WO2016061344A1 - Gsk3 inhibitors and their uses - Google Patents

Abstract

The present invention identifies a novel class of compounds that have demonstrated activity as inhibitors of GSK3 activity. The ability to inhibit GSK3 applies to administering the compounds to subjects with identified aberrant GSK3 activity, such as acute myeloid leukemia. The present invention also relates to the use of GSK3 inhibitors in combination with all trans retinoic acid (ATRA).

Description

GSK3 inhibitors and their uses

This application claims priority to US Provisional Patent Application 62/064,275, filed October 15, 2014, which is hereby incorporated by reference in its entirety.

Field Of The Invention

The present invention relates to new compounds of formula 1, as a free base or a pharmaceutically acceptable salt, solvate, or solvate of salt thereof, to pharmaceutical formulations containing said compounds and to the use of said compounds in therapy. The present invention also relates to the use of said compounds to preferentially inhibit partial activities of GSK3a and GSK33. The present invention further relates to the combination of compounds of formula 1 with all-trans retinoic acid (ATRA) for therapy. Further the present invention relates to the combination of any GSK3 inhibitor with ATRA for cancer therapy. Finally, the present invention relates to the use of GSK3 inhibitors in enhancing the a bility of Natural Killer cells to kill cancer cells and virally infected cells.

Background Of The Invention

Glycogen synthase kinase 3 (GSK3) is a constitutively active serine/threonine kinase, that is important in signaling pathways involved in a diverse range of biological processes such as the regulation of cell fate, protein synthesis, glycogen metabolism, cell mobility, proliferation and survival (1-3). There are two isoforms of GSK3 (a and β), however, all known chemical inhibitors block both forms. This kinase has been widely studied as a target for the treatment of diabetes, inflammation, and multiple neurological diseases, including Alzheimer's, stroke and bipolar disorders (Rayasam et al. Br J Pharmacol 2009; 156: 885-98; Doble et al. J Cell Sci 2003; 116: 1175-86; Grimes et al. Prog Neurobiol 2001; 65: 391-426; Hernandez et al. Mini Rev Med Chem 2009; 9: 1024-9). Recently, the therapeutic potential of GSK3 inhibitors in cancer has become an area of interest, mostly for solid tumors (Guzman et al. Blood 2007; 110: 4436-44; Zhou et al. Leuk Lymphoma 2008; 49: 1945-53; Martinez. Med Res Rev 2008; 28: 773-96). In the case of leukemia, GSK3 inhibitors were recently reported to have anti-proliferative activities, but specifically only in leukemic cells that possess MLL-translocation oncogene products (Wang et al. Nature 2008; 455: 1205-9). MLL-translocations are extremely rare in Acute Myeloid Leukemia (~5% of patients) and this study was focused on lymphoid leukemia's (a significantly different disease than AML) and did not examine any myeloid leukemic cells that lacked the MLL translocation. Our data with a panel of AML cells clearly shows that in contrast to results reported for

lymphoid leukemia, GSK3 inhibition in AML leads to anti-proliferative activity irrespective of MLL-status. One major concern with the clinical use of GSK3 inhibitors is the fear that they may lead to cancer development due to the induction of beta-catenin that has been well established to play an important role in the pathophysiology of colon cancer. GSK3 phosphorylates its substrates in two distinct fashions. For most substrates it requires a C- terminal priming phosphate for phosphorylation while for other substrates such as beta- catenin it does not (Frame et al. Mol Cell 2001; 7: 1321-7). The molecular studies that have been performed to understand these two pathways suggest that it is possible to selectively block one of the pathways.

Acute myeloid leukemia (AML) is a broad range of disorders that are all characterized by leukemic cells that have a differentiation arrest. AML can be classified morphologically according to the French-American-British criteria by the degree of differentiation as well as extent of cell maturation as M0-M7. Treatment for all subtypes of AML is very similar, except for acute promyelocytic leukemia (APL, M3 subtype). Traditional therapy involves combination systemic chemotherapy. Several different approaches are utilized; however, they usually involve an induction therapy with cytarabine and a second chemotherapeutic, such as daunorubicin or idarubicin and consolidation therapy with either a bone marrow transplant or additional chemotherapy. Besides significant side effects from the traditional chemotherapeutics, the efficacy of these agents in treating AML is poor.

To date the only exception to the poor treatment options for AML is the success of all- trans retinoic acid (ATRA) for one relatively uncommon subtype (5-10% of AM L), APL. Utilizing a com bination of ATRA and chemotherapy, the long-term survival and presumed cure of 75-85% of patients is possible. ATRA illustrates the great promise for new agents with greater efficacy and less toxicity. In fact, elderly patients with APL who cannot tolerate traditional chemotherapy can achieve complete remission with therapies that utilize ATRA. ATRA's success stems from the fact that AML is a clonal disease characterized by the arrest of differentiation of immature myeloid cells. ATRA overcomes this block in differentiation by forcing leukemic cells to terminally differentiate so that they are no longer capable of dividing. Unfortunately, APL is a rare subtype of AML and ATRA has not been found to be clinically useful for other subtypes.

Though many compounds have been shown to have some differentiation-inducing effects in vitro, their clinical utility has been limited by either suboptimal differentiation-inducing capacity and/or toxicity. For example, Vitamin D3 induces potent differentiation, however, it also causes severe hypercalcemia at the required dose. Treatments that promote the differentiation of immature myeloid cells hold considerable promise in improving the long term survival of AML patients while avoiding some of the toxicities of traditional chemotherapy. Treatment of leukemia could be revolutionized by novel compounds due to their potential to cure leukemia and provide elderly patients with alternative nontoxic regimens.

In addition to treating leukemia with small molecules, another approach for leukemia therapy as well as therapy for any cancer is treatment of patients with donor derived Natural Killer (N K) Cells. One of the major limitations of N K cell therapy for clinical use is that they do not have sufficient cell killing activity in many cases to exhibit clinical efficacy. We have found that inhibiting GSK3 in NK cells leads to a significant increase in the ability of these cells to kill cancer cells as well as virally infected cells.

Summary Of The Invention

The present invention provides a new class of compounds or therapeutic agents that can inhibit GSK3, which includes compounds that inhibit GSK3 represented by general formula I,

Wherein each of Xi, ...X₄ is independently N or C;

Ri is CI, Br, Me, OMe;

R₂, or R₃ is F, CI, Br, I, Me, Ethyl, CN, N H₂, N Me₂, OMe, N0₂, hydroxymethyl, CH₂OCOMe, HOCH Me₂, vinyl, Phenyl;

R₄ is cyclopropyl, furanyl; R₅ is cyclohexyl.

The invention also comprises the finding that inhibition of GSK3 with any GSK3 inhibitor in combination with all trans retinoic acid enhances the therapeutic benefits of either compound alone for cancer therapy.

In another aspect of the invention the use of these compounds can be for any condition in which GSK3 inhibition is associated with beneficial including cancer therapy, diabetes, neurological diseases and inflammatory diseases. The use may comprise administering an effective amount of the compounds of the present invention to a subject selected for or associated with GSK3 aberrant activity. The compounds may be administered alone or in combination with other known therapeutic compounds.

Another aspect of the invention provides the ability of the described compounds to preferentially inhibit only a portion of GSK3 activity therefore allowing GSK3 inhibition with only minimal upregulation of beta-catenin that may have potential deleterious effects.

The compounds described in the present invention can be used alone or in combination with other differentiating agents (such as ATRA) or other anti-proliferative agents or chemotherapeutic agents to treat cancer, such as leukemia. The myeloid differentiation agents can also be administered to a subject in conjunction with

myeloablative therapy, for example, prior to the subject receiving bone marrow

transplantation or stem cell therapy.

The compounds described in the present invention can also be provided in a pharmaceutical composition either alone or with one or more agents.

Brief Description of the Drawings

The foregoing and other features of the present invention will become apparent to those skilled in the art to which the present invention relates upon reading the following description with reference to the accompanying drawings, in which:

Figure 1 shows GSK3 inhibitors induce monocytic differentiation of HL-60 cells. A. SB415286 induces morphologic changes consistent with monocytic differentiation. After treatment for 4 days with SB415286, cytospin preparations were prepared and the cells were stained with Wright-Giemsa stain. B. Multiple GSK3 inhibitors induce N BT reduction activity consistent with myelomonocytic differentiation. H L-60 cells were treated with the indicated compounds for 4 days and then the NBT reduction assay was performed by stimulating cells with 200 ng/ml of PMA in the presence of 1 mg/ml N BT for 60 minutes. The percentage of N BT positive cells (blue cells) was calculated by counting at least 200 cells under a light microscope. C. GSK inhibitors induce immunophenotypic changes consistent with monocytic differentiation. After treatment for 4 days, H L-60 cells were stained with CDllb- PE and CD14-FITC, and flow cytometric analysis was performed.

