WO2014062454A1 - Compositions et procédés de traitement du cancer - Google Patents

Compositions et procédés de traitement du cancer Download PDF

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Publication number
WO2014062454A1
WO2014062454A1 PCT/US2013/064193 US2013064193W WO2014062454A1 WO 2014062454 A1 WO2014062454 A1 WO 2014062454A1 US 2013064193 W US2013064193 W US 2013064193W WO 2014062454 A1 WO2014062454 A1 WO 2014062454A1
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WIPO (PCT)
Prior art keywords
weel
inhibitor
chkl
neuroblastoma
cancer
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PCT/US2013/064193
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English (en)
Inventor
Kristina COLE
John Maris
Michael Russell
Yair BENITA
Jamie KUBICA
Stuart Denham SHUMWAY
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Merck Sharp & Dohme Corp.
Children's Hospital Of Philadelphia
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Publication of WO2014062454A1 publication Critical patent/WO2014062454A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/503—Pyridazines; Hydrogenated pyridazines spiro-condensed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems

Definitions

  • 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.
  • 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).
  • CHKl is an essential serine/threonine kinase involved in two cell cycle checkpoints, the intra-S and G2/M checkpoints.
  • 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).
  • 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
  • CHKl is necessary for unperturbed DNA replication and cell cycle coordination even in the absence of any exogenous insult.
  • 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 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).
  • 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).
  • 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).
  • CHK1 DNA damage response protein checkpoint kinase 1
  • 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
  • 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.
  • a WEEl inhibitor such as, WEEl-1 or a pharmaceutically acceptable salt thereof, or WEE 1-2 or a pharmaceutically acceptable salt thereof
  • a CHK1 inhibitor such as CHKl-1 or a pharmaceutically acceptable salt thereof
  • 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.
  • WEE1-A WEEl inhibitor
  • 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.
  • FIG. 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.
  • FIGs 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.
  • FIG. 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).
  • FIG 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
  • 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.
  • 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).
  • WEEl-1 WEE1 inhibitor
  • CHK1 inhibitor CHKl-1
  • SN-38 active metabolite of irinotecan
  • 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
  • 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.
  • CHK1 inhibitor 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
  • HU lmM hydroxyurea
  • WEEl-1 250nM
  • CHKl-1 500nM
  • SN-38 ⁇
  • Gem
  • 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.
  • WEEl WEEl
  • CHK1 CHKl-1
  • 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
  • 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.
  • FIG. 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.
  • FIGS 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).
  • WEEl-1 WEEl-1
  • CHKl-1 single-agent CHK1 inhibitor
  • FIG. 14B 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
  • 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.
  • neuroblastoma may be susceptible to therapies targeting the DNA damage response (DDR) pathway.
  • DDR DNA damage response
  • the synergistic anticancer activity of the CHK1 and WEE1 combination may also sensitize these tumors to allow the continued use of conventional therapeutic agents.
  • WEE1 and CHK1 inhibitors act synergistically when combined with other chemotherapy agents, such as, gemcitabine or irinotecan (SN-38).
  • SN-38 irinotecan
  • 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
  • CHK1 inhibitor is CHKl-1 or a
  • 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.
  • 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.
  • the invention in another embodiment, 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
  • 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.
  • the WEEl inhibitor is WEEl-1 or a pharmaceutically acceptable salt thereof.
  • the CHKl inhibitor is CHKl-1 or a pharmaceutically acceptable salt thereof.
  • the WEEl inhibitor is administered in a dose between 100 mg per day and 200 mg per day.
  • 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).
  • the CHKl inhibitor is administered in a dose from about 100 mg per day to 200 mg per day.
  • 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.
  • Successessive 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
  • 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.
  • 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.
  • treatment of cancer means that an anticancer agent is administered to a cancer patient so as to inhibit the growth of the cancer cells in the patient.
  • 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.
  • patient as referred to in this description means the recipient in need of medical intervention or treatment. Mammalian and non-mammalian patients are included.
  • 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.
  • gene marker 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.
