not limited to the biological disease of obesity, non-insulin-dependent diabetes mellitus, insulin dependency disease, affective disorder, neurodegenerative disease and obstacle and psychosis or obstacle.

Principle of the present invention and operation can be better understood in reference diagram and appended description.

Before in detail explaining at least a embodiment of the present invention, be appreciated that to the invention is not restricted to application of the present invention disclosed in detail in the following description or by the embodiment illustration.The present invention can have other embodiment or in various manners practice or operation.Be appreciated that also herein used wording and term are for the purpose described and can not be taken as restriction.

The parameter of a kind of responsible substrate-kinases identification is the element that is positioned in the substrate, and it often is called " identification motif ".As discussed above, GSK-3 has distinctive identification motif unlike other kinases, comprises the disclosed aminoacid sequence SX of SEQ ID NO:1 ₁X ₂X ₃S (p), wherein S is serine or threonine, X ₁, X ₂And X ₃Any aminoacid of respectively doing for oneself, and S (p) is the serine of phosphorylation or the threonine of phosphorylation.

Extensively provided its small peptide as the activity of substrate or inhibitor of empirical tests that a cover designs and synthesizes based on this identification motif in WO01/49709 and U.S. Patent Application Publication No. 20020147146, its disclosed full content is incorporated herein by reference.Based on these tests, many features of the active substrate of these peptides for GSK-3 or inhibitor have been determined.A kind of most important feature is that the phosphorylation residue of serine residue or threonine is that bound substrates and GSK-3 inhibitor are necessary in the motif.These tests have proved that further the part of these peptides is efficient and special GSK-3 inhibitor.These defined polypeptides are the substrate competition inhibitor.

The substrate of only identifying pre-phosphorylation (pre-phosphorylated) based on GSK-3 namely has the serine residue of phosphorylation or this discovery of substrate of threonine residues, and the GSK-3 substrate of supposing these pre-phosphorylations has makes itself and the interactional peculiar structure of GSK-3 catalytic core.Further hypothesis limits these peculiar structures and will make it possible to develop the micromolecule that can be used as GSK-3 substrate competition inhibitor.

Therefore searching simulation above in the micromolecule of the inhibition activity of described little GSK-3 inhibitor peptides, although delayed enforcement of the present invention, but determined stereochemical structure and the peculiar architectural feature of the small peptide substrate of hexamethyl phosphoric acid, and verified manyly have the chemical compound of these features as the activity of GSK-3 inhibitor.

Selected pre-phosphorylation small peptide p9CREB (ILSRRPS (p) YR, SEQ ID NO:2) as a kind of representative example of GSK-3 substrate.Measured the stereochemical structure of p9CREB and the stereochemical structure of corresponding non-phosphorylating PEPC REB (ILSRRPSYR, SEQ ID NO:3) by 2D NMR, partly described in detail at the following example.

Show that such as Fig. 1 a and 1b the p9CREB substrate of phosphorylation has definite structure in solution (Fig. 1 a), this corresponding non-phosphorylating PEPC REB does not show any peculiar structure (Fig. 1 b) thus.

From these results, prompting phosphate group in the peptide of phosphorylation interacts by tyrosine (Y8) and arginine (R4) cation-π one of its generation ' ring sample (loop-like) ' structure (is seen, table 2 and 3), and the result be positioned at outside the ring at the serine of phosphorylation of identification motif.This " bending " (" bended ") structure of substrate makes the serine of phosphorylation be easy to interact with the Binding Capacity bag of enzyme.

Really found the support to this prompting in the recent disclosed crystallization data of the GSK-3 that Dajani etc. (2001) describe.The substrate binding site of the crystallization data show GSK-3 of Dajani etc. comprises 3 positively charged residue A rg 96, Arg 180 and Lys 205 with the phosphate anion effect.

Find that based on these static that Fig. 2 is illustrated in " surface " (' surface ') of p9CREB peptide distributes.

When continuing imagination when of the present invention, thereby reason out the micromolecule that should produce according to following characteristic Design simulation GSK-3 substrate structure the substrate competition activity from described discovery above:

This molecule should comprise electronegative group, the preferably phosphoric acid base;

This electronegative group should be by tristearin steric hindrance (stearically hindered); With

This electronegative group should preferably have one or two positively charged group in one side or its both sides at least.

Based on top described, designed the general formula that suppresses the active potential chemical compound of GSK-3.As described in following embodiment part, namely having the preliminary experiment that the chemical compound of the simple structure of this formula carries out with " first generation " chemical compound has proved that these chemical compounds can suppress the GSK-3 activity, thereby the preliminary indication that suppresses this formula chemical compound of potential is provided.

Based on the top described chemical compound that has has also designed and synthesized more senior generation (more advancedgeneration), comprise noval chemical compound.As described in following embodiment part, the experiment of carrying out with these chemical compounds proves that further it can suppress glucose uptake in the active and further raising mice of the GSK-3 adipose cell, thereby has proved that the chemical compound according to this formula design is hopeful to produce depression effect and therapeutic effect.

Therefore, one aspect of the present invention provides a kind of chemical compound or its pharmaceutically acceptable salt with following general formula:

Wherein:

X, Y, Z and W independently are carbon atom or nitrogen-atoms separately;

A is alkyl or does not exist;

B is for having formula

Electronegative group, wherein L is selected from phosphorus atoms, sulphur atom, silicon atom, boron atom and carbon atom; Q, G and D independently are selected from oxygen and sulfur separately; And E is selected from hydroxyl, alkoxyl, aryloxy group, carbonyl, thiocarbonyl, O-carboxyl, sulfydryl, alkylthio group and arylthio or does not exist.

D is selected from hydrogen, alkyl, tri haloalkyl, cycloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, the heterolipid ring, halogen, hydroxyl, alkoxyl, aryloxy group, sulfydryl, alkylthio group, arylthio, sulfinyl, sulfonyl, cyano group, nitro, azo, amino-sulfonyl, carbonyl, ketone ester, thiocarbonyl, ester, ether, thioether, thiocarbamate, urea, thiourea, the O-carbamoyl, the N-carbamoyl, the O-thiocarbamoyl, the N-thiocarbamoyl, the C-acylamino-, the N-acylamino-, the C-carboxyl, the O-carboxyl, sulfonamido, the trihalomethyl group sulfonamido, amidino groups, guanidine radicals, guanidine alkylation, amino, aminoalkyl and hydrophobic part; And

R ₁, R ₂, R ₃And R ₄Independently be selected from separately hydrogen; lone pair electrons; alkyl; tri haloalkyl; cycloalkyl; thiazolinyl; alkynyl; aryl; heteroaryl; the heterolipid ring; halogen; hydroxyl; alkoxyl; aryloxy group; sulfydryl; alkylthio group; arylthio; sulfinyl; sulfonyl; cyano group; nitro; azo; amino-sulfonyl; carbonyl; ketone ester; thiocarbonyl; ester; ether; thioether; thiocarbamate; urea; thiourea; the O-carbamoyl; the N-carbamoyl; the O-thiocarbamoyl; the N-thiocarbamoyl; the C-acylamino-; the N-acylamino-; the C-carboxyl; the O-carboxyl; sulfonamido; the trihalomethyl group sulfonamido; amidino groups; the amidino groups alkyl; guanidine radicals; guanidine alkylation; amino; aminoalkyl; hydrazine and ammonium ion.

It will be appreciated by those skilled in the art that each substituent group (for example D, G, E and R ₁-R ₄) probability that is positioned to indicate the position depends on substituent quantivalence and chemical compatibility, the position of substitution and other substituent group.Thereby target of the present invention is to comprise all possible substituent group on any position.

Used term " alkyl " refers to comprise the saturated hydrocarbon resin of straight chain group and branched group herein.Preferred alkyl has 1-20 carbon atom.Whenever numerical range for example " 1-20 " described herein all means group and refers to that herein alkyl can contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms etc. until comprise 20 carbon atoms.The medium sized alkyl that more preferably has 1-10 carbon atom.Most preferably, unless indication is arranged in addition, has the low alkyl group of 1-4 carbon atom.This alkyl can be replacement or unsubstituted.If replace; this substituent group can be, for example hydroxy alkyl; tri haloalkyl; cycloalkyl; thiazolinyl; alkynyl; aryl; heteroaryl; the heterolipid ring; halogen; hydroxyl; alkoxyl; aryloxy group; sulfydryl; alkylthio group; arylthio; sulfinyl; sulfonyl; cyano group; nitro; azo; sulfonyl; sulfinyl; amino-sulfonyl; ketone ester; carbonyl; thiocarbonyl; ester; ether; thioether; thiocarbamate; urea; thiourea; the O-carbamoyl; the N-carbamoyl; the O-thiocarbamoyl; the N-thiocarbamoyl; the C-acylamino-; the N-acylamino-; the C-carboxyl; the O-carboxyl; the trihalomethyl group sulfonamido; amidino groups; the amidino groups alkyl; guanidine radicals; guanidine alkylation; amino; aminoalkyl; hydrazine and ammonium ion.

" cycloalkyl " refers to full carbon monocyclic groups or condensed ring (be a plurality of rings share adjacent carbon atom to) group, and wherein one or more rings do not have total conjugated π-electron system.The example of cycloalkyl is but is not limited to cyclopropane, Tetramethylene., Pentamethylene., cyclopentenes, cyclohexane extraction, cyclohexadiene, cycloheptane, cycloheptatriene and diamantane (obsolete).Cycloalkyl can be replacement or unsubstituted.If replace, this substituent group can be for example alkyl; hydroxy alkyl; tri haloalkyl; cycloalkyl; thiazolinyl; alkynyl; aryl; heteroaryl; the heterolipid ring; halogen; hydroxyl; alkoxyl; aryloxy group; sulfydryl; alkylthio group; arylthio; sulfinyl; sulfonyl; cyano group; nitro; azo; sulfonyl; sulfinyl; amino-sulfonyl; ketone ester; carbonyl; thiocarbonyl; ester; ether; carboxyl; thiocarboxyl group; thioether; thiocarbamate; urea; thiourea; the O-carbamoyl; the N-carbamoyl; the O-thiocarbamoyl; the N-thiocarbamoyl; the C-acylamino-; the N-acylamino-; the C-carboxyl; the O-carboxyl; sulfonamido; the trihalomethyl group sulfonamido; amidino groups; the amidino groups alkyl; guanidine radicals; guanidine alkylation; amino; aminoalkyl; hydrazine and ammonium ion.

The alkyl of definition during " thiazolinyl " refers to as mentioned, it is comprised of at least two carbon atoms and at least one carbon-to-carbon double bond.

The alkyl of definition during " alkynyl " refers to as mentioned, it is comprised of at least two carbon atoms and at least one carbon-to-carbon triple bond.

" aryl " refers to have the full carbon monocyclic groups of total conjugated π-electron system or condensed ring (be a plurality of rings share adjacent carbon atom to) group.The example of aryl is but is not limited to phenyl, naphthyl and anthryl.Aryl can be replacement or unsubstituted.If replace, this substituent group can be for example alkyl; hydroxy alkyl; tri haloalkyl; cycloalkyl; thiazolinyl; alkynyl; aryl; heteroaryl; the heterolipid ring; halogen; hydroxyl; alkoxyl; aryloxy group; sulfydryl; alkylthio group; arylthio; sulfinyl; sulfonyl; cyano group; nitro; azo; sulfonyl; sulfinyl; amino-sulfonyl; ketone ester; carbonyl; thiocarbonyl; ester; ether; carboxyl; thiocarboxyl group; thioether; thiocarbamate; urea; thiourea; the O-carbamoyl; the N-carbamoyl; the O-thiocarbamoyl; the N-thiocarbamoyl; the C-acylamino-; the N-acylamino-; the C-carboxyl; the O-carboxyl; sulfonamido; the trihalomethyl group sulfonamido; amidino groups; the amidino groups alkyl; guanidine radicals; guanidine alkylation; amino; aminoalkyl; hydrazine and ammonium ion.

" heteroaryl " refers to monocyclic groups or condensed ring (be a plurality of rings share adjacent carbon atom to) group, has one or more for example nitrogen, oxygen and sulphur atom in wherein one or more rings, and has in addition total conjugated π-electron system.The example of heteroaryl is but is not limited to pyrroles, furan, thiophene, imidazoles, oxazole, thiazole, pyrazoles, pyridine, pyrimidine, quinoline, isoquinolin and purine.Heteroaryl can be replacement or unsubstituted.If replace, this substituent group can be for example alkyl; hydroxy alkyl; tri haloalkyl; cycloalkyl; thiazolinyl; alkynyl; aryl; heteroaryl; the heterolipid ring; halogen; hydroxyl; alkoxyl; aryloxy group; sulfydryl; alkylthio group; arylthio; sulfinyl; sulfonyl; cyano group; nitro; azo; sulfonyl; sulfinyl; amino-sulfonyl; ketone ester; carbonyl; thiocarbonyl; ester; ether; carboxyl; thiocarboxyl group; thioether; thiocarbamate; urea; thiourea; the O-carbamoyl; the N-carbamoyl; the O-thiocarbamoyl; the N-thiocarbamoyl; the C-acylamino-; the N-acylamino-; the C-carboxyl; the O-carboxyl; sulfonamido; the trihalomethyl group sulfonamido; amidino groups; the amidino groups alkyl; guanidine radicals; guanidine alkylation; amino; aminoalkyl; hydrazine and ammonium ion.

