WO2012079079A1

WO2012079079A1 - Production of induced pluripotent stem cells

Info

Publication number: WO2012079079A1
Application number: PCT/US2011/064432
Authority: WO
Inventors: Junying Yuan; Dawei Ma; Junli Liu; Lihong Zhang
Original assignee: President And Fellows Of Harvard College; Shanghai Institute Of Organic Chemistry
Priority date: 2010-12-10
Filing date: 2011-12-12
Publication date: 2012-06-14

Abstract

The present disclosure relates to methods and compositions that improve the in vitro production of induced pluripotent stem cells through the use of compounds that promote degradation of p53. The disclosure also relates to compositions and methods for the treatment of cancer, pancreatitis and intracellular pathogens.

Description

Production of Induced Pluripotent Stem Cells

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/421, 865, filed December 10, 2010, the content of which is expressly incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under R37 AGO 12859 and DPI OD000580 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Induced pluripotent stem cells ("iPS cells") are pluripotent cells derived from non- pluripotent cells. Such cells have considerable promise because they carry the therapeutic and diagnostic potential of embryonic stem cells, but can be generated without the ethical complications associated the destruction of human embryos. Furthermore, since iPS cell- based therapies can be performed using previously differentiated cells from the particular subject to be treated, such therapies carry a reduced risk for transplant rejection or graft versus host disease compared to similar therapies that employ embryonic stem cells.

Despite their great therapeutic and diagnostic potential, development of iPS-based therapies has been held back by the fact that current methods for the production of iPS cells are inefficient. Recently, researchers have demonstrated that iPS cell generation can be improved through the disruption of p53 activity. See, e.g., Hong et al, Nature 460: 1132- 1135 (2009); Li et al, Nature 460: 1136-1139 (2009); Kawamura et al, Nature 460:1140- 1144 (2009); Utikal et al, Nature 460: 1145-1148 (2009); and Marion et al, Nature 460: 1149-1153 (2009), each of which is incorporated by reference in its entirety. Because p 3 is a tumor suppressor gene, it is critical that disruption of p 3 function during iPS cell development be transient, otherwise treatment with the resulting iPS cells would likely give rise to cancer. Thus, there exists a great need for novel compositions and methods that improve production of iPS cells through the transient inhibition of p53 function.

SUMMARY

Provided herein are compositions and methods for the improved production of iPS cells by enhancing degradation of p53 non-pluripotent cell populations. Also provided are novel compositions and methods of treating or preventing cancer, pancreatitis or disease caused by an intracellular pathogen.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 relates to identification of a small molecule inhibitor of autophagy by an image-based screen. A, structure of MBCQ. B, Quantitative analysis of LC3-GFP spot number per cell (a), spot size per cell (b), spot intensity per cell (c). The data are expressed as % of control vehicle treated cells. H4-LC3 cells were seeded in 96 well-plates and incubated with vehicle control (1% DMSO), 0.2μΜ rapamycin with or without ΙΟμΜ MBCQ for indicated time, fixed with 4% paraformaldehyde and stained with 4,6- diamidino-2-phenylindole (DAPI, 3μg/ml). Images of 1000 cells for each compound treatment were analyzed by ArrayScan HCS 4.0 Reader with a 20x objective (Cellomics, Pittsburgh, Pennsylvania).

Figure 2 depicts results relating to MBCQ inhibition of autophagy induced by starvation. Quantitative measurement of LC3-GFP spot number per cell (a), spot size per cell (b) and spot intensity per cell (c) using HCS and expressed as % of control. 3 -MA (1 OmM) or wortmannin (0.1 μΜ) were used as a positive control.

Figure 3 depicts electron microscopy analysis of the effect of MBCQ on autophagy. H4 cells were treated with 0.1% DMSO (vehicle), rapamycin (0.2μΜ), MBCQ (10μΜ), or MBCQ and rapamycin for 4h. The cells were processed and imaged by EM.

Figure 4 depicts approaches to the generation of MBCQ derivatives.

Figure 5 depicts results related to showing that active derivatives of MBCQ reduce the levels of LC3II in MEF cells. A, MEF cells were treated with DMSO (l%o), rapamycin (0.2μΜ) alone, or together with MBCQ (10μΜ), C43 (spautin) (10μΜ) or C71 (10μΜ), for 4h. The cell lysates were collected for western blotting using anti-LC3 antibody. B,

Electron microscopy confirmation of the autophagy inhibitory effects of C43 (spautin) on MEF cells. MEF cells were treated with vehicle control (l%o DMSO), and other indicated compounds for 4h. Rapamycin (0.2 μΜ) and C43 (spautin) (10 μΜ). Then the cells were fixed with glutaraldehyde and prepared the sample for EM assay. Bar, 1 : 11,000. Arrows indicate double and multi-membrane autophagosomic vesicles. : nucleus.

Figure 6 depicts results showing that MBCQ has little effect on H4 cell growth. A, H4 cells were treated with MBCQ (5 μΜ) for 5 days and harvested daily for cell number counting in the presence of trypan blue; B, H4 cells were treated with MBCQ (5 μΜ) for 24 h and 48 h, and then cells were fixed with 70% ethanol, stained with propidium iodide (40 μg/mL) and incubated with RNase (200 μg mL solution for 30 min. The cell cycle profile and possible apoptotic cell death were analyzed by flow cytometer.

Figure 7 depicts results showing that MBCQ and C43 (spautin) partially inhibit cell death of bax/bak D O cells induced by etoposide. A-C, Bax/bak DKO cells were treated with MBCQ (10 μΜ), or 3-MA (10 mM) in the presence of or absent etoposide (8 μΜ) for 8h or 24h. A, Cell survival as demonstrated by images. B, cell survival as demonstrated by MTT assay. C, cells were collected for western blotting using anti-LC3 antibody, a-tubulin was used as a control. D-F, Bax bak DKO cells were treated with spautin (10μΜ) or indicated concentration, in the presence of or absent etoposide (8μΜ) for 8h or indicated time. D, Cell survival as demonstrated by images and E, MTT assay. F, Cells were collected for western blotting using anti-LC3 antibody, α-tubulin was used as a control.

Figure 8 depicts results showing that MBCQ and C43 (spautin) reduce FYVE-RFP spots, but have no effect on the protein levels of FYVE-RFP. H4-FYVE cells were treated with DMSO (0.1%), MBCQ (10 μΜ) or C43 (spautin) (10μΜ) for indicated time. A, The images were analyzed by fluorescence microscopy and quantified by HCS after fixing in 4% paraformaldehyde and stained with 4,6-diamidino-2-phenylindole (DAPI, 3 μg/mL). Images of 1000 cells for each compound treatment were analyzed by ArrayScan HCS 4.0 Reader with a 20x objective (Cellomics, Pittsburgh, Pennsylvania). B, H4-FYVE cells were treated with DMSO (0.1%), RAPA (0.2 μΜ) alone, MBCQ (10μΜ) or C43 (spautin) (10μΜ) with or without RAPA (0.2uM) for 8h. The cell lysates were collected for western blotting using anti-RFP and anti-tubulin as a loading control.

Figure 9 depicts results showing that MBCQ and C43 (spautin) selectively reduce the cellular levels of PtdIns3P. MEF cells were treated with DMSO (0.1%), RAPA (0.2 μΜ) alone, A, MBCQ (10 μΜ) or B, C43 (spautin) (10 μΜ) with or without RAPA (0.2 μΜ) for 3h. The cellular Ptdlns species were extracted and applied onto polyvinylidene fluoride membrane. The levels of PtdIns3P were detected using GST-PX domain protein and anti-GST antibody.

Figure 10 depicts results showing that C43 (SPAYTIN) and its active derivatives selectively promote the degradation of Beclinl/Vps34/pl50 complex. A, C43 (spautin) is not a direct inhibitor of Vps34 enzymatic activity. The exogenous HA-Vps34 complex immunoprecipitated using anti-HA from 293T was incubated with Ptdlns in the presence of 2P-ATP in the absence or presence of indicated concentrations of C43 (spautin) and wortmannin (10 uM) for 10 min at room temperature. The product was analyzed by thin layer chromatography and autoradiography. In lane 1 , reaction buffer was used as negative control instead of Vps34/Beclin- 1 complex. B, Treatment of MBCQ, C29 and C43 (spautin) reduced the levels of exogenous Vps34 and Beclinl. 293T cells were transfected with HA-Vps34 and flag-Beclinl expression vectors. Twenty-four hours after the transfection, the cells were treated with indicated compounds for 12h. The cell lysates were analyzed by western blotting using anti-HA, anti-flag or anti-tubulin. C, MBCQ and C43 (spautin) reduce the levels of GFP-P150 protein. 293T cells were transfected with GFP- P150 vector. Twenty-four h after the transfection, the cells were treated with MBCQ (10 μΜ), C43 (spautin) (10 μΜ) for an additional 4h. The cell lysates were analyzed by western blotting using anti-GFP or anti-tubulin. D, MBCQ and C43 (spautin) reduce the levels of myc-Atgl4 protein. 293T cells were transfected with myc-Atgl4 vector. Twenty- four h after the transfection, the cells were treated with MBCQ (10 uM), C43 (spautin) (10 μΜ) for an additional 4h. The cell lysates were analyzed by western blotting using anti-myc or anti-tubulin. E, H4 cells were treated with Rapamycin (0.2 μΜ) with or without C43 (spautin) (10 μΜ) or 3-MA(10 mM) for 4 hrs, and DMSO (l o) was used as negative control. The cell lysates were harvested and analyzed by western blotting using: anti- Beclinl, anti-Atgl4, anti-Vps34 and anti-UVRAG. Anti-ot-tubulin was used as loading controls. F, 293T cells were treated with MBCQ or spautin in the presence of CHX to inhibit protein synthesis for indicated hrs and the cell lysates were analyzed by western blotting using anti-Beclinl . CHX (5μΜ), MBCQ (10μΜ), C43 (spautin) (10 μΜ). G, H4 cells were treated with Rapamycin (0.2 μΜ) with or without spautin (10μΜ) or 3-MA (lOmM) for 4 hrs, and DMSO (l%o) was used as negative control. The cell lysates were harvested and analyzed by western blotting using: anti-Beclinl and anti-LC3. Anti-a-tubulin was used as loading controls. H-M, 293T cells were transfected with indicated vectors. Twenty-four h after the transfection, the cells were treated with MBCQ (10 μΜ), C43 (spautin) (10 μΜ) or Rapamycin (0.2 μΜ) for an additional 4h. The cell lysates were analyzed by western blotting using indicated antibodies.

