WO2014062454A1 - Compositions and methods for treating cancer - Google Patents

Abstract

The instant invention relates to methods for the treatment of neuroblastoma by administering a combination of a WEE1 inhibitor and a CHK1 inhibitor, wherein the WEE1 inhibitor is WEE1-1 or a pharmaceutically acceptable salt thereof, or WEE1-2 or a pharmaceutically acceptable salt thereof, and the CHK1 inhibitor is CHK1-1 or a pharmaceutically acceptable salt thereof. In another embodiment, the invention relates to a method for treating a neuroblastoma patient, comprising administering a WEE1 inhibitor and a CHK1 inhibitor, wherein the cancer cells of said patient to be treated are characterized by amplified expression levels of MYCN.

Description

TITLE OF THE INVENTION

COMPOSITIONS AND METHODS FOR TREATING CANCER

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treating cellular proliferative disorders or disorders associated with WEEl kinase and CHKl kinase activity and for inhibiting WEEl kinase and CHKl kinase activity.

BACKGROUND OF THE INVENTION

Unlike broadly active chemotherapeutics which indescriminately kill dividing cells, select targeted cancer therapeuctics have demonstrated the potential to specifically eradicate cancer cells while sparing "normal" non-cancerous cells, resulting in clinical efficacy and minimized adverse side effects. Nevertheless, most single agent cancer therapies fail in the clinic due to underwhelming anti-tumor responses. Even in the few cases where targeted agent monotherapies have succeeded in treating solid tumors, the effect is usually transient and drug- resistant tumors quickly reemerge. One approach to improve clinical outcome of anti-cancer pharmaceuticals is the combination of two or more therapies, an approach that onclogists have utilized for decades with broadly active DNA-damaging agents. More recently the strategic pairing of targeted oncology agents has gained momentum with the hope of synergistic cytotoxicity, making the combination more effective in treating tumor cells than either single drug alone. Drug combinations are expected to take advantage of synthetic lethality or repressing compensatory feedback mechanisms that would otherwise allow cancer cells to survive effects of monotherapy. Optimal combinations might also delay onset of drug resistance by killing more tumor cells as well as by limiting alternate means of developed cellular resistance.

The maintenance of genomic integrity through error- free DNA replication and precisely timed cellular division is essential for the accurate transfer of genetic information to daughter cells. Flawless cell cycle progression is dependent upon the tightly regulated coordination between several cell cycle checkpoint proteins and the cyclin-dependent kinase (CDK) family of proteins. Malignant cells frequently exploit defects in DNA repair and/or cell cycle checkpoint pathways to stimulate aberrant cellular division, thereby gaining a distinct survival advantage (Malumbres, M. and Barbacid, M., Nature Rev. Cancer, 2009, 9: 153-166). Key regulators of DNA damage surveillance pathways, such as the checkpoint kinases ATR, CHKl and WEEl, recognize DNA aberrations and arrest cellular division until genomic integrity is restored (Sorensen, C.S. and Sylijuasen, R.G., Nucleic Acids Research. 201 1, 1-10; Reinhardt, H.C. and Yaffe, M.B., Curr. Opin. Cell Biol. 2009, 21 :245-255). Paradoxically the aberrant activity of DNA damage response proteins can also serve to dampen the replication stress generated by oncogenic transformation and thereby protect cancer cells (Xu, H., et al, Intl. J. Cancer, 201 1, 129(8): 1953-1962).

Both of the serine/threonine kinases CHKl and WEEl are overexpressed and/or aberrantly activated in several cancer types (Sorensen and Sylijuasen, 2011 ; Reinhardt and Yaffe, 2009; Xu et al, 201 1; Davies, K.D., et al, Cancer Biol. & Ther.. 201 1, 12:788-798; Mir, S.E., et al, Cancer Cell. 2010, 18:244-257; Wang, Y., et al, Cancer Biol. & Ther.. 2004, 3 :305; Hattori, H., et al, Mol. Cancer Ther.. 201 1, 10:670-678). (Smith, J., et al, Adv. Cancer Res.. 2010, 108:73-1 12). Abrogation of both CHKl and WEEl signaling forces mitotic progression with incompletely replicated or damaged DNA, leading to mitotic catastrophe and subsequent cell death (Mir et al, 2010; Castedo, M., et al, Oncogene. 2004, 23:2825-2837; Montano, R., et al, Mol. Cancer Ther.. 2012, 1 1 :427-438). This suggests that both CHKl and WEEl may serve as attractive molecular targets for pharmacologic intervention for cancer.

CHKl is an essential serine/threonine kinase involved in two cell cycle checkpoints, the intra-S and G2/M checkpoints. In response to DNA replication stress during S- phase of the cell cycle, CHKl activity prevents stalled replication forks from collapsing and causing genomic damage (Feijoo, C, et al, J. Cell Biology. 2001, 154(5):913-923; Abraham, R. T., Genes & Dev.. 2001, 15:2177-2196). Also, CHKl activity following DNA damage is necessary for arrest at the G2/M cell cycle boundary, preventing cells from prematurely entering mitosis before damaged DNA has been repaired (O'Connell, M.J., et al, Embo Journal

1997, 16(3):545-554; Liu, Q.H., et al, Genes & Development 2000, 14(12): 1448-59).

Importantly, CHKl is necessary for unperturbed DNA replication and cell cycle coordination even in the absence of any exogenous insult. As an example, conditional CHKl heterozygosity leads to abberant DNA replication, increased DNA damage, and premature mitosis in untreated murine mammary epithelial cells (Lam, M.H., et al, Cancer Cell. 2004, 6(l):45-59). Several publications describe the cytotoxic nature of CHKl knockdown or inhibition, either alone or in combination with DNA-damaging therapeutics, demonstrating preclinical proof of concept for CHKl targeted agents.

WEEl is an essential tyrosine kinase best recognized as a mitotic gatekeeper that phosphorylates and inactivates cyclin dependent kinase 1 (CDK1 = CDC2), the only

indispensible human cyclin dependent kinase (Malumbres, M., and Barbacid, M., Nature Reviews Cancer. 2009, 9(3): 153-166). As cells transition into mitosis, WEEl activity is reduced, allowing CDKl/cyclin Bl to intiate mitotic events. WEEl is therefore critical for properly timing cell division in unperturbed cells, and loss of WEEl results in chromosomal aneuploidy and accumulated DNA damage (Tominaga, Y., et al, Intl. J. Biol. Scl. 2006, 2(4): 161-170). Additionally, WEEl activity can be increased as a result of DNA damage, causing cells to arrest in G2 and allowing for repair of DNA lesions before beginning mitosis (Raleigh, J.M., and O'Connell, M.J., J. Cell Sci.. 2000, 1 13(10): 1727-1736). Recently, WEEl has been shown to be indispensible for genomic integrity specifically as cells traverse S-phase, describing a previously unrecognized role for WEEl in maintaining fidelity of DNA replication (Beck. H., et al, J. Cell Biology, 2010, 188(5):629-638). Knockdown of WEEl by siRNA led to rapid and S-phase specific accumulation of γΗ2ΑΧ, a phosphorylated histone protein that quantitatively represents DNA damage. Interfering with WEEl has been shown to repress cancer cell proliferation and lead to greater anti-tumor effects of DNA-damaging

chemotherapeutics than either single agent alone could achieve.

Neuroblastoma is a common pediatric tumor derived from the cells of the sympathetic nervous system that manifests with significant clinical heterogeneity (Maris, J. M., et al, Lancet, 2007, 369 (5979):2106-2120; Brodeur, G.M., Nature Rev. Cancer, 2003, 3 :203- 206; Maris, J.M., N. Eng. J. Medicine, 2010, 362:2202-2211). Patients are typically stratified into risk groups based upon several criteria at diagnosis, including age, tumor ploidy, MYCN amplification status and histological features (Maris, J.M., et al, 2007; Brodeur, G.M., 2003). Although low-risk patients are successfully treated with surgery, approximately 50% of all children with neuroblastoma are diagnosed with high-risk disease, requiring myeloablative chemotherapy followed by maintenance with retinoids and anti-GD2-based immunotherapy (Maris, J.M., 2010). Despite this intense multi-modal treatment regimen, half of these patients will eventually relapse and succumb to the disease and those that survive are typically burdened with treatment-related chronic illnesses (Hudson, M.M., et al, "Health Status of Adult Long- Term: A Report from the Childhood Cancer Survivor Study," J. Am.Medical Assn., 2003, 290: 1583-1592). The intense chemotherapy schedule and poor survival rate characteristic of high-risk and/or relapsed neuroblastoma underscores the need for novel therapies to successfully treat children burdened with this disease. Applicants recently identified, through a siRNA screen of the neuroblastoma kinome, that the DNA damage response protein checkpoint kinase 1 (CHK1) had disproportionate activity in neuroblastoma and was sensitive to single agent inhibition (Cole, K.A., et al, PNAS. 2011, 108:3336-3341).

SUMMARY OF THE INVENTION

The instant invention relates generally to methods for treating neuroblastoma by administering a therapeutically effective amount of the combination of a WEEl inhibitor and a

CHK1 inhibitor, wherein the WEEl inhibitor is WEEl-1 or a pharmaceutically acceptable salt thereof, or WEE 1-2 or a pharmaceutically acceptable salt thereof, and the CHK1 inhibitor is

CHKl-1 or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention herein is a method for treating a neuroblastoma patient, comprising administering a WEEl inhibitor, such as, WEEl-1 or a pharmaceutically acceptable salt thereof, or WEE 1-2 or a pharmaceutically acceptable salt thereof, and a CHK1 inhibitor, such as CHKl-1 or a pharmaceutically acceptable salt thereof, wherein the cancer cells of said patient are characterized by amplified MYCN expression. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphic illustration of the functional analysis conducted on the siRNA screen hits. MYCN regulated genes (linked by black lines to MYCN) were identified as signficantly over represented in the gene list. Hits indicating sensitivity are shown as (o) and those indicating resistance are shown as (·).

Figure 2 is a representation of the gene signature that correlated with sensitivity to a WEEl inhibitor (WEE1-A) in a panel of 93 lung cancer cell lines. Gene expression represented by the lighter color at the far right in the upper panel and upper right and lower left in the heat signature map indicates high gene expression, while the lighter color at the far left of the upper panel and upper left and lower right of the heat signature map indicates low gene expression.

Figure 3 is a graphic illustration of the functional analysis showing MYCN regulated genes as a significant cluster, showing the relationship of genes (·) that were highly expressed in sensitive cell lines.

Figures 4A - 4F are a representation of the gene expression heat map depicting analysis of tumors from animals that were exposed to radiation and a WEEl inhibitor (WEEl-1) at various dosing levels and treatment regimens.

Figure 5 is a graphic representation of the analysis of cluster number 5, which was enriched for genes known to be regulated by MYCN. Each dot represents a gene that was identified, while each edge represents group membership. For example, the mismatch repair group was found to be significant, but contained only three genes. However, two of the three genes were also known to be MYCN regulated. Thus, MYCN regulation was attributed to the observed gene response in that it was connected to the majority of genes.

Figure 6 is a graphic illustration that MYCN amplification predicts sensitivity to a WEEl inhibitor. MYCN amplified neuroblastoma cell lines: CHP-212, SK-N-DZ, IMR-32, SK- N-BE(2), and BE(2)-C. Unamplified cell lines: SK-N-SH, SH-SY5Y.

Figures 7A-7D illustrate that WEEl kinase was highly expressed in neuroblastoma. Figure 7 A is an illustration of the Western blot analysis of neuroblastoma cell lines. WEEl was highly expressed at the protein level and constitutively activated as compared to non-NB lines, such as, DAOY medulloblastoma cells or non-transformed RPE-1 cells.

Phosphorylated WEE I^s642 was present in the majority of neuroblastoma lines and often coincided with downstream phosphorylation of Cdc2^Y15 (Cdkl). Figure 7B is an illustration of a Western blot analysis showing that WEEl was highly expressed in diagnostic patient tumor samples, with increased WEEl activity in 67% (8 of 12) of tumors derived from high-risk patients (sample numbers 38, 58, 73, 193, 198, 969, 505, 260, 443, 495, 1000, and 1129), as compared to 28.5% (2 of 7) of tumors derived from low-risk patients (sample numbers 19, 66, 151, 415, 430, 1040, and 1 133). Figure 7C is an illustration of a neuroblastoma tissue microarray (TMA) stained positively for phospo- WEEl (S642), which illustrates representative staining for negative, low, intermediate, and high tumor risk groups. Figure 7D is a graphic representation of the higher expression levels of WEE 1 in high-risk, MYCN-amplified tumors, as compared to low-risk samples. HR = high-risk; LR = low-risk; INSS = International

Neuroblastoma Staging System.

Figures 8A-8D illustrate that the abrogation of either WEE1 or CHK1 signaling was cytotoxic to neuroblastoma cells. Figure 8A is a graphic illustration of siRNA-mediated depletion of CHK1 or WEE1 that resulted in a significant reduction in cell viability in representative neuroblastoma cell lines, Kelly, NLF, SKNAS. Figure 8B is a graphic illustrating that a majority of neuroblastoma cells (NB1691), as compared to cells derived from a retinal pigmented epithelium (RPE-1) used as a control, were sensitive to single-agent inhibition of WEE1 (WEEl-1) or CHK1 (CHKl-1) activity, with median IC50S of 300nM and 900nM, respectively (curve shifted <0.05 on x-axis to allow visualization where overlapped). Figure 8C is a graphic illustrating that sensitive neuroblastoma cell lines underwent apoptosis in response to CHK1 or WEE1 inhibition as evidenced by caspase 3/7 activation, whereas resistant cell lines, such as RPE-1 and NB- 1691, did not. (* = p < 0.05, ** = p < 0.01, *** = p < 0.001). Figure 8D is an illustration of the Western blot of the differential PARP cleavage to treatment with a WEE1 inhibitor (WEEl-1), a CHK1 inhibitor (CHKl-1), or an active metabolite of irinotecan (SN-38).

Figures 9A and 9B illustrate that murine neuroblastoma cell lines derived from MYCN transgenic mice were sensitive to CHK1/WEE1 inhibition. Figure 9A is a graphic illustration of cells homozygous (282) or heterozygous (844) for the MYCN oncogene that were derived from MYCN-transgenic murine tumors and that were found to be sensitive to a single- agent: CHK1 (CHKl-1 (top panel); WEE1 (WEEl-1) (bottom panel). Homozygous cells were twice as sensitive as their heterozygous counterparts. Figure 9B is an illustration of a Western blot analysis confirming target engagement 6 hours after treatment with increasing

concentrations of a WEE1 (WEEl-1) or a CHK1 (CHKl-1) inhibitor. Increasing

phosphorylation of CHK1^S345 has been shown to be a biomarker of CHK1 inhibition (Smith, J., et al . Adv. in Cancer Res.. 2010, 108:73-1 12).

Figure 1 OA- IOC illustrate that inhibition of both CHK1 and WEE1 resulted in accumulation of DNA double-strand breaks and mitotic catastrophe. Figure 10A is an illustration of abrogation of Cdc2 activity in BE2c cells treated with a WEE1 inhibitor (WEEl-1), with a concomitant increase in H2A.X phosphorylation, which was indicative of DNA damage. Figure 10B illustrates that inhibition of CHK1, using a CHK1 inhibitor (CHKl-1), resulted in only marginal increases in H2A.X phosphorylation. In combination with another chemotherapy agent (SN-38 or Gemcitabine), the CHK1 inhibitor (CHKl-1) rapidly (within 2 hours) induced double strand breaks as shown in the neuroblastoma cell line, NB-1643. Figure IOC illustrates that simultaneous inhibition of CHK1 and WEE1 (16 hours) resulted in robust H2A.x

phosphorylation and complete abolishment of Cdc2 signaling, suggesting the cells were progressing through mitosis with DNA damage, as shown in the NGP neuroblastoma cell line. HU = lmM hydroxyurea; WEEl-1 = 250nM, CHKl-1 = 500nM; SN-38 = ΙΟΟηΜ; Gem =

ΙΟΟηΜ gemcitabine)

Figures 1 1A and 1 IB illustrate that the growth of neuroblastoma xenografts was significantly impaired in response to CHK1/WEE1 combinatorial therapy. Figure 1 1A is a graphic illustration of the tumor burden in mice subcutaneously implanted with neuroblastoma xenografts and treated BID with 30 mg/kg/dose of a WEEl (WEEl-1) or a CHK1 (CHKl-1) inhibitor or both for 5 days for 2 weeks. A mixed linear analysis indicated a significant reduction in tumor burden in the combination treatment group in both neuroblastoma cell lines.

