EP1824468A2 - Inhibiteurs de glycogene synthase kinase-3 - Google Patents

Inhibiteurs de glycogene synthase kinase-3

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Publication number
EP1824468A2
EP1824468A2 EP05809234A EP05809234A EP1824468A2 EP 1824468 A2 EP1824468 A2 EP 1824468A2 EP 05809234 A EP05809234 A EP 05809234A EP 05809234 A EP05809234 A EP 05809234A EP 1824468 A2 EP1824468 A2 EP 1824468A2
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Prior art keywords
gsk
compound
atom
group
activity
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German (de)
English (en)
Inventor
Hagit Eldar-Finkelman
Moshe Portnoy
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Tel Aviv University Future Technology Development LP
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Tel Aviv University Future Technology Development LP
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/40—Esters thereof
    • C07F9/4003—Esters thereof the acid moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/4056—Esters of arylalkanephosphonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4192—1,2,3-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/12—Antidiuretics, e.g. drugs for diabetes insipidus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3882—Arylalkanephosphonic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6515—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having three nitrogen atoms as the only ring hetero atoms
    • C07F9/6518—Five-membered rings

Definitions

  • the present invention relates to novel compounds for inhibiting glycogen synthase kinase-3 (GSK-3) and their use in regulating biological conditions mediated by GSK-3 activity and, more particularly, to the use of these compounds in the treatment of biological conditions such as type II diabetes, neurodegenerative disorders and diseases and affective disorders.
  • GSK-3 glycogen synthase kinase-3
  • Protein kinases the enzymes that phosphorylate protein substrates, are key players in the signaling of extracellular events to the cytoplasm and the nucleus, and take part in practically any event relating to the life and death of cells, including mitosis, differentiation and apoptosis. As such, protein kinases have long been favorable drug targets. However, since the activity of protein kinases is crucial to the well being of the cell, while their inhibition oftentimes leads to cell death, their use as drug targets is limited. Although cell death is a desirable effect for anticancer drugs, it is a major drawback for most other therapeutics.
  • Glycogen synthase kinase-3 (GSK-3), a member of the protein kinases family, is a cytoplasmic proline-directed serine-threonine kinase that is involved in insulin signaling and metabolic regulation, as well as in Wnt signaling and the scheme of cell fate during embryonic development.
  • GSK-3 ⁇ and GSK-3 ⁇ Two similar isoforms of the enzyme, termed GSK-3 ⁇ and GSK-3 ⁇ , have been identified.
  • GSK-3 has long been considered as a favorable drug target among the protein kinase family since unlike other protein kinases, which are typically activated by signaling pathways, GSK-3 is normally activated in resting cells, and its activity is attenuated by the activation of certain signaling pathways such as those generated by the binding of insulin to its cell-surface receptor. Activation of the insulin receptor leads to the activation of protein kinase B (PKB, also called Akt), which in turn phosphorylates GSK-3, thereby inactivating it. The inhibition of GSK-3 presumably leads to the activation of glycogen synthesis.
  • PKA protein kinase B
  • the intricate insulin-signaling pathway is further complicated by negative-feedback regulation of insulin signaling by GSK-3 itself, which phosphorylates insulin-receptor substrate- 1 on serine residues (Eldar- Finkelman et al, 1997). Therefore, synthetic GSK-3 inhibitors might mimic the action of certain hormones and growth factors, such as insulin, which use the GSK-3 pathway. In certain pathological situations, this scheme might permit the bypassing of a defective receptor, or another faulty component of the signaling machinery, such that the biological signal will take effect even when some upstream players of the signaling cascade are at fault, as in non-insulin-dependent type II diabetes.
  • glycogen catabolism in cells is a critical biological function that involves a complex array of signaling elements, including the hormone insulin.
  • insulin exerts its regulatory effect by increasing the synthesis of glycogen by glycogen synthase (GS).
  • a key event in insulin action is the phosphorylation of insulin receptor substrates (IRS-I, IRS-2) on multiple-tyrosine residues, which results in simultaneous activation of several signaling components, including PI3 kinase (Myers et al, 1992)).
  • insulin receptor substrates IRS-I, IRS-2
  • PI3 kinase Myers et al, 1992
  • the activity of glycogen synthase is suppressed by its phosphorylation.
  • There is a marked decrease in glycogen synthase activity and in glycogen levels in muscle of type II diabetes patients (Shulman et al., 1990).
  • Insulin resistance is characterized by hyperinsulemia and hyperglycemia. Although the precise molecular mechanism underlying insulin resistance is unknown, defects in downstream components of the insulin signaling pathway are considered to be the cause.
  • Glycogen synthase kinase-3 (GSK-3) is one of the downstream components of insulin signaling. It was found that high activity of GSK-3 impairs insulin action in intact cells, by phosphorylating the insulin receptor substrate- 1 (IRS-I) serine residues (Eldar-Finkelman et al, 1997), and likewise, that increased GSK-3 activity expressed in cells results in suppression of glycogen synthase activity (Eldar- Finkelman et al, 1996). Further studies conducted in this respect uncovered that GSK-3 activity is significantly increased in epididymal fat tissue of diabetic mice (Eldar-Finkelman et al, 1999).
  • GSK-3 activity was detected in skeletal muscle of type II diabetes patients (Nickoulina et al, 2000). Additional recent studies further established the role of GSK-3 in glycogen metabolism and insulin signaling (for review see, Eldar-Finkelman, 2002Woodgett, 2001), thereby suggesting that the inhibition of GSK-3 activity may represent a way to increase insulin activity in vivo.
  • GSK-3 is also considered to be an important player in the pathogenesis of Alzheimer's disease.
  • GSK-3 was identified as one of the kinases that phosphorylate tau, a microtubule-associated protein, which is responsible for the formation of paired helical filaments (PHF), an early characteristic of Alzheimer's disease.
  • PHF paired helical filaments
  • abnormal hyperphosphorylation of tau is the cause for destabilization of microtubules and PHF formation.
  • GSK-3 phosphorylation directly affected tau ability to promote microtubule self-assembly (Mandelkow et al., 1992; Mulot et al., 1995).
  • GSK-3 is further linked with Alzheimer's disease by its role in cell apoptosis.
  • Glutamate-induced neuronal excitotoxicity is also believed to be a major cause of neurodegeneration associated with acute damage, such as in cerebral ischemia, traumatic brain injury and bacterial infection. Furthermore, it is believed that excessive glutamate signaling is a factor in the chronic neuronal damage seen in diseases such as Alzheimer's, Huntington's, Parkinson's, AIDS associated dementia, amyotrophic lateral sclerosis (AML) and multiple sclerosis (MS) (Thomas, 1995).
  • AML amyotrophic lateral sclerosis
  • MS multiple sclerosis
  • GSK-3 inhibitors are believed to be a useful treatment in these and other neurodegenerative disorders. Indeed, dysregulation of GSK-3 activity has been recently implicated in several CNS disorders and neurodegenerative diseases, including schizophrenia (Beasley et al., 2001), stroke, and Alzheimer's disease (AD) (Bhat and Budd, 2002; Lucas et al., 2001; Mandelkow et al., 1992). In view of the wide implication of GSK-3 in various signaling pathways, development of specific inhibitors for GSK-3 is considered both promising and important regarding various therapeutic interventions as well as basic research:
  • CREB cAMP response element binding protein
  • GSK-3 inhibitors were recently reported. Two structurally related small molecules SB-216763 and SB-415286 (Glaxo SmithKline Pharmaceutical) that specifically inhibited GSK-3 were developed and were shown to modulate glycogen metabolism and gene transcription as well as to protect against neuronal death induced by reduction in PI3 kinase activity (Cross et al., 2001; Coghlan et al., 2000). Another study indicated that Induribin, the active ingredient of the traditional Chinese medicine for chronic myelocytic leukemia, is 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 were identified by high throughput screening of chemical libraries. It is generally accepted that a major drawback of ATP-competitive inhibitors is their limited specificity (Davies et al., 2000).
  • the present inventors have now surprisingly found that compounds which are designed according to the unique features of the recognition motif of a GSK-3 substrate exhibit substrate competitive inhibition activity toward GSK-3 and can therefore be efficiently used in various applications where reducing the activity of GSK-3 is beneficial.
  • a compound comprising a negatively charged group and at least one amino moiety- containing group being covalently linked therebetween via a spacer, the spacer having a length, structure and flexibility selected for allowing at least one interaction between the negatively charged group and a first binding site in a catalytic domain of a GSK-3 and at least one interaction between the amino moiety-containing group and a second binding site in the catalytic domain of a GSK-3, such that the compound is capable of inhibiting a catalytic activity of a GSK-3.
  • X, Y, Z and W are each independently a carbon atom or a nitrogen atom;
  • A is the J;
  • B is the negatively charged group;
  • D is selected from the group consisting of hydrogen, alkyl, C 1 to C 6 substituted 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, 0-carbamyl, N-carbamyl, 0-thiocarbamyl, N- tliiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, trihalomethanesulfona
  • R 1 , R 2 , R 3 and R 4 is the amino moiety-containing group.
  • inhibiting the catalytic activity of a GSK-3 comprises diminishing a binding of a substrate to the catalytic domain.
  • the first binding site comprises at least one amino acid residue selected from the group consisting of arginine 180, arginine 96, and lysine 205.
  • the second binding site comprises at least one amino acid residue selected from the group consisting of aspartate 181, glutamate 97, aspartate 90, aspartate 181, glutamate 200, glutamine 89, tyrosine 215 and aspartate 95.
  • the compound further comprises a hydrophobic moiety that is capable of interacting with a third binding site of a GSK-3.
  • the third binding site is a part of the catalytic domain of the GSK-3.
  • the third binding site comprises at least one amino acid residue selected from the group consisting of isoleucine 217, phenylalanine 67 and tyrosine 215.
  • hydrophobic moiety forms a part of the spacer.
  • the length of the spacer ranges from 2 angstroms and 50 angstroms.
  • L is selected from the group consisting of a phosphor atom, a sulfur atom, a silicon atom, a boron atom and a 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, tbiocarbonyl, 0-carboxy, thiohydroxy, thioalkoxy and thioaryloxy or absent.
  • L is phosphor
  • Q, D and G are each oxygen and E is hydroxy.
  • the amino moiety-containing group comprises at least one positively charged group.
  • the at least one positively charged group is selected from the group consisting of ammonium ion and guanidinium ion.
  • the at least one positively charged group has a chemical structure derived from a side chain of a positively charged amino acid.
  • the positively charged amino acid is selected from the group consisting of arginine, lysine, histidine, proline and any derivative thereof.
  • the at least one amino moiety-containing group is selected from the group consisting of guanidino, guanidinoalkyl, amino, aminoalkyl, hydrazine, guanyl and guanyloalkyl.
  • At least one of the at least one amino moiety-containing group forms a part of the spacer.
  • the spacer comprises at least one cyclic moiety.
  • the at least one cyclic moiety is selected from the group consisting of an alicyclic, an aryl, a heteroaryl and a heteroalicyclic.
  • the spacer comprises at least two cyclic moieties.
  • At least two of the cyclic moieties are fused to one another.
  • 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.