Figure 11 depicts results showing that selected cancer cell lines are sensitive to MBCQ and its active derivatives under glucose free condition. BT549 cells were treated with indicated concentrations of C43 for 24h in normal DMEM (A) or under serum free condition(B). The cell viability was assayed by MTT or harvested for western blotting assay with anti-LC3(C). MCF-7 cells were treated with DMSO (l%o), C43(^M) in DMEM with (D)or without (E ) glucose, for 12h. The cell viability was assayed by MTT or images(F). And the cell lysates were analyzed by western blotting using anti-LC3 and a- tubulin was used as a loading control (G). Bcap-37 cells were treated with indicated concentrations of C43 for 24h in normal DMEM (H) or under serum free condition(I). The cell viability was assayed by MTT or images (J) And the cell lysates treated with C43 for indicated time were analyzed by western blotting using anti-PARP (L) or anti-LC3 (M)and a-tubulin was used as a loading contro.(K) Cell cycle profile of Bcap-37 treated with C43. Bcap-37 cells were treated with DMSO (0.1%) (left figure), C43 (ΙΟμΜ) (right figure) for 12h. The cells were then fixed with 70% ethanol, stained with propidium iodide (PI, 40 μg/mL) and treated with RNase enzyme (200 μg mL) solution for 30min in dark. Cell cycle profile and possible apoptotic death were statistics analyzed by flow cytometer.

Figure 12 depicts the results showing of experiments showing that spautin does not induce apoptosis in non-cancer cells. A-B, MDCK cells were treated with DMSO (l%o) and spautin at indicated concentration in DMEM with or without glucose for 24h. Cell survival as demonstrated by images (A) and MTT assay (B). C-D, Hs578Bst cells were treated with DMSO (l%o) and C43 as indicated concentration in DMEM with or without glucose for 24h. Cell survival as demonstrated by images (C) and MTT assay (D).

Figure 13 depicts results showing the effect of MBCQ and derivatives in vivo. (A) Mice were injected with rapamycin (10 mg/kg) alone as a positive control, or with C43 or MBCQ (40 mg/kg) intraperitoneally every hour for 4 h and then sacrificed at 5th h. The autophagy levels in liver were analyzed by western blotting using anti-LC3 antibody. (B) C43 reduces the levels of autophagy induced by cerulein. Rats were injected

intraperitoneally with cerulein (50 g/kg) alone or with C43 (40 mg/kg) hourly for 4 times. The rats were sacrificed at one h after the last injection and the pancreas were isolated for western blotting analysis using anti-LC3 and anti-tubulin (as a control).

Figure 14 depicts MBCQ derivatives that can inhibit autophagy. To calculate EC5₀,

H4-LC3 cells were seeded in 96 well-plates and cultured in the presence of compounds in different concentration for 24h, and then fixed with polyformate and stained with 4,6- diamidino-2-phenylindole (DAPI, 3 μg/ml). Images data were collected with an ArrayScan HCS 4.0 Reader with a 20x objective (Cellomics, Pittsburgh, Pennsylvania) for DAPI labeled nuclei and GFP-LC3, a marker for autophagy. The Spot Detector Bio-Application was used to acquire and analyze the images after optimization. Images of 1000 cells for each compound treatment were analyzed to obtain average cell number per field, fluorescence spot number, area and intensity per cell. DMSO and rapamycin were used as negative or positive control, respectively. The percentages of changes of LC3-GFP were calculated by dividing with that of DMSO treated samples. Each treatment was done in triplicate for mean and SD. The images were also analyzed using a conventional fluorescence microscope for visual inspection. The experiments were repeated three times Figure 15 depicts MBCQ derivatives with reduced or no ability to inhibit autophagy. To calculate EC50, H4-LC3 cells were seeded in 96 well-plates and cultured in the presence of compounds in different concentration for 24h, and then fixed with polyformate and stained with 4,6-diamidino-2-phenylindole (DAPI, 3 μg/ml). Images data were collected with an ArrayScan HCS 4.0 Reader with a 20x objective (Cellomics, Pittsburgh, Pennsylvania) for DAPI labeled nuclei and GFP-LC3, a marker for autophagy. The Spot Detector Bio-Application was used to acquire and analyze the images after optimization. Images of 1000 cells for each compound treatment were analyzed to obtain average cell number per field, fluorescence spot number, area and intensity per cell. DMSO and rapamycin were used as negative or positive control, respectively. The percentages of changes of LC3-GFP were calculated by dividing with that of DMSO treated samples. Each treatment was done in triplicate for mean and SD. The images were also analyzed using a conventional fluorescence microscope for visual inspection. The experiments were repeated three times.

Figure 16 depicts results of experiments showing that spautin promotes the degradation of Beclinl through proteasomal pathway. A, 293T cells were transfected with GFP-Beclinl and 24 hr after the transfection, the cells were treated with indicated compounds for an additional 24 hr. DMSO (l%o),MBCQ (10^iM),spautin (10μΜ), NH4C1 (lOmM), MG132 (5μΜ). The cell lysates were analyzed by western blotting using anti- GFP. B, 293T cells were transfected with GFP-Beclinl and HA-Ub expression vectors. Twenty- four hours after the transfection, the cells were treated with MG132 or spautin for 24 hours. The cell lysates were immunoprecipitated with anti-GFP antibody and the immunocomplexes were analyzed by western blotting using anti-HA antibody.

Figure 17 depicts the results of experiments demonstrating the effect of siRNA knockdown of USP3, USP10, USP13, USP16 and USP18 on the stability of selected autophagy proteins. H4 cells were transfected with indicated siR As for 72 hrs or treated with rapamycin (0.2 μΜ) or spautin (ΙΟμΜ) for 4 hrs, and non-target siRNA (N. T. siRNA) was used as negative control. The cell lysates were harvested and analyzed by western blotting using (Left): antibodies specific for the indicated proteins. Anti-a-tubulin was used as loading controls.

Figure 18 depicts the results of experiments demonstrating the effect of siRNA knockdown of USP3, USP10, USP13, USP16 and USP18 on the stability of USP proteins. H4 cells were transfected with indicated siRNAs for 72 hrs or treated with rapamycin (0.2 μΜ) or spautin (ΙΟμΜ) for 4 hrs, and non-target siRNA (N. T. siRNA) was used as negative control. The cell lysates were harvested and analyzed by western blotting using (Left): antibodies specific for the indicated proteins. Anti-a-tubulin was used as loading controls.

Figure 19 depicts the results of experiments demonstrating the effect of siRNA knockdown of USP3, USP10, USP13, USP 16, USP 18 and Beclinl on the stability of P53. H4 cells were transfected with the indicated siRNAs (3 for each USP) and treated with Rapamycin (0.2 μΜ) for 4 hrs and DMSO (1%) was used as a negative control. The cell lysates were harvested and analyzed by western blotting using: anti-p53 antibody or other indicated antibody. Anti-a-tubulin was used as loading controls.

Figure 20 depicts the results of experiments demonstrating that GFP-USP 10 and Myc-USP13 could indeed interact and that the interaction was inhibited in spautin-treated cells. 293T cells were transfected with GFP-USP10 (lane 1-4), Myc-USP13 (lane 2-4), MG132 (lane 3-4) and/or spautin (lane 4). The lysates were immunoprecipitated with anti- GFP antibody and the immunocomplexes were analyzed by western blot with the indicated antibody.

Figure 21 depicts the regulation of p53 by Vps34 complexes, USP10 and USP13. (A, C- F) H4 cells were transfected with indicated siRNAs for 72 hrs and treated with rapamycin (0.25 μΜ) or spautin- 1 (10 μΜ) for 4 h. The cell lysates were analyzed by western blotting using indicated antibodies. Anti-a-tubulin is a loading control. (B) H4 cells were incubated with MG132 (10 μΜ), NH₄C1 (lOmM) and spautin- 1 (10 μΜ) for 6 h. The cell lysates were analyzed by western blotting using indicated antibodies. Anti-tubulin is a control. (G) Heart and lung tissue of newborn BECN+/+ and BECN+/- mice were isolated and analyzed by western blotting using indicated antibodies. Anti-actin was used as a loading control.