For graphical purposes, mice removed from the study for excessive tumor burden had their last measurements carried forward. Arrows indicate end of treatment. (* = p < 0.05, *** = p <

0.0001) Figure 1 IB illustrates target engagement as verified by resection of Ebcl xenografts, where both CHK1 and Cdc2 activity was substantially reduced following four doses of a CHK1

(CHKl-1) or WEEl (WEEl-1) inhibitor, respectively.

Figures 12A and 12B illustrate that the expression and activity of WEEl is prevalent in neuroblastoma. Figure 12A illustrates a Western blot analysis of WEEl expression and activation (evidenced by phospho-Ser642) of a secondary panel of neuroblastoma cell lines.

Cdc2 activity was observed in all neuroblastoma lines. Figure 12B illustrates WEEl phosphorylation was generally much lower in a variety of adult cancer cell lines than that seen in neuroblastoma (far right lanes). PANC-1 = pancreactic carcinoma; T98G = glioblastoma multiforme; SK-OV-3 = ovarian carcinoma; H441 = lung adenocarcinoma; DAOY = medulloblastoma; RPE-1 = retinal pigmented epithelial; SK-N-SH = neuroblastoma; NB1643 = neuroblastoma.

Figures 13A-13F graphically illustrate that WEEl and CHK1 expression are elevated in high-risk, MYCN-amplified neuroblastoma. Primary tumors were obtained from 251 patients at diagnosis (221 high-risk, 30 low-risk; 68 MYCN amplified, 183 MYCN non- amplified), and were run on Affymetrix Human Exon 1.0 ST expression microarrays. Both WEEl (Figuresl3B and 13E) and CHK1 (Figures 13C and 13F) expression were expressed at a significantly higher rate in both the MYCN amplified and high-risk conditions.

Figures 14A and 14B illustrate that inhibition of both CHK1 and WEEl significantly impairs tumor growth in vivo. Mice were treated with either 30 mg/kg of a single agent WEEl inhibitor (WEEl-1), a single-agent CHK1 inhibitor (CHKl-1), or simultaneously with both inhibitors (WEEl-1 and CHKl-1) for two weeks (5 days on/2 days off). A mixed linear effects model was used to calculate the rate of tumor growth in each condition. Dual inhibition (CHK1 and WEEl) was efficacious in the NB1643 xenograph model (Figure 14A - top panel). Figure 14 B provides the slope and p-value for the impairment of tumor growth for WEEl and CHK1 expression in both MYCN amplified and high-risk conditions. Figures 15A and 15B graphically illustrate the higher mean activity of a WEE 1 inhibitor (WEEl-1) (Figure 15A) and a CHK1 inhibitor (CHKl-1) (Figure 15B) in

neuroblastoma cell lines (n = 7) relative to cell lines for other tumor types. DETAILED DESCRIPTION OF THE INVENTION

The preclinical studies discussed herein have shown that the combination of a WEE1 inhibitor with a CHK1 inhibitor results in synergistic inhibition of tumor growth in neuroblastoma cell lines. Even half maximum tolerated dosages (MTD) for either agent alone resulted in robust accumulation of DNA damage as evidenced by H2A.X phosphorylation, suggesting that the unique combination of a WEE1 and a CHK1 inhibitor induces DNA strand breakage, leading to mitotic disruption and ensuing apoptosis. Pharmacodynamic (PD) analysis in xenograft tumors supports this notion, showing an increase in both the percentage of DNA damage containing cells as well as the duration of the DNA damage signal. Consistent with the PD data, the data discussed herein demonstrates that the combination of WEE1 and CHK1 inhibitors leads to significant reduction in tumor burden in a murine xenograft model. As such, in vivo, the combination may inhibit cell growth at dosages much less than what is required for either agent alone to produce a similar effect.

Applicants herein have found that neuroblastoma may be susceptible to therapies targeting the DNA damage response (DDR) pathway. In that many relapsed neuroblastomas are refractory to conventional chemotherapy, the synergistic anticancer activity of the CHK1 and WEE1 combination may also sensitize these tumors to allow the continued use of conventional therapeutic agents. Applicants findings herein that show WEE1 is highly expressed in neuroblastoma, particularly in high-risk, MYCN-amplified tumors, provides a method for treating neuroblastoma by targeting this kinase for therapeutic intervention. In addition, not only is the combination of WEE1 and CHK1 inhibitors synergistic in themselves, these compounds act synergistically when combined with other chemotherapy agents, such as, gemcitabine or irinotecan (SN-38). Taken together, these data demonstrate the effectiveness of the dual treatment of a WEE1 and a CHK1 inhibitor which may result in great benefits for treating neuroblastoma.

Applicants have found that synergistically excellent anticancer activity can be achieved by using a WEE1 inhibitor with a CHK1 inhibitor, specifically wherein the WEE1 inhibitor is WEEl-1 or a pharmaceutically acceptable salt thereof, or WEE 1-2 or a

pharmaceutically acceptable salt thereof, and the CHK1 inhibitor is CHKl-1 or a

pharmaceutically acceptable salt thereof. Thus, the invention herein is directed to uses of this synergistic activity of WEE 1 and CHK1 and the combination of WEE 1 and CHK1 inhibitors to treat neuroblastoma.

Accordingly, the instant invention relates to methods for treating neuroblastoma with a WEE1 inhibitor and a CHK1 inhibitor, wherein the WEE1 inhibitor is WEEl-1 or a pharmaceutically acceptable salt thereof, or WEE 1-2 or a pharmaceutically acceptable salt thereof, and the CHKl inhibitor is CHKl-1 or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention relates to a method for treating a neuroblastoma patient, in need of treatment thereof, comprising administering a WEEl inhibitor, such as, WEEl-1 or a pharmaceutically acceptable salt thereof, or WEE 1-2 or a

pharmaceutically acceptable salt thereof, and a CHKl inhibitor, such as, CHKl-1 or a pharmaceutically acceptable salt thereof, wherein the cancer cells of said patients are characterized by amplified MY C expression.

In an embodiment of the invention, the WEEl inhibitor is WEEl-1 or a pharmaceutically acceptable salt thereof.

In another embodiment of the invention, the CHKl inhibitor is CHKl-1 or a pharmaceutically acceptable salt thereof.

In another embodiment of the invention, the WEEl inhibitor is administered in a dose between 100 mg per day and 200 mg per day. In an embodiment of the invention, the WEEl inhibitors may be dosed twice a day (BID) over the course of two and a half days (for a total of 5 doses) or once a day (QD) over the course of two days (for a total of 2 doses). In another embodiment of the invention, the CHKl inhibitor is administered in a dose from about 100 mg per day to 200 mg per day. In an embodiment of the invention, the CHKl inhibitor may be dosed once a day (QD) over either one or two days.

The WEEl inhibitor and the CHKl inhibitor can be prepared for simultaneous, separate or successive administration.

Reference to the preferred embodiments set forth above is meant to include all combinations of particular and preferred groups unless stated otherwise. The meanings of the terms used in this description are described below, and the invention is described in more detail hereinunder.

The term "simultaneous" as referred to in this description means that the pharmaceutical preparations of the invention are administered simultaneously in time.

The term "separate" as referred to in this description means that the pharmaceutical preparations of the invention are administered at different times during the course of a common treatment schedule.

The term "successive" as referred to in this description means that administration of one pharmaceutical preparation is followed by administration of the other pharmaceutical preparation; after administration of one pharmaceutical preparation, the second pharmaceutical preparation can be administered substantially immediately after the first pharmaceutical preparation, or the second pharmaceutical preparation can be administered after an effective time period after the first pharmaceutical preparation; and the effective time period is the amount of time given for realization of maximum benefit from the administration of the first

pharmaceutical preparation. The term "cancer" as referred to in this description includes various sarcoma and carcinoma and includes solid cancer and hematopoietic cancer. The solid cancer as referred to herein includes, for example, brain cancer, cervicocerebral cancer, esophageal cancer, thyroid cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, endometrial cancer, lung cancer, stomach cancer, gallbladder/bile duct cancer, liver cancer, pancreatic cancer, colon cancer, rectal cancer, ovarian cancer, choriocarcinoma, uterus body cancer, uterocervical cancer, renal pelvis/ureter cancer, bladder cancer, prostate cancer, penis cancer, testicles cancer, fetal cancer, Wilms' tumor, skin cancer, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's tumor, soft part sarcoma. On the other hand, the hematopoietic cancer includes, for example, acute leukemia, chronic lymphatic leukemia, chronic myelocytic leukemia, polycythemia vera, malignant lymphoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma.

The term "treatment of cancer" as referred to in this description means that an anticancer agent is administered to a cancer patient so as to inhibit the growth of the cancer cells in the patient. Preferably, the treatment results in some form of cancer growth regression or that the treatment delays or prevents the recurrence of the cancer. More preferably, the treatment results in complete disappearance of cancer.

The term "patient" as referred to in this description means the recipient in need of medical intervention or treatment. Mammalian and non-mammalian patients are included.

The term "amplified MYCN expression" or "amplified expression of MYCN" as referred to in this description means a cell, obtained from a cell line characterized as or from a patient diagnosed with neuroblastoma, having higher MYCN DNA, mRNA, or protein expression, or an increase in the number of copies of the MYCN gene, as compared to a cell, obtained from a cell line characterized as or from a patient not diagnosed with neuroblastoma, or a control cell. A control cell is a comparable cell that is not characterized as neuroblastoma or when referring to a dose response curve, is one that has not been treated with an inhibitor or therapeutic agent.

The term "gene marker" or "marker" or "surrogate" as referred to in this description means an entire gene, or a portion therof, such as an EST derived from that gene, the expression or level of which changes between certain conditions. Where the expression of the gene correlates with a certain condition, for example a drug treatment or a disease state, the gene is a marker for that condition. As used herein the term generally refers to the MYC- Neuroblastoma (MYCN) gene that generally exhibits elevated expression in neuroblastoma.

As used herein, the terms "measuring expression levels," "measuring gene expression level," or "obtaining an expression level" and the like, includes methods that quantify target gene expression level exemplified by a transcript of a gene, including microRNA

(miRNA) or a protein encoded by a gene, as well as methods that determine whether a gene of interest is expressed at all. Thus, an assay which provides a "yes" or "no" result without necessarily providing quantification of an amount of expression is an assay that "measures expression" as that term is used herein. Alternatively, the term may include quantifying expression level of the target gene expressed in a quantitative value, for example, a fold-change in expression, up or down, relative to a control gene or relative to the same gene in another sample, or a log ratio of expression, or any visual representation thereof, such as, for example, a "heatmap" where a color intensity is representative of the amount of gene expression detected. Exemplary methods for detecting the level of expression of a gene include, but are not limited to, Northern blotting, dot or slot blots, reporter gene matrix (see, for example, US 5,569,588), nuclease protection, RT-PCR, microarray profiling, differential display, SAGE (Velculescu et al, (1995), Science 270:484-87), Digital Gene Expression System (see WO2007076128;

WO2007076129), multiplex mRNA assay (Tian et al, (2004), Nucleic Acids Res. 32:el26), PMAGE (Kim et al, (2007), Science 316: 1481-84), cDNA-mediated annealing, selection, extension and ligation assay (DASL, Bibikova, et al, (2004), AJP 165: 1799-807), multiplex branched DNA assay (Flagella et al, (2006), Anal. Biochem. 352:50-60), 2D gel electrophoresis, SELDI-TOF, ICAT, enzyme assay, antibody assay, and the like.

WEE1 Inhibitors

In an embodiment of the invention, the WEE1 inhibitor of the instant invention is WEEl-1, the structure of which is as shown below.

WEEl-1

WEEl-1 is a WEE1 inhibitor which is useful for the treatment of cancer. WEE1- 1 is also known as 2-allyl-l-[6-(l-hydroxy-l-methylethyl)pyridin-2-yl]-6-{[4-(4- methylpiperazin- 1 -yl)phenyl] amino } - 1 ,2-dihydro-3 H-pyrazolo[3 ,4-d]pyrimidin-3 -one. WEE 1-1 has been described in U.S. Patent No.7, 834,019, and in PCT International Publication

WO2007/126122, WO 2007/126128 and WO2008/153207, which are incorporated by reference herein in their entirety. Crystalline forms of WEEl-1 are described in US Publication US2010- 0124544 and PCT International Publication WO2011/034743, which are incorporated by reference herein in their entirety.

In an embodiment of the invention, the WEE1 inhibitor of the instant invention is WEE 1-2, the structure of which is as shown below.

WEE 1-2 is a WEE1 inhibitor which is useful for the treatment of cancer. WEE1- 2 is also known as 3-(2,6-dichlorophenyl)-4-imino-7-[(2'-methyl-2',3'-dihydro-rH- spiro[cyclopropane-l,4'-isoquinolin]-7'-yl)amino]-3,4-dihydropyrimido[4,5-d]pyrimidin-2(lH)- one. WEE1-2 has been described in PCT International Publication WO2008/153207 and US Publication US201 1-0135601, which are incorporated by reference herein in their entirety. Crystalline forms of WEE 1-2 are described in International Publication WO2009/151997 and US Publication US201 1-0092520, which are incorporated by reference herein in their entirety.

CHK1 inhibitors

In an embodiment of the invention, the CHK1 inhibitor of the instant invention is CHKl-1, the structure of which is as shown below.

CHKl-1

CHKl-1 is a CHK1 inhibitor which is useful for the treatment of cancer. CHKl-1 is also known as (R)-(-)-6-Bromo-3-(l -methyl- lH-pyrazol-4-yl)-5-piperidin-3-yl-pyrazolo [1,5- a]pyrimidin-7-ylamine, or CHKl-1. CHKl-1 has been described in U.S. Patent No.7, 196,078, PCT International Publications WO2007/044449 and WO 201 1/1 19457, and uses are described in PCT International Publication WO2007/044441, which are incorporated by reference herein in their entirety.

In an embodiment of the invention, the CHK1 inhibitor of the instant invention is a CHK1 inhibitor which is useful for the treatment of cancer and is as described in PCT International Publication WO 2009/014637, which is incorporated by reference herein in its entirety.

The compounds of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: E.L. Eliel and S.H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1 119-1190), and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, all such stereoisomers being included in the present invention. In addition, the compounds disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the invention, even though only one tautomeric structure is depicted.

In the compounds described in the present invention, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present invention is meant to include all suitable isotopic variations of the compounds disclosed herein. For example, different isotopic forms of hydrogen (H) include protium (1H) and deuterium (2H). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples. Isotopically-enriched compounds disclosed herein can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and/or intermediates.

The WEE1 and CHK1 inhibitors of the instant invention may also exist as various crystals, amorphous substances, pharmaceutically acceptable salts, hydrates and solvates.

Further, the WEE1 and CHK1 inhibitors of the instant invention may be provided as prodrugs. In general, such prodrugs are functional derivatives of the WEE1 inhibitors of the instant invention that can be readily converted into compounds that are needed by living bodies.

Accordingly, in the method of treatment of various cancers in the invention, the term

"administration" includes not only the administration of a specific compound but also the administration of a compound which, after administered to patients, can be converted into the specific compound in the living bodies. Conventional methods for selection and production of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H.

Bundgaard, Elsevier, 1985, which is referred to herein and is entirely incorporated herein as a part of the present description. Metabolites of the compound may include active compounds that are produced by putting the compound in a biological environment, and are within the scope of the compound in the invention.

Dosing and Routes of Administration

With regard to the WEE1 inhibitors and CHK1 inhibitors of the invention, various preparation forms can be selected, and examples thereof include oral preparations such as tablets, capsules, powders, granules or liquids, or sterilized liquid parenteral preparations such as solutions or suspensions, suppositories, ointments and the like. The WEE1 inhibitors and CHK1 inhibitors are available as pharmaceutically acceptable salts. The WEE1 inhibitors and CHK1 inhibitors of the invention are prepared with pharmaceutically acceptable carriers or diluents.

