JP4954888B2 - Glycogen synthase kinase-3 inhibitor - Google Patents

Description

  The present invention relates to novel compounds for inhibiting glycogen synthase kinase-3 (GSK-3) and their use in the modulation of biological conditions mediated by GSK-3 activity, more particularly II It relates to the use of these compounds in the treatment of biological conditions such as type 2 diabetes, neurodegenerative disorders and neurodegenerative diseases, and affective disorders.

  Protein kinases are enzymes that phosphorylate protein substrates and play an important role in signaling extracellular events to the cytoplasm and nucleus, including cell life and death, including mitosis, differentiation and apoptosis Is involved in virtually all events related to Thus, protein kinases have long been a convenient drug target. While the activity of protein kinases is critical to the satisfactory state of the cell, on the other hand, its use as a drug target is limited because its inhibition often results in cell death. Although cell death is a desirable effect for anticancer drugs, it is a major drawback for most other therapeutic agents.

  Glycogen synthase kinase-3 (GSK-3) is a member of the protein kinase family and is involved in the signaling and metabolic regulation of insulin, as well as the cytoplasmic proline-directed serine − involved in Wnt signaling and cell fate schemes during embryonic development. Threonine kinase. Two similar isoforms of this enzyme termed GSK-3α and GSK-3β have been identified.

  GSK-3, unlike other protein kinases that are typically activated by signal transduction pathways, is normally activated in resting cells and its activity is due to insulin binding to its cell surface receptors. GSK-3 has long been regarded as a convenient drug target within the protein kinase family because it is weakened by the activation of certain signaling pathways, such as the resulting signaling pathway. Activation of the insulin receptor results in activation of protein kinase B (PKB, also called Akt), which in turn phosphorylates GSK-3, thereby deactivating GSK-3. Activate. Inhibition of GSK-3 is presumed to result in activation of glycogen synthesis. The complex insulin signaling pathway is further complicated by negative feedback regulation of insulin signaling by GSK-3 itself, which phosphorylates insulin receptor substrate-1 at serine residues (Eldar-Finkelman et al., 1997). .

  Thus, synthesized GSK-3 inhibitors can mimic the action of certain hormones and growth factors (eg, insulin, etc.) that use the GSK-3 pathway. In certain pathological conditions, this scheme allows the bypass of defective receptors, or other mis-components of signaling mechanisms, so that as in the case of non-insulin dependent type II diabetes Biological signals can be made effective even when some upstream participants in the signaling cascade are not normal.

  Regulation of glycogen catabolism in cells is a vital biological function that encompasses a complex arrangement of signaling elements, including the hormone insulin. Through various mediators, insulin exerts its regulatory effects by increasing the synthesis of glycogen by glycogen synthase (GS). An important event in insulin action is phosphorylation of insulin receptor substrates (IRS-1, IRS-2) at multiple tyrosine residues, resulting in the simultaneous activation of several signaling components including PI3 kinase (Myers et al., 1992). Similarly, glycogen synthase activity is inhibited by its phosphorylation. Significant reductions in glycogen synthase activity and glycogen concentration in muscle of type II diabetic patients have been observed (Sulman et al., 1990).

  One of the earliest changes associated with the development of type II diabetes (non-insulin dependent diabetes) is insulin resistance. Insulin resistance is characterized by hyperinsulinemia and hyperglycemia. The exact molecular mechanism that causes insulin resistance is unknown, but is thought to be due to defects in downstream components of the insulin signaling pathway.

  Glycogen synthase kinase-3 (GSK-3) is one of the downstream components of the insulin signaling pathway. The large activity of GSK-3 was found to impair the action of insulin in intact cells by phosphorylating the serine residue of insulin receptor substrate-1 (IRS-1) (Eldar-Finkelman et al., 1997) and similarly, increased GSK-3 activity expressed in cells was found to result in suppression of glycogen synthase activity (Eldar-Finkelman et al., 1996). Further studies conducted in this context revealed that GSK-3 activity is significantly increased in epididymal adipose tissue of diabetic mice (Eldar-Finkelman et al., 1999). Subsequently, increased GSK-3 activity was detected in skeletal muscle of type II diabetic patients (Nickoulina et al., 2000). Further recent studies have further elucidated the role of GSK-3 in glycogen metabolism and insulin signaling (for review, see Eldar-Finkelman, 2002; Woodgett, 2001). Thereby, it was suggested that inhibition of GSK-3 activity could be a way to increase insulin activity in vivo.

  GSK-3 is also thought to be an important participant in the pathogenesis of Alzheimer's disease. GSK-3 has been identified as one of the kinases that phosphorylate tau (microtubule binding protein) involved in the formation of paired helical filaments (PHF) (an early feature of Alzheimer's disease). Apparently, abnormal hyperphosphorylation of tau is responsible for microtubule destabilization and PHF formation. Despite the fact that several protein kinases have been shown to promote tau phosphorylation, only phosphorylation of GSK-3 is directly related to tau's ability to promote microtubule self-assembly. It was found to have an effect (Mandelkow et al., 1992; Mulot et al., 1995). Further evidence for the role of GSK-3 in this regard came from studies of cells that overexpress GSK-3 and from transgenic mice that specifically express GSK-3 in the brain . In both cases, GSK-3 resulted in the generation of the PHF-like epitope tau (Lucas et al., 2001).

  GSK-3 is further linked to Alzheimer's disease by its role in cellular apoptosis. Due to the fact that insulin is a neuronal survival factor and that insulin initiates its anti-apoptotic effect by activation of PI3 kinase and PKB, GSK-3, which is negatively regulated by these signaling components, is neuronal It was suggested to promote apoptosis. Several studies have actually confirmed this view and have shown that GSK-3 is extremely important in life and death decisions. Moreover, its apoptotic function has been shown to be independent of PI3 kinase. Overexpression of GSK-3 in PC12 cells caused apoptosis (Pap et al., 1998). Activation of GSK-3 in cerebellar granule neurons mediated migration and cell death (Tong et al., 2001). In human neuroblastoma SH-SY5Y cells, overexpression of GSK-3 promoted stauroaporin-induced cell apoptosis (Bijur et al., 2000).

  Studies where the relationship between GSK-3 inhibition and prevention of cell death showed that expression of Frat1, a GSK-3β inhibitor, was sufficient to rescue neurons from death induced by inhibition of PI3 kinase (Crowder et al., 2000).

  Another involvement of GSK-3 was detected in connection with affective disorders (ie bipolar disorder and manic depression). This link is based on the finding that lithium (the first mood stabilizer frequently used in bipolar disease) is a strong specific inhibitor of GSK-3 in the therapeutic concentration range used clinically (Klein Et al., 1996; Stambolic et al., 1996; Piel et al., 2001). This discovery has led to a series of studies initiated to reveal whether lithium can mimic loss of GSK-3 activity in cellular processes. Indeed, lithium activates glycogen synthesis (Cheng et al., 1983), β-catenin stabilization and accumulation (Stambolic et al., 1996), induction of axial replication in Xenopus embryos (Klein et al., 1996), As well as the protection of neuronal death (Bijur et al., 2000). Valproic acid (another commonly used mood stabilizer) has also been found to be an effective GSK-3 inhibitor (Chen et al., 1999). Taken together, these studies indicate that GSK-3 is a major in vivo target for lithium and valproic acid and thus has important implications in novel therapeutic treatment of affective disorders.

  One mechanism by which lithium and other GSK-3 inhibitors may act to treat bipolar disorder is the survival of neurons exposed to abnormally high levels of excitement induced by the neurotransmitter glutamate. To increase (Nonaka et al., 1998). Neuronal excitotoxicity induced by glutamate is also believed to be a major cause of neurodegeneration associated with acute injury in cerebral ischemia, traumatic brain injury and bacterial infections. Moreover, excessive glutamate signaling is chronic in diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, AIDS-related dementia, amyotrophic lateral sclerosis (AML), and multiple sclerosis (MS). It is believed to be a contributor to neuronal damage (Thomas, 1995).

  As a result, GSK-3 inhibitors are considered useful treatments in these and other neurodegenerative disorders. Indeed, dysregulation of GSK-3 activity has recently been associated with schizophrenia (schizophrenia) (Beasley et al., 2001), seizures and Alzheimer's disease (AD) (Bhat and Budd, 2002; Lucas et al., 2001; Mandelkow et al. 1992), and is associated with several CNS disorders and neurodegenerative diseases.

  In light of the widespread involvement of GSK-3 in various signaling pathways, the development of specific inhibitors of GSK-3 is promising and important not only in basic research but also in various therapeutic interventions It is considered to have meaning.

  As stated above, some mood stabilizers have been found to inhibit GSK-3. However, both inhibition of GSK-3 by lithium chloride (LiCl) (PCT International Patent Application Publication WO 97/41854) and inhibition of GSK-3 by purine inhibitors (PCT International Patent Application Publication WO 98/16528) have been reported. However, these inhibitors are not specific for GSK-3. In fact, these drugs have been shown to affect a number of signaling pathways and inhibit other cellular targets such as inositol monophosphatase (IMpath) and histone deacetylase (Berridge et al., 1989).

  Similarly, genetically engineered cAMP response element binding protein (CREB) (a known substrate of GSK-3) is added to other potential GSK-3 peptide inhibitors (Fiol et al., 1990). (Fiol et al., 1994). However, these substrates also only superficially inhibit GSK-3 activity.

  Other GSK-3 inhibitors have been recently reported. Two structurally related small molecules that specifically inhibit GSK-3 (SB-216763 and SB-415286) (Glaxo SmithKlein Pharmaceutical) have been developed, which regulate glycogen metabolism and gene transcription, and Have been shown to protect against neuronal death induced by reduced PI3 kinase activity (Cross et al., 2001; Coghlan et al., 2000). In another study, indirubin, an active ingredient of Kampo for chronic myeloid leukemia, was shown to be a GSK-3 inhibitor. However, indirubin also inhibits cyclic dependent protein kinase-2 (CDK-2) (Damiens et al., 2001). These GSK-3 inhibitors are ATP competitive and have been identified by high-throughput screening of chemical libraries. It is generally accepted that the major drawback of ATP competitive inhibitors is their limited specificity (Davies et al., 2000).

  General methodologies for developing specific peptides and other GSK-3 inhibitors are reported in WO 01/49709 and US Pat. No. 6,780,625. These documents are from the inventor and are hereby incorporated by reference as if fully set forth herein. This general methodology is based on defining the structural features of GSK-3 substrates and developing GSK-3 inhibitors according to these features. However, although these references define these structural features and teach various short peptides that effectively inhibit GSK-3 activity, these references are as GSK-3 inhibitors. It does not teach the design and synthesis of small molecules that can be useful. WO 2004/052414 by the inventor discloses that attaching a hydrophobic moiety to the end of a peptide GSK-3 inhibitor increases its inhibitory activity. This document is hereby incorporated by reference as if it had been fully described herein.

  However, peptides are intriguing pharmaceutical targets, but their use is often low in ability to penetrate biological membranes such as biological instability, immunogenicity, cell membranes and blood brain barrier (BBB) Limited by.

  Accordingly, it is widely recognized that there is a need for non-peptide compounds that inhibit GSK-3 activity that do not have the above limitations, and thus it would be very advantageous to have such compounds.

  The inventor has the advantage that compounds designed according to the specific features of the recognition motif of the GSK-3 substrate show substrate competition inhibitory activity against GSK-3 and thus reduce the activity of GSK-3. It has now surprisingly been found that it can be used effectively in various applications.

  Therefore, according to one aspect of the present invention, a compound comprising a negatively charged group and at least one amino moiety-containing group covalently bonded to a negatively charged group via a spacer, the spacer Has at least one interaction between the negatively charged group and the first binding site in the catalytic domain of GSK-3, and the amino moiety-containing group and the second binding site in the catalytic domain of GSK-3. Provided are compounds having a length, structure and flexibility selected to allow at least one interaction between them, whereby the compound can inhibit the catalytic activity of GSK-3.

式中、Ｘ，Ｙ，ＺおよびＷはそれぞれ独立して炭素原子または窒素原子であり；
ＡはＪであり；
Ｂは負に帯電した基であり、
Ｄは水素、アルキル、Ｃ_１〜Ｃ_６置換アルキル、トリハロアルキル、シクロアルキル、アルケニル、アルキニル、アリール、ヘテロアリール、複素脂環、ハロ、ヒドロキシ、アルコキシ、アリールオキシ、チオヒドロキシ、チオアルコキシ、チオアリールオキシ、スルフィニル、スルホニル、シアノ、ニトロ、アゾ、スルホンアミド、カルボニル、ケトエステル、チオカルボニル、エステル、エーテル、チオエーテル、チオカルバメート、尿素、チオ尿素、Ｏ−カルバミル、Ｎ−カルバミル、Ｏ−チオカルバミル、Ｎ−チオカルバミル、Ｃ−アミド、Ｎ−アミド、Ｃ−カルボキシ、Ｏ−カルボキシ、スルホンアミド、トリハロメタンスルホンアミド、グアニル、グアニロアルキル、グアニジノ、グアニジノアルキル、アミノ、アミノアルキル、ヒドラジンおよび疎水性部分からなる群から選択され；そして
Ｒ_１，Ｒ_２，Ｒ_３およびＲ_４はそれぞれ独立してアミノ部分含有基、水素、孤立電子対、アルキル、トリハロアルキル、シクロアルキル、アルケニル、アルキニル、アリール、ヘテロアリール、複素脂環、ハロ、ヒドロキシ、アルコキシ、アリールオキシ、チオヒドロキシ、チオアルコキシ、チオアリールオキシ、スルフィニル、スルホニル、シアノ、ニトロ、アゾ、スルホンアミド、カルボニル、ケトエステル、チオカルボニル、エステル、エーテル、チオエーテル、チオカルバメート、尿素、チオ尿素、Ｏ−カルバミル、Ｎ−カルバミル、Ｏ−チオカルバミル、Ｎ−チオカルバミル、Ｃ−アミド、Ｎ−アミド、Ｃ−カルボキシ、Ｏ−カルボキシ、スルホンアミド、トリハロメタンスルホンアミド、グアニル、グアニリノアルキル、グアニジノ、グアニジノアルキル、アミノ、アミノアルキル、ヒドラジンおよびアンモニウムイオンからなる群から選択され；
ただし、Ｘ，Ｙ，ＺおよびＷの少なくとも一つは窒素原子であるかおよび／またはＲ_１，Ｒ_２，Ｒ_３およびＲ_４の少なくとも一つはアミノ部分含有基である。 Excluded from the scope of the invention are compounds already described in WO 2005/000192. Therefore, excluded from the scope of the present invention are compounds having the general formula II or pharmaceutically acceptable salts thereof:

In which X, Y, Z and W are each independently a carbon atom or a nitrogen atom;
A is J;
B is a negatively charged group,
D is hydrogen, _alkyl, C 1 -C ₆ substituted alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryl Oxy, sulfinyl, sulfonyl, cyano, nitro, azo, sulfonamide, carbonyl, ketoester, thiocarbonyl, ester, ether, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- Thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, trihalomethanesulfonamide, guanyl, guaniloalkyl, guanidino, guanidinoalkyl, amino, aminoalkyl It is selected from the group consisting of hydrazine and a hydrophobic moiety; and R _{1, R 2,} R ₃ and R ₄ are amino moiety-containing groups each independently hydrogen, lone pair, alkyl, trihaloalkyl, cycloalkyl, alkenyl , Alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azo, sulfonamide, carbonyl, ketoester, thiocarbonyl Ester, ether, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, G Halo methanesulfonamide, guanyl, guanylyl Reno alkyl is selected guanidino, guanidinoalkyl, amino, aminoalkyl, from the group consisting of hydrazine and ammonium ions;
However, at least one of X, Y, Z and W is a nitrogen atom and / or at least one of R ₁ , R ₂ , R ₃ and R ₄ is an amino moiety-containing group.

  According to further features in preferred embodiments of the invention described below, inhibiting the catalytic activity of GSK-3 comprises reducing substrate binding to the catalytic domain.

  According to still further features in the described preferred embodiments the first binding site comprises at least one amino acid residue selected from the group consisting of arginine 180, arginine 96 and lysine 205.

  According to still further features in the described preferred embodiments the second binding site is selected from the group consisting of aspartic acid 181, glutamic acid 97, aspartic acid 90, aspartic acid 181, glutamic acid 200, glutamine 89, tyrosine 215 and asparagine 95. At least one amino acid residue.

  According to still further features in the described preferred embodiments the compound further comprises a hydrophobic moiety capable of interacting with the third binding site of GSK-3.

  According to still further features in the described preferred embodiments the third binding site is part of the catalytic domain of GSK-3.

  According to still further features in the described preferred embodiments the third binding site comprises at least one amino acid residue selected from the group consisting of isoleucine 217, phenylalanine 67 and tyrosine 215.

  According to still further features in the described preferred embodiments the hydrophobic moiety forms part of a spacer.

  According to still further features in the described preferred embodiments the spacer length is between 2 angstroms and 50 angstroms.

を有し、式中、Ｌはリン原子、イオウ原子、ケイ素原子、ホウ素原子および炭素原子からなる群から選択され；Ｑ^＊，ＧおよびＤ^＊はそれぞれ独立して酸素およびイオウからなる群から選択され；Ｅはヒドロキシ、アルコキシ、アリールオキシ、カルボニル、チオカルボニル、Ｏ−カルボキシ、チオヒドロキシ、チオアルコキシおよびチオアリールオキシからなる群から選択されるかまたは存在しない。 Negatively charged groups are formulas

Wherein L is selected from the group consisting of phosphorus atom, sulfur atom, silicon atom, boron atom and carbon atom; Q ^* , G and D ^* are each independently selected from the group consisting of oxygen and sulfur E is selected from the group consisting of hydroxy, alkoxy, aryloxy, carbonyl, thiocarbonyl, O-carboxy, thiohydroxy, thioalkoxy and thioaryloxy, or absent.

According to still further features in the described preferred embodiments L is phosphorus, Q ^* , D ^* and G are each oxygen and E is hydroxy.

  According to still further features in the described preferred embodiments the amino moiety-containing group comprises at least one positively charged group.

  According to still further features in the described preferred embodiments the at least one positively charged group is selected from the group consisting of ammonium ions and guanidinium ions.

  According to still further features in the described preferred embodiments the at least one positively charged group has a chemical structure derived from a side chain of positively charged amino acids.

  According to still further features in the described preferred embodiments the positively charged amino acid is selected from the group consisting of arginine, lysine, histidine, proline and any derivative thereof.

  According to still further features in the described preferred embodiments the at least one amino moiety-containing group is selected from the group consisting of guanidino, guanidinoalkyl, amino, aminoalkyl, hydrazine, guanyl and guaniloalkyl.

  According to still further features in the described preferred embodiments at least one of the at least one amino moiety-containing group forms part of a spacer.

  According to still further features in the described preferred embodiments the spacer is at least one cyclic moiety.

  According to still further features in the described preferred embodiments the at least one cyclic moiety is selected from the group consisting of alicyclic, aryl, heteroaryl and heteroalicyclic.

  According to still further features in the described preferred embodiments the spacer comprises at least two annular portions.

  According to still further features in the described preferred embodiments at least two of the annular portions are fused together.

  According to still further features in the described preferred embodiments at least two of the cyclic moieties are joined to each other via a linker.

  According to still further features in the described preferred embodiments the linker is selected from the group consisting of a bond, a heteroatom, a hydrocarbon chain and a hydrocarbon chain interrupted by at least one heteroatom.

を有し、式中、ｎは０〜１０の整数であり；
ｍは１〜６の整数であり；
Ｂは負に帯電した基であり；
Ｑは少なくとも一つのアミノ部分含有基の少なくとも一つであり；そして
Ｊ−（Ｓ）_１−（Ｓ）_２‐‐‐（Ｓ）_ｎ−Ｋはスペーサーであり；
Ｋはアリール、ヘテロアリール、脂環、または複素脂環からなる群から選択され；そして
ＪおよびＳ_１−Ｓ_ｎはそれぞれ独立して置換または非置換アリール、置換または非置換ヘテロアリール、置換または非置換脂環、置換または非置換複素脂環、結合、ヘテロ原子、置換または非置換炭化水素鎖および少なくとも一つのヘテロ原子で中断された置換または非置換炭化水素鎖からなる群から選択されるかまたは存在しない。 According to still further features in the described preferred embodiments the compound has the general formula I:

In which n is an integer from 0 to 10;
m is an integer from 1 to 6;
B is a negatively charged group;
Q is at least one of at least one amino moiety-containing group; and J- (S) _1- (S) _2- (S) _n -K is a spacer;
K is selected from the group consisting of aryl, heteroaryl, alicyclic, or heteroalicyclic; and J and S ₁ -S _n are each independently substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted Selected from the group consisting of a substituted alicyclic ring, a substituted or unsubstituted heteroalicyclic ring, a bond, a heteroatom, a substituted or unsubstituted hydrocarbon chain and a substituted or unsubstituted hydrocarbon chain interrupted by at least one heteroatom, or not exist.

  According to still further features in the described preferred embodiments m is an integer of 1-2.

  According to still further features in the described preferred embodiments n is an integer from 0-2.

According to still further features in the described preferred embodiments n is 2 and each of S ₁ , S ₂ and K is independently selected from the group consisting of aryl and heteroaryl.

  According to still further features in the described preferred embodiments J is a carbon hydrogen chain.

  According to still further features in the described preferred embodiments J is alkyl.

According to still further features in the described preferred embodiments S ₁ is aryl, S ₂ is heteroaryl and K is aryl.

According to still further features in the described preferred embodiments S ₁ is phenyl, S ₂ is triazole and K is phenyl.

According to still further features in the preferred embodiments described, at least one of J and S ₁ -S _n comprises at least one amino moiety-containing group.

  According to still further features in the described preferred embodiments the at least one amino moiety-containing group forms part of K.

At least one of J, (S) _1- (S) _n and K includes a hydrophobic moiety attached thereto.

  According to still further features in the described preferred embodiments the hydrophobic moiety is a fatty acid residue, a saturated alkylene chain having 4 to 30 carbon atoms, an unsaturated alkylene chain having 4 to 30 carbon atoms, an aryl , Selected from the group consisting of cycloalkyl and hydrophobic peptide sequences.

  According to still further features in the described preferred embodiments the fatty acid is selected from the group consisting of myristic acid, lauric acid, palmitic acid, stearic acid, oleic acid, arachidonic acid, linoleic acid and linolenic acid.

  According to still further features in the described preferred embodiments J is a hydrocarbon chain or absent and n is 0.

  According to yet another aspect of the present invention there is provided a pharmaceutical composition comprising, as an active ingredient, any of the above-mentioned compounds capable of inhibiting the activity of GSK-3 and a pharmaceutically acceptable carrier. .

  According to still further features in the described preferred embodiments the pharmaceutical composition is packaged in a packaging material and packaged for use in the treatment of biological conditions associated with GSK-3 activity as detailed below. It is identified by the surface of the material for use or the type in it.

  According to still further features in the described preferred embodiments the pharmaceutical composition further comprises at least one further active ingredient capable of altering the activity of GSK-3 as detailed below.

  According to yet another aspect of the present invention, there is provided a method of treating a biological condition associated with the activity of GSK-3, said method comprising a therapeutically effective amount of the above-mentioned in a subject in need thereof. Compound (comprising a negatively charged group and at least one amino moiety-containing group covalently bonded to the negatively charged group via a spacer, wherein the spacer comprises a negatively charged group and a GSK Allow at least one interaction between the first binding site in the catalytic domain of -3 and at least one interaction between the amino moiety-containing group and the second binding site in the catalytic domain of GSK-3 Administering a compound having a length, structure and flexibility selected to be).

  According to further features in the described preferred embodiments, the method according to this aspect of the invention further comprises co-administering at least one additional active ingredient capable of altering the activity of GSK-3.

  The further active ingredient can be an active ingredient that can inhibit the activity of GSK-3 or an active ingredient that can down-regulate the expression of GSK-3.

  According to another aspect of the present invention there is provided the use of a compound as described above in the treatment of a biological condition associated with the activity of GSK-3.

  According to yet another aspect of the invention, there is provided the use of a compound as described above for the preparation of a medicament for the treatment of a biological condition associated with the activity of GSK-3.

  The biological condition according to the present invention is preferably selected from the group consisting of obesity, non-insulin dependent diabetes, insulin dependent condition, affective disorder, neurodegenerative disease or neurodegenerative disorder, and psychotic disease or disorder. .

