SMALL MOLECULE MODULATORS OF GSK-3 ACTIVITY

RELATED APPLICATION/S

This application claims the benefit of priority under 35 USC § 119(e) of U.S. Provisional Patent Application No. 63/072,291 filed on August 31, 2020, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 88351 Sequence Listing.txt, created on 31 August 2021, comprising 10,034 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to novel glycogen synthase kinase-3 (GSK-3) modulators (e.g., inhibitors) and, more particularly, but not exclusively, to small molecule compounds that are usable in modulating an activity of GSK-3 (e.g., as substrate competitor inhibitors of GSK-3) and to the use of such compounds, for example, in the treatment of biological (e.g., medical) conditions associated with GSK-3 activity.

Protein kinases (PK) are important regulators of many biological processes, and represent an important class of targets for a diversity of human diseases and pathologies. Most protein kinase inhibitors developed to date are small molecules that compete with ATP binding of the kinase. This type of inhibitor, although powerful, often has limited specificity because the ATP binding site is highly conserved among protein kinases. Indeed, the vast majority of these inhibitors interact and cross-react with multiple members of the PK family; furthermore, they tend to induce drug resistance due to the formation of point mutations at the ATP binding site, a phenomenon that is likely to become an even greater challenge in the future.

A different type of protein kinase inhibitor, although not extensively studied, is the class of substrate competitive inhibitors (SCIs). SCIs interact with the less conserved (and consequently, more specific) substrate-binding cavity of the kinase, since they mimic unique interactions that occur in the substrate binding site. SCIs hold great promise as suitable therapeutics because they are highly selective, considered safe, and are less prone to drug-induced resistance. However, SCI peptides (that mainly function as ‘pseudosubstrates’) previously described for a number of protein kinases such as cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and calmodulin- dependent protein kinase, were shown to act only as weak inhibitors (IC ₅₀ values were in the mM range) [see, for example, Kemp et al. Methods Enzymol 201, 287-304 (1991); Soderling et al.. J Biol Chem 265, 1823-6 (1990); Eichholtz et al. J Biol Chem 268, 1982-6 (1993);’ and Brickeyet al. J Biol Chem 269, 29047-54 (1994)].

Identifying small-molecule SCIs is a challenge because the substrate binding site is large and shallow, complicating efficient drug design. Hence, SCI small molecules were barely developed.

Glycogen synthase kinase -3 (GSK-3) is recognized as an important target for drug discovery and its inhibition has been considered a promising therapeutic approach for treating several pathologies including neurodegenerative diseases, behavior and malignancies. In humans, GSK-3 is expressed as two isozymes, GSK-3α and GSK-3α (SEQ ID NO:1) which are encoded by two genes and share high homology in their catalytic domains. The mechanisms by which GSK-3 is thought to contribute to pathogenesis are diverse. These include phosphorylation of the microtubule-associated protein tau, destabilization of the Wnt signaling component β-catenin, regulation of multiple transcription factors such as NF-KB, activation of pro-inflammatory factors, and impairment of clearance pathways. Most of the GSK-3 inhibitors that were developed up to date, failed in the pre-clinical phase due to toxicity and side effects.

Some of the present inventors have previously reported of a novel class of substrate competitive inhibitors for GSK-3 [Plotkin et al. (2003) J. Pharmacol. Exp. Then, 974-980], designed based on the unique substrate-recognition motif of GSK-3 that includes a phosphorylated residue (usually serine) in the context of SXXXS(p) (SEQ ID NO:2) (where S is the target serine, S(p) is phosphorylate serine and X is any amino acid) [see also Woodgett & Cohen (1984) Biochim. Biophys. Acta. 788, 339-47; Fiol et al. (1987) J. Biol. Chem. 262, 14042-8]. Structural studies of GSK-3P identified a likely docking site for the phosphorylated residue; it is a positively charged binding pocket composed of Arg96, Argl80, and Lys205 [Dajani et al. (2001) Cell 105, 721-32; ter Haar et al. (2001) Nature Structural Biology 8, 593-6].

While further focusing on substrate recognition of GSK-3, three positions in the vicinity of the catalytic site (Phe67 in the P-loop, Gln89 and Asn95) were identified as important for GSK- 3 substrates binding [Houz et al. (2006) J. Biol. Chem. 281, 30621-30], and a cavity bordered by loop 89-QDKRFKN-95 (SEQ ID NO:3) located in the vicinity of the GSK-3β catalytic core, has also been identified as a substrate binding subsite.

In-silico modeling of the interaction of GSK-3 with its substrates pCREB and p9CREB, corroborated by mutation experiments, suggested that the substrates bind in the deep trough between the N- and C-terminal lobes of the kinase (Houz et al. 2006, supra). It was further suggested that the pre-phosphorylated S 133 _p residue is located in the phosphate binding pocket of GSK-3 formed by residues R96, R180 and K205, and the phosphorylation target S129 points toward the y phosphate of ATP.

The present inventors have developed a series of SCI peptides targeting GSK-3 and have shown that it is possible to optimize the potency and selectivity of such SCIs by manipulating the peptide sequence [Licht-Murava, A. et al. Science Signaling 9, ral 10 (2016); Licht-Murava et al. J. Mol. Biol. 408, 366-78 (2011)]. Combined computational and biochemical analysis identified the critical sites considered important for substrates and for inhibitor interactions and enabled SCIs to improve by adding new elements that strengthened interactions at the substrates’ binding sites, or formed new interactions with the binding site residues [Licht-Murava et al., 2011, 2016, supra]. Specifically, it was found that in addition to the ‘phosphate binding pocket’ (residues Arg 96, Arg 180, and Lys 205) that binds the pre-phosphorylated residue in GSK-3 substrates, the substrates/SCI peptides bind to a segment bordered by Gin 89-Asn 95, also termed the ’89-95’ loop (SEQ ID NO:3). In some cases, SCI peptides interact with a ‘hydrophobic patch’ (Vai 214, I1e 217, and Tyr 216) located in proximity to the GSK-3 -phosphate binding pocket. See, for example, Dajani, R. et al. Cell 105, 721-32 (2001); ter Haar, E. et al. Nature Structural Biology 8, 593-6 (2001); Ilouz. R. J. Biol. Chem. 281, 30621-30 (2006)].

Accordingly, several SCI peptides targeting GSK-3 were developed and their therapeutic potential was validated in several disease model systems including Alzheimer' s disease, multiple sclerosis, depressive behavior, and Fragile X syndrome. See, for example, vrahami, L. et al. J. Biol. Chem. 288, 1295-1306 (2013); Licht-Murava et al., 2011 (supra); Kaidanovich-Beilin et al. Biol. Psychiatry (2004); Pardo, M. et al. JCI Insight 2, e91782 (2017); Beurel, E. et al. Regulation of J. Immunol. 190, 5000-11 (2013)].

WO 2012/101599 describes studies conducted for identifying sites of GSK-3 that play an important role in binding GSK-3 substrates. In these studies, a role of Phe93, as well as of other amino acids within the 89-95 loop (SEQ ID NO:3) of a GSK-3 enzyme, in interacting with GSK- 3 substrates and hence with GSK-3 substrate competitive inhibitors, was uncovered, thereby indicating that a putative substrate competitive inhibitor should exhibit an interaction with the Phe93 residue, or with an equivalent amino acid thereof, in a GSK-3 enzyme Peptidic substrate competitive GSK-3 inhibitors were designed after the recognition motif of HSF while modifying the peptide’s hydrophobic nature by replacing hydrophilic polar amino acids by hydrophobic amino acids residues such as alanine and proline. Exemplary such substrate competitive inhibitors, which feature a hydrophobic amino acid residue at the first position upstream the phosphorylated serine or threonine residue, exhibited improved activity compared to, for example, L8O3 (SEQ ID NO:4).

WO 2012/101601 describes additional studies in which initial models obtained by rigid body docking of selected L8O3 conformers to GSK-3P with the geometric-electrostatichydrophobic version of MolFit followed by filtering based on statistical propensity measures and solvation energy estimates, were subjected to molecular dynamics (MD) simulations. These computations provided further understanding on the binding of the inhibitor. The computational model structures, supported by experimental data, have shown that a modified L8O3 peptide, which features a Phe residue at the C-terminus of L8O3, and which is termed L8O3F (SEQ ID NO:5), exhibits a substantially improved interaction with Phe 93, via its Pro8 and Phel2, and with the hydrophobic surface patch of GSK-3p.

Further in silico modeling suggested that while GSK-3 substrates interact with the positive pocket delimited by residues R96, R180 and K205, with the substrate binding cavity delimited by the P-loop and loop 89-95 (SEQ ID NO:3), with the protruding F93 residue, and with the hydrophobic surface patch opposite the P-loop, which consists of residues V214, Y216 and 1217, the inhibitor E8O3 (SEQ ID NO:4) uses only part of the sub-sites mentioned above: the prephosphorylated serine binds in the positive pocket but the other contacts are hydrophobic. Thus, L8O3 interacts with GSK-3 F93 and with the hydrophobic patch but it does not interact with the P-loop or with the substrate binding cavity.

Small molecules that are taught as acting as SCIs of GSK-3 are described, for example, in Marchena et al. Journal of Enzyme Inhibition and Medicinal Chemistry, 2017, Vol. 32, No. 1, 522-526 and in WO 2005/097117.

Compounds featuring an anthracenone-isoxazole core substituted by piperazine or piperidine and their use as tyrosine kinase inhibitors and/or in treating cancer are described in 2012/119079, U.S. Patent Application Publication No. 2015/128132, WO 2016/043975 and WO 2016/027465.

Additional background art includes WO 2014/207743, WO 2012/101599, WO 2012/101601, WO 2006/054298, WO 2005/000192, WO 2004/052404 and WO 01/49709, and Rippin et al., Int. J. Mol. Sci. 2020 Nov; 21(22): 8709, all being incorporated by reference as if fully set forth herein. SUMMARY OF THE INVENTION

The present inventors have uncovered small molecule compounds that are usable as substrate competitive inhibitors (SCIs) for the protein kinase GSK-3. As GSK-3 is a therapeutic target for multiple diseases, these compounds are usable in treating variable medical conditions associated with GSK-3 activity.

The small molecule compounds were uncovered by means of pharmacophore design and virtual screening, upon “translating” the interactions of GSK-3 with previously studied SCI peptides.

Embodiments of the present invention relate to small molecule compounds which are capable of acting as substrate competitive inhibitors of GSK-3, as described herein.

Embodiments of the present invention relate to small molecule compounds which are usable in modulating (e.g., inhibiting) an activity of GSK-3 and to uses thereof. The small molecule compounds are as described in the Examples section and in the following description and claims.

According to an aspect of some embodiments of the present invention there is provided a compound represented by Formula I: wherein:

Wi and W2 are each independently O or S;

L is or comprises a substituted or unsubstituted, linear or branched, alkyl, substituted or unsubstituted aryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heteroalicyclic, a substituted or unsubstituted heteroaryl, or is absent; Y is a negatively charged group; q is a positive integer representing the number of negatively charged groups attached to L or to NH;

X is an alicyclic or heteroalicyclic 5, 6, 7, or 8-membered ring; n is 0 or a positive integer that represents the number of substituents A of the X;

A is independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, haloalkyl, carboxylate, thiocarboxylate, amine, amide, carbamyl, thiocarbamyl, carbonyl, thiocarbonyl, alkoxy, aryloxy, hydroxy, thiol, thioalkoxy, thioaryloxy, alkenyl, alkynyl, cyano, nitro, azo, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, phosphoryl, ketoester, ether, thioether, urea, thiourea, guanyl, guanidino, carbohydrate, hydrazine, and hydrazide, or, alternatively, when n is greater than 1, two or more of the A substituents form at least one four-, five- or six-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring; m is 0 or a positive integer that represents the number of substituents B of the respective phenyl;

B is independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, haloalkyl, carboxylate, thiocarboxylate, amine, amide, carbamyl, thiocarbamyl, carbonyl, thiocarbonyl, alkoxy, aryloxy, hydroxy, thiol, thioalkoxy, thioaryloxy, alkenyl, alkynyl, cyano, nitro, azo, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, phosphoryl, ketoester, ether, thioether, urea, thiourea, guanyl, guanidino, carbohydrate, hydrazine, and hydrazide, or, alternatively, when m is greater than 1, two or more of the B substituents form at least one four-, five- or six-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring; and

D is hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, haloalkyl, carboxylate, thiocarboxylate, amine, amide, carbamyl, thiocarbamyl, carbonyl, thiocarbonyl, alkoxy, aryloxy, hydroxy, thiol, thioalkoxy, thioaryloxy, alkenyl, alkynyl, cyano, nitro, azo, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, phosphoryl, ketoester, ether, thioether, urea, thiourea, guanyl, guanidino, carbohydrate, hydrazine, or hydrazide, or a pharmaceutically acceptable salt thereof, provided that: when X is piperidine, n is a positive integer and at least one of the A substituents is or comprises an aryl or a heteroaryl; or when X is piperidine or piperazine, L is not an aryl or heteroaryl; or when X is piperazine and n is a positive integer, A is other than a cycloalkyl, the compound being for use in modulating (e.g., inhibiting) an activity of GSK-3. According to some of any of the embodiments described herein for Formula I, L is or comprises an alkyl (e.g., an unsubstituted linear or branched alkyl, preferably a lower linear alkyl).

According to some of any of the embodiments described herein for Formula I, the negatively charged group is selected from the group consisting of carboxylate, carbamate, thiocarbamate, phosphonate, phosphate, sulfonate, and sulfate, as these terms are defined herein.

According to some of any of the embodiments described herein for Formula I, Y is or comprises a carboxylate (e.g., one or more carboxylates).

According to some of any of the embodiments described herein for Formula I, Y is or comprises a phosphate or phosphonate.

According to some of any of the embodiments described herein for Formula I, X is selected from cyclohexane, piperazine and piperidine.

According to some of any of the embodiments described herein for Formula I, X is piperazine.

According to some of any of the embodiments described herein for Formula I, n is a positive integer and at least one A is an aryl (e.g., phenyl).

According to some of any of the embodiments described herein for Formula I, X is piperazine, n is a positive integer and at least one A is an aryl (e.g., phenyl).

According to some of any of the embodiments described herein for Formula I, n is greater than 1, and at least one another A is an aryl (e.g., phenyl).

According to some of any of the embodiments described herein for Formula I, when one or more of A is aryl, the aryl can independently be a substituted aryl (e.g., a substituted phenyl).

According to some of any of the embodiments described herein for Formula I, when one or more of A is aryl, the aryl can independently be substituted by one or more fluoro substituent(s).

According to some of any of the embodiments described herein for Formula I, when one or more of A is aryl, the aryl can independently be substituted by one or more of a carboxylate substituent, an alkoxy substituent and a hydroxy substituent.

According to some of any of the embodiments described herein for Formula I, D is hydrogen.

According to some of any of the embodiments described herein for Formula I, m is 0.

According to an aspect of some embodiments of the present invention there is provided a compound of Formula I as described herein in any of the respective embodiments and any combination thereof, for use in treating a medical condition associated with GSK-3 activity in a subject in need thereof. According to an aspect of some embodiments of the present invention there is provided a compound represented by Formula II: wherein:

Wi and W ₂ are each independently O or S;

L is or comprises a substituted or unsubstituted, linear or branched, alkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heteroalicyclic, a substituted or unsubstituted heteroaryl, or is absent; Y is a negatively charged group; q is a positive integer representing the number of negatively charged groups attached to L or to NH;

X is an alicyclic or heteroalicyclic 5, 6, 7, or 8-membered ring; n is 0 or a positive integer that represents the number of substituents A of the X;

A is independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, haloalkyl, carboxylate, thiocarboxylate, amine, amide, carbamyl, thiocarbamyl, carbonyl, thiocarbonyl, alkoxy, aryloxy, hydroxy, thiol, thioalkoxy, thioaryloxy, alkenyl, alkynyl, cyano, nitro, azo, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, phosphoryl, ketoester, ether, thioether, urea, thiourea, guanyl, guanidino, carbohydrate, hydrazine, and hydrazide, or, alternatively, when n is greater than 1, two or more As form at least one four-, five- or sixmembered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring; m is 0 or a positive integer that represents the number of substituents B of the respective phenyl; B is independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, haloalkyl, carboxylate, thiocarboxylate, amine, amide, carbamyl, thiocarbamyl, carbonyl, thiocarbonyl, alkoxy, aryloxy, hydroxy, thiol, thioalkoxy, thioaryloxy, alkenyl, alkynyl, cyano, nitro, azo, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, phosphoryl, ketoester, ether, thioether, urea, thiourea, guanyl, guanidino, carbohydrate, hydrazine, and hydrazide, or, alternatively, when m is greater than 1, two or more B substituents form at least one four-, five- or six-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring; and

D is hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, haloalkyl, carboxylate, thiocarboxylate, amine, amide, carbamyl, thiocarbamyl, carbonyl, thiocarbonyl, alkoxy, aryloxy, hydroxy, thiol, thioalkoxy, thioaryloxy, alkenyl, alkynyl, cyano, nitro, azo, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, phosphoryl, ketoester, ether, thioether, urea, thiourea, guanyl, guanidino, carbohydrate, hydrazine, or hydrazide, or a pharmaceutically acceptable salt thereof, provided that: n is greater than one, and at least two of the A substituents is or comprises an aryl or heteroaryl; and/or at least one of the negatively charged group(s) is other than carboxylate; and/or q is greater than 1.