Figure 22 depicts a H NMR spectra of A9.

Figure 23 depicts a Η NMR spectra of A30.

Figure 24 depicts a 1H NMR spectra of A36.

Figure 25 depicts the autophagy inhibition activity of two inventive compounds. Figure 26 depicts that Spautin-1 induces the death of selected cancer cell lines under non- proliferating conditions. (A) H4, MDA-MB-231 and MCF-7 cells were treated with 1 μΜ, 10 μΜ or 20 μΜ of spautin- 1 for 5 days and cell viability was determined with MTT assay. (B-D) MDA- MB-231 cells, SUM 159 cells and SKOV3 cells were treated with 20 μΜ of spautin-1 for indicated periods of time and imaged with phase contrast microscopy. (E) Indicated cell lines were treated with 20μΜ spautin-1 for 6 days and the cell viability was determined using ATP assay.

DETAILED DESCRIPTION

In an image-based screen for small molecule regulators of autophagy, an autophagy inhibitor, MBCQ, was identified. Extensive medicinal chemistry modification of MBCQ identified new derivatives, such as C43. In certain instances herein C43 is referred to as "spautin" (Specific and Potent Autophagy Inhibitor).

Autophagy, a cellular catabolic process, plays an important role in promoting cell survival under metabolic stress condition by mediating lysosomal-dependent turnover of intracellular constituents for recycling. Inhibition of autophagy has been proposed as a therapeutic strategy for the treatment of cancer, pancreatitis and intracellular pathogens. The compounds disclosed herein are therefore useful in the treatment of such diseases and disorders. Thus, in certain embodiments the instant invention relates to compositions and methods for the treatment of cancer, pancreatitis and intracellular pathogens.

Further characterization of MBCQ and the new derivatives revealed that the compounds also inhibited the deubiquitination of p53 by USP10. Blocking this

deubiquitination process resulted in enhanced p53 degradation and reduced levels of p53 in treated cells. It has recently been demonstrated that the efficiency of standard methods for the generation of iPS cells from non-pluripotent cells can be improved by blocking the p53 function in the non-pluripotent cells. The compounds described herein are therefore useful for enhancing iPS cell generation. Thus, in certain embodiments, the invention relates to methods of producing iPS cells that include the steps of: a) administering to non-pluripotent cells a p53 degradation enhancing compound described herein and b) culturing the cells under conditions and for a period of time such that at least a portion of the non-pluripotent cells become pluripotent.

Definitions

For convenience, certain terms employed in the specification, examples, and appended claims are collected here. All definitions, as defined and used herein, supersede dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non- limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.

This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non- limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

The definition of each expression, e.g., alkyl, m, n, and the like, when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

As used herein, "Oct4" refers to a naturally-occurring members of Octamer family of transcription factors, or variants thereof that maintain transcription factor activity, similar (within at least 50%, 80%, or 90% activity) to that of Oct4. In some embodiments, variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Oct4 such as listed in Genbank accession number NP 002692.2 (human Oct4) or NP 038661.1 (mouse Oct4).

As used herein, "Klf4" refers to a naturally-occurring member of the family of Kruppel-like factors (Klfs), zinc-finger proteins that contain amino acid sequences similar to those of the Drosophila embryonic pattern regulator Kruppel, or variants thereof that have transcription factor activity similar (within at least 50%>, 80%, or 90% activity) to that of Klf4. In some embodiments, variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Klf polypeptide family member such as to those listed in Genbank accession number CAX16088 (mouse Klf4) or CAX14962 (human Klf4).

As used herein, "Sox2" refers to a naturally-occurring member of the family SRY- related HMG-box (Sox) transcription factors, or variants thereof that have transcription factor activity similar (within at least 50%, 80%, or 90% activity) to that of Sox2. In some embodiments, variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Sox2, such as to those listed in Genbank accession number CAA83435 (human Sox2).

As used herein, "c-Myc" refers to a naturally-occurring member of members of the Myc family, or variants thereof that have transcription factor activity similar (within at least 50%, 80%, or 90% activity) to that of c-Myc. In some embodiments, variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring c-Myc, such as to those listed in Genbank accession number CAA2501 (human Myc).

As used herein, the term "pluripotent" or "pluripotency" refers to cells with the ability to give rise to progeny that can undergo differentiation, under the appropriate conditions, into cell types that collectively demonstrate characteristics associated with cell lineages from all of the three germinal layers (endoderm, mesoderm, and ectoderm).

Pluripotent stem cells can contribute to many or all tissues of a prenatal, postnatal or adult animal. A standard art-accepted test, such as the ability to form a teratoma in 8-12 week old SCID mice, can be used to establish the pluripotency of a cell population, however identification of various pluripotent stem cell characteristics can also be used to detect pluripotent cells.

It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.

The term "substituted" is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein below. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds. When "one or more" substituents are indicated, there may be, for example, 1, 2, 3, 4 or 5 substiuents.

The term "lower" when appended to any of the groups listed below indicates that the group contains less than seven carbons (i.e., six carbons or less). For example "lower alkyl" refers to an alkyl group containing 1-6 carbons.

For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The term "alkyl" means an aliphatic or cyclic hydrocarbon radical containing from 1 to 20, 1 to 15, or 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n- pentyl, isopentyl, neopentyl, n-hexyl, 2-methylcyclopentyl, and 1-cyclohexylethyl. The term "fluoroalkyl" means an alkyl wherein one or more hydrogens are replaced with fluorines.

The term "alkoxy" means an alkyl group bound to the parent moiety through an oxygen. The term "fluoroalkoxy" means a fluoroalkyl group bound to the parent moiety through an oxygen.

Selected Autophagy Inhibitors and Enhancers of p53 Degradation

One aspect of the invention relates to a compound represented by formula I:

I

or a pharmaceutically acceptable salt, biologically active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, wherein

n is 0, 1, 2, 3 or 4;

Y is -C(R¹)= or -N=; R is -H, lower alkyl, -CH₃, lower fluoroalkyl, -CH₂F, -CHF₂, -CF₃, -N0₂, -OH, -NH₂, -NH(lower alkyl), -N(lower alkyl)₂, or lower alkynyl;

R¹ is independently selected for each occurrence from the group consisting of -H, -F, -CI, -Br, -I, -NO2, -OH, -NH₂, -NH(lower alkyl), -N(lower alkyl)₂, -CH₃, -CF₃, - C(=0)(lower alkyl), -CN, -0(lower alkyl), -0(lower fluoroalkyl), -S(=0)(lower alkyl), -S(=0)₂(lower alkyl) and -C(=0)0(lower alkyl);

R² and R³ are independently selected from the group consisting of -H, lower alkyl, lower fluoroalkyl, lower alkynyl and hydroxyalkyl;

X is -0-, -S-, -N(H , -N(lower alkyl)-, -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-.

-CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂- or -CH₂CH₂CH₂CH₂CH₂CH₂-; and

Z is phenyl, pyridyl, vinyl, morphinyl, phenanthrolinyl, naphthyl, furyl or benzo[d]thiazolyl; and optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -0CF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -NO2, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4- methylene dioxy, and -C(=0)0(lower alkyl).

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, provided that the compound is not

or , wherein J is CI, OCHF₂,

OCH₂CH₃, OCH₂CF₃, 0(CH₂)₂CH₃, OCH(CH₃)₂, 0(CH₂)₃CH₃, or O(cyclopentyl).

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein n is 0. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein n is 1. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein n is 2. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein n is 3. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein n is 4.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Y is -C(R¹)=. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Y is -C(H)=.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R is -N=.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R is -H.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R is lower alkyl or lower fluoroalkyl.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R is -CH₃.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R is -CH2F, -CHF2 or -CF3.In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein at only one R¹ is -H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein only two R¹ are - H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein only three R¹ are -H.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein at least one R¹ is -NH₂, -CI, -N0₂, -I, or - OMe. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein at one R¹ is -NH₂, -CI, -NO₂, -I, or -OMe; and at least two R¹ are -H.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -CH3. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -H. In certain embodiments, the invention relates to any of the

aforementioned compounds and attendant definitions, wherein R² is hydroxyalkyl..

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R³ is -CH₃. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R³ is -H. In certain embodiments, the invention relates to any of the

aforementioned compounds and attendant definitions, wherein R³ is hydroxyalkyl.. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -Ci¼_; and R³ is H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -H; and R³ is -H.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein X is -0-, -S-, -N(H)-, -N(lower alkyl)- or - CH₂-. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein X is -N(H)- or -N(lower alkyl)-. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein X is -N(H)-.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein n is 0 or 1; X is -N(H)-; R² is -H; R³ is -H; and R is -H.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is 4-pyridyl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF3, and 3,4-methylene dioxy.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is morphinyl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, and 3,4-methylene dioxy.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is 2-furyl, optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH3, -OCF3, lower fluoroalkoxy, -F, -CI, -Br, -I, -NO2, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, and 3,4-methylene dioxy.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is 1-naphthyl or 2-napthyl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, and 3,4-methylene dioxy. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is benzo[d]thiazol-5-yl or benzo[d]thiazol- 6-yl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH , -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH (lower alkyl), -N(lower alkyl)₂, -CF₃, and 3,4-methylene dioxy.In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is phenyl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, and 3,4-methylene dioxy.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein n is 0 or 1 ; and Z is phenyl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH , -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4-methylene dioxy, and -C(=0)0(lower alkyl).