" heterolipid cyclic group " refers to have for example monocyclic groups or the condensed ring group of one or more atoms of nitrogen, oxygen and sulfur in wherein one or more rings.Also can have one or more pairs of keys in one or more rings.But, do not have total conjugated π-electron system in one or more rings.The heterolipid cyclic group can be replacement or unsubstituted.If replace, this substituent group can be for example lone pair electrons; alkyl; hydroxy alkyl; tri haloalkyl; cycloalkyl; thiazolinyl; alkynyl; aryl; heteroaryl; the heterolipid ring; halogen; hydroxyl; alkoxyl; aryloxy group; sulfydryl; alkylthio group; arylthio; sulfinyl; sulfonyl; cyano group; nitro; azo; sulfonyl; sulfinyl; amino-sulfonyl; ketone ester; carbonyl; thiocarbonyl; ester; ether; carboxyl; thiocarboxyl group; thioether; thiocarbamate; urea; thiourea; the O-carbamoyl; the N-carbamoyl; the O-thiocarbamoyl; the N-thiocarbamoyl; the C-acylamino-; the N-acylamino-; the C-carboxyl; the O-carboxyl; sulfonamido; the trihalomethyl group sulfonamido; amidino groups; the amidino groups alkyl; guanidine radicals; guanidine alkylation; amino; aminoalkyl; hydrazine and ammonium ion.Representative example is piperidines, piperazine, oxolane, Pentamethylene oxide., morpholino etc.

" lone pair electrons " refer to not participate in into the pair of electrons are of key.Lone pair electrons only exist when X, Y, Z or W are unsubstituted nitrogen-atoms.

" hydroxyl " refers to-OH.

" azo " refers to-N=N.

" alkoxyl " refer to-the O-alkyl and-the O-cycloalkyl, as defined herein.

" aryloxy group " refer to-the O-aryl and-the O-heteroaryl, as defined herein.

" sulfydryl " refers to-SH.

" alkylthio group " refer to-the S-alkyl and-the S-cycloalkyl, as defined herein.

" arylthio " refer to-the S-aryl and-the S-heteroaryl, as defined herein.

" carbonyl " refer to-C (=O)-and R ', wherein R ' is hydrogen, alkyl, thiazolinyl, cycloalkyl, aryl, heteroaryl (by encircling upper carbon atom bonding) or heterolipid cyclic group (by encircling upper carbon atom bonding), as defined herein.

" aldehyde " refers to carbonyl, and wherein R ' is hydrogen.

" thiocarbonyl " refer to-C (=S)-and R ', wherein R ' is the definition such as this paper R '.

" C-carboxyl " refer to-C (=O)-O-R ', wherein R ' is for as defined herein.

" O-carboxyl " refer to R ' C (=O)-O-, wherein R ' as defined herein.

" carboxylic acid " refers to the C-carboxyl, and wherein R is hydrogen.

" halogen " refers to fluorine, chlorine, bromine or iodine.

" trihalomethyl group " refers to-CX ₃, wherein X is halogen, as defined herein.

" trihalomethyl group sulfonyl " refers to X ₃CS (=O) ₂-, wherein X is halogen, as defined herein.

" sulfinyl " refer to-S (=O)-R ', wherein R ' is for as defined herein.

" sulfonyl " refer to-S (=O) ₂-R ', wherein R ' is for as defined herein.

" S-sulfonamido " refer to-S (=O) ₂-NR ' R ", and R ' as defined herein and R " be the definition such as this paper R '.

" N-sulfonamido " refer to R ' S (=O) ₂-NR ", wherein R ' and R " is for as defined herein.

" trihalomethyl group sulfonamido " refers to X ₃CS (=O) ₂NR ', wherein R ' and X are for as defined herein.

" O-carbamoyl " refer to-OC (=O)-NRR ", wherein R ' and R are " as defined herein.

" N-carbamoyl " refers to R " OC (=O)-and NR ', wherein R ' and R are " as defined herein.

" O-thiocarbamoyl " refer to-OC (=S)-NR ' R ", wherein R ' and R are " as defined herein.

" N-thiocarbamoyl " refers to R, and " (=S) NR ', wherein R ' and R are " as defined herein for OC.

" amino " refers to-NRR ", wherein R ' and R are " as defined herein.

The alkyl that is replaced by amino of definition during " aminoalkyl " refers to as mentioned.Preferred alkyl is terminal to be replaced by amino.

" C-acylamino-" refer to-C (=O)-NR ' R ", wherein R ' and R are " as defined herein.

" N-acylamino-" refer to R ' C (=O)-NR ", wherein R ' and R are " as defined herein.

" urea " refer to-NR ' C (=O)-NR " R " ', wherein R ' and R " as defined herein and R " ' such as R ' or the R " definition of this paper.

" guanidine radicals " refers to-R ' NC (=NR " ")-NR " R " ', wherein R ', R " and R " ' as defined herein and R " " as R ', the R " or R " of this paper ' definition.

" guanidine alkylation " refers to the alkyl that replaced by guanidine radicals in as defined herein the term.The preferred alkyl end is replaced by guanidine radicals.

" amidino groups " refer to R ' R " NC (=NR " ")-, wherein R ', R ", R " ' and R " " is as defined herein.

" amidino groups alkyl " refers to the alkyl that replaced by amidino groups in the term as defined herein.The preferred alkyl end is replaced by amidino groups.

" nitro " refers to-NO ₂

" cyano group " refers to-C=N.

Term " ketone ester " description-C (=O)-C (=O)-O-.

Term " thiourea " description-NR '-C (=S)-and NR '-, wherein R ' and R are " as defined herein.

NR '-NR described in term " hydrazine " ", wherein R ' and R are " as defined herein.

Term " ammonium ion " is described (NR ' R " R " ')+, wherein R ', R " and R " ' as defined herein.

Therefore the compounds of this invention namely is connected with aromatic ring (wherein X, Y, Z and W are carbon atom) or the hetero-aromatic ring (wherein at least a among X, Y, Z and the W is nitrogen-atoms) of electronegative group take a kind of rigid structure as the basis.Because the unique texture of this structure by providing the electronegative group that does not have stearic obstruction and have or identical geometry similar with phosphate to simulate the GSK-3 substrate, so these chemical compounds can suppress the GSK-3 activity.

Phrase used herein " electronegative group " refers to ionogen with " positively charged group ", typically has separately at least one negative or positive electrostatic charge after its ionization in aqueous medium.This electric charge group can its ionized form (ionized form) or ionizing before form (pre-ionized form) be present in the compounds of this invention.

Electronegative group of the present invention has the above structure of definition, thereby this positively charged group can be positive charge ion itself (such as ammonium ion) or any group (such as alkyl, cycloalkyl, aryl etc.) of being replaced by positive charge ion (such as secondary ammonium ion, tertiary amine ion or quaternary ammonium ion, Ionized aminoalkyl etc.).

Preferred electronegative group is phosphate, and L is phosphorus atoms in the following formula like this, Q, G and the D oxygen of respectively doing for oneself.Preferred, E is hydroxyl.Selectable, have another negative charge thereby hydroxyl is also ionizable.

Selectable, this electronegative group can be D2EHDTPA group, sulfate group or sulfonic acid group, boric acid base group (borate) or boride group (boronate) etc. in the following formula.

This electronegative group preferably is connected in aromatic ring/hetero-aromatic ring through alkyl, and A is alkyl in the following formula like this, preferred unsubstituted alkyl, and more preferably methyl.

The rigidity that is directly connected on the ring with electronegative group is opposite, and electronegative group is connected on the ring and can rotates freely through alkyl.The rotating freely property of this electronegative group is favourable because it interacts electronegative group and enzyme binding site easily.

Although should be noted that directly or indirectly to link on aromatic ring or the hetero-aromatic ring through simple program phosphate ester of the present invention or any other electronegative group to be effectively and to produce simple chemical compound on the structure, only synthesized so far a limited number of this chemical compound.These chemical compounds comprise pyridoxal 5-phosphate, benzyl phosphate ester, phenyl phosphate and a limited number of its derivant (pyridoxal 5-phosphate, the benzyl phosphate ester of replacement and the phenyl phosphate of replacement that for example replace).If do not have so far biological activity relevant with this chemical compound, just can not excite those of ordinary skills to prepare this chemical compound.But this aspect according to the present invention, chemical compound has been got rid of any present known compound that following formula comprises.

Such as above-mentioned points for attention, the basic structure of the compounds of this invention is aromatic ring or hetero-aromatic ring.

Yet since preferably have one and more preferably two positively charged groups that are positioned at electronegative group side, therefore preferred ring is hetero-aromatic ring, and at least a of X, Y, Z and W is nitrogen-atoms in following formula like this.Preferred Z or W are nitrogen-atoms.

Further at least two kinds of preferred X, Y, Z and W is nitrogen-atoms, and more preferably X and Y are that nitrogen-atoms or Z and W are nitrogen-atoms, and even more preferably Z and W are nitrogen-atoms.

Well-knownly in this area be, nitrogen-atoms is typical alkalescence in the aromatic ring under neutrallty condition, thereby and therefore it trends towards the protonated positively charged=NH that produces in biological environment ⁺-group.As noted before, it is preferred having one or two chemical compound that is positioned at this positively charged group of electronegative side.

In basic ring as selectable or except positively charged nitrogen atom, preferred R ₁, R ₂, R ₃And R ₄At least a for containing group of at least one amino part.

Phrase used herein " group that contains at least one amino part " refers to contain those above-mentioned groups (such as alkyl, cycloalkyl, aryl etc.) of one or more amino parts, term as defined herein.

The representative example that contains at least one amino part includes but not limited to amino, aminoalkyl, hydrazine, urea, thiourea, amidino groups, acylamino-, carbamoyl, guanidine radicals, guanidine alkylation and amidino groups alkyl, term as defined herein.

Well-knownly in this area be, free amine group is typical alkalescence under neutrallty condition, thereby and therefore it trends towards the protonated positively charged-NH that produces in biological environment ₃ ⁺-group.As noted before, it is preferred having one or two chemical compound that is positioned at this positively charged group of electronegative side.

Therefore, in the amino this group that partly preferably is present in as easy protonated part, amino nitrogen has in fact the part negative charge in the amino part.

Therefore the preferred embodiment that contains the group of at least one amino part includes but not limited to amino, aminoalkyl, hydrazine, amidino groups, amidino groups alkyl, guanidine radicals, guanidine alkylation and amidino groups alkyl, term as defined herein.

The group that contains at least one amino part can self or positively charged group and be present in the compounds of this invention, and wherein at least one amino part ionizes.

As indicated above, positively charged group of the present invention comprises ammonium ion, for example the representative example of positively charged group includes but not limited to, ammonium ion itself (protonated amino) and any as mentioned group with ammonium ion of middle definition, the alkyl that is for example replaced by ammonium ion, cycloalkyl or aryl, guanidine radicals, amidino groups, hydrazine etc.

Particularly preferred positively charged group is the chemical constitution that has derived from the positively charged amino acid side chain, for example lysine, arginine, histidine, proline and derivant thereof, and two kinds of front most preferably.

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

Preferred R ₁And R ₂Or R ₃And R ₄Contain at least one the amino part (for example positively charged group) just like the electronegative group of being positioned at of needs side.

Therefore, preferred chemical compound of the present invention has following formula:

Wherein m is the integer of 1-6; Q ₁And Q ₂Independent is carbon atom or nitrogen-atoms; And G and/or K respectively do for oneself and contain the group (for example positively charged group) of free amine group part.

As indicated above and in the further proof of the following example part, successfully synthesize according to the chemical compound lot of above-mentioned general formula design and to find that it suppresses GSK-3 active.Fig. 3 has shown the chemical constitution of these chemical compounds.Figure 12 and Figure 13 have shown the effectiveness of these chemical compounds as the GSK-3 activity inhibitor, and wherein Figure 14 a and 14b have proved that these chemical compounds are to the useful effect of glucose uptake in the mice adipose cell.

Such as demonstration in Figure 12 and Figure 13 and the following example part, some chemical compounds of having verified do not have positively charged group (for example GS-1 and GS-2), yet these chemical compounds performances are active to the inhibition of GSK-3.But, show further that such as Figure 12 and Figure 13 institute discovery has nitrogen-atoms in basic ring chemical compound is active higher inhibitor, thereby indicated the useful effect of this group.

Therefore, the other preferred chemical compound of the present invention chemical compound that is above-mentioned general formula, wherein X, Y, Z and the W carbon atom of respectively doing for oneself; And R ₃And R ₄At least a for containing the group (for example positively charged group) of an amino part; And D is hydrogen or alkyl.Respectively do for oneself carbon atom and W of X, Y and Z is nitrogen-atoms in the preferred chemical compound.

In the present invention's another preferred embodiment aspect this, described chemical compound has coupled hydrophobic part.

Describe in detail in PCT/IL03/10157 such as the inventor, find to be connected with the inhibition activity that hydrophobic part has improved peptide at the N-of GSK-3 inhibitor peptides end.

Since to be positioned at its C-terminal for the phosphorylation residue in the inhibitor peptides, infer that the compounds of this invention (identical with the inhibitor peptides situation) that comprises the hydrophobic part that is positioned electronegative group distal-most end will bring into play the inhibition activity that has improved.

Therefore, another aspect of the present invention provides has above described general formula, and wherein D is the chemical compound of hydrophobic part.

Used phrase " hydrophobic part " refers to be characterized as hydrophobic any material or residue herein.

Accept extensively such as this area, the covalently bound major part that arrives the material of another kind of material described in term " residue ", and chemical compound is as noted before herein.