According to some of any of the embodiments described herein for Formula II, Y is or comprises a phosphate.

According to some of any of the embodiments described herein for Formula II, q is 1.

According to some of any of the embodiments described herein for Formula II, A is an aryl and the aryl is substituted by one or more of hydroxy, alkoxy, thioalkoxy, thiol, phosphate, carboxylate, halo (e.g., fluoro), sulfate and sulfonate.

According to some of any of the embodiments described herein for Formula II, the aryl is substituted by one or more of hydroxy, alkoxy and carboxylate (e.g., carboxylic acid).

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the compound of Formula II as described herein and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present invention there is provided a compound of Formula II as described herein or the composition as described herein in any of the respective embodiments, for use in modulating (e.g., inhibiting) an activity of GSK-3. According to an aspect of some embodiments of the present invention there is provided a compound or a composition as described herein in any of the respective embodiments and any combination thereof for use in treating a medical condition associated with GSK-3 activity in a subject in need thereof.

According to an aspect of some embodiments of the present invention there is provided a process of preparing a compound of Formula II as described herein in any of the respective embodiments and any combination thereof.

According to an aspect of some embodiments of the present invention there is provided a compound represented by Formula III, as described herein in any of the respective embodiments.

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

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGs. 1A-1D (Background Art) present a structural model of GSK-3P (SEQ ID NO: bound to L8O3F peptide (SEQ ID NO:5) (FIG. 1A), dose response curves of GSK-3P inhibition by L8O3mts (SEQ ID NO:6) vs L8O3Fmts (SEQ ID NO:7) (FIG. IB), and Western blots (FIG. 1C) and a bar graph (FIG. ID) showing the in vivo effect of L8O3Fmts (SEQ ID NO:7) (60 pg/mouse) on hippocampus tau Phosphorylation (Ser396) (Results represent the mean of 5 animals ± SEM. *p<0.05).

FIG. 2 presents the pharmacophore model based on the structural model of the GSK-3 (SEQ ID NO: 1)/L8O3F (SEQ ID NO:5) complex. The pharmacophore is composed of two hydrogen bond acceptor features (Fl, F2: red vectors), one anionic feature (F3: red porcupine shape), and three hydrophobic features (F4, F5, F6: yellow spheres). Excluded volumes were placed in positions that are sterically claimed by the protein environment. GSK-3 interacting residues are marked.

FIGs. 3A-3B present data obtained in in vitro studies of the SCI hits uncovered in the first screening cycle. FIG. 3A presents the data obtained in in vitro kinase assays to measure GSK-3 activity conducted with ten selected hits (designated 1-1 - 7-70) at a concentration of 200 μM. Results are presented as the percentage of GSK-3 activity without inhibitor and represent the mean of three independent experiments. The table in the lower panel lists the IC ₅₀ values of the best performing compounds, 1-4, 1-6, and 7-7. FIG. 3B presents two-dimensional (2D) schematic presentations of the predicted interactions (within 4 A from the docked compound) of 7 -4, 1 -6, and 7-7 with the GSK-3 substrate binding site. Red, blue, and green spheres represent positively charged, negatively charged, and hydrophobic residues, respectively. Hydrogen bonds are denoted as pink arrows, 71-71 stacking is shown as green lines, and 71 -cation stacking is shown as red lines.

FIGs. 4A-4D present data obtained with SCI uncovered in the second screening cycle. FIG. 4A presents the data obtained in GSK-3 in vitro kinase assays conducted with selected hits (designated 2-7-2-77) at a concentration of 20 μM. Results are presented as the percentage of GSK- 3 activity without an inhibitor and represent the mean of three independent experiments. FIG. 4B presents the IC ₅₀ values of Compounds 2-7 and 2-2 as evaluated by dose inhibition curves. FIGs. 4C and 4D present computational docking Compounds of 2-7 (FIG. 4C) and 2-2 (FIG. 4D) into the GSK-3 substrate binding site. Key interactions formed between 2-7 and 2-2 and the GSK-3 binding site residues are indicated by dashed lines.

FIGs. 5A-5H present data obtained with SCI uncovered in the third screening cycle. FIG. 5A is a bar graph showing the data obtained in GSK-3 in vitro kinase assays conducted with selected top hits (designated 3-1-3-14) at a concentration of 20μM. Results are presented as the percentage of GSK-3 activity without inhibitor and represent the mean of three independent experiments. FIG. 5B presents dose inhibition curves of compounds 3-3, 3-7, and 3-8. The IC ₅₀ values are listed in the table at the right. FIGs. 5C and 5D present the docking of 3-7 (FIG. 5C) and 3-8 (FIG. 5D) into the GSK-3 substrate binding site. Key interactions that formed between 3.7 and 3.8 and between the binding site residues are indicated by dashed lines. FIGs. 5E and 5F present data obtained in in vitro kinase assays performed with the mutant F93A-GSK-3 (SEQ ID NO:8) in the presence of 20 μM of selected compounds according to the present embodiments or, 0.05 μM SB216763. FIG. 5G presents data obtained in in vitro kinase assays performed with GSK-3-WT (SEQ ID NO:1) and with the mutant F93A-GSK-3 (SEQ ID NO:8) in the presence of 20 μM of selected compounds according to the present embodiments or L8O3F (SEQ ID NO:5), or, 1 μM SB216763. Results are mean of three independent experiments ± SEM analyzed by one way ANOVA with Dunnett’s multiple comparisons. FIG. 5H presents the data obtained in CPA analysis, as described in the Examples section hereinbelow.

FIGs. 6A-6H present data obtained for the biological performance of Compound 3-8. FIG. 6A presents the immunoblot analysis showing the phosphorylation of IRS-1 (Ser ³³² ) and tyrosine phosphorylation of GSK-3α/β in CHO-IRS-1 cells treated with 3-8 for 4 hours at the indicated concentrations and stimulated with PMA for 30 minutes. All bands are from the same experiment and the same gel. FIG. 6B presents photographs of CHO cells transiently expressing GFP-0- catenin upon treatment with the indicated concentrations of 3-8 or with SB216763 (10 μM) for 4 hours. Fixed cells were imaged by fluorescent microscopy. β-catenin puncta were accumulated in the nucleus and are marked by white arrows. FIG. 6C is a bar graph showing the quantification of the GFP-β-catenin signal in the nucleus of the cells shown in FIG. 6B. Results are mean of 5-7 fields with a total amount of 80 cells +SEM *p<0.05, **p<0.01 as determined by Student’s t-test treated vs. control. FIG. 6D presents images of SH-SY5Y cells treated with the indicated concentrations of 3-8. Five cells were stained with LysoTracker Red and with Hoechst dye and imaged by confocal microscopy. Acidified lysosomes appear as red dots and nuclei are stained blue. FIG. 6E is a bar graph showing the quantification of number of lysosomes’ red puncta of FIG. 6D. Results are mean of 100-120 cells +SEM. **p<0.01, ***p<0.001 as determined by Student’s t-test treated vs. control. FIG. 6F present representative photos of embryonic spinal cord motor neurons in 96-well plates upon incubation with 5, 10, and 20 μM daily with 3-8 for six days. FIGs. 6G and 6H present comparative plots showing the changes in neurite length (FIG. 6G) and the number of branching points (FIG. 6H) during the six days of treatment. Results in both graphs represent the mean of 10 filed in 10 wells per time point. Statistics of neurite length: for experiments done with 5 μM 3-8, 24 hours, *p<0.05, 48 and 72 hours **p<0.0, 96-168 hours ***p<0.0. In the experiments conducted with 10 and 20 μM 3-8, 24 hours, ***p<0.01, other time intervals, ****p<0.0001. Statistics of branching points: for experiments done with 5μM 3-8, ** p<0.01 for all time point. I n experiments conducted with 3-8 at 10 and 20 μM, 24 hours, 48 hours, **p<0.01, 96-168, ****p<0.0001. Statistics was carried out with multiple Student’s t-test for treated vs. non-treated cells.

FIGs. 7A-7B present data obtained upon treating HEK-293 cells with 3-8 (10 μM) for 8 hours. FIG. 7A present Western Blot analysis. β-catenin was determined in the detergent soluble fraction (cytosol + membranes) and in the insoluble fraction (mainly nucleus) by immunoblot analysis using anti-β-catenin antibody. B-tubulin (cytosol) and B23 (nucleus) are shown for equal loading. FIG. 7B presents the densitometry analyses. Results are mean of three independent experiments +SEM. *p<0.05 by Student’s t-test.

FIGs. 7C-7D present data obtained upon treating SH-SY5Y cells with 3-8 or 1-8 (10 μM each). FIG. 7C presents confocal microscopy images of live cells stained with LysoTracker Red (red) and DAPI (blue). The ‘red dots’ are acidified lysosomes. FIG. 7D is a bar graph showing quantification of LysoTracker Red signal as mean of 100-120 cells +SEM. **p<0.01, by Student’s t-test treated vs. non-treated.

FIGs. 8A and 8B present the superposition of SCI molecules docked into GSK-3 substrate binding site. FIG. 8A presents the docking of 2-1, 2-2, 3-3, 3-7, 3-8 into the GSK-3 substrate binding site. The important binding residues within GSK-3, Phe 93, Arg 96, Arg 180 and Lys 205 are highlighted in black. Black circle marks the COO- moiety of molecules. FIG. 8B presents a table summarizing the number of interactions (+), type of interaction, and atom/substructure involved for compounds 2-1, 3-7 and 3-8 with each GSK-3 amino acid residue.

FIG. 8C presents a schematic illustration of the directions of an “electronic current”, exemplified for Compound 3-7 as described herein, dictated by the electron withdrawing effect of the electronegative atoms in the compound.

FIGs. 9A-9C present docking models of GSK-3 with Compounds 4-2 (FIG. 9A), 4-3 (FIG. 9B) and 4-4 (FIG. 9C) and show interactions of the compounds with the GSK-3 phosphate binding pocket, Phe 93 and Vai 214, and additional interactions with Phe 67, and with Lys 85 (for 4-3), or, Ser 66 (for 4-4). Interactions were analyzed by the Maestro software.

FIG. 9D presents a table summarizing the number of interactions (*), type of interaction, and atom/substructure involved for compounds 4-1, 4-2, 4-3, 4-4 and 4-5 with each GSK-3 amino acid residue. Green represents H-bond; blue represents stacking; black represents cation-7t; pink represents salt bridge. Ionic charges are indicated for indicated compounds.

FIGs. 10A-B presents data obtained in in vitro GSK-3 kinase assays with Compounds 4-1 to 4-5 compared with 3-8 (20 μM each). FIG. 10A is a bar graph showing the percentage of GSK- 3 activity without inhibitor (100 %) and represent the mean of three independent experiments ± SEM analyzed by one way ANOVA with Dunnett’s multiple comparisons. ****p<0.0001 treated vs. control (Ctrl). FIG. 10B presents dose response curves of GSK-3 inhibition using various concentrations of the tested compounds. Results are mean of three independent experiments. For 4-3 and 4-4 p <0.05 for all concentrations, for the rest of the molecules p<0.05 at concentrations >5 μM as determined by one-way ANOVA with Dunnett’s multiple comparisons. FIGs. 11A-11B present PCA analysis of Compounds 4-1 to 4-5 together with GSK-3-ATP competitive inhibitors listed in FIG. 11B. The first and second PCs which accounted for 81.8 % and 7.7 % of the original variance are shown at the X-axis and Y-axis respectively. Black circles represent the ATP competitive inhibitors; red circles represent the exemplary compounds according to some of the present embodiments.

FIG. 12 is a bar graph showing the data obtained in in vitro kinase assays performed with GSK-3 F93A mutant in the presence of Compounds 4-1 to 4-5 (20 μM). Results are mean of three independent experiments ± SEM, analyzed by one way ANOVA with Dunnett’s multiple comparisons, compounds vs control (Ctrl).

FIGs. 13A-B present representative Eadie-Hofstee plots for ATP competitive assays using two concentrations of 4-3 (FIG. 13A) and 4-4 (FIG. 13B) as indicated.

FIG. 14 is a bar graph showing data obtained in GSK-3 kinase assays performed with 4-3 and 4-4 (10 μM) in the presence of 0.05 % Triton xlOO. Results show that the detergent did not change the inhibition capacity of the compounds.

FIGs. 15A-15B present data obtained upon treating SH-SY5Y cells with increasing doses of 4-3, 4-4 or with the peptide inhibitor L807mts (SEQ ID NO:9) (20 μM) for 3 hours. FIG. 15A presents Western Blot analysis. Cytoplasmic β-catenin was collected after cell permeabilization with digitonin, and detected by immunoblot analysis using specific anti-β-catenin antibody. GAPDH is shown for equal loading. FIG. 15B is a bar graph showing densitometry analyses. Results are mean of three independent experiments +SEM. For doses 0.5, 1 and 5 μM p <0.05 by Student’s t-test treated vs. non-treated.

FIGs. 16A-16D present data showing lysosomal acidification in MEF cells. FIG. 16A presents confocal microscopy images of wild-type MEF (WT) and MEF KO/PS (KO/PS) cells stained in LysoTracker Red. It is shown that that lysosomal acidification is severely impaired in KO/PS cells. FIG. 16B presents confocal microscopy images of KO/PS cells treated with 4-3, 4-4 (5 μM), and L807mts (SEQ ID NO:9) (20 μM) for 3 hours. Live cells were stained with LysoTracker Red (red) and DAPI (blue) and imaged by confocal microscopy. FIG. 16C is a bar graph showing quantification of LysoTracker Red signal intensity. Results are mean of 100-120 cells +SEM. **p<0.01, ***<0.001 by Student’s t-test treated vs. non-treated. FIG. 16D presents data obtained upon treating MEF KO/PS cells with increasing doses of 4-3 or 4-4. Cathepsin D (CatD) (about 46 kDa) and its mature active form (mCatD) (about 26 kDa) were determine by immunoblot analysis using specific anti-CatD antibody. FIGs. 17A-F present the data obtained in experiments conducted for evaluating the biological activity of 4-4. FIG. 17A presents Western blot analysis showing the levels of cytoplasmic β-catenin in SH-SY5Y cells treated with 4-4 for 4 hours. FIG. 17B is a bar graph showing the densitometry analysis of SH-SY5Y cells treated with 4-4 for 4 hours. Results are mean of three independent experiments ± SEM * p < 0.05 by one-way ANOVA with Dunnett’s post hoc test. FIG. 17C presents images of hippocampal mouse neurons treated with 4-4 (5 μM), or CHIR99021 (CHIR, 10 μM) for 4 hours. Cells were co-stained with anti-β-catenin and anti-MAP2 antibodies. Images show overlapping β-catenin (green) and MAP2 (red) staining along with respective split channels (shown in grey). FIG. 17D is a bar graph showing the β-catenin signal as evaluated by Image J Colocalization-finder plugin. Results present the mean of 60 cells ± SEM, ** p < 0.01, *** p < 0.0001 by one-way ANOVA with Dunnett’s post hoc test. NT- non treated. FIG. 17E presents a Western Blot analysis showing the levels of phosphorylated tau (Ser 396), tau, and β-actin of hippocampal mouse neurons treated with 4-4 (20 μM) for 4 hours. FIG. 17F is a bar graph representing the densitometry analysis of ptau/p-actin. Results are mean of three independent experiments ± SEM using Student’s /-test. *p < 0.05. For all panels, Ctrl or 0 concentration represents cells treated with vehicle (DMSO/1 % Tween®80 at matched dilutions - 1:2000-4000).