  The affective disorder can be a unipolar disorder (eg, depression) or a bipolar disorder (eg, manic depression).

  The neurodegenerative disorder can result from an event selected from the group consisting of cerebral ischemia, stroke, traumatic brain injury and bacterial infection, or the neurodegenerative disorder can be Alzheimer's disease, Huntington's disease, Parkinson's disease, AIDS. It can be a chronic neurodegenerative disorder resulting from a disease selected from the group consisting of associated dementia, amyotrophic lateral sclerosis (AML) and multiple sclerosis.

  According to an additional aspect of the present invention, cells expressing GSK-3 are bound to the aforementioned compound (at least one amino moiety covalently bound to a negatively charged group and a negatively charged group via a spacer). Wherein the spacer comprises at least one interaction between the negatively charged group and the first binding site in the catalytic domain of GSK-3, and the amino moiety-containing group and the GSK- Contacting with an inhibitory effective amount of a compound having a length, structure and flexibility selected to allow at least one interaction between the second binding sites in the three catalytic domains A method for inhibiting the activity of GSK-3 is provided.

  According to still an additional aspect of the present invention there is provided the use of a compound as described above for inhibiting the activity of GSK-3. Inhibiting the activity of GSK-3 is preferably carried out by contacting cells expressing GSK-3 with an inhibitory effective amount of the aforementioned compounds.

  The activity may be phosphorylation activity and / or autophosphorylation activity.

  According to still an additional aspect of the present invention, there is provided a method for enhancing insulin signaling, wherein an insulin responsive cell is covalently bound to a compound as described above (a negatively charged group and a negatively charged group via a spacer). Wherein the spacer comprises at least one interaction between the negatively charged group and the first binding site in the catalytic domain of GSK-3; And compounds of length, structure and flexibility selected to allow at least one interaction between the amino moiety-containing group and the second binding site in the catalytic domain of GSK-3) There is provided a method comprising contacting with an appropriate amount.

  According to an additional aspect of the present invention there is provided the use of the above-mentioned compound for enhancing insulin signaling. Preferably, enhancing insulin signaling is performed by contacting insulin responsive cells with an effective amount of the compound described above.

  In each of these methods and / or uses, contacting the cells can be performed in vitro or in vivo.

  According to further features in the described preferred embodiments, the methods and / or uses according to these additional aspects of the invention each further comprise contacting the cell with at least one further active ingredient as described above.

  The present invention is currently known by providing newly designed non-peptide compounds for inhibiting GSK-3 activity that can be used efficiently in the treatment of various biological conditions. It has succeeded in dealing with the disadvantages of the form.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

  As used herein, the singular forms (“a”, “an”, and “the”) include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or the term “at least one compound” can encompass a plurality of compounds, including mixtures thereof.

  Throughout this disclosure, various aspects of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, descriptions of ranges such as 1-6 are specifically disclosed subranges (eg, 1-3, 1-4, 1-5, 2-4, 2-6, 3-6 etc.), and Should be considered as having individual numerical values (eg, 1, 2, 3, 4, 5 and 6) within the range. This applies regardless of the breadth of the range.

  Whenever a numerical range is indicated herein, it is meant to include any mentioned numerals (fractional or integer) included in the indicated range. The first indicated number and the second indicated number “within / between the range” and the first indicated number “from” to the second indicated number “to” The expression “range to / from” is used interchangeably and is meant to include the first indicated number, the second indicated number, and all fractions and integers in between.

The present invention is described herein by way of example only and with reference to the accompanying drawings. DETAILED DESCRIPTION Referring now more particularly to the drawings, the details shown are by way of example and for exemplary discussion of preferred embodiments of the invention and therefore the principles of the invention It is emphasized that it has been presented to provide what is considered to be the most useful and readily understood description of conceptual aspects. In this regard, it will be apparent to those skilled in the art, upon understanding the description in conjunction with the drawings, that some forms of the invention may actually be embodied, so that the structural details of the invention can be No attempt has been made to show in more detail than is necessary for a basic understanding of.
BRIEF DESCRIPTION OF THE FIGURES FIG. 1a-b represents a computer image of the 3D structure of peptides p9CREB (FIG. 1a) and CREB (FIG. 1b) as obtained by a 2D ¹ H-NMR study (hydrogen atoms shown). Not; the carbon skeleton is gray, the nitrogen atom is blue, the oxygen atom is red, and the phosphorus atom is yellow).
FIG. 2 is an image showing the electrostatic distribution of the p9CREB peptide based on the 3D structure of the peptide obtained by 2D ¹ H-NMR studies.
3a-b shows the chemical structures of phenyl phosphate, pyridoxal phosphate (P-5-P), GSC-1, GSC-2, GSC-3 and the new compounds GSC-4, GSC-5 and GSC-21 ( 3a) and the chemical structures of the new compounds GSC-6, GSC-7, GSC-8 and GSC-9 (FIG. 3b).
Figures 4a-b represent the ESI-MS (Figure 4a) and HPLC chromatogram (Figure 4b) of 3,5-bis (2-aminoethyl) benzyl phosphate (GSC-21).
FIG. 5 shows the GSK-3 inhibitory activity of phenylphosphate, GSC-1, GSC-2, GSC-3 and pyridoxal phosphate (P-5-P) in an in vitro inhibition assay using PGS-1 peptide substrates. Represents a comparative plot.
FIG. 6 represents a comparative plot showing GSK-3 inhibitory activity of GSC-1, GSC-2, GSC-3, GSC-4 and GSC-21 in an in vitro inhibition assay (the black circles indicate GSC-2, The red circle indicates GSC-1, the green circle indicates GSC-3, the blue circle indicates GSC-4, and the pink circle indicates GSC-21).
FIG. 7a-b shows GSC-1, GSC-2, GSC-3 and GSC-4 in an in vitro inhibition assay with p9CREB peptide substrate (FIG. 7a, black circle indicates GSC-2, blank circle indicates GSC -1, black triangles indicate GSC-3, black squares indicate GSC-4) and GSC-5, GSC-6 and GSC-7 (FIG. 7b, squares indicate GSC-5, triangles Represents a comparative plot showing GSK-3 inhibitory activity of GSC-6 and circles indicate GSC-7.
FIG. 8 represents a Lineweaver-Burk plot showing the inhibition of GSK-3 by GSC-7 at the indicated concentrations, represented by the incorporation of phosphate groups into p9CREB peptide substrate (CPM), GSC-7 Indicates a competitive specific inhibitor (results show one representative experiment from three experiments; each point is the average of duplicate samples).
FIG. 9 is an image of gel electrophoresis for CDK-2 kinase activity assayed in the presence of ³² P [γ-ATP] and histone H1 as substrate, GSC-4, GSC-5 and GSC-7. The absence of inhibitory activity is shown.
FIG. 10 shows the GSC-5 (hollow circle) and GSC-7 (solid circle) versus glycogen synthesis activity in C2C12 cells, shown as fold stimulation across cells treated with vehicle 0.1% HCl. Represents a plot that demonstrates the effect.
FIG. 11a-b shows [ ³ H] 2-deoxy in cells treated with GSC-4 and GSC-21 as fold activation over cells treated with peptide control (normalized to 1 unit). 12 is a bar graph showing the effect of GSC-21 (FIG. 11b) and GSC-4 (FIG. 11a) on glucose uptake in mouse adipocytes, represented by glucose uptake.
FIG. 12 represents a calculated simulation of the interaction between GSC-4 and GSK-3.
FIG. 13 represents a calculated simulation of the interaction between GSC-5 and GSK-3.
FIG. 14 represents a calculated simulation of the interaction between GSC-7 and GSK-3.
FIG. 15 represents a calculated simulation of the interaction between GSC-6 and GSK-3.
FIG. 16 represents a calculated simulation of the interaction between GSC-8 and GSK-3.
FIG. 17 represents a calculated simulation of the interaction between GSC-9 and GSK-3.
FIG. 18 represents the chemical structures of newly designed GSK-3 inhibitors, MP-1, MP-2, MP-3, MP-4, MP-5 and MP-6.
FIG. 19 represents a comparative plot showing the GSK-3 inhibitory activity of MP-1, MP-2, MP-3, MP-4, MP-5 and MP-6 in an in vitro inhibition assay using p9CREB peptide substrate.

  The present invention relates to novel non-peptide compounds that can inhibit GSK-3 activity and thus can be used in the treatment of biological conditions mediated by GSK-3. Specifically, the present invention relates to (i) a compound designed according to a pharmacophore coordination bond of a GSK-3 substrate (which may optionally be bound to a hydrophobic moiety), (ii) A pharmaceutical composition containing the compound, (iii) a method of using said compound to inhibit GSK-3 activity and to enhance insulin signaling, and (iv) obesity, non-insulin dependent diabetes, It relates to methods of using said compounds in the treatment of biological conditions such as, but not limited to, insulin dependent conditions, affective disorders, neurodegenerative diseases and neurodegenerative disorders, and psychotic diseases or disorders.

  The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

  Before describing at least one embodiment of the present invention in detail, it should be understood that the present invention is not limited in its application to the details set forth in the following description or the details illustrated by the examples. The invention is capable of other embodiments or of being practiced in various ways or being carried out in various ways. It should also be understood that the wording and terminology used herein is for description only and should not be considered limiting.

One parameter that has the role of recognizing substrate-kinase is an element located within the substrate, which is usually associated with a “recognition motif”. As mentioned above, GSK-3, unlike other kinases, has a specific recognition motif, which contains the amino acid sequence SX ₁ X ₂ X ₃ S (p) defined in SEQ ID NO: 1, where S Is serine or threonine, each of X ₁ , X ₂ and X ₃ can be any amino acid and S (p) is phosphorylated serine or phosphorylated threonine.

  Designed and synthesized based on this recognition motif as broadly taught in WO 01/49709 and US Pat. No. 6,780,625, which are hereby incorporated by reference as if fully set forth herein. A set of short peptides was tested for their activity as substrates or inhibitors. Based on these assays, a number of features that make these peptides active substrates or inhibitors for GSK-3 were determined. One of the most important features is that a phosphorylated serine or threonine residue in the motif is required for both substrate and inhibitor binding to GSK-3. These assays further showed that some of these peptides are very strong and specific inhibitors of GSK-3. These peptides have been defined as substrate competitive inhibitors.

  Based on the discovery that GSK-3 recognizes only pre-phosphorylated substrates, ie, substrates with phosphorylated serine or threonine residues, pre-phosphorylated GSK-3 substrates are those of GSK-3 It was hypothesized that it has a specific structure that allows it to interact with the catalyst core. Furthermore, it was hypothesized that determining this specific structure would allow the development of small molecules that could act as substrate competitive inhibitors of GSK-3.

  Thus, in the search for small molecules that mimic the inhibitory activity of the small GSK-3 peptide inhibitors described above, while practicing the present invention, the three-dimensional structure as well as the specific structure of the short phosphorylated peptide substrate The above characteristics were determined and a number of compounds characterized by these characteristics were tested for their activity as GSK-3 inhibitors.

  As a representative example of a GSK-3 inhibitor, a short pre-phosphorylated peptide p9CREB (ILSRRPS (p) YR, SEQ ID NO: 2) was selected. The three-dimensional structure of p9CREB as well as the three-dimensional structure of the corresponding non-phosphorylated peptide CREB (ILSRRPSYR, SEQ ID NO: 3) was determined by 2D NMR as detailed in the examples below (see Example 1). .

  As shown in FIGS. 1a and 1b, the phosphorylated p9CREB substrate has a defined structure in solution (FIG. 1a), but the corresponding non-phosphorylated peptide CREB does not show any specific structure. (FIG. 1b).

  In view of these results, it is suggested that the phosphate group in the phosphorylated peptide results in a “looped” structure through cation-π interaction between tyrosine (Y8) and arginine (R4) ( Tables 2 and 3), as a result, suggested that phosphorylated serine at the recognition motif is located outside the loop. Such a “bent” structure of the substrate makes phosphorylated serine accessible for interaction with the substrate binding pocket of the enzyme.

  Support for this suggestion was actually found in the recently published crystallization data for GSK-3 described by Dajani et al. (2001). The crystallization data of Dajani et al. Show that the substrate binding site of GSK-3 contains three positively charged residues Arg96, Arg180 and Lys205, and these residues interact with phosphate ions.

  FIG. 2 represents the electrostatic distribution on the “surface” of the p9CREB peptide based on these findings.

  While continuing to recall the present invention, from the above findings, small molecules that mimic the structure of GSK-3 substrate to exert substrate competitive inhibitory activity should be designed according to the following characteristics: It was derived:

  The molecule should contain a negatively charged group, preferably a phosphate group;

  Negatively charged groups should not be sterically hindered; and

  The negatively charged group is preferably adjacent by at least one side or both sides by one or two positively charged groups.

  Based on the above, a general formula for potential compounds to inhibit GSK-3 activity was designed (see Example 2). Preliminary experiments conducted with the “first generation” of these compounds, ie the compounds with the most simplified structure of this formula, as described in the Examples section below (see Example 3) Demonstrated the ability of these compounds to inhibit GSK-3 activity and thus provided preliminary suggestions for the inhibitory ability of compounds having such a formula.

  A more advanced generation of compounds, including novel compounds, was also designed and synthesized based on the above (see, eg, Example 2). As described in the Examples section below (see Example 3), experiments performed with these compounds further demonstrate their ability to inhibit GSK-3 activity, and further, mouse adipocytes Have demonstrated their ability to increase glucose uptake, thus demonstrating the reliable inhibitory and therapeutic effects exerted by compounds designed according to such a formula.

  This “first generation” compound family is based on a monoaryl or heteroaryl core (having one aryl or heteroaryl ring), which has a negatively charged group and one or two positively charged groups. Was bound. This family is also referred to collectively herein as GSC molecules.

  Additional studies on the pharmacophore binding sites of the catalytic domain of GSK-3 have been conducted to assess the interaction of these binding sites with the designed molecules and to interact strongly with these binding sites and thus substrate binding. This was done to further design small molecules that inhibit GSK-3 activity through competition with. In these studies, protein crystal data as taught by Ter Haar et al. (2001) was used as a model.

  As described in the Examples section below (see Example 4), the catalytic domain of GSK-3 is a functional group of small molecules (eg, negatively and / or positively charged groups, hydrophobic moieties). It has been derived that it contains various binding sites that can interact simultaneously. In order to achieve the maximum number and strength of interaction with these binding sites, small molecules (if not all) have a good proximity and orientation with the majority of the various functional groups to the binding sites. It was derived that it should be designed to be Thus, for example, small molecule designs should be considered where the distance between a negatively charged group and one or more positively charged groups is greater than that obtained with a single aromatic ring. Was derived.

  For this purpose, a second generation of compounds was designed, successfully prepared (see Example 5) and implemented (Example 6). These compounds are designed to include a spacer that binds a negatively charged group to one or more positively charged groups, which are believed to have a suitable length, structure and flexibility. It was therefore thought to allow strong interactions with various binding sites in the catalytic core of GSK-3. A representative example of such a “second generation” compound is a compound in which the spacer is composed of three cyclic moieties and the negatively and positively charged groups are arranged in a specific orientation relative to each other. As described in the Examples section below (see Example 6), experiments conducted with these compounds have shown their high ability to inhibit GSK-3 activity, relative to the functional groups. The effect of mechanical orientation was further demonstrated (see, eg, FIG. 19).

  Thus, according to one aspect of the present invention, there is provided a compound comprising a negatively charged group and at least one amino moiety-containing group covalently bonded to the negatively charged group via a spacer. The spacer comprises at least one interaction between a negatively charged group and a first binding site in the catalytic domain of GSK-3, and a second binding site in the catalytic moiety of the amino moiety-containing group and GSK-3. Have a length, structure and flexibility selected to allow at least one interaction with the compound, thereby allowing the compound to inhibit the catalytic activity of GSK-3. Excluded from the scope of this aspect of the invention are the compounds disclosed in WO 2005/000192 as described above.

  As used herein, the term “catalytic domain” describes the region of an enzyme where a catalytic reaction occurs. The term thus describes this part of the enzyme where the substrates and / or components participating in the catalytic reaction interact with the enzyme. In the context of the present invention, this term is specifically used to describe this part of the enzyme (GSK-3) to which the substrate binds during catalytic activity (eg phosphorylation). Thus, this term is referred to herein and in the art to be interchangeable with terms such as “substrate binding pocket”, “catalytic site”, “active site”, and the like.

  As used herein, the term “binding site” refers to a special in a catalytic domain that contains one or more reactive groups that can interact with a substrate and / or other components. Describe the site. Typically, the binding site consists of one or two amino acid residues and the interaction typically includes reactive groups on the side chains of these amino acids. As detailed below, during substrate binding, conformational changes in the catalytic domain of the enzyme occur to bring reactive groups into suitable proximity and orientation, allowing their interaction with functional groups on the substrate. To. Thus, as used herein, the term “binding site” encompasses amino acid residues that are arranged in proximity and orientation to allow such interactions.

  The interaction of various functional groups of a compound with various binding sites of the enzyme depends on the reactive groups participating in the interaction and their proximity and orientation to each other, for example, electrostatic interactions, hydrogen bonding It can be an interaction, a hydrophobic interaction, an aromatic interaction, a π-stacking interaction and the like.

  Exemplary electrostatic interactions include cation-anion interactions and acid-base interactions, such as between an ammonium cation and a carboxyl anion.

  Exemplary hydrogen bonding interactions include interactions between one or more component amine, hydroxyl or thiol hydrogens and other component oxygen, nitrogen and sulfur atoms, for example.

  Exemplary hydrophobic interactions include interactions between two or more hydrocarbon moieties such as alkyl, cycloalkyl and aryl.

  Exemplary aromatic interactions include interactions between two or more aromatic moieties such as aryl and heteroaryl, which are based on overlap in the aromatized molecular orbitals of these moieties.

  Exemplary π-stacking interactions include interactions between two or more parts containing π electrons (eg, unsaturated parts), which are based on the overlap in the π orbitals of these parts.

  As described briefly above and as is well known in the art, substrate-competitive enzyme inhibitors act by binding to the catalytic domain of the enzyme and are therefore bound to the enzyme during the catalytic process. Reduce the proportion of enzyme molecules. When an enzyme interacts with a substrate or inhibitor, the initial interaction quickly induces a conformational change in the enzyme, which enhances binding and brings the catalytic site closer to the functional group in the substrate or inhibitor . Enzyme-substrate / inhibitor interactions orient reactive groups present in both the enzyme and the substrate / inhibitor, bringing them close together with respect to each other. Binding of the substrate / inhibitor to the enzyme aligns the reactive groups so that the associated molecular orbitals overlap.

  Thus, an effective substrate competitive inhibitor is one in which the reactive group of the inhibitor is located sufficiently close to the corresponding reactive group (typically the side chain of an amino acid residue) in the catalytic domain of the enzyme. Thus, it should be designed to allow the presence of an effective concentration of inhibitor in the catalytic domain, and in addition, the reactive groups of the inhibitor are placed in the proper orientation to allow overlap It should be. In addition, the inhibitor should have a restriction on its conformational flexibility to avoid conformational changes that affect or weaken the interaction. It should contain structural elements known to be involved in the action.

  As mentioned above, initial studies have shown that the GSK-3 substrate has a negatively charged group and one or more positively charged groups, and that the negatively charged groups are arranged in a special spatial orientation. I found. Therefore, a substrate competitive GSK-3 inhibitor should include both of these groups and their relative orientation. Therefore, to allow strong interactions between these functional groups and reactive groups in the catalytic domain of GSK-3, inhibitors are optimal for these functional groups relative to the reactive group of the enzyme. Should be designed to be in close proximity and orientation. The proximity and orientation of these functional groups is, according to embodiments of the present invention, such that when each functional group interacts with a reactive group in the catalytic domain, each functional group has one or more competitive reactions in the enzyme binding site. It is determined by selecting spacers that bind these functional groups so that they are in optimal proximity and orientation to the sex group. Therefore, suitable spacers are suitable lengths, flexibility and structures that allow effective interaction in terms of proximity and orientation between each functional group and one or more comparable reactive groups. Would have.

  As noted above, previous studies on crystals of GSK-3 have shown that GSK-3 substrates have negatively charged groups (eg, phosphate groups) that interact with one or more of arginine 180, arginine 96 and lysine 205. Have been shown to interact with the catalytic domain of the enzyme (see Dajani et al., 2001 and Ter Haar et al., 2001). Thus, according to a preferred embodiment of the present invention, the first binding site (as described above, a negatively charged group can interact with this first binding site) is one of these amino acid residues. Including the above. Thus, the compounds are designed to allow interactions between negatively charged groups and one or more of these amino acid residues. These interactions are preferably electrostatic interactions. However, any other chemically comparable binding site in the catalytic domain of GSK-3 arranged in proximity and orientation that allows interaction with the negatively charged group of the inhibitor is within the scope of the present invention. It is in.

  As described in the Examples section below (see Example 4), molecular modeling studies of substrate competitive inhibitor interactions have shown that the amino acid moiety-containing group depends on the proximity and orientation of the aspartic acid 181 , Glutamic acid 97, aspartic acid 90, aspartic acid 181, glutamic acid 200, glutamine 89, tyrosine 215 and asparagine 95 have been shown to interact. Thus, according to a preferred embodiment of the present invention, the second binding site (as described above, the site and the amino moiety-containing group can interact) comprises one or more of these amino acid residues. Thus, the compounds are designed to allow interaction between the amino moiety-containing group and one or more of these amino acid residues. These interactions can be, for example, electrostatic interactions and / or hydrogen bonding interactions. However, any other chemically comparable binding site in the catalytic domain of GSK-3 arranged in close proximity and orientation to allow interaction with the amino moiety-containing group (s) of the inhibitor Within the scope of the invention.

  Although the compounds described herein preferably contain one negatively charged group, they may contain one or more (eg, two, three) amino moiety-containing groups. Thus, the second binding site described herein includes all of the reactive groups that interact with all of the amino moiety-containing groups.

  The compounds described herein can further include a hydrophobic moiety that can participate in the interaction of the compound with the enzyme. The hydrophobic moiety is attached to the spacer such that the length, structure and flexibility of the spacer allows its interaction with another (third) binding site of the enzyme. If desired or additionally, the hydrophobic moiety can form part of the spacer itself by selecting a hydrophobic spacer.

  Hydrophobic moiety interactions can occur within the catalytic domain of the enzyme and / or within another region of the enzyme. Thus, for example, these interactions can occur within hydrophobic patches present within the enzyme as previously reported by Dajani et al. (2001). In another example, these interactions can be with one or more of the amino acid residues isoleucine 217, phenylalanine 67, and tyrosine 215 in the catalytic domain of GSK-3. However, any other hydrophobic binding site in the catalytic domain of GSK-3 arranged in proximity and orientation that allows interaction with the hydrophobic portion of the inhibitor is within the scope of the invention. These interactions can be hydrophobic interactions, aromatic interactions and / or π-stacking interactions depending on the chemical structure of the moiety and binding site. Additional descriptions of hydrophobic moieties are defined below.

  The inventors have found that the optimum distance between the negatively charged group and the amino moiety-containing group is from about 2 angstroms to about 50 angstroms. Thus, according to a preferred embodiment of the present invention, the length of the spacer is from about 2 angstroms to about 50 angstroms, more preferably from about 2 angstroms to about 20 angstroms, and even more preferably from about 2 angstroms to about 10 angstroms. Angstrom. Thus, exemplary spacers will have a length of about 7-8 Angstroms.

  The inventors have further found that the flexibility of the spacer should preferably be limited. Accordingly, the spacer preferably includes one or more cyclic portions. Depending on the characteristics of GSK-3 and the nature of the functional group in the compound (eg, a negatively charged group, an amino moiety-containing group, a hydrophobic moiety), if the spacer contains two or more cyclic moieties, The moieties can be fused and / or unfused. If not fused, each of the two cyclic moieties is, but is not limited to, a bond, a heteroatom (eg, oxygen, nitrogen, sulfur) or a hydrocarbon chain (these terms are defined below). They can be linked to each other via a linker. The hydrocarbon chain can be interrupted by one or more heteroatoms (eg, oxygen, nitrogen, sulfur) if desired.

  The presence (if the cyclic moiety is not fused) or absence (if the cyclic moiety is fused) as well as the nature of the linker, as well as its nature, to the degree of flexibility required to allow the interaction described above Dependent. Accordingly, where flexibility is to be substantially limited, a rigid spacer comprising two or more fused annular portions is preferred. Alternatively, rigid spacers containing two or more aromatic moieties (aryl and / or heteroaryl) are preferred. Semi-rigid spacers that allow free rotation of one or more bonds are preferred where limited flexibility is required to some extent. In most cases, it is desirable to have a spacer that imparts at least some rotational properties to the compound that allows the functional group to readily interact with the reactive group in the binding site.