  • MYCN MYC- Neuroblastoma
  • measuring expression levels includes methods that quantify target gene expression level exemplified by a transcript of a gene, including microRNA
  • RNA Ribonucleic acid
  • a protein encoded by a gene as well as methods that determine whether a gene of interest is expressed at all.
  • 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.
  • 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.
  • the WEE1 inhibitor of the instant invention is WEEl-1, the structure of which is as shown below.
  • 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
  • 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.
  • the CHK1 inhibitor of the instant invention is CHKl-1, the structure of which is as shown below.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • WEE1 and CHK1 inhibitors of the instant invention may be provided as prodrugs.
  • 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.
  • 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.
  • 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.
  • 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.
  • pharmaceutically acceptable salt means ordinary, pharmaceutically acceptable salt.
  • the compound 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, ⁇ , ⁇ '-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.
  • 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.
  • excipients e.g., fats, beeswax, semi-solid and
  • Solid preparations can be prepared in the forms of tablet, capsule, granule and powder without any additives, or prepared using appropriate carriers (additives).
  • carriers 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 hydroxypropylmethylcellulose
  • other conventionally used additives such as gelatin, talc, plant oil and gum arabic.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • terapéuticaally effective amount 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.
  • a WEEl inhibitor is administered in doses ranging from about 100 mg per day to 250 mg per day.
  • a WEEl inhibitor is administered twice daily (BID), over the course of two and a half days, for a total of 5 doses.
  • a WEEl inhibitor is administered once daily (QD) over the course of two days, for a total of 2 doses.
  • 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.
  • a CHK1 inhibitor is administered in doses that range from about 100 mg per day to 200 mg per day.
  • a CHK1 inhibitor may be dosed once daily (QD) over either one or two days.
  • QD dosed once daily
  • a CHK1 inhibitor can be administered once a week.
  • a WEEl inhibitor can be administered 2 times per week.
  • a CHK1 inhibitor can be administered once a week.
  • a WEEl inhibitor can be administered 5 times per week.
  • a CHK1 inhibitor can be administered twice a week.
  • a WEEl inhibitor can be administered 2 times per week.
  • a CHK1 inhibitor can be administered twice a week.
  • a WEEl inhibitor can be administered 5 times per week.
  • the treatment of the present invention involves the combined administration of a WEEl inhibitor and a CHK1 inhibitor.
  • 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.
  • 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,
  • 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
  • hepatoma hepatocellular carcinoma
  • cholangiocarcinoma hepatocellular carcinoma
  • 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
  • skull skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges
  • meningiosarcoma 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
  • the WEE1 inhibitor and CHK1 inhibitor combination of the invention may also be useful in treating the following disease states: keloids and psoriasis.
  • 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.
  • 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).
  • 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.
  • hyperproliferative disorders such as, restenosis, inflammation, autoimmune diseases, and allergy/ asthma.
  • hypoinsulinism 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.
  • 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, ⁇ -secretase inhibitors, agents that interfere with receptor tyrosine kinases (RTKs) and agents that interfere with cell cycle checkpoints.
  • the mTOR inhibitor and ⁇ 3 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.
  • 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.
  • 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-d
  • hypoxia activated compound is tirapazamine.
  • proteosome inhibitors include but are not limited to lactacystin and MLN-341 (Velcade).
  • 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 6,288,23
  • 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, e
  • inhibitors of mitotic kinesins are described in Publications WO 2003/039460, WO 2003/050064, WO
  • 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.
  • histone deacetylase inhibitors include, but are not limited to,
  • “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.
  • PLK Polo-like kinases
  • 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 ⁇ 3001, 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- dich
  • 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.
  • HMG-CoA reductase inhibitors 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.
  • 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
  • GGPTase-II geranylgeranyl-protein transferase type-II (GGPTase-II, also called Rab GGPTase).
  • 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
  • Angiogenesis inhibitors refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism.
  • 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.
  • NSAIDs nonsteroidal anti-inflamm
  • 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).
  • 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).
  • 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.
  • 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 refer to compounds that inhibit RTKs and therefore mechanisms involved in oncogenesis and tumor progression.