FIG. 18 presents the chemical structures of Compounds 4-1, 4-2, 4-3, 4-4 and 4-5 and of additional newly designed SCIs of GSK-3 according to some embodiments of the present invention.

FIGs. 19A-19E present exemplary synthetic pathways for preparing Compound 4-1 (FIG. 19A), 4-2 (FIG. 19B), 4-3 (FIG. 19C), 4-4 (FIG. 19D) and 4-5 (FIG. 19E).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to novel glycogen synthase kinase-3 (GSK-3) modulators (e.g., inhibitors) and, more particularly, but not exclusively, to small molecule compounds that are usable in modulating an activity of GSK-3 (e.g., as substrate competitor inhibitors of GSK-3) and to the use of such compounds in, for example, the treatment of biological (e.g., medical) conditions associated with GSK-3 activity.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. The present inventors have devised a rational pharmacophore-based strategy, based on the previously described understanding of the mode of binding of substrate competitor inhibitor (SCI) peptides with GSK-3, and have uncovered SCI small molecules that target the GSK-3 substrate binding site.

The present inventors have designed a pharmacophore model based on the structural model of GSK-3 and a previously described peptide inhibitor, L8O3F (SEQ ID NO:5) (see, Background Art FIGs. 1A-C and FIG. 2), and screened a ‘drug-like’ database of about 6.3 million molecules. Following three successive iterations, potent compounds which share the same skeleton were identified. These compounds exhibited biological activity in inhibiting cellular GSK-3 in cell lines and in primary neurons. Based on the interactions of the identified compounds with GSK-3 (see, for example, FIGs. 8A-8B), the present inventors have designed and synthesized novel compounds featuring the same basic skeleton and have demonstrated the successfully performance of these compounds as substrate competitive inhibitors of GSK-3.

According to an aspect of some embodiments of the present invention there are provided compounds that are usable as SCI of GSK-3.

According to some embodiments of the present invention, the compounds are non-peptidic, small molecules, which interact with two or more amino acid residues in the catalytic binding site of GSK-3.

According to some embodiments of the present invention, the compounds interact with two or more of the following amino acid residues of GSK-3: Arg 96, Arg 180, Lys 205, Lys 85 and Phe 93 of a GSK-3 enzyme.

In some embodiments, a compound as described herein is such that is capable of interacting both with two or more of Arg 96, Arg 180, Lys 205, Lys 85 and Phe 93 of GSK-3, and with one or more of Phe 67, Gin 89, Asp 90, Lys 91, Arg 92, Lys 94 and Asp 95 of a GSK-3 enzyme.

In some embodiments, a compound as described herein is such that is capable of interacting with one or more, preferably two or more, more preferably three or more, e.g., with at least four, of Arg 96, Arg 180, Lys 205, Lys 85, Phe 93, Phe 67, Gin 89, Asp 90, Lys 91, Arg 92, Lys 94, Asp 95, and Vai 214 of a GSK-3 enzyme.

In some embodiments, a compound as described herein is such that is capable of interacting with one or more, preferably two or more, more preferably three or more, e.g., with four or all, of Arg 96, Arg 180, Lys 205, Lys 85, Phe 93, and Vai 214 of a GSK-3 enzyme. In some embodiments, a compound as described herein is such that is capable of interacting with one or more, preferably two or more, more preferably three or more, e.g., with all four, of Arg 96, Arg 180, Lys 205, and Phe 93 of a GSK-3 enzyme.

As used herein throughout, “GSK-3 enzyme”, which is also referred to herein simply as GSK-3, describes a polypeptide having an amino acid sequence of a known GSK-3 family member (e.g., GSK-3α or GSK-3β). Unless otherwise indicated, this term refers to a wild-type GSK-3 enzyme. A GSK-3 enzyme is identified by the EC number EC 2.7.11.26. While the amino acid of GSK-3 is highly conserved, a wild-type GSK-3 can be GSK-3 of a mammal (e.g., human) or of any other organism, including microorganisms. An amino acid sequence of an exemplary GSK- 3, human GSK-3β, is set forth in SEQ ID NO:1. A GSK-3 enzyme as used herein is homologous to SEQ ID NO:1 by at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 % or can be 100 % homologous.

By "wild-type" it is meant that the typical form of the enzyme as it occurs in nature, e.g., in an organism. A wild-type GSK-3 enzyme encompasses an enzyme isolated from an organism, a chemically synthesized enzyme and a recombinantly prepared enzyme.

Herein throughout, “a mutant of GSK-3 (or of GSK-3β)” is also referred to herein as “mutated GSK-3 enzyme” and is used to describe a polypeptide which differs from a corresponding wild-type GSK-3 (i.e., the starting point GSK-3) by at least one mutation (e.g., amino acid substitution).

A wild-type GSK-3 is as defined hereinabove for GSK-3.

A F93 mutant of GSK-3 is used herein to describe a mutated GSK-3 enzyme in which the amino acid substitution (mutation) is at position 93 of the 89-95 subunit. In most of the living organisms expressing GSK-3, this position corresponds to F93 (Phe93), as is in e.g., human GSK- 3β (SEQ ID NO: 1).

A F93A mutant of GSK-3 (which is also referred to herein as F93) is used herein to describe a mutated GSK-3 enzyme in which Phe residue at position 93 of the enzyme is substituted by Alanine (Ala or A).

The amino acid sequence of an exemplary F93A mutant of GSK-3 is as set of forth in SEQ ID NO:8. The GSK-3 mutant as used herein is homologous to SEQ ID NO:2 by at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 % or can be 100 % homologous, as long as the substitution at position 93 is maintained. Recombinant techniques, as described herein, are preferably used to generate the GSK-3 mutant. Alternatively, the mutant can be prepared by chemical synthesis, using, for example, solid phase synthesis as described herein.

Herein throughout, whenever a three-letter abbreviation of an amino acid is followed by a number it is meant the number of the indicated amino acid residue along the amino acid sequence downstream the N-terminus of the enzyme. The three-letter abbreviations described herein are as commonly used in the art.

By "position" it is meant a coordinate of the amino acid, whereby the indicated coordinate encompasses also an equivalent amino acid, as defined herein.

As used herein, an equivalent amino acid refers to an amino acid which is homologous (i.e., corresponding in position in either primary or tertiary structure) and/or analogous to a specific residue or portion thereof in a given sequence of GSK-3 or of a GSK-3 substrate (or inhibitor).

As used herein, the phrase "catalytic domain" describes a region of an enzyme, a GSK-3 as described herein, 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 embodiments, this phrase is particularly used to describe this part of an enzyme (a GSK-3 as described herein) 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.

As used herein, the phrases "binding site", “catalytic binding site” or “binding subsite”, which are used herein interchangeably, describe a specific site in the catalytic domain that includes one or more reactive groups through which the interactions of the enzyme with the substrate and/or an inhibitor can be effected. Typically, 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.

As is well known in the art, when an enzyme interacts with a substrate or an inhibitor, the initial interaction rapidly induces conformational changes in the enzyme and/or substrate and/or inhibitor that strengthen binding and bring enzyme’s binding 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. Thus, an inhibitor of an enzyme is typically associated with the catalytic domain of the enzyme such that the reactive groups of the inhibitor are positioned in sufficient proximity to corresponding reactive groups (typically side chains of amino acid residues) in the enzyme catalytic binding site, so as to allow the presence of an effective concentration of the inhibitor in the catalytic binding site and, in addition, the reactive groups of the inhibitor are positioned in a proper orientation, to allow overlap and thus a strong chemical interaction and low dissociation. An inhibitor therefore typically includes structural elements that are known to be involved in these interactions, and may also have a restriction of its conformational flexibility, so as to avoid conformational changes that would affect or weaken its association with the enzyme’s catalytic binding site.

In some of any of the embodiments described herein, the GSK-3 is GSK-3P (SEQ ID NO:1).

According to some embodiments of the present invention, the compounds comprise one or more negatively charged groups and one or more aromatic groups, and these groups are spatially arranged in a proximity and orientation that allows interactions of the one or more negatively charged groups with one or more of the Arg96, Arg 180, Lys85 and Lys205, or with one or more of the Arg96, Arg 180 and Lys205 residues (e.g., via electrostatic and/or hydrogen bond interactions), and of the one or more aromatic groups with Phe93 (via aromatic 7t-7t interactions), and optionally also with Phe67.

By “proximity and orientation” it is meant that an indicated group or moiety is sufficiently close and properly oriented so as to strongly interact with an indicated amino acid or two or more indicated amino acids within the catalytic domain of the enzyme, as described herein.

By "interacting" or “interact”, in the context of groups or moieties in a compound as described herein and an amino acid in the catalytic domain, it is meant a chemical interaction as a result of, for example, non-covalent interactions such as, but not limited to, hydrophobic interactions, including aromatic interactions, electrostatic interactions, Van der Waals interactions and hydrogen bonding.

According to some embodiments of the present invention, the compounds feature a rigid (e.g., planar) core structure to which the negatively charged group(s) and the aromatic group(s) are attached.

By “rigid core structure” it is meant that the compound has a skeleton (a chemical moiety to which one or more substituents are covalently attached) which is rigid, that is, it has a reduced number of free-rotating bonds. In some embodiments, the core structure has no more than one free -rotating bond, and in some embodiments, it has no free-rotating bonds. Preferably, the core structure is rigid and planar.

According to some of any of these embodiments, at least one of the negatively charged group(s) is attached to the rigid core via a flexible short chain (e.g., of 1 to 10, or 1 to 6, or 1 to 4, or 2 or 3, or 1 or 2, atoms in length).

According to some of any of these embodiments, the compounds described herein feature a dipole moment of at least 3.0, e.g., of 3.0, or of at least 3.5, or at least 3.8, or at least 4.0, or higher, e.g., of at least 4.2, at least 4.5 or even higher than 5.0. In some of these embodiments, the compounds feature electronegative groups and electron-withdrawing groups that are positioned throughout the planar core structure so as to feature a dipole moment as described herein and allow dipole-dipole interactions with amino acid residues in the GSK-3 binding site.

According to some of any of the embodiments described herein, the compounds comprise a rigid (e.g., planar) core structure which is, for example, a polycyclic structure that comprises two, three of more fused cyclic moieties (rings), at least some of the cyclic moieties being aromatic moieties, and which can optionally comprise one or more heteroatoms, preferably electronegative atoms such as nitrogen and/or oxygen, which allow for further interactions with, for example, positively charges amino acid residues such as the Arg96, Argl80, Lys85 and Lys205 residues of GSK-3.

According to some of any of the embodiments described herein, the compounds comprise a rigid (e.g., planar) core structure as described herein in any of the respective embodiments, to which are attached a flexible chain terminating with one or more negatively charged group(s) and a non-aromatic cyclic or heterocylic moiety, which is optionally substituted by, for example, one or more aromatic moieties (e.g., aryl such as phenyl).

According to some of any of the embodiments described herein, the compounds disclosed herein as substrate competitive inhibitors (SCIs) of GSK-3 are collectively represented by Formula

I:

wherein:

Wi and W2 are each independently NR’, CR’R”, O or S, with R’ and R” being as defined herein, and preferably Wi and W2 are each independently O or S, more preferably each is O;

L is a linking moiety which is or comprises a substituted or unsubstituted, linear or branched, alkyl, substituted or unsubstituted aryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heteroalicylic, a substituted or unsubstituted heteroaryl, or is absent;

Y is a negatively charged group; q is a positive integer representing the number of negatively charged groups attached to L or to NH (in case L is absent);

X is an alicyclic or heteroalicyclic 5, 6, 7, or 8-membered ring; n is 0 or is a positive integer that represents the number of substituents A of the X alicyclic or heteroalicyclic ring;

A is independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, haloalkyl, carboxylate, thiocarboxylate, amine, amide, carbamyl, thiocarbamyl, carbonyl, thiocarbonyl, alkoxy, aryloxy, hydroxy, thiol, thioalkoxy, thioaryloxy, alkenyl, alkynyl, cyano, nitro, azo, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, phosphoryl, ketoester, ether, thioether, urea, thiourea, guanyl, guanidino, carbohydrate, hydrazine, and hydrazide, or, alternatively, when n is greater than 1, two or more of the A substituents form at least one three-, four-, five- or six-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring; m is 0 or a positive integer that represents the number of substituents B of the respective phenyl;

B is independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, haloalkyl, carboxylate, thiocarboxylate, amine, amide, carbamyl, thiocarbamyl, carbonyl, thiocarbonyl, alkoxy, aryloxy, hydroxy, thiol, thioalkoxy, thioaryloxy, alkenyl, alkynyl, cyano, nitro, azo, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, phosphoryl, ketoester, ether, thioether, urea, thiourea, guanyl, guanidino, carbohydrate, hydrazine, and hydrazide, or, alternatively, when m is greater than 1, two or more of the B substituents form at least one three-, four-, five- or six-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring; and

D is hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, haloalkyl, carboxylate, thiocarboxylate, amine, amide, carbamyl, thiocarbamyl, carbonyl, thiocarbonyl, alkoxy, aryloxy, hydroxy, thiol, thioalkoxy, thioaryloxy, alkenyl, alkynyl, cyano, nitro, azo, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, phosphoryl, ketoester, ether, thioether, urea, thiourea, guanyl, guanidino, carbohydrate, hydrazine, or hydrazide, or a pharmaceutically acceptable salt thereof.

Compounds of Formula I as described herein (and also of Formula II and Formula III) all feature an anthracene rigid core structure.

According to some of any of the embodiments described herein, when X is piperidine, n is a positive integer and at least one of the A substituents of the piperidine is or comprises an aryl or a heteroaryl, as defined herein in any of the respective embodiments.

According to some of any of the embodiments described herein, when X is piperidine or piperazine, L is not or does not comprise an aryl or heteroaryl, as these terms are defined herein.

According to some of any of the embodiments described herein, when X is piperidine or piperazine, L is an alkyl as described herein in any of the respective embodiments. Alternatively, L is or comprises a cycloalkyl and does not comprise an aryl or a heteroaryl.

According to some of any of the embodiments described herein, when X is piperazine and n is a positive integer, at least one of the A substituents on the piperazine is other than a cycloalkyl.

According to some of any of the embodiments described herein, when X is piperazine and n is a positive integer, at least one, at least two or all of the A substituents on the piperazine is an aryl, for example, phenyl.

According to some of any of the embodiments described herein, a compound as described herein is for use in modulating (e.g., inhibiting) an activity of GSK-3.

According to some of any of the embodiments described herein, L is a linking moiety that is or comprises an alkyl (e.g., an unsubstituted linear or branched alkyl, preferably a lower linear alkyl of, for example, 1 to 10, preferably, 1 to 6, more preferably 1 to 4, more preferably 1, 2, 3, and more preferably 1 or 2, carbon atoms in length). In some of these embodiments, L is an unsubstituted alkyl. In some of these embodiments, L is a lower linear unsubstituted alkyl. According to some of any of the embodiments described herein, the negatively charged group is selected from the group consisting of carboxylate, carbamate, thiocarbamate, phosphonate, phosphate, sulfonate, and sulfate, or is as described hereinafter.

According to some of any of the embodiments described herein, the negatively charged group is carboxylate.

According to some of any of the embodiments described herein, the negatively charged group is phosphate.

According to some of any of the embodiments described herein, Y is or comprises a carboxylate (e.g., one or more carboxylates).

According to some of any of the embodiments described herein, Y is or comprises a phosphate (e.g., one or more phosphate groups).

For any of the compounds as described herein which comprise a negatively charged group as described herein, the compounds can be in an ionized form, and further comprise a cation, as defined herein for a pharmaceutically acceptable salt. Alternatively, the compounds can be in a non-ionized (e.g., pre-ionized), protonated form, in which the negative charge is protonated, as for example, in carboxylic acid, or in phosphonic or phosphoric acid, or sulfonic acid or sulfuric acid (for the respective negatively charged groups). When the negatively charged group is protonated, it is preferably ionizable under physiological conditions (de-protonated) to provide a negatively charged group as described herein.