  • K is selected from the group consisting of aryl, heteroaryl, alicylic, or heteroalicyclic;
  • J and S 1 -Sn are each independently selected from the group consisting of a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted alicylic, a substituted or unsubstituted heteroalicyclic, a bond, a heteroatom, a substituted or unsubstituted hydrocarbon chain, a substituted or unsubstituted hydrocarbon chain interrupted by at least one heteroatom, or absent.
  • n is an integer from 1 to 2.
  • n is an integer from 0 to 2.
  • n is 2 and each of S 1 , S 2 and K is independently selected from the group consisting of aryl and heteroaryl.
  • J is a hydrocarbon chain.
  • J is alkyl.
  • S 1 is aryl
  • S 2 is heteroaryl
  • K is aryl.
  • S 1 is phenyl
  • S 2 is triazole
  • K is phenyl
  • At least one of J and S 1 -Sn comprises at least one amino moiety-containing group.
  • the at least one amino moiety-containing group forms a part of the K.
  • At least one of J, (S) 1 -(S ⁇ and K comprises a hydrophobic moiety attached thereto.
  • the hydrophobic moiety is selected from the group consisting of a fatty acid residue, a saturated alkylene chain having between 4 and 30 carbon atoms, an unsaturated alkylene chain having between 4 and 30 carbon atoms, an aryl, a cycloalkyl and a hydrophobic peptide sequence.
  • 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.
  • J is a hydrocarbon chain or absent and n is 0.
  • a pharmaceutical composition that comprises, as an active ingredient, any of the compounds described hereinabove, which is capable of inhibiting an activity of GSK- 3, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is packaged in a packaging material and is identified in print, on or in the packaging material, for use in the treatment of a biological condition associated with GSK-3 activity, as is detailed hereinbelow.
  • the pharmaceutical composition further comprises at least one additional active ingredient that is capable of altering an activity of GSK-3, as is detailed hereinbelow.
  • a method of treating a biological condition associated with an activity of GSK-3 which is effected by administering to a subject in need thereof a therapeutically effective amount of a compound which comprises a negatively charged group and at least one amino moiety-containing group being linked therebetween via a spacer, wherein the spacer has a length, structure and flexibility suitable for enabling at least one interaction between the negatively charged group and a first binding site in the catalytic domain of a GSK-3 and at least one interaction between the amino moiety- containing group and a second binding site in the catalytic domain of a GSK-3, as is described hereinabove.
  • the method according to this aspect of the present invention further comprises co-administering to the subject at least one additional active ingredient, which is capable of altering an activity of GSK-3.
  • the additional active ingredient can be an active ingredient that is capable of inhibiting an activity of GSK-3 or an active ingredient that is capable of downregulating an expression of GSK-3.
  • the biological condition according to the present invention is preferably selected from the group consisting of obesity, non-insulin dependent diabetes mellitus, an insulin-dependent condition, an affective disorder, a neurodegenerative disease or disorder and a psychotic disease or disorder.
  • the affective disorder can be a unipolar disorder (e.g., depression) or a bipolar disorder (e.g., manic depression).
  • a unipolar disorder e.g., depression
  • a bipolar disorder e.g., manic depression
  • the neurodegenerative disorder can results from an event selected from the group consisting of cerebral ischemia, stroke, traumatic brain injury and bacterial infection, or can be a chronic neurodegenerative disorder that results from a disease selected from the group consisting of Alzheimer's disease, Huntington's disease, Parkinson's disease, AIDS associated dementia, amyotrophic lateral sclerosis (AML) and multiple sclerosis.
  • a disease selected from the group consisting of Alzheimer's disease, Huntington's disease, Parkinson's disease, AIDS associated dementia, amyotrophic lateral sclerosis (AML) and multiple sclerosis.
  • a method of inhibiting an activity of GSK-3 which comprises contacting cells expressing GSK-3 with an inhibitory effective amount of a compound which comprises a negatively charged group and at least one amino moiety-containing group being linked therebetween via a spacer, wherein the spacer has a length, structure and flexibility suitable for enabling at least one interaction between the negatively charged group and a first binding site in the catalytic domain of a GSK-3 and at least one interaction between the amino moiety-containing group and a second binding site in the catalytic domain of a GSK-3.
  • GSK-3 with an inhibitory effective amount of a compound as described herein.
  • the activity can be a phosphorylation activity and/or an autophosphorylation activity.
  • a method of potentiating insulin signaling which comprises contacting insulin responsive cells with an effective amount of which comprises a negatively charged group and at least one amino moiety-containing group being linked therebetween via a spacer, wherein the spacer has a length, structure and flexibility suitable for enabling at least one interaction between the negatively charged group and a first binding site in the catalytic domain of a GSK-3 and at least one interaction between the amino moiety-containing group and a second binding site in the catalytic domain of a GSK-3.
  • potentiating the insulin signaling is effected by contacting insulin responsive cells with an effective amount of a compound as described herein.
  • the contacting the cells can be effected in vitro or in vivo.
  • each of the methods and/or uses according to these additional aspects of the present invention further comprises contacting the cells with at least one an additional active ingredient, as is described hereinabove.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing newly designed, non-peptidic compounds for inhibiting GSK-3 activity, which can be efficiently used in the treatment of a variety of biological conditions.
  • 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, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • 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;
  • FIGs. 3a-b present the chemical structures of phenyl phosphate, pyridoxal phosphate (P-5-P), GSC-I, GSC-2, GSC-3 and of the novel compounds GSC-4, GSC- 5 and GSC-21 ( Figure 3a) and of the novel compounds GSC-6, GSC-7, GSC-8 and GSC-9 ( Figure 3b);
  • FIGs. 4a-b present the ESI-MS ( Figure 4a) and an HPLC chromatogram ( Figure 4) of 3,5-Bis(2-aminoethyl)benzyl phosphate (GSC-21);
  • FIG. 5 presents comparative plots demonstrating the GSK-3 inhibition activity of phenyl phosphate, GSC-I 1 GSC-2, GSC-3 and pyridoxal phosphate (P-5-P) in in vitro inhibition assays with PGS-I peptide substrate;
  • FIG. 6 presents comparative plots demonstrating the GSK-3 inhibition activity of GSC-I, GSC-2, GSC-3, GSC-4 and GS-21 in in vitro inhibition assays (Black circles denote GSC-2, red circles denote GSC-I, green circles denote GSC-3, blue circles denote GSC-4 and pink circles denote GSC-21); FIGs.
  • FIG. 7a-b present comparative plots demonstrating the GSK-3 inhibition activity of GSC-I, GSC-2, GSC-3 and GSC-4 (Figure 7a, black circles denote GSC-2, blanc circles denote GSC-I, black triangles denote GSC-3 and black rectangles denote GSC-4) and GSC-5, GSC-6 and GSC-7 ( Figure 7b, rectangles denote GSC-5, triangles denote GSC-6 and circles denote GSC-7) in in vitro inhibition assays with p9CREB peptide substrate;
  • FIG. 8 presents a Lineweaver-Burk plot showing the inhibition of GSK-3 by
  • GSC-7 at indicated concentrations, represented by phosphate incorporation into the p9CREB peptide substrate (CPM) and demonstrating that GSC-7 is a competitive specific inhibitor (Results show one representative experiment out of 3; each point is a mean of duplicate samples);
  • FIG. 9 is an image of a gel electrophoresis assay for CDK-2 kinase activity assayed in the presence of 32 P[y-ATP] and histone Hl as a substrate, and demonstrating the absence of inhibitory activity of GSC-4, GSC-5 and GSC-7;
  • FIG. 10 presents plots demonstrating the effect of GSC-5 (hollowed circles) and GSC-7 (filled circles) on glycogen synthase activity in C2C12 cells, shown as fold stimulation over the cells treated with vehicle 0.1 % HCL;
  • FIGs. lla-b are bar graphs demonstrating the effect of GSC-21 (Figure l ib) and GSC-4 (Figure Ha) on glucose uptake in mouse adipocytes, represented by the [ 3 H] 2-deoxy glucose incorporation in cells treated with GSC-4 and GSC-21 as fold activation over cells treated with a peptide control (normalized to 1 unit);
  • FIG. 12 presents a computed simulation of the interaction of GSC-4 with GSK-3
  • FIG. 13 presents a computed simulation of the interaction of GSC-5 with
  • FIG. 14 presents a computed simulation of the interaction of GSC-7 with GSK-3
  • FIG. 15 presents a computed simulation of the interaction of GSC-6 with GSK-3
  • FIG. 16 presents a computed simulation of the interaction of GSC-8 with GSK-3
  • FIG. 17 presents a computed simulation of the interaction of GSC-9 with GSK-3
  • FIG. 18 presents the chemical structures of the newly designed GSK-3 inhibitors MP-I, MP-2, MP-3, MP-4, MP-5 and MP-6; and
  • FIG. 19 presents comparative plots demonstrating the GSK-3 inhibition activity of MP-I, MP-2, MP-3, MP-4, MP-5 and MP-6 in in vitro inhibition assays with p9CREB peptide substrate.
  • the present invention is of novel, non-peptidic compounds, which are capable of inhibiting GSK-3 activity and can therefore be used in the treatment of biological conditions mediated by GSK-3.
  • the present invention is of (i) compounds that are designed according to the pharmacophoric coordinates of a GSK- 3 substrate, which may optionally have a hydrophobic moiety attached thereto; (ii) pharmaceutical compositions containing same; (iii) methods of using same for inhibiting GSK-3 activity and potentiating insulin signaling; and (iv) methods of using same in the treatment of biological conditions such as, but not limited to, obesity, non-insulin dependent diabetes mellitus, insulin-dependent conditions, affective disorders, neurodegenerative diseases and 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.
  • One of the parameters that are responsible for substrate-kinase recognition is an element located within the substrate, which is usually related to as a "recognition motif.
  • GSK-3 unlike other kinases, has a unique recognition motif, which includes the amino acid sequence SX 1 X 2 X 3 S(P), set forth in SEQ ID NO:1, where S is serine or threonine, each of X 1 , X 2 and X 3 is any amino acid, and S(p) is phosphorylated serine or phosphorylated threonine.
  • GSK-3 recognizes only pre-phosphorylated substrates, namely, substrates that have a phosphorylated serine or threonine residue. It was further hypothesized that determining this unique structure would enable the development of small molecules that could act as substrate competitive inhibitors of
  • the short pre- phosphorylated peptide ⁇ 9CREB (ILSRRPS( ⁇ )YR, SEQ ID NO:2) was selected.
  • the three-dimensional structures of p9CREB, as well as of the corresponding non- phosphorylated peptide CREB (ILSRRPSYR, SEQ ID NO:3) were determined by 2D NMR, as is detailed in the Examples section that follows (see, Example 1).
  • the phosphorylated p9CREB substrate has a defined structure in solution ( Figure Ia), whereby the corresponding non- phosphorylated peptide CREB does not exhibit any unique structure ( Figure Ib).
  • Figure 2 presents the electrostatic distribution on the 'surface' of the p9CREB peptide, based on these findings. While continuing to conceive the present invention, it was deduced from the findings described hereinabove that a small molecule that would mimic the structure of a GSK-3 substrate such that it would exerts substrate competitive inhibitory activity should be designed according to the following features:
  • the molecule should include a negatively charged group, preferably a phosphate group;
  • the negatively charged group should not be stearically hindered; and
  • the negatively charged group should preferably be flanked at least at one side or at both sides thereof by one or two positively charged groups.