Figure 2 illustrates GS87 has growth inhibitory effects on OCI cells. Figure 2A shows OCI inhibits the proliferation of H L-60 cells. Cells were treated with the indicated compounds for 5 d, and the number of cells present at specific times points was assessed by counting at least 200 cells with a hematocytometer. Results shown represent the number of OCI- treated cells present divided by the number of vehicle-treated cells at specific time points and are an average of three independent experiments. Figure 26 shows OCI induces alterations of the cell cycle. HL-60 cells were treated with the indicated compounds for 3 d, and the cells were stained with propidium iodide and analyzed by flow cytometry. Results are representative of three independent experiments. Figure 2C shows OCI leads to the up- regulation of p21. HL-60 cells were treated with the indicated compounds for 1, 3, or 5 d, and Western blot analysis was performed on the same membrane with p21 and β-actin antibodies. Figure 2D shows OCI inhibits colony formation in soft agar. OCI cells were incubated with GS87 or vehicle for 72 h, and the drug was washed off. An equal number of viable cells were added to soft agar, and colony formation was assessed after 10 d. Results are an average of two independent experiments performed in duplicate.

Figure 3 shows GSK3 inhibition synergizes with ATRA both in vitro and in vivo. Figure 3A shows GSK3 inhibitors synergize with ATRA to induce differentiation. Cells were treated with SB (15uM), AT (50nM with SB or ΙΟηΜ with GS87), GS87 (5uM or 15uM), or a com bination of GSK3 inhibitor and ATRA for 4 days and differentiation was assessed by NBT reduction, CDllb staining and morphology. Figure 3B shows GSK3 inhibition and ATRA synergize to inhibit colony formation. Cells were treated with vehicle, ATRA (10 or 50nM), GS87 (5 or 15uM), SB (15uM), Lithium (lOmM) or GSK3 inhibitor and ATRA for 72 hours and a colony assay was performed as described in figl. Figure 3C shows The combination of GSK3 inhibition and ATRA significantly inhibits the growth of AML tumors. Nude mice (5 per group) were injected with H L-60 cells bilaterally into the flank. Seven days after tumor cell inoculation, ATRA (15 mg/kg) was injected i.p. daily for 3 weeks and Lithium chow was fed to the mice or SB (5mg/kg) was injected i.p. This chow has been documented to lead to lithium blood levels of 0.7mM in mice. The average volume of tumors (mm³) measured on indicated days after treatment initiation is shown.

Figure 4 shows genetic studies confirm the role of GSK3 in AML differentiation. Figure 4A shows knockdown of GSK33 induces partial AML differentiation and sensitizes cells to ATRA and GSK3 inhibitor- mediated differentiation. H L-60 cells were infected with lentiviral hGSK3b or empty vector and stable cells were selected with puromycin. Confirmation of GSK3 knockdown is shown by western. The cells were treated with ATRA or SB for 4 days and the N BT reduction assay was performed. Figure 4B shows knockdown of GSK33 sensitizes cells to ATRA-mediated growth inhibition. Cells were treated with ATRA for 72 hours and the MTT assay was performed. Figure 4C shows overexpression of GSK3 S9A blocks GSK3 inhibitor-mediated differentiation. OCI cells were electroporated with a GSK3b S9A expression construct and selected in puromycin. Confirmation of GSK3 S9A overexpression is shown by western. The cells were treated with GS87 or ATRA for 4 days and the N BT reduction assay was performed.

Figure 5 shows GSK3 inhibition enhances NK cell activity. Figure 5A shows 6 Structurally distinct GSK3I enhance N K cell activity. NK cells were expanded and pre-treated with vehicle or GSK3I for 4hr. Cells were washed and incubated for 4hr with target OCI-AML3 leukemia cells and lysis was measured using a calcein am assay. Figure 5B shows GSK33 deficient mice exhibit hyperactive N K cells. NK cells isolated from 3 GSK33 deficient mice or wild-type controls were incubated with WEH I-3 cells and lysis was measured after 4hr. Figure 5C shows GSK3I enhances N K cell killing of 2 out of 3 pancreatic cell lines tested. N K cells were treated with GSK3I overnight and then incubated with the indicated cell lines for 4hr and assessed for lysis.

Figure 6 shows GSK3I treated N K cells exhibit high efficacy in mouse tumor studies. Primary human AML cells were injected into the femur of NSG mice (n=5 per group) and expanded N K cells treated with INV-117 or vehicle were injected into the mice i.v. weekly for 4 weeks. After 4 weeks, the mice were sacrificed and the percent leukemic cells in the bone marrow was assessed by flow cytometry using a human specific CD45 antibody.

Figure 7 shows mechanisms through which GSK3I hyperactivates N K cells. Figure 7A shows a model of how GSK3I activates N K cells. Figure 7B shows GSK3I N K cells exhibit enhanced binding to AML cells. N K cells and OCI-AML3 cells were labelled with different fluorescent dyes. The cells were incubated alone or together for 20min and run on flow cytometry to detect aggregates C. GSK3I N K cells produce 7 fold more TN Fct and 30% more IFNct than vehicle treated N K cells as measured by ELISA. D. Neutralization of TNF impairs the activity of GSK3I NK cells, but not vehicle treated N K cells against OCI-AML3 cells.

Figure 8 shows GSK3 plays a role in NK cell activity in AML. Figures 8A-B show NK cells from AML patients exhibit elevated GSK3b protein expression as compared to normal donors. N K cells were isolated from AML patients and normal donors (n=20 of each). Figure 8A shows GSK3b expression and Figure 8B shows phospho-GSK3b (p-GSK3b) expression were measured by flow cytometry. Data is represented as Mean fluorescence intensity (MFI). MFI = MFI GSK3b or pGSK3b/M FI Isotype. Figure 8C shows NK cells from AML patients exhibit dysfunctional cytotoxic activity. N K cells were isolated from AML patients and normal donors (n=20 of each) and their cytotoxic activity was tested against OCI-AML3 cells. Figures 8D-E show NK cells from AML patients exhibit increased cytotoxic activity against autologous and allogenic leukemia cells after GSK3 inhibition in vitro. N K cells isolated from AML patients, treated for 16hr with SB or vehicle, and co-cultured with Figure 8D shows autologous AML cells (n=8) or Figure 8E shows OCI-AML3 cells (n=12). Figure 8F shows GSK3 inhibition in vivo leads to enhanced NK cell cytotoxic activity. N K cells isolated from lithium treated patients were sorted out into lithium-low (Li-low, <0.6mM) and lithium-high (Li-high, >0.6mM) groups based on lithium blood levels. The NK cells were tested for cytotoxic activity against OCI-AM L3 cells. Figures 8G-H show GSK3 inhibition in vitro more

dramatically enhances NK cell cytotoxicity in patients with low circulating levels of lithium. The N K cells described in figlf were treated with vehicle or SB for 16hr, co-cultured with OCI-AM L3 cells and cytotoxicity was assessed. (p<0.001 * **, p<0.01 * *, ns=not significant p>0.05). Figure 9 shows structurally distinct GSK3 inhibitors enhance N K cell activity against AML cells. Figure 9A shows 5 structurally distinct GSK3 inhibitors lead to N K cell activation.

Expanded N K cells isolated from normal donors were pre-treated with vehicle or the indicated concentrations of the GSK3 inhibitors for 16hr. The N K cells were then incubated with OCI-AML3 cells and the calcein-AM assay was performed after 4hr. Figure 9B shows GSK3 inhibition leads to enhanced NK cell killing of a variety of primary AML samples.

Expanded N K cells were pretreated with vehicle, SB, or 117 for 16hr, incubated with primary AML patient leukemic cells and the calcein-AM assay was performed. Figure 9C shows N K cells from multiple N K cell donors all show activation after GSK3 inhibition. N K cells from three normal donors were expanded, pre-treated with vehicle, 117, or SB for 16hr. The cells were incubated for 4hr with OCI-AML3 cells and lysis was measured using the calcein AM assay. (p<0.001 ***, p<0.01 **, ns=not significant p>0.05). Cone. = Concentration.

Figure 10 shows genetic evidence of the role of GSK3 in N K cell activity against AML cells. Figure 10A shows Mouse N K cells with loss of GSK3b exhibit increased cytotoxic activity against AML cells. N K cells were isolated from GSK3b deficient mice or wild-type controls were incubated with WEH I-231 mouse cancer cells and target cell lysis was measured using the calcein-AM assay. Figures 10A-B show genetic manipulation of GSK3 in human N K cells modulates their cyototoxicity against AML cells. NK cells lentivirally transduced with empty vector, GSK3b shRNA (GSK3-b-KD), GSK3a shRNA (GSK3-a-KD), GSK3b (GSK3-b-over) or GSK3a (GSK3-a-over) were incubated with OCI-AML3 cells and target cell lysis was measured using the calcein-AM assay. (p<0.001 * **, p<0.01 **, p<0.05 *, ns=not significant p>0.05.

Figure 11 shows GSK3 inhibition leads to NK cell hyperactivation partially in a TN Fa- dependent fashion. Figure 11A shows GSK3 inhibition leads to a marked induction of TNFa from NK cells. Expanded N K cells were incubated with vehicle or SB for 16hr, co-cultured with OCI-AML3 cells for 2hr and then the indicated cytokine levels were measured in the conditioned medium by ELISA. Figure 11B shows Inhibition of TN Fa markedly impairs GSK3 inhibition-mediated N K cell cytotoxicity but not vehicle treated NK cell cytotoxicity.