According to some of any of the embodiments described herein, the cyclic moiety X is a five-, six-, or seven-membered ring.

According to some of any of the embodiments described herein, the cyclic moiety X is a six-membered ring.

According to some of any of the embodiments described herein, the cyclic moiety X is an alicyclic all-carbon moiety (a cycloalkyl, as described herein), and in some of these embodiments X is cyclohexane.

According to some of any of the embodiments described herein, the cyclic moiety X is a heteroalicylic moiety as described herein, and in some of these embodiments it is a nitrogencontaining heteroalicyclic moiety, for example, a piperazine or a piperidine.

According to some of any of the embodiments described herein, X is piperazine.

According to some of any of the embodiments described herein, n is a positive integer, such that X is a substituted moiety. According to some of any of the embodiments described herein, n is a positive integer and X is a substituted alicyclic, for example, a substituted cyclohexane.

According to some of any of the embodiments described herein, n is a positive integer and X is a substituted heteroalicyclic, for example, a substituted piperazine or a substituted piperidine.

According to some of these embodiments, at least one of the A substituent(s) is an aryl (e.g., phenyl).

According to some of these embodiments, n is 1 and the A substituent(s) is an aryl (e.g., phenyl).

According to some of any of the embodiments described herein, X is a substituted alicyclic, for example, a substituted cyclohexane, n is 1, and the A substituent is an aryl (e.g., phenyl).

According to some of any of the embodiments described herein, X is a substituted heteroalicyclic, for example, a substituted piperazine or a substituted piperidine, n is 1, and the A substituent is an aryl (e.g., phenyl). According to some of any of the embodiments described herein, X is piperazine, n is a positive integer and at least one A is an aryl (e.g., phenyl).

According to some of these embodiments, n is greater than 1, for example 2, and at least one another A is an aryl (e.g., phenyl).

According to some of any of the embodiments described herein, X is piperazine, n is 2 and each of the two A substituents is independently an aryl (e.g., phenyl). In some of these embodiments, both A substituents are phenyl.

According to some of any of the embodiments described herein, n is a positive integer and the at least one A substituent is a substituted aryl. In some of any of these embodiments, the aryl features an electronegative substituent such as, for example, hydroxy, alkoxy, carboxylate or halogen (e.g., fluoro), at a position that contributes to achieve a dipole moment as described herein. For example, in some of these embodiments, the aryl is substituted at the para position with respect to the position that is attached to X, and the substituent is an electronegative atom or group as described herein.

In exemplary embodiments, the aryl is substituted at the ortho and/or para positions by one or more electronegative substituents such as fluoro, hydroxy, carboxylate, sulfate, sulfonate, phosphate, phosphonate, etc.

In exemplary embodiments, X is a substituted alicyclic, for example, a substituted cyclohexane, n is 1, and the A substituent is a phenyl substituted at the ortho and/or para positions by one or more electronegative substituents such as fluoro, hydroxy, alkoxy, carboxylate, sulfate, sulfonate, phosphate, phosphonate, etc. In exemplary embodiments, X is a substituted heteroalicyclic, for example, a substituted piperazine or a substituted piperidine, n is 1, and the A substituent is a phenyl substituted at the ortho and/or para positions by one or more electronegative substituents such as fluoro, hydroxy, alkoxy, carboxylate, sulfate, sulfonate, phosphate, phosphonate, etc.

In some of these exemplary embodiments, the phenyl is substituted at the ortho and/or para positions by one or more electronegative substituents such as fluoro, hydroxy, alkoxy (e.g., methoxy), or carboxylate.

According to some of any of the embodiments described herein, D is hydrogen.

According to some of any of the embodiments described herein, m is 0.

According to an aspect of some embodiments of the present invention there are provided newly designed compounds, which exhibit structural features that may improve the interactions of the compound with the binding site of GSK-3.

The newly designed compounds can be collectively represented by Formula II: wherein:

Wi and W2 are as described herein for Formula I in any of the respective embodiments;

L is as described herein for Formula I in any of the respective embodiments;

Y is a negatively charged group, as described herein for Formula I in any of the respective embodiments, or a group that is capable of generating a negatively charged group at a physiological environment (a non-ionized, e.g., protonated (pre-ionized), negatively charged group, as described herein); q is a positive integer representing the number of negatively charged groups attached to L or to NH (in case L is absent); X is an alicyclic or heteroalicyclic 5, 6, 7, or 8-membered ring, as described herein for Formula I in any of the respective embodiments; n is 0 or a positive integer that represents the number of substituents A of the X;

A, B and D are each independently as described herein for Formula I in any of the respective embodiments; m is 0 or a positive integer that represents the number of substituents B of the respective phenyl, or a pharmaceutically acceptable salt thereof, provided that: n is greater than one, and at least two of the A substituents is or comprises an aryl or heteroaryl; and/or at least one of the negatively charged group(s) is other than carboxylate; and/or q is greater than 1.

Exemplary compounds of Formula II are presented in FIG. 18.

Compounds of Formula II can be in a protonated form, as described herein for Formula I, or in a de-protonated, ionized form, as described herein for Formula I, or in any other form that is capable of generating a negatively charged group as described herein, for example, as a prodrug as described herein.

According to some of any of the embodiments of Formula II, Y is or comprises a phosphate or a phosphate ester (e.g., phosphodiester).

According to some of any of the embodiments of Formula II, q is 1.

According to some of any of the embodiments of Formula II, n is at least 1 , A is aryl and the aryl is substituted at the ortho or para position with respect to X by an electronegative substituent as described herein. In some of these embodiments, the aryl (e.g., phenyl) is substituted by fluoro, alkoxy, carboxylate, and/or hydroxy. In some of these embodiments, the aryl (e.g., phenyl) is substituted by hydroxy (e.g., at the para position). In some of these embodiments, the aryl (e.g., phenyl) is substituted by a carboxylate (e.g., at the para position). In some of these embodiments, the aryl (e.g., phenyl) is substituted by an alkoxy such as methoxy (e.g., at the para position).

According to exemplary embodiments, compounds of Formula I or II can be collectively represented by Formula III, respectively:

wherein:

W i and W2 are as described herein for Formula I or II in any of the respective embodiments;

L is as described herein for Formula I or II in any of the respective embodiments;

Y is a negatively charged group, as described herein for Formula I or II in any of the respective embodiments, or a group that is capable of generating a negatively charged group at a physiological environment, as described herein; q is a positive integer representing the number of negatively charged groups attached to L or to NH (in case L is absent);

X is an alicyclic or heteroalicyclic 5, 6, 7, or 8-membered ring, as described herein for Formula I or II in any of the respective embodiments;

B and D are each independently as described herein for Formula I or II in any of the respective embodiments; m is 0 or a positive integer that represents the number of substituents B of the respective phenyl;

Z is an electronegative substituent as described herein in any of the respective embodiments, and/or for Formula II;

G is a substituent as described herein for a substituent of A in Formula I or II; and k is 0 or a positive integer represented the number of additional substituents on the phenyl, or a pharmaceutically acceptable salt thereof.

Herein throughout, whenever a substituent is not indicated, the compound may feature hydrogen at the indicated position. Syntheses and intermediate compounds:

Embodiments of the present invention further relate to processes of preparing compounds of Formula II or III as described herein, as depicted, for example, in FIGs. 19A-E, and to intermediate compounds usable in such processes, as also depicted, for example, in FIGs. 19A-E.

Generally, compounds of Formula II and III, according to some of the present embodiments, are prepared by coupling a compound of Formula IV:

Formula IV wherein:

Wi and W2, B, D, and m are as described herein for any of the respective embodiments of Formula II or III; and

Zi and Z2 are each independently as leaving group as described herein, with a compound of Formula V :

Formula V wherein each of E and q is as described herein for any of the respective embodiments of Formula II or III; and Y’ is a negatively charged group as described herein, or a protected form of the negatively charged group (e.g., an ester thereof), and with a compound of Formula VI:

Formula VI wherein X, A and n are as described herein for any of the respective embodiments of Formula II or III, and Z3 is a nucleophilic group or is absent. According to some embodiments of this aspect of the present invention, the process is effected by coupling a compound of Formula IV with a compound of Formula V, and then coupling the resulting intermediate with a compound of Formula VI.

Alternatively, the process is effected by coupling a compound of Formula IV with a compound of Formula VI, and then coupling the resulting intermediate with a compound of Formula V.

The coupling reactions are effected under conditions (e.g., temperature, reagents) known in the art for facilitating nucleophilic substitutions or addition-elimination, or any other coupling reactions.

According to some embodiments of this aspect of the present invention, X is a heteroalicyclic and Z3 is absent, such that the coupling is performed using the heteroatom of the heteroalicyclic as a nucleophilic group.

According to some embodiments of this aspect of the present invention, Y’ is a protected form (e.g., an ester form) of the negatively charged group, and the process further comprises, after the coupling of the compound of Formula IV with a compound of Formula V, and optionally after the coupling with a compound of Formula VI (either before or subsequent to the coupling of Formula IV with Formula V), de-protecting the formed intermediate so as to provide a negatively charged group.

According to some embodiments of this aspect of the present invention, A is a substituted aryl (e.g., a substituted phenyl), and the aryl is substituted by one or more of a hydroxy, or carboxylate, etc. In such cases, the respective coupling is with a compound of Formula VI*:

Formula VI* in which one or all of the A’ substituents is an aryl substituted by a protected form of a hydroxy, carboxylate, phosphate, phosphonate, sulfate, sulfonate, or any other protic substituents. For example, if A in Formula II is an aryl substituted by a protic electronegative group or if Z in Formula III is a protic electronegative group, A’ is Formula VI* is a protected form of this form, for example, an ester thereof.

In some of these embodiments, the process further comprises de-protecting to provide the respective substituent. According to an aspect of some embodiments of the present invention, there is provided a compound of Formula VII:

Formula VII wherein n, m, q, Wl, W2, L, X, A, and Y’ are as described herein in any of the respective embodiments and any combination thereof.

A compound of Formula VII can serve as an intermediate for providing a compound of Formula I, II or III as described herein, or can be used as a GSK-3 modulator (e.g., inhibitor), or in any of the methods and uses, as described herein in any of the respective embodiments.

Uses:

According to an aspect of some embodiments of the present invention there is provided a method of modulating (e.g., inhibiting) an activity of GSK-3, which is effected by contacting cells expressing GSK-3 with an effective amount of any of the compounds described herein (e.g., represented by Formula I or II or III).

As used herein, the term “effective amount” is the amount determined by such considerations as are known in the art, which is sufficient to modulate (e.g., reduce) the activity of GSK-3 by at least 5 %, at least 10 %, at least 20 %, at least 50 % and even at least 80 %, 90 % or by 100%. Typical assays for measuring kinase activity can be used for determining the (e.g., inhibitory) activity of the compounds as described herein.

The method according to this aspect of the present invention can be effected by contacting the cells with the compounds as described herein in vitro, ex vivo and in vivo.

Cells expressing GSK-3 can be derived from any biological sample, including, but not limited to, cell cultures or extracts thereof, enzyme preparations suitable for in vitro assays, biopsied material obtained from a mammal or extracts thereof, and samples of blood, saliva, urine, feces, semen, tears, spinal fluid, and any other fluids or extracts thereof. In some embodiments, the method according to these embodiments, utilizes the compounds as described herein as active agents in biological assays, and in particular, as GSK-3 (substrate competitive) inhibitors in such assays.

The method according to these embodiments of the present invention can be further effected by contacting the cells with an additional active ingredient that is capable of altering an activity of GSK-3, as is detailed hereinbelow.

By modulating (e.g., inhibiting) GSK-3 activity the compounds described herein may be effectively utilized for treating any biological condition that is associated with GSK-3.

Hence, according to another aspect of some embodiments of the present invention, there is provided a method of treating a biological condition associated with GSK-3 activity. The method, according to this aspect of the present invention, is effected by administering to a subject in need thereof a therapeutically effective amount of a compound as described herein.

The phrase “biological condition associated with GSK-3 activity” as used herein 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 aspects of the condition can be traced to the GSK-3 activity. Such a biological condition can also be regarded as a biological or medical condition mediated by GSK- 3.

Herein, the term “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 non-limiting examples, 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.

The term “administering” as used herein describes a method for bringing a compound as described herein 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 described herein can be administered via any route that is medically acceptable. The route of administration can depend on the disease, condition, organ or injury being treated. Possible administration routes include injections, by parenteral routes, such as intravascular, intravenous, intra-arterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, intracerebroventicular, intranasal or others, as well as via oral, nasal, ophthalmic, rectal or topical routes of administration, or by inhalation. Sustained release administration is also encompassed herein, by means such as, for example, depot injections or erodible implants, or by sustained release oral formulations (e.g., solid oral formulations). Administration can also be intra- articularly, intrarectally, intraperitoneally, intramuscularly, subcutaneously, or by aerosol inhalant. Where treatment is systemic, the compound can be administered orally, nasally 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.

In some embodiments, administration is effected nasally, namely via a nasal route of administration. A nasal administration can be effected either by intranasal injection or by means of a spray or liquid formulation that is administered nasally.

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. Thus, for example where inhibition of GSK-3 results in the arrest of phosphorylation of glycogen synthase, 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. Also, where 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, then the compound may be administered until polymerization of phosphorylated tau protein is substantially arrested. Levels of hippocampous β-catenin are also indicative for an effect on GSK-3 activity. 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.

As is discussed in detail hereinabove, 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.

By "insulin-dependent condition" it is meant any condition that is mediated by insulin and which is manifested or caused by reduced level of insulin or impaired insulin potentiation pathway. Exemplary such conditions include, but are not limited to, conditions that involve glucose intolerance and impaired glucose uptake, such as diabetes, including, for example, insulindependent diabetes and juvenile diabetes.

As GSK-3 inhibitors are known to inhibit differentiation of pre-adipocytes into adipocytes, in another example, the method of this aspect of the present invention can be used to treat obesity.

In yet another example, the method according to this aspect of the present invention can be used to treat diabetes including 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. Approximately 95% of all diabetic patients in the United States have non-insulin dependent, Type II diabetes mellitus (NIDDM), and, therefore, this is the form of diabetes that accounts for the great majority of medical problems. 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).

In another example, the method according to these embodiments 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).

As 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.

In one example, since inhibition of GSK-3 results in halting neuronal cell death, the method according to these embodiments 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.

In another example, since GSK-3 activity is implicated in various central nervous system disorders and neurodegenerative diseases, the method according to these embodiments 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.

As is discussed hereinabove, GSK-3 activity has particularly been implicated in the pathogenesis of Alzheimer’s disease. Hence, in one representative embodiment of the method described herein, there is provided a method of treating a patient with Alzheimer’s disease: A patient diagnosed with Alzheimer's disease is administered with a compound as described herein, which inhibits GSK-3 -mediated tau hyperphosphorylation, prepared in a formulation that crosses the blood brain barrier (BBB). 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 lysosome acidification, and therefore the method according to this aspect of embodiments of the present invention can be further used to treat conditions associated with lysosome acidification (e.g., conditions that involve impaired lysosome acidification).

GSK-3 has also been implicated with respect to psychotic disorders such as schizophrenia, and therefore the method according to this aspect of embodiments of the present invention can be further used to treat psychotic diseases or disorders, such as schizophrenia.

GSK-3 has also been implicated with respect to affective disorders. Therefore, in another example, 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).

GSK-3 has also been implicated with respect to cardiovascular conditions, and therefore, the compounds described herein can be further used to treat cardiovascular diseases or disorders.

Cardiovascular diseases and disorders include, but are not limited to, atherosclerosis, a cardiac valvular disease, stenosis, restenosis, in-stent-stenosis, myocardial infarction, coronary arterial disease, acute coronary syndromes, congestive heart failure, angina pectoris, myocardial ischemia, thrombosis, Wegener’s granulomatosis, Takayasu’s arteritis, Kawasaki syndrome, antifactor VIII autoimmune disease or disorder, necrotizing small vessel vasculitis, microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis, antiphospholipid syndrome, antibody induced heart failure, thrombocytopenic purpura, autoimmune hemolytic anemia, cardiac autoimmunity, Chagas’ disease or disorder, and anti-helper T lymphocyte autoimmunity.