  • 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
  • Specific anti-IGF-lR antibodies include, but are not limited to, dalotuzumab, figitumumab, cixutumumab, SHC 717454, Roche R1507, EM164 or Amgen AMG479.
  • NSAID's which are potent COX -2 inhibiting agents.
  • an NSAID is potent if it possesses an IC50 for the inhibition of COX-2 of ⁇ or less as measured by cell or microsomal assays.
  • NSAID's which are selective COX-2 inhibitors 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.
  • 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.
  • 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 ⁇ - 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
  • integrated circuit blockers refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the ⁇ ⁇ ⁇ 3 integrin, to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the ⁇ 5 integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the ⁇ ⁇ ⁇ 3 integrin and the ⁇ ⁇ ⁇ 5 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 ⁇ ⁇ ⁇ 6, « ⁇ 8 > « ⁇ , «2 ⁇ , ⁇ 5 ⁇ , «6 ⁇ , and ⁇ 6 ⁇ 4 integrins.
  • the term also refers to antagonists of any combination of ⁇ ⁇ ⁇ 3, ⁇ ⁇ ⁇ 5, ⁇ ⁇ ⁇ 6, ⁇ ⁇ ⁇ 8, ⁇ , «2 ⁇ , ⁇ 5 ⁇ , «6 ⁇ , an d ⁇ 6 ⁇ 4 integrins.
  • Combinations with compounds other than anti-cancer compounds are also encompassed in the instant methods.
  • combinations of the mTOR inhibitor and ⁇ 3 integrin antagonist combination of the instant invention with PPAR- ⁇ (i.e., PPAR-gamma) agonists and PPAR- ⁇ (i.e., PPAR-delta) agonists are useful in the treatment of certain malingnancies.
  • PPAR- ⁇ and PPAR- ⁇ are the nuclear peroxisome proliferator-activated receptors ⁇ and ⁇ .
  • the expression of PPAR- ⁇ 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..
  • PPAR- ⁇ agonists and PPAR- ⁇ / ⁇ 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
  • Another embodiment of the instant invention is the use of the presently disclosed compounds in combination with gene therapy for the treatment of cancer.
  • 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.
  • MDR inhibitors include inhibitors of p- glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar).
  • P-gp p- glycoprotein
  • 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.
  • 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.
  • neurokinin- 1 receptor antagonists especially 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.
  • an antidopaminergic such as, the phenothiazines (for example, prochlorperazine, fluphenazine, thioridazine and mesoridazine), metoclopramide or dronabinol.
  • 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;
  • 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
  • the WEE1 inhibitor and CHK1 inhibitor combination of the instant invention may also be administered with an agent useful in the treatment of anemia.
  • 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.
  • 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).
  • G-CSF human granulocyte colony stimulating factor
  • 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.
  • 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).
  • 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.
  • 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 ⁇ -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
  • 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.
  • employable are various radiations such as X-ray, ⁇ -ray, neutron ray, electron beam, proton beam; and radiation sources.
  • a linear accelerator is used for irradiation with external radiations, ⁇ -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
  • bendamustine hydrochloride (Treanda®); bevacuzimab (Avastin®); bexarotene capsules
  • Injection® doxorubicin liposomal
  • Doxil® doxorubicin liposomal
  • dromostanolone propionate dromostanolone®
  • dromostanolone propionate masterone injection®
  • Elliott's B Solution Elliott's B Solution®
  • epirubicin Ellence®
  • Epoetin alfa epogen®
  • eribulin mesylate Halaven®
  • 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
  • Leuprolide Acetate (Eligard®); (Lupron Depot®); (Viadur®); levamisole (Ergamisol®);
  • levoleucovorin Fusilev®
  • lomustine CCNU (CeeBU®)
  • meclorethamine nitrogen mustard
  • nelarabine (Arranon®); nilotinib hydrochloride monohydrate (Tasigna®); Nofetumomab
  • peginterferon alfa-2B (Sylatron®); pemetrexed disodium (Alimta®); pentostatin (Nipent®); pipobroman (Vercyte®); plerixafor injection (Mozobil®); plicamycin, mithramycin
  • Rasburicase Elitek®
  • Rituximab Rituxan®
  • romidepsin Istodax®
  • sorafenib (Nexavar®); streptozocin (Zanosar®); sunitinib maleate (Sutent®); talc (Sclerosol®); tamoxifen (Nolvadex®); temozolomide (Temodar®); temsirolimus (Torisel®); teniposide, VM-
  • 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.