Therefore, the residue of the preferred hydrophobic substance of hydrophobic part of the present invention, and preferably covalently is connected in above described chemical compound.

The representative example of the hydrophobic substance that can derive from hydrophobic part of the present invention includes but not limited to saturated alkylene chain, undersaturated alkylene chain, aryl, cycloalkyl and hydrophobic peptide sequence.

Used phrase " alkylene chain " refers to can be saturated or unsaturated hydrocarbon straight chain herein.This alkylene chain can be and replaces or unsubstituted, such as above description about alkyl, and is separated with such as nitrogen, oxygen, sulfur, phosphorus etc. of one or more hetero atoms (heterogamous) between can be further.This alkylene chain preferably includes at least 4 carbon atoms, more preferably at least 8 carbon atoms, and more preferably at least 10 carbon atoms and mat have up to 20,25 even 30 carbon atoms.

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

The preferred embodiment of the alkylene chain of this aspect of the present invention is to comprise the alkylene chain that carboxyl is fatty acid residue.

Available preferred fatty acid includes but not limited to have satisfied fatty acid or the unsaturated fatty acid that surpasses 10 carbon atoms in the context of this article, between preferred 12 carbon atom to 24 carbon atoms, such as but not limited to myristic acid, lauric acid, Palmic acid, stearic acid, oleic acid, linoleic acid, linolenic acid and arachidonic acid etc.

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

The representative example of hydrophobic amino acid residues includes but 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, or its any trim, as indicated above.

Selectable, hydrophobic amino acid residues can comprise by hydrophobic part being added any other amino acid residue of wherein modifying.

Used phrase " amino acid residue " is also interchangeable in this article herein is " aminoacid ", is described in the Amino Acid Unit in the polypeptide chain.Amino acid residue can be natural amino acid residue or modified amino acid residue in the hydrophobic peptide sequence, such as the phrase that defines hereinafter.

The amino acid residue of used phrase " natural amino acid residue " term describing as define in the preamble herein, it comprises 20 seed amino acids a kind of of occurring in nature discovery.

The amino acid residue such as the term that defines in the preamble described in used phrase " amino acid residue of modification " herein, and it is included in the natural amino acid residue that side chain is modified.Thisly be modified to known in the art and for example comprise, in side chain, add functional groups such as but not limited to hydroxyl, amino, carboxyl and phosphate.Unless therefore concrete indication is arranged in addition, this phrase comprises that the chemical modified amino acid comprises amino acid analogue (for example penicillamine, D-P), naturally occurring nonprotein (non-proteogenic) aminoacid (for example nor-leucine), and the chemosynthesis chemical compound with amino acid characteristics known in the art.Term " protein " refers to that this seed amino acid can the metabolic pathway through knowing be incorporated in the protein in cell.

Accordingly, used term " aminoacid " or " amino acids " are interpreted as and comprise these the 20 kinds natural aminoacid that exist herein; Those aminoacid are in vivo post translational modification usually, comprises for example hydroxyproline, phosphoserine and phosphothreonine; And other rare amino acid includes but not limited to AAA, hydroxylysine, isodensmosine, norvaline, nor-leucine and ornithine.And term " aminoacid " comprises D-aminoacid and L-aminoacid, and it is connected to the other aminoacid of at least a term as defined herein through peptide bond or peptide bond analog.

Because hydrophobic part is used for improving unpredictable activity, the known compound that is therefore replaced by hydrophobic part for example phenyl phosphate and pyridoxal 5-phosphate is also included within the scope of this respect of the present invention.

That discuss as mentioned and further proved in the following example part, based on the three dimensional structure design of GSK-3 substrate described the compounds of this invention and therefore be the potential substrate competition inhibitor of GSK-3 activity above.

Therefore, according to another aspect of the invention, provide the method that suppresses the GSK-3 activity, undertaken by the cell of expressing GSK-3 with the above described chemical compound contact that suppresses effective dose.

Used term " inhibition effective dose " is the amount that is enough to suppress the GSK-3 activity of considering mensuration by as known in the art herein.This activity can be phosphorylation activity and/or the autophosphorylation activity of GSK-3.

The method of this aspect of the present invention can be with described chemical compound in the external and/or body exposing cell carry out.This method can be further by carrying out with the further exposing cell of other active component that can change the GSK-3 activity described in detail below.

Suppressing the GSK-3 activity is a kind of approach that improves insulin active in the body.The high activity of GSK-3 infringement insulin function (Eldar-Finkelman etc., 1997) in intact cell.This infringement results from IRS-1 (IRS-1) serine residue by the GSK-3 phosphorylation.To type ii diabetes (non-insulin-dependent diabetes mellitus, NIDDM) patient's studies show that the Glycogensynthase activity significantly reduces among these patients, and detect reduction (Shulman etc., (1990) that insulin suppresses upstream regulation agent protein kinase B (PKB) activation of GSK-3; Nikoulina etc., (1997); Cross etc., (1995).Food rich in fat is induced in diabetes and the responsive mouse epididymis fatty tissue of obesity has the GSK-3 active (Eldar-Finkelman etc., 1999) that significantly improves.The active raising of the GSK-3 of cell inner expression causes the inhibition (Eldar-Finkelman etc., 1996) of Glycogensynthase activity.

The inhibition of GSK-3 activity thereby the useful method that is used for improving insulin active under the insulin dependency disease is provided.Like this, another other aspect of the present invention provides the method that strengthens the insulin signaling conduction, and it is undertaken by the compounds of this invention contact insulin sensitivity cell with the effective dose that above defines.

Used phrase " strengthening insulin signaling conducts " comprises that the phosphorylation that improves the Insulin receptor INSR downstream component reaches with curee or untreated cell are not compared raising glucose uptake speed herein.

The method of this aspect of the present invention can realize by exposing cell in or the body external with the compounds of this invention, and also can pass through to realize with the further exposing cell of insulin.

Give the interior insulin signaling conduction of conjugate enhancing body of the present invention and can be used as clinical terminal monitoring.In theory, the easiest method of observation patient's insulin potentiation is to carry out glucose tolerance test.After the fasting, give patient's glucose and by well known test determination glucose autoblood circulation release rate (namely by the cellular uptake glucose).Glucose clearance speed slowly (comparing with the health volunteer) will be indicated insulin resistance.Give insulin resistance patient inhibitor and do not treat the patient and compare and improved glucose uptake speed.Inhibitor can be given for a long time to the insulin resistance patient, and insulin in the blood circulation, glucose and leptin level (being generally high level) can be measured.Glucose level reduces will indicate inhibitor to strengthen insulin function.Only the reduction of insulin and leptin level not necessarily indicates insulin action to strengthen, but indication disease disease improves by other mechanism.

Chemical compound of the present invention mentioned above can effectively be used for the treatment of any biological disease relevant with GSK-3.

Therefore, the other aspect of the present invention provides the method for a kind for the treatment of biological disease relevant with the GSK-3 activity.According to this aspect of the invention, this method is undertaken by the chemical compound of the present invention of effective dose in the experimenter who needs it treatment mentioned above.

Phrase used herein " with the active relevant biological disease of GSK-3 " comprises any biological disease or medical conditions or the disorder of having differentiated GSK-3 effective active (normal or improper level).This disease or disorder can be by due to the GSK-3 activity or can be simply take GSK-3 as feature.Some aspects that the disease relevant with the GSK-3 activity means these diseases are traceable to the GSK-3 activity.

Term " treatment " comprises cancellation, basically suppresses, reduces or reverses disease or disorderly development herein, basically improves disease or disorderly clinical symptoms or basically prevent disease or disorderly clinical symptoms appearance.These effects can be by for example reducing type ii diabetes glucose uptake speed or prove by the neuronal cell of checking neurodegenerative disease is dead, as hereinafter describing in detail.

The cell that term used herein " gives " to describe the compounds of this invention and disease or disorderly impact is put together, and chemical compound can affect GSK-3 activity in these cells by this way.The compounds of this invention can give through medically acceptable any approach.Route of administration can maybe will be treated damage according to disease, disease and be decided.Possible route of administration comprises injection, through the parenteral approach for example in administration in intravascular administration, intravenous injection, intra-arterial injection, subcutaneous injection, intramuscular injection, the tumor, intraperitoneal, the ventricle, in the epidural, cerebrovascular or other approach, also comprise oral, per nasal, medicament for the eyes, per rectum, local application or through sucking.The present invention is also particularly including sustained-release administration, by the method for injecting or easily losing implants (erodibleimplants) such as durative action preparation.Also can be through intraarticular, internal rectum, intraperitoneal, intramuscular, subcutaneous, aerosol inhalation route administration.If systemic treatment, can be oral or parenteral give chemical compound, for example in intravenous, intramuscular, subcutaneous, the eye socket, in the capsule, administration in intraperitoneal or the brain, as long as provide with the composition forms that is suitable for chemical compound is effectively introduced target cell, as hereinafter describing in detail.

Phrase used herein " effective dose in the treatment " is described and to be given that individual amount is enough to cancel, basically suppresses, reduction or reverse and the active relevant disease progression of GSK-3, basically improves the clinical symptoms of disease or basically prevents the clinical symptoms appearance of this disease.The GSK-3 activity can be the GSK-3 kinase activity.Can directly measure amount of suppression by the inhibition of measuring the GSK-3 activity, perhaps for example when required effect during for the effect of the downstream activity of GSK-3 activity in to the approach that comprises DSK-3, can measure inhibition by measuring downstream effect.Like this, for example suppress GSK-3 and cause stopping of Glycogensynthase phosphorylation, the effect of described chemical compound can comprise the effect to insulin dependency or insulin relational approach, and gives described chemical compound in this point and can make glucose uptake bring up to optimum level.Equally, the inhibition of GSK-3 causes the disappearance of the protein phosphorylation of further biological activity needs, Protein tau for example, and can give described chemical compound until the polymerization of the Protein tau of phosphorylation stops basically.Thereby the inhibition of GSK-3 activity will partly depend on the approach that relates to the GSK-3 activity or the natural inhibition of process, and the depression effect of the GSK-3 activity that has in the biotic environment that provides.

With the amount that consists of the chemical compound of amount of suppression will depend in various degree the half life of chemical compound in this parameter such as chemical compound and usefulness thereof, the body, the development speed that will treat disease or biological disease, disease to the sensitivity of therapeutic dose or mode of administration, preparation, the doctor in charge to the assessment of medical conditions, and other correlative factor, and patient's substantially health status, and other consider for example former medication of other treatment, the inhibition activity that maybe will affect chemical compound maybe will affect any therapeutic co-administered of GSK-3 activity, or by the active approach that mediates of GSK-3.The expection amount of suppression will be quite wide scope and can measure by routine test.

As above discussing in detail, GSK-3 relate to multiple biological approach and therefore the method for this aspect of the present invention can be used for treating multiple biological disease, as hereinafter describing in detail.

GSK-3 relates to the insulin signaling pathway, and therefore in an example, the method for this aspect of the present invention can be used for treating any insulin dependency disease.

Because adipose cell (Pre-adipocytes) was divided into adipose cell before known GSK-3 inhibitor suppressed, in another example, the method for this aspect of the present invention can be used for treatment of obesity.

In another example, the method for this aspect of the present invention can be used for treating diabetes, particularly non-insulin-dependent diabetes mellitus.

Diabetes are a kind of multi-pathogenesis constitutional (heterogeneous primary disorder) disorder of carbohydrate metabolism, are usually directed to insulin deficit or insulin resistance or complicated Etiology factor that the two all has.The I type, the teenager morbidity, insulin dependent diabetes mellitus (IDDM) is present in the endogenous insulin secretion capacity seldom or the patient who does not have.These patients finally develop into extreme hyperglycemia and instant survival (immediate survival) relies on exogenous treating diabetes fully.The II type, or grow up morbidity or non-insulin-dependent diabetes mellitus be present in the patient who keeps some endogenous insulin secretion capacities, but the existing insulin deficit of thumping majority patient has again insulin resistance.Suffer from non-insulin-depending type, type ii diabetes (NIDDM) all insulin patients' of the U.S. about 95%, and therefore such diabetes are reasons of the difficult medical problem of thumping majority in the diabetes.Insulin resistance is that potential feature and this metabolic deficiency of NIDDM causes diabetic syndrome.Insulin resistance can be not enough owing to Expression of Insulin Receptor, the insulin binding affinity reduces or in any caused by abnormal (seeing U.S. Patent No. 5,861,266) of any step of insulin signaling pipeline.

The compounds of this invention can followingly be used for the treatment of the type ii diabetes of suffering from the type ii diabetes patient: give the chemical compound of effective dose on the patient treatment, and monitor for example blood sugar level of clinical indices (clinicalmarkers).The compounds of this invention is following type ii diabetes for the prevention experimenter further: give the chemical compound that the patient prevents upper effective dose, and monitor for example IRS-1 phosphorylation of clinical indices.

Measure the treatment of diabetes by the standard medical method.A target for the treatment of diabetes is that blood sugar level is dropped near normal level as far as possible safely.Usually the target of setting is 80-120 milligram/decilitre (mg/dl) and the front 100-140mg/dl that sleeps ante cibum.Individual physician can according to other factors for example patient's frequency that hypoglycemic reaction occurs this patent is arranged different target.Useful medical test comprises the check to blood sugar level in patient's blood and the urine, the check (HbA of glycosylated hemoglobin level (glycated hemoglobin level) _1cThe mensuration of the 2-3 in whole past monthly average blood sugar level, be 4-6% normal range), the check of cholesterol and fat level, and the check of urine protein level.This check is standard test well known by persons skilled in the art (referring to for example American Diabetes Association, 1998).Suffer from diabetic eye disease, kidney disease or sacred disease by less patient in the scheme and also can measure successful therapeutic scheme.