  In a preferred embodiment of the invention, the spacer comprises two or more, preferably three, cyclic moieties, which are covalently bonded to each other via a bond.

  In each of these embodiments, each of the cyclic moieties can be, for example, a carbocyclic moiety or a heterocyclic moiety.

  As used herein, a “carbocyclic moiety” describes a saturated or unsaturated cyclic moiety of carbon, while a “heterocyclic moiety” is one or more of nitrogen, oxygen, sulfur, phosphorus, silicon, etc. A saturated or unsaturated cyclic moiety containing a heteroatom of

  Carbocyclic moieties include cycloalkyl and aryl, and these terms are defined below. Heterocyclic moieties include heteroalicyclic and heteroaryl, these terms being defined below.

  Aromatic cyclic moieties (aryl and heteroaryl) are preferred to limit conformational changes in these structures because they are stiffer than non-aromatic cyclic moieties (cycloaryl and heteroalicyclic). However, combinations of aromatic and non-aromatic cyclic moieties can also allow desirable interactions.

  If the spacer comprises a heterocyclic moiety containing one or more nitrogen atoms, this moiety can act as an amino moiety-containing group that participates in interaction with the binding site.

  Where the spacer includes carbocyclic moieties and / or hydrocarbon chains, these moieties can act as hydrophobic and / or aromatic moieties that participate in interaction with the binding site, as described above.

  Thus, the spacer can be further selected to have a structure that allows or restricts its interaction with the binding site of the enzyme. Exemplary spacers having structures that allow interaction with a binding site include, but are not limited to, a hydrophobic moiety-containing spacer, an aromatic moiety-containing spacer, and an amino moiety-containing (eg, heterocyclic) spacer.

  However, the structure of the spacer can further influence its flexibility, stability and other features related to the interaction of the compound with the binding site in the catalytic domain of GSK-3.

  Accordingly, preferred compounds according to this embodiment include a rigid or semi-rigid spacer having a negatively charged group attached thereto. This structure mimics the specific structure of the GSK-3 substrate by providing a negatively charged group that is not configurationally inhibited and has a geometric structure similar or identical to the phosphate group, These compounds preferably inhibit GSK-3 activity through inhibition of substrate binding to the enzyme, as it allows interaction of negatively charged groups and amino moiety-containing groups with GSK-3 binding sites. be able to.

  The expressions “negatively charged group” and “positively charged group”, as used herein, refer to an ionizable group, which by ionization, usually in at least one negative medium. Or each has a positive charge. Charged groups can exist in their ionized or unionized form in the compounds described herein.

を有し、式中、Ｌはリン原子、イオウ原子、ケイ素原子、ホウ素原子および炭素原子からなる群から選択され；Ｑ^＊，ＧおよびＤ^＊はそれぞれ独立して酸素およびイオウからなる群から選択され；Ｅはヒドロキシ、アルコキシ、アリールオキシ、カルボニル、チオカルボニル、Ｏ−カルボキシ、チオヒドロキシ、チオアルコキシおよびチオアリールオキシ（これらの用語は以下で規定される）からなる群から選択されるかまたは存在しない。 The negatively charged group is, according to a preferred embodiment of the invention, the formula

Wherein L is selected from the group consisting of phosphorus atom, sulfur atom, silicon atom, boron atom and carbon atom; Q ^* , G and D ^* are each independently selected from the group consisting of oxygen and sulfur E is selected or present from the group consisting of hydroxy, alkoxy, aryloxy, carbonyl, thiocarbonyl, O-carboxy, thiohydroxy, thioalkoxy and thioaryloxy (these terms are defined below) do not do.

Preferably, the negatively charged group is a phosphate group, thus in the above formula, L is a phosphorus atom and each of Q ^* , G and D ^* is oxygen. More preferably, E is hydroxy. Alternatively, the hydroxy group can be ionized to have another negative electrostatic charge.

  Alternatively, the negatively charged group can be a thiophosphate group, a sulfate or sulfonate group, a borate or boronate group, etc., according to the above formula.

  The negatively charged group is preferably attached to the spacer via an alkyl group, wherein the alkyl is preferably unsubstituted alkyl, more preferably methyl.

  By attaching a negatively charged group to the ring via an alkyl group, the negatively charged group becomes a freely rotatable group. This is in contrast to its limited rotation when a negatively charged group is attached directly to the spacer. The free rotation of the negatively charged group is advantageous. This is because it allows negatively charged groups to easily interact with the enzyme binding site.

  The positively charged group is preferably derived from an amino moiety-containing group, said amino moiety-containing group being a compound in its ionized form (eg as ammonium or guanidinium ion) or as a free amine (form before ionization) Can exist inside.

  As used herein, the expression “amino moiety-containing group” refers to one or more amino moiety-containing groups, where the term amino moiety is as defined herein.

  Representative examples of amino moiety-containing groups include, without limitation, amino (amine), aminoalkyl, hydrazine, urea, thiourea, guanyl, amide, carbamyl, guanidino, guanidinoalkyl and guanylinoalkyl. As defined in the specification.

As is well known in the art, a free amino (amine) group is usually basic under neutral conditions, so in a biological environment it is protonated and positively charged —NH ₃ ⁺ There is a tendency to generate groups, for example. As noted above, compounds having one or two such positively charged groups adjacent to a negatively charged group at a suitable distance and orientation with respect to the enzyme binding site are preferred.

  Thus, it is preferred that the amino moiety be present in this group as a readily protonated moiety, i.e. the amino nitrogen has a substantial partial negative charge.

  Therefore, preferred representative examples of amino moiety-containing groups include, without limitation, amino (amine), aminoalkyl, guanyl, guanidino, guanidinoalkyl and guanylinoalkyl, which terms are as defined herein. belongs to.

  The amino moiety-containing group can be present as such or as a positively charged group in the compounds described herein, in which case at least one amino moiety is ionized.

  As described above, the positively charged group according to the present invention is such that a representative example of a positively charged group carries ammonium ion itself (protonated amino group) and ammonium ion (as defined above). The ammonium ion to include, without limitation, any group such as alkyl, cycloalkyl, or aryl substituted with an ammonium ion, and guanidino, guanyl, hydrazine, and the like.

  Particularly preferred are positively charged having a chemical structure derived from the side chain of positively charged amino acids (eg, lysine, arginine, histidine, proline and derivatives thereof, the first two being most preferred). It is a group.

  By “chemical structure derived from the side chain of a positively charged amino acid” is meant that the positively charged group has a similar or identical chemical structure to such side chain.

  Representative examples include guanidine and guanidinoalkyl (derived from arginine), amine and aminoalkyl (derived from lysine) and imidazole (derived from histidine), the first being currently most preferred.

式中、ｎは０〜１０の整数であり；
ｍは１〜６の整数であり；
Ｂは上述の負に帯電した基であり；
Ｑは上述のアミノ部分含有基の一つであり；そして
Ｊ−（Ｓ）_１−（Ｓ）_２‐‐‐（Ｓ）_ｎ−Ｋは上述のスペーサーであり；
Ｋはアリール、ヘテロアリール、脂環、または複素脂環からなる群から選択され；そして
ＪおよびＳ_１−Ｓ_ｎはそれぞれ独立して置換または非置換アリール、置換または非置換ヘテロアリール、置換または非置換脂環、置換または非置換複素脂環、結合、ヘテロ原子、置換または非置換炭化水素鎖および少なくとも一つのヘテロ原子で中断された置換または非置換炭化水素鎖（これらの用語は本明細書中で規定される）からなる群から選択されるかまたは存在しない。アリール、ヘテロアリール、シクロアルキル、複素脂環および炭化水素鎖が置換されている場合、置換基は以下に記述されるもののいずれかであることができる。 Preferred compounds according to this embodiment can be represented collectively by the general formula I:

Where n is an integer from 0 to 10;
m is an integer from 1 to 6;
B is a negatively charged group as described above;
Q is one of the amino moiety-containing groups described above; and J- (S) _1- (S) _2- (S) _n -K is the spacer described above;
K is selected from the group consisting of aryl, heteroaryl, alicyclic, or heteroalicyclic; and J and S ₁ -S _n are each independently substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted Substituted alicyclic, substituted or unsubstituted heteroalicyclic, bond, heteroatom, substituted or unsubstituted hydrocarbon chain and substituted or unsubstituted hydrocarbon chain interrupted by at least one heteroatom (these terms are used herein) Or selected from the group consisting of: When aryl, heteroaryl, cycloalkyl, heteroalicyclic and hydrocarbon chains are substituted, the substituents can be any of those described below.

  Thus, the spacer in these compounds consists of at least one cyclic moiety (K in Formula I above) to which 1 to 6 amino moiety-containing groups are attached. Preferably, one or two amino moiety-containing groups are attached to the cyclic moiety K, so m in formula I above is 1-2. Optionally, one of the amino moiety-containing groups shown as Q in Formula I above forms part of the cyclic moiety K as described above. In these cases, K is a heteroalicyclic ring or a heteroaryl, preferably a heteroaryl.

As is well known in the art, a nitrogen atom in an aromatic ring is usually basic under neutral conditions, and thus in a biological environment it is protonated and positively charged = NH ⁺ There is a tendency to generate groups.

Additional amino moiety-containing groups can also be present in the compound. Thus, at least one of J and S ₁ -S _n moiety can include one or more amino moiety-containing group. The amino moiety-containing group (s) can be attached to or form part of these moieties as described above.

A negatively charged group (B in Formula I above) can be directly attached to the cyclic moiety K (when n is 0 and J is not present in Formula I above). Optionally and preferably, the long spacer joins the negatively charged group B and the amino moiety containing group Q such that at least one of J and S ₁ -S _n is present in the spacer.

As described above, by selecting the number and structure of the components that make up the spacer (J, S ₁ -S _n and K in Formula I above), the length, structure and flexibility of the spacer can be Can be determined to allow the desired interaction.

Therefore, for example, a spacer having a rigid structure can be obtained by selecting a spacer in which each of J, S ₁ -S _n and K is a cyclic portion.

  By selecting a spacer in which the cyclic portion is an aromatic portion, a more rigid structure is obtained. By selecting a spacer in which two or more annular portions are fused, a more rigid structure can be obtained, and so on.

On the other hand, by at least one of J and S ₁ -S _n selects a spacer is a heteroatom, more flexible spacer is obtained. An even more flexible spacer can be obtained by selecting a spacer in which at least one of J and S ₁ -S _n is a hydrocarbon chain. An even more flexible spacer is obtained by selecting spacers where each of J and S ₁ -S _n is a heteroatom or a hydrocarbon chain.

  In another example, the number and size of the parts that make up the spacer determine the length of the spacer. If a long spacer is required to allow the desired interaction, n is greater than 5. If a short spacer is required, n is less than 5 and can be 0.

  As described in the Examples section below, GSK- reported by Ter Haar et al. (2001), which is hereby incorporated by reference as if all of its content was described herein. In molecular studies conducted on the basis of crystal data of 3, it was found that preferred substrate binding inhibitors of GSK-3 have spacers of about 7-8 angstroms in length.

  Based on these findings and additional results obtained for the activity of various compounds (see Examples 3 and 6), preferred compounds are 2 angstroms to 50 angstroms, more preferably 2 angstroms to 20 angstroms, and even more preferably. It was derived to include a spacer having a length of 2 angstroms to 10 angstroms.

  Taking into account that the average length of the cyclic part is 2 to 2.5 angstroms, the preferred inhibitor consists of 1 to 3 cyclic parts.

  Thus, in a preferred embodiment of the invention, n is an integer from 0-2.

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

  As mentioned above, negatively charged groups can be directly or indirectly attached to the spacer, the latter being preferred. Thus, J in Formula I above may not be present (if a negatively charged group is directly attached to the spacer) or that this group will readily interact with the first binding site described above. It can be a flexible group that allows.

Thus, preferably J is a hydrocarbon chain such as alkyl. Preferably J is a short alkyl (C ₁ -C ₆ alkyl), more preferably J is methyl.

  Based on the above preferred features, a representative family of compounds having the above formula I was designed and representative compounds of this family were successfully prepared. As shown in the Examples section below (see Example 6), these compounds (also referred to herein interchangeably as MP molecules or multiaryl / heteroaryl small molecules) are substrates for GSK-3. It has been found to be highly active as a competitive inhibitor.

式中、ＢおよびＱは上述の通りであり、Ｊはアルキルであり、Ｓ_１，Ｓ_ｎおよびＫのそれぞれはアリールまたはヘテロアリールである。 Accordingly, preferred compounds according to these embodiments of the present invention can be collectively represented by the following general formula III:

Wherein B and Q are as described above, J is alkyl, and each of S ₁ , _Sn and K is aryl or heteroaryl.

Exemplary compounds of this category include those in the above general formula wherein B is a phosphate group, J is alkyl (eg, methyl), S ₁ is aryl (eg, phenyl), and S ₂ is heteroaryl (eg, triazole) And K is aryl (eg, phenyl).

  The chemical structures of representative examples of these compounds are presented in FIG.

  The compounds described in WO 2005/000192 are described below, which are excluded from the scope of the present invention.

式中、Ｘ，Ｙ，ＺおよびＷはそれぞれ独立して炭素原子または窒素原子であり；
Ａは炭化水素鎖であるかまたは存在せず（上記Ｊに相当する）；
Ｂは上述のように負に帯電した基であり、
Ｄは水素、アルキル、トリハロアルキル、シクロアルキル、アルケニル、アルキニル、アリール、ヘテロアリール、複素脂環、ハロ、ヒドロキシ、アルコキシ、アリールオキシ、チオヒドロキシ、チオアルコキシ、チオアリールオキシ、スルフィニル、スルホニル、シアノ、ニトロ、アゾ、スルホンアミド、カルボニル、ケトエステル、チオカルボニル、エステル、エーテル、チオエーテル、チオカルバメート、尿素、チオ尿素、Ｏ−カルバミル、Ｎ−カルバミル、Ｏ−チオカルバミル、Ｎ−チオカルバミル、Ｃ−アミド、Ｎ−アミド、Ｃ−カルボキシ、Ｏ−カルボキシ、スルホンアミド、トリハロメタンスルホンアミド、グアニル、グアニジノ、グアニジノアルキル、アミノ、アミノアルキルおよび疎水性部分からなる群から選択され；そして
Ｒ_１，Ｒ_２，Ｒ_３およびＲ_４はそれぞれ独立して水素、孤立電子対、アルキル、トリハロアルキル、シクロアルキル、アルケニル、アルキニル、アリール、ヘテロアリール、複素脂環、ハロ、ヒドロキシ、アルコキシ、アリールオキシ、チオヒドロキシ、チオアルコキシ、チオアリールオキシ、スルフィニル、スルホニル、シアノ、ニトロ、アゾ、スルホンアミド、カルボニル、ケトエステル、チオカルボニル、エステル、エーテル、チオエーテル、チオカルバメート、尿素、チオ尿素、Ｏ−カルバミル、Ｎ−カルバミル、Ｏ−チオカルバミル、Ｎ−チオカルバミル、Ｃ−アミド、Ｎ−アミド、Ｃ−カルボキシ、Ｏ−カルボキシ、スルホンアミド、トリハロメタンスルホンアミド、グアニル、グアニリノアルキル、グアニジノ、グアニジノアルキル、アミノ、アミノアルキル、ヒドラジンおよびアンモニウムイオンからなる群から選択される。 These compounds are collectively represented by the general formula II or pharmaceutically acceptable salts thereof:

In which X, Y, Z and W are each independently a carbon atom or a nitrogen atom;
A is a hydrocarbon chain or absent (corresponding to J above);
B is a negatively charged group as described above,
D is hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, Nitro, azo, sulfonamide, carbonyl, ketoester, thiocarbonyl, ester, ether, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N- Selected from the group consisting of amide, C-carboxy, O-carboxy, sulfonamide, trihalomethanesulfonamide, guanyl, guanidino, guanidinoalkyl, amino, aminoalkyl and hydrophobic moieties; R _{1, R} 2, _{R 3} and _{R 4} are each independently hydrogen Te, lone pairs, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, Aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azo, sulfonamide, carbonyl, ketoester, thiocarbonyl, ester, ether, thioether, thiocarbamate, urea, thiourea, O-carbamyl , N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamido, trihalomethanesulfonamide, guanyl, guanylinoalkyl, guanidino, gua Jinoarukiru, amino, aminoalkyl, is selected from the group consisting of hydrazine and ammonium ions.

The feasibility of each of the substituents (eg, D, G, E, and R ₁ -R ₄ ) located at the indicated positions depends on the valence and chemical compatibility of the substituent, the substitution position, and other It will be appreciated by those skilled in the art that it depends on the substituent. Thus, the present invention is intended to encompass all possible substituents at any position.

  As noted above, spacers in these families of compounds include a cyclic moiety, preferably an aromatic ring (aryl) or a heteroaromatic ring (heteroaryl).

In one embodiment, the spacer is heteroaryl, so in formula III above, at least one of X, Y, Z and W is a nitrogen atom. As mentioned above, the nitrogen atom in an aromatic ring is usually basic under neutral conditions, so in a biological environment it tends to be protonated to produce a positively charged ═NH ⁺ group. is there. Thus, in such a spacer, the positively charged amino moiety-containing group forms part of the spacer. Since the amino moiety-containing group is preferably at a distance from the negatively charged group, Z or W is preferably a nitrogen atom.

  In another embodiment, at least two of X, Y, Z and W are nitrogen atoms, more preferably X and Y are nitrogen atoms or Z and W are nitrogen atoms, even more preferably Z And W is a nitrogen atom.

Instead of or in addition to the positively charged nitrogen atom in the spacer, preferably at least one of R ₁ , R ₂ , R ₃ and R ₄ is an amino moiety-containing group as described above.

Preferably, R ₁ and R ₂ or R ₃ and R ₄ are amino moiety-containing groups (eg, positively charged groups).

式中、ｍは１〜６の整数であり；Ｑ_１とＱ_２はそれぞれ独立して炭素原子または窒素原子であり；Ｇおよび／またはＫはそれぞれアミノ部分含有基（例えば正に帯電した基）である。 Accordingly, preferred compounds according to the present invention are those having the following general formulas IIA and IIB:

Wherein m is an integer from 1 to 6; Q ₁ and Q ₂ are each independently a carbon atom or a nitrogen atom; G and / or K are each an amino moiety-containing group (eg, a positively charged group) It is.

  As described above, and as further demonstrated in the Examples section below, a number of compounds are designed according to the above general formula and are successfully synthesized to exert high inhibitory activity on substrate binding to GSK-3. Was found. These compounds are also referred to interchangeably herein as GS molecules, GSC molecules or monoaryl / heteroaryl small molecules. The chemical structures of these compounds are shown in FIGS. 3a and 3b. The efficiency of these compounds as inhibitors of GSK-3 activity is shown, for example, in FIGS. 5-7, their specificity for GSK-3 is shown in Example 9, and glucose in mouse adipocytes Their advantageous effects on the uptake of are shown in FIGS. 11a and 11b.

  As shown in FIGS. 5-7 and as shown in the Examples section below, some of the tested compounds did not have positively charged groups (eg, GSC-1 and GSC-2 However, these compounds exhibit inhibitory activity against GSK-3. However, as further shown in FIGS. 5-7, compounds having a nitrogen atom in the basic ring were found to be more active inhibitors and thus show the advantageous effects of such groups.

Accordingly, additional preferred compounds according to this embodiment are compounds according to general formula II above, wherein each of X, Y, Z and W is a carbon atom; at least one of R ₃ and R ₄ is An amino moiety-containing group (eg, a positively charged group); a compound in which D is hydrogen or alkyl. More preferred compounds are those in which each of X, Y and Z is a carbon atom and W is a nitrogen atom.

  Direct or indirect attachment of a phosphate group or any other negatively charged group according to the present invention to an aromatic or heteroaromatic ring is accomplished via a simple procedure, resulting in a structurally simple compound However, it should be noted that only a very limited number of such compounds have ever been synthesized. These include pyridoxal phosphate, benzyl phosphate, phenyl phosphate and a limited number of their derivatives (eg substituted pyridoxal phosphate, benzyl phosphate and phenyl phosphate). Since no biological activity has ever been associated with such compounds, those skilled in the art were not motivated to provide such compounds. However, the compound according to this aspect of the invention excludes any currently known compound encompassed by the above formula.

式中、式ＡについてはＭはメチレンであるかまたは存在せず、Ｒａ，ＲｂおよびＴはそれぞれ水素であり、ＲｃおよびＲｄはそれぞれ独立して水素、−ＣＨ＝Ｎ−ＮＨ−Ｃ（＝ＮＨ）−ＮＨ_２，−ＣＨ_２−Ｎ（ＮＨ_２）−ＣＨ_３および−ＣＨ_２−ＮＨ−Ｃ（＝ＮＨ）−ＯＨからなる群から選択され、
式ＢについてはＭはメチレンであるかまたは存在せず、ＺおよびＷはそれぞれ独立して窒素または炭素であり、ＺおよびＷの少なくとも一つは窒素であり、Ｒｅ，Ｒｆ，Ｒｇ，ＲｈおよびＴはそれぞれ独立して水素、ニトロ、アルキル、ハロ、ヒドロキシおよびメチルからなる群から選択される。 In particular, compounds having the following general formulas A and B are excluded from the scope of this aspect of the invention:

Where, for Formula A, M is methylene or absent, Ra, Rb and T are each hydrogen, Rc and Rd are each independently hydrogen, —CH═N—NH—C (═NH ) —NH ₂ , —CH ₂ —N (NH ₂ ) —CH ₃ and —CH ₂ —NH—C (═NH) —OH,
For Formula B, M is methylene or absent, Z and W are each independently nitrogen or carbon, at least one of Z and W is nitrogen, Re, Rf, Rg, Rh and T Are each independently selected from the group consisting of hydrogen, nitro, alkyl, halo, hydroxy and methyl.

  As detailed in WO 2004/052404 (PCT / IL03 / 10157) by one of the inventors, binding a hydrophobic moiety to the N-terminus of a GSK-3 peptide inhibitor increases the inhibitory activity of the peptide. It has been found that Such increased activity can be attributed to the presence of a hydrophobic patch in the enzyme according to crystal data reported by Dajani et al. (2001), and thus the interaction of peptide inhibitors with this hydrophobic patch and It can be attributed to increased membrane permeability of the peptide inhibitor by a hydrophobic tail.

  Since the phosphorylated residue in the peptide inhibitor is located at its C-terminus, the compounds according to the invention comprising a hydrophobic moiety located far from the negatively charged group are as in the case of peptide inhibitors. It is believed that it will exhibit increased inhibitory activity.

  Thus, in preferred embodiments of this aspect of the invention, each of the compounds described herein has one or more hydrophobic moieties attached thereto. Preferably, the hydrophobic moiety is attached to a spacer, more preferably the hydrophobic moiety is a spacer at a position that allows interaction between this moiety and the binding site of GSK-3 as described above. Combined with

Thus, for example, according to this embodiment, in the compound having Formula I, at least one of J, (S) _1- (S) _n and K has a hydrophobic moiety attached thereto. Preferably K has a hydrophobic moiety attached thereto. Alternatively or additionally, _Sn has a hydrophobic moiety attached to it.

  In the compounds having formula II above, D is a hydrophobic moiety.

  The expression “hydrophobic component” as used herein refers to any substance or residue thereof characterized by hydrophobicity.

  As widely accepted in this field, the term “residue” refers to the major portion of a substance that is covalently bound to another substance (herein the compounds described hereinabove). Describe.

  Thus, the hydrophobic component according to the present invention is preferably a residue of a hydrophobic material, preferably covalently linked to the compounds described hereinabove.

  Representative examples of hydrophobic materials from which the hydrophobic component of the invention can be derived include, but are not limited to, saturated alkylene chains, unsaturated alkylene chains, aryls, cyclic alkyls, and hydrophobic peptide sequences. These terms are defined herein.

  The expression “alkylene chain” as used herein refers to a linear chain of hydrocarbons that can be saturated or unsaturated. The alkylene chain can be substituted or unsubstituted with respect to the alkyl group as described herein and can be interrupted by one or more heteroatoms such as nitrogen, oxygen, sulfur, phosphorus, etc. it can. The alkylene chain preferably comprises at least 4 carbon atoms, more preferably at least 8 carbon atoms, even more preferably at least 10 carbon atoms, up to 20, up to 25 and even up to 30 carbon atoms. Can have.