  • Example 2 Based on the above, a general formula of potential compounds for inhibiting GSK-3 activity has been designed (see, Example 2). As is described in the Examples section that follows (see, Example 3), preliminary experiments that were conducted with a 'first generation' of these compounds, namely, compounds having the most simplified structure of this formula, demonstrated the capability of these compounds to inhibit GSK-3 activity, thus providing a preliminary indication of the inhibitory potential of compounds having such a formula.
  • small molecules should be designed such that most, if not all, the various functionalities would be in a suitable proximity and orientation to the binding sites.
  • design of small molecules in which the distance between the negatively charged group and the one or more positively charged group(s) is greater than that obtained with a single aromatic ring should be considered.
  • Example 5 a second generation of compounds have been designed and successfully prepared (see, Example 5) and practiced (see, Example 6). These compounds were designed to include a spacer, linking the negatively charged group and one or more positively charged groups, which would have a suitable length, structure and flexibility, and hence would allow strong interactions with various binding sites in the catalytic core of GSK-3.
  • Representative examples of such 'second generation' compounds are compounds in which the spacer is composed of three cyclic moieties, and further in which the negatively and positively charged groups are positioned in certain orientations to one another.
  • experiments conducted with these compounds demonstrated their high capability to inhibit GSK-3 activity and further demonstrated the effect of the relative orientation between the functional groups (see, for example, Figure 19).
  • a compound which comprises a negatively charged group and at least one amino moiety-containing group being covalently linked therebetween via a spacer.
  • the spacer is designed to have a length, structure and flexibility selected for allowing at least one interaction between the negatively charged group and a first binding site in a catalytic domain of a GSK-3 and at least one interaction between the ammo moiety- containing group and a second binding site in the catalytic domain of a GSK-3, such that the compound is capable of inhibiting a catalytic activity of a GSK-3.
  • Excluded from the scope of this aspect of the present invention are the compounds disclosed in WO 2005/000192, as described hereinabove.
  • catalytic domain describes a region of an enzyme in which the catalytic reaction occurs. This phrase therefore describes this part of an enzyme in which the substrate and/or other components that participate in the catalytic reaction interacts with the enzyme. In the context of the present invention, this phrase is particularly used to describe this part of an enzyme (a GSK-3) to which the substrate binds during the catalytic activity (e.g., phosphorylation). This phrase is therefore also referred to herein and in the art, interchangeably, as “substrate binding pocket", “catalytic site” "active site” and the like.
  • binding site describes a specific site in the catalytic domain that includes one or more reactive groups through which the interactions with the substrate and/or other components can be effected.
  • the binding site is composed of one or two amino acid residues, whereby the interactions typically involve reactive groups at the side chains of these amino acids.
  • conformational changes of the catalytic domain of the enzyme occur so as to bring the reactive groups in suitable proximity and orientation, and allow their interaction with the functional groups of the substrate.
  • binding site encompasses those amino acid residues that are positioned in such proximity and orientation that allows such interaction.
  • the interactions of the various functional groups of the compound with the various binding sites of the enzyme can be, for example, electrostatic interactions, hydrogen bonding interactions, hydrophobic interactions, aromatic interactions, ⁇ - stacking interactions, and the like, depending on the reactive groups that participate in the interactions and their proximity and orientation to one another.
  • Exemplary electrostatic interactions include anion-cation interactions and acid-base interactions such as, for example, interactions between ammonium cation and carboxylate anion.
  • Exemplary hydrogen bonding interactions include interactions between hydrogens of amine, hydroxel or thiol of one or more component(s) and e.g., oxygen, nitrogen and sulfur atoms of other component(s).
  • 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 aryls and heteroaryls, which are based on overlap in the aromatized molecular orbitals of the moieties.
  • Exemplary ⁇ -stacking interactions include interactions between two or more moieties that contain ⁇ -electrons (e.g., unsaturated moieties), which are based on overlap in the ⁇ -orbitals of the moieties.
  • substrate competitive enzyme inhibitors act by binding to the catalytic domain of an enzyme and thus reducing the proportion of enzyme molecules that are bound to the enzyme during the catalytic process.
  • an enzyme interacts with a substrate or an inhibitor, the initial interaction rapidly induces conformational changes in the enzyme that strengthen binding and bring catalytic sites close to functional groups in the substrate or inhibitor.
  • Enzyme-substrate/inhibitor interactions orient reactive groups present in both the enzyme and the substrate/inhibitor and bring them into proximity with one another.
  • the binding of the substrate/inhibitor to the enzyme aligns the reactive groups so that the relevant molecular orbitals overlap.
  • an efficient substrate competitive inhibitor should be designed such that the reactive groups of the inhibitor would be positioned in sufficient proximity to corresponding reactive groups (typically side chains of amino acid residues) in the enzyme catalytic domain, so as to allow the presence of an effective concentration of the inhibitor in the catalytic domain and, in addition, the reactive groups of the inhibitor should be positioned in a proper orientation, to allow overlap. Still in addition, an inhibitor should have a restriction of its conformational flexibility, so as to avoid conformational changes that would affect or weaken the interactions and should include structural elements that are known to be involved in the interactions.
  • GSK-3 substrates have a negatively charged group and one or more positively charged groups, whereby the negatively charged group is positioned in a special spatial orientation.
  • Substrate competitive GSK-3 inhibitors should therefore include both these groups and the relative orientation thereof.
  • an inhibitor should be designed such that these functional groups would be in optimal proximity and orientation towards the enzyme's reactive groups.
  • the proximity and orientation of these functional groups are determined, according to the present embodiments, by selecting a spacer that links these functional groups such that when interacting with reactive groups in the catalytic domain, each functional group would be in an optimal proximity and orientation towards one or more compatible reactive groups in the enzyme binding sites.
  • a suitable spacer would therefore have suitable length, flexibility and structure that would allow efficient interaction, in terms of proximity and orientation, between each functional group and one or more compatible reactive group.
  • the first binding site (with which the negatively charged group can interact, as described hereinabove) comprises one or more of these amino acid residues.
  • the compounds are designed such that interactions between the negatively charged group and one or more of these amino acid residues are allowed. These interactions are preferably electrostatic interactions.
  • any other chemically compatible binding sites in the catalytic domain of GSK-3 that are arranged in proximity and orientation that allows interactions with the negatively charged group of the inhibitor are also within the scope of the present invention.
  • the amino moiety-containing group(s) interact with one or more of aspartate 181, glutamate 97, aspartate 90, aspartate 181, glutamate 200, glutamme 89, tyrosine 215 and aspartate 95.
  • the second binding site comprises one or more of these amino acid residues.
  • the compounds are designed such that interactions between the amino moiety-containing group(s) and one or more of these amino acid residues are allowed.
  • interactions can be, for example electrostatic interactions and/or hydrogen binding interactions.
  • any other chemically compatible binding sites in the catalytic domain of GSK-3 that are arranged in proximity and orientation that allows interactions with the amino moiety- containing group (s) of the inhibitor are also within the scope of the present invention.
  • the compounds described herein preferably comprise one negatively charged group, they may comprise one or more (e.g., two, three) amino moiety- containing groups.
  • the second binding site described herein includes all of those reactive groups that interact with ail of the amino moiety-containing groups.
  • the compounds described herein can further include a hydrophobic moiety that may participate in the interactions of the compound with the enzyme.
  • the hydrophobic moiety can be attached to the spacer such that the length, structure and flexibility of the spacer would allow its interactions with another (third) binding site of the enzyme.
  • the hydrophobic moiety can form a part of the spacer itself, by selecting a hydrophobic spacer.
  • the interactions of a hydrophobic moiety may be within the catalytic domain and/or within another region of the enzyme.
  • these interactions can be within a hydrophobic patch that is present within the enzyme, as was previously reported by Dajani et al. (2001).
  • 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 a GSK-3.
  • any other hydrophobic binding sites in the catalytic domain of GSK-3 that are arranged in proximity and orientation that allows interactions with the hydrophobic moiety of the inhibitor are also within the scope of the present invention.
  • These interactions can be hydrophobic interactions, aromatic interactions and/or ⁇ -stacking interactions, depending on the chemical structure of the moiety and the binding site. Additional description of hydrophobic moieties is set forth below.
  • an optimal distance between the negatively charged group and the amino moiety-containing group ranges from about 2 angstroms and about 50 angstroms.
  • the length of spacer ranges from about 2 angstroms and about 50 angstroms, more preferably from about 2 angstroms and about 20 angstroms, and more preferably from about 2 angstroms and about 10 angstroms.
  • An exemplary spacer would thus have a length of about 7-8 angstroms.
  • the spacer preferably comprises one or more cyclic moieties.
  • the moieties can be either fused and/or non-fused.
  • each two cyclic moieties can be linked to one another via linker such as, but not limited to, a bond, a heteroatom (e.g., oxygen, nitrogen, sulfur) or a hydrocarbon chain, as these terms are defined hereinbelow.
  • the hydrocarbon chain can optionally be interrupted by one or more heteroatoms (e.g., oxygen, nitrogen, sulfur and the likes).
  • the spacer comprises two or more, preferably three cyclic moieties, which are covalently linked to one another via a bond.
  • each of the cyclic moieties can be, for example, a carbocylic moiety or a heterocyclic moiety.
  • a “carbocylic moiety” describes an all-carbon, saturated or unsaturated cyclic moiety, whereby a “heterocyclic moiety” describes a saturated or unsaturated cyclic moiety that includes one or more heteroatoms such as nitrogen, oxygen, sulfur, phosphor, silicone and the like.
  • Carbocylic moieties include cycloalkyls and aryls, as these terms are defined hereinbelow.
  • Heterocyclic moieties include heteroalicyclics and heteroaryls, as these terms are defined hereinbelow.
  • Aromatic cyclic moieties (aryls and heteroaryls), by being more rigid than non- aromatic cyclic moieties (cycloalkyls and heteroalicyclics), are preferred due to the restriction of conformational changes in their structures. However, a combination of aromatic and non-aromatic cyclic moieties can also allow the desired interactions.
  • the spacer comprises a heterocyclic moiety that contains one or more nitrogen atoms
  • this moiety can serve as an amino moiety-containing group that participates in the interactions with the binding sites.
  • these moieties can serve as hydrophobic and/or aromatic moieties that participate in the interactions with the binding sites, as discussed hereinabove.
  • the spacer can be further selected so as to have a structure that would allow or restrict interactions thereof with binding sites of the enzyme.
  • Exemplary spacers that have a structure that allows interactions with binding sites include, without limitations, hydrophobic moiety-containing spacers, aromatic moiety- containing spacers and amino moiety-containing (e.g., heterocylic) spacers.
  • the structure of the spacer can further affect its flexibility, stability and other features that may be implicated with the interactions of the compound with the binding sites in the catalytic domain of GSK-3.
  • Preferred compounds according to the present embodiments therefore include a rigid or a semi-rigid spacer, to which a negatively charged group is attached.