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein n is 0 or 1; X is -N(H)-; and Z is phenyl optionally substituted with one or more substitutents selected from the group consisting of - CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH (lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4-methylene dioxy, and - C(=0)0(lower alkyl).

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein n is 0 or 1; X is -N(H)-; R² is -H; R³ is -H; and Z is phenyl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4-methylene dioxy, and -C(=0)0(lower alkyl).

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein n is 0 or 1; X is -N(H)-; R² is -H; R³ is -H; R is -H; and Z is phenyl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4- methylene dioxy, and -C(=0)0(lower alkyl). One aspect of the invention relates to a compound represented by formula II:

II

n is 0, 1, 2, 3 or 4;

R is -H, lower alkyl, -CH₃, lower fluoroalkyl, -CH₂F, -CHF₂, or -CF₃;

R¹ is independently selected for each occurrence from the group consisting of -H, - CH₃, -F, -CI, -Br, -I, and -N0₂;

R² and R³ are independently selected from the group consisting of -H, -CH₃ -CH2CH3, -CH2CH2CH3, and -CH(CH₃)₂;

R⁴, R⁵ and R⁸ are independently selected from the group consisting of -H, -CH₃, -CF₃, -OCH₃, -OCF₃, -F, -CI, -Br, and -I; and

R⁶ and R⁷ are independently selected from the group consisting of -H, -CH₃, -CF₃, -OCH₃, -OCF₃, -F, -CI, -Br, -I, and -C(=0)0(lower alkyl); or R⁶ and R⁷ taken together are OCH₂0-.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Y is -C(R¹)=.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Y is -C(H)=.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R is -CH₂F, -CHF₂ or -CF₃.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R¹ is -F. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R¹ is -CI. In certain embodiments, the invention relates to any of the

aforementioned compounds and attendant definitions, wherein R¹ is -Br. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R¹ is -I. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R¹ is -N0₂. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R¹ is -CH₃.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -CH_3, -CH₂CH₃, -CH₂CH₂CH₃ or -CH(CH₃)₂. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -CH₃.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R³ is -H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R³ is -CH₃, -CH₂CH₃, -CH₂CH₂CH₃ or -CH(CH₃)₂. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R³ is -CH₃.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁴ is -H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁴ is -F. In certain embodiments, the invention relates to any of the

aforementioned compounds and attendant definitions, wherein R⁴ is -CI. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁴ is -CH₃. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁴ is -OCH₃.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁵ is -H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁵ is -F. In certain embodiments, the invention relates to any of the

aforementioned compounds and attendant definitions, wherein R⁵ is -CI. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁵ is -CH₃. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁵ is -OCH₃.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -H, -F, -CI, -Br or -I. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -F. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -CI. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -Br. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -CH₃. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -CF₃. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -OCH₃. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -OCF₃. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -C(=0)0(lower alkyl). In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is - C(=0)OCH₃.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ and R⁷ taken together are -OCH₂O-.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁷ is -H, -F, -CI, -Br or -I. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁷ is -H.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁸ is -H.

One aspect of the invention relates to a compound, or a pharmaceutically acceptable salt, biologically active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer



One aspect of the invention relates to or a

pharmaceutically acceptable salt, biologically active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer thereof.

One aspect of the invention relates to a compound represented by formula III:

III

n is 0, 1, 2, 3 or 4;

Y is -C(R¹)= or -N=;

R is independently selected for each occurrence from the group consisting of -H, lower alkyl, -CH₃, lower fluoroalkyl, -CH₂F, -CHF₂, -CF₃, -N0₂, -OH, -NH₂, -NH(lower alkyl), -N(lower alkyl)₂, and lower alkynyl;

R¹ is independently selected for each occurrence from the group consisting of -H, -F, -CI, -Br, -I, -N0₂, -OH, -NH₂, -NH(lower alkyl), -N(lower alkyl)₂, -CH₃, -CF₃, - C(=0)(lower alkyl), -CN, -0(lower alkyl), -0(lower fluoroalkyl), -S(=0)(lower alkyl), -S(=0)₂(lower alkyl) and -C(=0)0(lower alkyl);

R² and R³ are independently selected from the group consisting of -H, lower alkyl, lower fluoroalkyl, lower alkynyl and hydroxyalkyl; X is -0-, -S-, -N(H , -N(lower alkyl)-, -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-.

Z is phenyl, pyridyl, vinyl, morphinyl, phenanthrolinyl, naphthyl, fiiryl or benzo[d]thiazolyl; and optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -NO2, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4- methylene dioxy, and -C(=0)0(lower alkyl).

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R is -CH3.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R is -CH₂F, -CHF₂ or -CF₃.In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein at only one R¹ is -H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein only two R¹ are - H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein only three R¹ are -H.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein at least one R¹ is -NH₂, -CI, -N0₂, -I, or - OMe. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein at one R¹ is -NH₂, -CI, -N0₂, -I, or -OMe; and at least two R¹ are -H.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -Ct¾. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -H. In certain embodiments, the invention relates to any of the

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R³ is -Ct¼. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R³ is -H. In certain embodiments, the invention relates to any of the

aforementioned compounds and attendant definitions, wherein R³ is hydroxyalkyl..

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -Ct¾_; and R³ is H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -H; and R³ is -H.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is 4-pyridyl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -NO₂, lower alkoxy, -NH(lower alkyl),

-N(lower alkyl)₂, -CF₃, and 3,4-methylene dioxy.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is morphinyl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH3, -OCF3, lower fluoroalkoxy, -F, -CI, -Br, -I, -NO2, lower alkoxy,

-NH(lower alkyl), -N(lower alkyl)₂, -CF₃, and 3,4-methylene dioxy.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is 2-furyl, optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl,

-OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl),

-N(lower alkyl)₂, -CF3, and 3,4-methylene dioxy.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is 1-naphthyl or 2-napthyl optionally substituted with one or more substitutents selected from the group consisting of -CH3, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy,

-NH(lower alkyl), -N(lower alkyl)₂, -CF₃, and 3,4-methylene dioxy.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is benzo[d]thiazol-5-yl or benzo[d]thiazol- 6-yl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH (lower alkyl), -N(lower alkyl)₂, -CF₃, and 3,4-methylene dioxy. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is phenyl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, and 3,4-methylene dioxy.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein n is 0 or 1 ; and Z is phenyl optionally substituted with one or more substitutents selected from the group consisting of -CH3, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy,

-NH(lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4-methylene dioxy, and -C(=0)0(lower alkyl). In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein n is 0 or 1; X is -N(H)-; and Z is phenyl optionally substituted with one or more substitutents selected from the group consisting of - CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH (lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4-methylene dioxy, and - C(=0)0(lower alkyl).

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein n is 0 or 1; X is -N(H)-; R² is -H; R³ is -H; R is -H; and Z is phenyl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4- methylene dioxy, and -C(=0)0(lower alkyl).

One aspect of the invention relates to a compound represented by formula IV:

IV

n is 0, 1, 2, 3 or 4;

Y is -C(R^X)= or -N=;

R is independently selected for each occurrence from the group consisting of -H, lower alkyl, -CH₃, lower fluoroalkyl, -CH₂F, -CHF₂, and -CF ;

R¹ is independently selected for each occurrence from the group consisting of -H, CH₃, -F, -CI, -Br, -I, and -N0₂; R² and R³ are independently selected from the group consisting of -H, -CH_3, -CH₂CH₃, -CH₂CH₂CH₃, and -CH(CH₃)₂;

R⁶ and R⁷ are independently selected from the group consisting of -H, -CH₃, -CF₃,

-OCH₃, -OCF₃, -F, -CI, -Br, -I, and -C(=0)0(lower alkyl); or R⁶ and R⁷ taken together are - OCH₂0-.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R¹ is -F. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R¹ is -CI. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R¹ is -Br. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R¹ is -I. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R¹ is - O₂. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R¹ is -CH3.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -CH₃, -CH₂CH₃, -CH₂CH₂CH₃ or -CH(CH₃)₂. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R² is -CH₃.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R³ is -H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R³ is -CH_3, -CH₂CH₃, -CH₂CH₂CH₃ or -CH(CH₃)₂. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R³ is -CH₃.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -H, -F, -CI, -Br or -I. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -H. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -F. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -CI. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -Br. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -CH3. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -CF₃. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -OCH3. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -OCF₃. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is -C(=0)0(lower alkyl). In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein R⁶ is - C(=0)OCH₃.

In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherei ⁸ is -H.