GSK-3 has also been implicated with respect to conditions (e.g., infections) associated with pathogenic parasites (e.g., malaria and trypanosomiasis), and therefore, the compounds described herein can be further used to treat a condition (e.g. infection) that is associated with a presence of a pathogenic parasite in a subject. Exemplary parasites include Acanthamoeba, Anisakis, Ascaris lumbricoid.es, Botfly, Balantidium coli, Bedbug, Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke, Loa, Paragonimus - lung fluke, Pinworm, Schistosoma, Strongyloides stercoralis, Mites, Tapeworm, Toxoplasma gondii, Trypanosoma, Whipworm, Wuchereria bancrofti and Plasmodium falciparum and related malaria-causing protozoan parasites.

Exemplary conditions caused by pathogenic parasites include, but are not limited to, Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis (caused by the Guinea worm), Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Taeniasis (cause of Cysticercosis), Toxocariasis, Toxoplasmosis, Trichinosis and Trichuriasis.

GSK-3 has also been suggested to be involved in stem cell maintenance and/or differentiation. Accordingly, the compounds described herein can be further utilized in the treatment of conditions in which transplantation of stem cells is used as part of the treatment. Such conditions include, for example, cancer and damaged tissues (treatable by tissue regeneration).

In some embodiments, the compounds described herein can be utilized for maintaining and/or differentiating stem cells. Thus, in some embodiments, there is provided a method of maintaining and/or differentiating stem cells, which is effected by contacting a compound as described herein with stem cells. In some embodiments, the contacting is effected ex-vivo. In some embodiments, the contacting is effected in the presence of a physiological medium, as acceptable for stem cells preparations. In some embodiments, the contacting is effected by placing stem cells in a suitable medium which further comprises a compound as described herein.

The method according to this aspect of the present invention can be further effected by coadministering to the subject one or more additional active ingredient(s) which is capable of altering an activity of GSK-3.

As used herein, “co-administering” describes administration of a compound as described herein 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 additional therapeutic agent, or may be accomplished by administration of the compound and the active agent 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.

Preferably, the additional active agent is capable of inhibiting an activity of GSK-3, such that the additional active agent can be any GSK-3 inhibitor other than the compounds described herein, and thus can be, as non-limiting examples, lithium, valproic acid and peptides or small molecules that are shown to inhibit GSK-3 activity as described herein.

Alternatively, the additional active agent can be an agent 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 mRNA or at the level of the protein. For example, a small interfering polynucleotide molecule which is designed to downregulate the expression of GSK-3 can be used as an additional active agent according to some embodiments 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. (Munshi CB, Graeff R, Lee HC, J Biol Chem 2002 Dec 20;277(51):49453-8), which includes duplex oligonucleotides which direct sequence specific degradation of mRNA through the previously described mechanism of RNA interference (RNAi) (Hutvagner and Zamore (2002) Curr. Opin. Genetics and Development 12:225-232). Further according to embodiments of the present invention there is provided a use of the compounds as described herein (e.g., of Formula I or II or III) in the manufacture of a medicament for treating a biological condition associated with GSK-3 activity, as described herein.

Further according to embodiments of the present invention there is provided a compound as described herein (e.g., of Formula I or II or III), for use in the treatment of a biological condition associated with GSK-3 activity, as described herein.

According to an aspect of some embodiments of the present invention there is provided a compound as described herein (e.g., of Formula I or II or III), for use in inhibiting an activity of GSK-3.

According to an aspect of some embodiments of the present invention there is provided a compound as described herein (e.g., of Formula I or II or III) for use in the treatment of a biological condition associated with GSK-3 activity.

According to an aspect of some embodiments of the present invention there is provided a method of inhibiting an activity of GSK-3, the method comprising contacting cells expressing GSK-3 with an effective amount of peptide compound as described herein (e.g., of Formula I or II or III).

According to an aspect of some embodiments of the present invention there is provided a use of peptide compound as described herein (e.g., of Formula I or II or III) in the manufacture of a medicament for inhibiting an activity of GSK-3 activity.

According to some embodiments of the invention, the activity is a phosphorylation activity and/or an autophosphorylation activity.

According to an aspect of some embodiments of the present invention there is provided a method of treating a biological condition associated with GSK-3 activity, the method comprising administering to a subject in need thereof a therapeutically effective amount of the compound as described herein (e.g., of Formula I or II or III).

According to an aspect of some embodiments of the present invention there is provided a use of the compound as described herein (e.g., of Formula I or II or III) in the manufacture of a medicament for treating a biological condition associated with GSK-3 activity.

According to some of any of the respective embodiments of the invention, the biological condition is associated with overexpression of GSK-3.

According to some of any of the respective embodiments of the invention, the biological condition is selected from the group consisting of obesity, non-insulin dependent diabetes mellitus, an insulin-dependent condition, an affective disorder, major depression, a neurodegenerative disease or disorder, a psychotic disease or disorder, a cardiovascular disease or disorder, a condition associated with a pathogenic parasite, and a condition treatable by stem cell transplantation and/or stem cells maintenance.

In any of the methods and uses described herein, the compounds as described herein can be utilized either per se, or, preferably, as a part of a pharmaceutical composition, which may further comprise a pharmaceutically acceptable carrier.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the compounds described herein (as active ingredient), or physiologically acceptable salts or prodrugs thereof, with other chemical components including but not limited to physiologically suitable carriers, excipients, lubricants, buffering agents, antibacterial agents, bulking agents (e.g. mannitol), antioxidants (e.g., ascorbic acid or sodium bisulfite), anti-inflammatory agents, antiviral agents, chemotherapeutic agents, anti-histamines and the like. The purpose of a pharmaceutical composition is to facilitate administration of a compound to a subject.

The term "active ingredient", which is also referred to herein interchangeably as "active agent" refers to a compound, which is accountable for a biological effect.

The terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a drug. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

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 compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. 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.l).

The pharmaceutical composition may be formulated for administration in either one or more of routes depending on whether local or systemic treatment or administration is of choice, and on the area to be treated. Administration may be done orally, nasally, by inhalation, or parenterally, for example by intravenous drip or intraperitoneal, subcutaneous, intramuscular or intravenous injection, or topically (including opthalmically, vaginally, rectally and intranasally).

Formulations for topical administration may include but are not limited to lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, pills, caplets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable.

Formulations for parenteral administration may include, but are not limited to, sterile solutions which may also contain buffers, diluents and other suitable additives. Slow release compositions are envisaged for treatment.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

The pharmaceutical composition may further comprise additional pharmaceutically active or inactive agents such as, but not limited to, an anti-bacterial agent, an antioxidant, a buffering agent, a bulking agent, a surfactant, an anti-inflammatory agent, an anti-viral agent, a chemotherapeutic agent and an anti-histamine.

According to an embodiment of the present invention, the pharmaceutical composition described herein is packaged in a packaging material and identified in print, in or on the packaging material, for use in the treatment of a medical condition associated with GSK-3 activity, as described herein.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. 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 accommodated 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.

In some embodiments, the pharmaceutical composition is identified for use in combination with an additional active agent, as described herein.

In some embodiments, the pharmaceutical composition further comprises an additional active agent as described herein, being co-formulated with the compound as described herein.

According to an aspect of some embodiments of the present invention there is provided a method of screening a plurality of non-peptidic compounds to thereby identify compounds capable of modulating (e.g., inhibiting) an activity of GSK-3, as described herein, the method comprising identifying compounds that feature a high score in computational docking into a catalytic domain of GSK-3, essentially as described herein and as is exemplified in the Examples section that follows.

According to an aspect of some embodiments of the present invention there is provided a method of computationally screening for a putative substrate-competitive non-peptidic inhibitor of GSK-3, substantially as described herein.

The screening methods as described herein can be used for identifying compounds capable of modulating (e.g., inhibiting) activity of other biological targets (e.g., enzymes, receptors).

According to some embodiments, the screening methods further comprise, upon identifying compounds that feature a high score in computational docking into the relevant domain in the biological target (e.g., GSK-3), testing the identified compounds for their activity. The testing can be performed using methods known in the art, and can be performed in vitro and/or in vivo. Exemplary assays are described in the Examples section that follows.

As used herein the term “about” refers to ± 10 % or ± 5 %.

The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

The term “consisting of’ means “including and limited to”.

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure. As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, 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.

The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

The term "hydrocarbon", as used herein, encompasses any moiety that is based on a linear and/or cyclic chain of carbons which are mainly substituted by hydrogens. A hydrocarbon can be a saturated or unsaturated moiety, and can optionally be substituted by one or more substituents, as described herein.

The term "alkyl" describes a saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; 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 2 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl is a lower alkyl having 2 to 6 carbon atoms. The alkyl group may be substituted or unsubstituted, as defined herein. The term "cycloalkyl" or “alicyclic” describes an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system. The cycloalkyl group may be substituted or unsubstituted.

The term "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. Representative examples are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane, morpholino and the like.

The term "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. The aryl group may be substituted or unsubstituted.

The term "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. Examples, without limitation, of heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or unsubstituted.

Whenever an alkyl, cycloalkyl, heteroalicyclic, aryl, heteroaryl or a hydrocarbon is substituted by one or more substituents, each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine.

A "hydroxy" group refers to an -OH group.

An "azide" group refers to a -N=N ⁺ =N“ group.

An "alkoxy" group refers to both an -O-alkyl and an -O-cycloalkyl group, as defined herein.

An "aryloxy" group refers to both an -O-aryl and an -O-heteroaryl group, as defined herein.

A "thiohydroxy" or “thiol” group refers to a -SH group.

A "thioalkoxy" group refers to both an -S-alkyl group, and an -S-cycloalkyl group, as defined herein. A "thioaryloxy" group refers to both an -S-aryl and an -S-heteroaryl group, as defined herein.

A "carbonyl" group refers to a -C(=O)-R’ group, where R’ is defined as hereinabove.

A "thiocarbonyl" group refers to a -C(=S)-R’ group, where R’ is as defined herein.

A "C-carboxy" group refers to a -C(=O)-O-R’ groups, where R’ is as defined herein.

An "O-carboxy" group refers to an R’C(=O)-O- group, where R’ is as defined herein.

An “oxo” group refers to a =0 group.

A “carboxylate” or "carboxyl" encompasses both C-carboxy and O-carboxy groups, as defined herein.

A "carboxylic acid" group refers to a C-carboxy group in which R’ is hydrogen.

A “thiocarboxy” or “thiocarboxylate” group refers to both -C(=S)-O-R’ and -O-C(=S)R’ groups.

An “ester” refers to a C-carboxy group wherein R’ is not hydrogen. Typically, R’ is an alkyl, cycloalkyl or aryl, as described herein.

An ester bond refers to a -O-C(=O)- bond.

A "halo" group refers to fluorine, chlorine, bromine or iodine.

A "sulfinyl" group refers to an -S(=O)-R’ group, where R’ is as defined herein.

A "sulfonyl" group refers to an -S(=O) ₂ -R’ group, where R’ is as defined herein.

A “sulfonate” group refers to an -S(=O) ₂ -O-R’ group, where R’ is as defined herein.

A "sulfonic acid" group refers to a sulfonate group in which R’ is hydrogen.

A “sulfate” group refers to an -O-S(=O) ₂ -O-R’ group, where R’ is as defined as herein.

A "sulfuric acid" group refers to a sulfate group in which R’ is hydrogen.

A “sulfonamide” or “sulfonamido” group encompasses both S-sulfonamido and N- sulfonamido groups, as defined herein.

An “S-sulfonamido” group refers to a -S(=O) ₂ -NR’R” group, with each of R’ and R” as defined herein.

An "N- sulfonamido" group refers to an R’S(=O) ₂ -NR” group, where each of R’ and R” is as defined herein.

An "O-carbamyl" group refers to an -OC(=O)-NR’R” group, where each of R’ and R” is as defined herein.

An "N-carbamyl" group refers to an R’OC(=O)-NR”- group, where each of R’ and R” is as defined herein.

A “carbamyl” or “carbamate” group encompasses O-carbamyl and N-carbamyl groups. A carbamate bond describes a -O-C(=O)-NR'- bond, where R' is as described herein.

An "O-thiocarbamyl" group refers to an -OC(=S)-NR’R” group, where each of R’ and R” is as defined herein.

An “N-thiocarbamyl” group refers to an R’OC(=S)NR”- group, where each of R’ and R” is as defined herein.

A “thiocarbamyl” or “thiocarbamate” group encompasses O-thiocarbamyl and N- thiocarbamyl groups.

A thiocarbamate bond describes a -O-C(=S)-NR'- bond, where R' is as described herein.

A "C-amido" group refers to a -C(=O)-NR’R” group, where each of R’ and R” is as defined herein.

An "N-amido" group refers to an R’C(=O)-NR”- group, where each of R’ and R” is as defined herein.

An “amide” group encompasses both C-amido and N-amido groups.

An amide bond describes a -NR'-C(=O)- bond, where R' is as defined herein.

A "urea" group refers to an -N(R’)-C(=O)-NR”R’” group, where each of R’ and R” is as defined herein, and R” ’ is defined as R’ and R’ ’ are defined herein.

A "nitro" group refers to an -NO ₂ group.

A "cyano" group refers to a -C=N group.

The term “halide”, as used herein, refers to the anion of a halo atom, i.e. F“, CT, Br“ and T.

The term “halo” or “halogen” refers to F, Cl, Br and I atoms as substituents.

The term “phosphonyl” or “phosphonate” describes a -P(=O)(OR’)(OR”) group, with R’ and R’ ’ as defined hereinabove.

A “phosphonic acid” is a phosphonate group is which each of R’ and R’ ’ is hydrogen.

The term “phosphate” describes an -O-P(=O)(OR’)(OR”) group, with each of R’ and R” as defined hereinabove.

A “phosphoric acid” is a phosphate group is which each of R’ and R” is hydrogen.

The term “phosphinyl” describes a -PR’R” group, with each of R’ and R” as defined hereinabove.

The term “thiourea” describes a -N(R’)-C(=S)-NR”- group, with each of R’ and R” as defined hereinabove.

Any of the compounds described herein can be in a form of a pharmaceutically acceptable salt thereof. The phrase "pharmaceutically acceptable salt" refers to a charged species of the parent compound and its counter ion, which is typically, but not necessarily, used to modify the solubility characteristics of the parent compound and/or to reduce any significant irritation to an organism by the parent compound, while not abrogating the biological activity and properties of the administered compound. An exemplary salt according to the present embodiments includes a compound featuring a negatively charged group as described herein in any of the respective embodiments and a counter ion, for example, an alkali metal ion.

The present invention further encompasses prodrugs, solvates and hydrates of the substances described herein.

As used herein, the term “prodrug” refers to an agent, which is converted into the active compound (the active parent drug) in vivo. Prodrugs are typically, but not necessarily, useful for facilitating the administration of the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility as compared with the parent drug in pharmaceutical compositions. Prodrugs are also often used to achieve a sustained release of the active compound in vivo. An example, without limitation, of a prodrug would be a compound, as described herein, having one or more carboxylic acid moieties, or one or more phosphoric or phosphonic acid moieties, which is administered as an ester (the “prodrug”). Such a prodrug is hydrolysed in vivo, to thereby provide the free compound (the parent drug). The selected ester may affect both the solubility characteristics and the hydrolysis rate of the prodrug. An example, without limitation, of a prodrug according to some embodiments of the present invention, includes a compound as described herein in any of the respective embodiments, in which the negatively charged group is masked, for example, in a form of an ester of a carboxylic acid or an ester of a phosphate or phosphonate group or an ester of a sulfonic or sulfuric acid. The selected ester is hydrolyzed in the vicinity of its target (e.g., GSK-3) by, for example, a suitable esterase, hydrolase, and/or acidic physiological conditions, to thereby regenerate the negatively charged group which interacts with the target as described herein.

The term “solvate” refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta- , hexa-, and so on), which is formed by a solute (the compound as described herein) and a solvent, whereby the solvent does not interfere with the biological activity of the solute. Suitable solvents include, for example, ethanol, acetic acid and the like.