Expanded N K cells were treated with vehicle or SB for 16hr, anti-TN F-receptor antibody (anti-TN F-R) and anti-TNF antibody (anti-TN F) were added to the indicated wells, the cells were co-cultured with OCI-AML3 cells and target cell lysis was measured using the calcein- AM assay. (p<0.001 * * *, p<0.01 * *, p<0.05 *, ns=not significant p>0.05).

Figure 12 shows GSK3 inhibition leads to NF-kB activation and its binding to the TN F promoter in N K cells. Figure 12A shows GSK3 inhibition leads to N F-kB activation in NK cells. N K cells were treated with the GSK3 inhibitor SB for the indicated times, cytoplasmic and nuclear lysates were prepared, and analyzed by Western blotting using indicated antibodies. Actin and hnRN PAl were used as internal loading controls for cytoplasmic and nuclear extracts. Tubulin was used as a control to examine the purity of nuclear extracts.. Figure 12B shows GSK3 inhibition in N K cells leads to NF-kB binding to the TN F promoter. Expanded N K cells were treated with SB for the indicated times. Nuclear extracts were prepared and analyzed by Oligonucleotide pull-down assay to examine N F-kB binding to the TNF promoter. Data presented is representative of 2 independent studies.

Figure 13 shows GSK3 inhibition modulates surface receptors on N K cells and target cells after co-culture and leads to enhanced conjugate formation. Figure 13A shows GSK3 inhibition leads to enhanced conjugate formation between N K and target AML cells.

Expanded N K cells labelled with CFSE (treated with either DMSO or SB overnight) were co- incubated with OCI-AML3 cells labelled with SNARF-1 dye. The cells were co-cultured and conjugate formation was analyzed using flow cytometry at the indicated times. As a control, isolated populations of N K cells and OCI-AML3 cells were analyzed. N K:AML ratio=2:l. This is a representative study of 3 independent experiments. Figure 13B shows GSK3 inhibition leads to induction of cell surface expression of active LFA-1 on N K cells. Expanded N K cells were treated with SB or vehicle for 16hr and the surface expression of active LFA-1 was assessed by flow cytometry. Data shown is the mean fluorescence intensity (MFI). MFI ratio = MFI LFA-l/MFI Isotype. Figure 13C shows GSK3 inhibition of N K cells leads to ICAM-1 induction on AM L cells after coculture in a TNF-dependent fashion. Expanded NK cells were treated with SB or DMSO for 16hr and then were co-cultured with OCI-AML3 cells with or without TN F neutralization (TN F-R and TN F antibodies). Data shown is the mean

fluorescence intensity (MFI). MFI ratio = MFI ICAM-1/MFI Isotype on gated OCAML3 cells. (p<0.001 ** *, p<0.01 * *). Figure 13D shows Model of GSK3 regulation of NK cell activity against AML. Figure 14 shows GSK3 inhibited N K cells show high activity in AM L mouse model systems. Figures 14A-B show GSK3 Inhibited N K cells lead to a dramatic reduction in leukemic burden in a circulating mouse model using an AM L cell line. NSG mice were injected with OCI-AM L3 cells followed by weekly injections of vehicle, expanded N K cells, or expanded N K cells that were pretreated with SB for 16hr. The percent human AM L cells (CD56- CD45+) were detected by flow cytometry from Figure 14A spleen and Figure 14B bone marrow after 6 weeks Figure 14C shows GSK3 Inhibited N K cells lead to a dramatic reduction in leukemic burden in a circulating mouse model using a primary AM L patient sample. NSG mice were injected with primary AM L cells followed by weekly injections of vehicle, expanded N K cells, or expanded N K cells that were pretreated with 117 for 16hr. The percent human AM L cells (CD56- CD45+) were detected by flow cytometry from the bone marrow after 5 weeks. (p<0.001 * * *, p<0.01 * *). Figure 14D shows GSK3 inhibited N K cells lead to increased survival of mice with AM L. NSG mice were injected with primary human AM L cells followed by four ( bi-weekly) injections of vehicle, expanded N K cells, or expanded N K cells pretreated with 117 for 16hr. Kaplan-Meier survival curves of mice are shown. Chi Square log rank test was done for statistical significance. p<0.01 * * for AM L+N K versus AM L+(N K+117) groups.

Detailed Description

The present invention relates to the synthesis of a novel class of specific GSK3 inhibitors and I I I :

Wherein each of Xi, X₂, X3, X4 is independently N or C;

Ri is CI, Br, Me, OMe;

R₂, or R₃ is F, CI, Br, I, Me, Ethyl, CN, N H₂, N Me₂, OMe, N0₂, hydroxymethyl, CH₂OCOMe, HOCH Me₂, vinyl, Phenyl;

R₄ is cyclopropyl, furanyl; R₅ is cyclohexyl.

E the following structures:



 The activity data for compounds 1-4 is shown in Table I. OCI-AML3 cells were treated with these compounds for 4 days and the N BT reduction assay performed. The nitroblue tetrazolium (NBT) reduction assay is a test that is highly specific to and has been used extensively as a measure of functional myelomonocytic differentiation for over 20 years (43- 54). The NBT reduction test measures the ability of cells to generate a respiratory burst, a function that is only present in differentiated cells. The percentage of cells that were N BT positive after treatment was determined for the following four compounds.

Table I

[NBT (%)]

Compound # 20 M 10 M

1 100 93

2 100 75

3 92 63

4 100 91

Compound 1 has been shown to potently inhibit GSK3 and GSK3 using an in vitro kinase assay.The IC₅₀ for compound 1 against GSK3 was 415.00 nM and for GSK3 was 528.20 nM. Staurosporine was used as the control compound.

Compound 1 has been shown to have highly selective kinase inhibition activity against GSK3. Table II shows its activity against a large panel of kinases using in vitro kinase assays (performed at Reaction Biology Corporation Malvern, PA).

Table II

ALK 110.09 106.31 1.40

ALK1/ACVRL1 107.22 98.45 5750.00

ARAF 107.32 109.57

ARK5/NUAK1 105.82 97.25 <1.0

ASK1/MAP3K5 103.72 98.48 31.02

Aurora A 99.60 100.42 2.35

Aurora B 101.73 101.33 6.33

AXL 101.94 96.42 13.92

BLK 98.31 102.01 1.95

BMX/ETK 99.62 95.36 19.33

BRAF 89.72 88.20

BRK 104.57 101.08 366.60

BRSK1 102.01 93.45 <1.0

BTK 110.27 102.50 8.63 c-Kit 96.22 95.89 38.98 c-MER 98.63 94.69 15.10 c-Met 106.62 103.95 684.80 c-Src 100.89 94.66 2.01