One aspect of the invention relates to

, or a

pharmaceutically acceptable salt, biologically active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer thereof. In certain embodiments, the invention relates to any of the aforementioned compounds and attendant definitions, wherein the compound is an autophagy inhibitor; and the EC5₀ of the autophagy inhibitor is less than about 100 nM.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound inhibits autophagy with an IC50 of less than about 10 μΜ. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound inhibits autophagy with an IC5₀ of less than about 5 μΜ. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound inhibits autophagy with an IC5₀ of less than about 1 μΜ. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound inhibits autophagy with an IC50 of less than about 750 nM. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound inhibits autophagy with an IC5₀ of less than about 500 nM. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound inhibits autophagy with an IC5₀ of less than about 250 nM. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound inhibits autophagy with an IC50 of less than about 100 nM.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound is an inhibitor of autophagy; and the compound does not inhibit PDE5.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound inhibits both autophagy and PDE5 the compound has an autophagy IC5₀ of between about 0.001 μΜ and about 10 uM; and the ratio of the PDE5 IC5₀ to the autophagy IC5₀ is between about 10 and about 50. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound inhibits both autophagy and PDE5; the compound has an autophagy IC5₀ of between about 0.001 μΜ and about 10 μΜ; and the ratio of the PDE5 IC5₀ to the autophagy IC5₀ is between about 50 and about 100. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound inhibits both autophagy and PDE5; the compound has an autophagy IC5₀ of between about 0.001 μΜ and about 10 μΜ; and the ratio of the PDE5 IC₅₀ to the autophagy IC₅₀ is between about 100 and about 1,000. Certain compounds of the invention which have acidic substituents may exist as salts with pharmaceutically acceptable bases. The present invention includes such salts. Examples of such salts include sodium salts, potassium salts, lysine salts and arginine salts. These salts may be prepared by methods known to those skilled in the art.

Certain compounds of the invention and their salts may exist in more than one crystal form and the present invention includes each crystal form and mixtures thereof.

Certain compounds of the invention and their salts may also exist in the form of solvates, for example hydrates, and the present invention includes each solvate and mixtures thereof.

Certain compounds of the invention may contain one or more chiral centers, and exist in different optically active forms. When compounds of the invention contain one chiral center, the compounds exist in two enantiomeric forms and the present invention includes both enantiomers and mixtures of enantiomers, such as racemic mixtures. The enantiomers may be resolved by methods known to those skilled in the art, for example by formation of diastereoisomeric salts which may be separated, for example, by

crystallization; formation of diastereoisomeric derivatives or complexes which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support for example silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where the desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step may be used to liberate the desired enantiomeric form. Alternatively, specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.

When a compound of the invention contains more than one chiral center, it may exist in diastereoisomeric forms. The diastereoisomeric compounds may be separated by methods known to those skilled in the art, for example chromatography or crystallization and the individual enantiomers may be separated as described above. The present invention includes each diastereoisomer of compounds of the invention and mixtures thereof.

Certain compounds of the invention may exist in different tautomeric forms or as different geometric isomers, and the present invention includes each tautomer and/or geometric isomer of compounds of the invention and mixtures thereof.

Certain compounds of the invention may exist in different stable conformational forms which may be separable. Torsional asymmetry due to restricted rotation about an asymmetric single bond, for example because of steric hindrance or ring strain, may permit separation of different conformers. The present invention includes each conformational isomer of compounds of the invention and mixtures thereof.

Certain compounds of the invention may exist in zwitterionic form and the present invention includes each zwitterionic form of compounds of the invention and mixtures thereof.

As used herein the term "pro-drug" refers to an agent which is converted into the parent drug in vivo by some physiological chemical process (e.g., a prodrug on being brought to the physiological pH is converted to the desired drug form). Pro-drugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmacological compositions over the parent drug. An example, without limitation, of a pro-drug would be a compound of the present invention wherein it is administered as an ester (the "pro-drug") to facilitate transmittal across a cell membrane where water solubility is not beneficial, but then it is metabolically hydrolyzed to the carboxylic acid once inside the cell where water solubility is beneficial. Pro-drugs have many useful properties. For example, a pro-drug may be more water soluble than the ultimate drug, thereby facilitating intravenous administration of the drug. A pro-drug may also have a higher level of oral bioavailability than the ultimate drug. After administration, the prodrug is enzymatically or chemically cleaved to deliver the ultimate drug in the blood or tissue.

Exemplary pro-drugs release an amine of a compound of the invention wherein the free hydrogen of an amine is replaced by (Ci-C6)alkanoyloxymethyl, l-((Ci- Ce)alkanoyloxy)ethyl, 1 -methyl- 1 -((C i-Ce)alkanoyloxy)ethyl, (C i - C6)alkoxycarbonyloxymethyl, N-(Ci-C6)alkoxycarbonylaminomethyl, succinoyl, (Ci- Ce)alkanoyl, a-amino(Ci-C4)alkanoyl, arylactyl and -aminoacyl, or a-aminoacyl-oc- aminoacyl wherein said a-aminoacyl moieties are independently any of the naturally occurring L-amino acids found in proteins, -P(0)(OH)₂, -P(0)(0(Ci-C6)alkyl)₂ or glycosyl (the radical resulting from detachment of the hydroxyl of the hemiacetal of a carbohydrate). I. Production of Induced Pluripotent Stem Cells

In certain embodiments, the invention relates to methods of producing iPS cells which include the steps of: a) administering to non-pluripotent cells a p53 degradation enhancing compound described herein and b) culturing the cells under conditions and for a period of time such that at least a portion of the non-pluripotent cells become pluripotent. In certain embodiments, the non-pluripotent cells are somatic cells, such as fibroblast cells. In some embodiments, the non-pluripotent cells are caused to express elevated levels of c- Myc, Klf4, Sox2 and/or Oct4. In some embodiments, the non-pluripotent cells are caused to express elevated levels of Oct4 and Sox2. In certain embodiments, the non-pluripotent cells are mammalian cells, such as human cells or mouse cells.

A large number of different methods and protocols have been established for inducing non-pluripotent mammalian cells into iPS cells. iPS cells are similar to embryonic stem cells ("ES cells") in morphology, proliferation, and pluripotency, judged by teratoma formation and chimaera contribution. The enhancers of p53 degradation described herein improve essentially any reprogramming protocol for generating iPS cells. Reprogramming protocols that can be improved include those involving introduction of one or more reprogramming transcription factors, such as Oct4, Sox2 Klf4 and/or a c-Myc. The reprogramming factors can be introduced into the cells, for example, by expression from a recombinant expression cassette that has been introduced into the target cell, or by incubating the cells in the presence of exogenous reprogramming transcription factor polypeptides such that the polypeptides enter the cell. Examples of methods for generating iPS cells can be found in U.S. Pat. Pub. Nos. 2009/0191159, 2010/0003757, 2010/0267141, 2010/0233804 and 2010/001840 1 , each of which is incorporated by reference in its entirety.

It has been demonstrated that retroviral transduction of fibroblasts with four transcription factors that are highly expressed in ES cells (Oct4, Sox2, KLF4 and c-Myc) generate iPS cells. See, Takahashi, K. & Yamanaka, S. Cell 126:663-676 (2006); Okita, K., Ichisaka, T. & Yamanaka, S, Nature 448:313-317 (2007); Wernig, M. et al. Nature 448:318-324 (2007); Maherali, N. et al. Cell Stem Cell 1 :55-70 (2007); Meissner, A., Wernig, M. & Jaenisch, R. Nature Biotechnol. 25 : 1 177-1181 (2007); Takahashi, K. et al. Cell 131 :861-872 (2007); Yu, J. et al. Science 318: 1917-1920 (2007); Nakagawa, M. et al. Nature Biotechnol. 26: 101-106 (2007); Wernig, M., Meissner, A., Cassady, J. P. &

Jaenisch, R. Cell Stem Cell. 2: 10-12 (2008), each of which are hereby incorporated by reference in their entirety. Notably, it has been demonstrated that disruption of p53 enables the production of iPS cells through transfection of only Socs2 and Oct4. Such methods are believed to be improved with the inclusion of enhancers of p53 degradation described herein.

To address the safety issues that arise from target cell genomes harboring integrated exogenous sequences, a number of modified genetic protocols have been further developed and can be used according to the present invention. These protocols produce iPS cells with reduced risks, and include non-integrating adenoviruses to deliver reprogramming genes (Stadtfeld, M., et al. Science yil^ S-^ ^ (2008)), transient transfection of reprogramming plasmids (Okita, ., et al. Science 322:949-953 (2008)), piggyBac transposition systems (Woltjen, ., et al, Nature 458:766-770 (2009), Yusa et al. Nat. Methods 6:363-369 (2009), Kaji, K., et al. Nature 458:771-775 (2009),), Cre-excisable viruses (Soldner, F. et al, Cell 136:964-977(2009)), and oriP/EBNAl-based episomal expression system (Yu, J., et al. (2009) Science DOI: 10.1126), each of which is hereby incorporated by reference in its entirety. In some embodiments, enhancers of p53 degradation described herein can be applied to any of the protocols described above.

As used herein, "non-pluripotent cells" refer to mammalian cells that are not pluripotent cells. Examples of such cells include differentiated cells as well as progenitor cells. Examples of differentiated cells include, but are not limited to, cells from a tissue selected from bone marrow, skin, skeletal muscle, fat tissue and peripheral blood.

Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T-cells.

In some embodiments where an individual is to be treated with the resulting pluripotent cells, the individual's own non-pluripotent cells are used to generate pluripotent cells according to the methods of the invention.

Cells can be from, e.g., humans or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, and bovines.

Cells to be induced to pluripotency can be cultured according to any method known in the art. General guidelines can be found in, e.g., Maherali, et al., Cell Stem Cell 3:595- 605 (2008).