  • this structure mimics the unique structure of a GSK-3 substrate by providing a negatively charged group which is not stearically hindered and has a geometrical structure similar or identical to a phosphate group, and further by allowing interactions of the negatively charged group and an amino moiety-containing group with binding sites of GSK-3, these compounds are capable of inhibiting GSK-3 activity, preferably via inhibition of the substrate binding to the enzyme.
  • negatively charged group and “positively charged group”, as used herein, refer to an ionizable group, which upon ionization, typically in an aqueous medium, has at least one negative or positive charge, respectively.
  • the charged groups can be present in the compounds described herein either in their ionized form or as a pre-ionized form.
  • the negatively charged group according to preferred embodiments of the
  • L is selected from the group consisting of a phosphor atom, a sulfur atom, a silicon atom, a boron atom and a 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, as these terms are defined hereinbelow, or absent.
  • the negatively charged group is a phosphate group, such that in the formula above L is a phosphor atom, whereby each of Q, G and D is oxygen.
  • E is hydroxy.
  • the hydroxy group can also be ionized so as to have another negative electrostatic charge.
  • the negatively charged group can be a thiophosphate group, sulfate or sulfonate group, a borate or boronate group and the like, according to the formula above.
  • the negatively charged group is preferably attached to the spacer via an alkyl group, preferably an unsubstituted alkyl, and more preferably a methyl.
  • the attachment of the negatively charged group to the ring via an alkyl group renders the negatively charged group a free rotatable group as opposed to its restricted roatatability when attached directly to the spacer.
  • the free rotatability of the negatively charged group is advantageous since it allows the negatively charged group to readily interact with the binding site of the enzyme.
  • the positively charged group is preferably derived from an amine moiety- containing group, which can be present in the compound in its ionized form (e.g., as an ammonium or guanidinium ion) or as a free amine (a pre-ionized form).
  • amino moiety-containing group refers to a group which contains one or more amino moieties, as this term is defined herein or to an amine per se.
  • amino moiety-containing groups include, without limitation, an amine, an aminoalkyl, hydrazine, urea, thiourea, guanyl, amido, carbamyl, guanidino, guanidinoalkyl and guanylinoalkyl, as these terms are defined herein.
  • a free amine group is typically basic under neutral conditions and therefore, at a biological environment, it tends to be protonated so as to produce a positively charged -NH 3 + group, for example.
  • a compound that has one or two of such positively charged groups flanking the negatively charged group in a suitable distance and orientation with respect to the binding sites of the enzyme is preferable.
  • the amino moiety is preferably present in this group as a readily- protonated moiety, that is, a moiety in which the amino nitrogen has a substantial partially negative charge.
  • amino moiety-containing groups therefore include, without limitation, an amine, an aminoalkyl, guanyl, guanylinoalkyl, guanidino, guanidinoalkyl and guanylinoalkyl, as these terms are defined herein.
  • amino moiety-containing groups can be present in the compounds described herein either as is or as positively charged groups, in which at least one of the amino moieties is ionized.
  • positively charged groups according to the present invention comprise an ammonium ion, such that representative examples of positively charged groups include, without limitation, an ammonium ion per se (a protonated amino group) and any group that bears an ammonium ion, as is defined hereinabove, such as an alkyl, cycloalkyl or aryl substituted by an ammonium ion, guanidino, guanyl, hydrazine and the like.
  • positively charged groups that have a chemical structure derived from a side chain of a positively charged amino acid, e.g., lysine, arginine, histidine, proline and derivatives thereof, with the first two being the most preferred.
  • a chemical structure derived from a side chain of a positively charged amino acid it is meant that the positively charged group has a similar or identical chemical structure as such a side chain.
  • Representative examples include guanidine and guanidinoalkyl (derived from arginine), amine and aminoalkyl (derived from lysine) and imidazole (derived from histidine), with the first being the presently most preferred.
  • B is a negatively charged group, as described hereinabove;
  • Q is one of the amino moiety-containing group(s) described hereinabove;
  • K is selected from the group consisting of aryl, heteroaryl, alicylic, or heteroalicyclic;
  • J and S 1 -Sn are each independently selected from the group consisting of a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted alicylic, a substituted or unsubstituted heteroalicyclic, a bond, a heteroatom, a substituted or unsubstituted hydrocarbon chain, a substituted or unsubstituted hydrocarbon chain interrupted by at least one heteroatom, as these terms are defined herein, or absent.
  • the substituents can be any of those described hereinbelow.
  • the spacer in these compounds is comprised of at least one cyclic moiety (K in Formula I above) to which 1-6 amino moiety-containing groups are attached.
  • K in Formula I above a cyclic moiety
  • one or two amino moiety-containing groups are attached to the cyclic moiety K, such that m in Formula I above ranges from 1 to 2.
  • one of the amino moiety-containing groups, denoted as Q in Formula I above forms a part of the cyclic moiety K, as is detailed hereinabove.
  • K is a heteroalicyclic or a heteroaryl, preferably heteroaryl.
  • Additional amino moiety-containing groups can also be present within the compound.
  • at least one of the J and S 1 -Sn moieties can comprise one or more amino moiety-containing group(s).
  • the amino moiety-containing group(s) can be attached to these moieties or form a part thereof, as described hereinabove.
  • a longer spacer links the negatively charged group B and the amino moiety-containing group Q, such that at least one of J and S 1 -Sn is present within the spacer.
  • the length, structure and flexibility of the spacer can be determined so as to allow the desired interactions described hereinabove.
  • the number and size of the moieties composing the spacer determines the length of the spacer. In cases where a lengthy spacer is required in order to allow the desired interactions, n is greater than 5. In cases where a shorter spacer is required, n is lower than 5 and can also be 0.
  • preferred compounds comprise a spacer that have a length that ranges from 2 angstroms and 50 angstroms, preferably from 2 angstroms and 20 angstroms and more preferably from 2 angstroms and 10 angstroms.
  • n is an integer from
  • n is 2 and each of S 1 , S 2 and K is independently selected from the group consisting of aryl and heteroaryl.
  • the negatively charged group can be attached to the spacer either directly or indirectly, with the latter being preferred.
  • J in Formula I above can be absent (when the negatively charged group is directly attached to the spacer) or a flexible group that would allow this group to readily interact with the first binding site described hereinabove.
  • J is a hydrocarbon chain such as alkyl.
  • J is a short alkyl (C 1 -C 6 alkyl) and more preferably it is methyl.
  • J is alkyl and each of S 1 , Sn and K is aryl or heteroaryl.
  • Exemplary compounds in this category are those having the general Formula above in which B is phosphate, J is alkyl (e.g., methyl), S 1 is aryl (e.g., phenyl), S 2 is heteroaryl (e.g., triazole) and K is aryl (e.g., phenyl).
  • X, Y, Z and W are each independently a carbon atom or a nitrogen atom;
  • A is a hydrocarbon chain or absent (corresponds to J above);
  • B is a negatively charged group as described hereinabove;
  • D is selected from the group consisting of 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, 0-thiocarbamyl, N-thiocarba
  • R 1 , R 2 , R 3 and R 4 are each independently selected from the group consisting of hydrogen, a lone pair of electrons, 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-amido, N-amido, C- carboxy, O-carboxy, sulfonamido, trihal
  • the spacer in these family of compounds comprises a cyclic moiety and preferably an aromatic ring (aryl) or a heteroaromatic ring (heteroaryl).
  • the spacer is a heteroaryl, such that in Formula III above, at least one of X, Y, Z and W is a nitrogen atom.
  • a positively charged amino moiety-containing group forms a part of the spacer. Since it was found that the amino moiety-containing group should preferably be in a certain distance from the negatively charged group, preferably, Z or W is a nitrogen atom.
  • At least two of X, Y, Z and W are nitrogen atoms, more preferably either X and Y are nitrogen atoms or Z and W are nitrogen atoms, and even more preferably Z and W are nitrogen atoms.
  • R 1 , R 2 , R 3 and R 4 are amino moiety-containing groups, as described hereinabove.
  • R 1 and R 2 or R 3 and R 4 are amino moiety-containing groups
  • preferred compounds according to the present invention are those having the following general Formulae IIA and HB:
  • Formula IIA Formula IIA wherein m is an integer from 1 to 6; each of Q 1 and Q 2 is independently a carbon atom or a nitrogen atom; and G and/or K are each an amino moiety-containing group (e.g., a positively charged group).
  • each of the compounds described herein has one or more hydrophobic moieties attached thereto.
  • the hydrophobic moiety is attached to the spacer and further preferably, the hydrophobic moiety is attached to the spacer in such a position that would allow interaction between this moiety and a binding site of a GSK-3, as discussed hereinabove.
  • At least one of J, (S)i-(S)n and K comprises a hydrophobic moiety attached thereto.
  • K has a hydrophobic moiety attached thereto.
  • Sn has a hydrophobic moiety attached thereto.
  • D is a hydrophobic moiety.
  • hydrophobic moiety refers to any substance or a residue thereof that is characterized by hydrophobicity.
  • the term “residue” describes a major portion of a substance that is covalently linked to another substance, herein the compound described hereinabove.
  • a hydrophobic moiety according to the present invention is preferably a residue of a hydrophobic substance, and is preferably covalently attached to the compound described hereinabove.
  • hydrophobic substances from which the hydrophobic moiety of the present invention can be derived include, without limitation, a saturated alkylene chain, an unsaturated alkylene chain, an aryl, a cycloalkyl and a hydrophobic peptide sequence, as these terms are defined herein.
  • alkylene chain refers to a hydrocarbon linear chain, which can be saturated or unsaturated.
  • the alkylene chain can be substituted or unsubstituted, as is described herein with respect to an alkyl group, and can be further interrupted by one or more heteroatoms such as nitrogen, oxygen, sulfur, phosphor and the like.
  • the alkylene chain preferably includes at least 4 carbon atoms, more preferably at least 8 carbon atoms, more preferably at least 10 carbon atoms and may have up to 20, 25 and even 30 carbon atoms.
  • the hydrophobic moiety of the present invention can therefore comprise a residue of the hydrophobic substances described hereinabove.
  • a preferred example of an alkylene chain according to this aspect of the present invention is an alkylene chain that comprises a carboxy group, namely, a fatty acid residue(s).
  • Preferred fatty acids that are suitable for use in the context of the present invention include, without limitation, saturated or unsaturated fatty acids that have more than 10 carbon atoms, preferably between 12 and 24 carbon atoms, such as, but not limited to, myristic acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid and more.
  • the hydrophobic moiety can be a hydrophobic peptide sequence.
  • the hydrophobic peptide sequence preferably includes between 2 and 15 amino acid residues, more preferably between 2 and 10 amino acid residues, more preferably between 2 and 5 amino acid residues, in which at least one amino acid residue is a hydrophobic amino acid residue.
  • hydrophobic amino acid residues include, without limitation, an alanine residue, a cysteine residue, a glycine residue, an isoleucine residue, a leucine residue, a valine residue, a phenylalanine residue, a tyrosine residue, a methionine residue, a proline residue and a tryptophan residue, or any modification thereof, as is described hereinabove.
  • the hydrophobic amino acid residue can include any other amino acid residue, which has been modified by incorporation of a hydrophobic moiety thereto.