CAMKla 101.85 99.85 2.55

CAMKK1 101.12 106.29 50.92

CDKl/cyclin B 89.29 86.67 2.77

CDKl/cyclin A 105.02 102.07 6.94

CDK2/cyclin A 95.94 94.26 <1.0

CDK2/cyclin E 104.33 100.55 2.16

CDK3/cyclin E 104.23 100.35 5.24

CDK4/cyclin Dl 96.76 101.68 26.85

CDK4/cyclin D3 94.22 100.99 60.55

CDK5/p25 98.12 102.13 2.77

CDK5/p35 106.82 99.76 1.91

CDK6/cyclin Dl 101.38 97.14 6.88

CDK6/cyclin D3 90.65 100.11 36.89 CDK7/cyclin H 101.20 101.93 285.00

CDK9/cyclin Tl 93.83 95.74 21.98

CDK9/cyclin K 91.15 95.76 27.82

CHK1 101.56 114.14 <1.0

CKlal 85.35 80.34 5417.00

CKld 103.49 100.12 5554.00

CLK1 101.61 96.08 98.50

COT1/MAP3K8 104.89 93.94

CSK 97.83 101.61 17.19

CTK/MATK 93.70 93.22 722.10

DAPK1 81.31 83.70 5.28

DCAMKL2 94.04 101.83 39.51

DDR2 99.44 100.78 32.35

DMPK 106.63 107.60 64.50

DRAK1/STK17A 101.21 106.64 5.67

DYRKI/DYRKIA 97.39 93.58 21.84

EGFR 107.70 103.65 161.50

EPHA1 87.53 105.49 53.78

ERBB2/HER2 115.36 116.35 17.14

ERK1 95.48 92.40 4722.00

ERK2/MAPK1 106.60 99.69 >20000

FAK/PTK2 95.18 93.91 16.44

FER 94.11 97.11 <1.0

FES/FPS 95.13 96.73 1.32

FGFR1 90.53 104.26 <1.0

FGR 96.92 94.57 <1.0

FLT1/VEGFR1 91.37 98.92 4.90

FLT3 105.47 105.71 <1.0

FMS 100.27 102.56 1.29

FRK/PTK5 89.02 104.04 34.81

FYN 96.48 102.27 <1.0 GCK/MAP4K2 101.72 104.73 <1.0

GRK2 106.87 99.52 809.40

GSK3a 15.12 17.80 3.71

GSK3b 23.41 28.34 12.06

Haspin 97.19 102.87 12.95

HCK 95.58 96.21 5.53

HGK/MAP4K4 90.77 94.13 <1.0

HIPK1 100.22 97.79

IGF1R 105.52 106.62 48.10

IKKa/CHUK 102.19 98.50 282.70

IKKb/IKBKB 96.76 98.78 1016.00

IR 85.43 101.90 55.06

IRAKI 106.69 110.81 57.13

IRR/INSRR 100.02 109.98 15.46

ITK 94.05 92.89 6.06

JAK1 99.19 92.18 <1.0

JAK2 107.32 125.41 <1.0

JNK1 113.16 110.89 6241.00

KDR/VEGFR2 101.74 103.41 25.81

KHS/MAP4K5 102.70 101.77 <1.0

LCK 97.32 104.49 5.57

LIMK1 93.00 93.28 3.80

LKB1 107.09 101.43 83.32

LOK/STK10 96.85 100.76 1.23

LRRK2 102.73 99.68 27.87

LYN 97.95 102.49 <1.0

MAPKAPK2 95.58 100.42 40.93

MAPKAPK3 101.18 100.38 3341.00

MAPKAPK5/PRAK 98.76 103.14 316.80

MARK1 105.77 103.68 <1.0

MEK1 97.04 101.99 452.90 MEK2 97.42 99.84 171.80

MEKK2 96.42 100.81 5.09

MEKK3 103.94 93.03 13.93

MELK 105.44 97.09 <1.0

MINK/MINK1 103.29 108.02 3.31

MKK6 93.22 100.78 83.73

MLCK/MYLK 101.43 94.38 26.14

MLK1/MAP3K9 107.08 97.63 <1.0

MLK2/MAP3K10 95.12 96.00 1.86

MLK3/MAP3K11 95.84 94.74 3.17

MNK1 99.19 95.69 17.32

MRCKa/CDC42BPA 100.92 97.90 11.35

MRCKb/CDC42BPB 107.38 103.55 7.99

MSK1/RPS6KA5 92.37 94.21 <1.0

MSSK1/STK23 101.19 95.99 2806.00

MST1/STK4 100.86 103.64 <1.0

MUSK 95.69 93.77 174.00

NEK1 100.16 103.63 13.85

NIK/MAP3K14 110.60 109.19 1668.00

NLK 105.22 104.98 130.10

0SR1/0XSR1 97.94 89.58 80.41

P38a/MAPK14 106.68 101.56

P38g 95.94 95.31 5288.00

P70S6K/RPS6KB1 95.19 103.52 <1.0

PAK1 102.84 101.80 <1.0

PASK 101.27 94.16 10.26

PBK/TOPK 106.08 111.90 133.50

PDGFRa 98.29 104.43 1.50

PDK1/PDPK1 113.08 105.95 12.58

PHKgl 98.84 95.25 2.39

PIM1 93.83 96.62 2.02 PKA 82.55 84.93 <1.0

PKCa 95.52 100.21 <1.0

PKCbl 96.50 103.25 2.97

PKCd 92.81 88.74 2.03

PKCepsilon 93.53 100.51 <1.0

PKCeta 102.37 97.97 <1.0

PKCiota 98.49 91.50 5.37

PKCtheta 106.20 105.59 7.27

PKCzeta 101.09 100.91 60.61

PKD2/PRKD2 104.26 104.39 <1.0

PKGla 104.56 96.82 <1.0

PKN1/PRK1 108.04 102.58 <1.0

PLK1 81.60 84.45 262.10

PRKX 60.45 59.22 1.13

PYK2 94.01 93.53 3.85

RAF1 97.70 93.54

RET 100.59 108.70 1.98

RIPK2 91.11 86.55 326.80

ROCK1 94.74 98.46 <1.0

RON/MST1R 101.69 100.00 320.00

ROS/ROS1 100.25 94.51 <1.0

RSK1 100.97 102.94 <1.0

RSK2 107.43 106.14 <1.0

SGK1 101.43 106.83 13.92

SIK2 85.66 96.58 36.98

SLK/STK2 104.01 101.10 6.60

SNARK/NUAK2 108.59 104.67 19.10

SRMS 92.67 103.70 13650.00

SRPK1 101.43 89.03 24.28

STK16 104.27 107.23 205.20

STK22D/TSSK1 100.39 97.21 <1.0 STK25/YSK1 102.28 106.25 10.76

STK33 99.40 99.37 30.95

STK39/STLK3 100.81 94.62 19.15

SYK 100.25 101.05 <1.0

TAK1 102.26 99.33 37.21

TAOK1 91.72 91.92 <1.0

TBK1 95.86 99.67 1.62

TEC 96.61 93.22 876.90

TGFBR2 110.86 107.58

TIE2/TEK 106.07 97.19 39.80

TRKA 99.04 96.71 1.05

TSSK2 97.10 107.98 2.61

TTK 90.10 98.84 28.96

TXK 99.48 102.12 19.16

TYK1/LTK 105.43 100.37 7.16

TYK2 98.27 98.45 <1.0

TYR03/SKY 98.54 99.84 3.20

ULK1 102.34 99.38 6.32

VRK1 97.73 92.84 4880.00

WEE1 89.95 90.87 15400.00

WNK2 106.38 103.81 2800.00

YES/YES1 101.10 98.56 1.52

ZAK/MLTK 99.43 93.57 >20000

ZAP70 90.60 103.71 10.84

ZIPK/DAPK3 99.94 96.90 4.79

Synthesis of the Compounds

The synthesis of compounds 1-4 were carried out according to Scheme 1. A general procedure for the syntheses was used (Kubota et al. O. Chem. Pharm. Bull. 1970, 18: 1696).

Scheme 1. General synthesis of compounds 1-4.

The synthesis of compounds presented in examples was carried out according to Scheme 2. General procedure used for the synthesis of compounds presented as examples in this invention.

Synthesis of representative exemplary compounds of this invention

Compound B: Sodium nitrite (l.llg, 16.17 mmol) in water (5 ml) was added with stirring over 15 min to a cold (0 °C) solution of A (2.18 g,14.7 mmol) in concentrated hydrochloric acid (7 ml) and ice (10 g). The mixture was stirred at 0 °C for a further 20 min. and the cold solution was added over 30 min to a stirred solution (room temperature) of potassium iodide (12.2 g, 73 mmol) in water (15 ml). The mixture was left standing at room temperature overnight and extracted with dichlormethane (2x50 ml). The combined organic extracts were washed successively with 10% NaOH, water, and dried (Na₂S0₄). Evaporation of the solvent and silica gel column chromatography of the residue (Hexanes/EtOAc) gave iodide B (3.46 g, 80%).

Compound C: To a solution of B (2.59 g, 10 mmol) in ethanol (20 ml) was added 8- hydroxyquinoline (8 mg, 0.05 mmol). Hydroxylamine hydrochloride (1.56 g, 23mmol) and sodium carbonate (1.79 g, 17 mmol), each dissolved in water (10 ml), were sequentially added over a period of 20 min, then the reaction mixture was heated to reflux for 2 hr. After cooling the mixture, solvent was removed in vacuo, and silica gel chromatography of the residue (Hexanes/EtOAc) gave C (1.81 g, 62%).

Compound D: To a solution of C (0.96 g, 3.3 mmol) in acetone (20 ml) chloroacetyl chloride (0.37 g, 3.3 mmol) was added and the mixture was stirred at rt for 30 min. Acetone was evaporated and the residue was washed with sodium bicarbonate solution (5 ml) and water (10 ml) to give the crude solid of compound D, which was dried and used directly for the next step.

Compound E: Compound D (0.74 g, 2 mmol) was suspended in o-xylene (20 ml) and refluxed for 2 hr. After cooling the mixture, the o-xylene solution was directly loaded on to silica gel column, elution with hexane/EtOAc gave compound E.

Compound G: A mixture of F (0.77 g, 5 mmol), KOH (0.28 g, 5 mmol) and carbon disulfide (4 ml) in ethanol (50 ml) was refluxed for 12 hr. The solution was then

concentrated, cooled and acidified with diluted HCI. The solid mass that separated out was filtered, washed with ethanol, and dried to give G, which was used directly for the next step.

Compound H : A mixture of G (20 mg, 0.1 mmol), KOH (28 mg, 0.5 mmol) and E (35 mg, 0.1 mmol) in DMSO (2 ml) was stirred at rt for 1 hr. EtOAc (10 ml) was added to the reaction mixture, and the solution was washed with water (5 ml x 2). The EtOAc extract solution was evaporated and the residue was subjected to silica gel chromatography to give H (30 mg, 62%). ¹HN MR (CDCI₃): δ 8.52 (d, J=7 Hz, 2H), 8.21 (d, J=2 Hz, 1H), 8.02 (d, J=7 Hz, 2H), 7.73 (dxd, J=2, 9Hz, 1H), 6.80 (d, J=9 Hz, 1H), 4.76 (s, 2H), 3.92 (s, 3H).

Uses of the compounds

In an aspect of this invention, these compounds or therapeutic agents can be used to induce differentiation of immature myeloid cells. The present invention provides in part methods of administering the compounds of the present invention to a su bject. The compounds may be formulated as active therapeutic agents or salts thereof. Agents in accordance with the invention have a high-potency a nd low toxicity in ma mmalian su bjects and can be used in the treatment of myeloid disorders, such as myeloproliferative disorders, acute myeloid leukemia and auto-immune disease, to induce and/or promote

differentiation of the myeloid cells. The agents can also be used as a myeloa blative agent in conjunction with bone marrow transplantation and stem cell therapies.