  Thus, the hydrophobic component of the present invention can include the residues of the hydrophobic materials described hereinabove.

  Preferred examples of alkylene chains according to this aspect of the invention are those containing carboxyl groups, ie fatty acid residues.

  Preferred fatty acids suitable for use in connection with the present invention include, but are not limited to, saturated or unsaturated fatty acids having more than 10 carbon atoms (preferably 12 to 24 carbon atoms). Examples include, but are not limited to, myristic acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, and the like.

  Alternatively, the hydrophobic component according to the invention can comprise a hydrophobic peptide sequence. The hydrophobic peptide sequence according to the present invention preferably comprises 2 to 15 amino acid residues, more preferably 2 to 10 amino acid residues, more preferably 2 to 5 amino acid residues. Wherein at least one amino acid residue is a hydrophobic amino acid residue.

  Representative examples of hydrophobic amino acid residues include, but are not limited to, alanine residues, cysteine residues, glycine residues, isoleucine residues, leucine residues, valine, as described hereinabove. Residues, phenylalanine residues, tyrosine residues, methionine residues, proline residues and tryptophan residues, or any modification thereof are included.

  Alternatively, a hydrophobic amino acid residue can include any other amino acid residue that has been modified by incorporation of a hydrophobic moiety thereto.

  Preferably, the hydrophobic amino acid sequence comprises at least two, more preferably at least five hydrophobic amino acid residues, which are more preferably linked to one another, and thus the contiguous sequence within the hydrophobic amino acid sequence. give.

  Examples of hydrophobic amino acid sequences (which are interchangeably referred to in the art as “membrane permeable sequences” or “MPS”) can be found, for example, in Hagiwer (1999).

  The expression “amino acid residue” as used herein, interchangeably referred to herein as “amino acid”, describes an amino acid unit within a polypeptide chain. The amino acid residues in the hydrophobic peptide sequence can be either natural amino acid residues or modified amino acid residues (the expressions of which are defined herein below).

  As used herein, the expression “natural amino acid residue” describes an amino acid residue comprising any of the 20 amino acids found in nature (this term is defined herein above). .

  As used herein, the expression “modified amino acid residue” describes an amino acid residue comprising a natural amino acid that has been modified in its side chain (this term is defined herein above). . Such modifications are widely known in the art and include, for example, the incorporation of functional groups such as, but not limited to, hydroxy, amino, carboxy and phosphate groups in the side chain. included. Thus, unless expressly indicated otherwise, this expression includes chemically modified amino acids including amino acid analogs (eg, penicillamine, 3-mercapto-D-valine, etc.), naturally occurring non-proteinogenic amino acids (eg, And chemically synthesized compounds having properties known in the art to be characteristic of amino acids. The term “non-protein-forming” indicates that amino acids cannot be taken up by proteins in cells by well-known metabolic pathways.

  Thus, as used herein, the term “amino acid (s)” refers to the 20 naturally occurring amino acids; such amino acids that are often post-translationally modified in vivo (including For example, hydroxyproline, phosphoserine and phosphothreonine); and other unusual amino acids, including 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine and ornithine It is understood to include (but not limited to). Furthermore, the term “amino acid” encompasses both D-amino acids and L-amino acids linked by a peptide bond or peptide bond analog to at least one additional amino acid as the term is defined herein.

  Since the hydrophobic moiety provides increased unpredictable activity, known compounds substituted with the hydrophobic moiety (phenylphosphate, pyridoxal phosphate, and other known compounds described above) are also included in this aspect of the invention. Included in the range.

  Listed below are definitions of various chemical moieties cited in this application.

  As used herein, the term “bond” refers to a single bond, a double bond or a triple bond.

  As used herein, the expression “hydrocarbon chain” and the term “hydrocarbon” refer to moieties composed primarily of carbon and hydrogen atoms. Thus, a hydrocarbon chain includes one or more of alkyl, alkenyl, alkynyl, cycloalkyl and aryl, these terms as defined herein, each of which is unsubstituted as described herein. Or it can be substituted. The hydrocarbon chain can be further interrupted by one or more heteroatoms as defined herein.

  The term “heteroatom” as used herein refers to any atom that is not carbon but is capable of forming a covalent bond with one or more carbon atoms. Exemplary heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, phosphorus, silicon, boron, and the like.

  The term “alkyl” as used herein refers to a saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range, such as “1-20” is mentioned herein, it means that the group (in this case an alkyl group) is 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. Of up to 20 carbon atoms. More preferably, the alkyl group is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl group is a lower alkyl having 1 to 4 carbon atoms. Alkyl groups can be substituted or unsubstituted. When substituted, the substituent is, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryl Oxy, sulfinyl, sulfonyl, cyano, nitro, azo, sulfonamide, ketoester, carbonyl, thiocarbonyl, ester, ether, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- Thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, trihalomethanesulfonamide, guanyl, guanylinoalkyl, guanidino, guanidinoalkyl, amino, aminoalkyl, It may be hydrazine and ammonium ions.

  As used herein, the term “cycloalkyl” or “alicyclic” refers to an all-carbon monocyclic group or fused ring in which one or more of the rings do not have a fully conjugated π-electron system (ie, A ring that shares a pair of adjacent carbon atoms). Non-limiting examples of cycloalkyl groups include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene and adamantane. Cycloalkyl groups can be substituted or unsubstituted. When substituted, the substituents are, for example, alkyl, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, Thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azo, sulfonamide, ketoester, carbonyl, thiocarbonyl, ester, ether, carboxy, thiocarboxy, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl , O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamido, trihalomethanesulfonamide, guanyl, guanylinoalkyl, g Nijino, guanidino alkyl, amino, aminoalkyl, it is a hydrazine and ammonium ions.

  The term “alkenyl” group refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon double bond.

  The term “alkynyl” group refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon triple bond.

  The term “aryl” refers to a monocyclic or fused polycyclic (ie, ring sharing adjacent pairs of carbon atoms) groups consisting of all carbons with a fully conjugated pi-electron system. Non-limiting examples of aryl groups include phenyl, naphthalenyl and anthracenyl. Aryl groups can be substituted or unsubstituted. When substituted, the substituents are, for example, alkyl, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, Thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azo, sulfonamide, ketoester, carbonyl, thiocarbonyl, ester, ether, carboxy, thiocarboxy, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl , O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamido, trihalomethanesulfonamide, guanyl, guanylinoalkyl, g Nijino, guanidino alkyl, amino, aminoalkyl, it is a hydrazine and ammonium ions.

  The term “heteroaryl” group includes, for example, monocyclic groups having one or more atoms in the ring (s), such as nitrogen, oxygen and sulfur, and further having a fully conjugated π-electron system Or a fused ring (ie, a ring sharing adjacent pairs of carbon atoms) groups are included. Non-limiting examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. A heteroaryl group can be substituted or unsubstituted. When substituted, the substituents are, for example, alkyl, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, Thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azo, sulfonamide, ketoester, carbonyl, thiocarbonyl, ester, ether, carboxy, thiocarboxy, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl , O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamido, trihalomethanesulfonamide, guanyl, guanylinoalkyl, g Nijino, guanidino alkyl, amino, aminoalkyl, it is a hydrazine and ammonium ions.

  The term “heteroalicyclic” group refers to a monocyclic or fused ring group having in the ring (s) one or more atoms such as nitrogen, oxygen and sulfur. The ring can also have one or more double bonds. However, the ring does not have a fully conjugated pi-electron system. Heteroalicyclic groups can be substituted or unsubstituted. When substituted, the substituent may be, for example, a lone pair, alkyl, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy , Thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azo, sulfonamide, ketoester, carbonyl, thiocarbonyl, ester, ether, carboxy, thiocarboxy, thioether, thiocarbamate, urea, thiourea, O-carbamyl , N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, trihalomethanesulfonamide, guanyl, guanylino Alkyl, guanidino, guanidinoalkyl, amino, aminoalkyl, be a hydrazine and ammonium ions. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholino and the like.

  The term “lone electron pair” refers to an electron pair that is not involved in binding. A lone pair exists only when X, Y, Z or W is an unsubstituted nitrogen atom.

  The term “hydroxy” group refers to an —OH group.

  The term “azo” group refers to a —N═N group.

  The term “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl group, as defined herein.

  The term “aryloxy” group refers to both an —O-aryl group and an —O-heteroaryl group, as defined herein.

  The term “thiohydroxy” group refers to a —SH group.

  The term “thioalkoxy” group refers to both an —S-alkyl and an —S-cycloalkyl group, as defined herein.

  The term “thioaryloxy” group refers to both an —S-aryl and an —S-heteroaryl group, as defined herein.

  The term “carbonyl” group refers to a —C (═O) —R ′ group, where R ′ is hydrogen, alkyl, alkenyl, cycloalkyl, aryl, (ring Heteroaryl (bonded through carbon) or heteroalicyclic (bonded through ring carbon).

  The term “aldehyde” group refers to a carbonyl group where R ′ is hydrogen.

  The term “thiocarbonyl” group refers to a —C (═S) —R ′ group, where R ′ is as defined herein for R ′.

  The term “C-carboxy” group refers to a —C (═O) —O—R ′ group, where R ′ is as defined herein.

  The term “O-carboxy” group refers to a R′C (═O) —O— group, wherein R ′ is as defined herein.

  The term “carboxylic acid” group refers to a C-carboxyl group where R ′ is hydrogen.

  The term “halo” group refers to fluorine, chlorine, bromine or iodine.

The term “trihalomethyl” group refers to a —CX ₃ group, where X is a halo group as defined herein.

The term “trihalomethanesulfonyl” group refers to a X ₃ CS (═O) ₂ — group, where X is a halo group as defined herein.

  The term “sulfinyl” group refers to a —S (═O) —R ′ group, where R ′ is as defined herein.

The term “sulfonyl” group refers to a —S (═O) ₂ —R ′ group, where R ′ is as defined herein.

The term “S-sulfonamido” group is a —S (═O) ₂ —NR′R ″ group, where R ′ is as defined herein and R ″ is defined as R ′. Is).

The term “N-sulfonamido” group refers to an R ′S (═O) ₂ —NR ″ group, where R ′ and R ″ are as defined herein.

The term “trihalomethanesulfonamido” group refers to a X ₃ CS (═O) ₂ NR′— group, where R ′ and X are as defined herein.

  The term “O-carbamyl” group refers to a —OC (═O) —NR′R ″ — group, where R ′ and R ″ are as defined herein.

  The term “N-carbamyl” group refers to an R ″ OC (═O) —NR′— group, where R ′ and R ″ are as defined herein.

  The term “O-thiocarbamyl” group refers to a —OC (═S) —NR′R ″ group, where R ′ and R ″ are as defined herein.

  The term “N-thiocarbamyl” group refers to an R ″ OC (═S) NR′— group, where R ′ and R ″ are as defined herein.

  The term “amino” group refers to a —NR′R ″ group, where R ′ and R ″ are as defined herein.

  The term “aminoalkyl” group refers to an alkyl substituted with an amino group, as defined herein. Preferably the alkyl is terminated by an amino group.

  The term “C-amido” group refers to a —C (═O) —NR′R ″ group, where R ′ and R ″ are as defined herein.

  The term “N-amido” group refers to a R′C (═O) —NR ″ group, where R ′ and R ″ are as defined herein.

  The term “urea” group refers to a —NR′C (═O) —NR ″ R ′ ″ group, where R ′ and R ″ are as defined herein, and R ′ ″ is R As defined as either 'or R ″).

  The term “guanidino” group refers to a —R′NC (═NR ″ ″) — NR ″ R ′ ″ group, where R ′, R ″ and R ′ ″ are as defined herein. And R ″ ″ is as defined as either R ′, R ″ or R ′ ″).

  The term “guanidinoalkyl” group refers to an alkyl group substituted with a guanidino group, as these terms are defined herein. Preferably, the alkyl group is terminated by a guanidino group.

  The term “guanyl” group is an R′R ″ NC (═NR ″ ″) — group, where R ′, R ″, R ′ ″ and R ″ ″ are defined herein. Is).

  The term “guanylinoalkyl” group refers to an alkyl group substituted with a guanyl group, as these terms are defined herein. Preferably, the alkyl group is terminated by a guanyl group.

The term “nitro” group refers to a —NO ₂ group.

  The term “cyano” group refers to a —C≡N group.

  The term “ketoester” refers to the group —C (═O) —C (═O) —O—.

  The term “thiourea” refers to a —NR′—C (═S) —NR ″ — group, where R ′ and R ″ are defined as described above.

  The term “hydrazine” refers to the group NR′—NR ″, where R ′ and R ″ are as defined herein.

The term “ammonium ion” refers to (NR′R ″ R ′ ″) ⁺ , where R ′, R ″ and R ′ ″ are as defined herein.

The term “guanidinium ion” is — (R′NC (═NR ″ ″) — NR ″ R ′ ″ R ″ ″) ⁺ group (where R ′, R ″, R ′ ″). And R ″ ″ are as defined herein).

  The invention further encompasses pharmaceutically acceptable salts of the compounds described herein.

  The expression “pharmaceutically acceptable salt” refers to the charged species of the parent compound and its counterion. Examples of pharmaceutically acceptable salts include, but are not limited to, compounds containing an ammonium or guanidinium cation and an anion of an acid (eg, HCl, trifluoroacetic acid (TFA), etc.). As described above and further shown in the Examples section below, each of the compounds described above is based on the three-dimensional structure of the GSK-3 substrate, and thus potential substrate competitive inhibitors of GSK-3 activity. It is.

  Thus, according to another aspect of the present invention, there is provided a method of inhibiting the activity of GSK-3, which comprises inhibiting cells expressing GSK-3 from an inhibitory effective amount of any one of the aforementioned compounds. Achieved by contacting with.

  The term “inhibitory effective amount” as used herein is an amount determined by considerations as known in the art that is sufficient to inhibit the activity of GSK-3. . The activity may be phosphorylation and / or autophosphorylation activity of GSK-3.

  The method according to this aspect of the invention can be achieved by contacting a cell with a compound in vitro and / or in vivo. This method can be further achieved by contacting the cell with an additional active ingredient capable of altering the activity of GSK-3, as described hereinabove.

  Inhibition of GSK-3 activity is one way to increase insulin activity in vivo. The large activity of GSK-3 reduces insulin action in intact cells (Eldar-Finkelman et al., 1997). This decrease results from the phosphorylation of the serine residue of insulin receptor substrate-1 (IRS-1) by GSK-3. Studies conducted in patients with type II diabetes (non-insulin dependent diabetes mellitus, NIDDM) show that glycogen synthase activity is significantly reduced in these patients and that protein kinase B (PKB) by insulin (GSK-3) It has been shown that reduced activation of (upstream regulators) is also detected (Sulman et al. (1990); Cross et al. (1995)). Mice susceptible to diabetes and obesity induced by a high-fat diet have significantly increased GSK-3 activity in epididymal adipose tissue (Eldar-Finkelman et al., 1999). Increased GSK-3 activity was expressed in cells resulting in suppression of glycogen synthase activity (Eldar-Finkelman et al., 1996).

  Thus, inhibition of GSK-3 activity provides a useful method for increasing insulin activity in an insulin dependent state. Thus, according to another aspect of the present invention, there is provided a method for enhancing insulin signaling, which comprises treating insulin responsive cells with any one of the compounds of the present invention as described above. Achieved by contacting with an effective amount as defined in

  As used herein, the expression “enhance insulin signaling” includes increased phosphorylation of downstream components of the insulin receptor and the rate of glucose uptake compared to glucose uptake in untreated subjects or cells. Increase.

  The method according to this aspect of the invention can be achieved by contacting a cell with a compound of the present invention in vitro or in vivo, and can also be accomplished by further contacting the cell with insulin.

  The in vivo enhancement of insulin signaling resulting from the administration of the compounds described herein can be monitored as a clinical endpoint. In principle, the easiest way to observe insulin enhancement in a patient is to perform a glucose tolerance test. After fasting, glucose is given to the patient and the rate of disappearance of glucose from the blood circulation (ie glucose uptake by cells) is measured by assays well known in the art. The slow rate of glucose clearance (when compared to healthy subjects) indicates insulin resistance. Administration of inhibitors to insulin resistant patients increases the rate of glucose uptake when compared to untreated patients. Inhibitors can be administered to insulin resistant patients over longer periods of time, and insulin concentration, glucose concentration and leptin concentration (usually high) in the blood circulation can be measured. A decrease in glucose concentration indicates that the inhibitor has enhanced insulin action. Decreased insulin and leptin levels alone may not necessarily show enhanced insulin action, but rather show improvement in disease state by other mechanisms.

  The compounds described above can be effectively utilized to treat any biological condition associated with GSK-3.

  Thus, according to another aspect of the invention, a method of treating a biological condition associated with GSK-3 activity is provided. This method according to this aspect of the invention is achieved by administering to a subject in need thereof a therapeutically effective amount of any one of the compounds described hereinabove.

  As used herein, the expression “biological condition associated with GSK-3 activity” refers to any organism for which effective GSK-3 activity is identified, even at normal or abnormal levels. Includes a medical or medical condition or disorder. Such a condition or disorder can be caused by GSK-3 activity or simply characterized by GSK-3 activity. That a state is associated with GSK-3 activity means that some aspect of that state can be located in GSK-3 activity.

  As used herein, the term “treating” refers to preventing, substantially inhibiting, slowing, or reversing the progression of a condition or disorder, or clinical of a condition or disorder. Substantially improving clinical symptoms or substantially preventing the appearance of clinical symptoms of a condition or disorder. These effects can be manifested, for example, by reducing glucose uptake rates for type II diabetes or by stopping neuronal cell death for neurodegenerative disorders, as described in detail herein below.

  As used herein, the term “administering” refers to a compound of the invention and a cell affected by a condition or disorder, such that the compound can affect GSK-3 activity in these cells. Describes how to join together in style. The compounds of the present invention can be administered by any medically acceptable route. The route of administration may depend on the disease, condition or injury being treated. Possible routes of administration include injection, parenteral routes such as intravascular, intravenous, intraarterial, subcutaneous, intramuscular, intratumoral, intraperitoneal, intraventricular, intraepithelial or intraventricular, as well as oral Nasal, ocular, rectal or topical routes, or by inhalation. Sustained release administration is also specifically included in the present invention by such means as depot injection or erodible implants. Administration can also be intra-articular, rectal, intraperitoneal, intramuscular, subcutaneous, or can be effected by aerosol inhalation. When treatment is systemic, the compound can be administered orally or as long as provided in a suitable composition to achieve introduction of the compound into the target cell, as described in detail herein below. It can be administered parenterally (eg, intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally or intracisternally).

  As used herein, the expression “therapeutically effective amount” is intended to prevent, substantially inhibit, slow or reverse the progression of a condition associated with GSK-3 activity. Or an amount administered to an individual that is sufficient to substantially improve the clinical symptoms of such condition or to substantially prevent the appearance of clinical symptoms of such condition Describe. The GSK-3 activity can be GSK-3 kinase activity. The amount of inhibition can be determined directly by measuring inhibition of GSK-3 activity, or, for example, the desired effect is activity downstream of GSK-3 activity in a pathway involving GSK-3. Inhibition can be measured by measuring downstream effects. Thus, for example, where inhibition of GSK-3 results in termination of glycogen synthase phosphorylation, the effect of the compound may include an effect on an insulin-dependent pathway or an insulin-related pathway, It can be administered where glucose uptake increases to an optimal level. Alternatively, if inhibition of GSK-3 results in the absence of phosphorylation of a protein (eg, tau protein) required for further biological activity, the compound is a multimer of phosphorylated tau protein. Can be administered until crystallization is substantially stopped. Thus, inhibition of GSK-3 activity depends, in part, on the nature of the inhibited pathway or process involving GSK-3 activity, and inhibition of GSK-3 activity has in certain biological situations. Depends on effect.

  The amount of a compound that constitutes an inhibitory amount depends on the compound and its potency, the half-life of the compound in the body, the rate of progression of the disease or biological condition being treated, the responsiveness of the condition to the therapeutic dose or pattern of administration, formulation, medicine Evaluation of the clinical situation by the attending physician, and other relevant factors, and generally the patient's health and other considerations (eg, prior administration of other therapeutic agents or any effect on the inhibitory activity of the compound) Such as co-administration of therapeutic agents, or co-administration of any therapeutic agents that have an effect on GSK-3 activity, or pathways mediated by GSK-3 activity). The amount of inhibition is expected to fall within a relatively broad range that can be determined by routine testing.

  As discussed in detail hereinabove, GSK-3 is involved in various biological pathways, and thus the method according to this aspect of the invention is described in detail herein below. As such, it can be used in the treatment of various biological conditions.

  GSK-3 is involved in the insulin signaling pathway, and thus in one example, the method according to this aspect of the invention can be used to treat any insulin dependent condition.

  In another example, the method according to this aspect of the invention may be used to treat obesity because GSK-3 inhibitors are known to inhibit the differentiation of preadipocytes into adipocytes. Can do.

  In yet another example, the method according to this aspect of the invention can be used to treat diabetes, particularly to treat non-insulin dependent diabetes.

  Diabetes is a heterogeneous primary disorder of carbohydrate metabolism due to a number of developmental factors that are commonly associated with insulin deficiency or insulin resistance or both. Type I (juvenile) insulin-dependent diabetes exists in patients who have little endogenous insulin secretory capacity. These patients develop extreme hyperglycemia and are completely dependent on exogenous insulin treatment for immediate survival. Type II or adult-onset or non-insulin dependent diabetes exists in patients who retain some endogenous insulin secretory capacity. However, the majority are both insulin deficient and insulin resistant. About 95% of all diabetic patients in the United States have insulin-independent type II diabetes (NIDDM), and this is therefore the form of diabetes that accounts for the majority of medical problems. Insulin resistance is a fundamental unique feature of NIDDM, and this metabolic defect results in diabetes syndrome. Insulin resistance can be due to insufficient expression of the insulin receptor, decreased insulin binding affinity, or some abnormality at any stage along the insulin signaling pathway (see US Pat. No. 5,861,266). ).

  The compounds of the present invention can be used to treat type II diabetes in type II diabetic patients as described below. In this case, a therapeutically effective amount of the compound is administered to the patient and clinical markers (eg, blood glucose concentration) are monitored. The compounds of the present invention can further be used to prevent type II diabetes in a subject, as described below. In this case, a prophylactically effective amount of the compound is administered to the patient and clinical markers (eg, phosphorylation of IRS-1) are monitored.

Treatment of diabetes is determined by standard medical methods. The goal of diabetes treatment is to reduce the sugar concentration to near normal values in a safe manner. A generally set target is 80-120 milligrams / deciliter (mg / dl) before meals and 100-140 mg / dl at bedtime. A particular physician may set different goals for a patient depending on other factors, such as how often the patient has a hypoglycemic response. Useful medical tests include tests on the patient's blood and urine to measure blood glucose levels, glycated hemoglobin levels (HbA _1c ; an indicator of mean blood glucose levels over the past 2 to 3 months, a normal range of 4 % To 6%), tests for cholesterol and fat levels, and tests for urine protein levels. Such tests are standard tests known to those skilled in the art (see, for example, American Diabetes Assocation (1998)). A successful treatment program can also be determined by the fact that few patients are receiving a program for diabetic eye disease, kidney disease or neurological disease.

  Accordingly, in one particular embodiment of the method according to this aspect of the invention, a method of treating non-insulin dependent diabetes is provided. In this case, the patient is diagnosed at an early stage of non-insulin dependent diabetes. The compounds of the present invention are formulated in enteric capsules. Patients are instructed to take one tablet after each meal for the purpose of stimulating the insulin signaling pathway, so that glucose metabolism obviates conditions that require the administration of exogenous insulin Controlled by level.

  As discussed further hereinabove and as demonstrated in WO 2004/052404, GSK-3 inhibition is associated with affective disorders. Thus, in another example, the method according to this aspect of the invention may be used to treat affective disorders such as unipolar disorders (eg, depression) and bipolar disorders (eg, manic depression). it can.

  Since GSK-3 is also considered to be an important participant in the pathogenesis of neurodegenerative disorders and neurodegenerative diseases, the method according to this aspect of the present invention is further adapted to treat a variety of such disorders and diseases. Can be used for

  In one example, inhibition of GSK-3 results in stopping neuronal cell death, so the method according to this aspect of the invention can be used to treat a neurodegenerative disorder resulting from an event that causes neuronal cell death. it can. Such an event can be, for example, cerebral ischemia, stroke, traumatic brain injury or bacterial infection.