Therefore, in the particular of the method for the present invention aspect this, provide the method for the treatment of non-insulin-dependent diabetes mellitus: make a definite diagnosis the patient at the non-insulin-dependent diabetes mellitus commitment.The compounds of this invention is made enteric coated capsule.The patient directly takes a slice at every turn after meal be used for to stimulate the insulin signaling pathway, thereby and control carbohydrate metabolism level reach the level that need not give exogenous insulin.

Further discuss as mentioned and the exercise question of same Applicant be that the instant application of conduct of " Glycogen SynthaseKinase-3 Inhibitors " is verified in the pct international patent application of on the same day application, the GSK-3 inhibition is relevant with affective disorder.Therefore, in another example, the method for this aspect of the present invention can be used for treating affective disorder for example unipolar disorders (for example depression) and bipolar disorder (for example manic depressive illness).

Because think that GSK-3 also is important participation material in the pathogenesis of neurodegenerative disorders and disease, the method for this aspect of the present invention can further be used for the treatment of various such disorders and disease.

In an example, stop neuronal cell dead because suppress GSK-3, the method for this aspect of the present invention can be used for treating the neurodegenerative disorders that is caused by the event that causes neuronal cell death.This event can be, and for example cerebral ischemia, apoplexy, traumatic brain injury or antibacterial infect.

In another example, because the GSK-3 activity relates to multiple central nervous system disorder and neurodegenerative disease, the method for this aspect of the present invention can be used for treating multiple chronic neurodegenerative disease such as but not limited to Alzheimer's disease, Huntington Chorea, parkinson disease, the relevant dementia of AIDS, amyotrophic lateral sclerosis (AML) and multiple sclerosis.

Discuss as mentioned, the GSK-3 activity is particularly related to the pathogenesis of Alzheimer's disease.Therefore, in a representative embodiment of the method for the present invention aspect this, treatment Alzheimer patient's method is provided: suffer from the compounds of this invention that the Alzheimer patient suppresses the tau hyperphosphorylation of GSK-3-mediation to making a definite diagnosis, it is made and passes blood brain barrier (BBB) preparation.The protein monitoring phosphorylation polymer that periodic analysis separates from patient's brain cell, because this protein exists with the form of tau phosphorylation on the SDS-PAGE gel, known is the feature that disease occurs and develops.The phosphorylation form of the Protein tau that the dosage that must adjust chemical compound exists with minimizing.

Therefore GSK-3 also relates to mental case such as schizophrenia, and the method for this aspect of the present invention can further be used for the treatment of for example schizophrenia of psychosis or disorder.

The method of this aspect of the present invention can be undertaken by jointly giving one or more other active component that can change the GSK-3 activity of experimenter.

" jointly giving " used herein describes the compounds of this invention with other active component (being also referred to as active medicine or medicine) coupling.This other active component can be is of value to any medicine for the treatment of patient disease.Jointly give and to give simultaneously, for example by giving the mixture of chemical compound and medicine, maybe can separately give chemical compound and active medicine, for example at short notice.Jointly give also to comprise and give successively chemical compound and one or more other medicines.Can give to give other medicine before or after the chemical compound.Dosage treatment can be single dose therapeutic regimen or multiple dose therapeutic regimen.

Other active component can be insulin.

Preferred other active component can suppress the GSK-3 activity, so the other active component of the present invention can be any GSK-3 inhibitor except the compounds of this invention, the small peptide GSK-3 inhibitor described in WO01/49709, PCT/IL03/01057 and the U.S. Patent Application Publication No. 20020147146A1 for example.Selectable, the GSK-3 inhibitor can be, for example lithium, valproic acid and/or lithium ion.

Selectable, other active component can be can reduce the active component that GSK-3 expresses.

The medicine that downward modulation GSK-3 expresses refers to affect any medicine that GSK-3 synthesizes (deceleration) or degraded (acceleration) in the mRNA level or at protein level.For example, be designed for the little interference polynucleotide molecule of reducing the GSK-3 expression and can be used as active component other in embodiment of the present invention.

A kind of example that can reduce the little interference polynucleotide molecule of GSK-3 expression is siRNA or siRNA, for example, and morpholino antisense oligonucleotide (Munshi CB, Graeff R, Lee HC, J Biol Chem 2002Dec 20 that Munshi etc. describe; 277 (51): 49453-8), it comprises that the RNA by former description disturbs (RNAi) mechanism (Hutvagner anmore (2002) Curr.Opin.Genetics and Development 12:225-232) to instruct the double chain oligonucleotide of the particular sequence degraded of mRNA.

Phrase used herein " double chain oligonucleotide " refers to oligonucleotide structure or by strand self complementary nucleotide chain or the analogies by at least two complementary nucleotide chain formation." double chain oligonucleotide " of the present invention can be comprised of double-stranded RNA (dsRNA), DNA RNA hybrid, single stranded RNA (ssRNA), the RNA (be partially purified RNA, substantially pure RNA) that separates, synthetic RNA and the RNA of recombinant production.

Preferred specific little interference double chain oligonucleotide of the present invention is a kind of oligoribonucleotide that mainly is comprised of ribonucleic.

Www.ambion.comIn the manufacturing specification of the double chain oligonucleotide that can mediate rna disturbs is provided.

Therefore, little interference polynucleotide molecule of the present invention can be RNAi molecule (rnai molecule).

Selectable, little interference polynucleotide molecule can be the oligonucleotide for example special antisense molecule of GSK-3-or ribozyme molecule (rybozyme molecule), hereinafter further describes.

Antisense molecule is oligonucleotide, contains two or more chemically unique zones, and each is at least one nucleotide composition freely.These oligonucleotide typically contain at least one zone, and wherein oligonucleotide is modified so that oligonucleotide improves the resistance to the ribozyme degraded, increase cellular uptake and/or raising to the target polynucleotide binding affinity.The other zone of oligonucleotide can be used as the substrate of the enzyme that can shear RNA:DNA or RNA:RNA crossbred.This example comprises RNase H, and it is the cell endonuclease of shearing the RNA chain of RNA:DNA duplex.Therefore, activate RNase H and cause shearing the RNA target, thereby significantly improve the effectiveness of oligonucleotide inhibition of gene expression.Natural, when using chimeric oligonucleotide, compare with the phosphorothioate deoxy-oligonucleotide that hybridizes to identical target area, usually can obtain equal result with short oligonucleotide more.By gel electrophoresis and if needed, but the shearing of associated nucleotide hybridization technique conventional sense RNA target known in the art.

Antisense molecule of the present invention can consist of the combinative structure of the oligonucleotide of two or more aforesaid oligonucleotide, modification.Provide the representative United States Patent (USP) of the preparation of this hybrid structure to include but not limited to, 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 and 5,700,922, each full content is incorporated herein by reference.

Ribozyme (rybozyme) molecule is increasingly extensive for the sequence-specific inhibition of gene expression by shearing mRNA.Several ribozyme sequences can be fused to oligonucleotide of the present invention.These sequences include but not limited to that specificity suppresses the ANGIOZYME of VEGF-R (vascular endothelial growth factor receptor) formation of key component in the blood vessel development ways, with the ribozyme for selective destruction hepatitis C virus (HCV) RNA design be HEPTAZYME (RybozymePharmaceuticals, Incorporated-WEB home page).

Further selectable, little interference polynucleotide molecule of the present invention can be the DNA ribozyme.

The DNA ribozyme is the catalytic nucleic acid molecule of strand.The Universal Model (" 10-23 " model) of DNA ribozyme has been proposed." 10-23 " DNA ribozyme has the catalytic domain of 15 deoxyribonucleotides, and both sides are the substrate differential threshold of two 7-9 deoxyribonucleotides of respectively doing for oneself.Such DNA ribozyme can be effectively at purine: the pyrimidine node is sheared its substrate RNA (Santoro, S.W.﹠amp; Joyce, G.F.Proc.Natl, Acad.Sci.USA 199; For rev of DNAzyme is referring to Khachigian, LM Curr Opin Mol Ther 2002; 4:119-21).

Joyce etc. are at U.S. Patent number 6,326,174 interior synthetic, the structure of genetic engineering DNA ribozyme of identification strand and double-stranded target shearing site and the examples of amplification of disclosing.Recently the DNA ribozyme of observing for the Human uPAR similar Design suppresses the urokinase receptor expression, and (Itoh etc., 20002, Abstract 409, AnnMeeting Am Soc Gen Ther successfully to suppress in vivo the colon cancer cell transfer Www.asgt.org).In another application, express in the leukaemia with the inhibition oncogene of the DNA ribozyme success of bcr-abl oncogene complementation, and in CML and ALL situation, reduce the relapse rate of autologous bone marrow transplantation.

Can be according to any oligonucleotide synthesis method known in the art oligonucleotide that for example enzymatic is synthetic or the solid phase synthesis generation designs according to the inventive method.The equipment and the reagent that are used for solid phase synthesis are commercially available, for example available from Applied Biosystems.This synthetic any other method also can adopt; The reality of oligonucleotide is synthetic to be fully in those skilled in the art's limit of power.

Since be effective medicine, and because the active component that usually requires to treat individual effective dose is used in treatment, the compounds of this invention preferably is present in the Pharmaceutical composition as active component, and described Pharmaceutical composition further comprises to be made chemical compound conveniently be treated individuality and may promote active component to be discharged into the pharmaceutically acceptable carrier of target tissue or cell.

Therefore, another other aspect of the present invention provides and has comprised as the compounds of this invention of active component and the Pharmaceutical composition of pharmaceutically acceptable carrier.

Hereinafter, phrase " pharmaceutically acceptable carrier " and " the upper acceptable carrier of physiology " refer to the patient is not had significant stimulation and do not weaken the biological activity of the chemical compound that gives and carrier or the diluent of biological property.The example of carrier for but be not limited to the mixed liquor of propylene glycol, saline, emulsion, organic solvent and water.

This paper term " excipient " refers to join make in the Pharmaceutical composition and gives more easily inert substance of chemical compound.The example of excipient includes but not limited to calcium carbonate, calcium phosphate, various saccharide and various starch, cellulose derivative, gelatin, vegetable oil and Polyethylene Glycol.

Pharmaceutically acceptable carrier can comprise further that other medicines are such as but not limited to absorption delay agent, antibacterial, antifungal, antioxidant, binding agent, buffer agent, filler, cation lipid agent (cationic lipid agents), coloring agent, diluent, disintegrating agent, dispersant, emulsifying agent, excipient, flavoring agent, fluidizer, isotonic agent, liposome, microsome, solvent, sweeting agent, viscosity aromatics modifier, wetting agent and skin penetration promoter.

At " Remington ' s Pharmaceutical Sciences, " Mack PublishingCo., Easton, PA can find drug preparation technique and medicine-feeding technology in the latest edition, and it is incorporated herein by reference.

Suitable route of administration can comprise, for example oral, rectum, through mucous membrane, pass medicine or parenteral is passed medicine through skin, enteral, comprise intramuscular injection, subcutaneous injection and intramedullary injection, also comprise intrathecal injection, the directly interior injection of ventricle, intravenous injection, peritoneal injection, nasal injection or intraocular injection.

Can make Pharmaceutical composition of the present invention by method well known in the art, for example by routine mixing, dissolving, granulation, lozenge manufacturing, water fly, emulsifying, encapsulate, embedding (entrapping) or lyophilization.

Available conventional method uses one or more to promote chemical compound to make the Pharmaceutical composition that the pharmaceutically pharmaceutically acceptable carrier preparation that comprises excipient and adjuvant of spendable preparation meets purposes of the present invention.Compositions can be made into delivery form for example aerosol pass medicine form, aqueous solution, bolus, capsule, colloid, slow releasing agent, durative action preparation, solvable powder, drop, Emulsion, erodable (erodible) implant, gel, gel capsule, granule, injection, can absorb solution, inhalation solution, lotion, oil solution, pill, suppository, ointment, suspensoid, slow releasing preparation, syrup, tablet, tincture, medicinal external emulsifiable paste, transdermal delivery form.Determine suitable preparation according to selected route of administration.

Be that injection uses, the compounds of this invention can be made into aqueous solution, preferably at the buffer of physical compatibility for example Hank ' s solution, Ringer's solution or contain or do not contain for example normal saline of propylene glycol, Polyethylene Glycol of organic solvent.Be mucosal, use penetrating agent in the preparation.This penetrating agent is as known in the art.

Be oral administration, can be combined with pharmaceutically acceptable carrier well known in the art by chemical compound and be easy to the preparation chemical compound.This carrier can make the compounds of this invention make tablet, pill, lozenge, capsule, liquid preparation, gel, syrup, unguentum, suspensoid etc., and it is oral to be used for the patient.The pharmaceutical formulation that can use the solid excipient preparation to orally use is chosen wantonly and grind the gained mixture after being added suitable required adjuvant, and the preparation granulate mixture is to obtain tablet or lozenge nuclear.Suitable excipient especially filler for example steamed bun stuffed with sugar draw together lactose, sucrose, mannitol or sorbitol; Cellulose preparation for example corn starch, wheaten starch, rice starch, potato starch, gelatin, tragakanta, methylcellulose, hydroxypropyl emthylcellulose, sodium carboxymethyl cellulose (sodium carbomethylcellulose) and/or physiology is gone up for example polyvinylpyrrolidone (PVP) of acceptable macromolecular material.Can add disintegrating agent, for example crosslinked polyvinylpyrrolidone, agar or alginic acid or its salt sodium alginate for example if needed.