The term "pharmaceutically acceptable salt" as referred to in this description means ordinary, pharmaceutically acceptable salt. For example, when the compound has a hydroxyl group, or an acidic group such as a carboxyl group and a tetrazolyl group, then it may form a base-addition salt at the hydroxyl group or the acidic group; or when the compound has an amino group or a basic heterocyclic group, then it may form an acid-addition salt at the amino group or the basic heterocyclic group.

The base-addition salts include, for example, alkali metal salts such as sodium salts, potassium salts; alkaline earth metal salts such as calcium salts, magnesium salts;

ammonium salts; and organic amine salts such as trimethylamine salts, triethylamine salts, dicyclohexylamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, procaine salts, Ν,Ν'-dibenzylethylenediamine salts.

The acid-addition salts include, for example, inorganic acid salts such as hydrochlorides, sulfates, nitrates, phosphates, perchlorates; organic acid salts such as maleates, fumarates, tartrates, citrates, ascorbates, trifluoroacetates; and sulfonates such as

methanesulfonates, isethionates, benzenesulfonates, p-toluenesulfonates.

The term "pharmaceutically acceptable carrier or diluent" refers to excipients [e.g., fats, beeswax, semi-solid and liquid polyols, natural or hydrogenated oils, etc.]; water (e.g., distilled water, particularly distilled water for injection, etc.), physiological saline, alcohol (e.g., ethanol), glycerol, polyols, aqueous glucose solution, mannitol, plant oils, etc.); additives [e.g., extending agent, disintegrating agent, binder, lubricant, wetting agent, stabilizer, emulsifier, dispersant, preservative, sweetener, colorant, seasoning agent or aromatizer, concentrating agent, diluent, buffer substance, solvent or solubilizing agent, chemical for achieving storage effect, salt for modifying osmotic pressure, coating agent or antioxidant], and the like.

Solid preparations can be prepared in the forms of tablet, capsule, granule and powder without any additives, or prepared using appropriate carriers (additives). Examples of such carriers (additives) may include saccharides such as lactose or glucose; starch of corn, wheat or rice; fatty acids such as stearic acid; inorganic salts such as magnesium metasilicate aluminate or anhydrous calcium phosphate; synthetic polymers such as polyvinylpyrrolidone or polyalkylene glycol; alcohols such as stearyl alcohol or benzyl alcohol; synthetic cellulose derivatives such as methylcellulose, carboxymethylcellulose, ethylcellulose or

hydroxypropylmethylcellulose; and other conventionally used additives such as gelatin, talc, plant oil and gum arabic.

These solid preparations such as tablets, capsules, granules and powders may generally contain, for example, 0.1 to 100% by weight, and preferably 5 to 98% by weight, of the mTOR inhibitor, based on the total weight of each preparation. Liquid preparations are produced in the forms of suspension, syrup, injection and drip infusion (intravenous fluid) using appropriate additives that are conventionally used in liquid preparations, such as water, alcohol or a plant-derived oil such as soybean oil, peanut oil and sesame oil.

In particular, when the preparation is administered parenterally in a form of intramuscular injection, intravenous injection or subcutaneous injection, appropriate solvent or diluent may be exemplified by distilled water for injection, an aqueous solution of lidocaine hydrochloride (for intramuscular injection), physiological saline, aqueous glucose solution, ethanol, polyethylene glycol, propylene glycol, liquid for intravenous injection (e.g., an aqueous solution of citric acid, sodium citrate and the like) or an electrolytic solution (for intravenous drip infusion and intravenous injection), or a mixed solution thereof.

Such injection may be in a form of a preliminarily dissolved solution, or in a form of powder per se or powder associated with a suitable carrier (additive) which is dissolved at the time of use. The injection liquid may contain, for example, 0.1 to 10% by weight of an active ingredient based on the total weight of each preparation.

Liquid preparations such as suspension or syrup for oral administration may contain, for example, 0.1 to 10% by weight of an active ingredient based on the total weight of each preparation.

Each preparation in the invention can be prepared by a person having ordinary skill in the art according to conventional methods or common techniques. For example, a preparation can be carried out, if the preparation is an oral preparation, for example, by mixing an appropriate amount of the compound of the invention with an appropriate amount of lactose and filling this mixture into hard gelatin capsules which are suitable for oral administration. On the other hand, preparation can be carried out, if the preparation containing the compound of the invention is an injection, for example, by mixing an appropriate amount of the compound of the invention with an appropriate amount of 0.9% physiological saline and filling this mixture in vials for injection.

The components of this invention may be administered to mammals, including humans, either alone or, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice. The components can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.

Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular patient undergoing treatment. In some instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved. Further information about suitable dosages is provided below.

The term "administration" and variants thereof (e.g., "administering" a compound) in reference to a component of the invention means introducing the component or a prodrug of the component into the system of the animal in need of treatment. When a component of the invention or prodrug thereof is provided in combination with one or more other active agents (e.g., the WEEl inhibitor), "administration" and its variants are each understood to include concurrent and sequential introduction of the component or prodrug thereof and other agents.

As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

The term "therapeutically effective amount" as used herein means that amount of active compound or pharmaceutical agent that elicits a biological or medicinal response in a tissue, system, animal or human, that is being sought by a researcher, veterinarian, medical doctor or other clinician. This includes combination therapy involving the use of multiple therapeutic agents, such as a combined amount of a first and second treatment where the combined amount will achieve the desired biological response. The desired biological response is partial or total inhibition, delay or prevention of the progression of cancer including cancer metastasis; inhibition, delay or prevention of the recurrence of cancer including cancer metastasis; or the prevention of the onset or development of cancer (chemoprevention) in a mammal, for example a human.

A suitable amount of a WEEl inhibitor is administered to a patient undergoing treatment for cancer. In an embodiment, a WEEl inhibitor is administered in doses ranging from about 100 mg per day to 250 mg per day. In an embodiment of the invention, a WEEl inhibitor is administered twice daily (BID), over the course of two and a half days, for a total of 5 doses. In another embodiment of the invention, a WEEl inhibitor is administered once daily (QD) over the course of two days, for a total of 2 doses.

In an embodiment of the invention, a WEEl inhibitor can be administered 5 times per week. In another embodiment of the invention, a WEEl inhibitor can be administered 2 times per week.

A suitable amount of a CHK1 inhibitor is administered to a patient undergoing treatment for cancer. In an embodiment, a CHK1 inhibitor is administered in doses that range from about 100 mg per day to 200 mg per day. In an embodiment of the invention, a CHK1 inhibitor may be dosed once daily (QD) over either one or two days. In an embodiment of the invention, a CHK1 inhibitor can be administered once a week. In another embodiment of the invention, a WEEl inhibitor can be administered 2 times per week.

In an embodiment of the invention, a CHK1 inhibitor can be administered once a week. In another embodiment of the invention, a WEEl inhibitor can be administered 5 times per week.

In an embodiment of the invention, a CHK1 inhibitor can be administered twice a week. In another embodiment of the invention, a WEEl inhibitor can be administered 2 times per week.

In an embodiment of the invention, a CHK1 inhibitor can be administered twice a week. In another embodiment of the invention, a WEEl inhibitor can be administered 5 times per week.

In a broad embodiment, the treatment of the present invention involves the combined administration of a WEEl inhibitor and a CHK1 inhibitor. The combined

administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.

Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992). The WEEl inhibitor may precede, or follow administration of the CHK1 inhibitor or may be given simultaneously therewith. The clinical dosing of the therapeutic combination of the present invention is likely to be limited by the extent of any adverse reactions.

Additional indications

In addition to the treatment of neuroblastoma, the WEEl inhibitor and CHK1 inhibitor combination may also be useful for the treatment of the following cancers: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma,

rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal

adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colon, colorectal, rectal; Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate

(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma,

hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxo fibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges

(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,

schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli- Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplasia syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; and Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma. Thus, the term "cancerous cell" as provided herein, includes a cell afflicted by any one of the above-identified conditions.

The WEE1 inhibitor and CHK1 inhibitor combination of the invention may also be useful in treating the following disease states: keloids and psoriasis.

Further included within the scope of the invention is a method of treating or preventing a disease in which angiogenesis is implicated, which is comprised of administering to a mammal in need of such treatment a therapeutically effective amount of the combination of the present invention. Ocular neovascular diseases are an example of conditions where much of the resulting tissue damage can be attributed to aberrant infiltration of blood vessels in the eye (see WO 2000/30651, published 2 June 2000). The undesirable infiltration can be triggered by ischemic retinopathy, such as that resulting from diabetic retinopathy, retinopathy of prematurity, retinal vein occlusions, etc., or by degenerative diseases, such as the choroidal

neovascularization observed in age-related macular degeneration. Inhibiting the growth of blood vessels by administration of the present compounds would therefore prevent the infiltration of blood vessels and prevent or treat diseases where angiogenesis is implicated, such as ocular diseases like retinal vascularization, diabetic retinopathy, age-related macular degeneration, and the like.

Further included within the scope of the invention is a method of treating or preventing a non-malignant disease in which angiogenesis is implicated, including but not limited to: ocular diseases (such as, retinal vascularization, diabetic retinopathy and age-related macular degeneration), atherosclerosis, arthritis, psoriasis, obesity and Alzheimer's disease (Dredge, et al, Expert Opin. Biol. Ther., 2002, 2(8):953-966). In another embodiment, a method of treating or preventing a disease in which angiogenesis is implicated includes: ocular diseases (such as, retinal vascularization, diabetic retinopathy and age-related macular degeneration), atherosclerosis, arthritis and psoriasis.

Further included within the scope of the invention is a method of treating hyperproliferative disorders, such as, restenosis, inflammation, autoimmune diseases, and allergy/ asthma.

Further included within the scope of the invention is the use of the instant combination to coat stents and, therefore, the use of the instant compounds on coated stents for the treatment and/or prevention of restenosis (WO 2003/032809).

Further included within the scope of the invention is the use of the instant combination for the treatment and/or prevention of osteoarthritis (WO 2003/035048).

Further included within the scope of the invention is a method of treating hypoinsulinism.

Exemplifying the invention is the use of the WEE1 inhibitor and CHK1 inhibitor combination described above in the preparation of a medicament for the treatment of neuroblastoma.

Additional anti-cancer agents

The WEE1 inhibitor and CHK1 inhibitor combination of the instant invention is also useful in combination with additional therapeutic, chemotherapeutic and anti-cancer agents. Further combination with the WEE1 inhibitor and CHK1 inhibitor combination of the instant invention with therapeutic, chemotherapeutic and anti-cancer agents are within the scope of the invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology by V.T. Devita and S. Hellman (editors), 6^th edition (February 15, 2001), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved. Such additional agents include the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors and other angiogenesis inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, inhibitors of cell proliferation and survival signaling, bisphosphonates, aromatase inhibitors, siR A therapeutics, γ-secretase inhibitors, agents that interfere with receptor tyrosine kinases (RTKs) and agents that interfere with cell cycle checkpoints. The mTOR inhibitor and ανβ3 integrin antagonist combination of the instant invention may be particularly useful when co-administered with radiation therapy.

"Estrogen receptor modulators" refers to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of mechanism. Examples of estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381,

LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-l-oxopropoxy-4-methyl-2-[4-[2-(l- piperidinyl)ethoxy]phenyl]-2H-l-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate, 4,4'- dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

"Androgen receptor modulators" refers to compounds which interfere or inhibit the binding of androgens to the receptor, regardless of mechanism. Examples of androgen receptor modulators include finasteride and other 5a-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.

"Retinoid receptor modulators" refers to compounds which interfere or inhibit the binding of retinoids to the receptor, regardless of mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, a- difluoromethylornithine, ILX23-7553, trans-N-(4'-hydroxyphenyl) retinamide, and N-4- carboxyphenyl retinamide.

"Cytotoxic/cytostatic agents" refer to compounds which cause cell death or inhibit cell proliferation primarily by interfering directly with the cell's functioning or inhibit or interfere with cell myosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors/microtubule-stabilizing agents, inhibitors of mitotic kinesins, histone deacetylase inhibitors, inhibitors of kinases involved in mitotic progression, inhibitors of kinases involved in growth factor and cytokine signal transduction pathways, antimetabolites, biological response modifiers, hormonal/anti-hormonal therapeutic agents, haematopoietic growth factors, monoclonal antibody targeted therapeutic agents, topoisomerase inhibitors, proteosome inhibitors, ubiquitin ligase inhibitors, and aurora kinase inhibitors.

Examples of cytotoxic/cytostatic agents include, but are not limited to, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide, cis-aminedichloro(2- methyl-pyridine)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans, trans)-bis-mu- (hexane-l,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum (II)]tetrachloride, diarizidinylspermine, arsenic trioxide, l-(l l-dodecylamino-10-hydroxyundecyl)-3,7- dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston, 3 '-deamino-3 '-morpholino-13-deoxo-10- hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN10755, 4-demethoxy-3-deamino- 3-aziridinyl-4-methylsulphonyl-daunorubicin (see WO 00/50032), Raf kinase inhibitors (such as Bay43-9006) and mTOR inhibitors, such as ridaforolimus, everolimus, temsirolimus, sirolimus or a rapamycin-analog.

An example of a hypoxia activated compound is tirapazamine.

Examples of proteosome inhibitors include but are not limited to lactacystin and MLN-341 (Velcade).

Examples of microtubule inhibitors/microtubule-stabilizing agents include paclitaxel, vindesine sulfate, 3 ',4'-didehydro-4'-deoxy-8'-norvincaleukoblastine, docetaxol, rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881, BMS-184476, vinflunine, cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L- proline-t-butylamide, TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and 6,288,237) and BMS-188797. In an embodiment the epothilones are not included in the microtubule inhibitors/microtubule-stabilising agents.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3',4'-0-exo-benzylidene-chartreusin, 9-methoxy-N,N- dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H) propanamine, l-amino-9-ethyl-5-fluoro-2,3- dihydro-9-hydroxy-4-methyl- 1 H, 12H-benzo [de]pyrano [3 ' ,4 ' :b,7] -indolizino[ 1 ,2b]quinoline- 10, 13(9H, 15H)dione, lurtotecan, 7-[2-( -isopropylamino)ethyl]-(20S)camptothecin, BNP1350, BNPI1 100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2'- dimethylamino-2'-deoxy-etoposide, GL331, N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6- dimethyl-6H-pyrido[4,3-b]carbazole-l-carboxamide, asulacrine, (5a, 5aB, 8aa,9b)-9-[2-[N-[2- (dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydro0xy-3,5-dimethoxyphenyl]- 5,5a,6,8,8a,9-hexohydrofuro(3',4' :6,7)naphtho(2,3-d)-l,3-dioxol-6-one, 2,3-(methylenedioxy)-5- methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium, 6,9-bis[(2- aminoethyl)amino]benzo[g]isoguinoline-5, 10-dione, 5-(3-aminopropylamino)-7,10-dihydroxy-2- (2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5, l-de]acridin-6-one, N-[l- [2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide, N-(2- (dimethylamino)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy- 7H-indeno[2, l-c] quinolin-7-one, and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the human mitotic kinesin KSP, are described in Publications WO 2003/039460, WO 2003/050064, WO

2003/050122, WO 2003/049527, WO 2003/049679, WO 2003/049678, WO 2004/039774, WO 2003/079973, WO 2003/09921 1, WO 2003/105855, WO 2003/106417, WO 2004/037171, WO 2004/058148, WO 2004/058700, WO 2004/126699, WO 2005/018638, WO 2005/019206, WO 2005/019205, WO 2005/018547, WO 2005/017190, US 2005/0176776. In an embodiment inhibitors of mitotic kinesins include, but are not limited to, inhibitors of KSP, inhibitors of MKLP 1, inhibitors of CENP-E, inhibitors of MCAK, and inhibitors of Rab6-KIFL.

Examples of "histone deacetylase inhibitors" include, but are not limited to,

SAHA, TSA, oxamflatin, PXD101, MG98 and scriptaid. Further reference to other histone deacetylase inhibitors may be found in the following manuscript; Miller, T.A., et ah, J. Med. Chem., 2003, 46(24):5097-5116.

"Inhibitors of kinases involved in mitotic progression" include, but are not limited to, inhibitors of aurora kinase, inhibitors of Polo-like kinases (PLK; in particular inhibitors of PLK-1), inhibitors of bub-1 and inhibitors of bub-Rl. An example of an "aurora kinase inhibitor" is VX-680.