The agents in accordance with the present invention can be used alone or in com bination with other differentiation inducing agents such as ATRA, anti-proliferative agents, and/or chemotherapeutic agents for the treatment of proliferative and/or other neoplastic disorders.

In an aspect of the present invention, these GSK3 inhibitors can be used to treat any conditions or diseases in which GSK3 inhibitors are useful including cancer, inflammatory diseases, neurological disorders and diabetes.

In another aspect of the present invention, any GSK3 inhibitor can be com bined with ATRA to treat cancer as the com bination of these agents leads to greater activity than the use of either agent alone.

In another aspect of the present invention, these compounds can be used to preferentially inhibit a portion of GSK3 inhibitors. In this way, it is possible to block GSK3 activities without significantly upregulating beta-catenin.

These GSK3 inhibitor agents in accordance with the present disclosure can be administered in a therapeutically effective amount to a patient or su bject with a disorder characterized by myeloproliferation. These disorders can include, for example,

myeloproliferative diserorders, such as leukemia, and immunity related diseases. I n addition, they can be used for any disorder in which GSK3 inhibitors have been found to be useful including dia betes, neurological diseases, and inflammatory diseases.

The term "therapeutic" refers to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of disease. For example, treatment of a patient by administration of myeloid differentiation agent of the present invention encompasses chemoprevention in a patient susceptible to developing myeloid leukemia (e.g., at a higher risk, as a result of genetic predisposition, environmental factors, or the like) and/or in cancer survivors at risk of cancer recurrence, as well as treatment of a myeloid leukemia patient by inhibiting or causing regression of a disorder or disease.

"Effective amounts", in terms of each of the foregoing methods, are amounts of the myeloid differentiation agent effective to induce or promote differentiation of the immature myeloid cells in the subject being treated without being cytotoxic to the subject.

The agents of the present invention can be provided in the form of pharmaceutical compositions. The pharmaceutical compositions can be administered to any mammal that can experience the beneficial effects of the myeloid differentiation inducing agents or GSK3 inhibition of the present invention. Foremost among such animals are humans, although the present invention is not intended to be so limited.

The pharmaceutical compositions of the present invention can be administered by any means that achieve their intended purpose. For example, administration can be by parenteral, subcutaneous, intravenous, intraarticular, intrathecal, intramuscular, intraperitoneal, or intradermal injections, or by transdermal, buccal, oromucosal, ocular routes or via inhalation. Alternatively or concurrently, administration can be by the oral route. Particularly preferred is oral administration. The dosage administered will be dependent upon the age, health, and weight of the patient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

In addition, the pharmacologically active compounds can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active agents into preparations that can be used pharmaceutically. The pharmaceutical preparations of the present invention are manufactured, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active agents with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders, such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents can be added, such as the above-mentioned starches and also carboxymethyl-starch, cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings, that, if desired, are resistant to gastric juices. For this purpose, concentrated saccharide solutions can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol, and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, are used. Slow-release and prolonged-release formulations may be used with particular excipients such as methacrylic acid-ethylacrylate copolymers, methacrylic acid-ethyl acrylate copolymers, methacrylic acid-methyl methacrylate copolymers and methacrylic acid-methyl methylacrylate copolymers. Dye stuffs or pigments can be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.

Other pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the active compounds in the form of granules that may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids such as fatty oils or liquid paraffin. In addition, stabilizers may be added.

Formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts and alkaline solutions. Especially preferred salts are maleate, fumarate, succinate, S,S tartrate, or R,R tartrate. In addition, suspensions of the active compounds as appropriate oily injection suspensions can be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides or polyethylene glycol-400 (the compounds are soluble in PEG-400). Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.

In a further aspect of the invention, the GSK3 inhibitors can be used in combination and adjunctive therapies for treating proliferative disorders.

The phrase "combination therapy" embraces the administration of the myeloid differentiation inducing agents and a therapeutic agent as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of these therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the com bination selected). "Combination therapy" is intended to embrace administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally. Alternatively, for example, all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection. The sequence in which the therapeutic agents are administered is not narrowly critical. "Combination therapy" also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients (such as, but not limited to, a second and different therapeutic agent) and non-drug therapies (such as, but not limited to, surgery or radiation treatment). Where the combination therapy further comprises radiation treatment, the radiation treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the radiation treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

The phrase "adjunctive therapy" encompasses treatment of a subject with agents that reduce or avoid side effects associated with the combination therapy of the present invention, including, but not limited to, those agents, for example, that reduce the toxic effect of anticancer drugs, e.g., bone resorption inhibitors, cardioprotective agents; prevent or reduce the incidence of nausea and vomiting associated with chemotherapy, radiotherapy or operation; or reduce the incidence of infection associated with the administration of myelosuppressive anticancer drugs.

In another aspect of the invention, the therapeutic agents administered in com bination therapy with the GSK3 inhbitors can comprise at least one anti-proliferative agent selected from the group consisting of a chemotherapeutic agent, an antimetabolite, an antitumorgenic agent, an antimitotic agent, an antiviral agent, an antineoplastic agent, an immunotherapeutic agent, and a radiotherapeutic agent.

The phrase "anti-proliferative agent" can include agents that exert antineoplastic, chemotherapeutic, antiviral, antimitotic, antitumorgenic, and/or immunotherapeutic effects, e.g., prevent the development, maturation, or spread of neoplastic cells, directly on the tumor cell, e.g., by cytostatic or cytocidal effects, and not indirectly through

mechanisms such as biological response modification. There are large numbers of antiproliferative agent agents available in commercial use, in clinical evaluation and in preclinical development, which could be included in the present invention by combination drug chemotherapy. For convenience of discussion, anti-proliferative agents are classified into the following classes, subtypes and species: ACE inhibitors, alkylating agents, angiogenesis inhibitors, angiostatin, anthracyclines/DNA intercalators, anti-cancer antibiotics or antibiotic-type agents, antimetabolites, antimetastatic compounds, asparaginases, bisphosphonates, cG MP phosphodiesterase inhibitors, calcium carbonate, cyclooxygenase-2 inhibitors, DHA derivatives, DNA topoisomerase, endostatin, epipodophylotoxins, genistein, hormonal anticancer agents, hydrophilic bile acids (URSO), immunomodulators or immunological agents, integrin antagonists, interferon antagonists or agents, MMP inhibitors, miscellaneous antineoplastic agents, monoclonal antibodies, nitrosoureas, NSAIDs, ornithine decarboxylase inhibitors, pBATTs, radio/chemo sensitizers/protectors, retinoids,selective inhibitors of proliferation and migration of endothelial cells, selenium, stromelysin inhibitors, taxanes, vaccines, and vinca alkaloids.

The major categories that some anti-proliferative agents fall into include antimetabolite agents, alkylating agents, antibiotic-type agents, hormonal anticancer agents, immunological agents, interferon-type agents, and a category of miscellaneous antineoplastic agents. Some anti-proliferative agents operate through multiple or unknown mechanisms and can thus be classified into more than one category.

Any GSK3 inhibitors in accordance with the present invention can allow the com bination therapeutic agents and therapies of the present invention to be administered at a low dose, that is, at a dose lower than has been conventionally used in clinical situations.

A benefit of lowering the dose of the combination therapeutic agents and therapies of the present invention administered to a mammal includes a decrease in the incidence of adverse effects associated with higher dosages. For example, by the lowering the dosage of a chemotherapeutic agent such as methotrexate, a reduction in the frequency and the severity of nausea and vomiting will result when compared to that observed at higher dosages. Similar benefits are contemplated for the compounds, compositions, agents and therapies in combination with the inhibitors of the present invention.

By lowering the incidence of adverse effects, an improvement in the quality of life of a patient undergoing treatment for cancer is contemplated. Further benefits of lowering the incidence of adverse effects include an improvement in patient compliance, a reduction in the number of hospitalizations needed for the treatment of adverse effects, and a reduction in the administration of analgesic agents needed to treat pain associated with the adverse effects.

Alternatively, the methods and combination of the present invention can also maximize the therapeutic effect at higher doses.

When administered as a combination, the therapeutic agents can be formulated as separate compositions which are given at the same time or different times, or the therapeutic agents can be given as a single composition.

This invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patent applications, patents, and published patent applications cited throughout this application are hereby incorporated by reference in their entirety.