Provide lozenge nuclear (dragee cores) with suitable coating.For this purpose, can use to choose wantonly and contain arabic gum, Pulvis Talci, polyvinylpyrrolidone, carbopol gel, Polyethylene Glycol, titanium dioxide, lacquer solution and suitable organic solvent or the concentrated sugar solution of solvent mixture.Can add dyestuff or pigment as differentiating or the different active component dosage that form of expression to tablet or dragee coatings.

The Pharmaceutical composition that can orally use comprises that pushing that gelatin makes agree with (push-fit) capsule, also comprises softness, seal capsule that the plasticizer of gelatin and for example glycerol or sorbitol is made.Push and agree with capsule and can contain active component, with filler lactose for example, binding agent is starch for example, and lubricant is Pulvis Talci or magnesium stearate and optional stabilizing agent composition mixture for example.In soft capsule, active component solubilized or be suspended in the suitable liquid, for example fatty oil, liquid paraffin or liquid macrogol.In addition, can add stabilizing agent.All oral Preparations should be made the dosage that is suitable for selected route of administration.

Be the buccal administration, compositions can adopt tablet that conventional method makes or the form of lozenge.

For passing through inhalation, for example dichlorodifluoromethane, Arcton 11, dichlorotetra-fluoroethane or carbon dioxide give the compounds of this invention with aerosol form easily from pressurized package or nebulizer release thereby use suitable propellant.In the pressurised aerosol situation, can determine that dosage unit is to discharge the amount through metering by the valve that provides.Being used for for example gelatine capsule of inhaler or insufflator and cartridge case can be made into and contain for example mixture of powders of lactose or starch of this composition and suitable powder substrate.

Chemical compound described herein can be made into the parenteral form, for example by bolus injection or continuous infusion.Ejection preparation can unit dose, for example in ampoule or in multi-dose container and the form of optional additional antiseptic exist.Compositions can be suspension, solution or the emulsion that solvent is oiliness or aqueous, and can contain reagent preparation for example suspending agent, stabilizing agent and/or dispersant.

The Pharmaceutical composition that is used for parenteral comprises the aqueous solution of water-soluble form chemical compound.In addition, the suspensoid of chemical compound can be made into suitable oily injection suspension form.Suitable lipophilic solvent or carrier comprise for example Oleum Sesami of fatty oil, or synthetic fatty acid ester for example ethyl oleate, triglyceride or liposome.Moisture injection suspension can contain the material that is improved suspensoid viscosity, for example sodium carboxymethyl cellulose, sorbitol or glucosan.Choose wantonly, this suspensoid also can contain suitable stabilizing agent or the medicine that is improved the active component dissolubility and prepares highly concentrated solution.

Selectable, chemical compound also can be made into powder type, uses front with for example aseptic apirogen water reconstruct of suitable carrier.

The compounds of this invention for example also can use conventional suppository bases, and for example cocoa butter or other glyceride are prepared as rectum with compositions for example suppository or enema,retention.

Described Pharmaceutical composition also can comprise colloid bearer or the excipient of suitable solid herein.The example of this carrier or excipient includes but not limited to for example Polyethylene Glycol of calcium carbonate, calcium phosphate, various saccharide, starch, cellulose derivative, gelatin and macromolecular material.

The Pharmaceutical composition that is suitable for using in the context of the invention comprises the compositions of the amount that contains the chemical compound that effectively accomplishes the end in view.In particular, effective dose refers to that the amount useful effect of chemical compound is the symptom of disease or prolongation curee's life cycle in the treatment.

The effective dose of measuring in the treatment is those skilled in the art's knacks, especially according to disclosing of providing in detail herein.

For any active component is used in the inventive method, can at first estimate the upper effective dose for the treatment of or dosage from cell culture and/or active animal test.This data is useful dosage among the Accurate Measurement people more.

Dosage can change according to dosage form and the used route of administration used.Each doctor can consider that patient's situation selects appropriate preparation, route of administration and dosage (see for example Fingl, etc., 1975, in " The Pharmacological Basis of Therapeutics ", Ch.1 is p.1).

If needed, the present composition may reside in packing or the distributor, the medicine box of FDA approval for example, and it can contain one or more unit dosage forms that contains chemical compound.Manner of packing for example comprises for example blister package of metal forming or plastic foil (blister pack).Packing or distributor can add the medication instruction book.Packing or distributor also can add the points for attention relevant with container, stipulate manufacturer, drug use or sale with the form of government department's approval, composition forms or human or the form for animals of points for attention reflection government permission.This points for attention for example can have FDA Food and Drug Administration's approval to be used for the label of prescription drugs or the approved products plug-in unit is arranged.The compositions that comprises the compounds of this invention that suitable pharmaceutical carrier is advanced in preparation also can prepare, put into suitable container, and the indication of labelling treatment.The indication of indication on label can comprise, any biological disease relevant with the GSK-3 activity of for example above listing.

Therefore, Pharmaceutical composition of the present invention can be packaged in the packaging material and on described packaging material or in stamp sign, be used for the treatment of or the active relevant biological disease of prevention and GSK-3.

As noted before, Pharmaceutical composition of the present invention can further comprise the other active component that can disturb the GSK-3 activity.

According to the test that is not intention restriction the following example of the present invention, another object of the present invention, advantage and novel feature to those of ordinary skills will become obviously.In addition, there is experiment support each different embodiments of the present invention as noted before and that the following claims part is claimed and each aspect in the following example.

Embodiment

With reference to the following example, in unrestriced mode the present invention is described with above description.

Material and experimental technique

Material:

Peptide, Genemed Synthesis Inc synthesizes (San Francisco, CA).

Radioactive substance is available from Amersham Ltd.

Phenyl phosphate and pyridoxal 5-phosphate (being also referred to as P-5-P herein) derive from Sigma (Israel).

Except as otherwise noted, all reagent and solvent all obtain (for example Sigma, Acros, Aldrich) and press the supplying method use from commercial source.

According to program known in the art synthetic GS-1, GS-2 and GS-3, as hereinafter describing in detail.

New compound GS-4, GS-5 and the GS-21 of designing and synthesizing as mentioned below.

Measure the 3D structure of GSK-3 substrate by NMR spectroscopy and Structure Calculation:

Use a kind of small peptide of phosphorylation in these researchs, it copys the known GSK-3 substrate CREB that is expressed as p9CREB, and reaching two kinds of other peptides is unphosphorylated peptide 9CREB and variant 9ECREB, wherein S ¹Replace with glutamic acid (its intention simulation charged group), and list in lower tabulation 1.Determine that by the time-history analysis (Time courseanalyses) of GSK-3 Phosphorylated Peptide it is that p9CREB is the GSK-3 substrate that the peptide of phosphorylation is only arranged, and 9CREB and 9ECREB be fully by GSK-3 phosphorylation (data do not show), is the absolute demand of GSK-3 institute thereby again indicate the serine of phosphorylation.

Table 1

Peptide	Sequence	?SEQ?ID?NO：
			p9CREB	IL SRRPS(p)YR	?2
9CREB	IL SRRPSYR	?3
			9ECREB	IL SRRPEYR	?4

Use follow procedure to pass through 2D ¹H NMR spectrum, measured p9CREB (Fig. 1 a), the 3D structure of 9CREB (Fig. 1 b) and 9ECREB (not shown):

By containing 10%D ₂The dissolved freeze-dried powder end is for the preparation of each peptide solution of structural research in the water of O.On Bruker Avance DMX spectrometer with 600.13MHz's ¹H proton frequency (proton frequency) obtains the 2D-NMR spectrum.At the water signal carrier frequency (carrier frequency) is set, between relaxation period, reaches by low power radiation inhibition carrier frequency by using the WATERGATE method.Because the quick exchange under more ambient temperature, optimization experiment temperature (280K) to be reducing population mean, and preserves optimal spectral resolution.(carry out the spectrum width of all experiments and record 12ppm in TPPI or the state-TPPI), and true (real) t of 4K in the phase sensitivity pattern ₂Data point (data points) and 512 t ₁-increment.Use MLEV pulse train and the NOESY experiment of incorporation time between 100 milliseconds-750 milliseconds of 150 milliseconds of incorporation times to collect the two-dimentional same Nuclear Data that comprises TOCSY.Typically, relaxation delay was respectively 1.5 and 2.0 seconds in TOCSY and NOESY experiment.In ROESY measured, be set be 400 milliseconds and power spin-locking (lock) persistent period was 3.4KHz.All spectrums are proofreaied and correct with tetramethylsilane.

Use Bruker XWINNMR software (Bruker Analytische Messtechnik, GmbH, 2.7 editions) deal with data.(INDY R4000and INDIGO2R10000) processes, calculates and analyze all data at silicon graphics work station (Silicon Graphicsworkstations).Before Fourier transformation, use the apodization (apodization) of the indirect zero filling of tieing up and free induction decay (freeinduction decay) by tieing up Sine-squared conversion window function at all to improve spectral resolution.By the further phasing spectrum of the automatic multinomial baseline correction (automatic polynomial baseline correction) of using the Bruker exploitation.

According to the continuous dispensing method that Wuthrich uses Bruker software program AURELIA (Bruker AnalyticGmbH, 2.7 editions) to develop, resonance distributes based on the TOCSY that measures under the same experimental conditions and NOESY spectrum.

The NOE spacing suppresses the NOESY spectrum that (distance restraints) derives from 450 milliseconds of lower records.The best mixing time of NOE signal strength measuring p9CREB peptide sample relatively in the series of experiments that in 100 milliseconds of-750 milliseconds of scopes, changes by incorporation time.Selected incorporation time makes NOE increase maximum and nothing significantly increases the spin diffusion.This value is used for the non-phosphorylating analog and tests to keep identical experiment condition.Use r ^-6Dependency the peak volume integral be converted to spacing suppress and use known tyrosine aromatic ring two adjacent protons 2.47

The spacing record.Suppressing to be divided into strong (1.8-2.5A), medium (1.8-3.5A) and weak (1.8-5.0A) three classes.The upper limit that relates to methyl suppresses to add the experience correction of 0.5A.

Use XPLOR (3.856 editions) by hybrid distance geometry-dynamical simulated annealing computation structure.With square-well potential (square-well potential) and 50Kcal/mol The constant force constant introduce the NOE energy.3,000 1 fsec steps during simulated annealing comprises under the 1000K 1,500 3 fsec steps and is cooled to 300K.At last, using the conjugate gradient energy minimization to make construction minimizes is 4000 iteration (iterations).Use INSIGHTII (molecular model system 97.0 editions, Molecular Simulations, Inc.) to manifest and analyze the MR-derived structure.Use PROCHECK to estimate its quality.

Analytical method:

Obtain H spectrum, C spectrum, F spectrum and P spectrum nuclear magnetic resoance spectrum and with ppm (δ) report at Bruker AMX 500 spectrometers or Brucker AV 300 spectrometers.With the interior mark of tetramethylsilane (TMS) as the H spectrum, phosphoric acid is as the interior mark of P spectrum, and solvent peak is as the reference peak of C spectrum and F spectrum.

Obtain mass spectrum at Finnigan LCQ Duo LC-MS ion trap electrospray ionization (ESI) mass spectrograph.

Use the Analtech silica gel plate to carry out thin layer chromatography (TLC) and the development of ultraviolet (UV) rayed, or dye at 0.2

wt%

1,2,3-indantrione monohydrate butanol solution.

By Quantitative Technologies, Inc. (Whitehouse, NJ) technology is carried out elementary analysis.

Use Hypersil BDS C18 post, 4.6 * 150mm, 5um, column temperature room temperature, detector@220nm and Application standard solvent gradient program obtain HPLC and analyze, and be 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

The A=0.1%TFA aqueous solution

The B=0.1%TFA acetonitrile solution

Body outer suppressioning test:

Under prescribed concentration, the restructuring rabbit GSK-3 β (Eldar-Finkelman etc., 1996) of purification and peptide substrates PGS-1 (YRRAAVPPSPSLSRHSSPSQS (p) EDEEE) (SEQ IDNO:1) and phenyl phosphate, pyridoxal 5-phosphate (P-5-P), GS-1, GS-2, GS-3, GS-5 or GS-21 (structural formula is shown in Fig. 3) are hatched together.Reactant mixture comprises Tris 50mM (pH=7.3), 10mM MgAc, ³²P[γ-ATP] (100 μ M), 0.01% beta-mercaptoethanol and 30 ℃ were hatched 10 minutes.Reaction object point (p81) on cellulose phosphate paper, with the 100mM phosphoric acid washing and calculate radioactivity (such as Eldar-Finkelman etc., 1996 descriptions).

Glucose uptake in the ex vivo adipocytes: such as digesting from epididymis buccal pad (fat pad) separating mouse adipose cell (Lawrence etc., 1977) with 0.8mg/ml collagenase (Worthington Biochemical) of former description.The buccal pad that digested is by blood nylon filter and with containing 1% bovine serum albumin (Fraction V, Boehringer Mannheim, Germany), Krebs-bicarbonate buffer (pH=7.4) washed cell of 10mM HEPES (pH=7.3), 5mM glucose and 200nM adenosine 3 times.Cell and GS-5 and GS-21 were hatched 2.5 hours under the concentration in indication, then add the 2-deoxidation [ ³H] glucose (0.5 μ ci/vial) hatched 10 minutes.Centrifuge cell stops experiment through dinonyl phthalate (ICN, USA).Then measure by liquid scintillation analyzer (Packard) ³H.By add radioactive substance added in front 30 minutes Cytochalasin B (50 μ M) measure the 2-deoxidation-[ ³H] the non-specific picked-up of glucose.