"Antiproliferative agents" includes antisense R A and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and ΓΝΧ3001, and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-methylidenecytidine, 2'- fluoromethylene-2 ' -deoxycytidine, N- [5 -(2,3 -dihydro-benzofuryl)sulfonyl] - ' -(3 ,4- dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero- B-L-manno-heptopyranosyl]adenine, aplidine, ecteinascidin, troxacitabine, 4-[2-amino-4-oxo- 4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][l,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-flurouracil, alanosine, l l-acetyl-8-(carbamoyloxymethyl)-4-formyl-6- methoxy-14-oxa-l, l l-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-yl acetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase, 2'-cyano-2'-deoxy-N4-palmitoyl-l-B-D- arabino furanosyl cytosine, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, and trastuzumab.

Examples of monoclonal antibody targeted therapeutic agents include those therapeutic agents which have cytotoxic agents or radioisotopes attached to a cancer cell specific or target cell specific monoclonal antibody. Examples include Bexxar.

"HMG-CoA reductase inhibitors" refers to inhibitors of 3-hydroxy-3- methylglutaryl-CoA reductase. Examples of HMG-CoA reductase inhibitors that may be used include, but are not limited to, lovastatin (MEVACOR®; see U.S. Patent Nos. 4,231,938, 4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Patent Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; see U.S. Patent Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL®; see U.S. Patent Nos. 5,354,772, 4,911,165, 4,929,437, 5,189, 164, 5, 1 18,853, 5,290,946 and 5,356,896), atorvastatin (LIPITOR®; see U.S. Patent Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952) and cerivastatin (also known as rivastatin and BAYCHOL®; see US Patent No. 5, 177,080). The structural formulas of these and additional HMG-CoA reductase inhibitors that may be used in the instant methods are described at page 87 of M. Yalpani, Cholesterol Lowering Drugs, Chemistry & Industry, 1996, pp. 85-89, and US Patent Nos. 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefor the use of such salts, esters, open-acid and lactone forms is included within the scope of this invention.

"Prenyl-protein transferase inhibitor" refers to a compound which inhibits any one or any combination of the prenyl-protein transferase enzymes, including farnesyl-protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I), and

geranylgeranyl-protein transferase type-II (GGPTase-II, also called Rab GGPTase).

Examples of prenyl-protein transferase inhibitors can be found in the following publications and patents: WO 96/30343, WO 97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/291 19, WO 95/32987, U.S. Patent No. 5,420,245, U.S. Patent No. 5,523,430, U.S. Patent No. 5,532,359, U.S. Patent No. 5,510,510, U.S. Patent No. 5,589,485, U.S. Patent No. 5,602,098, European Patent Publ. 0 618 221, European Patent Publ. 0 675 1 12, European Patent Publ. 0 604 181, European Patent Publ. 0 696 593, WO 94/19357, WO

95/08542, WO 95/1 1917, WO 95/12612, WO 95/12572, WO 95/10514, U.S. Patent No.

5,661, 152, WO 95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO 96/00736, U.S. Patent No. 5,571,792, WO 96/17861, WO 96/33159, WO 96/34850, WO 96/34851, WO

96/30017, WO 96/30018, WO 96/30362, WO 96/30363, WO 96/31 11 1, WO 96/31477,

WO 96/31478, WO 96/31501, WO 97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO 98/02436, and U.S. Patent No. 5,532,359. For an example of the role of a prenyl-protein transferase inhibitor on angiogenesis, see, European J. of Cancer, 1999, 35(9): 1394-1401.

"Angiogenesis inhibitors" refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism. Examples of angiogenesis inhibitors include, but are not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-l/KDR (VEGFR2), inhibitors of epidermal-derived, fibrob last-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, interferon-a, interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors, including nonsteroidal anti-inflammatories (NSAIDs), like aspirin and ibuprofen, as well as selective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib (PNAS, 1992, 89:7384; JNCI. 1982, 69:475; Arch. Opthalmol, 1990, 108:573; Anal Rec. 1994, 238:68; FEBS Letters. 1995, 372:83: Clin. Oithop.. 1995. 313 :76: J. Mol. Endocrinol. 1996, 16:07; Jpn. J. Pharmacol. 1997, 75: 105; Cancer Res.. 1997, 57: 1625; Cell, 1998, 93 :705; Intl. J. Mol Med.. 1998, 2:715; J. Biol Chem., 1999. 274:91 16), steroidal anti-inflammatories (such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, 6-0-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin- 1, angiotensin II antagonists (see, Fernandez, et al, J. Lab. Clin. Med.. 1985, 105: 141-145), and antibodies to VEGF (see, Nature Biotechnology. 1999, 17:963-968); Kim, et al, Nature. 1993, 362:841-844; WO 2000/44777; and WO 2000/61186).

Other therapeutic agents that modulate or inhibit angiogenesis and may also be used in combination with the compounds of the instant invention, include agents that modulate or inhibit the coagulation and fibrinolysis systems (see, review in Clin. Chem. La. Med.. 2000, 38:679-692). Examples of such agents that modulate or inhibit the coagulation and fibrinolysis pathways include, but are not limited to, heparin (see, Thromb. Haemost, 1998, 80: 10-23), low molecular weight heparins and carboxypeptidase U inhibitors (also known as, inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIa]) (see, Thrombosis Res.. 2001, 101 :329-354). TAFIa inhibitors have been described in PCT International Publication WO 2003/013526.

"Agents that interfere with cell cycle checkpoints" refer to compounds that inhibit protein kinases that transduce cell cycle checkpoint signals, thereby sensitizing the cancer cell to DNA damaging agents. Such agents include inhibitors of ATR, ATM, the CHK11 and CHK12 kinases and cdk and cdc kinase inhibitors and are specifically exemplified by 7-hydroxy- staurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

"Agents that interfere with receptor tyrosine kinases (RTKs)" refer to compounds that inhibit RTKs and therefore mechanisms involved in oncogenesis and tumor progression. Such agents include inhibitors of c-Kit, Eph, PDGF, Flt3 and c-Met. Further agents include inhibitors of RTKs as described by Bume- Jensen and Hunter, Nature. 2001, 41 1 :355-365.

"Inhibitors of cell proliferation and survival signaling pathway" refer to compounds that inhibit signal transduction cascades downstream of cell surface receptors. Such agents include inhibitors of serine/threonine kinases (including but not limited to inhibitors of Akt such as described in WO 02/083064, WO 02/083139, WO 02/083140, US 2004-0116432, WO 02/083138, US 2004-0102360, WO 03/086404, WO 03/086279, WO 03/086394, WO 03/084473, WO 03/086403, WO 2004/041162, WO 2004/096131, WO 2004/096129, WO 2004/096135, WO 2004/096130, WO 2005/100356, WO 2005/100344, US 2005/029941, US 2005/44294, US 2005/43361, WO 2006/135627, WO 2006/091395, WO 2006/110638), inhibitors of Raf kinase (for example BAY-43-9006 ), inhibitors of MEK (for example CI- 1040 and PD-098059), inhibitors of mTOR (for example Wyeth CCI-779), and inhibitors of PI3K (for example LY294002).

Specific anti-IGF-lR antibodies include, but are not limited to, dalotuzumab, figitumumab, cixutumumab, SHC 717454, Roche R1507, EM164 or Amgen AMG479.

As described above, the combinations with NSAID's are directed to the use of NSAID's which are potent COX -2 inhibiting agents. For purposes of this specification an NSAID is potent if it possesses an IC50 for the inhibition of COX-2 of ΙμΜ or less as measured by cell or microsomal assays.

The invention also encompasses combinations with NSAID's which are selective COX-2 inhibitors. For purposes of this specification NSAID's which are selective inhibitors of COX-2 are defined as those which possess a specificity for inhibiting COX-2 over COX-1 of at least 100 fold as measured by the ratio of IC50 for COX-2 over IC50 for COX-1 evaluated by cell or microsomal assays. Such compounds include, but are not limited to, those disclosed in U.S. Patent 5,474,995, U.S. Patent 5,861,419, U.S. Patent 6,001,843, U.S. Patent 6,020,343, U.S. Patent 5,409,944, U.S. Patent 5,436,265, U.S. Patent 5,536,752, U.S. Patent 5,550,142, U.S. Patent 5,604,260, U.S. 5,698,584, U.S. Patent 5,710, 140, WO 94/15932, U.S. Patent 5,344,991, U.S. Patent 5, 134, 142, U.S. Patent 5,380,738, U.S. Patent 5,393,790, U.S. Patent 5,466,823,U.S. Patent 5,633,272,and U.S. Patent 5,932,598, all of which are hereby incorporated by reference.

Inhibitors of COX-2 that are particularly useful in the instant method of treatment are: 3 -phenyl -4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and 5-chloro-3-(4-methylsulfonyl) phenyl-2-(2-methyl-5-pyridinyl)pyridine, or a pharmaceutically acceptable salt thereof.

Compounds that have been described as specific inhibitors of COX-2 and are therefore useful in the present invention include, but are not limited to, the following: parecoxib, BEXTRA® and CELEBREX® or a pharmaceutically acceptable salt thereof.

Other examples of angiogenesis inhibitors include, but are not limited to, endostatin, ukrain, ranpirnase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]- l-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino-l-[[3,5-dichloro-4- (4-chlorobenzoyl)phenyl] methyl] - 1 Η- 1 ,2,3 -triazole-4-carboxamide,CM 101, squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl- bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(l,3- naphthalene disulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (SU5416).

As used above, "integrin blockers" refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the α_νβ3 integrin, to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the ανβ5 integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the α_νβ3 integrin and the α_νβ5 integrin, and to compounds which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells. The term also refers to antagonists of the α_νβ6, «νβ8_> «ΐβΐ, «2βΐ, α5βΐ, «6βΐ, and α6β4 integrins. The term also refers to antagonists of any combination of α_νβ3, α_νβ5,α_νβ6, α_νβ8, αΐβΐ, «2βΐ, α5βΐ, «6βΐ, ^and α6β4 integrins.

Some specific examples of tyrosine kinase inhibitors include N-

(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide, 3-[(2,4-dimethylpyrrol-5- yl)methylidenyl)indolin-2-one, 17-(allylamino)-17-demethoxygeldanamycin, 4-(3-chloro-4- fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline, N-(3- ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine, BIBX1382, 2,3,9, 10, 11, 12- hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-lH-diindolo[l,2,3-fg:3',2', - kl]pyrrolo[3,4-i][l,6]benzodiazocin-l-one, SH268, genistein, STI571, CEP2563, 4-(3- chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethane sulfonate, 4-(3-bromo- 4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 4-(4' -hydroxyphenyl)amino-6,7- dimethoxyquinazoline, SU6668, STI571A, N-4-chlorophenyl-4-(4-pyridylmethyl)-l- phthalazinamine, and EMD121974.

Combinations with compounds other than anti-cancer compounds are also encompassed in the instant methods. For example, combinations of the mTOR inhibitor and ανβ3 integrin antagonist combination of the instant invention with PPAR-γ (i.e., PPAR-gamma) agonists and PPAR-δ (i.e., PPAR-delta) agonists are useful in the treatment of certain malingnancies. PPAR-γ and PPAR-δ are the nuclear peroxisome proliferator-activated receptors γ and δ. The expression of PPAR-γ on endothelial cells and its involvement in angiogenesis has been reported in the literature (see, J. Cardiovasc. Pharmacol. 1998, 31 :909-913; J. Biol Chem.. 1999. 274:9116-9121; Invest. Ophthalmol Vis. ScL. 2000. 41 :2309-2317). More recently, PPAR-γ agonists have been shown to inhibit the angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibit the development of retinal neovascularization in mice (Arch. Ophthamol. 2001; 1 19:709-717). Examples of PPAR-γ agonists and PPAR- γ/α agonists include, but are not limited to, thiazolidinediones (such as DRF2725, CS-01 1, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544, 2344, KRP297, NP0110, DRF4158, 622, GI262570, PNU182716, DRF552926, 2-[(5,7-dipropyl-3- trifluoromethyl-l,2-benzisoxazol-6-yl)oxy]-2-methylpropionic acid (disclosed in USSN

09/782,856), and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy) phenoxy)propoxy)-2-ethylchromane- 2-carboxylic acid (disclosed in USSN 60/235,708 and 60/244,697).

Another embodiment of the instant invention is the use of the presently disclosed compounds in combination with gene therapy for the treatment of cancer. For an overview of genetic strategies to treat cancer, see, Hall, et al, Am. J. Hum. Genet.. 1997, 61 :785-789 and Kufe, et ah, Cancer Medicine. 5th Ed, , B.C. Decker, Hamilton, 2000, pp 876-889. Gene therapy can be used to deliver any tumor suppressing gene. Examples of such genes include, but are not limited to, p53, which can be delivered via recombinant virus-mediated gene transfer (see, U.S. Patent No. 6.069.134). a uPA/uPAR antagonist (Gene Therapy. 1998, 5(8): 1 105- 13), and interferon gamma (J. Immunol. 2000, 164:217-222).

The compounds of the instant invention may also be administered in combination with an inhibitor of inherent multidrug resistance (MDR), in particular MDR associated with high levels of expression of transporter proteins. Such MDR inhibitors include inhibitors of p- glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar). A compound of the present invention may be employed in conjunction with antiemetic agents to treat nausea or emesis, including acute, delayed, late-phase, and anticipatory emesis, which may result from the use of a compound of the present invention, alone or with radiation therapy. For the prevention or treatment of emesis, a compound of the present invention may be used in conjunction with other anti-emetic agents, especially neurokinin- 1 receptor antagonists, 5HT3 receptor antagonists, such as ondansetron, granisetron, tropisetron, and zatisetron, GABAB receptor agonists, such as baclofen, a corticosteroid such as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten or others such as disclosed in U.S. Patent Nos. 2,789, 118, 2,990,401, 3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, an antidopaminergic, such as, the phenothiazines (for example, prochlorperazine, fluphenazine, thioridazine and mesoridazine), metoclopramide or dronabinol. In another embodiment, conjunctive therapy with an anti-emesis agent selected from a neurokinin- 1 receptor antagonist, a 5HT3 receptor antagonist and a corticosteroid is disclosed for the treatment or prevention of emesis that may result upon administration of the instant compounds.

Neurokinin- 1 receptor antagonists of use in conjunction with the compounds of the present invention are fully described, for example, in U.S. Patent Nos. 5, 162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595, 5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719, 147;

European Patent Publication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430 771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0 512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517 589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0 545 478, 0 558 156, 0 577 394, 0 585 913,0 590 152, 0 599 538, 0 610 793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0 707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733 632 and 0 776 893; PCT International Patent Publication Nos. WO 90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151, 92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330, 93/00331, 93/01 159, 93/01 165, 93/01169, 93/01170, 93/06099, 93/091 16, 93/10073, 93/14084, 93/141 13, 93/18023, 93/19064, 93/21155, 93/21181, 93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429, 94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165, 94/10167, 94/10168, 94/10170, 94/1 1368, 94/13639, 94/13663, 94/14767, 94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309, 95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549, 95/1 1880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129, 95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418, 95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094, 96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304, 96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553, 97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084, 97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529, 2 268 931, 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293 169, and 2 302 689. The preparation of such compounds is fully described in the aforementioned patents and publications, which are incorporated herein by reference.

In an embodiment, the neurokinin- 1 receptor antagonist for use in conjunction with the compounds of the present invention is selected from: 2-(R)-(l-(R)-(3,5-bis

(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-lH,4H-l,2,4-triazolo) methyl)morpholine, or a pharmaceutically acceptable salt thereof, which is described in U.S. Patent No. 5,719, 147.

The WEE1 inhibitor and CHK1 inhibitor combination of the instant invention may also be administered with an agent useful in the treatment of anemia. Such an anemia treatment agent is, for example, a continuous erythropoiesis receptor activator (such as, Epoetin alfa).

The WEE1 inhibitor and CHK1 inhibitor combination of the instant invention may also be administered with an agent useful in the treatment of neutropenia. Such a neutropenia treatment agent is, for example, a hematopoietic growth factor which regulates the production and function of neutrophils such as a human granulocyte colony stimulating factor, (G-CSF). Examples of a G-CSF include filgrastim.

The WEE1 inhibitor and CHK1 inhibitor combination of the instant invention may also be administered with an immunologic-enhancing drug, such as levamisole, isoprinosine and Zadaxin.