In some embodiments, the cells are cultured in contact with feeder cells. Exemplary feeder cells include, but are not limited to fibroblast cells, e.g., mouse embryonic fibroblast (MEF) cells. Methods of culturing cells on feeder cells are known in the art.

In some embodiments, the cells are cultured in the absence of feeder cells. Cells, for example, can be attached directly to a solid culture surface (e.g., a culture plate), e.g., via a molecular tether. Exemplary molecular tethers include, but are not limited to, matrigel, an extracellular matrix (ECM), ECM analogs, laminin, fibronectin, or collagen. Those of skill in the art however will recognize that this is a non-limiting list and that other molecules can be used to attach cells to a solid surface. Methods for initial attachment of the tethers to the solid surface are known in the art.

II. Novel Compositions and Therapeutic Methods

Pharmaceutical Compositions

One or more compounds of this invention can be administered to a human patient by themselves or in pharmaceutical compositions where they are mixed with biologically suitable carriers or excipient(s) at doses to treat or ameliorate a disease or condition as described herein. Mixtures of these compounds can also be administered to the patient as a simple mixture or in suitable formulated pharmaceutical compositions. For example, one aspect of the invention relates to pharmaceutical composition comprising a therapeutically effective dose of a compound of formula I or II, or a pharmaceutically acceptable salt, biologically active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer thereof; and a pharmaceutically acceptable diluent or carrier.

As used herein, a therapeutically effective dose refers to that amount of the compound or compounds sufficient to result in the prevention or attenuation of a disease or condition as described herein. Techniques for formulation and administration of the compounds of the instant application may be found in references well known to one of ordinary skill in the art, such as "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition.

Suitable routes of administration may, for example, include oral, eyedrop, rectal, transmucosal, topical, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

Alternatively, one may administer the compound in a local rather than a systemic manner, for example, via injection of the compound directly into an edematous site, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with endothelial cell-specific antibody.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g.,

dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral administration by injection, e.g., bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly or by intramuscular injection). Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethysulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Many of the compounds of the invention may be provided as salts with

pharmaceutically compatible counterions (i.e., pharmaceutically acceptable salts). A

"pharmaceutically acceptable salt" means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound or a prodrug of a compound of this invention. A "pharmaceutically acceptable counterion" is an ionic portion of a salt that is not toxic when released from the salt upon administration to a recipient. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.

Acids commonly employed to form pharmaceutically acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well as organic acids such as para-toluenesulfonic, salicylic, tartaric, bitartaric, ascorbic, maleic, besylic, fumaric, gluconic, glucuronic, formic, glutamic, methanesulfonic, ethanesulfonic, benzenesulfonic, lactic, oxalic, para- bromophenylsulfonic, carbonic, succinic, citric, benzoic and acetic acid, and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,

dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4- dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephathalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, .beta.-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, naphthalene -2-sulfonate, mandelate and the like salts. Preferred pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid.

Suitable bases for forming pharmaceutically acceptable salts with acidic functional groups include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributyl amine; pyridine; N-methyl,N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkyl amines), such as mono-, bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N-di alkyl-N-(hydroxy alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine, or tri-(2- hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art.

Therapeutic Methods

One aspect the invention provides a method for inhibiting autophagy in a subject for whom inhibition of autophagy is beneficial, comprising administering to the subject a compound of the invention such that autophagy activity in the subject is altered and treatment or prevention is achieved. In certain embodiments, the subject is a human.

The term "treating" as used herein, encompasses the administration and/or application of one or more compounds described herein, to a subject, for the purpose of providing prevention of or management of, and/or remedy for a condition. "Treatment" for the purposes of this disclosure, may, but does not have to, provide a cure; rather,

"treatment" may be in the form of management of the condition. When the compounds described herein are used to treat unwanted proliferating cells, including cancers,

"treatment" includes partial or total destruction of the undesirable proliferating cells with minimal destructive effects on normal cells. A desired mechanism of treatment of unwanted rapidly proliferating cells, including cancer cells, at the cellular level is apoptosis.

The term "preventing" as used herein includes either preventing or slowing the onset of a clinically evident unwanted cell proliferation altogether or preventing or slowing the onset of a preclinically evident stage of unwanted rapid cell proliferation in individuals at risk. Also intended to be encompassed by this definition is the prevention or slowing of metastasis of malignant cells or to arrest or reverse the progression of malignant cells. This includes prophylactic treatment of those at risk of developing precancers and cancers. Also encompassed by this definition is the prevention or slowing of restenosis in subjects that have undergone angioplasty or a stent procedure.

The term "subject" for purposes of treatment includes any human or animal subject who has been diagnosed with, has symptoms of, or is at risk of developing a disorder wherein inhibition of autophagy would be beneficial. For methods of prevention the subject is any human or animal subject. To illustrate, for purposes of prevention, a subject may be a human subject who is at risk of or is genetically predisposed to obtaining a disorder characterized by unwanted, rapid cell proliferation, such as cancer. The subject may be at risk due to exposure to carcinogenic agents, being genetically predisposed to disorders characterized by unwanted, rapid cell proliferation, and so on. Besides being useful for human treatment, the compounds described herein are also useful for veterinary treatment of mammals, including companion animals and farm animals, such as, but not limited to dogs, cats, horses, cows, sheep, and pigs.

One aspect of the invention relates to a method of treating or preventing cancer, comprising the step of administering to a subject in need thereof a therapeutically effective amount of one or more compounds of formula I or II, or a pharmaceutically acceptable salt, biologically active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer thereof.

Suppression of autophagy has been proposed to be a new anticancer therapy by promoting radiosensitization and chemosensitization. In an animal model of cancer therapy, inhibition of therapy-induced autophagy either with shR A against a key autophagy gene ATG5 or with anti-malarial drug chloroquine enhanced cell death and tumor regression of Myc-driven tumors in which either activated p53 or alkylating chemotherapy was used to drive tumor cell death (Amaravadi, R. ., et al., Autophagy inhibition enhances therapy- induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest, 2007. 117(2): p. 326-36). Chloroquine causes a dose-dependent accumulation of large autophagic vesicles and enhances alkylating therapy-induced cell death to a similar degree as knockdown of ATG5. In the case of chronic myelogenous leukemia (CML), chloroquine markedly enhanced death of a CML cell line, K562, induced by imatinib. Furthermore, imatinib- resistant cell lines, BaF3/T315I and BaF3/E255K, can be induced to die by co-treatment with imatinib and chloroquine. These studies suggest that inhibiting autophagy may potentiate conventional chemotherapy.

The National Cancer Institute alphabetical list of cancer includes: Acute

Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma,

Childhood; AIDS-Related Lymphoma; AIDS-Related Malignancies; Anal Cancer;

Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma/Malignant Glioma,

Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer,

Childhood; Breast Cancer, Male; Bronchial Adenomas/Carcinoids, Childhood; Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical;

Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central Nervous System

Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral

Astrocytoma/Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma, Childhood Brain Stem; Glioma, Childhood Visual Pathway and

Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin's Lymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma,

Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic

Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS-Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T- Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's, Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin's, Adult; Lymphoma, Non- Hodgkin's, Childhood; Lymphoma, Non-Hodgkin's During Pregnancy; Lymphoma,

Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Malignant

Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplasia Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma; Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood; Non-Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer;

Oropharyngeal Cancer; Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood; Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile

Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma (Osteosarcoma)/Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer

(Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of,

Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and

Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macroglobulmemia; and Wilms' Tumor. The methods of the present invention may be useful to treat such types of cancer.

Another aspect of the invention relates to a method of treating or preventing acute pancreatitis, comprising the step of administering to a subject in need thereof a

therapeutically effective amount of one or more compounds of formula I or II, or a pharmaceutically acceptable salt, biologically active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer thereof.

Pancreatitis is an inflammation of the pancreas mediated by the release of digestive enzymes that eventually lead to the destruction of the organ itself. Pancreatitis can be a severe, life-threatening illness with many complications. In severe cases, bleeding, tissue damage to the heart, lungs and kidneys, and infection may occur. About 80,000 cases of acute pancreatitis occur annually in the United States; about 20 percent of them are severe. There is no known treatment for pancreatitis. The current approaches for managing pancreatitis involve waiting for it to resolve on its own and the treatment of heart, lungs and kidney complications if that occur.

Autophagy has been shown to play an important role in mediating cellular damage induced by acute pancreatitis. Autodigestion of the pancreas by its own prematurely activated digestive proteases is believed to be important for the onset of acute pancreatitis. Although lysosomal hydrolases are known to play a key role in pancreatic trypsinogen activation, it remains unclear where and how trypsinogen meets these lysosomal enzymes. Recently, autophagy has been proposed to play a key role in the release of pancreatitic digestive enzymes in animal models of pancreatitis (Hashimoto, D., et al., Involvement of autophagy in trypsinogen activation within the pancreatic acinar cells. J Cell Biol, 2008. 181(7): p. 1065-72; and Ohmuraya, M. and K. Yamamura, Autophagy and acute pancreatitis: a novel autophagy theory for trypsinogen activation. Autophagy, 2008. 4(8): p. 1060-2.) In Atg5-/- mice, which are defective for a key autophagy gene Atg5, the severity of acute pancreatitis induced by cerulein is greatly reduced with a significantly decreased level of trypsinogen activation. Thus, activation of autophagy may exert a detrimental effect in pancreatic acinar cells by mediating the activation of trypsinogen to trypsin. Inhibition of autophagy may provide a unique opportunity for blocking trypsinogen activation in acute pancreatitis. Development of an autophagy inhibitor may provide a first-in-class inhibitor for acute pancreatitis.