Biological activity for the compounds of the invention

Through screening known kinase inhibitors to identify pathways that activate AML differentiation, small molecule inhibitors of GSK3 were identified as AML differentiation agents. Utilizing H L-60 cells, 5 separate small molecule GSK3 inhibitors demonstrate evidence of monocytic differentiation as measured by N BT reduction, morphology (ex. increased cytoplasm, vacuoles, altered nuclear morphology), and the upregulation of myelomonocytic cell surface makers CDllb (fig 1 and data not shown). The NBT reduction assay is a method to quantitate myelomonocytic differentiation. This commonly used assay provides a highly specific functional measure for differentiation as it detects the respiratory burst capacity, a process that only occurs in differentiated cells (21-25). While all the GSK3 inhibitors induce differentiation, the differentiation activity is significantly higher with our optimized compound, GS87 (compound 1). The differentiation activity was not limited to H L-60 cells as GSK3 inhibitors were found to induce the differentiation of a vast array of different AML cell lines including THP1, U937, Ml, N B4, MONOMAC3, OCI-AML3 (OCI), and WEHI3 as well as AML patient samples by N BT reduction, morphology and

immunophenotyping (figure 1 and data not shown). Of note, GS87 exhibits high differentiation activity in many AML cell lines such as H L-60, OCI, TH P1 cells (fig lb-c and fig3a). Please note that most experiments described in this application were performed with SB, GS87 and Lithium with very similar results (except for the fact that GS87 has higher differentiation activity). Besides differentiation, GSK3 inhibition led to significant growth inhibition of AML cells. Utilizing a panel of 10 different AML cell lines, the average IC50 of GS87 was 7.5 M and SB 22.5 M at 72 hours after treatment using the MTT assay. There was no relationship between the MLL status of the cells and the growth inhibition as all lines (3 had MLL-translocations) exhibited extremely similar IC₅₀'s. As the primary goal of AML differentiation therapy is to permanently prevent the growth of AM L cells, colony assays were performed to test for irreversible growth arrest after GSK3-inhibition. For this assay, AML cells were exposed to drug for 3 days, drug was washed off and an equal number of viable cells were plated in soft agar. At optimal doses of GSK3 inhibitors for differentiation, nearly complete prevention of colony growth was observed in several AML cell lines that were tested with SB, GS87 and to a lesser extent Lithium (fig Id for SB and fig3b for GS87 and Li colony assays).

EXAMPLES

Identification of GS87, a novel GSK3 inhibitor optimized for AML differentiation:

Though existing GSK3 inhibitors induce differentiation, these inhibitors exhibit only moderate activity. A novel GSK3 inhibitor, GS87, was identified which exhibits high differentiation activity (figlb). GS87 contains a basic four ring structure with 2 pyridine groups that were found to be important for this high activity.

GS87 is a novel GSK3 inhibitor:

As GS87 has only minor structural resemblances to other GSK3 inhibitors, we confirmed that it is truly a GSK3 inhibitor. Similar to other known GSK3 inhibitors, GS87 leads to the inhibitory Ser9 phosphorylation of GSK33. Interestingly, however, it only leads to an extremely weak upregulation of β-catenin as compared to other GSK3 inhibitors like SB. β-catenin is phosphorylated by GSK3 under normal conditions leading to its proteasomal degradation (fig 2). In addition, commercial kinase specificity profiling (using radioactive in vitro kinase assays) of GS87 was performed (Reaction Biology Corp). This screening demonstrated that GS87 is among the most specific GSK3 inhibitors ever reported as it demonstrated significant inhibition of GSK3a and GSK33 (IC₅₀ 415nM and 521nM

respectively) while it had significantly less activity on a panel of almost 200 other kinases at ΙΟμΜ. Though GS87 was found to exhibit extremely high AML differentiation activity, similar, to other GSK3 inhibitors, GS87 exhibits low in vitro cytotoxicity on normal cells. While GS87 leads to irreversible growth arrest and differentiation of leukemic cells at ~15 μηη, other cell types such as fibroblasts, human lymphocytes, and endothelial cells exhibit no evidence of cell death at concentrations up to 50μΜ. As discussed earlier GSK3 inhibitors in general have been well tolerated in early clinical trials.

GSK3 inhibitors dramatically synergize with ATRA both in vitro and in vivo:

As differentiation agents would likely be used clinically as a component of combination therapy, the ability of GSK3 inhibitors to synergize with other clinically used AML agents was tested. Low doses of GS87 and SB that alone lead to little differentiation were found to synergize with low dose ATRA (ΙΟηΜ for GS87 and 50nM for SB) in inducing differentiation, growth inhibition, and irreversible growth arrest (as measured by colony assays) of a wide range of AML cell lines and patient samples (fig3 and data not shown). Please note the Bliss method was used to assess synergy. Interestingly, the combination of ATRA and GSK3 inhibition differentiates H L-60 cells into neutrophils in contrast to monocytes which occurs with GSK3 inhibition alone (fig3a). High dose ATRA (ΙμΜ) on the other hand differentiates H L-60 cells into neutrophils (26). Besides cell based studies, GSK3 inhibitors and ATRA also showed synergy in preventing tumor growth in a mouse xenograft model system. While neither ATRA nor GSK3 inhibition alone (with Lithium or SB) had a major effect on tumor growth, the combination led to significant reductions in tumor growth (fig3c). This work suggests that GSK3 inhibition is a promising strategy to achieve a long held goal in AML therapy of enhancing the clinical activity of ATRA in non-APL leukemia.

Genetic Evidence of GSK3-inhibition mediated AML differentiation:

Though the studies with small molecule GSK3 inhibitors provide strong evidence for the role of GSK3 in AML differentiation, we knocked down GSK3 in several AML cell lines (H L-60, THP1, OCI and U937) to provide genetic evidence of its role in AML differentiation. The reduction in GSK33 levels resulted in an increase in the basal levels of differentiation as well as an enhanced sensitivity to ATRA-mediated (as well as SB-mediated) differentiation as measured by N BT reduction and immunophenotyping in all cell lines (fig 4a and data not shown). The increase in differentiation with SB treatment suggests a potential role for GSK33 whose levels were not affected by GSK33 knockdown. Besides differentiation, GSK3 knockdown cells also exhibited greater sensitivity to ATRA-mediated growth inhibition (fig 4b). We also tested the effect of overexpressing a mutant form of GSK33 (ser-9 mutated to ala) as this site renders the kinase resistant to inactivation (27). Overexpression of this mutant GSK3 in OCI cells blocked GS87-mediated, but not ATRA-mediated differentiation further demonstrating GSK3 inhibition directly leads to AML differentiation (fig 4c).

In addition to the activity of GSK3 inhibitors directly on leukemic cells, we found that pre-treatment of the N K cells ex vivo with 6 structurally distinct GSK3 inhibitors (GSK3I) results in a dramatically enhanced killing of a wide variety of cancer cell lines including AML cell lines and primary patient samples (fig5a and data not shown). Importantly, this enhanced N K cell cytotoxic activity only requires a short exposure (~ 4hr) of the NK cells to GSK3I ex vivo. We have focused on the compound 117 for most studies though similar results can be found with a wide variety of GSK3 inhibitors.

In addition to pharmacologic inhibitors, we confirmed the role of GSK3 by knocking out GSK33 in NK cells from mice as well as knocking down the expression of GSK33 in human N K cells. Both genetic approaches confirmed that GSK3 deficiency leads to a significantly enhanced ability of NK cells to kill AML cells (fig5b and data not shown). Not only does GSK3I enhance N K cell activity against leukemic cells, but similar results have been found using a wide range of hematologic (ex. AML, ALL, myeloma) and many non- hematologic (ex. colon, pancreas, osteosarcoma) cancer cells (fig5c and data not shown).

Besides exhibiting activity in vitro, these human N K cells show dramatic activity against human AML cells in multiple mouse AML model systems (fig6 and data not shown). Utilizing a circulating model of primary human AML in NSG mice, we found our GSK3I- treated N K cells led to a significant reduction in leukemic burden in the mouse bone marrow. Importantly similar to the in vitro studies, our cells performed significantly better in vivo than the expanded NK cells which are in the process of being tested in numerous clinical trials for hematologic and non-hematologic malignancies as well as several AML drugs such as cytarabine.

Besides efficacy studies, this work has elucidated how GSK3I enhances N K cell activity as depicted in fig7a. GSK3I leads to a dramatic increase in adhesion of N K cells to target cells as demonstrated by a flow cytometric adhesion assay (49% vs 83% after 20 min incubation) as well as live cell imaging (fig7b and data not shown). Consistent with the increased adhesion, as seen in fig7c GSK3I N K cells as well as target cells (after co- incubation) exhibit increased expression of essential NK cell-target adhesion molecules (9) including L-selectin (on N K cells) and ICAM (on target cells). L-selectin also is important in the trafficking of N K cells to tumor cells (10).The induction of ICAM on target cells is due to a marked induction in TN Fct production from the N K cells upon incubation with target cells (fig7c and data not shown). TNFct neutralization impairs the N K activity of the GSK3I- inhibited NK cells but not vehicle treated cells (fig7d). GSK3I also leads to changes in the NK cells that enhance activity such as increased expression of granzyme and perforin and secretion of IFNy (fig7d and data not shown). Activity in Natural Killer Cells

Natural killer cells from Acute Myeloid Leukemia patients (AML-N K) show a dramatic impairment in their cytotoxic activity. The exact reasons for this dysfunction are not well characterized. We found that Glycogen Synthase Kinase beta (GSK33) expression was elevated in AML-NK cells compared to normal donors. Interestingly, GSK3 overexpression in normal N K cells impaired their ability to kill AML cells while GSK3 inhibition through genetic and pharmacologic approaches enhanced their cytotoxic activity. In N K cells, GSK3 inhibition leads to a rapid activation of N F-κΒ, enhanced binding of N F-κΒ to the TNFct promoter, increased TNF-ct production, up-regulation of adhesion molecules and increased conjugate formation between N K/AML cells resulti ng in increased killing of AML cells. Finally, GSK3 inhibited NK cells showed significant efficacy in human AML mouse models. Overall, our work has revealed a mechanistic insight into the dysfunction of AML-N K cells as well as a novel strategy for NK cell therapy.