The WEE1 inhibitor and CHK1 inhibitor combination of the instant invention may also be useful for treating or preventing cancer, including bone cancer, in combination with bisphosphonates (understood to include bisphosphonates, diphosphonates, bisphosphonic acids and diphosphonic acids). Examples of bisphosphonates include but are not limited to: etidronate (Didronel), pamidronate (Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate (Zometa), ibandronate (Boniva), incadronate or cimadronate, clodronate, EB-1053, minodronate, neridronate, piridronate and tiludronate including any and all pharmaceutically acceptable salts, derivatives, hydrates and mixtures thereof.

The WEE1 inhibitor and CHK1 inhibitor combination of the instant invention may also be useful for treating or preventing breast cancer in combination with aromatase inhibitors. Examples of aromatase inhibitors include but are not limited to: anastrozole, letrozole and exemestane.

The WEE1 inhibitor and CHK1 inhibitor combination of the instant invention may also be useful for treating or preventing cancer in combination with siRNA therapeutics.

The WEE1 inhibitor and CHK1 inhibitor combination of the instant invention may also be administered in combination with γ-secretase inhibitors and/or inhibitors of NOTCH signaling. Such inhibitors include compounds described in WO 01/90084, WO 02/30912, WO 01/70677, WO 03/013506, WO 02/36555, WO 03/093252, WO 03/093264, WO 03/093251, WO 03/093253, WO 2004/039800, WO 2004/039370, WO 2005/030731, WO 2005/014553, USSN 10/957,251, WO 2004/089911, WO 02/081435, WO 02/081433, WO 03/018543, WO

2004/031 137, WO 2004/031 139, WO 2004/031 138, WO 2004/101538, WO 2004/101539 and WO 02/47671 (including LY-450139).

The WEE1 inhibitor and CHK1 inhibitor combination of the instant invention may also be useful for treating or preventing cancer in combination with inhibitors of Akt. Such inhibitors include compounds described in, but not limited to, the following publications: WO 02/083064, WO 02/083139, WO 02/083140, US 2004-0116432, WO 02/083138, US 2004- 0102360, WO 03/086404, WO 03/086279, WO 03/086394, WO 03/084473, WO 03/086403, WO 2004/041 162, WO 2004/096131, WO 2004/096129, WO 2004/096135, WO 2004/096130, WO 2005/100356, WO 2005/100344, US 2005/029941, US 2005/44294, US 2005/43361.WO 2006/135627, WO 2006091395, WO 2006/1 10638).

The WEE1 inhibitor and CHK1 inhibitor combination of the instant invention may also be useful for treating or preventing cancer in combination with PARP inhibitors.

Radiation therapy itself means an ordinary method in the field of treatment of cancer. For radiation therapy, employable are various radiations such as X-ray, γ-ray, neutron ray, electron beam, proton beam; and radiation sources. In a most popular radiation therapy, a linear accelerator is used for irradiation with external radiations, γ-ray.

The WEE1 inhibitor and CHK1 inhibitor combination of the instant invention may also be useful for treating cancer in further combination with the following therapeutic agents: abarelix (Plenaxis depot®); abiraterone acetate (Zytiga®); (Actiq®); aldesleukin (Prokine®); Aldesleukin (Proleukin®); Alemtuzumab (Campath®); alfuzosin HC1

(UroXatral®); alitretinoin (Panretin®); allopurinol (Zyloprim®); altretamine (Hexalen®); amifostine (Ethyol®); anastrozole (Arimidex®); (Anzemet®); (Anexsia®); aprepitant

(Emend®); arsenic trioxide (Trisenox®); asparaginase (Elspar®); azacitidine (Vidaza®);

bendamustine hydrochloride (Treanda®); bevacuzimab (Avastin®); bexarotene capsules

(Targretin®); bexarotene gel (Targretin®); bleomycin (Blenoxane®); bortezomib (Velcade®); (Brofenac®); busulfan intravenous (Busulflex®); busulfan oral (Myleran®); cabazitaxel (Jevtana®); calusterone (Methosarb®); capecitabine (Xeloda®); carboplatin (Paraplatin®); carmustine (BCNU®, BiCNU®); carmustine (Gliadel®); carmustine with Polifeprosan 20 Implant (Gliadel Wafer®); celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil

(Leukeran®); cinacalcet (Sensipar®); cisplatin (Platinol®); cladribine (Leustatin®, 2-CdA®); clofarabine (Clolar®); cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide (Cytoxan Injection®); cyclophosphamide (Cytoxan Tablet®); cytarabine (Cytosar-U®); cytarabine liposomal (DepoCyt®); dacarbazine (DTIC-Dome®); dactinomycin, actinomycin D

(Cosmegen®); Darbepoetin alfa (Aranesp®); dasatinib (Sprycel®); daunorubicin liposomal (DanuoXome®); daunorubicin, daunomycin (Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); decitabine (Dacogen®); degarelix (Degarelix®); Denileukin diftitox (Ontak®); denosumab (Xgeva®); dexrazoxane (Zinecard®); docetaxel (Taxotere®); doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®, Rubex®); doxorubicin (Adriamycin PFS

Injection®); doxorubicin liposomal (Doxil®); dromostanolone propionate (dromostanolone®); dromostanolone propionate (masterone injection®); Elliott's B Solution (Elliott's B Solution®); epirubicin (Ellence®); Epoetin alfa (epogen®); eribulin mesylate (Halaven®); erlotinib

(Tarceva®); estramustine (Emcyt®); etoposide phosphate (Etopophos®); etoposide, VP- 16

(Vepesid®); everolimus (Afinitor®); exemestane (Aromasin®); fentanyl buccal (Onsolis®); fentanyl citrate (Fentora®); fentanyl sublingual tablets (Abstral®); Filgrastim (Neupogen®); floxuridine (intraarterial) (FUDR®); fludarabine (Fludara®); fluorouracil, 5-FU (Adrucil®); flutamide (Eulexin®); fulvestrant (Faslodex®); gefitinib (Iressa®); gemcitabine (Gemzar®); gemtuzumab ozogamicin (Mylotarg®); goserelin acetate (Zoladex Implant®); goserelin acetate

(Zoladex®); granisetron (Kytril Solution®) (Sancuso®); histrelin acetate (Histrelin implant®); human papillomavirus bivalent vaccine (Cervarix®); hydroxyurea (Hydrea®); Ibritumomab

Tiuxetan (Zevalin®); idarubicin (Idamycin®); ifosfamide (IFEX®); imatinib mesylate

(Gleevec®); interferon alfa 2a (Roferon A®); Interferon alfa-2b (Intron A®); ipilimumab (Yervoy®); irinotecan (Camptosar®); (Kadian®); ixabepilone (Ixempra®); lapatinib (Tykerb®); lenalidomide (Revlimid®); letrozole (Femara®); leucovorin (Wellcovorin®, Leucovorin®);

Leuprolide Acetate (Eligard®); (Lupron Depot®); (Viadur®); levamisole (Ergamisol®);

levoleucovorin (Fusilev®); lomustine, CCNU (CeeBU®); meclorethamine, nitrogen mustard

(Mustargen®); megestrol acetate (Megace®); melphalan, L-PAM (Alkeran®); mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®); mesna (Mesnex tabs®); methotrexate (Methotrexate®); methoxsalen (Uvadex®); mitomycin C (Mutamycin®); mitomycin C (Mitozytrex®); mitotane

(Lysodren®); mitoxantrone (Novantrone®); nandrolone phenpropionate (Durabolin-50®);

nelarabine (Arranon®); nilotinib hydrochloride monohydrate (Tasigna®); Nofetumomab

(Verluma®); ofatumumab (Arzerra®); ondansetron (Zuplenz®); Oprelvekin (Neumega®); (Neupogen®); oxaliplatin (Eloxatin®); paclitaxel (Paxene®); paclitaxel (Taxol®); paclitaxel protein-bound particles (Abraxane®); palifermin (Kepivance®); palonosetron (Aloxi®);

pamidronate (Aredia®); panitumumab (Vectibix®); pazopanib (Votrient®); pegademase

(Adagen (Pegademase Bovine)®); pegaspargase (Oncaspar®); Pegfilgrastim (Neulasta®);

peginterferon alfa-2B (Sylatron®); pemetrexed disodium (Alimta®); pentostatin (Nipent®); pipobroman (Vercyte®); plerixafor injection (Mozobil®); plicamycin, mithramycin

(Mithracin®); porfimer sodium (Photofrin®); pralatrexate injection (Folotyn®); procarbazine

(Matulane®); (Quadramet®); quadrivalent human papillomavirus (types 6, 1 1, 16, 18) recombinant vaccine (Gardasil®); quinacrine (Atabrine®); raloxifene hydrochloride (Evista®);

Rasburicase (Elitek®); Rituximab (Rituxan®); romidepsin (Istodax®); sargramostim

(Leukine®); Sar gramostim (Prokine®); secretin (SecreFlo®); sipuleucel-T (Provenge®);

sorafenib (Nexavar®); streptozocin (Zanosar®); sunitinib maleate (Sutent®); talc (Sclerosol®); tamoxifen (Nolvadex®); temozolomide (Temodar®); temsirolimus (Torisel®); teniposide, VM-

26 (Vumon®); (Temodar®); testolactone (Teslac®); thalidomide (Thalomid®); thioguanine, 6- TG (Thioguanine®); thiotepa (Thioplex®); topotecan (Hycamtin®); toremifene (Fareston®); Tositumomab (Bexxar®); Tositumomab/I-131 tositumomab (Bexxar®); Trastuzumab (Herceptin®); (Trelstar LA®); tretinoin, ATRA (Vesanoid®); triptorelin pamoate (Trelstar Depot®); (UltraJect®); Uracil Mustard (Uracil Mustard Capsules®); valrubicin (Valstar®); vandetanib (Vandetanib®); vinblastine (Velban®); vincristine (Oncovin®); vinorelbine (Navelbine®); vorinostat (Zolinza®); (Zofran ODT®); and zoledronate (Zometa®).

All patents, publications and pending patent applications identified are hereby incorporated by reference.

The abbreviations used herein have the following tabulated meanings.

Abbreviations not tabulated below have their meanings as commonly used unless specifically stated otherwise.

TFA Trifluoroacetic Acid

THF Tetrahydrofuran

The WEE1 and CHK1 inhibitors of the instant invention can be prepared according to the following examples, using appropriate materials. The specific anticancer agents illustrated in the examples are not, however, to be construed as forming the only genus that is considered as the invention. The illustrative Examples below, therefore, are not limited by the anticancer agents listed or by any particular substituents employed for illustrative purposes. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. All temperatures are degrees Celsius unless otherwise noted.

EXAMPLES

EXAMPLE 1

Preparation of WEE 1 - 1

Production of 2-allyl- 1 -\6-( 1 -hydroxy- 1 -methylethyl)pyridin-2-yl^"|-6- { [4-(4-methylpiperazin- 1 - vDphenyllamino} - 1 ,2-dihydro-3H-pyrazolor3 ,4-d1pyrimidin-3-one

Step 1) Production of 2-(6-bromo-2-pyridinyl)-2-propanol:

In a nitrogen atmosphere, 30 mL of 3 M methylmagnesium iodide/diethyl ether was added to 300 mL of diethyl ether solution of 8.72 g of methyl 6-bromopyridine-2- carboxylate. Water and 2 N hydrochloric acid were added to the reaction liquid, and extracted with ethyl acetate. This was washed with aqueous saturated sodium hydrogencarbonate solution and saturated saline water, and dried with anhydrous magnesium sulfate. The solvent was evaporated away under reduced pressure to obtain 8.51 g of crude 2-(6-bromo-2-pyridinyl)-2- propanol as a yellow oily substance. iH-NMR (400 MHz, CDC13) δ: 7.56 (1H, t, J=7.8 Hz), 7.38 (1H, dd, J=7.8, 1.0 Hz), 7.36 (1H, dd, J=7.8, 1.0 Hz), 1.55(6H, s). ESI-MS Found:

m/z[M+H]+ 216, 218. Step 2) Production of 2-allyl-l-[6-(l-hydroxy-l-methylethyl)-2-pyridinyl]-6-

(methylthio)-l,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one:

12.89 g of the entitled compound was obtained in the same manner as in

Preparative Example 1-1, for which, however, the compound obtained in the above reaction was used in place of 2-iodopyridine used in Preparative Example 1-1. iH-NMR (400 MHz, CDCI3) δ: 8.95 (IH, s), 7.91 (IH, t, J=8.0 Hz), 7.76 (IH, d, J=7.3 Hz), 7.40 (IH, dd, J=7.8, 1.0 Hz), 5.70 (IH, ddt, J=17.1, 10.2, 6.3 Hz), 5.06 (IH, dd, J=10.2, 1.0 Hz), 4.93 (IH, dd, J=17.1, 1.2 Hz), 4.81 (2H, d, J=6.3 Hz), 2.59 (4H, s), 1.59 (6H, s). ESI-MS Found: m/z[M+H]+ :358. Step 3) Production of 2-allyl-l-[6-(l -hydroxy- l-methylethyl)pyridin-2-yl]-6- {[4-(4- methylpiperazin- 1 -yl)phenyl] amino} - 1 ,2-dihydro-3H-pyrazolo[3 ,4-d]pyrimidin- 3 -one:

817 mg of m-chloroperbenzoic acid (> 65%) was added to toluene (20 mL) solution of 1.10 g of the above produce, and stirred fro 20 minutes. 1.61 mL of N,N- diisopropylethylamine and 706 mg of 4-(4-methylpiperazin-l-yl)aniline were added to the reaction liquid, and stirred overnight. Aqueous saturated sodium hydrogencarbonate solution was added to the reaction liquid, extracted with ethyl acetate, washed with saturated saline water, and dried with anhydrous magnesium sulfate. The solvent was evaporated away, and the residue was purified through basic silica gel column chromatography (hexane/ethyl acetate = 1/1 to 0/1, ethyl acetate/ethanol = 98/2). After concentrated, this was recrystallized from ethyl acetate to obtain 1.20 g of the entitled compound as a yellow solid. iH-NMR (400 MHz, CDCI3) δ: 8.83

(IH, s), 7.86 (IH, dd, J=8.0, 7.8 Hz), 7.75 (IH, d, J=7.3 Hz), 7.49 (IH, brs), 7.48 (2H, d, J=9.0 Hz), 7.34 (IH, d, J=7.4 Hz), 6.93 (2H, d, J=9.0 Hz), 5.70 (IH, ddt, J=17.2, 10.0, 6.5 Hz), 5.04 (IH, d, J=10.0 Hz), 4.94 (IH, d, J=17.2 Hz), 4.74 (2H, d, J=6.5 Hz), 3.26 (4H, t, J=4.8 Hz), 2.73 (4H, brs), 2.44 (3H, s), 1.59 (6H, s). ESI-MS Found: m/z[M+H]+ 501.