  • the hydrophobic amino acid sequence comprises at least two and more preferably at least 5 hydrophobic amino acid residues, which, further preferably, are attached to one another, so as to provide a consecutive sequence thereof within the hydrophobic amino acid sequence.
  • hydrophobic amino acid sequences which are also referred to in the art interchangeably as “membrane permeable sequences” or “MPS”, are found, for example, in Hagiwer (1999).
  • amino acid residue which is also referred to herein, interchangeably, as “amino acid” describes an amino acid unit within a polypeptide chain.
  • amino acid residues within the hydrophobic peptide sequence can be either natural or modified amino acid residues, as these phrases are defined hereinafter.
  • natural amino acid residue describes an amino acid residue, as this term is defined hereinabove, which includes one of the twenty amino acids found in nature.
  • modified amino acid residue describes an amino acid residue, as this term is defined hereinabove, which includes a natural amino acid that was subjected to a modification at its side chain.
  • modifications are well known in the art and include, for example, incorporation of a functionality group such as, but not limited to, a hydroxy group, an amino group, a carboxy group and a phosphate group within the side chain.
  • This phrase therefore includes, unless otherwise specifically indicated, chemically modified amino acids, including amino acid analogs (such as penicillamine, 3-mercapto-D-valine), naturally-occurring non- proteogenic amino acids (such as norleucine), and chemically-synthesized compounds that have properties known in the art to be characteristic of an amino acid.
  • proteogenic indicates that the amino acid can be incorporated into a protein in a cell through well-known metabolic pathways.
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor ⁇ leucine and ornithine.
  • amino acid includes both D- and L- amino acids which are linked via a peptide bond or a peptide bond analog to at least one addition amino acid as this term is defined herein.
  • hydrophobic moiety provides for enhanced unpredictable activity
  • known compounds such as phenyl phosphate and pyridoxal phosphate and other known compounds described hereinabove, which are substituted by a hydrophobic moiety, are also included within the scope of this aspect the present invention.
  • bond describes a single bond, a double bond or a triple bond.
  • hydrocarbon chain and the term “hydrocarbon” describes a moiety that is mainly composed of carbon and hydrogen atoms.
  • a hydrocarbon chain can therefore include one or more of alkyl, alkenyl, alkynyl, cycloalkyl and aryl, as these terms are defined herein, whereby each can be unsubstituted or substituted, as described herein.
  • the hydrocarbon chain can further be interrupted by one or more heteroatoms, as defined herein.
  • heteroatom describes any atom which is not carbon but which can form a covalent bond with one or more carbon atoms.
  • exemplary heteroatoms include, without limitation, nitrogen, oxygen, sulfur, phosphor, silicone, boron and the like.
  • alkyl describes a saturated aliphatic hydrocarbon including straight chain and branched chain groups.
  • the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; e.g., "1-20", is stated herein, it implies that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. More preferably, the alkyl is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or unsubstituted.
  • the substituent group can be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfmyl, sulfonyl, cyano, nitro, azo, sulfonyl, sulfinyl, sulfonamide, ketoester, carbonyl, thiocarbonyl, ester, ether, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, 0-carboxy, trihalomethanesulfonamido, gu
  • cycloalkyl or "alicyclic” describes an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system.
  • examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane.
  • a cycloalkyl group may be substituted or unsubstituted.
  • the substituent group can be, for example, alkyl, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfmyl, sulfonyl, cyano, nitro, azo, sulfonyl, sulfinyl, sulfonamide, ketoester, carbonyl, thiocarbonyl, ester, ether, carboxy, thiocarboxy, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,
  • alkynyl describes an alkyl group, as defined hereinabove, which consists of at least two carbon atoms and at least one carbon-carbon triple bond.
  • aryl describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system.
  • aryl groups are phenyl, naphthalenyl and anthracenyl.
  • the aryl group may be substituted or unsubstituted.
  • the substituent group can be, for example, alkyl, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azo, sulfonyl, sulfinyl, sulfonamide, ketoester, carbonyl, thiocarbonyl, ester, ether, carboxy, thiocarboxy, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,
  • heteroaryl describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system.
  • heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.
  • the heteroaryl group may be substituted or unsubstituted.
  • the substituent group can be, for example, alkyl, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfmyl, sulfonyl, cyano, nitro, azo, sulfonyl, sulfmyl, sulfonamide, ketoester, carbonyl, thiocarbonyl, ester, ether, carboxy, thiocarboxy, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,
  • heteroalicyclic describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur.
  • the rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system.
  • the heteroalicyclic may be substituted or unsubstituted.
  • the substituted group can be, for example, lone pair electrons, alkyl, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfmyl, sulfonyl, cyano, nitro, azo, sulfonyl, sulfmyl, sulfonamide, ketoester, carbonyl, thiocarbonyl, ester, ether, carboxy, thiocarboxy, thioether, thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, C-amido, N-amido, C-carboxy,
  • lone pair of electrons describes a pair of electrons that are not participating in a bond. The lone pair of electrons is present only when X, Y, Z or W is an unsubstituted nitrogen atom.
  • hydroxy describes an -OH group.
  • alkoxy describes both an -O-alkyl and an -O-cycloalkyl group, as defined herein.
  • aryloxy describes both an -O-aryl and an -O-heteroaryl group, as defined herein.
  • thiohydroxy describes a -SH group.
  • thioalkoxy describes both an -S-alkyl group, and an -S-cycloalkyl group, as defined herein.
  • thioaryloxy describes both an -S-aryl and an -S-heteroaryl group, as defined herein.
  • aldehyde describes a carbonyl group, where R' is hydrogen.
  • carboxylic acid describes a C-carboxyl group in which R is hydrogen.
  • halo describes fluorine, chlorine, bromine of iodine.
  • trihalomethyl describes a -CX 3 group wherein X is a halo group as defined herein.
  • trihalomethanesulfonamido describes an X 3 CS ⁇ O) 2 NR'- group, where R' and X are as defined herein.
  • R are as defined herein.
  • R are as defined herein.
  • amino describes an -NR'R" group where R' and R" are as defined herein.
  • aminoalkyl describes an alkyl, as defined hereinabove, substituted by an amino group. Preferably, the alkyl terminates by the amino group.
  • C-amido describes a -C(O)-NR 5 R" group, where R' and R" are as defined herein.
  • guanidinoalkyl describes an alkyl group substituted by a guanidino group, as these terms are defined herein.
  • the alkyl group terminates by the guanidino group.
  • guanyloalkyl describes an alkyl group substituted by a guanyl group, as these terms are defined herein.
  • the alkyl group terminates by the guanyl group.
  • nitro describes a -NO 2 group.
  • cyano describes a -C ⁇ N group.
  • hydrazine describes a NR' -NR" group, with R' and R" as defined hereinabove.
  • ammonium ion describes a -(NR'R"R'") + , where R', R" and R'" as defined hereinabove.
  • phrases "pharmaceutically acceptable salt” refers to a charged species of the parent compound and its counter ion.
  • An example, without limitation, of a pharmaceutically acceptable salt would be a compound that comprises an ammonium or guanidinium cation and an anion of an acid such as, for example, HCl, trifluoroacetic acid (TFA) and the like.
  • an acid such as, for example, HCl, trifluoroacetic acid (TFA) and the like.
  • TFA trifluoroacetic acid
  • each of the compounds described hereinabove is designed based on the three-dimensional structure of a GSK-3 substrate and is therefore potential substrate competitive inhibitor of GSK-3 activity.
  • a method of inhibiting an activity of GSK-3 which is effected by contacting cells expressing GSK-3 with an inhibitory effective amount of any one of the compounds described hereinabove.
  • the term "inhibitory effective amount” is the amount determined by such considerations as are known in the art, which is sufficient to inhibit the activity of GSK-3.
  • the activity can be a phosphorylation and/or autophosphorylation activity of GSK-3.
  • the method according to this aspect of the present invention can be effected by contacting the cells with the compounds in vitro and/or in vivo. This method can be further effected by further contacting the cells with an additional active ingredient that is capable of altering an activity of GSK-3, as is detailed hereinbelow.
  • GSK-3 inhibition is a way to increase insulin activity in vivo.
  • High activity of GSK-3 impairs insulin action in intact cells (Eldar-Finkelman et al, 1997). This impairment results from the phosphorylation of insulin receptor substrate- 1 (IRS-I) serine residues by GSK-3.
  • IRS-I insulin receptor substrate- 1
  • Studies performed in patients with type II diabetes show that glycogen synthase activity is markedly decreased in these patients, and that decreased activation of protein kinase B (PKB), an upstream regulator of GSK-3, by insulin is also detected (Shulman et al, (1990); Cross et al, (1995).
  • PDB protein kinase B
  • mice susceptible to high fat diet-induced diabetes and obesity have significantly increased GSK-3 activity in epididymal fat tissue (Eldar-Finkelman et al, 1999). Increased GSK-3 activity expressed in cells resulted in suppression of glycogen synthase activity (Eldar- Finkelman et al, 1996).
  • Inhibition of GSK-3 activity therefore provides a useful method for increasing insulin activity in insulin-dependent conditions.
  • a method of potentiating insulin signaling which is effected by contacting insulin responsive cells with an effective amount, as is defined hereinabove, of any one of the compounds described hereinabove.
  • the phrase "potentiating insulin signaling" includes an increase in the phosphorylation of insulin receptor downstream components and an increase in the rate of glucose uptake as compared with glucose uptake in untreated subjects or cells.
  • the method according to this aspect of the present invention can be effected by contacting the cells with the compound of the present embodiments, in vitro or in vivo, and can be also effected by further contacting the cells with insulin.
  • Potentiation of insulin signaling, in vivo, resulting from administration of the compounds described herein, can be monitored as a clinical endpoint.
  • the easiest way to look at insulin potentiation in a patient is to perform the glucose tolerance test. After fasting, glucose is given to a patient and the rate of the disappearance of glucose from blood circulation (namely glucose uptake by cells) is measured by assays well known in the art. Slow rate (as compared to healthy subject) of glucose clearance will indicate insulin resistance.
  • the administration of an inhibitor to an insulin-resistant patient increases the rate of glucose uptake as compared with a non-treated patient.
  • the inhibitor may be administered to an insulin resistant patient for a longer period of time, and the levels of insulin, glucose, and leptin in blood circulation (which are usually high) may be determined. Decrease in glucose levels will indicate that the inhibitor potentiated insulin action. A decrease in insulin and leptin levels alone may not necessarily indicate potentiation of insulin action, but rather will indicate improvement of the disease condition by other mechanisms.
  • the compounds described hereinabove, can be effectively utilized for treating any biological condition that is associated with GSK-3.
  • a method of treating a biological condition associated with GSK-3 activity is effected by administering to a subject in need thereof a therapeutically effective amount of the any of the compounds described hereinabove.
  • biological condition associated with GSK-3 activity includes any biological or medical condition or disorder in which effective GSK-3 activity is identified, whether at normal or abnormal levels.
  • the condition or disorder may be caused by the GSK-3 activity or may simply be characterized by GSK-3 activity. That the condition is associated with GSK-3 activity means that some aspect of the condition can be traced to the GSK-3 activity.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition or disorder, substantially ameliorating clinical symptoms of a condition or disorder or substantially preventing the appearance of clinical symptoms of a condition or disorder.