Another aspect of the invention relates to a method of treating or preventing a disease caused by an intracellular pathogen, comprising the step of administering to a subject in need thereof a therapeutically effective amount of one or more compounds of formula I or II, or a pharmaceutically acceptable salt, biologically active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer thereof. See, for example, US Patent Application Publication No. 2009/0111799 to Chen et al. (hereby incorporated by reference in its entirety).

Recent studies have established a role for autophagy in cellular defense against intracellular pathogens including bacteria, such as Mycobacterium tuberculosis,

Streptococcus pyogenes, Shigella spp. and Salmonella typhimurium, as well as viruses and protozoa which use autophagosomes to proliferate. The execution of autophagy is regulated by upstream signal transduction systems that are influenced by largely physiological factors such as nutrient status, growth factors/cytokines, and hypoxia. The pharmacological induction of autophagy is a therapeutic strategy in which this effector of innate immunity would be triggered or amplified to defend against intracellular pathogens.

Another aspect of the invention relates to a method of inactivating a

deubiquitinating protease complex comprising the step of contacting the deubiquitinating protease complex with one or more compounds of formula I or II; wherein the

deubiquitinating protease complex comprises USP3 and USP10. Such methods can be used to ameliorate any condition which is caused by or potentiated by the activity of the deubiquitinating protease complex.

Combination Therapy

In one aspect of the invention, a compound of the invention, or a pharmaceutically acceptable salt thereof, can be used alone or in combination with another therapeutic agent to treat diseases such cancer and pancreatitis. It should be understood that the compounds of the invention can be used alone or in combination with an additional agent, e.g., a therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent that is art-recognized as being useful to treat the disease or condition being treated by the compound of the present invention. The additional agent also can be an agent that imparts a beneficial attribute to the therapeutic composition e.g., an agent that affects the viscosity of the composition.

The combination therapy contemplated by the invention includes, for example, administration of a compound of the invention, or a pharmaceutically acceptable salt thereof, and additional agent(s) in a single pharmaceutical formulation as well as administration of a compound of the invention, or a pharmaceutically acceptable salt thereof, and additional agent(s) in separate pharmaceutical formulations. In other words, coadministration shall mean the administration of at least two agents to a subject so as to provide the beneficial effects of the combination of both agents. For example, the agents may be administered simultaneously or sequentially over a period of time.

It should further be understood that the combinations included within the invention are those combinations useful for their intended purpose. The agents set forth below are illustrative for purposes and not intended to be limited. The combinations, which are part of this invention, can be the compounds of the present invention and at least one additional agent selected from the lists below. The combination can also include more than one additional agent, e.g., two or three additional agents if the combination is such that the formed composition can perform its intended function.

For example, one aspect of the invention relates to the use of small molecule autophagy inhibitors (e.g. those of formula I or II) in combination with an anti- angiogenesis inhibitors for the treatment of cancers. It is known that anti-angiogenesis inhibitors have the promise to inhibit tumor growth by suppressing the growth of blood vessels in tumors which are required for supporting tumor survival and growth. For example, the angiostatic agent endostatin and related chemicals can suppress the building of blood vessels and reduce tumor growth. Several hundred clinical trials of anti-angiogenesis drugs are now under way. In tests with patients, anti-angiogenesis therapies are able to suppress tumor growth with relatively few side effects. However, anti-angiogenesis therapy alone may not be insufficient to prolong patient survival; combination with a conventional chemotherapy may therfore be beneficial. Specifically, autophagy inhibitors may provide a new option to work alone or in combination with anti-angiogenesis therapy. Endostatin has been shown to induce autophagy in endothelial cells by modulating Beclin 1 and beta-catenin levels (Nguyen, T.M., et al, Endostatin induces autophagy in endothelial cells by modulating Beclin 1 and beta-catenin levels. J Cell Mol Med, 2009). As disclosed herein, it has been found that inhibition of autophagy selectively kills a subset of cancer cells under starvation condition. Therefore, it is proposed that anti-angiogenesis therapy may induce additional metabolic stress to sensitize cancer cells to autophagy inhibitors, which are not normally cytotoxic. Thus, a combination of anti-angiogenesis therapy and anti-autophagy therapy may provide a new option for treatment of cancers without cytotoxicity to normal cells (Ramakrishnan, S., et al., Autophagy and angiogenesis inhibition. Autophagy, 2007. 3(5): p. 512-5).

Non-limiting examples of anti-angiogenesis agents with which a compound of the invention of the invention can be combined include, for example, the following:

bevacizumab (Avastin®), carboxyamidotriazole, TNP-470, CM101, IFN-a, IL-12, platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonists, angiostatic steroids with heparin, Cartilage-Derived Angiogenesis Inhibitory Factor, matrix metalloproteinase inhibitors, angiostatin, endostatin, 2-methoxyestradiol, tecogalan, thrombospondin, prolactin, ανβ3 inhibitors and linomide.

In addition, as described in US Patent Application Publication No. 2008/0269259 to Thompson et al. (hereby incorporated by reference in its entirety), autophagy inhibitors can be used to treat a subject who has been identified as having a glycolysis dependent cancer by combining one or more autophagy inhibitors with one or more anti-cancer compounds which converts glycolysis dependent cancer to cells incapable of glycolysis. Examples of anti-cancer compounds which convert glycolysis dependent cancer to cells incapable of glycolysis: Alkylating Agents; Nitrosoureas; Antitumor Antibiotics; Corticosteroid Hormones; Anti-estrogens; Aromatase Inhibitors; Progestins; Anti-androgens; LHRH agonists; Kinase Inhibitors; and Antibody therapies; for example, busulfan, cisplatin, carboplatin, chlorambucil, cyclophosphamide, ifosfamide, dacarbazine (DTIC), mechlorethamine (nitrogen mustard), melphalan, carmustine (BCNU), lomustine (CCNU), dactinomycin, daunorubicin, doxorubicin (Adriamycin), idarubicin, mitoxantrone, prednisone, dexamethasone, tamoxifen, fulvestrant, anastrozole, letrozole, megestrol acetate, bicalutamide, flutamide. leuprolide, goserelin, gleevac, Iressa, Tarceva, Herceptin, Avastin, L-asparaginase and tretinoin. Dosage

As used herein, a "therapeutically effective amount" or "therapeutically effective dose" is an amount of a compound of the invention or a combination of two or more such compounds, which inhibits, totally or partially, the progression of the condition or alleviates, at least partially, one or more symptoms of the condition. A therapeutically effective amount can also be an amount which is prophylactically effective. The amount which is therapeutically effective will depend upon the patient's size and gender, the condition to be treated, the severity of the condition and the result sought. For a given patient, a therapeutically effective amount can be determined by methods known to those of skill in the art.

For any compound used in a method of the present invention, the therapeutically effective dose can be estimated initially from cellular assays. For example, a dose can be formulated in cellular and animal models to achieve a circulating concentration range that includes the IC5₀ as determined in cellular assays (i.e., the concentration of the test compound which achieves a half-maximal inhibition). In some cases it is appropriate to determine the IC5₀ in the presence of 3 to 5% serum albumin since such a determination approximates the binding effects of plasma protein on the compound. Such information can be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the maximum tolerated dose (MTD) and the ED₅₀ (effective dose for 50% maximal response). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between MTD and ED₅₀. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al, 1975, in "The Pharmacological Basis of

Therapeutics", Ch. 1 pi). In the treatment of crises, the administration of an acute bolus or an infusion approaching the MTD may be required to obtain a rapid response. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the kinase modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using the MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90% until the desired amelioration of symptoms is achieved. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

Kits

The compounds and compositions of the invention (e.g., compounds and compositions of formula I or II) may, if desired, be presented in a kit (e.g., a pack or dispenser device). The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for use of the compound in any method described herein. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition. Instructions for use may also be provided.

EXEMPLIFICATION

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1: Isolation of a Small Molecule Inhibitors of Autophagy

To explore the mechanism of autophagy and identify additional small molecules that can activate it, a high-throughput image-based screen for autophagy regulators was developed. This system takes advantage of the localization of light chain 3 coupled to GFP (LC3-GFP) to autophagosomal membrane upon induction of autophagy (Zhang, L., Yu, J., Pan, H., Hu, P., Hao, Y., Cai, W., Zhu, H., Yu, A.D., Xie, X., Ma, D., et al. (2007). Small molecule regulators of autophagy identified by an image-based high-throughput screen. Proc Natl Acad Sci U S A 104, 19023-19028). Mammalian LC3, the ortholog of yeast ATG8, has been shown to mark autophagosome membrane specifically (Kabeya, Y.,

Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y., and Yoshimori, T. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19, 5720-5728; and Mizushima, N., and Yoshimori, T. (2007). How to interpret LC3 immunoblotting. Autophagy 3, 542- 545). The number of LC3-GFP-positive autophagosomes per cell is very low under normal growth conditions but is rapidly increased upon serum starvation or the addition of rapamycin. Compounds that increase cellular levels of LC3-GFP, however, are not necessarily able to increase degradative activity of autophagy. Instead, the increases of LC3-GFP may be associated with cell death or may be a result of lysosomal defect and thus associated with blockage of autophagy.