Preparative Example 1-1

Production of 2-allyl-6-(methylthio)-l-pyridin-2-yl-3H-pyrazolo[3,4-d]pyrimidin-3-one:

2.4 mL of Ν,Ν'-dimethylethylenediamine was added to 1,4-dioxane (50 mL) solution of 4.44 g of 2-allyl-6-(methylthio)-l,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one, 3.80 g of copper(I) iodide, 5.33 g of 2-iodopyridine and 3.80 g of potassium carbonate, and stirred overnight at 95°C. The reaction liquid was cooled, aqueous ammonia was added thereto and extracted with ethyl acetate, washed with saturated saline water and dried with anhydrous magnesium sulfate. The solvent was evaporated away under reduced pressure, and crystallized with ethyl acetate to obtain 5.15 g of the entitled compound as a white solid. lH-NMR (400

MHz, CDCI3) δ: 8.94 (IH, s), 8.52 (IH, d, J=5.1 Hz), 7.90 (2H, d, J=3.5 Hz), 7.29-7.25 (IH, m),

5.68 (IH, ddt, J=17.0, 10.2, 6.3 Hz), 5.05 (IH, d, J=10.2 Hz), 4.91 (IH, d, J=17.0 Hz), 4.85 (IH, d, J=6.3 Hz), 2.58 (3H, s). EXAMPLE 2

Preparation of CHKl-1

Production of (R)-(-)-6-Bromo-3-(l -methyl- lH-pyrazol-4-yl)-5-piperidin-3-yl-pyrazolo[l .5- alpyrimidin-7-ylamine

Preparative Example 2- 1

Step 1) Method A: Phosphorus oxychloride (6.92 g, 45.1 mmol, 1.5 eq.) was cooled to

0°C and then added drop-wise to anhydrous DMF (3.50 mL, 45.2 mmol, 1.5 eq.) at 0°C. The colorless DMF solution soon becomes orange. The mixture was stirred for 1 hour at room temperature and then heated to 80°C. 1 -Methyl- IH-pyrazole (2.5 mL, 30.2 mmol) is then added drop-wise to the reaction, and the resulting mixture was stirred 3 hours at 95°C. The reaction was then quenched by slow addition to ice (40 g) via Pasteur pipette. The pH of the resulting solution was 2, and it was raised to 5 by slowly adding 12N aqueous sodium hydroxide solution (11.2 mL total). The resulting aqueous solution was extracted with dichloromethane (3 x 40 mL). At this point, the pH of the aqueous layer had dropped to 3, therefore additional 12 N NaOH solution (1 mL) was added to bring the pH to 6. The aqueous layer was then extracted further with ether (4 x 40 mL). The combined extracts were then dried over sodium sulfate, filtered and concentrated (at about 40-50°C). After drying for 30 minutes under vacuum, a brown oil was recovered (3.79 g) which NMR indicated consisted of a mixture of product, starting material and DMF (52 wt %, 22 wt %, and 26 wt %, respectively). The calculated yield of was 59% and the calculated yield for recovery of unreacted starting material, was 34%. This crude material may be used without further purification in the next step.

Method B: Phosphorus oxychloride (46.7 g, 304.51 mmol, 1.0 eq.) was added dropwise to a stirred solution of 1 -methyl- IH-pyrazole (25 g, 304.51 mmol) at 0°C in anhydrous DMF (62 mL, 800.69 mmol, 2.63 eq.). The solution was then heated to 100°C and stirred for 2.5 hours. After cooling, the reaction was quenched with ice-water (400 mL), basified with aqueous sodium hydroxide solution to pH 8, and extracted with dichloromethane (4 x 1L). The combined extracts were dried over sodium sulfate, filtered and concentrated to give a brown oil (32g). This was then partially purified by silica gel chromatography eluting with ether followed by 95% ether-ethyl acetate to yield a yellow oil (23 g) containing 7 wt% DMF. Step 2) Potassium ?-butoxide (23.47 g, 199.1 mmol, 2.44 eq.) was suspended in anhydrous DME (90 mL) and cooled to - 60°C. Tosyl methyl isocyanide (23.76 g, 121.7 mmol, 1.49 eq.) was dissolved in anhydrous DME (75 mL) and the solution was added drop-wise to the potassium ?-butoxide solution over 20 minutes. After stirring for 20 minutes between - 60 and - 55°C, the aldehyde from Step 1 in anhydrous DME (55 mL) was added over 23 minutes. The reaction was stirred for one hour at - 55 to - 50°C, and then methanol (90 mL) was added. The cooling bath was removed, and after stirring for 5 minutes in air, the reaction flask was immersed in an oil bath preheated to 85°C. The reaction was stirred for 1 hour. After cooling, the mixture was concentrated and the resulting tan solid was dissolved in water (180 mL) with acetic acid (9 mL). This was extracted with ethyl acetate (3 x 250 mL), and these extracts were combined, washed with brine (100 mL), dried over sodium sulfate, filtered and concentrated to yield a brown oil (13.71 g). This oil was dissolved in dichloromethane and purified by silica-gel chromatography using a gradient from 0% to 15% dichloromethane-acetone to yield a bright yellow oil in 63% yield (7.89 g).

Preparative Example 2-2

Step 1) The compound from Preparative Example 2-1 (8.00 g, 66.17 mmol) and ethyl formate (1 1.3 mL, 139.9 mmol, 2.11 eq.) were dissolved in anhydrous DME (35 mL) and added drop-wise to a suspension of potassium-?-butoxide (1 1.88g of 95%, 100.77 mmol, 1.52 equiv.) in anhydrous DME (85 mL) in an open pressure tube. After addition was complete, the tube was sealed and stirred at 85°C for 18 hours. After cooling, the resulting thick suspension was diluted with water (300 mL) to yield a solution of pH 8, and was extracted with ethyl acetate (3 x 300 mL). These extracts were discarded, and the aqueous solution was then acidified to pH 4 with 8N aqueous hydrochloric acid (2 mL) resulting in the formation of a white precipitate. This suspension was extracted with ethyl acetate (3 x 700 mL). The combined extracts were washed with brine, dried with sodium sulfate, filtered and concentrated to yield a yellow-white solid (8.98 g, 93% yield).

Step 2) The formyl acetonitrile from Step 1 (10.97 g, 73.63 mmol) was suspended in absolute ethanol (400 mL), and hydrazine monohydrochloride (10.67 g, 156 mmol, 2.12 equiv.) was then added. The mixture was stirred 15 hours at 90°C to yield an orange solution with a large amount of a fine yellow precipitate. After briefly allowing the reaction to cool, 7N ammonia/methanol (25 mL, 175 mmol) was added and the mixture was stirred for 20 minutes. The mixture was filtered to remove the precipitated solid. The filtrate solution was then concentrated to yield a yellow-white solid weighing 17.70 g. This solid was then loaded dry on a chromatography column and purified eluting with 10% methanol-dichloromethane (5 volumes) followed by 10% to 15% 7N-ammonia/methanol-dichloromethane (7 volumes) to yield an off- white solid (1 1.35 g, 95% yield). ^XH NMR (DMSO-d₆): δ 1 1.4 (s, 1H), 7.76 (s, 1H), 7.54 (s, 1H), 7.48 (s, 1H), 4.54 (s, 2H), 3.79 (s, 3H).

Preparative Example 2-3

Step 1) A solution of N-Boc-(R)-nipecotic acid (2.0 g, 8.72 mmol) in THF (26 mL) was treated with l,l '-carbonyldiimidazole (1.41 g, 1.0 equiv.). The solution was stirred at 25°C for 18 hours. Saturated NaCl (50 mL) was added. The aqueous layer was extracted with Et₂0 (3 x 25 mL). The Et₂0 layer was washed with a 5% aqueous NaHCC solution (50 mL) and saturated NaCl (50 mL). The organic layer was dried (MgS0₄), filtered and concentrated under reduced pressure to provide the product (2.2 g, 90.7%) as a white solid. Step 2) A solution of LiHMDS (15.8 mL of a 1.0M solution in THF, 2.0 equiv.) in THF

(24 mL) was cooled to -78°C and treated with CH₃CN (0.83 mL, 2.0 equiv.) dropwise. The solution was stirred at -7°C for 1 hour. To this solution was added the solution of acyl imidazole from Step 1 (2.2 g) in THF (24 mL) dropwise over 10 minutes. The solution was stirred at -78°C for 0.5 hours and quenched by the addition of saturated NH₄C1 (100 mL). The aqueous layer was extracted with Et₂0 (3 x 50 mL). The combined organic layer was dried (Na₂S0₄), filtered and concentrated under reduced pressure. Purification by an Analogix purification system using a RediSep 40g column (0-50% ethyl acetate-hexanes gradient) afforded the product (1.25 g, 63%) as a pale yellow oil. Preparative Example 2-4

A solution of the compound from Preparative Example 2-3 (0.23 g, 0.93 mmol) and the compound from Preparative Example 2-2 (0.12 g, 1.0 equiv.) in EtOH (0.37 mL) was heated at 45°C for 18 hours. The solution was cooled to 25°C. Preparative thin layer chromatography (20% acetone-CH₂Cl₂) afforded the product (0.26 g, 70 %) as a white solid.

Preparative Example 2-

To a solution of the compound from Preparative Example 2-4 (0.79g, 1.0 equiv.) in CH₃CN/CH₂C1₂ (10 mL, 1 : 1) was added a solution of NBS (0.34 mg, 0.95 equiv.) in CH₃CN (2 mL) over 10 minutes. When TLC analysis showed complete consumption of starting material, the mixture was concentrated under reduced pressure. The crude product was dissolved in CHCI₃ and washed sequentially with H₂0 (3 x 3 mL) and brine (1 x 3 mL). The organic layer was dried over Na₂S0₄, filtered and concentrated under reduced pressure to afford a light orange solid (0.92 g, 96% yield). LCMS: M⁺ = 476.

Preparative Example 2-

To a solution of the compound from Preparative Example 2-5 (l .Og, 1.0 equiv.) in CH₂C1₂ (20 mL) at 0°C was added TFA dropwise. The solution was stirred at 0°C for 30 minutes and warmed to room temperature for 15 minutes. The mixture was concentrated under reduced pressure adding several portions of CH₂C1₂ to azeotrope trace TFA. The resulting oil was dried under high vacuum for 1 hour and treated with 7M NH3 in MeOH (50 mL) and stirred for 4 hours at room temperature. The resulting solution was concentrated under reduced pressure and the crude product purified by Analogix BSR pump using a 40g Isco column on 35% speed using a gradient of 20: 1 CH₂Cl₂/MeOH to 40: 1 CH₂C1₂/7M NH₃ in MeOH to 20: 1 CH₂C1₂/7M NH₃ in MeOH to afford pure product. LCMS: M⁺ = 376 EXAMPLE 3

Preparation of WEE 1-2

Production of 3-(2,6-dichlorophenyl -4-imino-7-r(2'-methyl-2',3'-dihvdro-rH-spiro

[cyclopropane-l ^'-isoquinolinl-7'-yl)aminol-3^

A 1-butanol solution of 1.5 g of 7-chloro-3-(2,6-dichlorophenyl)-4-imino- dihydropyrimido[4,5-d]pyrimidin-2(lH)-one obtained in Preparative Example 3-1, 1 g of 2'- methyl-2',3'-dihydro-rH-spiro[cyclopropane-l,4'-isoquinolin]-7'-amine obtained in Preparative Example 3-2, and 0.83 g of p-toluene sulfonic acid monohydrate was stirred at 90°C for 15 minutes. The reaction liquid was cooled, diluted with chloroform, and the organic layer was washed with aqueous saturated sodium bicarbonate solution and then saturated saline water, and dried with anhydrous magnesium sulfate, filtered, and the solvent was evaporated away. Thus obtained, the roughly -purified product was purified through basic silica gel column

chromatography to obtain 3-(2,6-dichlorophenyl)-4-imino-7-[(2'-methyl-2',3'-dihydro-rH- spiro[cyclopropane-l,4'-isoquinolin]-7'-yl)amino]-3,4-dihydropyrimido[4,5-d]pyrimidin-2(lH)- one. This was dissolved in a mixed solvent of chloroform/methanol, and 1.5 equivalents of aqueous hydrochloric acid solution was added thereto, and stirred at room temperature for 5 minutes. Then, the solvent was evaporated away, and the residue was washed with ethyl acetate to obtain 1.5 g (yield, 64 %) of 3-(2,6-dichlorophenyl)-4-imino-7-[(2'-methyl-2',3'-dihydro-l'H- spiro[cyclopropane-l,4'-isoquinolin]-7'-yl)amino]-3,4-dihydropyrimido[4,5-d]pyrimidin-2(lH)- one dihydrochloride as a yellow solid. iH-NMR (400 MHz, DMSO-d6) δ: 1 1.83 (1H, brs),

10.05 (1H, brs), 9.10 (1H, s), 8.88 (1H, s), 7.79-7.68 (1H, m), 7.63-7.59 (2H, m), 7.47 (1H, t, J=8.2 Hz), 7.38 (1H, d, J=8.3 Hz), 6.63 (1H, d, J=8.5 Hz), 3.59 (2H, s), 2.44 (2H, s), 2.32 (3H, s), 0.90-0.81 (4H, m) ESI-MS Found: m/z [M+H]+ 494

Preparative Example 3-1

Production of 7-chloro-3-(2,6-dichlorophenyl)-4-imino-3,4-dihydropyrimido[4,5-d]pyrimidin-

2(lH)-one 1.12 g of sodium hydride was added to an N,N-dimethylformamide (35 mL) solution of 3.0 g of 4-amino-2-chloropyrimidine-5-carbonitrile, and stirred at room temperature for 5 minutes. 4.38 g of 2,6-dichlorophenyl isocyanate was added to the reaction liquid, and stirred at room temperature for 1 hour. Ethyl acetate and aqueous 1 N hydrochloric acid solution were added to the reaction solution, and the organic layer was separated. This was washed with saturated saline water, dried with anhydrous magnesium sulfate, and the solvent was evaporated away. The precipitated solid was solidified with a mixed solvent of methanol/ethyl acetate and taken out through filtration to obtain 3.8 g of the entitled compound as a white solid. lH-NMR (400 MHz, DMSO-d6) δ: 9.33 (1H, s), 7.66 (2H, d, J=8.2 Hz), 7.53 (1H, t, J=8.2 Hz) ESI-MS Found: m/z [M+H] 342 Preparative Example 3-2

Production of 2'-methyl-2',3'-dihydro- H-spiro[cyclopropane-l,4'-isoquinolin]-7'-amine

Step 1) Production of methyl l-(2-cyanophenyl)cyclopropanecarboxylate:

1.5 g of tetra-n-butylammonium bromide, 6.5 g of 1,2-dibromoethane and 20 mL of aqueous 50 % sodium hydroxide solution were added to a toluene (40 mL) solution of 4.0 g of methyl 2-cyanophenylacetate, and stirred at room temperature for 1 hour. Water was added to the reaction liquid, and extracted with ethyl acetate. The organic layer was washed with saturated saline water, dried with anhydrous magnesium sulfate, and the solvent was evaporated away under reduced pressure. The crude product was purified through silica gel column chromatography (hexane/ethyl acetate) to obtain 3.0 g of the entitled compound as a colorless compound. lH-NMR (400 MHz, CDCI3) δ: 7.66 (1H, dd, J=7.6, 1.2 Hz), 7.55 (1H, td, J=7.6, 1.2 Hz), 7.43-7.36 (2H, m), 3.66 (3H, s), 1.82 (2H, q, J=3.7 Hz), 1.30 (2H, q, J=3.7 Hz)

ESI-MS Found: m/z [M+H] 202

Step 2) Production of methyl l-[2-(aminomethyl)phenyl]cyclopropanecarboxylate

monohy drochloride :

1.6 g of 10 % palladium-carbon was added to an ethanol (50 mL) solution of 2.95 g of the compound obtained in the above reaction Step 1), and stirred in a hydrogen atmosphere under 2 atmospheric pressure at room temperature for 3 hours. The palladium-carbon was removed through filtration, the filtrate was concentrated under reduced pressure, and the crude product was washed with diethyl ether to obtain 3.2 g of the entitled compound as a colorless solid. iH-NMR (DMSO-d6) δ: 8.47 (2H, s), 7.55 (IH, d, J=6.8 Hz), 7.38 (3H, td, J=7.2, 2.1

Hz), 7.36-7.29 (2H, m), 4.04 (2H, d, J=4.9 Hz), 3.54 (3H, s), 1.61-1.56 (2H, m), 1.33-1.29 (2H, m) ESI-MS Found: m/z [M+H] 206

Step 3) Production of r,2'-dihydro-3'H-spiro[cyclopropane-l,4'-isoquinolin]-3'-one:

4 mL of aqueous 5 N sodium hydroxide solution was added to a methanol (50 mL) solution of 3.2 g of the compound obtained in the above reaction Step 2), and stirred at room temperature for 30 minutes. This was neutralized with aqueous 1 N hydrochloric acid added thereto, and methanol was evaporated away under reduced pressure. The residue was diluted with water, and extracted three times with ethyl acetate. The organic layer was washed with saturated saline water, dried with anhydrous magnesium sulfate, and the solvent was evaporated away under reduced pressure to obtain 2.1 g of the entitled compound as a colorless solid. iH-NMR (CDCI3) δ: 7.23 (IH, td, J=7.8, 1.1 Hz), 7.18 (IH, td, J=7.3, 1.1 Hz), 7.10 (IH, dd, J=7.3, 1.0 Hz), 6.73 (IH, dd, J=7.8, 1.0 Hz), 4.69 (2H, d, J=1.5 Hz), 1.85 (2H, q, J=3.7 Hz), 1.24 (2H, q, J=3.7 Hz) ESI-MS Found: m/z [M+H] 174