  • These effects may be manifested, for example, by a decrease in the rate of glucose uptake with respect to type II diabetes or by halting neuronal cell death with respect to neurodegenerative disorders, as is detailed hereinbelow.
  • administering describes a method for bringing the compound of the present invention and cells affected by the condition or disorder together in such a manner that the compound can affect the GSK-3 activity in these cells.
  • the compounds of the present invention can be administered via any route that is medically acceptable.
  • the route of administration can depend on the disease, condition or injury being treated. Possible administration routes include injections, by parenteral routes, such as intravascular, intravenous, intra-arterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, intracerebrovascular or others, as well as oral, nasal, ophthalmic, rectal, topical, or by inhalation.
  • Sustained release administration is also specifically included in the invention, by such means as depot injections or erodible implants.
  • Administration can also be intra-articularly, intrarectally, intraperitoneally, intramuscularly, subcutaneously, or by aerosol inhalant.
  • the compound can be administered orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally or intracisternally, as long as provided in a composition suitable for effecting the introduction of the compound into target cells, as is detailed hereinbelow.
  • the phrase "therapeutically effective amount", as used herein, describes an amount administered to an individual, which is sufficient to abrogate, substantially inhibit, slow or reverse the progression of a condition associated with GSK-3 activity, to substantially ameliorate clinical symptoms of a such a condition or substantially prevent the appearance of clinical symptoms of such a condition.
  • the GSK-3 activity can be a GSK-3 kinase activity.
  • the inhibitory amount may be determined directly by measuring the inhibition of a GSK-3 activity, or, for example, where the desired effect is an effect on an activity downstream of GSK-3 activity in a pathway that includes GSK-3, the inhibition may be measured by measuring a downstream effect.
  • the effects of the compound may include effects on an insulin-dependent or insulin-related pathway, and the compound may be administered to the point where glucose uptake is increased to optimal levels.
  • the inhibition of GSK-3 results in the absence of phosphorylation of a protein that is required for further biological activity, for example, the tau protein
  • the compound may be administered until polymerization of phosphorylated tau protein is substantially arrested. Therefore, the inhibition of GSK-3 activity will depend in part on the nature of the inhibited pathway or process that involves GSK-3 activity, and on the effects that inhibition of GSK-3 activity has in a given biological context.
  • the amount of the compound that will constitute an inhibitory amount will vary depending on such parameters as 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 dose of treatment or pattern of administration, the formulation, the attending physician's assessment of the medical situation, and other relevant factors, and in general the health of the patient, and other considerations such as prior administration of other therapeutics, or co ⁇ administration of any therapeutic that will have an effect on the inhibitory activity of the compound or that will have an effect on GSK-3 activity, or a pathway mediated by GSK-3 activity. It is expected that the inhibitory amount will fall in a relatively broad range that can be determined through routine trials.
  • GSK-3 is involved in various biological pathways and hence, the method according to this aspect of the present invention can be used in the treatment of a variety of biological conditions, as is detailed hereinunder.
  • GSK-3 is involved in the insulin signaling pathway and therefore, in one example, the method according this aspect of the present invention can be used to treat any insulin-dependent condition.
  • GSK-3 inhibitors are known to inhibit differentiation of pre-adipocytes into adipocytes, and therefore, in another example, the method of this aspect of the present invention can be used to treat obesity.
  • the method according to this aspect of the present invention can be used to treat diabetes and particularly, non-insulin dependent diabetes mellitus.
  • Diabetes mellitus is a heterogeneous primary disorder of carbohydrate metabolism with multiple etiologic factors that generally involve insulin deficiency or insulin resistance or both.
  • Type I juvenile onset, insulin-dependent diabetes mellitus, is present in patients with little or no endogenous insulin secretory capacity. These patients develop extreme hyperglycemia and are entirely dependent on exogenous insulin therapy for immediate survival.
  • Type II or adult onset, or non-insulin- dependent diabetes mellitus, occurs in patients who retain some endogenous insulin secretory capacity, but the great majority of them are both insulin deficient and insulin resistant.
  • NIDDM non-insulin dependent, Type II diabetes mellitus
  • Insulin resistance is an underlying characteristic feature of NIDDM and this metabolic defect leads to the diabetic syndrome. Insulin resistance can be due to insufficient insulin receptor expression, reduced insulin-binding affinity, or any abnormality at any step along the insulin signaling pathway (see U.S. Patent No. 5,861,266).
  • the compounds of the present invention can be used to treat type II diabetes in a patient with type II diabetes as follows: a therapeutically effective amount of the compound is administered to the patient, and clinical markers, e.g., blood sugar level, are monitored.
  • clinical markers e.g., blood sugar level
  • the compounds of the present invention can further be used to prevent type II diabetes in a subject as follows: a prophylactically effective amount of the compound is administered to the patient, and a clinical marker, for example IRS-I phosphorylation, is monitored.
  • Treatment of diabetes is determined by standard medical methods. A goal of diabetes treatment is to bring sugar levels down to as close to normal as is safely possible. Commonly set goals are 80-120 milligrams per deciliter (mg/dl) before meals and 100-140 mg/dl at bedtime.
  • a particular physician may set different targets for the patent, depending on other factors, such as how often the patient has low blood sugar reactions.
  • Useful medical tests include tests on the patient's blood and urine to determine blood sugar level, tests for glycated hemoglobin level (HbA lc ; a measure of average blood glucose levels over the past 2—3 months, normal range being 4-6 %), tests for cholesterol and fat levels, and tests for urine protein level. Such tests are standard tests known to those of skill in the art (see, for example, American Diabetes Association, 1998).
  • a successful treatment program can also be determined by having fewer patients in the program with diabetic eye disease, kidney disease, or nerve disease.
  • a method of treating non-insulin dependent diabetes mellitus a patient is diagnosed in the early stages of non-insulin dependent diabetes mellitus.
  • a compound of the present invention is formulated in an enteric capsule.
  • the patient is directed to take one tablet after each meal for the purpose of stimulating the insulin signaling pathway, and thereby controlling glucose metabolism to levels that obviate the need for administration of exogenous insulin.
  • the method according to this aspect of the present invention can be used to treat affective disorders such as unipolar disorders (e.g., depression) and bipolar disorders (e.g., manic depression).
  • affective disorders such as unipolar disorders (e.g., depression) and bipolar disorders (e.g., manic depression).
  • GSK-3 is also considered to be an important player in the pathogenesis of neurodegenerative disorders and diseases
  • the method according to this aspect of the present invention can be further used to treat a variety of such disorders and diseases.
  • the method according to this aspect of the present invention can be used to treat a neurodegenerative disorder that results from an event that cause neuronal cell death.
  • Such an event can be, for example, cerebral ischemia, stroke, traumatic brain injury or bacterial infection.
  • the method according to this aspect of the present invention can be used to treat various chronic neurodegenerative diseases such as, but not limited to, Alzheimer's disease, Huntington's disease, Parkinson's disease, AIDS associated dementia, amyotrophic lateral sclerosis (AML) and multiple sclerosis.
  • various chronic neurodegenerative diseases such as, but not limited to, Alzheimer's disease, Huntington's disease, Parkinson's disease, AIDS associated dementia, amyotrophic lateral sclerosis (AML) and multiple sclerosis.
  • a method of treating a patient with Alzheimer's disease A patient diagnosed with Alzheimer's disease is administered with a compound of the present invention, which inhibits GSK-3-mediated tau hyperphosphorylation, prepared in a formulation that crosses the blood brain barrier (BBB).
  • BBB blood brain barrier
  • the patient is monitored for tau phosphorylated polymers by periodic analysis of proteins isolated from the patient's brain cells for the presence of phosphorylated forms of tau on an SDS-PAGE gel known to characterize the presence of and progression of the disease.
  • the dosage of the compound is adjusted as necessary to reduce the presence of the phosphorylated forms of tau protein.
  • GSK-3 has also been implicated with respect to psychotic disorders such as schizophrenia, and therefore the method according to this aspect of the present invention can be further used to treat psychotic diseases or disorders, such as schizophrenia.
  • the method according to this aspect of the present invention can be further effected by co-administering to the subject one or more additional active ingredient(s) which is capable of modulating an activity of GSK-3.
  • co-administering describes administration of a compound according to the present invention in combination with the additional active ingredient(s) (also referred to herein as active or therapeutic agent).
  • the additional active agent can be any therapeutic agent useful for treatment of the patient's condition.
  • the co-administration may be simultaneous, for example, by administering a mixture of the compound and the therapeutic agents, or may be accomplished by administration of the compound and the active agents separately, such as within a short time period.
  • Co-administration also includes successive administration of the compound and one or more of another therapeutic agent.
  • the additional therapeutic agent or agents may be administered before or after the compound. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the additional active ingredient can be insulin.
  • the additional active ingredient is capable of inhibiting an activity of GSK-3, such that the additional active ingredient according to the present invention can be any GSK-3 inhibitor other than the compounds of the present invention, e.g., a short peptide GSK-3 inhibitor as described in WO 01/49709, WO 2004/052404 and U.S. Patent No. 6,780,625.
  • the GSK-3 inhibitor can be, for example, lithium, valproic acid and/or lithium ion.
  • the additional active ingredient can be an active ingredient that is capable of downregulating an expression of GSK-3.
  • An agent that downregulates GSK-3 expression refers to any agent which affects GSK-3 synthesis (decelerates) or degradation (accelerates) either at the level of the HiRNA or at the level of the protein.
  • a small interfering polynucleotide molecule which is designed to down regulate the expression of GSK-3 can be used as an additional active ingredient according to this embodiment of the present invention.
  • An example for a small interfering polynucleotide molecule which can down- regulate the expression of GSK-3 is a small interfering RNA or siRNA, such as, for example, the morpholino antisense oligonucleotides described by in Munshi et al.
  • RNAi RNA interference
  • duplex oligonucleotide refers to an oligonucleotide structure or mimetics thereof, which is formed by either a single self- complementary nucleic acid strand or by at least two complementary nucleic acid strands.
  • the "duplex oligonucleotide” of the present invention can be composed of double-stranded RNA (dsRNA), a DNA-RNA hybrid, single-stranded RNA (ssRNA), isolated RNA (i.e., partially purified RNA, essentially pure RNA), synthetic RNA and recombinantly produced RNA.
  • the specific small interfering duplex oligonucleotide of the present invention is an oligoribonucleotide composed mainly of ribonucleic acids.
  • the small interfering polynucleotide molecule according to the present invention can be an RNAi molecule (RNA interference molecule).
  • a small interfering polynucleotide molecule can be an oligonucleotide such as a GSK-3-specific antisense molecule or a rybozyme molecule, further described hereinunder.
  • Antisense molecules are oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target polynucleotide.
  • An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNArDNA or RNA:RNA hybrids.
  • An example for such includes RNase H, which is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region.
  • RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
  • the antisense molecules of the present invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, as described above.
  • Representative U.S. patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
  • Rybozyme molecules are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs.
  • rybozyme sequences can be fused to the oligonucleotides of the present invention. These sequences include but are not limited ANGIOZYME specifically inhibiting formation of the VEGF-R (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway, and HEPTAZYME, a rybozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, (Rybozyme Pharmaceuticals, Incorporated - WEB home page).