Natural killer (N K) cells are lymphocytes that kill malignant or virally infected cells without antigen-specific receptor recognition. Due to their high activity in specifically killing cancer cells, efforts have been made to utilize ex vivo expanded donor N K cells for cancer therapy. While N K cells have been used to target numerous malignancies, hematologic malignancies including Acute Myeloid Leukemia (AML) have shown particular potential for this approachl. In fact, the use of haploidentical N K cells has been found to be successful for treating at least some AML patients.

NK cells lead to specific killing of cancer cells due to the expression of a variety of activating (e.g. NKG2D) and inhibitory receptors (eg. Killer Inhibitory Receptors) on their surface. These receptors interact with specific ligands on target cells and the balance of these activating and inhibitory signals determines if cell killing occurs. Cancer cells commonly upregulate ligands for N K cell activating receptors such as MICA/B and downregulate ligands for inhibitory receptors such as HLA class-15. This H LA downregulation avoids T cell detection making many cancer cells paradoxically sensitive to N K cell killing.

NK cells exert anti-tumor effects through both direct cytotoxic effects and cytokine production. N K cell mediated killing of malignant cells depends on several discrete steps that ultimately lead to the polarization and exocytosis of lytic granules towards the target cell. The contact between N K and target cells is the first step and is established through N K cell receptors and adhesion molecules. Engagement of Lymphocyte function-associated antigen 1 (LFA-1) by its ligand, Intercellular adhesion molecule (ICAM-1), on target cells is one such interaction resulting in the sta ble adhesion of N K cells to their target cells a nd is sufficient to induce the polarization of lytic granules in resting N K cells. Another important step is cytokine production by N K cells including I FN-γ and TN F-ct. The exact role of these cytokines in N K cell cytotoxic function is not yet fully clear.

N K cells in AM L patients are known to exhibit significant defects in cytotoxic activity and to be markedly reduced in num ber. Recent studies showed that downregulation of activating receptors on N K cells, particularly N KG2D and the natural cytotoxicity receptors (NCRs) N Kp46 and N Kp30, and defective AM L-N K synapse formation are partially responsible for the N K cell dysfunction. However, specific signaling alterations leading to these functional changes are not clear.

In an effort to understand the dysregulation of N K cells in AM L patients, part of the present invention identified that Glycogen Synthase Kinase beta (GSK3-3) protein levels are upregulated in N K cells from AM L patients as compared to normal donors. Importantly for purposes of adoptive cell therapy, N K cells from both AM L patients as well as normal donors show a significant enhancement in cytotoxic activity after GSK3 inhibition. GSK3 is a serine threonine protein kinase that plays a central role in a number of key signaling pathways such as Wnt/3-catenin and N FKB as well as biological processes such as cellular

proliferation, inflammation and apoptosisl3. GSK3 has previously been shown to be a promising target in AM L cells as GSK3 inhibitors lead to the growth inhibition and differentiation of leukemic cells. I nterestingly, N K cells from patients with X-linked lymphoproliferative (XLP) fail to phosphorylate GSK3 following N K receptor 2B4 (CD244) stim ulation and this has been reported to be partially responsible for the N K cell defects observed in these patientsl6. In addition, the drug enzastaurin which is an activator of GSK3 as well as inhibitor of protein kinase CP was found to suppress N K cell cytotoxic activity. Here we demonstrate that GSK3 plays an importa nt role in the a bility of N K cells to target AM L and that the ex vivo inhibition of GSK3 is an attractive strategy for adoptive N K cell therapy for leukemia.

NK cells from AML patients exhibit high levels of GSK33 and low cytotoxic activity

N K cells from AM L patients are known to exhibit significant functional defects that are implicated in AML development, progression and relapse. Though phenotypic cha nges in these cells have been described, specific causes for this dysfunction are less clear. As GSK3 protein expression is known to be elevated in cancer cellsl8-25, it was tested if GSK3 protein expression is altered in NK cells from AM L patients. Interestingly, NK cells from AML patients express higher levels of GSK33 as compared to NK cells from normal donors (Figure 8A). No difference in GSK33 phosphorylation (serine-9) was detected (Figure 8B). In addition, as previously reported N K cells from AM L patients exhibit a reduced ability to kill target AML cells as compared to normal donor NK cells (Figure 8C).

Next, it was tested if the impaired N K cell function in AML patients could be overcome by GSK3 inhibition. Interestingly after treatment with a specific GSK3 inhibitor SB415286 4 (SB), N K cells showed enhanced efficacy in killing their own AML cells (Figure 8D). These AML patient derived N K cells also showed an enhanced ability to kill allogeneic AML cells after GSK3 inhibition (Figure 8E). These results suggest that high GSK3 activity may directly contribute to N K cell dysfunction in AML and that GSK3 inhibition may significantly enhance the efficacy of N K cell therapy for AML patients.

To begin to explore if regulating GSK3 activity in vivo can impact N K cell activity, the fact that Lithium is currently an FDA approved GSK3 inhibitor that is used in patients with bipolar disease was utilized. It has previously been reported that Lithium levels slightly lower than lmM are necessary in vivo in order to significantly inhibit GSK3. The activity of N K cells isolated from patients taking lithium was tested. Interestingly, NK cells from patients with high circulating levels (>0.6mM) of lithium sufficient to impair GSK3 demonstrate significantly higher cytotoxic activity than NK cells from individuals with low lithium levels (<0.6mM) that are known not lead to significant GSK3 inhibition (Figure 8F). Further when these N K cells were treated with additional GSK3 inhibitor ex vivo, a much more dramatic increase in cytotoxic activity was observed in the Lithium low group suggesting that their GSK3 activity was not optimally inhibited in vivo (4/5 patients in lithium low group vs 2/6 in lithium high group demonstrated statistically significant enhanced activity, Figure 8G-H). These results with lithium treated patients suggest that treatment of AML patients with GSK3 inhibitors may have therapeutic benefit by enhancing NK cell activity. The elevated GSK3 protein in N K cells from AML patients in conjunction with studies demonstrating that GSK3 inhibition increases NK cell cytotoxic activity towards AML cells, defines a new mechanism for N K cell dysfunction in AML. GSK3 inhibition markedly enhances normal donor NK cell cytotoxicity towards AML cells

While the use of donor N K cells for cancer is an area of intense interest, these N K cells are limited by sub-optimal cytotoxic activity that limits their clinical potential. Due to the observation that targeting GSK3 enhances the ability of NK cells to kill AML cells, the potential of using GSK3 inhibition as an N K cell hyperactivation strategy for adoptive cell therapy was explored. Pretreatment of N K cells with several structurally distinct GSK3 inhibitors resulted in enhanced killing of AML cells. Importantly, this enhanced NK cell cytotoxic activity only involves a short ex vivo exposure (16 hours) to GSK3 inhibitors.

Therefore, this strategy does not require a patient to be exposed to the high doses of GSK3 inhibitors that are necessary for potent kinase inhibition as well as NK cell hyperactivation. Five structurally distinct GSK3 inhibitors led to dose dependent enhancement of N K cell activity against an AML cell line, OCI-AML3 and primary AML patient samples (Figure 9A). To appreciate the ability of GSK3 inhibition to enhance N K cell-mediated killing in AM L, a panel of primary AML cells from different patients was tested. The GSK3-inhibited N K cells demonstrated a significant enhancement in cytotoxic activity in 4 of 5 samples tested (Figure 9B).

As N K cells are highly heterogeneous even among normal donors, we also tested the impact of GSK3 inhibition using a panel of different N K cells from normal donors. All 5 donor N K cells tested showed increased cytotoxicity after GSK3 inhibition towards AML cells as compared to vehicle treated cells (Figure 9C). These results indicate that inhibition of GSK3 can significantly augment normal donor NK cell cytotoxicity activity against AML cells.

Genetic evidence for the role of GSK3 in NK cell cytotoxic activity

To further assess the importance of GSK3 in N K cell-mediated killing of AML cells, genetic approaches were utilized. As the loss of GSK33 is embryonic lethal, we crossed floxed-GSK33 mice to Vav/Cre mice which led to a loss of GSK33 expression in

hematopoietic cells. N K cells derived from these mice showed a significantly increased cytotoxic activity against the mouse cancer cell line, WEHI-231 as compared to wild-type age matched mice (Figure 10A). N K cells lacking GSK33 exhibited ~3 fold more killing than wild- type NK cells (30-40% vs 10-15%). Of note all reported small molecule GSK3 inhibitors impact both GSK3a and GSK33 making it difficult to discern the importance of specific isoforms in N K cell activity. These results using GSK33 deficient mice demonstrate that GSK33 plays an important role in N K cell activity.