Step 4) Production of 7'-nitro- ,2'-dihydro-3'H-spiro[cyclopropane-l,4'-isoquinolin]-3'- one:

1.3 g of potassium nitrate was gradually added to a sulfuric acid (60 mL) solution of 2.1 g of the compound obtained in the above reaction 3), taking 5 minutes, and further stirred at room temperature for 10 minutes. The reaction liquid was poured into ice water, the precipitated crystal was taken out through filtration, and washed with water to obtain 2.4 g of the entitled compound as a yellow solid. lH-NMR (CDCI3) δ: 8.09 (IH, dd, J=8.8, 2.4 Hz), 8.01

(IH, t, J=2.4 Hz), 6.86 (IH, d, J=8.8 Hz), 6.30 (IH, s), 4.78 (2H, d, J=1.5 Hz), 2.01 (2H, q, J=4.1 Hz), 1.35 (2H, q, J=4.1 Hz) ESI-MS Found: m/z [M+H] 219

Step 5) Production of 7'-nitro- ,2'-dihydro-3'H-spiro[cyclopropane-l,4'-isoquinoline]:

With cooling with ice, 6.3 g of boron trifluoride-diethyl ether complex was added to a tetrahydrofuran suspension of 1.3 g of sodium borohydride, and stirred for 1 hour. A tetra- hydrofuran (100ml) solution of 2.4 g of the compound obtained in the above reaction Step 4) was added to the reaction liquid, and heated under reflux for 2 hours. The reaction liquid was cooled, and then neutralized with aqueous saturated sodium bicarbonate solution. The solvent was evaporated away under reduced pressure, the residue was dissolved in ethanol, 5 N hydrochloric acid was added to it, and heated under reflux for 1 hour. The reaction liquid was cooled, then the solvent was evaporated away under reduced pressure, and the residue was neutralized with aqueous potassium carbonate solution. The aqueous layer was extracted with chloroform, the organic layer was dried with anhydrous magnesium sulfate, and the solvent was evaporated away under reduced pressure to obtain the entitled compound. ESI-MS Found: m/z [M+H] 205

Step 6) Production of 2'-methyl-7'-nitro-2',3'-dihydro- H-spiro[cyclopropane-l,4'- isoquinoline]:

1.5 g of sodium cyanoborohydride was added to a methanol (50 mL) solution of the compound (2.3g) obtained in the above reaction Step 5), 2.7 mL of aqueous 37 %

formaldehyde solution and 0.7 mL of acetic acid, and stirred at room temperature for 15 hours. The reaction liquid was neutralized with aqueous saturated sodium bicarbonate solution, and methanol was evaporated away under reduced pressure. The residue was diluted with water and extracted three times with chloroform. The organic layer was dried with anhydrous magnesium sulfate, the solvent was evaporated away under reduced pressure, and the crude product was purified through silica gel column chromatography (hexane/ethyl acetate) to obtain 1.7 g of the entitled compound as a colorless solid. iH-NMR (CDCI3) δ: 7.97 (1H, dd, J=8.8, 2.4 Hz), 7.91

(1H, d, J=2.4 Hz), 6.78 (1H, d, J=8.8 Hz), 3.77 (2H, s), 2.57 (2H, s), 2.48 (3H, s), 1.16-1.12 (2H, m), 1.10-1.06 (2H, m) ESI-MS Found: m/z [M+H] 219

Step 7) Production of 2'-methyl-2',3'-dihydro-rH-spiro[cyclopropane-l,4'-isoquinolin]-7'- amine:

800 mg of 10 % palladium-carbon was added to an ethanol (20 mL) solution of 1.7 g of the compound obtained in the above reaction Step 6), and stirred in a hydrogen atmosphere under 1 atmospheric pressure at room temperature for 15 hours. Palladium-carbon was removed through filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified through basic silica gel column chromatography (hexane/ethyl acetate) to obtain 1.1 g of the entitled compound as a colorless solid. iH-NMR (CDCI3) δ: 6.50-

6.48 (2H, m), 6.38-6.36 (1H, m), 3.61 (2H, s), 3.50 (2H, s), 2.49 (2H, s), 2.42 (3H, s), 0.91 (2H, dd, J=6.3, 4.6 Hz), 0.81 (2H, dd, J=6.3, 4.6 Hz) ESI-MS Found: m/z [M+H] 189

EXAMPLE 4

Materials and Methods

A. Curated data used for analysis

To allow efficient and reliable analysis of large data sets, gene and protein interaction data were obtained from three commercial databases: GeneGo Metabase (GeneGo Inc., San Diego, CA), Ingenuity (Ingenuity Systems Inc., Redwood City, CA) and NetPro (Molecular Connections, Brookville, NY). Each database includes detailed information on each interactions and the scientific reference from which it was extracted. Using the PubMed reference ID numbers, the three databases were integrated. References that supported more than ten interactions were discarded and, from the remaining, only interactions supported by at least two references were retained. These interactions were separated into two types: expression related, i.e. gene A affects the expression of gene B, and non-expression related, i.e.

phosphorylation, cleavage, binding, etc.

Each gene A in the expression related data set, a "gene set A" was defined as a set of all genes that affected the expression of gene A or that were affected by gene A. For example, all genes that affected the expression of MYCN or that were affected by MYCN were grouped into a single gene set that was used for further statistical analysis. B. Enrichment analysis

To identify regulators of genes that were differentially expressed in an experiment, a statistical analysis was performed in R (R Project for Statistical Computing, available from the Institute for Statistics and Mathematics, Univeristy of Economics and Business, Vienna, Austria) using a hyper-geometric distribution with the following command: phyper(x-l, m, n-m, k and lower.tail = FALSE), where x is the number of genes from an experiment that are members of the gene set, m is the number of genes in the gene set, n is the total number of unique genes in all gene sets, and k is the number of genes from an experiment that have an entry in any gene set. This method results in a p-value that is indicative of the level of significance for the observation.

This approach typically results in multiple significant gene sets due to redundancy. To remove any redundancy this approach was employed iteratively, which requires that one save each time the gene set that scored as the most significant and remove the associated genes from the list. As a result, at each step, the analysis was repeated with a shorter gene list until no gene set was significant.

This approach was also employed for data obtained from other sources, such as, GeneOntology and GeneGo pathways.

C. siRNA Transfection

Transfections were performed in triplicate as previously described (Mosse, Y.P., et al, Nature. 2008, 455:930-935) using ON-TARGET SMARTpool siRNAs

(ThermoScientific,® Waltham, MA) specific for GAPDH, PLK1, WEE1 and CHK1. Cell viability was quantified at 72 hours by use of Cell Titer-Glo® luminescent assay (Promega, Madison, WI). Gene knockdown was confirmed to be >90% by quantitative real-time PCR.

D. Immunohistochemistry

Following standard antigen retrieval protocol, a phospho WEE1 antibody (#4910, Cell Signaling Technology, Beverly. MA ) was used to stain formalin fixed paraffin embedded sections at a 1 : 1000 dilution for 1 hour at room temperature. Slides were again rinsed, then incubated with biotinylated anti-Rabbit IgG (BA-1000, Vector Laboratories, Burlingame, CA) at a 1 :200 dilution for 30 minutes at room temperature, followed by avidin biotin complex (PK- 6100, Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature. Slides were then rinsed and incubated with DAB (Cytomation K3468, DAKO, An Agilent Technologies

Company, Carpinteria, CA). Counterstaining was performed for 1 minute in Harris Hematoxylin (6765001, ThermoFisher Scientific, Waltam, MA). Slides were rinsed and dehydrated through a series of ascending concentrations of ethanol and xylene, then cover-slipped. After drying, slides were digitally scanned at 20X magnification on an Aperio OS slide scanner (Aperio

Technologies Inc., Vista, CA).

E. Pharmacological Inhibition

The CHK1 inhibitor (CHKl-1) and the WEE1 inhibitor (WEEl-1) were provided by Merck & Co., Inc. Twenty-four hours after plating, cells were treated in triplicate over a four-log dose range (10 -10,000 nM) and a DMSO control. Cells were cultured for 72 hours and cell viability was measured using Cell Titer-Glo® assays (Promega, Madison, WI). IC50 determination was made using a non-linear log inhibitor versus normalized response curve fit function (GraphPad Software, Inc., La Jolla, CA). Caspase activation assays were performed at 16 hours and quantified by use of the Caspase-Glo® 3/7 assay (Promega, Madison, WI).

F. Combination Studies

Following single-agent IC50 determination, neuroblastoma cells were plated in duplicate in 96-well plates and treated with two agents at doses ranging in a 2-fold difference above and below each individual IC50 (i.e., 0.25X, 0.5X, IX, 2X and 4X). Combination indices were determined using CalcuSyn software (Biosoft, Intl., Palo Alto, CA) via the Chou-Talalay method (Chou, T.-C, Cancer Res., 2010, 70(2):440-446). All combination studies were repeated at least once (total of n > 4 for each cell line).

G. Western Blotting

Cell lysates were prepared as described previously (Mosse, Y.P., et ah, Nature. 2008, 455:930-935). Neuroblastoma cell lines or primary tumor lysates (40 μg) were separated on 4-12% gradient polyacrylamide gels via SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA). Primary antibody dilutions included 1 : 1,000 CHK1, CHK1^S296,

WEE1, WEE1^S642, p-H2A.X^(sl39), Cdc2^(Y15) and 1 :3,000 β-actin (Cell Signaling, Beverly, MA).

H. In Vivo Studies

CB 17SC-M SCID -/- mice (Taconic, Hudson, NY) were used to propagate subcutaneously implanted neuroblastoma xenografts. Caliper measurements were obtained, and tumor volumes were calculated using the formula, (π/6) x d², where d represents the mean diameter. Once the tumor was greater than 200 mm³, mice bearing neuroblastoma tumors were randomized to treatment arms of: 1) 30 mg/kg/dose twice daily i.p. CHKl-1, 2) 30 mg/kg/dose twice daily p.o. WEEl-1, 3) the two compounds combined, or 4) vehicle control administered for five consecutive days for two weeks. Tumors were measured twice weekly for a total of 28 days or until tumor volume reached 3cm³. The Children's Hospital of Philadelphia Institutional Animal Care and Use Committee approved all animal studies.

I. Statistical Analysis

Group comparisons were determined with a two-tailed t-test. For the xenograft studies, a linear mixed effects model was used to test the difference in the rate of tumor volume changing over time between different the vehicle group and treatment groups.

EXAMPLE 5

MYCN expression as a sensitivity marker for WEE1 inhibition

The WEE1 inhibitor WEEl-1 is a cytotoxic drug with potential to treat human neoplasms. Identification of which human tumor subtypes are especially sensitive to WEE1 inhibition can be used to improve the therapeutic benefit of WEEl-1 by allowing greater antitumor efficacy within the tolerated range of drug exposure. Preclinical experiments were conducted to identify correlative markers, which in turn, led to the identification of MYCN expression as a marker for cells that are sensitive to inhibition of WEE 1. The preclinical experiments that illustrated this relationship are summarized as follows. A. Genome wide siRNA screen

To identify genes that mediate sensitivity or resistance to WEE1 inhibition, a genome wide siRNA screen was conducted in a TOV21G ovarian cancer cell line harboring stable shRNA targeting p53. The screen included over 20,000 genes using pooled siRNA in the presence of a suboptimal dose of a WEE 1 inhibitor (WEEl-A), the structure of which is shown below and is p emcitabine.

Two follow up screens were conducted on the hits from the primary screen and each gene was tested with up to seven different individual siRNAs. This experiment resulted in 203 genes that sensitized or provided resistance to the WEE1 inhibitor and gemcitabine combination. An enrichment analysis was employed to identify cellular processes or groups of genes that are highly represented in the gene list of 203 hits. MYCN was identified as a known regulator of 13 genes that provide resistance upon knock down and 16 genes that enhance sensitivity. Figure 1 graphically illustrates the genes that were correlated with sensitivity (o) and resistance (·).

A computational functional analysis of these genes (Example 4A), identified MYCN as a known regulator of fourteen genes that provide resistance upon knock down and fifteen genes that enhance sensitivity (Figure 1). B. Drug response in cell lines

A panel of 93 lung cancer cell lines were assembled and evaluated to identify a gene signature that correlated with sensitivity for WEE 1. Cells were treated with a WEE1 inhibitor (WEEl-A) at 370 nM for 72 hours. Cell viability was assessed relative to DMSO treated cell lines and normalized to a 0- 1 scale where 0 indicates complete death and 1 complete viability (data not shown).

Each cell line was profiled using gene expression microarrays (Affymetrix® Microarray Solution, Affymetrix, Santa Clara, CA) in basal state (no drug treatment). To identify genes that predict response to WEEl-1, a Spearman rank correlation was computed (Spearman, C, Amer. J. Psychol, 1904, 15:72-101) for each probe on the array and the drug response. Genes showing no expression across the panels or a standard deviation of 0 were discarded. The remaining genes were correlated to sensitivity of a WEE 1 inhibitor (WEEl-A) at 450nM. The top 200 correlated genes and top 200 anti-correlated genes were defined as a gene expression signature of WEE 1 sensitivity. The 200 best correlated probes and 200 best anti- correlated probes defined a signature of response to a WEEl inhibitor (WEEl -A). Figure 2 illustrates gene expression levels of genes in the signature. Cell lines were sorted from left to right by WEEl (WEEl -A) sensitivity and the rows were sorted by correlation with sensitivity.

Genes comprising the WEEl-1 signature were subject to a computational enrichment analysis using a hyper-geometric distribution test (Example 4B), where MYCN, together with ZFX, was identified as the most significantly over represented in the gene signature (Figure 3). The list of MYCN regulated genes was obtained by combining multiple licensed databases (Example 4A). C. Animal studies of WEEl inhibitor and radiation

A human tumor xenograft model was used to evaluate the synergistic changes in gene expression upon administration of therapeutic radiation and a WEEl inhibitor (WEEl-1). Tumor bearing mice were divided into six groups: vehicle, WEEl (WEEl-1) treatment, radiation treatment, simultaneous treatment with radiation and WEEl-1, and delayed radiation treatment following WEEl-1 treatment. Xenograft tumor tissue was collected at varying time points after treatment and gene expression data was obtained for subsequent analysis. As shown in Figure 4, across the various treatment groups, gene expression patterns allowed for division into six clusters using a -means clustering analysis (MacQueen, J.B., "Some Methods for Classification and Analysis of Multivariate Observations," Proceedings of 5^th Berkely Symposium on

Mathematical Statistics and Probability, U. California Press, 1967, pp. 281-297). An enrichment analysis (Example 4B) conducted for each cluster confirmed that one of the clusters, cluster 5, included genes that were known to be primarily regulated by MYCN (Figure 5).

D. Data integration

The experiments described above (Examples 5A-5C) all identified MYCN as a gene that was an important component of WEEl inhibitor sensitivity. It has previously been reported that MYCN was amplified and associated with poor outcomes in neuroblastoma (Weiss, W. A., et al, EMBO J.. 1997, 16(1 1):2985-2995; Westermark, U.K., et al, Seminars Cancer Biol, 2011, 21(4):256-266). In addition, MYCN has been associated with centrosome hyper- amplification in neuroblastoma (Slack, A.D., et al, Cancer Res.. 2007, 67(6):2448-2455;

Sugihara, E., et al, , Oncogene, 2004, 23(4): 1005-9). Taken together, the data herein suggested that cancers associated with MYCN gene amplification and, specifically, neuroblastoma, may be amenable to treatment with a WEEl inhibitor.

EXAMPLE 6

MYCN amplification predicts sensitivity to WEEl and combination with CHK1

To confirm that MYCN amplified neuroblastoma cells were predictive of sensitivity to WEEl, Applicants assembled neuroblastoma cell lines with and without MYCN amplification, that is, neuroblastoma cells with multiple copies of the MYCN gene, that were also verified by the presence of absence of the MYCN protein. Cells were treated with titrations of a WEEl inhibitor (WEEl-1) and scored for WEEl-1 EC50 values in a 72 hour proliferation assay (Figure 6). The EC50 values are presented in nM values and are a measure of the concentration of drug (i.e., WEEl-1) required to achieve 50% of the maximal anti-proliferative effect, such that lower EC50 values typify greater sensitivity. The three most sensitive cell lines

(CHP-212, SKNDZ, and IMR-32) were identified as having MYCN amplification. Four of the five MYCN amplified cell lines, CHP-212, SKNDZ, IMR-32, and SKNBE(2), were also considered sensitive to treatment with a WEEl inhibitor (WEEl-1) (EC50 < 200 nM), while only one of the two neuroblastoma cell lines without MYCN amplification was considered sensitive to a WEEl inhibitor (WEEl-1) (SKNSH, EC₅₀ = 145 nM). Table 1 shows the average EC50 value in nM for the neuroblastoma cell lines evaluated.