  In another example, since GSK-3 activity is associated with various central nervous system disorders and neurodegenerative diseases, the method according to this aspect of the invention may be used for various chronic neurodegenerative diseases such as Alzheimer's disease, It can be used to treat Huntington's disease, Parkinson's disease, AIDS dementia, amyotrophic lateral sclerosis (AML), multiple sclerosis, and the like.

  As discussed hereinabove, GSK-3 activity is particularly implicated in the pathogenesis of Alzheimer's disease. Accordingly, in one exemplary embodiment of a method according to this aspect of the invention, a method of treating an Alzheimer's disease patient is provided. In this case, to a patient diagnosed with Alzheimer's disease, a compound of the invention that inhibits GSK-3 mediated hyperphosphorylation of tau is prepared and administered in a formulation that crosses the blood brain barrier (BBB). . Patients can regularly analyze proteins isolated from their brain cells for the presence of phosphorylated forms of tau on SDS-PAGE gels known to characterize the presence and progression of the disease. Monitored for tau phosphorylated multimers. The dosage of the compound is adjusted as necessary to reduce the presence of the phosphorylated form of tau protein.

  GSK-3 has also been implicated for psychotic disorders such as schizophrenia. Thus, the method according to this aspect of the invention can further be used to treat psychotic diseases or disorders such as schizophrenia.

  The method according to this aspect of the invention can further be achieved by co-administering to the subject one or more additional active ingredients capable of altering the activity of GSK-3.

  As used herein, “co-administer” administers a compound according to the present invention in combination with a further active ingredient, which is also indicated herein as an active agent or therapeutic agent. Describe that. The additional active agent can be any therapeutic agent that is useful for treating the patient's condition. Co-administration can be simultaneous, for example, by administering a mixture of the compound and the therapeutic agent, or can be accomplished by administering the compound and the active agent separately, such as within a short period of time. Simultaneous administration also includes sequential administration of the compound and one or more of the other therapeutic agents. The additional therapeutic agent (s) can be administered before or after the compound. Dosage treatment can be a single dose schedule or a multiple dose schedule.

  The further active ingredient can be insulin.

  Preferably, the further active ingredient is described in any GSK-3 inhibitor, for example WO 01/49709, WO 2004/052404 and US Pat. No. 6,780,625, wherein the further active ingredient according to the invention is different from the compounds of the invention. The activity of GSK-3 can be inhibited such that it can be a short peptide GSK-3 inhibitor. Alternatively, the GSK-3 inhibitor can be, for example, lithium, valproic acid and / or lithium ion.

  Alternatively, the further active ingredient may be an active ingredient capable of down-regulating GSK-3 expression.

  Agents that down-regulate GSK-3 expression affect (slow down) GSK-3 synthesis, either at the level of mRNA or protein, or affect (promote) GSK-3 degradation. Indicates any drug. For example, small interfering polynucleotide molecules designed to down regulate the expression of GSK-3 can be used as a further active ingredient according to this aspect of the invention.

  An example of a small interfering polynucleotide molecule capable of down-regulating GSK-3 expression is the previously described mechanism of RNA interference (RNAi) (Hutvagner and Zamore (2002), Curr. Opin. Genecs and Development). 12: 225-232), including double-stranded oligonucleotides that allow sequence-specific degradation of mRNA, eg, Munshi et al. (Munshi CB, Graeff R, Lee HC, J Biol Chem, 2002 (Dec, 20): 277 (51): 49453-8), which are small interfering RNAs or siRNAs such as morpholino antisense oligonucleotides.

  As used herein, the expression “double-stranded oligonucleotide” refers to an oligonucleotide structure formed by either one self-complementary nucleic acid strand or at least two complementary nucleic acid strands or a mimetic thereof. . A “double-stranded oligonucleotide” of the present invention is a double-stranded RNA (dsRNA), a DNA-RNA hybrid, a single-stranded RNA (ssRNA), an isolated RNA (ie, partially purified RNA, essentially Can be composed of pure RNA), synthetic RNA, and recombinantly produced RNA.

  Preferably, the specific small interfering duplex oligonucleotide of the present invention is an oligonucleotide composed mainly of ribonucleic acid.

  Descriptions for making double stranded oligonucleotides capable of mediating RNA interference are available at www. ambion. com.

  Thus, small interfering polynucleotide molecules according to the present invention can be RNAi molecules (RNA interference molecules).

  Alternatively, the small interfering polynucleotide molecule can be an oligonucleotide, such as a GSK-3-specific antisense molecule or ribozyme molecule, further described herein below.

  Antisense molecules are oligonucleotides that contain two or more chemically distinct regions each composed of at least one nucleotide. These oligonucleotides have at least one modified oligonucleotide so as to confer to the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target polynucleotide. The region typically contains. Additional regions of the oligonucleotide can serve as substrates for enzymes that can cleave RNA: DNA hybrids or RNA: RNA hybrids. An example for such an enzyme is RNase H, a cellular endonuclease that cleaves the RNA strand of an RNA: DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly increasing the efficiency of oligonucleotide inhibition of gene expression. As a result, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used compared to phosphorothioate deoxyoligonucleotides that hybridize to the same target region. it can. Cleavage of the RNA target can be detected by conventional methods by gel electrophoresis and, if necessary, by associated nucleic acid hybridization techniques known in the art.

  The antisense molecules of the present invention can be formed as a composite structure of two or more oligonucleotides (modified oligonucleotides) as described above. Representative US patents that teach the preparation of such hybrid structures include, but are not limited to, US Pat. Nos. 5,013,830, 5,149,977, 5,222,0007, 5,256,775, 5,366,878, No. 5403711, No. 5491133, No. 5565350, No. 5623065, No. 5562355, No. 5562356 and No. 5700922, each of which is incorporated herein by reference in its entirety. Incorporated).

  Ribozyme molecules are increasingly being used for sequence-specific inhibition of gene expression by cleavage of mRNA. Several ribozyme sequences can be fused to the oligonucleotides of the invention. These sequences include, but are not limited to, ANGIOZYME, which specifically inhibits the formation of VEGF-R (vascular endothelial growth factor receptor), an important component in the angiogenic pathway, and hepatitis C virus (HCV) RNA The ribozyme HEPTAZYME (Rybozyme Pharmaceuticals, Incorporated, WEB homepage) designed to selectively destroy the ribosomes is included.

  Additionally or alternatively, the small interfering polynucleotide molecule according to the present invention may be a DNAzyme.

  DNAzymes are single-stranded catalytic nucleic acid molecules. A general model for the DNAzyme (“10-23” model) has been proposed. The “10-23” DNAzyme has a catalytic domain consisting of 15 deoxyribonucleotides, flanked by two substrate recognition domains, each consisting of 7-9 deoxyribonucleotides. This type of DNAzyme can effectively cleave its substrate RNA at the purine: pyrimidine junction (Santoro, SW & Joyce, GF Proc. Natl, Acad. Sci. USA 199; DNAzyme (See Khachigian, LM Curr Opin Mol Ther 2002: 4: 119-21).

  An example of the construction and amplification of engineered synthetic DNAzymes that recognize single and double stranded target cleavage sites is disclosed in US Pat. No. 6,326,174 (Joyce et al.). A similarly designed DNAzyme directed to the human urokinase receptor has recently been observed to successfully inhibit the expression of the urokinase receptor and inhibit colon cancer cell metastasis in vivo (Itoh et al., 2002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org). In another application, a DNAzyme complementary to the bcr-abl oncogene successfully inhibited oncogene expression in leukemia cells and reduced relapse rates in autologous bone marrow transplants in the case of CML and ALL. .

  Oligonucleotides designed in accordance with the teachings of the present invention can be made according to any oligonucleotide synthesis method known in the art, such as enzymatic synthesis or solid phase synthesis. Instruments and reagents for performing solid phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis can also be used. The actual synthesis of the oligonucleotide is well within the ability of one skilled in the art.

  While being a highly efficient therapeutic agent, and also in therapeutic applications, it is often required to administer an effective amount of the active ingredient to the individual being treated. The described compounds are preferably pharmaceutical to facilitate administration of the compound to the individual being treated and possibly to facilitate entry of the active ingredient into the targeted tissue or cells. In a pharmaceutical composition further comprising an acceptable carrier, it is contained as an active ingredient.

  Thus, according to yet a further aspect of the present invention there is provided a pharmaceutical composition comprising any one of the compounds described herein as an active ingredient and a pharmaceutically acceptable carrier.

  Hereinafter, the expressions “pharmaceutically acceptable carrier” and “physiologically acceptable carrier” do not cause significant irritation to a subject and do not inhibit the biological activity and properties of the administered compound. Or a diluent is shown. Non-limiting examples of carriers include polyethylene glycol, saline, emulsions and mixtures of organic solvents and water.

  As used herein, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

  The pharmaceutically acceptable carrier further includes other agents such as absorption retardants, antibacterial agents, antifungal agents, antioxidants, binders, buffering agents, bulking agents, cationic lipid agents, coloring agents, Diluents, disintegrants, dispersants, emulsifiers, excipients, flavoring agents, lubricants, isotonic agents, liposomes, microcapsules, solvents, sweeteners, viscosity modifiers, wetting agents, skin penetration enhancers, etc. Can be included).

  Various techniques for drug formulation and administration can be found in “Remington's Pharmaceutical Sciences” (Mack Publishing Co., Easton, PA, latest edition), which is incorporated herein by reference. . Suitable routes of administration include, for example, oral delivery, rectal delivery, transmucosal delivery, transdermal delivery, intestinal delivery or parenteral delivery (including intramuscular, subcutaneous and intramedullary injection, and subarachnoid injection). Injection, direct intraventricular injection, intravenous injection, intraperitoneal injection, intranasal injection or intraocular injection).

  The pharmaceutical composition of the present invention can be obtained by various processes well known in the art, for example, conventional mixing, dissolving, granulating, dragee preparation, grinding, emulsifying, encapsulating, entrapping or lyophilizing. It can be manufactured by a process.

  Accordingly, a pharmaceutical composition used in accordance with the present invention comprises one or more pharmaceutically acceptable carriers containing excipients and adjuvants that facilitate the processing of the compound into a preparation that can be used as a medicament. It can be used and formulated in a conventional manner. Composition is aerosol delivery form, aqueous solution, bolus, capsule, colloid, delayed release, depot, dissolvable powder, drop, emulsion, erodible implant, gel, gel capsule, granule, injection solution, ingestion Formulated in delivery forms such as possible solutions, inhalable solutions, lotions, oil solutions, pills, suppositories, salves, suspensions, sustained release, syrups, tablets, tinctures, topical creams, transdermal delivery forms can do. Proper formulation is dependent upon the route of administration chosen.

  For injection, the compounds of the present invention are preferably in physiologically compatible buffers (eg, Hanks's solution, Ringer's solution) in aqueous solutions with or without organic solvents (eg, propylene glycol, polyethylene glycol, etc.). Or in physiological saline buffer, etc.). For transmucosal administration, penetrants are used in the formulation. Such penetrants are generally known in the art.

  For oral administration, the compounds can be readily formulated by combining the compounds with pharmaceutically acceptable carriers that are widely known in the art. Such carriers allow the compounds of the present invention to be formulated as tablets, pills, dragees, capsules, solutions, gels, syrups, slurries and suspensions, etc. that are taken orally by the patient. For orally used pharmacological preparations, solid excipients are used, and the resulting mixture is optionally ground to obtain tablets or dragee cores, with suitable adjuvants added if desired Later, the mixture of granules can be processed and made. Suitable excipients include, among others, fillers such as sugars including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxy There are physiologically acceptable polymers such as propylmethylcellulose, sodium carbomethylcellulose; and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

  Dragee cores are provided with suitable coatings. For this purpose, high-concentration sugar solutions can be used, in which case the sugar solution is optionally gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solution and suitable Organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings to characterize the quantity of active ingredient or to characterize different combinations of active ingredient quantities.

  Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules contain the active ingredients in admixture with fillers (eg lactose etc.), binders (eg starch etc.), lubricants (eg talc or magnesium stearate) and optionally stabilizers can do. In soft capsules, the active ingredients can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

  For buccal administration, the composition can take the form of tablets or lozenges formulated in a conventional manner.

  For administration by inhalation, the compounds according to the invention are suitable for aerosol spray presentations from pressurized packs or nebulizers by the use of suitable propellants (eg dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide). Conveniently delivered in form. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges used in inhalers or insufflators, eg capsules and cartridges made of gelatin, containing a powder mixture of the ingredients and a suitable powder base (eg lactose or starch etc.) it can.

  The compounds described herein can be formulated for parenteral administration, eg, by bolus injection or continuous infusion. Injectable formulations may be presented in unit dosage form, eg, in ampoules or multi-dose containers, optionally with preservatives added. The composition can be a suspension or solution or emulsion in an oily vehicle or an aqueous vehicle, and can contain compounding agents such as suspending, stabilizing and / or dispersing agents. .

  Pharmaceutical compositions for parenteral administration include aqueous solutions of the compounds in water-soluble form. Alternatively, suspensions of the compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate), triglycerides or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

  Alternatively, the compound can be in the form of a powder configured with a suitable vehicle (eg, sterile pyrogen-free water) prior to use.

  The compounds of the present invention can also be formulated in rectal compositions such as suppositories or retention enemas, using, eg, conventional suppository bases such as cocoa butter or other glycerides.

  The pharmaceutical compositions described herein can also include a suitable solid of gel phase carrier or excipient. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

  Pharmaceutical compositions suitable for use in connection with the present invention include compositions wherein the compound is contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount is effective to affect the symptoms of the condition of the subject being treated or effective to prolong the survival of the subject being treated. It means the amount of a compound.

  Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

  For any active ingredient used in the method of the invention, the therapeutically effective amount or dose can be estimated initially from activity assays in cell cultures and / or animals. Such information can be used to more accurately determine useful doses in humans.

  The dosage can vary depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, eg, Fingl et al., 1975, “The Pharmaceutical Basis of Therapeutics”, Chapter 1, page 1. )

  The compositions of the invention can be provided in packs or dispenser devices, such as FDA approved kits, which can contain one or more unit dosage forms containing the compound, if desired. The pack can include, for example, metal foil or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration. The pack or dispenser can also be accompanied by a notice attached to the container in a form stipulated by a government authority that regulates the manufacture, use or sale of the pharmaceutical product. In this case, such notification reflects the form of the composition or approval by the authorities for administration to humans or animals. Such notice can be, for example, a label approved by the US Food and Drug Administration for prescription drugs, or an approved product package insert. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier can also be prepared, placed in a suitable container and labeled to treat the indication. Suitable conditions indicated on the label may include, for example, any of the biological conditions associated with GSK-3 activity as indicated herein above.

  Accordingly, the pharmaceutical composition of the present invention is packed into a packaging material for use in the treatment or prevention of biological conditions associated with GSK-3 and is identified by the surface of the packaging material or the type in the packaging material. Can be done.

  The pharmaceutical compositions of the present invention can further comprise additional active ingredients that can interfere with the activity of GSK-3, as described hereinabove.

  Further objects, advantages and novel features of the present invention will become apparent to those skilled in the art upon review of the following examples, which are not intended to be limiting. Also, each of the various embodiments and aspects of the invention as described hereinabove and as claimed in the claims section is found experimental support in the following examples.

  Reference is now made to the following examples. The following examples, together with the above description, illustrate the invention in a non-limiting manner.

material:
Peptides are available from Genemed Synthesis Inc. (San Francisco, CA).

  The radioactive material is Amersham Ltd. Purchased from.

  Phenyl phosphate and pyridoxal phosphate (also referred to as P-5-P) were obtained from Sigma (Israel).

  All other reagents and solvents were obtained from commercial sources (eg, Sigma, Acros, Aldrich) and used as supplied unless otherwise indicated.

  GSC-1, GSC-2 and GSC-3 were synthesized according to procedures known to those skilled in the art detailed below.

  The synthesis of the new compounds GSC-4, GSC-5, GSC-6, GSC-7 and GSC-21 were designed and carried out as described below.

  The synthesis of the novel compounds MP1-MP6 was designed and performed as described below.

Example 1
Determination of 3D structure of GSK-3 substrate by NMR spectroscopy and structure calculation based on it:
Peptides tested:
A small phosphorylated peptide (named p9CREB) patterned according to the known GSK-3 substrate CREB and two additional peptides (unphosphorylated peptides 9CREB and S ¹ mutants substituted with glutamic acid 9ECREB) (which is thought to mimic charged groups) was used in these studies and is listed in Table 1 below.

  Time course analysis of peptide phosphorylation by GSK-3 shows that only the phosphorylated peptide p9CREB is a substrate for GSK-3, and 9CREB and 9ECREB completely fail to be phosphorylated by GSK-3 And thus again showed that phosphorylated serine is an essential requirement for GSK-3.

Determination of the 3D structure of the tested peptides:
Determination of the 3D structure of p9CREB (FIG. 1a), 9CREB (FIG. 1b) and 9ECREB (not shown) by 2D ¹ H NMR spectroscopy was performed using the following procedure:

Solutions of each peptide were prepared by dissolving the lyophilized powder in water containing 10% D ₂ O. 2D-NMR spectra were obtained on a Bruker Avance DMX spectrophotometer at a ¹ H proton frequency of 600.13 MHz. The carrier frequency was set to the water signal and was suppressed by applying the WATERGATE method or by using low power irradiation during the relaxation period. The experimental temperature (280 ° K) was optimized to reduce population averaging due to faster exchange at more ambient temperatures and to preserve the best possible spectral resolution. All experiments were performed in phase sensitive mode (TPPI or States-TPPI) and were recorded with a spectral width of 12 ppm, a true t ₂ data point of 4K and a 512 t ₁ -increase. The two-dimensional homonuclear data collected included TOCSY using MLEV pulse sequences with a mixing time of 150 msec, and NOESY experiments with a mixing time in the range of 100-750 msec. Typically, the relaxation delay was 1.5 seconds and 2.0 seconds in the TOCSY and NOESY experiments, respectively. In ROESY measurement, the spin lock period was set to 400 ms with an output of 3.4 KHz. All spectra were calibrated against tetramethylsilane.

  Data was processed using Bruker XWIN NMR software (Bruker Analystique Messtechnik, GmbH, version 2.7). All data processing, calculations and analysis were performed on a Silicon Graphics workstation (INDY R4000 and INDIGO2R10000). Free induction delay apodization with shifted doubled sinusoidal functions in both dimensions and indirect dimension zero filling were applied prior to the Fourier transform to increase spectral resolution. The spectrum was further phase corrected by applying automatic polynomial baseline correction developed by Bruker.

  Resonance assignment was based on TOCSY and NOESY spectra measured under the same experimental conditions according to the sequential assignment method developed by Wetherich using the Bruker software program AURELIIA (Bruker Analytical GmbH, version 2.7).

The NOE distance constraint was derived from the NOESY spectrum recorded at 450 ms. This optimal mixing time was determined for p9CREB peptide samples by comparing NOE signal intensities in a series of experiments with mixing times ranging from 100 msec to 750 msec. The selected mixing time gave the maximum NOE build-up with no significant contribution from spin diffusion. This value was used for non-phosphorylated analog experiments to maintain the same experimental conditions. The integrated peak value was converted to a distance constraint using the r- ⁶ dependency, and a known distance between two adjacent protons of the tyrosine aromatic ring of 2.47 was used for calibration. The constraints were classified as strong (1.8-2.5 Å), moderate (1.8-3.5 Å) and weak (1.8-5.0 Å). An empirical correction of 0.5 Å was added to the upper bond for constraints involving methyl groups.

The structure was calculated by dynamic simulated annealing using hybrid distance geometry-XPLOR (version 3.856). NOE energy was derived as the square well potential with a constant force constant of 50Kcal / mol · Å ^2. The simulated annealing consisted of a 1500 3f second step at 1000K and a 3000 1f second step during cooling to 300K. Finally, the structure was minimized using a conjugate gradient energy minimization method for 4000 iterations. INSIGHT II (Molecular Modeling System version 97.0, Molecular Simulations, Inc.) was used for visualization and analysis of structures derived from NMR. Their quality was evaluated using PROCHECK.

result:
Tables 2 and 3 below represent the structural coordinate data used to input into the structural analysis software for visualization of 3D structures.

  From the resulting 3D structure represented in FIGS. 1 a and 1 b, it was observed that only the phosphorylated peptide has a defined structural conformation. As shown in FIG. 1a, for p9CREB, phosphorylation results in a significant “bend” of the peptide backbone, bringing Tyr8 and Arg4 close together, forming a “loop structure”, thereby allowing phosphorylated residues to be Turn outward. This conformation minimizes interference of positively charged residues (Arg4 and Arg5) on the one hand with the enzyme's catalytic binding pocket and, on the other hand, makes phosphorylated serine readily accessible to the enzyme. This structural analysis provides an explanation for the specific substrate recognition of GSK-3. Thus, the design of small molecules that mimic the structure represented here provides a way to obtain selective inhibitors for GSK-3.

Example 2
Chemical synthesis instrument data of monoaryl / heteroaryl GSK-3 inhibitor (GSC small molecule):
Proton, carbon, fluorine and phosphorus nuclear magnetic resonance spectra were obtained on a Bruker AMX 500 spectrophotometer or a Brucker AV 300 spectrophotometer and reported in ppm (σ). Tetramethylsilane (TMS) was used as an internal standard for the proton spectrum, phosphoric acid was used as the internal standard for the phosphorus spectrum, and the solvent peak was used as a reference peak for the carbon and fluorine spectra.

  Mass spectroscopic spectra were obtained on a Finnigan LCQ Duo LC-MS ion capture electron scatter ionization (ESI) mass spectrometer.

  Thin layer chromatography (TLC) was performed using Analtech silica gel plates and visualized by staining the plates with 0.2 wt% ninhydrin in butanol or by ultraviolet (UV) light.

  Elemental analysis is performed by Quantitative Technologies, Inc. (Whitehouse, NJ).

HPLC analysis was obtained using a Hypersil BDS C18 Column, 4.6 × 150 mm, 5 μm with a detector operating at ambient column temperature and at 220 nm, using the standard solvent gradient program shown below as the mobile phase.

Preparation of GSK-3 inhibitors:
Based on the 3D structure determination described above, a series of small molecules that act as potential selective inhibitors of GSK-3 were designed and successfully prepared as shown below.

A. Synthesis of p-methylbenzyl phosphate (GSC-2):
The general route for the synthesis of GSC-2 is represented by Equation 1 below.

Preparation of di-tert-butyl, p-methylbenzyl phosphate:
1H-tetrazole solution (0.45 M in acetonitrile, 20 ml, 9 mmol, 3 eq) was diluted with 4-methylbenzyl alcohol (0.4 gram, 3.3 mmol, 1.1 eq) in dry THF (3 ml). -Added in one portion to a stirred solution of tert-butyldiisopropyl phosphoramidite (0.95 ml, 0.83 grams, 3 mmol, 1 eq). The mixture was stirred for 15 minutes at 20 ° C. The mixture was cooled to −40 ° C. (by dry ice / acetonitrile) and 85% m-chloroperbenzoic acid (mCPBA) in dichloromethane (4 ml) (0.81 grams, 4 mmol, 1.3 eq in 1 ml dichloromethane). The solution was quickly added while the reaction temperature was kept below 0 ° C. The solution was warmed to room temperature and stirred for 5 minutes at 20 ° C., after which 10% aqueous NaHSO ₃ solution (10 ml) was added and the mixture was stirred for an additional 10 minutes. The mixture was then extracted with ether (70 ml) and the aqueous phase was discarded. The ether phase was washed with 10% aqueous NaHSO ₃ (2 × 20 ml) and saturated aqueous 5% NaHCO ₃ (2 × 20 ml), dried over sodium sulfate and filtered. The organic filtrate is evaporated and the residue is purified by chromatography on a silica gel column with an EtOAc / hexane 1:15 mixture as eluent to give the product (di-tert-butyl, p-methylbenzyl phosphate) and starting material. Was obtained and used without further separation.