Result of the test

The 3D structure determination of GSK-3 substrate:

Lower tabulation 2 and table 3 have provided the structure coordinate data that are used for showing in the input structure analysis software 3D structure.

Observe the gained 3D structure that Fig. 1 a and 1b show, only have the peptide of phosphorylation to have the structure conformation of restriction.P9CREB as shown in Fig. 1 a, phosphorylation adds to one of peptide main chain significant " turnover ", makes Tyr 8 and Arg 4 more approaching, and form " ring structure " thus the residue of phosphorylation point to outside the ring.This configuration has reduced the interference of positively charged residue (Arg 4 and Arg 5) to the catalyzed combination bag of enzyme on the one hand, makes on the other hand the serine of phosphorylation easily near enzyme.This structural analysis provides the GSK-3 explanation of peculiar substrate identification.The micromolecule of this structure of design simulation, thus the method that obtains potential GSK-3 selective depressant is provided.

Chemosynthesis:

Synthesizing of the p-methyl benzyl of phosphoric acid ester (GS-1):

Following scheme 1 has been described the routine of GS-1 and has been synthesized.

Scheme 1

The preparation of the p-methyl benzyl of di(2-ethylhexyl)phosphate-tert-butyl ester: the 4-xylyl alcohol (0.4g in stir, 3.3mmol, 1.1 equivalent) and two-tert-butyl diisopropylphosphoramidite (0.95ml, 0.83g, 3mmol, 1 equivalent) (0.45M is dissolved in the acetonitrile, 20ml once to add 1H-TETRAZOLE solution in anhydrous THF (3ml) solution, 9mmol, 3 equivalents).Stirred the mixture under 20 ℃ 15 minutes.Mixture is cooled to-40 ℃ (adopting dry ice/acetonitrile), (0.81g is dissolved in the 1ml dichloromethane to keep adding rapidly below 0 ℃ 85% metachloroperbenzoic acid (mCPBA) in reaction temperature, 4mmol, 1.3 equivalents) dichloromethane (4mL) solution.Make solution be warming up to room temperature and 20 ℃ lower stir 5 minutes after, add 10%NaHSO ₃Aqueous solution (10ml) also continued to stir the mixture 10 minutes.Then use ether (70ml) extracting mixture and aqueous phase discarded.Use 10%NaHSO ₃Aqueous solution (2 * 20ml), and 5%NaHCO ₃Saturated aqueous solution (2 * 20ml) washing ether phases, dry and filtration on sodium sulfate.Evaporate organic filtrate and pass through chromatography purification residue as eluent at silicagel column with 1: 15 mixture of EtOAc/ hexane, make the mixture of product (the p-methyl benzyl of di(2-ethylhexyl)phosphate-tert-butyl ester) and raw material, do not need further to separate directly to 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)：?

137.0(Ar)，129.0(Ar)，127.7(Ar)，82.3(c)，683(CH ₂O)，29.8(OtBu)，21.1(CH ₃).

³¹P?NMR(81.3MHz，CDCl ₃)：?

The preparation of the p-methyl benzyl of phosphoric acid ester: add the solution of HCl (4M is dissolved in the dioxane, 2ml, 8mmol, 2.6 equivalents) and dioxane (6ml) under 20 ℃ to the p-methyl benzyl of the di(2-ethylhexyl)phosphate-tert-butyl of gained ester, and by TLC monitoring reaction.Treat the complete hydrolysis lower evaporation dioxane that namely reduces pressure, and (2 * 15ml) to remove excessive benzylalcohol raw material residue (15ml) soluble in water and with the ether washing.The lower evaporating solvent of decompression and dry (prolonged high vacuum drying) gained clarification of the long-time high vacuum of warp grease slowly become colorless solid, make the end-product of 0.18g (30%).

¹H?NMR(200MHzD 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)：?

134.0(Ar)，129.2(Ar)，128.0(Ar)，68.0(CH ₂O)，20.1(CH ₃).

Synthesizing of benzyl phosphate ester (GS-2):

It is synthetic that hereinafter scheme 2 has been described the routine of benzyl phosphate ester.

Scheme 2

The preparation of di(2-ethylhexyl)phosphate-tert-butyl benzyl ester: the benzylalcohol (0.34ml in stir, 3.3mmol, 1.1 equivalent) and two-tert-butyl diisopropylphosphoramidite (0.95ml, 0.83g, 3mmol, 1 equivalent) (0.45M is dissolved in the acetonitrile, 20ml once to add 1H-TETRAZOLE solution in anhydrous THF (3ml) solution, 9mmol, 3 equivalents).Mixture stirred 15 minutes and then is cooled to-40 ℃ (adopting dry ice/acetonitriles) for 20 ℃ times, reaction temperature keeps adding rapidly below 0 ℃ 85%mCPBA, and (0.81g is dissolved in the 1ml dichloromethane, 4mmol, 1.3 equivalents) dichloromethane (DCM) is solution (4mL).Make solution be warming up to room temperature and 20 ℃ lower stir 5 minutes after, add 10%NaHSO ₃Aqueous solution (10ml) and continued to stir the mixture 10 minutes.Then use ether (70ml) extracting mixture and aqueous phase discarded.Use 10%NaHSO ₃Aqueous solution (2 * 20ml), and 5%NaHCO ₃Saturated aqueous solution (2 * 20ml) washing ether phases, dry and filtration on sodium sulfate.The organic filtrate of evaporation drying and with 1: 15 mixture of EtOAc/ hexane as eluent at silicagel column by the chromatography purification residue, make the mixture of product (di(2-ethylhexyl)phosphate-tert-butyl benzyl ester) and raw material, need not be further purified direct use.

¹HNMR(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)：?

The preparation of benzyl phosphate ester: add the solution of HCl (4M is dissolved in the dioxane, 2ml, 8mmol, 2.6 equivalents) and dioxane (6ml) under 20 ℃ to the di(2-ethylhexyl)phosphate of gained-tert-butyl benzyl ester, and react by the TLC monitoring.Treat the complete hydrolysis lower evaporation dioxane that namely reduces pressure, and (2 * 15ml) to remove excessive benzylalcohol raw material residue (15ml) soluble in water and with the ether washing.The lower evaporating solvent of decompression and dry (prolonged high vacuumdrying) gained clarification of the long-time high vacuum of warp oily slowly become colorless solid, make the end-product of 0.17g (30%).

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)：?

Synthesizing of phosphoric acid 3-pyridine methyl ester (GS-3):

Following scheme 3 has been described the routine of phosphoric acid 3-pyridine methyl ester and has been synthesized.

Scheme 3

The preparation of di(2-ethylhexyl)phosphate-tert-butyl 3-pyridine methyl ester: the 3-pyridine radicals methanol (0.31ml in stir, 3.3mmol, 1.1 equivalent) and two-tert-butyl diisopropylphosphoramidite (0.95ml, 0.83g, 3mmol, 1 equivalent) (0.45M is dissolved in the acetonitrile, 20ml once to add 1H-TETRAZOLE solution in anhydrous THF (3ml) solution, 9mmol, 3 equivalents).Then 20 ℃ of lower stirrings of mixture were cooled to-40 ℃ (adopting dry ice/acetonitrile) in 15 minutes, kept adding rapidly below 0 ℃ DCM (4mL) solution of 85%mCPBA (0.81g is dissolved in the 1ml dichloromethane, 4mmol, 1.3 equivalents) in reaction temperature.Make solution be warming up to room temperature and 20 ℃ lower stir 5 minutes after, add 10%NaHSO ₃Aqueous solution (10ml) also continued to stir the mixture 10 minutes.Then use ether (70ml) extracting mixture and aqueous phase discarded.Use 10%NaHSO ₃Aqueous solution (2 * 20ml), and 5%NaHCO ₃Saturated aqueous solution (2 * 20ml) washing ether phases, dry and filtration on sodium sulfate.The organic filtrate of evaporation drying is also used CHCl ₃1: 1 mixture of/hexane passes through the chromatography purification residue as eluent at silicagel column, makes the mixture of di(2-ethylhexyl)phosphate-tert-butyl 3-pyridine methyl ester and raw material, need not be further purified direct use.

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).

¹³C?NMR(50.4MHz，CDCl ₃)：? 148.1(Ar)，135.3(Ar)，134.3(Ar)，123.1(Ar)，77.9(c)，64.1(CH ₂O)，29.6(OtBu).

³¹P?NMR(81.3MHz，CDCl ₃)：?

The preparation of phosphoric acid 3-pyridine methyl ester: add the solution of HCl (4M is dissolved in the dioxane, 2ml, 8mmol, 2.6 equivalents) and dioxane (6ml) under 20 ℃ to the di(2-ethylhexyl)phosphate of gained-tert-butyl 3-pyridine methyl ester, and react by the TLC monitoring.Treat the complete hydrolysis lower evaporation dioxane that namely reduces pressure, and residue (15ml) soluble in water and with ether washing (2 * 15ml).The lower evaporating solvent of decompression makes the end-product of 0.19g (30%).

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).

¹³C?NMR(100.8MHz，D2O)：? 139.9(Ar)，139.0(Ar)，126.8(Ar)，62.8(CH ₂O).

³¹P?NMR(162MHz，D ₂O)：?

Phosphoric acid 3,5-two (2-amino-ethyl) benzyl ester (GS-21) synthetic:

Design and implementation following scheme 4 the conventional strategy of synthetic GS-21 and the purification process of end-product are described.By the synthetic phosphoric acid 3 that obtains 5% gross production rate of 8 steps, 5-two (2-amino-ethyl) benzyl ester (GS-21).Also prepared corresponding trifluoroacetate.

Scheme 4

Differentiated that from low-cost

raw material

1,3 5-benzenetricarboxylic acid trimethyl ester is through 4 steps reaction obtainable key intermediate benzylalcohol intermediate (square case 4), as hereinafter describing in detailed description and the scheme 5.

Scheme 5

According to the method for Johns (Tetrahedron Lett.1988,29,2369-2372), in the presence of tetrazolium, introduce phosphonate moiety by pure and mild two-tert-butyl diisopropylphosphoramidite reaction.The gained phosphite ester generates corresponding phosphate ester by metachloroperbenzoic acid (mCPBA) oxidation immediately without separating.Under controlled conditions by using trifluoroacetic acid to obtain amine and the phosphate ester of whole deprotections.Then obtain its trifluoroacetate.The latter through recrystallization then spent ion exchange resin process and obtain enough pure required product, typical about 90% (by AUC of HPLC) is such as following scheme 6 descriptions.

Classify down the detailed description of synthetic method as:

The preparation of 1,3,5-three (hydroxymethyl) benzene: 3 liters round-bottomed flask also assembles splash bar, the charging hopper of top formula and the reflux condenser that is full of lithium aluminium hydride reduction (49.7grams, 1.31mol) and anhydrous THF (500ml) under the nitrogen is housed.The slow reflux of gained suspension and anhydrous THF (1.0 liters) solution that drips 1,3,5-benzenetricarboxylic acid trimethyl-ester (100.0g, 0.40mol) are to keep gentle reflux (3 hours).Stir gained Lycoperdon polymorphum Vitt suspension under refluxing then externally cooled off in the ice-water bath in lasting 7 hours.By dripping water (50ml, 45 minutes), then add 15%NaOH (50ml slowly flows out) and add again at last water (150ml slowly flows out) the excessive lithium aluminium hydride reduction of hydrolysis.Stirred the gained suspension 14 hours under the room temperature.Solids removed by filtration and under high vacuum concentrated filtrate obtaining colorless oil, thereby it slowly solidifies and provides 1,3 of white solid, 5-three (hydroxymethyl) benzene (62.3g, 94%).Fig. 4 a-b shows ¹H NMR and ¹³C NMR spectrum is consistent with this specified structure.

The preparation of 3,5-two (bromomethyl) benzylalcohol: 2 liters of round-bottomed flasks of assembling magnetic splash bar, charging hopper be equipped with 1,3,5-three (hydroxymethyl) benzene [33.7g, 0.20mol) and anhydrous acetonitrile (750ml).Slowly add while stirring trimethylammonium bromide silane (TMSBr) 979.0ml, 0.60mol to the gained suspension) liquid stream.The white slurry becomes the slurry of brown and thickness.Reactant mixture is heated to 40 ℃ to be continued 25 minutes and makes it become settled solution.TLC analyzes (90: 10 methylene chloride/methanol, UV illumination is developed, raw material Rf value 0.07, product Rf value 0.77) judgement and reacts completely.Decompression is lower removes solvent to obtain brown pastel.By column chromatography (silica gel, 0%-5%MeOH/CH ₂Cl ₂) the purification crude product.Obtain 3 of white solid, 5-two (bromomethyl) benzylalcohol (33.5g, 57%).Fig. 5 a-b shows ¹H NMR and ¹³C NMR spectrum is consistent with this specified structure.