The term “hydrate” refers to a solvate, as defined hereinabove, where the solvent is water.

The phrase “negatively charged group”, as used herein, refer to an ionizable group, which upon ionization, typically in an aqueous medium, has at least one negative 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 some embodiments of the present invention, has the formula wherein V 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 U are each independently selected from the group consisting of oxygen and sulfur; and 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.

For example, when the negatively charged group is a phosphate group, in the formula above V is a phosphor atom, and each of Q, G and U is oxygen. E can be hydroxy.

For example, when the negatively charged group is a carboxylate group, in the formula above, V is a carbon atom, Q and E are absent, and each of G and U is oxygen.

As used herein throughout, the phrase “leaving group” describes a labile atom, group or chemical moiety that readily undergoes detachment from an organic molecule during a chemical reaction, while the detachment is facilitated by the relative stability of the leaving atom, group or moiety thereafter. Typically, any group that is the conjugate base of a strong acid can act as a leaving group. Representative examples of suitable leaving groups according to the present embodiments therefore include, without limitation, carboxylate (e.g., acetate), thiocarboxylate, sulfate (e.g., tosylate, mesylate), sulfonate (e.g., triflate), sulfinate, thiosulfate, thiosulfonate, thiosulfinate, sulfoxide, alkoxy, halogen (preferably bromo or iodo), amine, sulfonamide, carbamate, thiocarbamate, azide, phosphonyl, phopshinyl, phosphate, cyanate, thiocyanate, nitro and cyano, as these terms are defined herein.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

MATERIALS AND EXPERIMENTAL METHODS

Compounds: Compounds selected by virtual screening were purchased from MolPort Riga, Latvia, that supplied compounds from ChemDiv Inc., San Diego, CA USA, Specs, Zoetermeer, The Netherlands, and Enamine Ltd. NJ, USA. Compounds 3-7 and 3-8 were purchased in three different batches to validate results. Validation of compounds 3-7 and 3-8 was conducted in house by NMR, high-resolution mass spectrometry, and HPLC (data not shown).

Compounds 4-1, 4-2, 4-3, 4-4 and 4-5 were synthesized as described herein.

Compounds were dissolved in DMSO for in vitro studies and with DMSO/1 % Tween®80 for studies in cells.

In vitro kinase assays : An ELISA-based assay was developed in our laboratory to measure GSK-3 activity. In brief, 96-well microplates coated with avidin were bound to biotin-labelled IRS- 1 peptide substrate [Liberman, Z. & Eldar-Finkelman, H. J. Biol. Chem. 280, 4422-8 (2005)]. A GSK-3 assay solution (0.5 μg GSK-3β, 20 mM Tris (pH 7.3), 10 mM MgCl ₂ , and 10 μM ATP) was added to each well, together with the candidate molecule at indicated concentrations, and the plates were incubated for 15 minutes at 30 °C. After having been washed, the plates were incubated with a specific anti-phospho IRS-1 antibody (Ser ³³² ) (custom service) (1:2000), followed by HRP- secondary antibody (1:8,000). The signal was developed with TMB solution, stopped with H ₂ SO ₄ , and was monitored in a plate reader (580 nm).

Cells: CHO-IRS-1 cells were grown in F12 medium supplemented with 10 % fetal calf serum (FCS), 5 mM L-glutamine, and 0.5 mg/ml Penicillin Streptomycin. SH-SY5Y cells were grown in RPMI 1640 medium supplemented with 10 % fetal calf serum (FCS), 5 mM L-glutamine, and 0.04 % gentamycin. For western blot analysis, after treatment with the indicated molecules, cells were collected and lysed in ice-cold buffer G (20 mM Tris-HCl, 10 % glycerol, 1 mM EDTA, 1 mM EGTA, 0.5 % Triton X100, 0.5 mM orthovanadate, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM NaF, 1 mM benzaminidine, and protease inhibitors aprotenin, leupeptine, and pepstatin A). Cell extracts were centrifuged at 14,000xg for 20 minutes, and supernatants were collected. Protein concentrations were determined by Bradford assays. In some experiments, CHO cells were transiently transfected with GFP-β-catenin plasmid using Lipofectamine 2000 transfected reagent. The cells were fixed with 4 % PFA and visualized by fluorescence microscopy. GFP-β-catenin signal intensity in the cell nucleus was analyzed by image J measuring integrated density.

Western blot analysis: Equal amounts of proteins (30-50 pg) were subjected to SDS PHAGE gel electrophoresis and transferred to nitrocellulose membranes, and immunoblotted with anti-phospho IRS-1 (Ser ³³² ), anti-β-catenin antibody (Transduction Laboratories, NJ, US), tau, phospho-tau (Ser ³⁹⁶ ), β-actin, (all from Cell Signaling), B23, or Cathepsin D (Cell Signaling, MA, US), followed by incubation with HRP-linked anti-rabbit, or anti-mouse IgG (Cell signaling and Jackson Immune Research, PA, US, respectively). Densitometry analysis of respected bands was analyzed with Image J software. Representative protein bands are taken from the same gel, and each gel represent three independent experiments.

Lysosome staining and live-cell imaging-. Cells were treated with indicated concentration of the tested compound for 4 hours and then incubated with 50 nM LysoTracker Red (Molecular Probes) for 12 minutes at 37 °C. Live cells were imaged on a laser scanning confocal microscope (Leica TCS SP5 II) with x63.0 and N.A 1.40 objective lens. Lysosomes' puncta were analyzed with Imaris BITPLANE using the spots detection feature.

Immunostaining:

Primary neuron cells were treated with the tested compound (5 μM), CHIR99021 (10 μM) (Merck, MA, USA), or, vehicle (DMSO/1 % Tween®80) for 4 hours. Cells were fixed with 4 % PFA and immunostained with anti-Pcatenin and anti-MAP2 (Sypnactic System, Goettingen, Germany, A neuronal marker) antibodies. B-catenin signal was evaluated by Image J software using co-localization finder plugin (www(dot)//imagej(dot)nih.gov/ij/plugins/colocalization- finder(dot)html) that calculated the ratio of Pcatenin over MAP2 and DAPI in each cell. The average ratio that was determined for control vehicle-treated cells was set to 1 and respective folds of treated cells were calculated accordingly.

Primary neurons: Primary spinal motor neurons were isolated from ICR mice at embryonic day 12.5 (E12.5). In brief, E12.5 mouse embryos were collected in IX HBSS buffer, trypsinized, and consequently put in L-15 medium (Life Technologies) containing 0.4 % BSA and 2 %-10 % DNAse. Cells were centrifuged through a 4 % BSA cushion and then resuspended in Complete Neurobasal Medium (CNB) containing 2 % Horse serum, 2 % B27 Supplement, 1 % Glutamax, 1 % Penicillin-Streptomycin, 0.5 % 2-mercaptoethanol, 1 ng/mL BDNF, B-250, 1 ng/mL GDNF, and 0.25 ng/mL CNTF. Motor neurons were isolated by centrifugation through Optiprep gradient (10.4 % Optiprep (Sigma-Aldrich), 10 mM Tricine, 4 % glucose) for 20 minutes at 760 x g. Neurons were then plated in 96-well plates (5000 cells per well) pre-coated with poly D-L- ornithine and Laminin with complete Neurobasal medium. Phase contrast microscopic images were automatically taken using the IncuCyte Live Cell Imaging System once a day for 6 days. Neurite length was automatically analyzed with IncuCyte’ s NeuroTrackl.l software. Two independent experiments were performed using five well repeats and ten well repeats, respectively, from each indicated condition. All animal experiments were performed under the supervision and approval of the Tel- Aviv University Committee for Animal Ethics.

PC A Analysis:

A principal component analysis (PCA) was performed for a small subset of all compounds tested in this work together with 29 selected GSK-3-ATP competitive inhibitors retrieved from the CheMBL database (listed in FIG. 11B). Each compound was characterized by a total of 1875 descriptors (1444 ID and 2D descriptors and 431 3D descriptors) calculated by the PaDEL- Descriptor software (www(dot)yapcwsoft(dot)com/dd/padeldescriptor/). The resulting descriptors matrix was submitted to Principal Component Analysis (PCA) as implemented in Canvas (Schrodinger Release 2017-4: Canvas, Schrodinger, LLC, New York, NY, USA, 2017). The first and second PCs accounted for 81.8% and 7.7% of the original variance, respectively.

Statistical analyses: Statistical analyses were performed with GraphPad Prism 7 software.

Data are shown as means ± SEM of three independent experiments or as indicated in the figure legends. The unpaired Student’s /-test was used for comparison of assays incubated with compound vs. control and of cells treated with 3-8 vs. non-treated cells, p < 0.05 was considered significant.

Additional experimental methods are described within the below Examples.

EXAMPLE 1

Design of a pharmacophore model based on the GSK-3/SCI peptide binding model

The present inventors have used the previously developed and validated SCI peptide binding mode to GSK-3 as a basis for a reliable pharmacophore model.

In brief, previously uncovered GSK-3 SCI peptides are short phosphorylated peptides derived from the unique substrate-recognition motif of GSK-3, S ¹ XXXS ² (p) (SEQ ID NO:2) (where S ¹ is the GSK-3 -phosphorylation site and S ² is the phosphorylated priming site) and are patterned after the GSK-3 substrate heat shock factor- 1. An exemplary peptide designated as L8O3F (KEAPPSPPQS(p)PF; SEQ ID NO:5) was found to be a potent GSK-3 SCI inhibitor. This peptide constitutes an improved version of the ‘original’ L8O3 peptide inhibitor (KEAPPSPPQS(p)P; SEQ ID NO:4). Background Art FIG. 1A shows that the addition of Phenylalanine to the C-terminal end of L8O3 (SEQ ID NO:4) strengthens its interaction with Phe 93 located at the ‘89-95’ loop (SEQ ID NO:3), a critical site for substrate binding. In addition, the GSK-3 (SEQ ID NO:1)/L803F (SEQ ID NOG) structural model identified Tyr 216, Vai 214, and IIe 217, which formed an ‘hydrophobic patch’, which is important for substrate/inhibitor interaction. The priming site (phosphorylated serine) of L8O3F (SEQ ID NOG) interacted with the phosphate-binding pocket of the kinase (Arg 96, Arg 180, and Lys 205). In vitro GSK-3 kinase assays confirmed that L8O3F (SEQ ID NOG), or its cell-permeable form, L8O3Fmts (e.g., myristic acid is attached to its N-terminal; SEQ ID NOG), is a better GSK-3 inhibitor with a 10-fold increase in potency, compared with the parent L8O3mts (SEQ ID NOG) (see, Background Art FIG. IB). L8O3Fmts (SEQ ID NOG) was also shown to be a potent inhibitor in cells and animals. For example, treating mice with L8O3Fmts (SEQ ID NOG) reduced the phosphorylation levels of the microtubule-associated protein tau (Ser 396), a well-known GSK-3 target, in the mouse hippocampus (see, Background Art FIGs. 1C and ID).

Based on the above data, the present inventors conceived that molecules that mimic the binding mode of SCIs such as L8O3F (SEQ ID NOG) with GSK-3 would function as potent SCI molecules and have sought for such molecules.

To this end, a structure-based pharmacophore model was generated, based on the GSK-3 (SEQ ID NO:1)/L803F (SEQ ID NOG) binding model described above.

The pharmacophore model consisted of six features (F1-F6): two hydrogen bond acceptors (Fl, F2), one anionic feature (F3), and three hydrophobic features (F4-F6), as shown in FIG. 2. These features correspond to the interaction of L8O3F with the GSK-3-binding site residues Arg 96, Lys 205 (hydrogen bond acceptors), Arg 96, Arg 180, Lys 205(anionic), and Phe 93, pTyr 216, and IIe 217 (hydrophobic).

Exclusion volumes were also derived, based on the protein environment. The initial pharmacophore was found to be too large to be fitted by small molecule compounds and thus, its size was reduced by setting up the pharmacophore feature corresponding to the hydrophobic interaction within the hydrophobic patch (F6) as optional.

The structure-based pharmacophore model, based on the GSK-3 peptide complex, was generated using LigandScout 4.0 [Wolber, G. & Langer, T. J Chem Inf Model 45, 160-9 (2005)]. The pharmacophore model consisted of six features (F1-F6): two hydrogen bond acceptors (Fl, F2), one anionic feature (F3), and three hydrophobic features (F4-F6). Exclusion volumes were also derived, based on the protein environment. The initial pharmacophore was found to be too large to be fitted by small molecule compounds and thus, the pharmacophore feature (F6) corresponding to the hydrophobic interaction with pTyr 216 or IIe 217 was set up as optional.

EXAMPLE 2

Identification of GSK- 3 SCIs (First cycle)

The pharmacophore model described in Example 1 herewin was successfully assessed for its ability to recognize the active conformation of L803F peptide and was used to virtually screen a database of about 6.36 million commercially available compounds from the ZINC database [Irwin, J. J. & Shoichet, B.K. ZINC— a free database of commercially available compounds for virtual screening. J Chem Inf Model 45, 177-82 (2005)]. Twenty-five conformers were generated for each compound in the database using the OMEGA [Hawkins et al. Conformer generation with OMEGA: algorithm and validation using high quality structures from the Protein Databank and Cambridge Structural Database. J Chem Inf Model 50, 572-84 (2010)] conformer generator to create a multi -conformer structure database.

Screening resulted in 3680 hits, which were subsequently docked into the GSK-3 site. L803F was also included in the docking procedure and the filtration procedure discussed below to validate the screening method.

Prior to docking, hits were prepared by LigPrep [Shelley, J.C. et al. Epik: a software program for pK (a) prediction and protonation state generation for drug-like molecules. J Comput Aided Mol Des 21, 681-91 (2007)] as implemented in Maestro (Schrodinger, USA) at pH = 7 +/' - 0.2 with OPLS 2005 FF, including tautomeric variations.

Docking was performed using Extra-Precision (XP) Glide [Friesner, R.A. et al. J Med Chem 49, 6177-96 (2006)].

The resulting binding mode were filtered using interaction fingerprints based on the known critical interactions formed between L803F and GSK-3, namely, those involving Arg 96, Arg 180, Lys 205, and Phe 93.

Filtration resulted in 1024 compounds, including the positive reference L803F. The ten best compounds, as ranked by GlideScore, as presented in Table 1 below, were selected for biological testing.

Thus, the pharmacophore model was used to screen the “in stock” portion of the ZINC database consisting of about 6.36 million compounds and to select those having features compatible with the model. Screening identified 3680 hits that matched at least five pharmacophoric features. These hits were subsequently docked into the GSK-3 substrate binding site using Glide, and ranked using GlideScore.

The ten best molecules, termed 1-1-1-10 (see, Table 1 below) were selected for purchasing and their inhibitory activity was tested in GSK-3β in vitro kinase assays, as described in the Materials and Methods section hereinabove.

Table 1

(Table 1; Cont.)

In vitro kinase assays to measure GSK-3 activity were conducted with the ten selected hits shown in Table 1 at a concentration of 200 μM. Results are presented as the percentage of GSK-3 activity without inhibitor and represent the mean of three independent experiments. Results were significant for 1-4, 1-5, 1-6, 1-7, p values were in the range of 0.01-0.001 by one way ANOVA with Dunnett’s multiple comparisons.

In the kinase assays conducted, three hits, namely, 1-4, 1-6, and 1-7 exhibited 70-80 % inhibition at a concentration of 200 μM, as shown in FIG. 3A. Detailed dose response curves revealed an IC ₅₀ value in the range of 80-200 p M (as shown in the table in the lower panel in FIG. 3 A, which lists the IC ₅₀ values of the best performing compounds, 1-4, 1-6, and 7-7).

Two-dimensional (2D) schematic presentations of the predicted interactions (within 4 A from the docked compound) of 1-4, 1-6, and 1-7 with the GSK-3 substrate binding site are presented in FIG. 3B. Red, blue, and green spheres represent positively charged, negatively charged, and hydrophobic residues, respectively. Hydrogen bonds are denoted as pink arrows, 71-71 stacking is shown as green lines, and 71 -cation stacking is shown as red lines.