Preparation of p-methylbenzyl phosphate: A solution of HCl (4M in dioxane, 2 ml, 8 mmol, 2.6 eq) and dioxane (6 ml) was added to the resulting di-tert-butyl, p-methylbenzyl phosphate. Added at 20 ° C. and the reaction was monitored by TLC. Once hydrolysis was complete, the dioxane was evaporated under reduced pressure and the residue was dissolved in water (15 ml) and washed with ether (2 × 15 ml) to remove excess benzyl alcohol starting material. The solvent was then evaporated under reduced pressure, and the resulting clear oil slowly changed to a colorless solid with extended high vacuum drying, yielding 0.18 grams (30%) of the final product.

B. Synthesis of benzyl phosphate (GSC-1):
The general route for the synthesis of GSC-1 is represented by Equation 2 below.

The general synthesis of benzyl phosphate is represented by Formula 2 below.

Preparation of di-tert-butylbenzyl phosphate:
1H-tetrazole solution (0.45 M in acetonitrile, 20 ml, 9 mmol, 3 eq) was added to benzyl alcohol (0.34 ml, 3.3 mmol, 1.1 eq) and di-tert-butyl in dry THF (3 ml). To a stirred solution of diisopropyl phosphoramidite (0.95 ml, 0.83 grams, 3 mmol, 1 equiv) was added in one portion. The mixture was stirred for 15 minutes at 20 ° C., after which the mixture was cooled to −40 ° C. (by dry ice / acetonitrile). A solution of 85% mCPBA (0.81 grams, 4 mmol, 1.3 eq in 1 ml dichloromethane) in dichloromethane (DCM) (4 ml) was added rapidly while the reaction temperature was kept below 0 ° C. The solution was warmed to room temperature and stirred for 5 minutes at 20 ° C., after which 10% aqueous NaHSO ₃ solution (10 ml) was added and the mixture was stirred for an additional 10 minutes. The mixture was then extracted with ether (70 ml) and the aqueous phase was discarded. The ether phase was washed with 10% aqueous NaHSO ₃ (2 × 20 ml) and saturated aqueous 5% NaHCO ₃ (2 × 20 ml), dried over sodium sulfate and filtered. The organic layer is evaporated and the residue is purified by chromatography on a silica gel column with an EtOAc / hexane 1:15 mixture as eluent to give a mixture of product (di-tert-butylbenzyl phosphate) and starting material. This was used without further purification.

Preparation of benzyl phosphate: A solution of HCl (4M in dioxane, 2 ml, 8 mmol, 2.6 eq) and dioxane (6 ml) was added to the resulting di-tert-butylbenzyl phosphate at 20 ° C. and the reaction Was monitored by TLC. Once hydrolysis was complete, the dioxane was evaporated under reduced pressure and the residue was dissolved in water (15 ml) and washed with ether (2 × 15 ml) to remove excess benzyl alcohol starting material. The solvent was then evaporated under reduced pressure, and the resulting clear oil slowly changed to a colorless solid upon prolonged high vacuum drying, yielding 0.17 grams (30%) of the final product.

C. Synthesis of 3-pyridylmethyl phosphate (GSC-3):
The general route for the synthesis of 3-pyridylmethyl phosphate is represented by Formula 3 below.

Preparation of di-tert-butyl 3-pyridylmethyl phosphate:
1H-tetrazole solution (0.45M in acetonitrile, 20 ml, 9 mmol, 3 eq) was added to 3-pyridylmethanol (0.31 ml, 3.3 mmol, 1.1 eq) and di-tert in dry THF (3 ml). -Added in one portion to a stirred solution of butyl diisopropyl phosphoramidite (0.95 ml, 0.83 grams, 3 mmol, 1 equiv). The mixture was stirred for 15 minutes at 20 ° C., after which the mixture was cooled to −40 ° C. (by dry ice / acetonitrile). A solution of 85% mCPBA (0.81 grams, 4 mmol, 1.3 eq in 1 ml DCM) in DCM (4 ml) was then quickly added while the reaction temperature was kept below 0 ° C. The solution was warmed to room temperature and stirred for 5 minutes at 20 ° C., after which 10% aqueous NaHSO ₃ solution (10 ml) was added and the mixture was stirred for an additional 10 minutes. The mixture was then extracted with ether (70 ml) and the aqueous phase was discarded. The ether phase was washed with 10% aqueous NaHSO ₃ (2 × 20 ml) and saturated aqueous 5% NaHCO ₃ (2 × 20 ml), dried over sodium sulfate and filtered. The organic filtrate is evaporated and the residue is purified by chromatography on a silica gel column with a mixture of CHCl ₃ / hexane 1: 1 as eluent, the product (di-tert-butyl 3-pyridylmethyl phosphate) and starting material Was obtained and used without further purification.

Preparation of 3-pyridylmethyl phosphate: A solution of HCl (4M in dioxane, 2 ml, 8 mmol, 2.6 eq) and dioxane (6 ml) was added to the resulting di-tert-butyl 3-pyridylmethyl phosphate to 20 Added at 0 C and the reaction was monitored by TLC. Once the hydrolysis was complete, dioxane was evaporated under reduced pressure and the residue was dissolved in water (15 ml) and washed with ether (2 × 15 ml). The solvent was then evaporated under reduced pressure to yield 0.19 grams (30%) of the final product.

D. Synthesis of 3,5-bis (2-aminoethyl) benzyl phosphate (GSC-21):
A general method for the synthesis of GSC-21 represented by Formula 4 below, as well as a purification protocol for the final product, was designed and implemented. 3,5-bis (2-aminoethyl) benzyl phosphate (GSC-21) was obtained in an overall yield of 5% by an 8-step synthesis. The corresponding trifluoroacetate salt was also prepared.

The benzyl alcohol intermediate (see Formula 4) is obtained in four steps from an inexpensive starting material, trimethyl 1,3,5-benzenetricarboxylate, as detailed below and as represented in Formula 5. Identified as an important intermediate that can.

The phosphate moiety was then introduced by reacting the alcohol with di-tert-butyldiisopropyl phosphoramidite in the presence of tetrazole according to the method of Johnson (Tetrahedron Lett. 1988, 29, 2369-2372). Immediate oxidation with m-chloroperbenzoic acid (mCPBA) without isolation of the phosphite produced the corresponding phosphate ester. Global deprotection of amines and phosphates was achieved by using trifluoroacetic acid under controlled conditions. The material was then obtained as its trifluoroacetate salt. The latter was recrystallized prior to treatment with the ion exchange resin to yield the desired product with the appropriate purity (usually about 90%) (AUC by HPLC) as shown in Equation 6 below.

The following is a detailed description of the synthesis.
Preparation of 1,3,5-tris (hydroxymethyl) benzene: A 3 liter round bottom flask equipped with an overhead stirrer, additional funnel and reflux condenser was charged with lithium aluminum hydride (49.7 grams, 1 .31 mol) and anhydrous THF (500 ml) were charged under a nitrogen atmosphere. The resulting suspension was slowly heated to reflux, and a solution of trimethyl-1,3,5-benzenetricarboxylate (100.0 grams, 0.40 mol) in anhydrous THF (1.0 liter) was present. While maintaining a gentle reflux (3 hours). The resulting gray suspension was stirred at reflux for an additional 7 hours and cooled with an external ice-water bath. Excess lithium aluminum hydride was hydrolyzed by the dropwise addition of water (50 ml, 45 minutes), and then 15% NaOH (50 ml, slow stream) and finally more water (150 ml, slow stream). The resulting suspension was stirred at ambient temperature for 14 hours. The solids are filtered and the filtrate is concentrated under high vacuum to give a colorless oil that slowly solidifies to 1,3,5-tris (hydroxymethyl) benzene (62 as a white solid). Yielded 3 grams, 94%). ¹ H NMR and ¹³ C NMR spectra were consistent with the assigned structure.

Preparation of 3,5-bis (bromomethyl) benzyl alcohol: In a 2 liter round bottom flask equipped with a magnetic stir bar and additional funnel, add 1,3,5-tris (hydroxymethyl) benzene (33.7 grams, 0.20 mol) and anhydrous acetonitrile (750 ml) were charged. To the resulting suspension, bromotrimethylsilane (TMSBr) (979.0 ml, 0.60 mol) was added as a slow stream with stirring. The white slurry turned brown and had a viscosity. The reaction mixture was then heated to 40 ° C. for 25 minutes, resulting in a clear solution. Completion of the reaction was judged by TLC analysis (90:10 methylene chloride / methanol, UV visualization, starting material Rf 0.07, product Rf 0.77). The solvent was removed under reduced pressure to yield a brown paste. The crude material was purified by column chromatography (silica _{gel, 0-5% MeOH / CH 2 Cl} 2). 3,5-Bis (bromomethyl) benzyl alcohol (33.5 grams, 57%) was obtained as a white solid. ¹ H NMR and ¹³ C NMR spectra were consistent with the assigned structure.

Preparation of 3,5-bis (cyanomethyl) benzyl alcohol: To a 1 liter round bottom flask equipped with an overhead stirrer, additional funnel and reflux condenser, was added 3,5-bis (bromomethyl) benzyl alcohol (33. 1 gram, 0.11 mol) and methanol (400 ml) were charged. The resulting clear solution was heated to reflux. A solution of sodium cyanide (16.2 grams, 0.33 mol) in water (25 ml) was added slowly. Heating is continued for 6 hours under reflux, after which the completion of the reaction is confirmed by TLC analysis (95: 5 methylene chloride / methanol, UV visualization, starting material Rf is 0.62, product Rf is 0.38) Was judged by. The reaction mixture was cooled to ambient temperature and the solvent was removed under reduced pressure to yield a brown paste. The latter was ground with MTBE (6 × 100 ml). The MTBE extracts were combined and the solvent was removed under reduced pressure. Thus obtained yellow oil is then purified by column chromatography (silica _{gel, 0-5% MeOH / CH 2 Cl} 2) was purified by. 3,5-bis (cyanomethyl) benzyl alcohol (18.7 grams, 89%) was obtained as a light brown oil that slowly changed to a waxy white solid. A 1 gram sample was removed and purified via column chromatography to give a purified sample. ¹ H NMR and ¹³ C NMR spectra were consistent with the assigned structure.

Preparation of 3,5-bis (aminoethyl) benzyl alcohol: A sample of 3,5-bis (cyanomethyl) benzyl alcohol (8.0 grams, 0.04 mol) was divided into three, 2.5-3.0 grams each. Portions were filled into separate 500 ml Parr bottles, followed by ethanol (100 ml) and aqueous NaOH (1.2 grams in 5 ml water). Raney Ni (50% suspension in water, 1.2 grams) was added to the resulting solution. The mixture was hydrogenated on a Parr shaker at 30 psi. The reaction was monitored by ¹ H NMR and judged complete after 3 hours. The catalyst was filtered through a pad of diatomaceous earth, which was washed with ethanol (200 ml). The filtrates from all three reactions were combined and the solvent was removed under reduced pressure to give 3,5-bis (aminoethyl) benzyl alcohol (14.16 grams) as a brown paste. The ¹ H NMR spectrum of the product showed the presence of 25% (w / w) ethanol. No significant change in ethanol content was observed when the sample was dried under high vacuum for an extended time. This material was used in the next step of the synthesis without any further purification.

Preparation of 3,5-bis (tert-butoxycarbonylaminoethyl) benzyl alcohol: dissolved in THF (590 ml) in a 3-neck 3 liter round bottom flask equipped with a magnetic stir bar, thermometer and gas inlet adapter 3,5-bis (aminoethyl) -1-hydroxymethylbenzyl alcohol (29.4 grams) and 2N aqueous NaOH (590 ml) were charged. To the stirred mixture, di-tert-butyl dicarbonate (59.4 grams, 272 mmol) was added in one portion. The mixture was heated to 45 ° C. for 4 hours. The resulting solution was cooled to ambient temperature and volatile organics were removed by vacuum. Methanol (600 ml) was added to the resulting water mixture. The stirred solution was heated to 45 ° C. for 2 days to selectively hydrolyze the tert-butoxycarbonate moiety while storing the carbamate. After cooling to ambient temperature, the solution was freed of volatile components and the aqueous mixture was extracted with chloroform (3 × 600 ml). The organic layers were combined, washed with brine (600 ml) and concentrated. After drying under high vacuum, crude 3,5-bis (tert-butoxycarbonylaminoethyl) benzyl alcohol (24.88 grams) was obtained as a pure white solid in 67% yield. ^{The 1} H NMR spectrum was consistent with the assigned structure. The crude material was used without further purification.

Preparation of protected 3,5-bis (2-aminoethyl) benzyl phosphate: In a 500 ml round bottom flask equipped with a magnetic stir bar and an additional funnel, add 3,5-bis (tert-butoxycarbonylaminoethyl). ) Benzyl alcohol (2.3 grams, 0.0058 mol) and anhydrous methylene chloride (45 ml) were charged. The resulting solution was cooled to about 5 ° C. in an ice-water bath. Di-tert-butyldiisopropyl phosphoramidite (4.5 ml, 0.0144 mol) in anhydrous acetonitrile (45 ml) was added as a slow stream from an additional funnel. Next, a solution of tetrazole (1.0 gram, 0.0144 mol) in a 1: 1 mixture of anhydrous acetonitrile / anhydrous methylene chloride (90 ml) was slowly added (15 minutes). The resulting white suspension was stirred at about 5 ° C. for 1 hour and reaction completion was confirmed by TLC analysis (95: 5 chloroform / isopropyl alcohol, visualized by staining in ninhydrin, starting material Rf 0.23, formation The Rf of the product was judged by 0.30). The solvent was removed under reduced pressure to give a paste, which was dissolved in anhydrous methylene chloride (75 ml) and cooled in a dry ice / acetonitrile bath. A solution of mCPBA (1.3 grams, 0.0144 mol) in anhydrous methylene chloride (50 ml) was added in one portion. The resulting mixture was stirred for 1 hour, warmed to ambient temperature (1 hour) and stirred for an additional 30 minutes. The reaction mixture was then washed successively with 1.0 M aqueous sodium thiosulfate solution (100 ml) and saturated sodium bicarbonate (2 × 100 ml). The organic extract was dried over anhydrous sodium sulfate and filtered. The solvent was removed under reduced pressure to give the crude phosphate as a yellow oil, which was then purified by column chromatography (silica gel, 0-5% MeOH / CH ₂ Cl ₂ ). Protected 3,5-bis (2-aminoethyl) benzyl phosphate (2.1 grams, 61%) was obtained as a viscous colorless oil. The ¹ H NMR, ¹³ C NMR and ³¹ P NMR spectra of the product were consistent with the assigned structure.

Preparation of 3,5-bis (2-aminoethyl) benzyl phosphate TFA salt: A 250 ml round bottom flask equipped with a magnetic stir bar was charged with protected phosphate (2.9 grams, 0.0049 mol), anhydrous Dichloromethane (30 ml) and trifluoroacetic acid (30 ml) were charged. The resulting clear solution was stirred at ambient temperature for 3 hours. Completion of the reaction was judged by ¹ H NMR and ³¹ P NMR analysis. Removal of the solvent under reduced pressure gave a viscous orange oil that was dissolved in methanol (7.5 ml) and added to diethyl ether (500 ml) with stirring to precipitate the product. Produced. The resulting slurry was stirred for 1 hour at ambient temperature and then the solid was allowed to settle. The clear solution was decanted from the top and the product was triturated with ether (2 × 100 ml). The clear solution was decanted each time a solid was precipitated. The product was finally dried in a vacuum oven for 108 hours at 55 ° C. and then further 192 hours at 65 ° C. to give 3,5-bis (2-aminoethyl) benzyl phosphate TFA salt (1 Yielded .78 grams, 71%). ¹ H NMR, ¹³ C NMR and ³¹ P NMR spectra were consistent with the assigned structure. ¹ H NMR spectrum showed the presence of 8.5% (w / w) ether in the product. This ether proved extremely difficult to remove and the material was characterized as hygroscopic.

Preparation of 3,5-bis (2-aminoethyl) benzyl phosphate (GSC-21): three equipped with overhead mechanical stirrer, thermometer, 1 liter additional pressure equalizing funnel and gas inlet adapter A 5-liter round bottom flask with a neck is charged with a solution of 3,5-bis (tert-butoxycarbonylaminoethyl) benzyl alcohol (24.88 grams, 63.15 mmol) in anhydrous dichloromethane (1 l) under a nitrogen atmosphere. It was done. The reaction mixture was cooled with an ice / brine bath. Di-tert-butyldiisopropyl phosphoramidite (49.8 ml, 157.9 mmol) in anhydrous acetonitrile (1 liter) was passed through a pressure equalizing funnel at such a rate that the reaction temperature was maintained below 6 ° C. Added. Tetrazole (351 ml of a 0.45 M solution in acetonitrile, 157.9 mmol) was diluted with anhydrous acetonitrile (150 ml) and anhydrous dichloromethane (500 ml) at a rate such that the temperature was maintained below 6 ° C. Added via funnel. After the addition was complete, the flask was moved into a cold bath and the reaction mixture was stirred for 1 hour. The flask was then cooled to -35 ° C with a dry ice / acetonitrile bath. A solution of 3-chloroperoxybenzoic acid (18.4 grams, 82.1 mmol) in anhydrous dichloromethane (500 ml) was added in one portion. The mixture was warmed to ambient temperature and then stirred for 2 hours. The solution was poured into a solution of Na ₂ S ₂ O ₃ (20 grams) and K ₂ CO ₃ (50 grams) in water (1.5 liters). The resulting biphasic mixture had a pH of 11. After stirring for 15 minutes, volatile organics were removed in vacuo and the aqueous layer was extracted with chloroform (4 × 750 ml). The combined organic layers were dried over magnesium sulfate, filtered and concentrated to a yellow oil (69 grams). The crude material was purified by column chromatography (silica gel, MTBE / heptane, 6: 4). The combined fractions containing the product were combined and concentrated to yield a light yellow oil (32.6 grams). Of this oil, 28.6 grams were dissolved in dichloromethane (287 ml, 10 vol) and charged into a 1 liter round bottom flask equipped with a 500 ml additional pressure equalizing funnel and a magnetic stir bar. Trifluoroacetic acid (287 ml, 10 vol) was added rapidly via an additional pressure equalizing funnel. The resulting solution was stirred for 5 hours. After being concentrated and dried overnight under high vacuum, a dark orange oil (37.88 grams) was obtained. The residue was dissolved in water (57 ml, 1.5 vol) and added dropwise to stirred methanol (90 vol) to give a precipitate. After stirring for 30 minutes, the solid was allowed to settle for 1 hour and the liquid was decanted. The remaining liquid was removed in vacuo to give 13.72 grams of solid. The material was dissolved in water (68 ml, 5 volumes) and added into Dowex 50WX8-200 ion exchange resin (137 grams). The column was washed with water (550 ml, 40 volumes). The product was eluted with 3: 1 aqueous MeOH / NH ₄ OH (2 liters, 145 volumes). The methanol fraction was concentrated under reduced pressure to yield a pure white solid. The solid was dissolved in a minimum amount of water and added to stirred methanol (40 vol). The precipitate was collected via filtration and dried overnight under vacuum. The resulting powder was ground with water (7 volumes). After filtration and drying under high vacuum, the final product (2.0 grams) was obtained as a white powder. The filtrate was concentrated and the residue was triturated with water (5 volumes). After filtration and drying under high vacuum, a second group of products (0.9 grams) was obtained. The two lots were combined and blended for 10 minutes. 3,5-Bis (2-aminoethyl) benzyl phosphate (GSC-21) was obtained as a white powder. The ¹ H NMR, ³¹ P NMR and ¹³ C NMR spectra of the product were consistent with the assigned structure.

The mass spectrum of the final product represented in FIG. 4a showed a molecular peak at m / z of 275 [C ₁₁ H ₁₉ N ₂ O ₄ P + H] ⁺ . The HPLC chromatogram represented in FIG. 4b (obtained using Method A above) showed a purity of 96.7% of the product. The final product was characterized as non-hygroscopic.

  Using similar methods, novel compounds GSC-4 and GSC-5 were synthesized as follows:

E. Synthesis of 3- (guanidinomethyl) benzyl phosphate (GSC-5):
The general synthesis of GSC-5 as the trifluoroacetate salt is represented by the following formula 7:

Preparation of 3- (aminomethyl) benzyl alcohol: A solution of 3- (hydroxymethyl) benzonitrile in THF was slowly added into a refluxing solution of LiAlH ₄ in THF under vigorous stirring, under a nitrogen atmosphere. Maintained. The solution was heated at reflux overnight, after which time water was slowly added dropwise to stop the reaction (until no further evolution of H ₂ became apparent). The THF was evaporated under reduced pressure and ether / acidified water was added. The ether phase was discarded. The aqueous phase was washed with ether and the organic phase was discarded. NaOH was added until the pH of the aqueous phase reached pH7. The solution is extracted three times with THF, dried over MgSO ₄ and evaporated under reduced pressure to give a pale yellow residue which begins with ethyl acetate and ends with a 1: 1 mixture of ethyl acetate: MeOH. Was purified by chromatography on a silica gel column to give the intermediate in 40% yield.

Preparation of 3- (N, N′-bis-BOC-guanidinomethyl) benzyl alcohol: 3- (aminomethyl) benzyl alcohol, 3- (N, N′-bis (tert-butoxycarbonyl) -S in dry DMF The solution of methylisothiourea and triethylamino was stirred overnight at room temperature, then an ether / water mixture was added and the organic layer was separated, while the aqueous layer was extracted with ether. The extract was washed with water, dried over MgSO ₄ and evaporated under reduced pressure The crude product was flash chromatographed using a gradient eluent starting with hexane and ending with a 1: 5 mixture of ethyl acetate: hexane. To give a yield of 85%.

Preparation of di-tert-butyl 3- (N, N′-bis-BOC-guanidinomethyl) benzyl phosphate:
1H-tetrazole solution (0.45 M in acetonitrile, 20 ml, 9 mmol, 3 eq) was combined with 3- (N, N′-bis-BOC-guanidinomethyl) benzyl alcohol (1 eq) in dry THF (3 ml). To a stirred solution of di-tert-butyldiisopropyl phosphoramidite (1.42 ml, 1.24 grams, 4.5 mmol, 1.5 equiv) was added in one portion. The mixture was stirred for 30 minutes at 20 ° C., then the mixture was cooled to −40 ° C. (by dry ice / acetonitrile). A solution of 85% mCPBA (1.25 grams, 6.15 mmol, 2.0 equiv in 1.5 ml DCM) in DCM (4 ml) was added rapidly while the reaction temperature was kept below 0 ° C. The solution was allowed to warm to room temperature and stirred for 20 minutes before 10% aqueous NaHSO ₃ (10 ml) was added and the mixture was stirred for an additional 5 minutes. The mixture was then extracted with ether (70 ml) and the aqueous phase was discarded. The ether phase was washed with 10% aqueous NaHSO ₃ (2 × 20 ml) and saturated aqueous 5% NaHCO ₃ (2 × 20 ml), dried over sodium sulfate and filtered. The organic filtrate is evaporated and the residue is purified by chromatography on a silica gel column using a gradient eluent of EtOAc / hexane 1: 9 to 1: 5 to give a mixture of phosphate ester product and benzyl alcohol starting material. This was further purified by chromatography on a silica gel column using a gradient eluent of CHCl ₃ : MeOH 30: 1 to 20: 1 to give pure di-tret-butyl 3- (N, N′-bis- BOC-guanidinomethyl) benzyl phosphate was given in 70% yield.

Preparation of 3- (guanidinomethyl) benzyl phosphate trifluoroacetate salt: A solution of 25% trifluoroacetic acid (TFA) in DCM was added to di-tert-butyl 3- (N, N′-bis-BOC-guanidinomethyl). ) Added to benzyl phosphoric acid at 20 ° C. and the reaction mixture was stirred for 18 hours. The solvent and TFA were then evaporated under reduced pressure and the residue was dissolved in water and washed with ether. The solvent was then evaporated under reduced pressure to give the pure product in 60% yield.

  Trifluoroacetic acid can be removed or replaced, for example with HCl, using procedures well known in the art to give free guanidine or the hydrochloride salt of the compound, for example.

F. Synthesis of 3-guanidinobenzyl phosphate (GSC-4):
The general synthesis of GSC-4 as the trifluoroacetate salt is represented by the following formula 8:

Preparation of 3- (N, N′-bis-BOC-guanidino) benzyl alcohol: N, N′-bis (tert-butoxycarbonyl) -S-methylisothiourea (1.32 grams, 4.4 mmol, 1.1 equiv. ), Mercury chloride (1.22 grams, 4.4 mmol, 1.1 eq.) And triethylamine (1.72 ml, 12 mmol, 3 eq.) In 3-dimethylbenzyl alcohol (DMF) in dry dimethylformamide (DMF). 0.5 grams, 4 mmol, 1.0 eq.) And the reaction mixture was stirred at room temperature for 5 hours. The mixture was then extracted with ether / water and the organic layer was washed with saturated aqueous NH ₄ Cl and brine. The aqueous layer was extracted with ether. The combined ether solution was dried over MgSO ₄ and evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel using a gradient eluent from hexane to 40:60 ethyl acetate: hexane to give the intermediate in 60% yield.