  • VEGF-R Vascular Endothelial Growth Factor receptor
  • HCV Hepatitis C Virus
  • a small interfering polynucleotide molecule can be a DNAzyme.
  • DNAzymes are single-stranded catalytic nucleic acid molecules.
  • a general model (the "10-23” model) for the DNAzyme has been proposed.
  • "10-23" DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each.
  • This type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions (Santoro, S. W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, LM Curr Opin MoI Ther 2002;4: 119-21).
  • DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al., 20002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org). In another application, DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL.
  • Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis.
  • Equipment and reagents for executing solid-phase synthesis are commercially available from, for example,
  • the compounds described herein are preferably included, as active ingredients, in a pharmaceutical composition which further comprises a pharmaceutically acceptable carrier for facilitating administration of a compound to the treated individual and possibly to facilitate entry of the active ingredient into the targeted tissues or cells.
  • a pharmaceutical composition which comprises, as an active ingredient, any of the compounds described herein and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier any of the compounds described herein and a pharmaceutically acceptable carrier.
  • physiologically acceptable carrier refers to a carrier or a diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound.
  • carriers are propylene glycol, saline, emulsions and mixtures of organic solvents with water.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • the pharmaceutical acceptable carrier can further include other agents such as, but not limited to, absorption delaying agents, antibacterial agents, antifungal agents, antioxidant agents, binding agents, buffering agents, bulking agents, cationic lipid agents, coloring agents, diluents, disintegrants, dispersion agents, emulsifying agents, excipients, flavoring agents, glidants, isotonic agents, liposomes, microcapsules, solvents, sweetening agents, viscosity modifying agents, wetting agents, and skin penetration enhancers.
  • agents such as, but not limited to, absorption delaying agents, antibacterial agents, antifungal agents, antioxidant agents, binding agents, buffering agents, bulking agents, cationic lipid agents, coloring agents, diluents, disintegrants, dispersion agents, emulsifying agents, excipients, flavoring agents, glidants, isotonic agents, liposomes, microcapsules, solvents, sweetening agents, viscosity modifying agents
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, transdermal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the compound into preparations which can be used pharmaceutically.
  • the composition can be formulated in a delivery form such as an aerosol delivery form, aqueous solution, bolus, capsule, colloid, delayed release, depot, dissolvable powder, drops, emulsion, erodible implant, gel, gel capsule, granules, injectable solution, ingestible solution, inhalable solution, lotion, oil solution, pill, suppository, salve, suspension, sustained release, syrup, tablet, tincture, topical cream, transdermal delivery form.
  • a delivery form such as an aerosol delivery form, aqueous solution, bolus, capsule, colloid, delayed release, depot, dissolvable powder, drops, emulsion, erodible implant, gel, gel capsule, granules, injectable solution, ingestible solution, inhalable solution, lotion, oil solution, pill, suppository, salve, suspension, sustained release, syrup, tablet, tincture, topical cream, transdermal delivery form.
  • Proper formulation is dependent upon the route of administration chosen.
  • the compound of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer with or without organic solvents such as propylene glycol, polyethylene glycol.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer with or without organic solvents such as propylene glycol, polyethylene glycol.
  • organic solvents such as propylene glycol, polyethylene glycol.
  • penetrants are used in the formulation. Such penetrants are generally known in the art.
  • the compound can be formulated readily by combining the compound with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compound of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • disintegrating agents may 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.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active ingredient doses.
  • compositions which 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.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compound according to the present invention is conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the ingredient and a suitable powder base such as lactose or starch.
  • the compound described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the compound in water-soluble form. Additionally, suspensions of the compound may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredient to allow for the preparation of highly concentrated solutions.
  • the compound may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water
  • the compound of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions herein described may also comprise suitable solid of gel phase carriers or excipients.
  • suitable solid of gel phase carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.
  • compositions suitable for use in context of the present invention include compositions wherein the compound is contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to affect symptoms of a condition or prolong the survival of the subject being treated.
  • 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 may vary depending upon 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 e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1
  • P-I)- Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as a FDA approved kit, which may contain one or more unit dosage forms containing the compound.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • Suitable conditions indicated on the label may include, for example, any of the biological conditions associated with GSK-3 activity listed hereinabove.
  • the pharmaceutical composition of the present invention can be packaged in a packaging material and identified in print, on or in the packaging material, for use in the treatment or prevention of a biological condition associated with GSK-3.
  • the pharmaceutical composition of the present invention can further comprises an additional active ingredient that is capable of modulating an activity of GSK-3, as is described hereinabove. Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
  • Peptides were synthesized by Genemed Synthesis Inc. (San Francisco, CA). Radioactive materials were purchased from Amersham Ltd. Phenyl phosphate and pyridoxal phosphate (also referred to herein as P-5-P) were obtained from Sigma (Israel).
  • GSC-I, GSC-2 and GSC-3 were synthesized according to procedures known in the art, as is detailed hereinunder.
  • a solution of each peptide was prepared by dissolving lyophilized powder in water containing 10% D 2 O. 2D-NMR spectra were acquired at the 1 H proton frequency of 600.13 MHz on a Bruker Advance DMX spectrometer. The carrier frequency was set on the water signal and it was suppressed by applying either a WATERGATE method or by low-power irradiation during the relaxation period.
  • the experimental temperature (280° K) was optimized in order to reduce population averaging due to the fast exchange at more ambient temperatures, while preserving the best possible spectral resolution.
  • Resonance assignment was based on the TOCSY and NOESY spectra measured at the same experimental conditions, according to the sequential assignment methodology developed by W ⁇ thrich using the Bruker software program AURELIA (Bruker Analytic GmbH, version 2.7).
  • the NOE distance restraints were derived from NOESY spectra recorded at 450 msec. This optimal mixing time was determined for the p9CREB peptide sample by comparing the NOE signal intensities in a series of experiments with mixing times varying from 100 msec to 750 msec. The chosen mixing time gave maximal NOE buildup with no significant contribution from spin diffusion. This value was used for the non-phosphorylated analog experiment in order to maintain identical experimental conditions. Integrated peak volumes were converted into distance restraints using a r '6 dependency and the known distance of 2.47 A between the two adjacent protons of the tyrosine aromatic ring was used for calibration.
  • the restraints were classified into strong (1.8-2.5 A), medium (1.8-3.5 A) and weak (1.8-5.0 A). An empirical correction of 0.5 A was added to the upper bound for restraints involving methyl groups.
  • the structures were calculated by hybrid distance geometry - dynamical simulated annealing using XPLOR (version 3.856). The NOE energy was introduced as a square- well potential with a constant force constant of 50 Kcal/mol-A 2 . Simulated annealing consisted of 1500 3 fsec steps at 1000 K and 3000 lfsec steps during cooling to 300 K. Finally, the structures were minimized using conjugate gradient energy minimization for 4000 iterations. INSIGHTII (Molecular Modeling System version 97.0, Molecular Simulations, Inc.) was used for visualization and analysis of the NMR-derived structures. Their quality was assessed using PROCHECK. Results:
  • Tables 2 and 3 below present the structural coordinate data that was used for inputting into structure analysis software for visualization of the 3D structures.
  • GSK-3 The design of small molecules that mimic the structure presented here thus provides a method for obtaining selective inhibitors for GSK-3.
  • TMS Tetramethylsilane
  • phosphoric acid was used as an internal standard for phosphorus spectra
  • solvent peak was used as the reference peak for carbon and fluorine spectra.
  • Thin-layer chromatography was performed using Analtech silica gel plates and visualized by ultraviolet (UV) light, or by staining the plates in 0.2 wt % ninhydrine in butanol.
  • Elemental analysis was performed by Quantitative Technologies, Inc. (Whitehouse, NJ). HPLC analyses were obtained using a Hypersil BDS Cl 8 Column, 4.6 x 150 mm, 5 ⁇ m, at ambient column temperature and with a detector operating at 220 nm, using, as a mobile phase, a standard solvent gradient program, as follows:
  • the mixture was cooled to -40 °C (by means of dry ice/acetonitrile), and a solution of 85 % m-chloroperbenzoic acid (mCPBA) (0.81 gram in 1 ml dichloromethane, 4 mmol, 1.3 equivalents) in dichloromethane (4 ml) was rapidly added while the reaction temperature was kept below 0 °C.
  • mCPBA m-chloroperbenzoic acid
  • the solution was allowed to warm up to room temperature and after stirring for 5 minutes at 20 °C, 10 % aqueous NaHSCb (10 ml) was added and the mixture was stirred for a further 10 minutes.
  • the mixture was then extracted with ether (70 ml) and the aqueous phase discarded.
  • the ethereal phase was washed with 10 % aqueous NaHSO 3 (2 x 20 ml), 5 % saturated aqueous NaHCO 3 (2 x 20 ml), dried on sodium sulfate and filtered.
  • the organic filtrate was evaporated and the residue was purified by chromatography on a silica gel column, using a mixture of EtOAc/hexanes 1 : 15 as eluent, to give a mixture of the product (di-tert-butyl, p-methyl benzyl phosphate) and the starting material, which was used without further separation.
  • the ethereal phase was washed with 10 % aqueous NaHSO 3 (2 x 20 ml), 5 % saturated aqueous NaHCO 3 (2 x 20 ml), dried over sodium sulfate and filtered.
  • the organic layer was evaporated and the residue was purified by chromatography on a silica gel column using a mixture of EtOAc/hexanes 1:15 as eluent, to give a mixture of the product (di-tert-butyl, benzyl phosphate) and the starting material, which was used without further purification.
  • the benzyl alcohol intermediate (see, Scheme 4) was identified as a key intermediate obtainable in four steps from the inexpensive starting material trimethyl 1,3,5-benzenetricarboxylate, as is detailed hereinbelow and is depicted in Scheme 5.
  • Bis(cyanomethyl)benzyl alcohol (8.0 grams, 0.04 mol) was divided in three parts and each 2.5- to 3.0-grams portion was charged into separate 500-ml Parr bottles, followed by ethanol (100 ml), and aqueous NaOH (1.2 grams in 5 ml of water). To the resulting solution was added Raney Ni (50 % suspension in water, 1.2 grams). The mixture was hydrogenated at 30 psi on a Parr shaker. The reaction was monitored by 1 H NMR and judged complete after 3 hours. The catalyst was filtered on a pad of diatomaceous earth and the diatomaceous earth pads washed with ethanol (200 ml).
  • Di-tert-butyl diisopropylphosphoramidite (49.8 ml, 157.9 mmol) in anhydrous acetonitrile (1 liter) was added via the pressure-equalizing addition funnel at such a rate that the reaction temperature was maintained ⁇ 6 °C.
  • 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) and added via the pressure-equalizing addition funnel at such a rate that the temperature was maintained below 6 °C.
  • Trifluoroacetic acid (287 ml, 10 volumes) was added rapidly via the pressure-equalizing addition funnel. The resulting solution was stirred for 5 hours. After concentrating and drying overnight under high vacuum, a thick orange oil (37.88 grams) was obtained. The residue was dissolved in water (57 ml, 1.5 volumes) and added dropwise into stirred methanol (90 volumes) yielding a precipitate. After stirring for 30 minutes, the solids were allowed to settle for 1 hour and the liquid was decanted off. The remaining liquid was removed in vacuum giving 13.72 grams of solid. The material was dissolved in water (68 ml, 5 volumes) and loaded onto Dowex 50WX8-200 ion-exchange resin (137 grams).