As shown in FIG. 3B, the three compounds were of diverse structures; however, they all shared a carboxylic acid moiety (see, FIG. 3B, red arrows). According to the docking analysis shown in FIG. 3B, this negatively charged acidic functionality enabled H-bonding and ionic interactions with one or two of the basic residues that form the phosphate binding pocket of GSK- 3. This novel observation indicated for the first time that a non-phosphorylated group can mimic the ‘primed’ phosphorylation site of the GSK-3-substrates.

As further shown in FIG. 3B, all three compounds interacted with the positively charged residues at the phosphate binding pocket, but also showed additional and different interactions. Thus, compound 1-4 formed H-bonding with GSK-3-Arg 96 and GSK-3-Gly 202 through the carbonyl of the anthracenone ring and the amine at the carboxylic chain, respectively. Compound 1-6 formed H-bonding with GSK-3-Asn 95 and Glu 97 through its phenyl hydroxyl group and ketone. Apparently both Asn 95 and Glu 97 are important elements in kinase activity: Asn 95 is a residue within the ’89-95’ loop, an important substrate binding pocket, and Glu 97 is a highly conserved residue in protein kinases located at the conserved aC-helix, which is responsible for phosphoryl transfer [louz et al. J. Biol. Chem. 281, 30621-30 (2006); Bhat, R. et al. J. Biol. Chem. 278, 45937-45 (2003)]. Compound 1-6 also formed an H-bonding and a 71-cation interaction with GSK-3-Arg 96 through the carbonyl of the anthracenone ring and the thiophene ring, respectively. Compound 7-7 formed H-bonding with GSK-3-Val 114 through its carboxylic group, and its bromo-aromatic ring formed stacking interactions with Phe 93. Both Phe 93 and Vai 114 were previously shown to play a role in GSK-3-substrate/inhibitor interactions [Licht-Murava et al. J. Mol. Biol. 408, 366-78 (2011)]. Taken together, the identified molecules captured binding elements similar to those of L8O3F with GSK-3. Furthermore, they exhibited binding with residues that are not part of the pharmacophore features, but play a role in the kinase activity of GSK-3, thus supporing the practiced approach for idetifying potential SCIs.

EXAMPLE 3

Identification of GSK-3 SCIs (Second cycle)

Compounds 1-4, 1-6, and 1-7 were used as starting points, to find additional SCIs with improved activity. A second search was conducted to find in the ZINC Database analogues with 90 % similarity to 1-4, 1-6, and 1-7. The hits (241 analogues for 1-4, 11 analogues for 1-6, and 21 analogues for 1-7), were docked into the GSK-3 substrate binding site and ranked, as described hereinabove, by GlideScore.

Two methods of docking were used: Glide XP and Induced Fit docking (IFD) in which residues within 10 A of any of the resulting top 20 ligand poses (from the initial docking) were subjected to a conformational search and minimization, whereas residues outside this region were kept fixed [Sherman et al. J Med Chem 49, 534-53 (2006)]. The final 20 new receptor conformations were taken forward for redocking with Glide XP. The binding affinity of each complex was evaluated by GlideScore, and was based on a combination of GlideScore and visual inspection of the resulting binding modes.

Based on GlideScore and visual inspection of the resulting binding modes, 30 compounds were selected, from which 11 available compounds, presented in Table 2 below, were purchased.

Table 2

(Table 2; Cont.)

As can be seen in Table 2, all compounds possessed a carboxylic acid moiety, indicating that this moiety is needed for binding (as indeed was shown in the previous cycle). As can further be seen, the best scoring compounds are 1 -4 analogues typically bearing an anthracenone-isoxazole core. Compounds 2-1 and 2-2 were predicted to be the best binders.

GSK-3 in vitro kinase assays were conducted with the compounds shown in Table 2 at a concentration of 20 μM. The results are presented in FIG. 4A, as the percentage of GSK-3 activity without an inhibitor and represent the mean of three independent experiments. Results were significant for 2-1, 2-2, 2-3, 2-4, 2-6, where p values were in the range of 0.01-0.001 by one way ANOVA with Dunnett’s multiple comparisons.

As shown in FIG. 4A, the in vitro kinase assays confirmed that out of the eleven compounds tested, Compounds 2-1 and 2-2 exhibited about 70 % inhibition at a concentration of 20 μM, a significant improvement, compared with the performance of first cycle compounds such as 1-4, 1- 6, and 1-7.

The IC ₅₀ values of 2-1 and 2-2 were evaluated by dose inhibition curves, and are shown in FIG. 4B, with estimated IC ₅₀ values of 11 and 21 μM for 2-1 and 2-2, respectively.

FIGs. 4C and 4D presents the docking of 2-1 (FIG. 4C) and 2-2 (FIG. 4D) into the GSK-3 substrate binding site. Key interactions formed between 2-1 and 2-2 and the GSK-3 binding site residues are indicated by dashed lines.

As shown in FIGs. 4C-D, the carboxylic acid moiety of 2-1 and 2-2 interacted with Arg 96 and Arg 180 or Arg 180 and Lys 205, respectively. In 2-1 the carboxylic acid moiety also formed H-bonding with Vai 214. Furthermore, the anthracenone-isoxazole core formed 7t-7t stacking interactions with Phe 93, and the O and N atoms at the isoxazole ring formed H-bonding with Arg 96. Compound 2-2 did not interact with Phe 93, but its benzoic acid moiety formed 7t-cation interactions with Arg 96, and the ester group formed H-bonding with Lys 85.

Although Lys 85 was not part of the pharmacophore features, it has been shown to be a critical element in ATP binding [Houz et al., 2006, supra). Thus, it appeared that 2-1 exhibited a binding mode similar to the previously described SCI peptide(s), whereas 2-2 possesses a different binding set that includes interactions with Lys 85 and Gly 202, which are assumed to contribute to the inhibition potency of 2-2.

These data further supports the practiced approach.

EXAMPLE 4

Identification of GSK-3 SCIs (Third cycle)

The results of the second cycle showed a preference for an anthracenone-isoxazole core attached to a piperazine ring as SCIs. Thus, to identify compounds with yet improved activity, a third search cycle was conducted for compounds featuring a substructure composed of an anthracene substructure attached to piperazine/cyclohexane.

From this search, 137 compounds were obtained, from which only 15 had a negative charge that could mimic the phosphorylated moiety of L8O3F and that interacted with the positive pocket of GSK-3 (Arg 96, Arg 180, and Lys 205). Fourteen of these compounds, presented in Table 3 elow and numbered from 3-1 to 3-14, were commercially available and were used for further IFD flexible docking for identification of binding mode and for experimental testing.

GSK-3 in vitro kinase assays were conducted with compounds 3-1-3-14 at a concentration of 20 μM. The results are presented in FIG. 5A, as the percentage of GSK-3 activity without inhibitor and represent the mean of three independent experiments. All results were statistically significant except for 3-1 and 3-12 for which p values were in the range of 0.01-0.0001 by one way ANOVA with Dunnett’s multiple comparisons FIG. 5B presents dose inhibition curves for compounds 3-3, 3-7 and 3-8, with the respective IC ₅₀ values listed in the table therein.

In vitro GSK-3 kinase assays indicated that most of the purchased compounds could indeed inhibit GSK-3 at 20 μM, further indicating that extending the molecular core by adding a piperazine/cyclohexane moiety improves the binding. The obtained IC ₅₀ values presented in FIG. 5B and Table 3 below showed that 3-3, 3-7, and 3-8 were the best inhibitors, having a IC ₅₀ value in the single digit μM range, a significant improvement of about 2-fold, compared with the second cycle molecules.

Table 3

(Table 3; Cont.)

The docking of 3-7 and 3-8 into the GSK-3 substrate binding site is presented in FIGs. 5C and 5D, respectively. Key interactions that formed between 3-7 and 3-8 and between the binding site residues are indicated by dashed lines. In both compounds the carboxylic acid moiety interacts with all three residues of the phosphate binding pocket (Arg 96 and Arg 180, Lys 205), and Phe 93 of GSK-3 forms 71-71 stacking interactions with the isoxazole ring. In 3.7 (upper panel), Lys 85 interacts with the metoxy group (MOE).

Docking analysis predicted that 3-3 forms fewer interactions with GSK-3, compared with 3-7 and 3-8 (not shown).

The docking models shown in FIGs. 5C-D could clearly explain the improvement in 3-7 and 3-8. Unlike SCIs from the first and second cycles, the carboxylic acid moiety of 3-7 and 3-8 formed H-bonding and ionic interactions with all three positively charged residues, Arg 96, Arg 180, and Lys 205 of the phosphate binding pocket. In both compounds, the anthracenone-isoxazole core formed 71-71 stacking interactions with Phe 93, and the heteroatoms of the isoxazole ring (O and N) formed H-bonding with Arg 96. Compound 3-7 also interacted with Lys 85 through its methoxy group. Thus, there was a good correlation between the number of interactions, the binding energy of the molecule docked in the active site, and the compound’s potency. To further verify that compounds 3-7 and 3-8 act as substrate-competitive inhibitors, the sensitivity of 3-7 and 3-8 to increasing concentrations of ATP was tested and the results were compared with the ATP competitive GSK-3 inhibitor SB216763 [Coghlan, M.P. et al. Chem Biol 7, 793-803 (2000)].

The ability of compounds 3-7 and 3-8 to interact with Phe 93 was also tested. To this end, a GSK-3 mutant F93A-GSK-3 (SEQ ID NO:8) in which Phe 93 was replaced by alanine (F93A), was used. FIGs. 5E and 5F present the data obtained in in vitro kinase assays performed with the mutant F93A-GSK-3 (SEQ ID NO:8) in the presence of the indicated compounds, L8O3F (SEQ ID NO:5) (20 μM each), or SB216763 (denoted SB; 1 or 0.5 μM). Results are mean of three independent experiments ± SEM analyzed by one way ANOVA with Dunnett’s multiple comparisons. ** p < 0.01 with inhibitor vs no inhibitor.

As shown therein, 3-7 and 3-8 inhibited GSK-3 in the presence of 10-500 μM ATP, while SB216763 gradually lost its ability to inhibit GSK-3 with increasing doses of ATP.

The obtained data show that 3-7 and 3-8 were not effective in inhibiting F93A, in contrast to SB216763.

FIG. 5G summarizes the data obtained in in vitro kinase assays with GSK-3-WT (SEQ ID NO:1) and a GSK-3 mutant F93A-GSK-3 (SEQ ID NO:8) for selected compounds, compared with L8O3F (SEQ ID NO:5) and SB216763.

A principal component analysis (PCA) was conducted to determine whether the SCI compounds are chemically distinct from other known GSK-3 inhibitors. This was performed to confirm that the discovered compounds are unique. Briefly, PCA projects a dataset originally described in a high-dimensional space into a 2-dimensional space while keeping, as much as possible, the original distribution of the data points (i.e., the distances between them). The analysis was performed on all the retrieved compounds together with representative ATP competitive GSK- 3 inhibitors including SB-216763, CHIR98014, AR-A014418, VP2.51, Bio6, Kenpaullone, and 1- Azakenpaullone (listed in FIG. 11B).

FIG. 5H presents the data obtained in PCA analysis of the compounds retrieved in the three cycles and representative GSK-3-ATP competitive inhibitors listed in FIG. 11B. The first and second PCs accounted for 81.8 % and 7.7 % of the original variance and are shown at the X-axis and Y-axis, respectively. Black circles represent the ATP competitive inhibitors, green circles represent exemplary compounds retrieved in the first cycle, red circles represent exemplary compounds retrieved in the second cycle, and blue circles represent exemplary compounds retrieved in the third cycle. The analysis clearly showed that the SCI compounds occupy a chemical space distinct from that occupied by the ATP competitive inhibitors. The PC A analysis also revealed the successive focusing toward the active compounds region obtained through the three search cycles.

Thus, novel compounds that act as SCIs for GSK-3 were uncovered based on the SCI strategy described herein.

EXAMPLE 5

Biological profiling

Compound 3-8 was tested in biological systems.

The ability of 3-8 to reduce the phosphorylation of the GSK-3 target, the insulin receptor substrate-1 (IRS-1), at serine 332 (Ser ³³² ) was determined in Chinese hamster cells (CHO) expressing IRS-1. According to this scenario, the ‘priming site’ on IRS-1 is Ser ³³⁶ , which is phosphorylated by PKC ⁵⁴ . Thus, CHO-IRS-1 cells were first incubated with 3-8 and then cells were stimulated with phorbol-ester (PMA, 30 min) to initiate IRS-l-Ser ³³⁶ phosphorylation.

CHO-IRS-1 cells were treated with 3-8 (4 hours) at the indicated concentrations and were stimulated with PMA for 30 minutes. Phosphorylation of IRS-1 (Ser ³³² ) and tyrosine phosphorylation of GSK-3α/β were determined by immunoblot analysis. All bands are from the same experiment and the same gel.

The obtained data is presented in FIG. 6A and show that 3-8 significantly reduced IRS-1 phosphorylation of Ser ³³² in the PMA-treated cells. Phosphorylation at Tyr 271/216 at GSK-3a and GSK-3β respectively, slightly decreased by 3-8, which could further indicate that 3-8 does not act as an ATP competitive inhibitor, since phosphorylation of these sites reflects autophosphorylation [Hughes et al. EMBO J. 12, 803-8 (1993)].

Another ‘classical’ target of GSK-3 is β-catenin, a Wnt signaling component, which upon phosphorylation by GSK-3, undergoes proteolytic degradation. Therefore, inhibition of GSK-3 would result in stabilization of β-catenin. To observe the impact of 3-8 on β-catenin, CHO cells that were transiently transfected in GFP-β-catenin plasmid were used.

CHO cells transiently expressing GFP-β-catenin were treated with the indicated concentrations of 3-8 or with SB216763 (10 μM) for 4 hours. GFP signal was imaged in fixed cells by fluorescence microscopy and after treatment with 3-8, and the results are presented in FIG. 6B. β-catenin punctate were accumulated in the nucleus and are marked by white arrows. Quantification of the GFP-β-catenin signal in the nucleus is shown in FIG. 6C. Results are mean of 5-7 fields with a total amount of 80 cells +SEM *p<0.05, **p<0.01 as determined by Student’s t-test treated vs. control.

The GFP-β-catenin signal was accumulated in the cell nucleus and appeared as punctate that were larger and more intense in the 3-8 treated cells. Treating with SB216763, an ATP competitive GSK-3 inhibitor showed similar results, as shown in the bar graph in FIG. 6C.

Lysosomal acidification is also a target regulated by GSK-3 through the autophagy and endocytic pathways [Azoulay-Alfaguter et al. (2015) Oncogene 34, 4613-4623; Avrahami et al. (2020) Cell Signal 71, 109597]. Lysosomes are acidified organelles and their acidification is crucial for their proper function. Inhibition of GSK-3 triggers lysosome acidification.

Thus, an additional assay was performed in SH-SY5Y human neuroblastoma cells that were treated with 3-8 and then stained with LysoTracker Red, a dye that specifically stains acidified lysosomes.

SH-SY5Y cells were treated with the indicated concentrations of 3-8. Live cells were stained with LysoTracker Red and with Hoechst dye and imaged by confocal microscopy. Acidified lysosomes appear as red dots and nuclei are stained blue. The obtained data is shown in FIG. 6D. Quantification of number of lysosomes’ red puncta is shown in FIG. 6E. Results are mean of 100-120 cells +SEM. **p<0.01, ***p<0.001 as determined by Student’s t-test treated vs. control.

As shown in FIG. 6D, an increase in the number of acidified lysosomes was readily detected in the 3-S-treated cells, whereby compound 1 -8, which was shown as a non-potent GSK-3 inhibitor, did not affect the lysosomes.

GSK-3 has been reported as a critical regulator of shape, morphogenesis and axon dynamics in developing embryonic neurons. The ability of 3-8 to impact growth of embryonic neurons was tested. To this end, spinal cord motor neurons prepared from E12.5 mouse embryos were cultured in 96-well plates and incubated with various doses of 3-8 for six days. Neurite outgrowth was monitored every day in IncuCyte automated live-cell imaging; data were collected and analyzed.