The preparation of 3,5 two (cyano methyl) benzylalcohol: splash bar, charging hopper and the reflux condenser of 1 liter round-bottomed flask assembling top formula, to wherein adding 3,5-two (bromomethyl) benzylalcohol (33.1g, 0.11mol) and methanol (400ml).Gained settled solution reflux.The aqueous solution (25ml) that slowly adds Cyanogran. (16.2grams, 0.33mol).Under backflow, continued heating 6 hours until TLC analysis (95: 5 methylene chloride/methanol, UV illumination is developed, raw material Rf value 0.62, product Rf value 0.38) judgement reacts completely.Reactant mixture is cooled under room temperature and the decompression and removes solvent to obtain brown pastel.(6 * 100ml) grind pastel with MTBE.Merge the lower solvent of removing of MTBE extract and decompression.Then by column chromatography (silica gel, 0%-5%MeOH/CH ₂Cl ₂) the thus obtained yellow oil of purification.Obtain 3 of light brown oily, 5-two (cyano methyl) benzylalcohol (18.7g, 89%), it slowly becomes wax-like white solid.Take out the 1g sample and make sterling through the column chromatography purification.Fig. 6 a-b shows ¹H NMR and ¹³C NMR spectrum is consistent with this specified structure.

3, the preparation of 5-two (amino-ethyl) benzylalcohol: 3,5-two (cyano methyl) benzylalcohol (8.0g, 0.04mol) sample is divided in the 500-ml Parr bottle that 3 parts and every 2.5g-3.0g partly pack into independent, then adds ethanol (100ml) and NaOH aqueous solution (1.2g is dissolved in the 5ml water).Add in the gained solution Raney Ni (50% aqueous suspension, 1.2g).On the Parr agitator with the 30psi hydrogenated mixture.Pass through after 3 hours ¹H NMR monitoring reaction and judgement react completely.Wash the kieselguhr pad with kieselguhr pad filtering catalyst and with ethanol (200ml).Merge the filtrate of whole 3 reactions and under reduced pressure remove solvent to obtain the brown pastel (14.16g) of 3,5-two (amino-ethyl) benzylalcohol.The product that Fig. 7 shows ¹The ethanol of the bright existence 25% of H NMR stave (w/w).Prolongation sample drying time under high vacuum is observed ethanol content does not have significant change.Material need not any further purification and is used for next step synthesis step.

3, the preparation of 5-two (uncle-butoxy carbonyl amino-ethyl) benzylalcohol: 3 necks of assembling magnetic splash bar, thermometer and air inlet connections, 3 liters, round-bottomed flask are equipped with and are dissolved in 3 of THF (590ml) and 2N NaOH (590ml) aqueous solution, 5-two (amino-ethyl)-1-hydroxymethyl benzylalcohol (29.4 g).In the mixture that is just stirring, once add two-tert-butyl bicarbonate (59.4g, 272mmol).Mixture heated to 45 ℃ continues 4 hours.Gained solution is cooled to room temperature and removes volatile organic matter by vacuum equipment.Add methanol (600ml) to the gained aqueous mixtures.But heating positive agitating solution to 45 ℃ continues 2 days with selective hydrolysis uncle-butoxy carbonate moiety keeps carbamate.The volatile ingredient that has been cooled to after the room temperature solution removal, and with chloroform (3 * 600ml) extracting mixed aqueous solutions.Merge organic layer, with saline (600ml) washing and concentrated.Under the high vacuum after the drying, obtain the thick 3 of productive rate 67%, 5-two (uncle-butoxy carbonyl amino-ethyl) benzylalcohol (24.88g) off-white color solid.Fig. 8 shows ¹H NMR spectrum is consistent with this specified structure.Be not further purified and use this thick product.

The phosphoric acid 3 of protection; the preparation of 5-two (2-amino-ethyl) benzyl ester: 500 milliliters of round-bottomed flasks of assembling magnetic splash bar, charging hopper are equipped with 3; 5-two (uncle-butoxy carbonyl amino-ethyl) benzylalcohol (2.3g, 0.0058mol) and anhydrous methylene chloride (45ml).Cooling gained solution is to about 5 ℃ in ice-water bath.Anhydrous acetonitrile (45ml) the solution liquid stream that slowly adds two-tert-butyl diisopropylphosphoramidite (4.5ml, 0.0144mol) from charging hopper.Then slowly add the solution that (15 minutes) tetrazolium (1.0g, 0.0144mol) is dissolved in 1: 1 mixture of anhydrous acetonitrile/anhydrous methylene chloride (90ml).Judge about 5 ℃ of lower stirring gained white suspension 1 hour and by the TLC analysis react completely (95: 5 chloroform/isopropyl alcohols, raw material Rf value 0.23, product Rf value 0.30 are developed in 1,2,3-indantrione monohydrate dyeing).The lower solvent of removing of decompression is to obtain pastel, and it is dissolved in anhydrous methylene chloride (75ml) and cooling in dry ice/acetonitrile is bathed.Anhydrous methylene chloride (50ml) solution that adds immediately mCPBA (1.3g, 0.0144mol).Stirred the gained mixture 1 hour, and made to be warmed up to room temperature (1 hour) and restir 30 minutes.Aqueous solution (100ml) and saturated sodium bicarbonate (2 * 100ml) continuous washing reactant mixtures with 1.0M sodium thiosulfate.Dry organic extract and filtration on anhydrous sodium sulfate.The lower solvent of removing of decompression is to obtain the raw phosphoric acid ester of yellow oily, then by column chromatography (silica gel, 0%-5%MeOH/CH ₂Cl ₂) purification.Obtain the phosphoric acid 3 of the protection of thickness, colorless oil, 5-two (2-amino-ethyl) benzyl ester (2.1g, 61%).Fig. 9 a-c shows it ¹H NMR and ¹³C NMR spectrum is consistent with this specified structure.

Phosphoric acid 3, the preparation of 5-two (2-amino-ethyl) benzyl ester tfa salt: 250 milliliters of round-bottomed flasks of assembling magnetic splash bar are equipped with phosphate ester (2.9g, 0.0049mol), anhydrous methylene chloride (30ml) and the trifluoroacetic acid (30ml) of protection.Stirred the gained settled solution 3 hours under the room temperature.By ¹H NMR and ³¹P NMR analyzes judgement and reacts completely.The lower solvent of removing of decompression obtains the thickness orange oil, and it is dissolved under methanol (7.5ml) and the stirring and joins in the ether (500ml), obtains the precipitation of product.Stirred the gained slurry 1 hour under the room temperature, then make the solid sedimentation.Decant settled solution from top and use ether (2 * 100ml) grinding products.Make the solid sedimentation at every turn and outwell gently settled solution.Product at last in vacuum drying oven 55 ℃ lower dry 108 hours, then 65 ℃ lower in addition dry 192 hours to obtain phosphoric acid 3, the white solid of 5-two (2-amino-ethyl) benzyl ester tfa salt (1.78g, 71%).Figure 10 a-c shows ¹HNMR, ¹³CNMR and ³¹P NMR spectrum is consistent with this specified structure. ¹H NMR spectrum shows the ether that has 8.5% (w/w) in the product.Proved that this ether is very difficult to remove and the feature tool hygroscopicity of material.Figure 10 d shows that the mass spectrum of product shows that the molecule peak is at m/z 275[C ₁₁H ₁₉N ₂O ₄P+H] ⁺

Phosphoric acid 3, the preparation of 5-two (2-amino-ethyl) benzyl ester (GS-21): be equipped with boost 3 necks, 5 liters, round-bottomed flask of dynamic balance charging hopper and air inlet connections of top formula mechanical agitator, thermometer, 13 under the nitrogen is housed, anhydrous methylene chloride (11) solution of 5-two (uncle-butoxy carbonyl amino-ethyl) benzylalcohol (24.88g, 63.15mmol).With ice/brine bath reaction mixture.Anhydrous acetonitrile (1 liter) solution that adds two-tert-butyl diisopropylphosphoramidite (49.8ml, 157.9mmol) through the pressure balance charging hopper with the maintenance reaction temperature less than 6 ℃ speed.(the 0.45M acetonitrile solution of 351ml 157.9mmol), adds with the speed of maintenance reaction temperature less than 6 ℃ through the pressure balance charging hopper to dilute tetrazolium with anhydrous acetonitrile (150ml) and anhydrous methylene chloride (500ml).Add finish after, flask is put in the psychrolusia and stirred reaction mixture 1 hour.Then adopt dry ice/acetonitrile to bathe and flask is cooled to-35 ℃.Anhydrous methylene chloride (500ml) solution that once adds 3-chloroperoxybenzoic acid (18.4g, 82.1mmol).Mixture is warming up to room temperature, then stirs 2 hours.Solution is poured into Na ₂S ₂O ₃(20g) and K ₂CO ₃In the aqueous solution (50g) (1.5 liters).The pH of gained biphase mixture is 11.Stirring 15 minutes final vacuums removes organic volatile and uses chloroform (4 * 750ml) extracting water layers.The dry organic layer that merges on magnesium sulfate filters and the concentrated yellow oil (69g) that obtains.The gained crude product is by column chromatography purification (silica gel, MTBE/ heptane, 6: 4).Merge and contain product and the faint yellow oily thing of simmer down to (32.6g) that mixes flow point (Mixed fractions).The oil of a 28.6-g is dissolved in dichloromethane (287ml, 10 times of volumes) and packs into and is equipped with in 1 liter of round-bottomed flask of 500-ml pressure balance charging hopper and magnetic stirring bar.Add rapidly trifluoroacetic acid (287ml, 10 times of volumes) through the pressure balance charging hopper.Gained solution stirring 5 hours.Concentrated and dried overnight obtains dense orange (37.88g) under the vacuum.Residue is dissolved in water (57ml, 1.5 times of volumes) and is added drop-wise to methanol (90 volume) the generation precipitation of stirring.Stir after 30 minutes, make solid sedimentation 1 hour and decant liquid.Remove the solid that residual liquid obtains 13.72g in the vacuum.This material (68ml, 5 times of volumes) soluble in water also is loaded on (loadedonto) Dowex 50WX8-200 ion exchange resin (137g).Water (550ml, 40 times of volumes) flushing pillar.With 3: 1 MeOH/NH ₄OH aqueous solution (2 liters, 145 times of volumes) eluted product.The lower concentrated methanol part of decompression is to produce beige solid.This solid is dissolved in the water of minimum and joins (40 times of volumes) in the methanol that is just stirring.Dried overnight under collecting precipitation and the vacuum after filtration.Gained powder water grinds (7 times of volumes).After the drying, obtain the end-product (2.0g) into white powder under filtration and the high vacuum.Concentrated filtrate and water (5 times of volumes) grind residue.Filter and the high vacuum drying after, obtain second take turns product [0.9g).Merge this twice product and mixed 10 minutes.Obtain to be the phosphoric acid 3 of white powder, 5-two (2-amino-ethyl) benzyl ester (GS-21).The product that Figure 11 a-c shows ¹H NMR, ³¹P NMR and ¹³C NMR spectrum is consistent with the structure of this appointment.The mass spectrum that Figure 11 d shows shows that the molecule peak carries 275[C ₁₁H ₁₉N ₂O ₄P+H] ⁺The HPLC chromatogram that Figure 11 e shows (using said method A to obtain) shows that product purity is 96.7%.End-product be characterized as non-hygroscopic.

Use same policy, following new compound GS-4 and the GS-5 of having synthesized:

Synthesizing of phosphoric acid 3-(guanidine radicals methyl) benzyl ester (GS-4):

Following scheme 7 has been described as the routine of the GS-4 of its trifluoroacetate synthetic:

Scheme 7

GS-4 (tfa salt)

The preparation of 3-(amino methyl) benzylalcohol: remain under the nitrogen, under the vigorous stirring to LiAlH ₄The reflux solution of THF solution add the THF solution of 3-(hydroxymethyl) benzonitrile.Vlil is spent the night, and then slowly drips water quencher reaction (until there is not H ₂Further release).The lower evaporation THF of decompression also adds ether/acidifying water.Discard the ether phase.Wash water and discard organic facies with ether.Add NaOH until ph value of aqueous phase reaches pH 7.With THF extracted solution 3 times, at MgSO ₄The lower evaporation of upper drying and decompression uses gradient eluent to finish from the initial mixed liquor to 1: 1 ethyl acetate: MeOH of ethyl acetate to make faint yellow residue, makes its purification by chromatograph on silicagel column, obtains the intermediate of 40% productive rate.

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(8，2H，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) benzylalcohol: stir 3-(amino methyl) benzylalcohol under the room temperature, (N, the anhydrous DMF solution of N '-two (uncle-butoxy carbonyl)-S-methyl isothiourea and triethylamine spends the night 3-.Then add ether/water mixed liquid and separate organic facies, and use the ether extraction water layer.Wash the organic extract of merging with water, at MgSO ₄The lower evaporation of upper drying and decompression.Use gradient eluent initial to 1: 1 ethyl acetate from hexane: the mixed liquor of hexane finishes, and by flash chromatography purification of crude product, obtains 85% productive rate.

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.