Preparation of di-tert-butyl 3- (N, N′-bis-BOC-guanidino) benzyl phosphate:
1H-tetrazole solution (0.45M in acetonitrile, 18.4 ml, 8.3 mmol, 3 eq) was added to 3- (N, N′-bis-BOC-guanidino) benzyl alcohol (1) in dry THF (3 ml). Gram, 2.8 mmol, 1 eq) and di-tert-butyldiisopropyl phosphoramidite (1.13 ml, 3.6 mmol, 1.3 eq) were added in one portion. The mixture was stirred at 20 ° C. for 30 minutes, after which the mixture was cooled to −40 ° C. (by dry ice / acetonitrile). A solution of 85% mCPBA (0.85 grams, 4.20 mmol, 1.5 eq in 1.5 ml DCM) in DCM (4 ml) was added rapidly while the reaction temperature was kept below 0 ° C. . The solution was allowed to warm to room temperature and stirred for 20 minutes before 10% aqueous NaHSO ₃ (10 ml) was added and the mixture was stirred for an additional 10 minutes. The mixture was then extracted with ether (50 ml) and the aqueous phase was discarded. The ether phase was washed with 10% NaHSO ₃ solution (2 × 20 ml) and saturated aqueous NaHCO ₃ (2 × 20ml), dried over sodium sulfate and filtered. The solvent was evaporated and the residue was purified by chromatography on a silica gel column using a gradient eluent of EtOAc / hexane 10:90 to 30:70 to give the protected product in 60% yield.

Preparation of trifluoroacetate salt of 3-guanidinobenzyl phosphate: A solution of 25% TFA (1.5 ml) in DCM (4.5 ml) was added to di-tert-butyl-3- (N, N′-bis- To BOC-guanidino) benzyl phosphate (0.3 grams, 0.54 mmol, 1 eq) was added at 20 ° C. and the reaction mixture was stirred for 18 hours. The solvent and TFA were then evaporated under reduced pressure and the residue was dissolved in water and washed with ether. The solvent under a reduced pressure is evaporated (lyophilizer), the pure product was given in 40% yield _{(C 10 H 13 F 3 N} 3 O 6 P; Mw = 359.2 grams / mol).

  Trifluoroacetic acid can be removed or replaced, for example with HCl, using procedures well known in the art to give free guanidine or the hydrochloride salt of the compound, for example.

G. Synthesis of 3- (guanidinoethyl) benzyl phosphate (GSC-6):
The general synthesis of GSC-6 as the trifluoroacetate salt is represented by Formula 9 below:

Preparation of methyl 3- (cyanomethyl) benzoate:
A mixture of methyl 3- (bromomethyl) benzoate (0.6 grams, 2.6 mmol), potassium cyanide (0.35 grams, 5.2 mmol) and 60 mg 18-crown-6 in 6 ml acetonitrile was vigorously stirred at room temperature for 24 hours. Stirred. The mixture was filtered, the filtrate was concentrated to half volume, water was added to the remaining mixture and extraction was performed with dichloromethane. The organic phase was dried over MgSO ₄ and the solvent was evaporated to give the product as a yellow liquid, which was used without purification.

Preparation of 3- (2-aminoethyl) benzyl alcohol: To a solution of methyl-3- (cyanomethyl) benzoate (4 grams, 0.023 mol, 1 molar equivalent) in 5 ml dry THF was added 2M LiBH ₄ ( 60 ml, 5 molar equivalents) and (MeO) ₃ B (3 ml, 1 molar equivalent) were added under a nitrogen atmosphere. The reaction mixture was stirred and heated at 70-75 ° C. for 16-18 hours. After cooling, THF was evaporated, _2N H 2 SO ₄ was added to the residue (to acidify the solution to pH 1-2). The resulting solution was washed with ether and added to the aqueous phase until powdered K ₂ CO ₃ reached a pH of 7. NaCl was then added and the solution was extracted with THF. The organic phase was dried over MgSO ₄ and the solvent was evaporated to give 2.3 grams (67%) of pure product, which was used without purification.

Preparation of 3- (N, N′-bis-BOC-guanidinoethyl) benzyl alcohol: 3- (aminoethyl) benzyl alcohol in dry DMF (25 ml) (2.3 grams, 0.015 mol, 1 molar equivalent) , N, N′-bis (tert-butoxycarbonyl) -S-methylisothiourea (4.8 grams, 0.016 mol, 1.1 molar equivalent), mercury chloride (4.5 grams, 0.016 mol, 1.1 Molar equivalents) and triethylamine (7 ml, 0.048 mol, 3 molar equivalents) were stirred at room temperature overnight under a nitrogen atmosphere. Then ethyl acetate (100 ml) was added and the suspension was filtered over Celite. The filtrate was washed with water, saturated aqueous NH ₄ Cl and brine, dried over MgSO ₄ and the solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography (using a gradient eluent of a mixture of EtOAc / hexanes 10:90 to 30:70) to give 1.9 grams (32%) of pure product.

Preparation of di-tert-butyl 3- (N, N′-bis-BOC-guanidinoethyl) benzyl phosphate: 1H-tetrazole solution (0.45M in acetonitrile, 30 ml, 14 mmol, 3 molar equivalents) was added to dry THF 3- (N, N′-bis-BOC-guanidinoethyl) benzyl alcohol (1.8 grams, 4.6 mmol, 1 molar equivalent) and di-tert-butyldiisopropyl phosphoramidite (2.2 ml) in (8 ml). (1.9 grams, 7 mmol, 1.5 molar equivalents) was added all at once under a nitrogen atmosphere. The mixture was stirred for 50 minutes at room temperature and then cooled to -40 ° C. A solution of 77% mCPBA (1.8 grams, 9.2 mol, 2 molar equivalents) in DCM (10 ml) was added rapidly while the reaction temperature was kept below 0 ° C. The solution was warmed to room temperature and stirred for 30 minutes before 10% aqueous NaHSO ₃ (20 ml) was added and the mixture was stirred for an additional 10-15 minutes. The mixture was then washed with 10% NaHSO ₃ , 5% NaHCO ₃ and brine and dried over sodium sulfate. The organic layer was evaporated and the residue was purified by flash chromatography using a gradient eluent of EtOAc / hexanes 10:90 to 30:70 to give impure product. Further purification by chromatography with isocratic CHCl ₃ gave the pure product in 35% yield.

Preparation of 3- (guanidinoethyl) benzyl phosphate hydrochloride: di-tert-butyl 3- (N, N′-bis-BOC-guanidinoethyl) benzyl phosphate (100 mg) was added to HCl in ether (5 ml). Dissolved in 1M solution and the solution was stirred at room temperature for 5-6 hours. The ether was then evaporated, water was added to the residue and the resulting solution was washed with ether. The aqueous solution was then lyophilized to give 40 mg (72%) of product.

H. Synthesis of 3,5-bisguanidinobenzyl phosphate (GSC-7):
GSC-7 was prepared as follows:
Preparation of 3,5-bis (N, N′-bis-BOC-guanidino) benzyl alcohol: 3,5-diaminobenzyl alcohol dihydrochloride (2.2 grams, 0.01 mol, 1.mol in dry DMF (30 ml)). 0 mole equivalent) to a solution of N, N′-bis (tert-butoxycarbonyl) -S-methylisothiourea (6.6 grams, 0.023 mole, 2.3 mole equivalent), mercury chloride (6.2 grams, 0.023 mol, 2.3 molar equivalents) and triethylamine (10 ml, 0.07 mol, 3 molar equivalents) were added and the reaction mixture was stirred at room temperature overnight (about 18 hours). Ethyl acetate was then added and the resulting suspension was filtered through Celite. The organic phase was washed with H ₂ O, saturated NH ₄ Cl and brine, dried over MgSO ₄ and the solvent was evaporated under reduced pressure. The crude product is purified by flash chromatography on silica gel (using a gradient eluent of 20% EtOAc / hexanes to 30% EtOAc / hexanes) to give 6.0 grams (96%) of product. It was.

Preparation of di-tert-butyl 3,5-bis (N, N′-bis-BOC-guanidino) benzyl phosphate: 1H-tetrazole solution (0.45M in acetonitrile, 20 ml, 0.009 mol, 3 molar equivalents) Was prepared from 3,5-bis (N, N′-bis-BOC-guanidino) benzyl alcohol (2 grams, 0.003 mol, 1 molar equivalent) and di-tert-butyldiisopropylphosphorymi in dry THF (5 ml). To a stirred solution of date (1.15 ml, 0.0036 mol, 1.2 molar equivalent) was added all at once under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 50-60 minutes and then cooled to −40 ° C. (by dry ice / acetonyl). A solution of 77% mCPBA (0.8 grams, 0.0045 mol, 1.5 molar equivalents) in DCM (5 ml) was added rapidly while the reaction temperature was kept below 0 ° C. The reaction mixture was then warmed to room temperature and stirred for 30 minutes. Then 10% NaHSO ₃ aqueous solution (10 ml) was added and the mixture was stirred for an additional 10-15 minutes. The mixture was extracted with ether (20-80 ml) and the ether phase was washed with 10% aqueous NaHSO ₃ solution and brine, dried over MgSO ₄ and filtered. The solvent was evaporated and the residue was chromatographed on a silica gel column (using a gradient eluent of EtOAc / hexane 10:90 to 30:70 mixture) to give impure product. Further purification by chromatography with isocratic CHCl ₃ gave the pure product in 40% yield.

Preparation of 3,5-bisguanidinobenzyl phosphate dihydrochloride: 1M solution of HCl in ether (35 ml) is di-tert-butyl 3,5-bis (N, N′-bis-BOC-guanidino) benzyl phosphorus. To the acid (0.45 grams, 0.57 mmol) was added and the resulting mixture was stirred at room temperature for 22-24 hours, during which time a white precipitate formed. The ether was then decanted, the precipitate was washed 3-4 times with dry ether, evaporated, and the residue was dried under high vacuum to give 0.13 grams (65%) of the final product.

Example 3
Activity assay method:
In Vitro Inhibition Assay: Purified recombinant rabbit GSK-3β (Eldar-Finkelman et al., 1996) was transformed into peptide substrate PGS-1 (YRRAAVPPSPSLSRHSSSPSQS (p) EDEEE) (SEQ ID NO: 1) or peptide substrate p9CREB (SEQ ID NO: 2) And phenyl phosphate, pyridoxal phosphate (P-5-P), GSC-1, GSC-2, GSC-3, GSC-4, GSC-5, GSC-6, GSC-7 or GSC-21 (structural formula Were incubated at the indicated concentrations. The reaction mixture was added with Tris 50 mM (pH 7.3), 10 mM MgAc, ³² P [γ-ATP] (100 μM), 0.01% β-mercaptoethanol and incubated at 30 ° C. for 10 minutes. The reaction was dropped onto cellulose phosphate paper (p81), washed with 100 mM phosphoric acid, and the radioactivity was counted (as described in Eldar-Finkelman et al., 1996).

  The effects of GSC-4, GSC-5 and GSC-7 (100 and 200 μM) on other protein kinases are similar to those described above and together with the reaction mixture containing histone H1 substrate (5 μg) CDK-2 (1 unit ) Was incubated. Reactions were boiled with SDS sample buffer, separated by gel electrophoresis, and radiographed.

  Activation of glycogen synthase by GSK-3 inhibitor: Activation of glycogen synthase acts as a good marker for inhibition of GSK-3. Our previous data showed that GSK-3 peptide inhibitors activate glycogen synthase. Therefore, activation of glycogen synthase by GSC-5 and GSC-7 was tested in C2C12 myotubes. C2C12 cells were treated with GSC-5 and GSC-7 at the indicated concentrations for 2.5 hours, after which the lysate supernatant was assayed for glycogen synthase activity. Glycogen synthase activity in cells treated with vehicle DMSO (0.1% DMSO) was normalized to 1 unit and was observed for glycogen synthase activity observed in cells treated with GSK-3 inhibitors. Values are expressed as fold stimulation for cells treated with vehicle 0.1% HCl.

Glucose uptake in isolated adipocytes: Mouse adipocytes were obtained from epididymal fat by digestion with 0.8 mg / ml collagenase (Worthington Biochemical) as previously described (Lawrence et al., 1977). Isolated from pad. The digested fat pad is passed through a nylon mesh and the cells contain 1% bovine serum albumin (fraction V, Boehringer Mannheim, Germany), 10 mM HEPES (pH = 7.3), 5 mM glucose and 200 mM adenosine. Washed 3 times with Krebs dicarbonate buffer (pH 7.4). Cells were incubated with GSC-4 and GSC-21 at the indicated concentrations for 2.5 hours, then 2-deoxy [ ³ H] glucose (0.5 μci / vial) was added for 10 minutes. The assay was stopped by centrifuging the cells through dinonyl phthalate (ICN, USA). ³ H was then quantified by a liquid scintillation analyzer (Packard). Nonspecific uptake of 2-deoxy- [ ³ H] glucose was determined by adding cytochalasin B (50 μM) 30 minutes before the addition of radioactive material.

result:
In vitro inhibition assay:
In a preliminary inhibition assay, the GSK-3 inhibitory activity of the known compounds phenyl phosphate, pyridoxal phosphate, GSC-1, GSC-2 and GSC-3 was tested as described above. The results presented in FIG. 5 show that all tested compounds exerted inhibitory activity against GSK-3, the phosphate derivative of pyridine, ie pyridoxal phosphate, and the phosphate derivative of GSC-3 phenyl ( It was shown to be more active than phenylphosphoric acid, GSC-1 and GSC-2).

  In further inhibition assays, GSK-3 inhibitory activity of GSC-1, GSC-2, GSC-3, GSC-4 and GSC-21 was tested. The activity of GSK-3 phosphorylating PGS-1 peptide substrates was measured in the presence of the indicated concentrations of these compounds. The results shown in FIG. 6 represent the percentage of GSK-3 activity compared to control incubations without inhibitor, and are the mean ± SEM of two independent experiments, each point assayed in triplicate.

  As shown in FIG. 6, the tested compounds were found to be very active in inhibiting GSK-3 activity (IC50 value of 1-5 mM), with GSC-3 and GSC-4 being the most active Compound. These results show that the presence of one or more nitrogen atoms in or adjacent to the ring (eg, directly attached to the ring atom) can be attributed to the newly designed small molecule's GSK-3 inhibitory activity. It may suggest that it is a feature that can affect (increase GSK-3 inhibitory activity).

  In still further inhibition assays, GSK-3 inhibitory activity of GSC-1, GSC-2, GSC-3, GSC-4, GSC-5, GSC-6 and GSC-7 was tested. The activity of GSK-3 that phosphorylates the p9CREB peptide substrate was measured in the presence of the indicated concentrations of these compounds. The results shown in FIGS. 7a and 7b represent the percentage of GSK-3 activity compared to control incubations without inhibitor, and are the mean ± SEM of two independent experiments, each point being assayed in triplicate. It was.

  As shown in FIGS. 7a-7b, the tested compounds were found to be very active in inhibiting GSK-3 activity (1-50 mM IC50 value), with GSC-4 and GSC-7 being It was the most active compound (IC50 = 0.5 Mm). These results indicate that one or more nitrogen atoms in or adjacent to the ring (eg, directly attached to the ring atom) can affect the GSK-3 inhibitory activity of the newly designed small molecule. We further support the suggestion that it is a feature that can be given (can increase GSK-3 inhibitory activity). These results further support the suggestion that the presence of the guanidine moiety is a feature that can affect the GSK-3 inhibitory activity of newly designed small molecules (can increase GSK-3 inhibitory activity).

  The kinetic properties of the inhibitor were studied by measuring the initial rate as a function of substrate concentration at several inhibitor concentrations. The Lineweaver-Burk plot of GSK-3 inhibition by these inhibitors confirmed the hypothesis that they are substrate competitive inhibitors (data not shown). FIG. 8 represents a Lineweaver-Burk plot of GSK-3 inhibition by gsc-7 as an exemplary inhibitor, represented by phosphate group incorporation (CPM) into the p9CREB peptide substrate. The results show one representative experiment from three. Each point is an average of two samples.

The selectivity of the novel compounds for GSK-3 was measured by evaluating the inhibition of CDK-2, a kinase closely related to GSK-3. Therefore, CDK-2 activity was assayed in the presence of ³² P [γ-ATP] and histone H1 as a substrate. GSC-4, GSC-5 and GSC-7 were added at a final concentration of 2 mM. The results are presented in FIG. 9, clearly showing that no inhibition of histone H1 phosphorylation was observed, thus suggesting a high selectivity of the compound for GSK-3.

  Activation of glycogen synthase by GSK-3 inhibitor: Activation of glycogen enzyme activity in C2C12 cells treated with GSC-5 and GSC-7 was assayed as described above. Glycogen synthase activity in cells treated with vehicle DMSO (0.1% DMSO) is normalized to 1 unit and is a GSK-3 inhibitor (GS4 (hollow circle), GS6 (solid circle)) The values for glycogen synthase activity observed in cells treated with 1 are expressed as fold stimuli for cells treated with vehicle 0.1% HCl.

  The results are shown in FIG. 10 and clearly show that both GSC-5 (hollow circle) and GSC-7 (solid circle) activated glycogen synthase 1.5 and 1.8 times, respectively. .

Glucose uptake:
The ability of the newly designed compounds GSC-4 and GSC-21 to promote glucose uptake was tested in mouse primordial adipocytes as described above. The relative [ ³ H] 2-deoxyglucose uptake observed in untreated adipocytes was normalized to 1 unit and [ ³ H] 2 in adipocytes treated with GSC-4 or GSC-21 -Values obtained for deoxyglucose are expressed as fold activation against cells treated with peptide control, and are the mean ± SEM of 6 independent experiments, each point assayed in triplicate.

  The results presented in FIG. 11a (GSC-4) and FIG. 11b (GSC-21) show that GSC-21 increased glucose uptake by 2.5 and 1.7 fold at concentrations of 5 μM and 0.5 μM, respectively. Indicates. A somewhat reduced effect was observed in the presence of GSC-4, which increased glucose uptake approximately 2-fold at a concentration of 10 μM. Figures 11a and 1! As further shown in b, the activation of glucose uptake achieved by GSC-4 and GSC-21 was comparable to that achieved in the presence of 100 nM insulin. These results indicate that these newly designed compounds can act as insulin mimetics in increasing insulin signaling and in treating diseases mediated by GSK-3 such as diabetes. Prove that you have

Example 4
Stimulation of GSC inhibitor interaction with the GSK-3 catalytic domain Simulated annealing is a molecular modeling method used to find stable conformations of proteins. The study experimented with the interaction of the above-described newly designed GSK-3 inhibitors (also referred to herein as GSC molecules) with the GSK-3 catalytic domain, by Ter Haar et al. (2001). Based on protein crystal data for GSK-3 as taught.

  Simulated annealing uses repeated heating and cooling of the system to find the best energy minimum of the system. In this study, an additional parameter, “protein-ligand binding energy”, was added to the consideration of selecting a new starting point at each interval of heating.

experimental method:
Complex preparation: The structure of GSK subunit B as taught by Ter Haar et al. (2001) was used as the starting model for the calculations. Hydrogen atoms were added to the structure and incomplete residues were fixed. Since the phosphate group is located between Arg96 and Arg180 according to the structure described in Ter Haar et al. (2001), the phosphate groups of the small (GSC) molecules tested were manually changed between Arg96 and Arg180. Inserted between. Four conformers with different orientations of each small molecule were generated manually as a starting point while maintaining the phosphate group position as a common foundation. Calculations were performed independently of each other. The system was then relaxed by minimization, in which ligands, hydrogen atoms, side chains and water molecules within a radius of 15 mm from the phosphate group atoms were freed.

Simulated annealing: Simulated annealing included the following steps:
Equilibration of 1000 steps to 1000 ° K;
7500 step simulation at 1000 ° K (wherein every 500 steps coordinates are collected for structural diversity, which is done a total of 15 times);
Energy minimization of the 15 structures;
Calculation of the interaction between the minimized enzyme and the ligand structure described above;
Selecting the best interacting structure from the 15 minimized structures to repeat the dynamic simulation described above; and repeating the dynamic simulation, minimization, calculation and selection 15 times.

  During minimization and dynamics, water molecules and side chains within a radius of 15 cm from hydrogen atoms, substrate analogs and phosphate group atoms were released.

  The distal part of the enzyme (greater than 15 cm) and the protein backbone were immobilized.

  Cut-off: 9.5 A distance dependent; each step was 1 fs.

  The calculation is performed in Accelrys inc. The program discover3 / Insight II.

  The program Discover3 was used for minimization and dynamics. The force field used to calculate the energy of the system was CVFF.

  Three parallel methods were selected to generate a good repertoire of interactions between GSK-3 and its ligand. Numerous conformational starting points and molecular simulations at high temperatures are well known methods used. In addition, a simulated annealing process that emphasizes the enzyme-ligand binding energy during the simulation was used.

  The combined approach has generated many conformations. For each starting point, the conformation with the lowest binding energy was evaluated in the last (15th) simulation cycle. The binding energies were compared and the best one was selected. In some cases, more than one conformation was analyzed for each ligand.

  During the simulation, the ligand was released and moved. In all simulations, the phosphate group interaction between arginine residues 180 and 96 did not brake. In most simulations, lysine 205 did not maintain interaction with the phosphate group and formed a salt bridge with glutamic acid 211.

result:
The data obtained in this study is shown in FIGS. These figures show the interaction between the best conformation of the analyzed compound (inhibitor) and the amino acids in the catalytic domain of GSK-3, as determined by the simulated annealing described above. The dominant interaction between the analyzed compound and the catalytic domain is the electrostatic interaction between the amine or guanidine moiety of the inhibitor and the side chain acid moiety of amino acids (eg glutamate and aspartate) or the hydroxyl group of tyrosine. And / or hydrogen bonding interactions, and aromatic and / or hydrophobic interactions between the aromatic portion of the inhibitor and the aromatic or hydrocarbon side chain of an amino acid (eg, phenylalanine, tyrosine and isoleucine). Taking into account the insertion of the phosphate moiety between Arg96 and Arg180, it was found that most of these interactions are with Ile217, Tyr215, Phe67, Asp181, Glu97, Asp90, Asp181 and Glu200. Phe67 is an important binding moiety in the binding site of the enzyme, which appears to be a door that closes over the binding site.

  Table 4 below shows the distances between the various atoms of the analyzed inhibitors and the various amino acid residues of the enzyme that interact with these atoms. The number (indicated by Å) is the short distance between the inhibitor and the residue.

  GSC-4 interaction with GSK-3: As shown in FIG. 12, the best conformation of GSC-4 (3-guanidinyl benzyl phosphate) is the hydrophobic interaction with Ile217 and Tyr216 and An electrostatic interaction with Asp181 and optionally a hydrogen bond with the hydroxyl group of Tyr215 was demonstrated.

  Interaction of GSC-5 with GSK-3: As shown in FIG. 13, the best conformation of this compound showed a low interaction with acidic groups Glu97, Asp90 or Asp181. Significant interaction with Phe67 was observed.

  Interaction of GSC-7 with GSK-3: As shown in FIG. 14, since the arm of the guanide group bonded to the aromatic core is too short, most of the interaction of this compound is aromatic with Phe67. And the interaction with acidic groups (eg Asp90 and Glu97) was weak, especially in the conformation with the lowest binding energy (3.48 to Asp90 and 4.92 from Glu97).

  Interaction of GSC-6 with GSK-3: As shown in FIG. 15, the guanidine group is bonded to the ring through an ethylene spacer as compared to GSC-4 in which the guanidine group is directly bonded to the aromatic ring. In GSC-6, the interaction of the guanidine group with an acidic group (for example, Asp181) is firm. Aromatic interactions with Phe67 and Tyr216 are also observed.