Embryonic spinal cord motor neurons in 96-well plates were incubated with 5, 10, and 20 μM daily with 3-8 for six days. Neurite outgrowth was monitored daily. FIG. 6F presents representative photos of cells (control and treated with 3-8 (20μM)) at day 6. FIGs. 6G and 6H are bar graphs showing changes in neurite length and the number of branching points during the six days of treatment. Results in both graphs represent the mean of 10 filed in 10 wells per time point. Statistics of neurite length: for experiments done with 5 μM 3-8, 24 hours, *p<0.05, 48 and 72 hr **p<0.0, 96-168 hr ***p<0.0. In the experiments conducted with 10 and 20 μM 3-8, 24 hr, ***p<0.01, other time intervals, ****p<0.0001. Statistics of branching points: for experiments done with 5p,M 3-8, ** p<0.01 for all time point. In experiments conducted with 3-8 at 10 and 20 p.M, 24 hr, 48 hr, **p<0.01, 96-168, ****p<0.0001. Statistics were carried out with multiple Student’s t-test for treated vs. non-treated cells.

As can be clearly seen in FIGs. 6F and 6G, 3-8 prevented ‘normal’ neurite outgrowth causing about 14 % reduction in neuron length at day 6. This was accompanied by a reduction of about 28 % in the formation of neurite branching points (FIG. 6H). These results demonstrate the role of GSK-3 in regulating the growth of embryonic motor neurons.

To ensure the potential biological activity the ability of Compound 3-8 to inhibit cellular GSK-3 was tested. β-catenin amount, a major Wnt signaling component, is a well-established GSK-3 target. β-catenin undergoes proteolytic degradation upon phosphorylation by GSK-3, and alterations in β-catenin amounts or cellular distribution is a ‘good’ marker for inhibition of GSK- 3.

To this end, SH-SY5Y cells were treated with 3-8 and β-catenin levels were determined in the detergent-soluble and detergent-insoluble fractions that included the cytoplasm and membranes, and cells’ nuclei respectively. As can be seen in FIGs. 7A and 7B, treatment with 3-8 increased β-catenin levels in both cellular fractions, with major changes in the nuclear fraction. This likely reflected the translocation of β-catenin to the cell nucleus upon activation (and stabilization) by 3-8.

Lysosomal acidification is another target regulated by GSK-3 through the autophagy and endocytic pathways, as further discussed hereinbelow. Lysosomes are acidified organelles and their acidification is crucial for their proper function.

SH-SY5Y cells were treated with 3-8 and live cells we stained with LysoTracker Red, a dye that stains acidified lysosomes. As shown in FIGs. 7C and 7D, 3-8 increased the number of acidified lysosomes as indicated by increased ‘red puncta’ in the 3-8-treated cells (FIG. 7C). Compound 1-8 that did not inhibit GSK-3 was used as a negative control (FIG. 7D).

EXAMPLE 6

Intermediate Remarks

Small-molecule substrate competitive inhibitors (SCIs) present an appealing alternative to the more classical ATP competitive inhibitors because of their potentially improved pharmacological profile. GSK-3 SCI peptides were described in previous studies, and the present inventors used structural models of the GSK-3/SCI peptide complex as a basis for searching for molecules that mimic the interactions of the SCI peptide.

Analyses of the compounds uncovered in the screening tests that were conducted provided some insights into the structural/element requirements for SCI inhibitors. Our requirement is of a flexible chain bearing a carboxylic acid moiety, which enables interactions with three amino acids (Arg 96, Arg 180, and Lys 205) that facilitate interactions with the ‘primed’ phosphorylated substrates of GSK-3. The anthracenone-isoxazole substructure (5-amino-6/7-anthra[ 1 ,9- (z/Jisoxazol-6) appeared to be the preferred element in most compounds. The isoxazole ring within this core formed significant interactions with Phe 93 and Arg 96 because of its aromaticity and its heteroatoms O and N bear non-bonding electrons. However, except for the isoxazole ring, the anthracenone-isoxazole core did not form specific interactions with GSK-3. Without being bound by any particular theory, it is assumed that the planar unsaturated aromatic nature of this core may play an important role in the spatial arrangement of the molecule within the enzyme binding pocket.

Shorter chains bearing carboxylic acid (2 methylenes vs 3), were shown to be preferred, as can be concluded, for example, by comparing 3-8 with 2-1.

A strong dipole moment formed by the fluorine or the methoxy group attached to the aromatic ring in 3-8 or 3-7, respectively, is assumed to play an important role in maneuvering the molecule deeply into the binding site. When the fluorine is attached at the ortho position, as in 3- 3, the dipole moment direction is changed (inflection by at least 70 °), and thereby adversely affect the potency.

FIG. 8 A presents superposition of the exemplary compounds 2-1, 2-2, 3-3, 3-7, 3-8 docked into GSK-3 substrate binding site. The important binding residues within GSK-3, Phe 93, Arg 96, Arg 180 and Lys 205 are highlighted in black. Black circle marks the COO’ moiety of molecules. As can be seen, there is an overlap in the position of the carboxylic acid moiety of all molecules that enables binding with the phosphate binding pocket. The anthracenone-isoxazole core in 2-2 and 3-3 is positioned differently in comparison to that of the rest of the molecules that show a similar alignment across the GSK-3 binding core. This observation may correlate with the inferior performance of 2-2 and 3-3. Taken together, this study directs towards the structural features required for effective GSK-3 SCIs.

FIG. 8B presents a table summarizing the number of interactions (+), type of interaction, and atom/substructure involved for compounds 2-1, 3-7 and 3-8 with each GSK-3 residue. Red = H-bond, green = Halogen bond, blue stacking. As can be seen, all compounds formed salt bridges with one or more of the residues within the phosphate binding pocket (Arg 96, Arg 180, Lys 205) through CO ₂ H moiety.

The data presented herein indicate that the compounds as described herein feature an activity that is attributed, at least on part to their strong interactions with specific amino acids inside the active site: Arg 96, Phe 93, Lys 205, Argl80, Val214 and Lys 85.

Exemplary such interactions include one, two or more of the following: Strong ionic interactions between the positively charged Arg 96 or Arg 146 and the carboxylic acid moiety; Strong hydrogen bonds between the heteroatoms of the isoxazole ring and Arg 96; Strong 7t-7t interactions between the isoxazole ring and the aromatic ring of Phe 93; Strong ionic interactions between the carboxylic acid moiety and Lys 205; Van-der-Waals interaction between the carboxylic acid and Vai 180; Hydrogen bonding between an OMe substituent (if present, e.g., compound 3-7) and Lys 85. As expected one observes a good correlation between the number of interactions, the binding energy of the molecule docked in the active site and the compound potency.

The attachment of a negatively charged group such as carboxylate to the core via a flexible chain appears to allow its interaction with 4 amino acids. A shorter chain (2 methylenes vs 3) appears to be preferred. The isoxazole/isothiazole ring plays a role due to its aromaticity and its heteroatoms O and N bearing non-bonding (lone pair) electrons.

Without being bound by any particular theory, it is assumed that the planar core structure affects the way the molecule enters the active site, directed by dipole-dipole interactions toward its lowest energetic position. As shown schematically in FIG. 8C, the electron withdrawing effect of the electronegative atoms O, N, CO ₂ , induces “an electronic current” along the molecule. Approaching the enzyme, the molecule behaves as a magnet bearing two negative partial charges attracted by the positive centers deeply inside the active site, where it interacts with one or more of Arg 96, Arg 146, Lys 205 and Lys 85.

EXAMPLE 7 Additional Designs

Design and Syntheses:

Based on the data obtained in the screening assays and following biological assays conducted, the present inventors have designed additional compounds which exhibit structural features identified as required for interacting with the binding site of GSK-3, as discussed in Example 6 hereinabove. FIG. 18 presents the chemical structures of exemplary such compounds.

The new compounds were designed based upon the structure of Compound 3-8, while introducing one or more of the following modifications:

The negatively charged carboxylic acid moiety was replaced with a phosphate group (PO ₃ H ₂ ), to better mimic the ‘native’ phosphorylation in GSK-3 substrates (see, FIG. 18; Compounds 4-2 and 4-4), or with a sulfate or sulfonate group;

The fluorine substituent of the aromatic ring was replaced with a negatively charged group such as CO ₂ H, OH (see, FIG. 18; Compounds 4-3 and 4-5), or sulfate or sulfonate;

An additional fluorine substituent was introduced to the aromatic ring (see, FIG. 18; Compound 4-1), to allow additional interactions with the enzyme.

Representative docking models of GSK-3 with 4-2, 4-3 and 4-4 are shown in FIGs. 9A-C. FIG. 9D presents a table summarizing the number of interactions (*), type of interaction, and atom/substructure involved for compounds 4- 1, 4-2, 4-3, 4-4 and 4-5 with each GSK-3 amino acid residue. Green represents H-bond; blue represents stacking; black represents cation pink represents salt bridge. Ionic charges are indicated for indicated compounds. As can be seen in FIGs. 9A-D, the new compounds showed similar docking poses as those produced with 3-8. As conceived, replacing the carboxylic acid moiety with the PO ₃ H ₂ moiety lead to interaction of the latter with the phosphate binding pocket, and the anthracenone-isoxazole core formed stacking interactions with Phe 93.

As can be further seen, Compounds 4-3 and 4-4 formed additional interactions with residues Phe 67, Lys 85 or Ser 66 through their CO ₂ H or OH aryl substituents. These amino acid residues play important roles in GSK-3-substrate binding or- catalytic activity [Ilouz, et al. (2006) J. Biol. Chem. 281, 30621-30630; Smith and Rayment, (1996) Biophys J 70, 1590-1602]. The additional interactions observed with 4-3 and 4-4 should contribute to their inhibitory capacity.

Compounds 4-1, 4-2, 4-3, 4-4, and 4-5 were synthesized and further tested for their activity.

The synthetic schemes of these compounds are presented in FIGs. 19A-E. All structures were verified by NMR and MS (data not shown).

Activity assays:

In vitro kinase assays, performed as described hereinabove, confirmed that the new compounds inhibited GSK-3 and were even better inhibitors as compared to 3-8, as shown in FIGs. 10A-B. Collectively, compounds 4-3 and 4-4 acted as excellent inhibitors, showing IC ₅₀ values of about 1-4 μM. PCA analysis, shown in FIG. 11A further confirmed that the new compounds are chemically distinct from other ATP competitive GSK-3 inhibitors as listed in FIG. 1 IB.

To further asses the binding properties of the new compounds, kinase assays were performed with the GSK-3 F93A mutant, as described hereinabove. As shown in FIG. 12, the new compounds were unable to inhibit F93A, indicating that an interaction with Phe 93 is required for inhibition.

To eliminate the possibility that 4-3 or 4-4 compete with ATP, Michaelis Menten ATP competitive assays were performed and data was analyzed with Eadie-Hofstee plots. FIGs. 13A- B present exemplary plots. The ‘parallel’ lines produced by the Eadie-Hofstee plots indicated that 4-3 and 4-4 do not compete with ATP.

Further experiments were conducted to rule out the possibility of formation of colloidal aggregates of the small molecules, that may result in a non-specific aggregate-enzyme inhibition (as described, for example, in Owen et al. (2012) ACS Chem Biol 7, 1429-1435), GSK-3 kinase assays were conducted with increasing concentrations of Triton xlOO that disrupts colloidal aggregates. Inhibition of GSK-3 by 4-3 or 4-4 was not affected by the presence of 0.01-0.05 % detergent. FIG. 14 presents the data obtained with 0.05 % Triton xlOO. These results confirmed that inhibition of GSK-3 was specific.

Biological applications:

The effect of Compounds 4-3 and 4-4 on lysosomes acidification was tested, as described hereinabove.

SH-SY5Y cells were treated with increasing doses of 4-3 or 4-4, as described hereinabove. To better detect changes in cytoplasmic levels of β-catenin, the cells were permeabilized with the di gitonin and β-catenin levels were determined in the collected sup. As shown in FIGs. 15A-B, treatment with 1-5 μM concentrations of the tested compounds were sufficient to activate β-catenin in the cytoplasm.

Without being bound by any particular theory, it is assumed that the reduction in cytoplasmic β-catenin is response to the higher doses of the compounds reflects its translocation to the nucleus and cell membranes.

The activity of Compounds 4-3 and 4-4 in repairing lysosomal acidification involved in pathological conditions was also tested.

‘Correct’ lysosomal function is vital for normal cell homeostasis, and impairments in lysosomal acidification was detected in pathological disorders [see, for example, Nixon, R. A. (2013) Nat Med 19, 983-997; Rubinsztein et al. Nat. Rev. Drug Discov. 11, 709-730]. It was previously shown that presenilin proteins (PSEN), transmembrane proteins with y-secretase activity and cardinal genes in Alzheimer’s disease (AD) pathogenesis [Cruts et al. (1996) Hum. Mol. Genet. 5 Spec No, 1449-1455], are essential players in lysosomal acidification. Dysfunction of PSEN proteins impaired the autophagic-lysosomal cellular clearance activity [Lee et al. (2011) Cell 141, 1146-1158; Cataldo et al. (2004) J. Neuropathol. Exp. Neurol. 63, 821-830].

MEF KO-PS cells (MEF cells lacking PSEN1/2 that showed severe deficiency in lysosomal acidification as described in Lee et al. 2011 supra and shown in FIG. 16A) were treated with indicated concentration of the tested compounds for 4 hours and then incubated with 30-50 nM LysoTracker Red (Molecular Probes Thermofisher, MA, USA) for 12 minutes at 37 °C. Live cells were imaged on a laser scanning confocal microscope (Leica TCS SP5 II) with x40 and N.A 1.25 objective lens). Lysosomes’ puncta were analyzed with Imaris BITPLANE using the spots detection feature, or, by ImageJ software using the area integrated intensity measurement. Analysis included 100-120 cells taken from three independent experiments. As a positive control the previously reported GSK-3 SCI peptide, L807mts (SEQ ID NO:9), which was shown to trigger lysosomal acidification, was used [Licht-Murava et al. (2016) Science Signaling 9, ral lO].

As shown in FIGs. 16B and 16C, treatment of MEF KO-PS cells with 4-3 or 4-4 significantly restored lysosomal acidification. These results further correlated with the ability of 4-3 and 4-4 to increase the active form of cathepsin D (CatD), a principal lysosomal protease [Gieselmann et al. (1985) J. Biol. Chem. 260, 3215-3220], as shown in FIG. 16D. Upon acidification of lysosomes, CatD (46kDa) is cleaved to a mature active products (mCatD).

Collectively, these results show that the newly designed GSK-3 SCI compounds have biological activity in cells, and further suggest that 4-3 and 4-4 may serve as initial templates for the design of novel ‘pro ’-lysosome-acidification drugs that can be useful in treating pathological conditions with impaired cellular homeostasis and cellular clearance.

In further studies, potential alterations in β-catenin levels following treatment with 4-4 was evaluated. Human neuroblastoma SH-SY5Y cells were treated with increasing concentrations of 4-4, and the levels of β-catenin were determined in the cytoplasmic fraction (prepared after cell permeabilization and sup collection), this to avoid possible “masking” of membrane-associated β- catenin. The results are shown in FIGs. 17A and 17B and indicated that treatment with 7-5 μM of 4-4 was sufficient to activate β-catenin in the cytoplasm. The reproducible reduction in cytoplasmic β-catenin in response to higher doses (10 μM) is probably a consequence of β-catenin translocation to the nucleus. The compounds were not toxic to cells within the concentrations tested (1-20 μM) and at 24-72 hours post-treatment. Compound 4-4 was further tested in neurons, which represent a “more relevant” physiological cell system. Mouse hippocampal primary neurons were prepared from 1-day old pups, and were treated with 4-4. or, CHIR9920, a known ATP competitive inhibitor. The cells were then immunostained for β-catenin. As shown in FIGs. 17C and 17D, a significant increase in β-catenin levels was detected in the soma and dendrites of neurons treated with 4-4 or CHIR9920.

FIGs. 17E and 17F show that phosphorylation of tau (Ser 396), a known GSK-3 substrate in neurons was reduced following treatment with 4-4.

Together, these results indicated that 4-4 can inhibit cellular GSK-3.

Pharmacological properties:

Compound 4-4 was subjected to pharmacological tests. These included inhibition of hERG potassium channels (to test possible cardiac arrest), impact on hepatic CYP activities, stability in microsomes, and stability in plasma. The results are summarized in Table 4. The data indicates that 4-4 has pharmacological properties within acceptable safety margins, and hence is qualified as a future drug.

Table 4

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.