  • GSC-5 3-(guanidinomethy) benzyl phosphate
  • the ethereal phase was washed with 10 % aqueous NaHSO 3 (2 x 20 ml) and saturated aqueous NaHCO 3 (2 x 20 ml), dried over sodium sulfate and filtered.
  • the organic filtrate was evaporated and the residue was purified by chromatography on a silica gel column using a gradient eluent of ethyl acetate/hexanes 1:9 to 1:5), to give a mixture of the phosphate ester product and the benzyl alcohol starting material, which 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-tert-butyl, 3-(N,N'-bis-BOC- guanidinomethy) benzyl phosphate in 70 % yield.
  • the trifluoroacetic acid can be removed or replace by, for example, HCl, using procedures well known in the art, to give the free guanidine or, for example, a hydrochloride salt of the compound.
  • GSC-4 3-guanidinobenzyl phosphate
  • the mixture was thereafter 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 of hexanes to 40:60 ethyl acetaterhexanes), to give the intermediate in 60 % yield.
  • the ethereal phase was washed with 10 % aqueous NaHSO 3 (2 x 20 ml) and saturated aqueous NaHCO 3 (2 x 20 ml), dried over MgSO 4 and filtered.
  • the solvent was evaporated and the residue was purified by chromatography on a silica gel column using a gradient eluent of ethyl acetate/hexanes 10:90 to 30:70), to give the protected product in 60 % yield.
  • the trifluoroacetic acid can be removed or replace by, for example, HCl, using procedures well known in the art, to give the free guanidine or, for example, a hydrochloride salt of the compound.
  • GSC-7 was prepared as follows:
  • the resulting mixture was stirred for 50-60 minutes at room temperature, then cooled to -40 0 C (by means of dry ice/acetonitrile) and a solution of 77 % m-CPBA (0.8 grams, 0.0045 mol, 1.5 molequivalent) in 5 ml dichloromethane was rapidly added while maintaining the reaction temperature below 0 0 C.
  • the reaction mixture was thereafter allowed to warm to room temperature and was stirred for 30 minutes. 10 % aqueous NaHSO 3 (10 ml) was then added and the mixture stirred for additional 10-15 minutes.
  • Activation of glycogen synthase by GSK-3 inhibitors Activation of glycogen synthase can serve as a good marker for inhibition of GSK-3.
  • activation of glycogen synthase by GSC-5 and GSC-7 was tested in C2C12 myotubes. C2C12cells were treated with GSC-5 and GSC-7 for 2.5 hours at indicated concentrations and lysate supernatants were thereafter assayed for glycogen synthase activity.
  • glycogen synthase in cells treated with vehicle DMSO (0.1 % DMSO) was normalized to 1 unit and the values for glycogen synthase activity observed in cells treated with GSK-3 inhibitors are presented as fold stimulation over the cells treated with vehicle 0.1 % HCL.
  • bovine serum albumin Fraction V, Boehringer Mannheim, Germany
  • GSK-3 inhibition activity of GSC-I, GSC-2, GSC-3, GSC-4, and GSC-21 was tested.
  • the ability of GSK-3 to phosphorylate PGS-I peptide substrate was measured in the. presence of indicated concentrations of these compounds.
  • the results, presented in Figure 6, represent the percentage of GSK-3 activity as compared with a control incubation without inhibitors and are mean of 2 independent experiments ⁇ SEM, where each point was assayed in triplicate.
  • the selectivity of the novel compounds towards GSK-3 was measured by evaluating the inhibition of CDK-2, a kinase closely related to GSK-3.
  • CDK-2 activity was assayed in the presence of 32 P[ ⁇ -ATP] and histone Hl as a substrate.
  • GSC-4, GSC-5 and GSC-7 were added at a final concentration of 2 mM. The results are presented in Figure 9 and clearly show that no inhibition of histone Hl phosphorylation was observed, thus indicating high selectivity of the compounds towards GSK-3.
  • Activation of glycogen synthase by GSK-3 inhibitors Activation of glycogen synthase activity in C2C12 cells treated with GSC-5 and GSC-7 was assayed as described hereinabove.
  • the activity of glycogen synthase in cells treated with vehicle DMSO (0.1% DMSO) was normalized to 1 unit and the values for glycogen synthase activity observed in cells treated with GSK-3 (GS4 (hollow circles) GS6 (filled circles) are presented as fold stimulation over the cells treated with vehicle 0.1% HCL. The results are presented in Figure 10 and clearly show that both GSC-5
  • GSC-7 filled circles activated glycogen synthase by 1.5 and 1.8 fold, respectively.
  • Glucose Uptake The ability of the newly designed compounds GSC-4 and GSC-21 to promote glucose uptake was tested in mouse primary adipocytes as described hereinabove. The relative [ 3 H] 2-deoxy glucose incorporation observed in non-treated adipocytes was normalized to 1 unit and the values obtained for [ 3 H] 2- deoxy glucose in adipocytes treated with GSC-4 or GSC-21 are presented as fold activation over cells treated with the peptide control, and are the mean of 6 independent experiments ⁇ SEM, where each point was assayed in triplicate.
  • Simulated annealing is a molecular modeling method that is used to find stable conformations of proteins. The study examined the interaction of the newly designed
  • GSK-3 inhibitors described above also referred to herein as GSC molecules
  • GSK-3 catalytic domain and was based on protein crystallography data of GSK-3, as taught by Ter Haar et al. (2001).
  • Simulated annealing uses a repetitive heating and cooling of the system to find the best energetic minima of the system.
  • an additional parameter, the "Protein - Ligand Binding Energy" was added to the consideration of electing a new starting point in each interval of heating.
  • Stimulated annealing included the following steps:
  • the hydrogen atoms, the substrate analogue and the side chains and water molecules that are in a 15 A radius from the phosphate atom were set free.
  • Cut off 9.5 A distance dependent; each step was lfs.
  • the combined approach produced many conformations. For each starting point, the conformation with lowest binding energy in the last (15th) simulation cycle was evaluated. The binding energy was compared and the best one was chosen. In some cases more then one conformation for each ligand was analyzed.
  • FIGs 12-17 show the interactions between the best conformation of the analyzed compound (inhibitor) and the amino acids in the catalytic domain of GSK-3, as determined by the stimulated annealing described above.
  • the predominant interactions between the analyzed compounds and the catalytic domain include electrostatic and/or hydrogen bonding interactions between the amine or guanidine moiety of the inhibitor and an acidic moiety at a side chain of an amino acid (e.g., of glutamic acid and aspartic acid) or the hydroxyl group of tyrosine, as well as aromatic and/or hydrophobic interactions between an aromatic moiety of the inhibitor and an aromatic or hydrocarbon side chain of an amino acid (e.g., phenylalanine, tyrosine and isoleucine).
  • an amino acid e.g., of glutamic acid and aspartic acid
  • aromatic and/or hydrophobic interactions between an aromatic moiety of the inhibitor and an aromatic or hydrocarbon side chain of an amino acid e.g., phenylalan
  • Phe67 is an important binding moiety in the binding site of the enzyme and, moreover, it is like a door closing on the binding site.
  • Table 4 hereinunder presents the distances between various atoms of the analyzed inhibitor and the various amino acid residues of the enzyme which interact with these atoms.
  • the number (presented in A) is the shorter distance between the inhibitor and the residue.
  • GSC-4 in which a guanidine group is attached directly to the aromatic ring
  • the interaction of the guanidine group in GSC-6, in which the guanidine group is attached to the ring via an ethylene spacer, with the acid groups (e.g., Aspl81) is tight.
  • Aromatic interactions with Phe67 and Tyr216 are also observed.
  • Interaction of GSC-8 with GSK-3 As shown in Figure 16, GSC-8 perfectly fits the binding site. The best conformation includes one guanido arm that points to As ⁇ 90 (1.71 A) and the other to Glu97 (1.88 A), and additional interactions with Phe67 (3.6), Arg96 (2.06 A) and Argl ⁇ O (2.63 A).
  • the complex interaction has a good binding energy.
  • GSC-9 perfectly fits the binding site, with increased number of interactions.
  • the best conformation includes one guanido arm that points to Asp90 (3.43 A) and the other to Glu97 (2.38 A).
  • the guanido groups interact with additional residues such as Glu200, Gln89 and the backbone o Leu88, such that the binding is strengthened. Phe67, though not strongly interacting, seems to close on the binding site.
  • the reaction mixture was thereafter poured into water (8 ml) and extracted three times with diethylether (3 x 8 ml). The combined organic layers were washed with water and brine (8 ml), the aqueous phase was re-extracted two times with diethylether (2 x 8 ml), and the combined organic layers were dried with MgSO 4 and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (using a gradient eluent of hexanes and a 20:80 ethyl acetaterhexanes mixture).
  • a 50 ml round bottomed flask was charged with a stirring bar, 25 ml 2N HCl, and aminobenzylalcohol (2.15 grams, 17.5 mmol, 1 molequivalent). The solution was cooled to -5°C in a salt-ice bath. An ice-cold solution of sodium nitrite (1.45 gram, 21 mmol, 1.2 molequivalent) in 5 ml water was slowly added over five minutes such that the temperature of the reaction did not rise above -3 °C. After five minutes, 125 mg urea was added to destroy the excess nitrous acid.
  • the solution of the resulting diazonium salt was then added during five minutes to a stirred ice-cold solution of sodium azide (2.28 grams , 35 mmol, 2 molequivalent) and sodium acetate (4.20 grams, 51 mmol, 3 molequivalent) in 25 ml water.
  • the mixture was stirred for 2 hours at 0 °C, and the dark oily product was extracted with diethyl ether (2 x 50ml).
  • the ethereal solution was washed with IN NaOH (2 x 50ml) and water (2 x 50ml), dried over MgSO 4 , and evaporated to dryness. The obtained compound was sufficiently pure and was used without further purification.
  • CuSO 4 (0.02 gram, 0.125 mmol, 0.5 molequivalent) and Cu wires, were added to a solution of a N-(emynylphenyl),N',N'-bis(fert-butoxycarbonyl) guanidine (0.07 gram, 0.212 mmol, 1 molequivalent) and a di-fer/-butyl, azidobenzyl phosphate (0.07 gram, 0.205 mmol, 1 molequivalent) in DMF (5 ml) and the resulting mixture was stirred under nitrogen atmosphere overnight.
  • This compound was obtained as described hereinabove, except that the mixing was continued for 22 hours.
  • ATOM 62 CD ARG 4 2.627 -2.865 4.571 1.00 0.00
  • ATOM 63 HDl ARG 4 1.720 -2.327 4.798 1.00 0.00

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Abstract

La présente invention a trait à de nouveaux composés destinés à permettre des interactions avec des sites de liaison de la GSK-3 et donc capables d'inhibition de l'activité de la GSK-3, grâce à l'inhibition de liaison de substrat. L'invention a également trait à des compositions pharmaceutiques comportant de tels composés et à des procédés d'utilisation de tels composés dans le traitement de conditions médiées par la GSK-3.
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