  Interaction of GSC-8 with GSK-3: As shown in FIG. 16, GSC-8 is perfectly compatible with the binding site. The best conformation is one guanide arm pointing to Asp90 (1.71 Å) and the other arm pointing to Glu97 (1.88 Å), and Phe67 (3.6 Å), Arg96 (2.06 Å) and Arg180 (2 .63Å) with additional interaction. Complex interactions have good binding energy. Since two side-by-side guanidino groups are attached to the aromatic ring via a methylene spacer, these groups can interact simultaneously with Asp90 and Glu97. It should be noted that GSC-8 has a better interaction than GSC-7 where the guanidine group is directly attached to the ring.

  Interaction of GSC-9 with GSK-3: As shown in FIG. 17, GSC-9 is perfectly matched to the binding site and has an increased number of interactions. The best conformation includes one guanide arm pointing to Asp90 (3.43 Å) and the other arm pointing to Glu97 (2.38 Å). Although this interaction is not robust, the guanide group interacts with additional residues such as Glu200, Glu89 and the skeleton of Leu88, thus strengthening the bond. Phe67 does not interact strongly, but appears to close over the binding site.

  It should be noted that other conformations of this kind (Asp90, Glu97) have good energy and have a stronger interaction with the carboxyl group.

  Other interactions with the GSK-3 binding site: In addition to the interaction of the engineered GSC molecule with the catalytic binding site of GSK-3, this study will show which interactions are optimally bound to this site It had the role of determining what was required. Overall, it was found that the desired inhibitor would interact with Phe67, Glu97, Glu89 and Asn95. These findings further support the unique features of GSK-3. Although Glu97 is conserved among protein kinases and is important in catalytic activity, the roles of Phe67, Gln89 and Asn95 are novel and unique to GSK-3.

  Therefore, additional work has been done to design new compounds that can interact with these residues and thus lead to specific inhibition of GSK-3.

Example 5
Chemical Synthesis of Multiaryl / Heteroaryl GSK-3 Inhibitors (MP Small Molecules) Based on the molecular modeling studies described above, we designed and successfully prepared a new family of GSK-3 inhibitors. There, the multiaryl and / or heteroaryl containing skeleton acts as a spacer between the phosphate group moiety and the amino containing moiety (eg, guanidine).

  General procedures and data for each step in the synthetic pathway of representative compounds of this family are defined below. The chemical structure of the compound is represented in FIG.

Experimental data:
Proton, carbon and phosphorus nuclear magnetic resonance spectra were obtained on a Bruker AVANCE 200 or 400 MHz spectrometer and reported in ppm (δ). Unless otherwise noted, tetramethylsilane (TMS) was used as an internal standard for proton spectra; phosphoric acid was used as an internal standard for phosphorus spectra; solvent peaks were reference peaks for carbon and fluorine spectra Used as.

  Mass spectra were obtained on a Micromass VG autospec M250.

Ｎ，Ｎ′−ビス（ｔｅｒｔ−ブトキシカルボニル）−Ｓ−メチルイソチオ尿素（１．３２グラム、４．４ｍｍｏｌ、１．１モル当量）、塩化水銀（１．２２グラム、４．４ｍｍｏｌ、１．１モル当量）、およびトリエチルアミン（１．７２ｍｌ、１２．０ｍｍｏｌ、３．０モル当量）は、乾燥ＤＭＦ（１５ｍｌ）中のヨードアニリン（０．８８グラム、４．０ｍｍｏｌ、１．０モル当量）に添加され、生じた混合物は室温で５時間撹拌された。次に混合物は酢酸エチル（６０ｍｌ）に注がれ、セライトを通して濾過された。有機層は水（５０ｍｌ）、飽和ＮＨ_４Ｃｌ水溶液（５０ｍｌ）および塩水で洗浄され、ＭｇＳＯ_４で乾燥された。溶媒は減圧下で蒸発された。粗化合物は十分純粋であり、さらなる精製なしに使用された。 Preparation of N- (iodophenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine:
The general procedure for the preparation of N- (iodophenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine is represented by Formula 10 below.

N, N'-bis (tert-butoxycarbonyl) -S-methylisothiourea (1.32 grams, 4.4 mmol, 1.1 molar equivalent), mercury chloride (1.22 grams, 4.4 mmol, 1.1 mole) Equivalents), and triethylamine (1.72 ml, 12.0 mmol, 3.0 molar equivalents) were added to iodoaniline (0.88 grams, 4.0 mmol, 1.0 molar equivalents) in dry DMF (15 ml). The resulting mixture was stirred at room temperature for 5 hours. The mixture was then poured into ethyl acetate (60 ml) and filtered through celite. The organic layer was washed with water (50 ml), saturated aqueous NH ₄ Cl solution (50 ml) and brine and dried over MgSO ₄ . The solvent was evaporated under reduced pressure. The crude compound was pure enough and was used without further purification.

Ｎ−（２−ヨードフェニル），Ｎ′，Ｎ″−ビス（ｔｅｒｔ−ブトキシカルボニル）グアニジン：
この化合物は、反応混合物の撹拌を４日間続けることのみによって上述のようにして得られた。
収率：８５％

Ｎ−（４−ヨードフェニル），Ｎ′，Ｎ″−ビス（ｔｅｒｔ−ブトキシカルボニル）グアニジン：
収率：８５％

Using this general procedure, the following compounds were prepared:
N- (3-iodophenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine:
Yield: 85%

N- (2-iodophenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine:
This compound was obtained as described above only by stirring the reaction mixture for 4 days.
Yield: 85%

N- (4-iodophenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine:
Yield: 85%

Preparation of N- (TMS-ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine:
The general procedure for the preparation of N- (TMS-ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine is represented by Formula 11 below.

Pd (PPh ₃ ) ₂ Cl ₂ (16 mg, 0.02 mmol, 0.02 molar equivalent), CuI (8 mg, 8 mL, dimethylamine (1.8 ml, 14.8 mmol, 16 equivalents) and dimethylformamide (0.6 ml) 0.05 mmol, 0.04 molar equivalents), trimethylsilylacetylene (0.20 ml, 1.2 mmol, 1.1 molar equivalents) solution of N- (iodophenyl), N ′, N ″ -bis (tert-butoxy Carbonyl) guanidine (0.50 grams, 1.1 mmol, 1 molar equivalent) was added and the resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere, after which the reaction mixture was poured into water (8 ml). And extracted three times with diethyl ether (3 × 8 ml) The combined organic layers were washed with water and brine (8 ml) and the aqueous phase was diethyl . Ether are re-extracted twice with (2 × 8 ml), combined organic layers were dried over MgSO _4, concentrated under reduced pressure the residue (hexane and eighty past eight p.m. ethyl acetate: gradient eluant hexane mixture Purified by flash chromatography on a silica gel column.

Ｎ−（２−ＴＭＳ−エチニルフェニル），Ｎ′，Ｎ″−ビス（ｔｅｒｔ−ブトキシカルボニル）グアニジン：
この化合物は、撹拌を７日間続けることのみによって上述のようにして得られた。
収率：８０％

Ｎ−（４−ＴＭＳ−エチニルフェニル），Ｎ′，Ｎ″−ビス（ｔｅｒｔ−ブトキシカルボニル）グアニジン：
収率：８０％

Using this general procedure, the following compounds were prepared:
N- (3-TMS-ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine:
Yield: 80%

N- (2-TMS-ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine:
This compound was obtained as described above only by continuing stirring for 7 days.
Yield: 80%

N- (4-TMS-ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine:
Yield: 80%

Preparation of N- (ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine:
The general procedure for the preparation of N- (ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine is represented by Formula 12 below.

A solution of N- (TMS-ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine (97 mg, 0.24 mmol, 1 molar equivalent) in THF (3 ml) was −5 under nitrogen atmosphere. Stirred (with ice: salt bath) at 0 C. TBAF (1.0 M in THF, 0.4 ml, 0.4 mmol, 1.6 molar equivalents) was added dropwise over 7 minutes and the mixture was allowed to warm to room temperature. The solvent was evaporated under reduced pressure and the product was extracted with ethyl acetate (6 ml) The organic phase was washed 3 times with water (3 × 15 ml) and washed with brine. , Dried over MgSO ₄ and evaporated under reduced pressure The crude product was pure enough and used without further purification.

Ｎ−（２−エチニルフェニル），Ｎ′，Ｎ″−ビス（ｔｅｒｔ−ブトキシカルボニル）グアニジン：
収率：８５％

Ｎ−（４−エチニルフェニル），Ｎ′，Ｎ″−ビス（ｔｅｒｔ−ブトキシカルボニル）グアニジン：
収率：８５％

Using this general procedure, the following compounds were prepared:
N- (3-ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine:
Yield: 85%

N- (2-ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine:
Yield: 85%

N- (4-ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine:
Yield: 85%

Preparation of azidobenzyl alcohol:
The general procedure for the preparation of azidobenzyl alcohol is represented by formula 13 below.

A 50 ml round bottom flask was charged with a stir bar, 25 ml of 2N HCl and aminobenzyl alcohol (2.15 grams, 17.5 mmol, 1 molar equivalent). The solvent was cooled to −5 ° C. in a salt ice bath. An ice-cold solution of sodium nitride (1.45 grams, 21 mmol, 1.2 molar equivalents) in 5 ml water was added slowly over 5 minutes so that the temperature of the reaction solution did not rise above -3 ° C. After 5 minutes, 125 mg of urea was added to destroy excess nitrous acid. The resulting diazonium salt solution was then added to a stirred ice of sodium azide (2.28 grams, 35 mmol, 2 molar equivalents) and sodium acetate (4.20 grams, 51 mmol, 3 molar equivalents) in 25 ml water. Added to the cold solution for 5 minutes. The mixture was stirred at 0 ° C. for 2 hours and the dark oily product was extracted with diethyl ether (2 × 50 ml). The solution containing ether was washed with 1N NaOH (2 × 50 ml) and water (2 × 50 ml), dried over MgSO ₄ and evaporated to dryness. The resulting compound was sufficiently pure and was used without further purification.

４−アジドベンジルアルコール：
収率：９０％

Using this general procedure, the following compounds were prepared:
3-azidobenzyl alcohol:
Yield: 83%

4-azidobenzyl alcohol:
Yield: 90%

Preparation of di-tert-butyl, azidobenzyl phosphate:
The general procedure for the preparation of di-tert-butyl, azidobenzyl phosphate is represented by formula 14 below.

1H-tetrazole solution (0.45M in acetonitrile, 44.7 ml, 20.2 mmol, 3 molar equivalent) was added to azidobenzyl alcohol (1.0 gram, 6.7 mmol, 1 molar equivalent) in dry THF (7 ml). And di-tert-butyldiisopropyl phosphoramidite (3.17 ml, 10.1 mmol, 1.3 molar equivalents) were added in one portion. The mixture was stirred at 20 ° C. for 30 minutes, after which the mixture was cooled to −40 ° C. (by dry ice / acetonitrile). A solution of 85% mCPBA (1.45 grams, 10.05 mmol, 1.5 eq in 2.4 ml DCM) in DCM (9 ml) was added rapidly while the reaction temperature was kept below 0 ° C. . The reaction mixture was warmed to room temperature and stirred for 20 minutes, after which 10% aqueous NaHSO ₃ (24 ml) was added and the mixture was stirred for an additional 10 minutes. The mixture was then extracted with ether (120 ml) and the aqueous phase was discarded. The ether phase was washed with 10% NaHSO ₃ solution (2 × 48 ml) and saturated aqueous NaHCO ₃ (2 × 48ml), dried over sodium sulfate and filtered. The solvent was evaporated and the residue was chromatographed on a silica gel column (using a gradient eluent of hexane to EtOAc / hexane 40:60 mixture) to give the product.

ジ−ｔｅｒｔ−ブチル、４−アジドベンジルリン酸：
収率：１２％

Using this general procedure, the following compounds were prepared:
Di-tert-butyl, 3-azidobenzyl phosphate:
Yield: 10%

Di-tert-butyl, 4-azidobenzyl phosphate:
Yield: 12%

Dipolar cycloaddition of azidobenzyl phosphate and N- (ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine:
The general procedure for the dipolar cycloaddition of azidobenzyl phosphate and N- (ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine is represented by Formula 15 below.

CuSO ₄ (0.02 grams, 0.125 mmol, 0.5 molar equivalent) and Cu wire were prepared from N- (ethynylphenyl), N ′, N ″ -bis (tert-butoxycarbonyl) guanidine in DMF (5 ml). (0.07 grams, 0.212 mmol, 1 molar equivalent) and a solution of di-tert-butyl, azidobenzyl phosphate (0.07 grams, 0.205 mmol, 1 molar equivalent) and the resulting mixture is nitrogen After stirring overnight under atmosphere, the solution was extracted with ethyl acetate (10 ml), the organic layer was washed three times with brine, and the aqueous layer was re-extracted twice with ethyl acetate (2 × 10 ml). The combined organic phases were concentrated under reduced pressure and the residue was chromatographed on silica gel (using a gradient eluent of hexane to 60:40 ethyl acetate / hexane mixture). Purified by chromatography to give the protected addition product.

保護された３−（４−（２−グアニジノフェニル）−１，２，３−トリアゾール−１−イル）ベンジルリン酸塩酸塩（保護された２−Ｇｕａ−３−Ｐｈｏｓ）：
この化合物は、反応混合物が４日間撹拌されたことを除いては上述のようにして得られた。
収率：５０％

保護された３−（４−（４−グアニジノフェニル）−１，２，３−トリアゾール−１−イル）ベンジルリン酸塩酸塩（保護された４−Ｇｕａ−３−Ｐｈｏｓ）：
収率：７３％

保護された４−（４−（３−グアニジノフェニル）−１，２，３−トリアゾール−１−イル）ベンジルリン酸塩酸塩（保護された３−Ｇｕａ−４−Ｐｈｏｓ）：
収率：９０％

保護された４−（４−（２−グアニジノフェニル）−１，２，３−トリアゾール−１−イル）ベンジルリン酸塩酸塩（保護された２−Ｇｕａ−４−Ｐｈｏｓ）：
この化合物は、反応混合物が４８時間撹拌されたが反応はなお完了しなかったことを除いては上述のようにして得られた。
収率：４３％

保護された４−（４−（４−グアニジノフェニル）−１，２，３−トリアゾール−１−イル）ベンジルリン酸塩酸塩（保護された４−Ｇｕａ−４−Ｐｈｏｓ）：
収率：７５％

Using this general procedure, the following compounds were prepared:
Protected 3- (4- (3-guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate (protected 3-Gua-3-Phos):
Yield: 85%

Protected 3- (4- (2-guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate (protected 2-Gua-3-Phos):
This compound was obtained as described above except that the reaction mixture was stirred for 4 days.
Yield: 50%

Protected 3- (4- (4-guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate (protected 4-Gua-3-Phos):
Yield: 73%

Protected 4- (4- (3-guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate (protected 3-Gua-4-Phos):
Yield: 90%

Protected 4- (4- (2-guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate (protected 2-Gua-4-Phos):
This compound was obtained as described above except that the reaction mixture was stirred for 48 hours but the reaction was still not complete.
Yield: 43%

Protected 4- (4- (4-guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate (protected 4-Gua-4-Phos):
Yield: 75%

Preparation of MP-1 to MP-6 (deprotection of t-Bu and BOC groups):
The general procedure for deprotecting the t-Bu and BOC groups to give the final product is represented by Equation 16 below.

  A solution of HCl (4M in dioxane, 2 ml, 8 mmol, 2.6 molar equivalents) and dioxane (6 ml) was added to the protected compound at 20 ° C. and the resulting mixture was stirred overnight. Dioxane was then evaporated under reduced pressure to give the final product.

３−（４−（２−グアニジノフェニル）−１，２，３−トリアゾール−１−イル）ベンジルリン酸塩酸塩（２−Ｇｕａ−３−Ｐｈｏｓ、ＭＰ３）：
収率：７２％

３−（４−（４−グアニジノフェニル）−１，２，３−トリアゾール−１−イル）ベンジルリン酸塩酸塩（４−Ｇｕａ−３−Ｐｈｏｓ、ＭＰ４）：
収率：７２％

４−（４−（３−グアニジノフェニル）−１，２，３−トリアゾール−１−イル）ベンジルリン酸塩酸塩（３−Ｇｕａ−４−Ｐｈｏｓ、ＭＰ５）：
収率：７５％

４−（４−（２−グアニジノフェニル）−１，２，３−トリアゾール−１−イル）ベンジルリン酸塩酸塩（２−Ｇｕａ−４−Ｐｈｏｓ、ＭＰ６）：
この化合物は、混合が２２時間続けられたことを除いては上述のようにして得られた。
収率：７６％

４−（４−（４−グアニジノフェニル）−１，２，３−トリアゾール−１−イル）ベンジルリン酸塩酸塩（４−Ｇｕａ−４−Ｐｈｏｓ、ＭＰ２）：
収率：７５％

Using this general procedure, the following compounds were prepared (see Figure 18 for the chemical structure of the compounds):
3- (4- (3-Guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate hydrochloride (3-Gua-3-Phos, MP1):
Yield: 70%

3- (4- (2-Guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate hydrochloride (2-Gua-3-Phos, MP3):
Yield: 72%

3- (4- (4-Guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate hydrochloride (4-Gua-3-Phos, MP4):
Yield: 72%

4- (4- (3-Guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate hydrochloride (3-Gua-4-Phos, MP5):
Yield: 75%

4- (4- (2-Guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate hydrochloride (2-Gua-4-Phos, MP6):
This compound was obtained as described above except that mixing was continued for 22 hours.
Yield: 76%

4- (4- (4-Guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate hydrochloride (4-Gua-4-Phos, MP2):
Yield: 75%

Example 6
GSK-3 inhibition by MP molecules The ability of GSK-3 to phosphorylate p9CREB peptide substrate was measured in the presence of the indicated concentrations of MP molecules using the protocol described above in Example 3. The results shown in FIG. 19 represent the percentage of GSK-3 activity in control cultures where the inhibitor is omitted. Results are the mean ± SEM of two independent experiments, with each point assayed twice.

  As shown in FIG. 19, MP-1 and MP-4 were found to have the IC50 value of about 1 mM and to show the best inhibitory activity.

  It will be appreciated that certain features of the invention described in separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, the various features of the invention described in a single embodiment for the sake of brevity can also be provided separately or in appropriate subcombinations.

  While the invention has been described in terms of specific embodiments thereof, it will be apparent to those skilled in the art that there are many alternatives, modifications and variations. Accordingly, the present invention is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, and patent applications cited in this application are hereby incorporated by reference in their entirety as if each individual publication, patent, and patent application were specifically and individually cited. To do. Furthermore, citation or confirmation in this application should not be considered as a confession that it can be used as prior art to the present invention.

FIGS. 1a-b represent computer images of the 3D structure of peptides p9CREB (FIG. 1a) and CREB (FIG. 1b) as obtained by 2D ¹ H-NMR studies. FIG. 2 is an image showing the electrostatic distribution of p9CREB peptide based on the 3D structure of the peptide obtained by 2D ¹ H-NMR studies. FIG. 3a shows the chemical structure of phenyl phosphate, pyridoxal phosphate (P-5-P), GSC-1, GSC-2, GSC-3 and the chemical structures of the new compounds GSC-4, GSC-5 and GSC-21. Represents. FIG. 3b shows the chemical structures of phenyl phosphate, pyridoxal phosphate (P-5-P), GSC-1, GSC-2, GSC-3 and the new compounds GSC-6, GSC-7, GSC-8 and GSC- 9 represents the chemical structure. FIG. 4a represents the ESI-MS of 3,5-bis (2-aminoethyl) benzyl phosphate (GSC-21). FIG. 4b represents the HPLC chromatogram of 3,5-bis (2-aminoethyl) benzyl phosphate (GSC-21). FIG. 6 represents a comparative plot showing GSK-3 inhibitory activity of phenylphosphate, GSC-1, GSC-2, GSC-3 and pyridoxal phosphate (P-5-P) in an in vitro inhibition assay using PGS-1 peptide substrates . FIG. 4 represents a comparative plot showing GSK-3 inhibitory activity of GSC-1, GSC-2, GSC-3, GSC-4 and GSC-21 in an in vitro inhibition assay. GSC-1, GSC-2, GSC-3 and GSC-4 in an in vitro inhibition assay using p9CREB peptide substrate (FIG. 7a, black circle indicates GSC-2, blank circle indicates GSC-1, black The triangles indicate GSC-3, the black squares indicate GSC-4) and GSC-5, GSC-6 and GSC-7 (FIG. 7b, the squares indicate GSC-5 and the triangles indicate GSC-6) , Circles indicate GSK-3 inhibitory activity of GSC-7). Represents a Lineweaver-Burk plot showing inhibition of GSK-3 by GSC-7 at the indicated concentrations, represented by incorporation of phosphate groups into p9CREB peptide substrate (CPM), where GSC-7 is competitively specific That it is a chemical inhibitor. ^{32 is} a gel electrophoresis image of CDK-2 kinase activity assayed in the presence of ³² P [γ-ATP] and histone H1 as a substrate, showing the inhibitory activity of GSC-4, GSC-5 and GSC-7. Indicates non-existence. Demonstrating the effect of GSC-5 (hollow circle) and GSC-7 (solid circle) on glycogen synthesis activity in C2C12 cells, shown as fold stimulation over cells treated with vehicle 0.1% HCl Represents a plot. FIG. 11a-b shows [ ³ H] 2-deoxy in cells treated with GSC-4 and GSC-21 as fold activation over cells treated with peptide control (normalized to 1 unit). 12 is a bar graph showing the effect of GSC-21 (FIG. 11b) and GSC-4 (FIG. 11a) on glucose uptake in mouse adipocytes, represented by glucose uptake. Fig. 4 represents a calculated simulation of the interaction between GSC-4 and GSK-3. Fig. 4 represents a calculated simulation of the interaction between GSC-5 and GSK-3. Fig. 4 represents a calculated simulation of the interaction between GSC-7 and GSK-3. Fig. 4 represents a calculated simulation of the interaction between GSC-6 and GSK-3. Fig. 4 represents a calculated simulation of the interaction between GSC-8 and GSK-3. Fig. 4 represents a calculated simulation of the interaction between GSC-9 and GSK-3. 1 represents the chemical structures of newly designed GSK-3 inhibitors, MP-1, MP-2, MP-3, MP-4, MP-5 and MP-6. FIG. 6 represents a comparative plot showing GSK-3 inhibitory activity of MP-1, MP-2, MP-3, MP-4, MP-5 and MP-6 in an in vitro inhibition assay using p9CREB peptide substrate.

SEQ ID NO: 1 is the GSK-3 recognition motif consensus sequence.
SEQ ID NOs: 2 to 4 are synthetic peptide sequences.

Claims (9)

And a negatively charged phosphate group, a compound containing at least one guanidino group covalently bonded to the charged phosphate groups negatively via a spacer, the spacer, the phosphate group and GSK-3 catalyst To allow at least one interaction between the first binding site in the domain and at least one interaction between the guanidino group and a second binding site in the catalytic domain of GSK-3. A compound having a selected length, structure and flexibility, whereby the compound is capable of inhibiting the catalytic activity of GSK-3 and having the following formula :

Wherein the phosphate group and the guanidino group are attached at any position on their respective phenyl rings.   3- (4- (3-guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate, 4- (4- (4-guanidinophenyl) -1,2,3-triazole- 1-yl) benzyl phosphate, 3- (4- (2-guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate, 3- (4- (4-guanidinophenyl) ) -1,2,3-triazol-1-yl) benzyl phosphate, 4- (4- (3-guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate, 2. The compound of claim 1 selected from the group consisting of and 4- (4- (2-guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate hydrochloride.   The compound according to claim 1, which is 3- (4- (3-guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate hydrochloride.   The compound according to claim 1, which is 3- (4- (4-guanidinophenyl) -1,2,3-triazol-1-yl) benzyl phosphate hydrochloride. A pharmaceutical composition comprising as an active ingredient a compound according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier. 6. A pharmaceutical composition according to claim 5 , which is packaged in a packaging material and identified by the surface of the packaging material or type in it for use in the treatment of biological conditions associated with GSK-3 activity. object. The pharmaceutical composition according to claim 5 , further comprising at least one further active ingredient capable of altering the activity of GSK-3. Wherein the biological condition is described obesity, non-insulin dependent diabetes mellitus, insulin-dependent conditions, in claim 6 which is selected from the group consisting of affective disorders, neurodegenerative diseases or disorders, and psychotic diseases or disorders Pharmaceutical composition. 6. A pharmaceutical composition according to claim 5 , which is packaged in a packaging material and identified by the surface of the packaging material or type in it for use in enhancing insulin signaling.

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