  • Step 1) Phosphorus oxychloride (6.92 g, 45.1 mmol, 1.5 eq.) was cooled to
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 Et 2 0 (3 x 25 mL). The Et 2 0 layer was washed with a 5% aqueous NaHCC solution (50 mL) and saturated NaCl (50 mL).
  • Step 2 A solution of LiHMDS (15.8 mL of a 1.0M solution in THF, 2.0 equiv.) in THF
  • 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.
  • the roughly -purified product was purified through basic silica gel column
  • GeneGo Metabase GeneGo Inc., San Diego, CA
  • Ingenuity Ingenuity Systems Inc., Redwood City, CA
  • NetPro Molecular Connections, Brookville, NY.
  • Each database includes detailed information on each interactions and the scientific reference from which it was extracted.
  • 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.
  • 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.
  • 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
  • CHK1 inhibitor CHKl-1
  • WEE1 inhibitor WEEl-1
  • CHKl-1 and WEE1 inhibitor 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).
  • 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).
  • 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 ⁇ g) 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 ,
  • mice (Taconic, Hudson, NY) were used to propagate subcutaneously implanted neuroblastoma xenografts. Caliper measurements were obtained, and tumor volumes were calculated using the formula, ( ⁇ /6) x d 2 , where d represents the mean diameter.
  • 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 3cm 3 . The Children's Hospital of Philadelphia Institutional Animal Care and Use Committee approved all animal studies.
  • 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
  • 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.
  • WEEl-A WEE 1 inhibitor
  • 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).
  • 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).
  • WEEl-A WEE1 inhibitor
  • Each cell line was profiled using gene expression microarrays (Affymetrix® Microarray Solution, Affymetrix, Santa Clara, CA) in basal state (no drug treatment).
  • Affymetrix® Microarray Solution Affymetrix, Santa Clara, CA
  • 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.
  • WEEl-A WEE 1 inhibitor
  • 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.
  • 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.
  • 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
  • Example 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;
  • MYCN amplification predicts sensitivity to WEEl and combination with CHK1
  • 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).
  • WEEl-1 a WEEl inhibitor
  • 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 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.
  • 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.
  • 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.).
  • 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.
  • a panel of 581 tumor cell lines was screened with either a WEEl inhibitor (WEEl-1) or a CHKl inhibitor (CHKl-1).
  • WEEl-1 WEEl-1
  • CHKl-1 CHKl inhibitor
  • 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.
  • EC50 values was not possible among all cell lines evaluated, relative sensitivities were compared by percent viability at fixed concentrations of either drug.
  • Neuroblastoma harbors elevated WEE I s642 phosphorylation
  • Neuroblastoma is sensitive to targeted CHKl/WEEl inhibition
  • CHK1 and WEEl inhibition acts synergistically with chemotherapy in neuroblastoma cells
  • 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).
  • 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).
  • a separate cohort of mice bearing xenografts from the Ebc-1 neuroblastoma line was used to confirm target engagement of CHKl and WEEl inhibition.

Abstract

La présente invention concerne des procédés pour le traitement d'un neuroblastome par administration d'une combinaison d'un inhibiteur de WEE1 et un inhibiteur de CHK1, l'inhibiteur de WEE1 étant WEE1-1 ou un sel pharmaceutiquement acceptable de celui-ci, ou WEE1-2 ou un sel pharmaceutiquement acceptable de celui-ci, et l'inhibiteur de CHK1 étant CHK1-1 ou un sel pharmaceutiquement acceptable de celui-ci. Dans un autre mode de réalisation, l'invention concerne un procédé pour traiter un patient atteint de neuroblastome, comprenant l'administration d'un inhibiteur de WEE1 et un inhibiteur de CHK1, les cellules cancéreuses dudit patient à traiter étant caractérisées par des niveaux d'expression de MYCN amplifiés.
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