Besides utilizing a genetic approach in mice, it was further assessed the impact of GSK3 in human NK cells through overexpression or knockdown of GSK3. As shown in Figure 10B, knockdown of either GSK3a or GSK33 enhanced N K cell mediated killing of OCI-AML3 cells. Similarly, overexpression of both isoforms decreased N K activity (Figure IOC). The lower level of N K cell activity in these genetic studies as compared to those using small molecule inhibitors suggest that the simultaneous inhibition of both isoforms is necessary for maximal effect.

GSK3 inhibition enhances TNFa production by NK cells through NF-KB activation

In order to explore mechanisms through which GSK3 inhibition enhances N K cell activity, N K cell cytokine production was assessed. N K cells produce a wide variety of cytokines and chemokines including IFNy, TN Fa and IL-1027,28. It was identified that GSK3 inhibitor treatment of NK cells with SB leads to a marked induction in TN Fa levels (>6 fold) and a modest increase in IFNy (Figure 11A). In contrast, there was no change in TGF3 or IL- 10 levels (Figure 11A). TN F neutralization markedly impairs the cytotoxic activity of the GSK3-inhibited NK cells (Figure 11B). Importantly, TN Fa neutralization did not impact the activity of control NK cells suggesting that TN Fa plays an important role specifically in GSK3 inhibitor-mediated N K cell hyperactivation. As TN Fa itself is known to lead to cancer cell death, it was tested if the levels of TNFa produced could lead to direct leukemia cell death. TN Fa treatment did not lead to evidence of AML cell death at the doses and time points tested demonstrating that the cell killing is mediated by the N K cells.

Since TNFa plays a major role in GSK3 inhibitor-mediated N K cell activity, it was further explored mechanisms leading to its induction. As TNFa is an NF-κΒ target gene, the impact of GSK3 inhibition on N F-κΒ signaling in N K cells was checked. An increase in nuclear translocation of the N F-κΒ subunits p65, c-Rel and p50 in SB 6 treated N K cells was observed (Figure 12A), whereas DMSO treated N K cells did not show any N F-κΒ activation at the time points tested. In order to demonstrate that the NF-κΒ activation in N K cells is indeed responsible for the observed TN F induction, a nucleotide pull down assay using 4 known N F- KB binding regions in the TN Fa promoter29 was performed. It was found that p65; p50 and c-Rel binding to the TNFa promoter were enhanced following GSK3 inhibition (Figure 12B). Interestingly, it was observed enrichment of two distinct NF-κΒ complexes; 1. A p65/p50 complex binding to the κΒΙ region and 2. A complex containing p65, p50 and c-Rel binding to the KB2a region. Inducible c-Rel occupancy was found to be unique to the KB2a region. The varying binding intensity of the N F-κΒ subunits to the four regions in the TN F promoter shows that the binding is inducible and specific to the cognate DNA sequences.

Increased target-effector cell conjugate formation after co-culture of AML cells with GSK3 inhibited NK cells

N K cell killing requires conjugate formation between NK and target AML cells. This interaction forms N K cell immunological synapses that lead to the activation of a cascade of intracellular signals leading to target cell Iysis30. To determine the effect of GSK3 inhibitors on N K cell conjugate formation, separately labelled AML and N K cells were co-incubated. Interestingly, GSK3 inhibitor treated N K cells led to a dramatic increase in conjugate formation with AML cells as compared to vehicle control N K cells (Figure 13A).

As conjugate formation involves the interaction of cell surface molecules on N K and target cells, the expression of a variety of N K cell surface receptors as well as

granzyme/perforin which directly mediates killing after an overnight incubation of N K cells with GSK3 inhibitor was measured. It was observed a modest increase in perforin and granzyme expression in GSK3 inhibited NK cells as compared to control cells. While significant changes in the expression of most cell surface receptors tested was not identified, LFA-1 (active form) and ICAM-1 were induced on the NK cells and AML cells respectively (Figure 13B-C). LFA-1 and ICAM-1 expression on NK cells and AML cells respectively is reported to play an important role in AM L-N K conjugate formation.

To delineate the role of TN Fct in this conjugate formation, ICAM-1 and LFA-1 expression on AML and N K cells respectively after TN F neutralization was checked.

Neutralization of TN Fct decreased ICAM expression in AML cells as well as impaired NK cell killing (Figures 13C and 11B). Though LFA-1 is not regulated by TN Fct, it has been previously shown that recycling of other integrins and their expression on the cell surface is regulated by GSK3. This study supports the model that GSK3 inhibition activates N F- κΒ which leads to TN Fct production, induction of cell surface receptors, enhanced conjugate formation and subsequent AML cell killing (Figure 13D). GSK3 inhibited NK cells also shows increased killing of AML cells in a mouse model

In order to assess the potential of GSK3 inhibited N K cells for AML therapy, multiple models of human AML in immunodeficient mice were utilized. It was found that GSK3 inhibitor treated N K cells (SB and 117) led to a significant reduction in leukemic cell burden in the mouse bone marrow and spleen at the end of the study period as compared to control treated NK cells (Figure 14). In the first model the N K cells were tested against the OCI cell line (Figure 14A-B) and in the second model the cells were tested against a primary human AML sample (Figure 14C). For the second model, survival in addition to disease burden at a defined endpoint was also assessed. A significant improvement in mouse survival when utilizing 117 pre-treated N K cells as compared to control treated N K cells was observed. (Figure 14D). Importantly, similar to the in vitro studies, the GSK3 inhibitor treated cells performed significantly better in vivo than the vehicle treated and ex vivo expanded N K cells which have previously been tested in clinical trials for hematologic and non-hematologic malignancies.

NK cells have long been known to play a major role in immune monitoring to prevent the development and progression of cancer. Unfortunately, NK cells in AML patients are known to exhibit significant defects in number and cytotoxic activity that are implicated in AML development, progression and relapse. Though phenotypic changes in these cells have been described, specific molecular causes for this dysfunction are less clear. It has for example been reported that tumor microenvironment factors such as PGE2, TGFpi and IL- 10 may play a role. Further alterations in N K cell surface receptor expression have been reported such as a reduction in NKG2D expression as well as changes in conjugate formation.

These studies show that GSK3 plays an important role in N K cell cytotoxic activity against AML. Both genetic and pharmacologic manipulation of GSK3 was found to impact the ability of N K cells to kill AML cells. Interestingly, it was also observed that elevated GSK33 expression in NK cells from AML patients that are functionally defective. These studies suggest that this elevated GSK33 protein in N K cells from AML patients may contribute to N K cell dysfunction in AML.

Interestingly it has previously been reported that GSK33 protein is upregulated in a wide variety of cancer cells including leukemia and that GSK3 may play a role in disease progression. The work here demonstrates that this dysregulation of GSK3 protein expression is not limited to cancer cells. GSK3 inhibition in AML cells is known to enhance AML differentiation and growth inhibition. In fact GSK3 inhibitors are currently being tested clinically for cancer due to their direct anti-cancer activities [NCT01632306] [NCT01287520] [NCT01214603]. In contrast to these previous studies, this study demonstrates that GSK3 inhibition not only impacts AML cells directly, but also hyperactivates NK cells and leads to AML cell killing. Though the study has focused on AML, it is likely that GSK3 is an important mediator of N K cell activity in other disease contexts.

It is understood that the invention may embody other specific forms without departing from the spirit or central characteristics thereof. The disclosure of aspects and embodiments, therefore, are to be considered as illustrative and not restrictive. While specific embodiments have been illustrated and described, other modifications may be made without significantly departing from the spirit of the invention. All references cited herein, including patents, patent applications and non-patent literature are hereby incorporated by reference in their entirety.

Claims

What is Claimed: Having described the invention, the following is claimed:

1. A method of inducing myeloid differentiation in a subject comprising:

administering to the subject a therapeutically effective amount of at least one GSK3 kinase inhibitor.

2. The method of claim 1, wherein said GSK3 kinase inhibitors have the following

wherein each of Xi, ...X₄ is independently N or C;

Ri is CI, Br, Me, OMe;

R₂, or R₃ is F, CI, Br, I, Me, Ethyl, CN, NH₂, NMe₂, OMe, N0₂, hydroxymethyl, CH₂OCOMe, HOCHMe₂, vinyl, Phenyl;

R₄ is cyclopropyl, furanyl;

R₅ is cyclohexyl.

3. The method comprising administering all-trans retinoic acid in combination with any GSK3 kinase inhibitor to enchance the activity of either agent alone

4. A method of treating any disease for which GSK3 inhibitors have been shown to be clinically useful (cancer, diabetes, autoimmune disease etc) using compounds having the general formula (I).

5. A method of preferentially inhibiting only a portion of GSK3 kinase activity such that this inhibition does not result in significant beta-catenin upregulation.

6. A composition of matter comprising a compound having the following general formula I, II or III

wherein each of XI, ...X4 is independently N or C;

Rl is CI, Br, Me, OMe;

R2, or R3 is F, CI, Br, I, Me, Ethyl, CN, N H2, NMe2, OMe, N02, hydroxymethyl, CH20COMe, HOCH Me2, vinyl, Phenyl;

R4 is cyclopropyl, furanyl;

R5 is cyclohexyl.

7. A composition of matter having a general formula selected from a group consisting

45

47