Di(2-ethylhexyl)phosphate-tert-butyl group 3-(N, N '-two-BOC-guanidine radicals methyl) preparation of benzyl ester: the 3-(N in stir, N-two-BOC-guanidine radicals methyl) benzylalcohol (1 equivalent) and two-tert-butyl diisopropylphosphoramidite (1.42ml, 1.24g, 4.5mmol, 1.5 equivalents) anhydrous THF (3ml) solution in once add 1H-TETRAZOLE solution (0.45M be dissolved in the acetonitrile, 20ml, 9mmol, 3 equivalents).Stirred the mixture under 20 ℃ 30 minutes, then mixture is cooled to-40 ℃ (adopting dry ice/acetonitrile), DCM (4mL) solution that keeps adding rapidly below 0 ℃ 85%mCPBA (1.25g is dissolved in 1.5ml DCM, 6.15mmol, 2.0 equivalents) in reaction temperature.After making solution be warming up to room temperature and stirring 20 minutes, add 10%NaHSO ₃Aqueous solution (10ml) also continued to stir the mixture 5 minutes.Then use ether (70ml) extracting mixture and aqueous phase discarded.Use 10%NaHSO ₃Aqueous solution (2 * 20ml), and 5%NaHCO ₃Saturated aqueous solution (2 * 20ml) washing ether phases, dry and filtration on sodium sulfate.The organic filtrate of evaporation drying is also used ethyl acetate/hexane 1: 9-1: 5 gradient eluent by the chromatography purification residue, makes the mixture of phosphate product and benzylalcohol raw material at silicagel column, uses CHCl ₃: MeOH 30: 1-20: 1 gradient eluent is further purified by chromatography on silicagel column, makes the pure di(2-ethylhexyl)phosphate of 70% productive rate-tert-butyl group 3-(N, N '-two-BOC-guanidine radicals methyl) benzyl ester.

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(162Mz，CDCl ₃)：δ＝-9.3.

Phosphoric acid 3-(guanidine radicals methyl) benzyl ester, the preparation of trifluoroacetate: to two-tert-butyl group, 3-(N, N '-two-BOC-guanidine radicals methyl) benzyl phosphate ester added the DCM solution of 25% trifluoroacetic acid (TFA) and stirred reaction mixture 18 hours under 20 ℃.Then the lower evaporating solvent of decompression and TFA, residue is soluble in water and wash with ether.Then the lower evaporating solvent of decompression makes the clean product of 60% productive rate.

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.

Synthesizing of phosphoric acid 3-guanidine radicals benzyl ester (GS-5):

Following scheme 8 has been described as the routine of the GS-5 of its trifluoroacetate synthetic:

Scheme 8

N, N-two (tertbutyloxycarbonyl)

-S-methyl isothiourea

GS-5TFA salt

3-(N, N '-two-BOC-guanidine radicals) preparation of benzylalcohol: anhydrous dimethyl formamide (DMF) solution to 3-aminobenzyl alcohol (0.5g, 4mmol, 1.0 equivalents) adds N, N '-two (uncle-butoxy carbonyl)-S-methyl isothiourea (1.32g, 4.4mmol, 1.1 equivalents), mercuric chloride (1.22gram, 4.4mmol, 1.1 equivalent) and triethylamine (1.72ml, 12mmol, 3 equivalents) solution, stirred reaction mixture is 5 hours under the room temperature.Then also use saturated NH with ether/water extracting mixture ₄Cl aqueous solution and salt water washing organic layer.Use the ether extraction water layer.At MgSO ₄The diethyl ether solution that the lower evaporation of upper drying and decompression merges.Use is from hexane to 40: 60 ethyl acetate: the gradient eluent of hexane by flash chromatography purification of crude product, makes the intermediate of 60% productive rate at silicagel column.

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.

Di(2-ethylhexyl)phosphate-tert-butyl 3-(N, N '-two-BOC-guanidine radicals) preparation of benzyl ester: the 3-(N in stir, N '-two-BOC guanidine radicals) benzylalcohol (1g, 2.8mmol, 1 equivalent) and two-tert-butyl diisopropylphosphoramidite (1.13ml, 3.6mmol, 1.3 (0.45M is dissolved in the acetonitrile once to add 1H-TETRAZOLE solution in anhydrous THF (3ml) solution equivalent), 18.4ml, 8.3mmol, 3 equivalents).Stirred the mixture under 20 ℃ 30 minutes, then mixture is cooled to 40 ℃ (adopting dry ice/acetonitrile), DCM (4mL) solution that keeps adding rapidly below 0 ℃ 85%mCPBA (0.85g is dissolved in 1.5ml DCM, 4.20mmol, 1.5 equivalents) in reaction temperature.Make solution be warming up to room temperature, stir after 20 minutes, add 10%NaHSO ₃Aqueous solution (10ml) also continued to stir the mixture 10 minutes.Then use ether (50ml) extracting mixture and aqueous phase discarded.Use 10%NaHSO ₃Aqueous solution (2 * 20ml) and NaHCO ₃Saturated aqueous solution (2 * 20ml) washing ether phases, dry and filtration on sodium sulfate.The organic filtrate of evaporation drying is also used ethyl acetate/hexane 10: 90-30: 70 gradient eluent by the chromatography purification residue, makes the product of the protection of 60% productive rate at silicagel column.

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.

Phosphoric acid 3-guanidine radicals benzyl ester, the preparation of trifluoroacetate: under 20 ℃ to di(2-ethylhexyl)phosphate-tert-butyl group 3-(N, N '-two-BOC-guanidine radicals) the DCM solution of benzyl ester (0.3g, 0.54mmol, 1 equivalent) adding 25% trifluoroacetic acid (TFA) and stirred reaction mixture are 18 hours.Then the lower evaporating solvent of decompression and TFA, residue is soluble in water and wash with ether.Then lower (lyophilizer) evaporating solvent of decompression makes the clean product of 40% productive rate.(C ₁₀H ₁₃F ₃N ₃O ₆P；Mw＝359.2g/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.

Body outer suppressioning test:

As noted before in the inhibition test trial test, empirical tests the GSK-3 of known compound phenyl phosphate, pyridoxal 5-phosphate, GS-1, GS-2 and GS-3 suppress active.It is active that Figure 12 shows that the result shows that the chemical compound of all checkings all has for the inhibition of GSK-3, and the phosphate derivative of pyridine is that pyridoxal 5-phosphate and GS-3 are stronger than phosphate derivative (phenyl phosphate, GS-1 and the GS-2) activity of benzene.

The GSK-3 that has verified GS-1, GS-2, GS-3, GS-5 and GS-21 in other inhibition test suppresses active.In the presence of these chemical compounds of indication concentration, measured the ability of GSK-3 phosphorylation PGS-1 peptide substrates.Result's representative that Figure 13 shows is hatched the percentage rate of comparing the GSK-3 activity with the contrast that does not have inhibitor and is the meansigma methods ± SEM of 2 independent trialss, and wherein each some replication is 3 times.

As shown in figure 13, the chemical compound of all checkings finds that all the GSK-3 of high activity suppresses active (the IC50 value of 1-5mM), and GS-3 and GS-5 are the highest chemical compounds of activity.These results can point out in ring or on its adjoining position the existence of (for example attachment band atom) one or more nitrogen-atoms be can affect the micromolecular GSK-3 of (raising) new design to suppress active feature.

Glucose uptake:

Newly-designed chemical compound GS-5 and the GS-21 of having verified in mice initial stage adipose cell as noted before promotes the ability of glucose uptake.In untreated adipose cell, observe relatively [ ³H] 1,5-anhydroglucitol is standardized as 1 unit, and obtain in the adipose cell of processing with GS-5 or GS-21 [ ³H] value of 1,5-anhydroglucitol is as providing with peptide control treatment cytoactive multiple, and be the meansigma methods ± SEM of 6 independent experiments, wherein each some replication is 3 times.

The result demonstrates GS-21 among Figure 14 a (GS-5) and Figure 14 b (GS-21), improves respectively 2.5 times of glucose uptakes and 1.7 times under 5 μ M and 0.5 μ M concentration.Observe the effect of slight reduction in the presence of GS-5, it improves about 2 times of glucose uptake when 10 μ M concentration.As further showing among Figure 14 a and the 14b, the glucose uptake effect active and that the existence of 100nM insulin is issued to that reaches by GS-5 and GS-21 is suitable.These results have proved that further these newly-designed chemical compounds are strengthening for example ability of diabetes of insulin signaling conduction and treatment GSK-3 mediated disorders as insulin-mimickers.

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

$CDIH

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 O.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 8.630 -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 HB12 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 50 C 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 S8 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 0.69S -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 139 CE2 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.OO 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 150 CB 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 HH11 ARG 9 -8.742 -5.408 -1.904 1.00 0.00

ATOM 164 HH12 ARG 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 HH21 ARG 9 -10.615 -5.476 -5.429 1.00 0.00

ATOM 167 HH22 ARG 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，bondg，angles，improper，vdw，noe，cdih

REMARK?energies：104.733，3.47295，70.7767，3.51384，6.64866，

20.3204，$?CDIH

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 -1.358 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 O.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

AT0M 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 SBR 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 -4.798 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 6.3S4 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 140 CD2 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

For the purpose of being interpreted as knowing, some feature of the present invention is described in the context of the embodiment of separating, and also can be combined in the single embodiment provides.Otherwise for for purpose of brevity, various features of the present invention is described in the context of single embodiment, also can provide respectively or provide with any suitable time combination.

Although described the present invention in conjunction with its particular, many changes, modifications and variations are apparent for a person skilled in the art.Accordingly, comprise that all this changes, modifications and variations all have a mind to be included in the spirit and broad range of claims.The full content of all publications, patent and patent application that this description is mentioned is incorporated herein by reference, its scope with indicate one by one each independent publication, patent or patent application incorporated herein by reference identical.In addition, the quoting or differentiate should not be construed as and admit that this list of references is as prior art of the present invention of any list of references among the application.

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Sequence table

＜110〉(the Tel Aviv University Future Technology of Tel Aviv University Future Tec

Development?L.P.)

＜120〉glycogen synthase kinase-3 inhibitors

<130>CPCH0564607P

<140>

＜141〉27-June-2004

<150>US?60/482,719

＜151〉27-June-2003

<150>US?60/528,495

＜151〉11-December-2003

<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 natural aminoacid that exists

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Ser of phosphorylation or Thr

<400>1

Xaa?Xaa?Xaa?Xaa?Xaa

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

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

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

5

Claims

1. chemical compound or its pharmaceutically acceptable salt with following general formula:

Wherein:

X, Y, Z and the W carbon atom of respectively doing for oneself;

A is (CH ₂) _m, wherein m is the integer of 1-6;

B is electronegative bound phosphate groups;

D in the described general formula, R ₁And R ₂Be respectively hydrogen; With

R ₃Hydrogen and R ₄Be selected from guanidine radicals and guanidine radicals (C _1-4) alkyl; Perhaps R ₃And R ₄Amino (C respectively does for oneself _1-4) alkyl.

2. the chemical compound of claim 1, described chemical compound can suppress the activity of GSK-3.

3. the chemical compound of claim 1, this chemical compound is selected from

Or its pharmaceutically acceptable salt.

4. Pharmaceutical composition, described Pharmaceutical composition comprise as each chemical compound and pharmaceutically acceptable carrier among the claim 1-3 of active component.

5. the Pharmaceutical composition of claim 4, described Pharmaceutical composition be packaged in the packaging material and on described packaging material or in stamp sign, be used for the treatment with the active relevant biological disease of GSK-3.

6. the Pharmaceutical composition of claim 5, wherein said biological disease is selected from obesity, non-insulin-dependent diabetes mellitus, insulin dependency disease, affective disorder, neurodegenerative disease or obstacle and psychosis or obstacle.

7. the Pharmaceutical composition of claim 6, wherein said affective disorder is selected from single phase property obstacle and two-phase sexual disorders.

8. the Pharmaceutical composition of claim 7, wherein said single phase property obstacle is depression.

9. the Pharmaceutical composition of claim 7, wherein said two-phase sexual disorders is manic depressive illness.

10. the Pharmaceutical composition of claim 6, wherein said neurodegenerative disease is by being selected from due to the event that cerebral ischemia, apoplexy, traumatic brain injury and antibacterial infect.

11. the Pharmaceutical composition of claim 6, wherein said neurodegenerative disease are chronic neurodegenerative disease.

12. the Pharmaceutical composition of claim 11, wherein said chronic neurodegenerative disease is by due to the disease that is selected from Alzheimer's disease, Huntington Chorea, parkinson disease, the relevant dementia of AIDS, amyotrophic lateral sclerosis (AML) and multiple sclerosis.

13. the Pharmaceutical composition of claim 4, described Pharmaceutical composition are packaged in the packaging material and on described packaging material or in stamp sign, be used for to suppress the activity of GSK-3.

14. the Pharmaceutical composition of claim 4, described Pharmaceutical composition are packaged in the packaging material and on described packaging material or in stamp sign, be used for to improve glucose uptake speed.

15. the Pharmaceutical composition of claim 4, described Pharmaceutical composition are packaged in the packaging material and on described packaging material or in stamp sign, be used for the treatment of diabetes.

16. the Pharmaceutical composition of claim 4, described Pharmaceutical composition further comprise at least a other active component that can change the GSK-3 activity.

17. the Pharmaceutical composition of claim 16, wherein said other active component can suppress the activity of GSK-3.

18. the Pharmaceutical composition of claim 16, the expression that wherein said other active component can be reduced GSK-3.

19. the chemical compound of claim 1 or its pharmaceutically acceptable salt are for the preparation of the purposes in the medicine of blood sugar lowering level.

20. the purposes of claim 19, wherein R ₃And R ₄Be selected from separately guanidine radicals, guanidine radicals (C _1-4) alkyl and amino (C _1-4) alkyl.

21. the purposes of claim 19, described medicine further comprises insulin.

22. the purposes of claim 19, described chemical compound is selected from

Or its pharmaceutically acceptable salt.

23. the chemical compound of claim 1 or its pharmaceutically acceptable salt for the preparation of the treatment diabetes medicine in purposes.

24. the purposes of claim 23, described chemical compound is selected from

Or its pharmaceutically acceptable salt.

25. the purposes of claim 23, described medicine further comprises insulin.