Table 1

This data supported the hypothesis that cancers associated with the MYCN gene and, specifically, neuroblastoma, were amenable to treatment with a WEEl inhibitor. Without wishing to be bound by any theory, Applicants believe that neuroblastoma cells having MYCN amplification are more likely than not to be sensitive to WEEl, such that they may be more amenable to treatment with a WEEl inhibitor, such as WEEl-1. As such, MYCN expression may be used as a surrogate marker to identify neuroblastoma cells, i.e. neuroblastoma patients, who are most likely to respond to treatment with a WEEl inhibitor, such as WEEl-1.

Similarly, it has been shown that MYCN amplified neuroblastoma cells were sensitive to CHKl phosphorylation and inhibition (Cole, K., et al, PNAS. 201 1, 108(8):3336- 3341). CHKl inhibitor sensitivity correlated with total MYCN protein levels, with concomitant growth inhibition of neuroblastoma cells when inhibited (Id.). As such, MYCN expression may also be used as a surrogate marker to identify neuroblastoma cells, i.e. neuroblastoma patients, who are most likely to respond to treatment with a CHKl inhibitor, such as CHKl-1.

As a confirmation of this sensitivity, a panel of 581 tumor cell lines was screened with either a WEEl inhibitor (WEEl-1) or a CHKl inhibitor (CHKl-1). Cells were analyzed for proliferation using a CellTiter-Glo® (Promega, Madison, WI) luminescent assay 96 hours after treatment and viability was calculated as counts in treated wells relative to DMSO control treated wells. In that the determination of EC50 values was not possible among all cell lines evaluated, relative sensitivities were compared by percent viability at fixed concentrations of either drug. The mean viability of the neuroblastoma lines (n = 7) was the lowest among all twenty one tumor types examined to both a WEE1 inhibitor (WEEl-1) (at 450 nM) and a CHK1 inhibitor (CHKl-1) (at 1.1 μΜ) (Figures 15A and 15B). Further, when neuroblastoma cells lines were treated with the WEEl-1 and CHKl-1 in combination, synergistic inhibition of cell proliferation was observed (data not shown). EXAMPLE 7

Neuroblastoma harbors elevated WEE I^s642 phosphorylation

It has been previously shown that CHK1, a DNA damage response kinase, was highly expressed and aberrantly activated in neuroblastoma, leading to cellular dependence on this pathway (Cole, K., et ah, 201 1). In order to elucidate possible mechanisms underlying sensitivity to CHK1 inhibition, Applicants further evaluated WEE1, a serine/threonine kinase that signals through a pathway complimentary to CHK1. In a manner similar to CHK1, robust expression of WEE 1 protein levels with extensive phosphorylation was observed in a comprehensive panel (n=18) of neuroblastoma lines. In addition, canonical signal transduction through the WEE1 pathway was intact in these neuroblastoma lines, as phosphorylation of Cdc2 at Y15 often pheno-copied WEE1 activation (Figures 7A and 12A). In contrast, substantial WEE1 expression or activation was not detected in DAOY medulloblastoma or in TERT immortalized non-transformed, retinal pigmented epithelial (RPE-1) cells (Figure 12B).

Similarly, among several adult tumor types investigated, very low basal WEE1 activity was observed in comparison to that for neuroblastoma (Figure 12B).

To exclude the possibility that robust WEE1 activity was solely an artifact of

DNA damage induction during cell culturing, Applicants examined the WEE1 status in patient primary tumor samples obtained at diagnosis. As shown in Figures 7B, 7 of 12 high risk samples (58.3%) had appreciable p-WEEl expression, as compared to 2 of 7 low risk patient samples (28.5%). These findings were further supported by immunohistochemical evidence of WEE1 phosphorylation (Ser642) obtained by staining neuroblastoma tumor microarrays, representing tumors from 91 patients (Figure 7C). It was again observed that high-risk neuroblastoma tumors had increased levels of WEE1 phosphorylation as compared to low risk tumors (p < 0.0001) (Figures 7C and 7D). Lastly, quantification of WEE 1 mRNA expression levels in a large number of neuroblastoma tumor samples (n=251) confirmed that, like CHK1, there was significantly higher expression in high-risk, MYCN-amplified tumors (Figures 13B and 13E). Taken together, this data demonstrated that WEE1 expression and activity was significantly elevated in neuroblastoma, particularly as to MYCN-amplified, high-risk disease patient samples. EXAMPLE 8

Neuroblastoma is sensitive to targeted CHKl/WEEl inhibition

To ascertain the contribution of WEEl signaling in neuroblastoma, Applicants depleted WEEl in three neuroblastoma lines using siRNA. Depletion of WEEl substantially inhibited the growth and viability of neuroblastoma cell lines (Figure 8A). In order to assess the effect of pharmacologic kinase inhibition, small molecular inhibitors of CHK1 (CHKl-1) and WEEl (WEEl-1) were evaluated for neuroblastoma cell growth and viability. The majority of the neuroblastoma cell lines evaluated were sensitive to single-agent inhibition of CHK1 (82%) or WEEl (91%), with median IC50S of roughly 900nM and 300nM, respectively (Figure 8B). It was subsequently confirmed that the observed decrease in cell viability was due primarily to apoptosis, rather than cell cycle arrest, as both activation of caspase 3/7 and cleavage of poly ADP ribosome polymerase (PARP) was observed in four sensitive neuroblastoma lines (Figures 8C and 8D, respectively).

Two murine tumor lines derived from a MYCN transgenic mouse model were also tested to examine the consequence of MYCN expression on the potency of these inhibitors (Weiss, W.A., et al, EMBO J., 1997, 16:2985-2995). Both single-agent WEEl and CHK1 inhibition were cytotoxic in these MYCN-derived lines, with the MYCN-homozygous line roughly twice as sensitive to both CHK1 and WEEl inhibition than the heterozygous line, demonstrating a transgene-dosage effect (Figure 9A). Without wishing to be bound by any theory, Applicants believe that these results support the hypothesis that MYCN-driven replicative stress may underlie aberrant activation of the DNA damage response (DDR) pathway in neuroblastoma, resulting in a dependence on CHKl/WEEl signaling to maintain cell viability. Indeed, siRNA-mediated depletion of WEEl significantly impaired cell viability and the majority of neuroblastoma cells demonstrated single-agent sensitivity to WEEl or CHK1 inhibition, with IC50S in physiologically attainable ranges (Figure 9A). Target engagement in these murine cells was confirmed, with the expected Ataxia telangiectasia and Rad3 related kinase (ATR) compensatory CHK1^S345 phosphorylation in response to administration of a CHK1 inhibitor (CHKl-1) (Leung-Pineda, V., et al, Mol. & Cell. Biol, 2006, 26:7529-7538) and a corresponding decrease in Cdc2^Y15 phosphorylation following exposure to a WEEl inhibitor (WEEl -1) (Figure 9B).

EXAMPLE 9

CHK1 and WEEl inhibition acts synergistically with chemotherapy in neuroblastoma cells

To assess chemopotentiation in neuroblastoma, combination strategies utilizing gemcitabine and SN-38 (the active metabolite of irinotecan) were evaluated across a panel of neuroblastoma cell lines. Gemcitabine, although not used for the treatment of neuroblastoma (Wagner-Bohn, A., et al, Anti-Cancer Drugs, 2006, 17:859-865), has been previously shown to be chemosensitized by CHKl and WEE1 inhibition (Xu, H., et al, Intl. J. Cancer, 201 1, 129(8): 1953-1962; Hiral, H., et al, Mol. Cancer Ther.. 2009, 8:2992-3000), whereas irinotecan has been used clinically for treatment of relapsed neuroblastoma (Bagatell, R., et al, I

Clin.Oncology. 2011, 29:208-213). Nearly all of the neuroblastoma cell lines evaluated herein were found to demonstrate synergy in a variety of combinations (Table 2). Dual inhibition of CHKl and WEE1 was highly synergistic in nearly every cell line - equivalent to, or exceeding many of the chemotherapy combinations. In support of these findings, recent data from Davies et al. demonstrated that dual CHK1/WEE1 inhibition was synergistic in several different cancer cell lines and suggested that further studies be done to identify sensitive cancer cell types or if p53 status dependency was necessary for sensitivity (Davies, K.D., et al, Cancer Biol. & Ther.. 201 1, 12:788-796).

Prior to combination experiments, single-agent IC50S were calculated for both SN-38 (the active metabolite of irinotecan) and gemcitabine for the neuroblastoma cell line panel (n=10) using a 4-log dose-response cell viability assay with a 72 hour time point. Ten neuroblastoma cell lines were evaluated for synergistic interactions between a WEE1 (WEEl-1) and a CHKl (CHKl-1) inhibitor in combination with these chemotherapeutic agents, to generate a combination index (CI) value denoting the level of observed synergy (Table 2). Combination indices were determined by increasing concentrations of both inhibitors simultaneously (based on multiples of each inhibitor's individual IC50, utilizing the Chou-Talalay method (Chou, T.-C, Cancer Res.. 2010, 70(2):440-46). Nearly all of the neuroblastoma lines exhibited a pronounced synergistic effect (denoted by a CI value <0.7) when combining these inhibitors with either SN- 38 (7 out of 10 cell lines for both WEEl-1 and CHKl-1) or gemcitabine (8 out of 10 cell lines for WEEl-1, 10 out oflO cell lines for CHKl-1) (Table 3). In addition, potent cytotoxicity was evident in almost all neuroblastoma lines when combining a WEE1 (WEEl-1) and a CHKl (CHKl-1) inhibitor (9 out of 10 cell lines), suggesting that dual inhibition of CHKl and WEE1 was a robust synergistic combination.

Table 2

CI Value Symbol Observed Synergy

<0.1 +++++ very strong

0.1 - 0.3 ++++ strong

0.3 - 0.7 +++ synergy

0.7 - 0.85 ++ moderate synergy

0.85 - 0.9 + slight synergism

0.9 - 1.1 additive

>1.1 - antagonistic

Table 3

EXAMPLE 10

Simultaneous inhibition of CHKl and WEEl results in substantial DNA double strand breakage

Applicants examined pathway signaling following pharmacologic disruption in an effort to elucidate downstream events responsible for the synergistic cytotoxicity in the CHKl (CHKl -1)/WEE1 (WEEl-1) combination group (Figures 1 OA- IOC). There was substantial inhibition of Cdc2 phosphorylation/Cdc2^Y15 signaling downstream of WEEl following administration of WEEl-1. In addition, inhibition of WEEl resulted in a modest accumulation of DNA double-strand breaks as evidenced by sustained phosphorylation of histone H2A.X (Figure 10A). Similarly, although inhibition of CHKl alone resulted in phosphorylation of histone H2A.X, CHKl-1 in combination with chemotherapy potentiated the DNA damaging aspect of both SN-38 and gemcitabine (Figure 10B). The combination of WEEl-1 and CHKl-1 resulted in greater than additive histone H2A.X phosphorylation (lane 6), than either inhibitor alone (lanes 4 and 7), and caused DNA damage equivalent to that induced by the cytotoxic chemotherapeutic agents (Figure IOC). Moreover, despite the presence of DNA damage, the neuroblastoma cells were unable to inhibit the cell cycle through Cdc2 phosphorylation, suggesting that mitotic progression was proceeding in the presence of damaged DNA, leading to mitotic catastrophe and resultant apoptosis. EXAMPLE 11

Dual CHK1/WEE1 inhibition is efficacious in vivo

Based on the dual efficacy of the CHKl (CHKl-1) and WEEl (WEEl-1) inhibitors in vitro, the WEEl and CHKl inhibitor combination was administered to mice bearing neuroblastoma tumors. Mice harboring xenografts from NB-1643 or SKNAS cell lines were treated with a vehicle control, single-agent WEEl-1, single-agent CHKl-1, or the combination of WEEl- 1 and CHKl-1 (5 days/week for two weeks), which were generally well tolerated. Mice receiving both the WEEl and CHKl inhibitors had a significant reduction in tumor growth rate as compared to control mice receiving vehicle alone (p < 0.0001 for NB-1643, p < 0.05 for SKNAS) (Figures 1 1A and 14A). Mice with NB-1643 tumors demonstrated significant growth inhibition with single-agent WEEl-1 (p = 0.042), whereas mice harboring SKNAS tumors responded to single-agent CHKl-1 (p = 0.023). A separate cohort of mice bearing xenografts from the Ebc-1 neuroblastoma line was used to confirm target engagement of CHKl and WEEl inhibition. Tumor resection following 48 hours of treatment (4 doses) with these inhibitors showed the expected reduction of CHKl (S296) autophosphorylation in the CHKl-1 group, as well as abrogation of Cdc2 (Y15) phosphorylation in the WEEl-1 treated mice (Figure 1 IB). These in vivo markers of pathway inhibition mimic those seen in our in vitro experiments and provide a rationale for the biological effect of tumor growth inhibition seen in these experiments.

Claims

WHAT IS CLAIMED IS:

1. A method of treating neuroblastoma with a WEEl inhibitor and a CHKl inhibitor, wherein the WEEl inhibitor is WEEl-1 or a pharmaceutically acceptable salt thereof, or WEEl-2 or a pharmaceutically acceptable salt thereof, and the CHKl inhibitor is CHKl-1 or a pharmaceutically acceptable salt thereof.

2. A method of claim 1 for treating neuroblastoma in a patient, in need of treatment thereof, comprising administering to said patient a therapeutically effective amount of a WEEl inhibitor and a CHKl inhibitor, wherein the WEEl inhibitor is WEEl-1 or a pharmaceutically acceptable salt thereof, or WEEl-2 or a pharmaceutically acceptable salt thereof, and the CHKl inhibitor is CHKl-1 or a pharmaceutically acceptable salt thereof.

3. The method of Claim 2 wherein the WEEl inhibitor is WEEl-1 or a pharmaceutically acceptable salt thereof.

4. The method of Claim 2 wherein the CHKl inhibitor is CHKl-1 or a pharmaceutically acceptable salt thereof.

5. The method of Claim 2 wherein the WEEl inhibitor is WEEl-1 or a pharmaceutically acceptable salt thereof, and the CHKl inhibitor is CHKl-1 or a

pharmaceutically acceptable salt thereof.

6. The method of Claim 5 wherein the WEEl inhibitor is administered in a dose between 100 mg and 250 mg.

7. The method of Claim 6 wherein the WEEl inhibitor is administered five times, over the course of two and a half days.

8. The method of Claim 6 wherein the WEEl inhibitor is administered once a day, over the course of two days.

9. The method of Claim 5 wherein the CHKl inhibitor is administered in a dose between 100 mg and 200 mg per day.

10. The method of Claim 9 wherein the CHKl inhibitor is administered once a day, over the course of two days.

1 1. A method for treating a neuroblastoma patient, in need of treatment thereof, comprising administering a therapeutically effective amount of a WEEl inhibitor and a CHKl inhibitor, wherein the WEEl inhibitor is with WEEl-1 or a pharmaceutically acceptable salt thereof, or WEEl -2 or a pharmaceutically acceptable salt thereof, and the CHKl inhibitor is CHKl-1 or a pharmaceutically acceptable salt thereof, and wherein the cancer cells of said patient to be treated are characterized by amplified expression of MYCN.

12. The method of Claim 1 1 wherein the WEEl inhibitor is WEEl-1 or a pharmaceutically acceptable salt thereof.

13. The method of Claim 12 wherein the CHKl inhibitor is CHKl-1 or a pharmaceutically acceptable salt thereof.

14. The method of Claim 12 wherein the WEEl inhibitor is WEEl-1 or a pharmaceutically acceptable salt thereof, and the CHKl inhibitor is CHKl-1 or a

pharmaceutically acceptable salt thereof.

15. A kit to characterize a neuroblastoma patient as having amplified expression of MYCN comprising a detection agent capable of detecting the expression product of MYCN in a test sample.