In a screen of 480 known bioactive compounds, a LC3-GFP -based high throughput image screen was coupled with a low throughput assay for long-lived protein degradation which allowed for the identification compounds which could specifically induce autophagic degradation from those that nonspecifically increase levels of LC3-GFP as a result of causing cellular damage or by blocking downstream lysosomal functions. The results of the screen led to the identification of eight compounds, seven of which were FDA-approved drugs, that can induce autophagy and promote long-lived protein degradation without causing obvious cellular injury (Zhang, L., Yu, J., Pan, FL, Hu, P., Hao, Y., Cai, W., Zhu, H., Yu, A.D., Xie, X., Ma, D., et al. (2007). Small molecule regulators of autophagy identified by an image-based high-throughput screen. Proc Natl Acad Sci U S A 104, 19023-19028).

In this screen, a known bioactive compound, MBCQ (Figure 1A), previously known as a PDE5 inhibitor (MacPherson, J.D., Gillespie, T.D., Dunkerley, H.A., Maurice, D.H., and Bennett, B.M. (2006). Inhibition of phosphodiesterase 5 selectively reverses nitrate tolerance in the venous circulation. J Pharmacol Exp Ther 317, 188-195), was identified as having autophagy inhibitor activity. Stimulation of LC3-GFP-H4 cells with rapamycin (0.2 μΜ) led to increases in the levels of LC3-GFP as expected. The presence of MBCQ inhibited both basal levels as well as rapamycin stimulated LC3-GFP. The reduction of LC3-GFP dots was obvious at 1 hr after the addition of MBCQ and rapamycin compared to that of rapamycin alone. Quantitative analysis of LC3-GFP dots using high throughput microscopy (Figure IB). The treatment of MBCQ reduced the number, spot size as well as spot intensity of LC3-GFP dots compared to the control or to rapamycin treatment alone. The intensity of LC3-GFP was measured both in the presence of both rapamycin and

MBCQ together versus that of rapamycin alone, and the IC50 of MBCQ was determined to be 0.788 μηι, which is about 10,000 fold more potent than the commonly used type III PtdIns3P kinase inhibitor, 3-methyl-adenine (3-MA), which has the working concentration of lO mM.

To confirm the inhibition of autophagy by MBCQ, H4-LC3 cells, 293T cells and mouse embryonic fibroblast cells were treated with MBCQ and the levels of endogenous LC3II were measured by western blot.. Consistent with the inhibitory activity of MBCQ, the levels of LC3II were consistently reduced in MBCQ and rapamycin co-treated H4-LC3, 293T and MEF cells compared to that of rapamycin alone. Consistent with LC3-GFP analysis (Figure IB), the levels of LC3II were significantly lower after treatment with rapamycin and MBCQ for lh compared to that of rapamycin alone.

To determine the effect of MBCQ on starvation induced autophagy, H4-LC3-GFP cells were cultured in Hanks buffer for lh, which was sufficient to induce autophagy as demonstrated by the increases in the levels of LC3-GFP dots (Figure 2). In the presence of MBCQ (5 μΜ), starvation induced autophagy is significantly reduced Quantitative measurement of the LC3-GFP spot number, spot size and spot intensity confirmed that starvation induced autophagy is inhibited by MBCQ (5 μΜ) or positive controls of 3-MA (10 mM) or wortmannin (0.1 μΜ).

The ultra-structure of cells treated with rapamycin was determined in the presence or absence of MBCQ. It was found that the cells treated with MBCQ alone for 4 h are morpho logically similar to that control treated with vehicle (1% DMSO). Treatment of rapamycin led to the formation of a large numbers of autophagosomes with characteristic double membrane. Such double membrane autophagosomes were conspicuously absent in cells treated with rapamycin and MBCQ together (Figure 3).

Example 2: Structure Activity Relationship (SAR) of MBCQ

MBCQ is a 4-heteroatom-substituted quinazoline compound. For the purposes of the SAR, the structure of MBCQ was divided into three parts - parts A, B and C - as shown in Figure 4A. In part A, different substituents were introduced into 6-position: halogens, electron- deficient groups (e.g., nitro and methyl sulfonyl group), and electron-rich groups (e.g. methoxy and amino group); halogens were introduced into 7-position; halogens were introduced into both 6- and 8-position; and methyl or amino group were introduced into 2- position.

For part B, the nitrogen was replaced with an oxygen or sulfur atom; the methylene chain was extended; and a branch point (i.e. substitution) was added to the methylene chain.

In part C, the effects of different aromatic cycles were investigated, including:

4-pyridinyl, morpholinyl, and substituted and unsubstituted phenyl. Substituted phenyl substituents included both electron- withdrawing groups (e.g., halogen, nitro, and trifluoromethyl group) substituted phenyl 5) and electron-donating groups (e.g. amino, methoxy group).

A total of 194 compounds with above modifications on the MBCQ structure were synthesized and their activities in inhibiting autophagy were analyzed.

The SAR results can be summarized as follows (see also Figure 4B):

(1) The nature of substituents on 6-position of quinazoline is critical for activity. Electron-withdrawing substituents (e.g. nitro or fluorous group) improves the activity (e.g. C29 in Figure 16). The compounds with electron-donating substituents (e.g. amino group) on 6-position has no activity (e.g. C71 in Figure 14). Compounds without substituents on 6- position have moderate activity.

(2) Substituents on 7- and 8- position have negative effect on activity. For example, when the quinazoline is mono-substituted on 7- or 8- position, the compound loses activity (e.g. C83), and the same as compounds that are bis-substitued with chloro group both on 6- and 8-position (e.g. CI 9, C20).

(3) Steric hindrance on the part A impedes activity (e.g. C68, C01).

(4) When heteroatom in the part B is O or S, no activity was detected (e.g. ClOl,

C45).

(5) Compounds lose activity when the benzene in part C is replaced with

morpholine or furan (e.g. C78, C54).

(6) Compounds with 4-CF3, 4-NO2 or 4-pyridine in the part C exhibit no activity

(e.g. CI 5). When there are substituents on 3-, 4- and 5-position simultaneously, no activities were detected (e.g. CI 5). (7) High activity was observed when heteroatom in the part B was nitrogen, which linked with 1-3 carbons (e.g. C16, C51 and C13). No activity was detected when more than three methylene units are in the chain linking part A and part C (e.g. C30, C49). In addition, bulky substituents on the branch chain leads to no activity (e.g. C81, C86 and C94). Further, no appreciable effect on activity was detected with different optical configuration (R or S) on branch chain (e.g. C69 and C84, C76 and C77).

Among the MBCQ derivatives synthesized and analyzed for their autophagy inhibiting activity, 44 compounds exhibited autophagy inhibitory activity similar or above that of MBCQ (Figure 16). At the same time, a number of compounds were identified, such as C71 and C82, which are similar to MBCQ structurally but have no autophagy inhibitory activity and were used as negative controls in subsequent experiments (Figure 15).

To confirm the inhibitory activity on autophagy, mouse embryo fibroblasts (MEF) cells were treated with C29, C43 or C71 for 4 hours in the presence or absence of rapamycin and the levels of autophagy were determined by LC3 western blotting. The treatment of C43 or C29, but not the negative control C71 , inhibited autophagy induced by rapamycin. (Figure A).

The effect of C29 and C43 on autophagy was further confirmed by electron microscopy. In rapamycin treated MEF cells, numerous autophagosome vesicles with double membranes were observed, as well as many vesicles with multi-membrane as expected (Figure 5B). In cells treated with rapamycin and C29 or C43, autophagosomes are largely absent as that is in vehicle treated cells (Figure 5).

Example 3: MBCQ Inhibits Selective Cell Death Models Involving Autophagy

To characterize the effect of MBCQ on cellular activity, the effect of MBCQ on cell survival and cell cycle was determined as outlined below. H4 cells were treated with MBCQ (5 μΜ) for 5 days and harvested daily for cell number counting in the presence of trypan blue. As shown in Figure 6A, the treatment of MBCQ had no effect on cell proliferation. The cell cycle profile and possible apoptotic cells in H4 cells treated with MBCQ (5 μΜ) for 24 h and 48 h was also determined. As shown in Figure 6B, MBCQ has no detectable effect on cell cycle distribution.

Autophagy has been proposed to contribute to cell death in a number of apoptotic deficient cell types. For example, bax/bak double deficient mouse embryonic fibroblast cells (DKO mefs) are highly resistant to apoptosis (Wei, M.C., Zong, W.X., Cheng, E.H., Lindsten, T., Panoutsakopoulou, V., Ross, A.J., Roth, K.A., MacGregor, G.R., Thompson, C.B., and orsmeyer, S.J. (2001). Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292, 727-730). Stimulation of bax/bak DKO mefs with etoposide has been shown to induce cell death in part through autophagy induction (Shimizu, S., Kanaseki, T., Mizushima, N., Mizuta, T., Arakawa-Kobayashi, S., Thompson, C.B., and Tsujimoto, Y. (2004). Role of Bcl-2 family proteins in a non- apoptotic programmed cell death dependent on autophagy genes. Nat Cell Biol 6, 1221- 1228). To test if MBCQ may inhibit cell death of bax/bak DKO cells induced by etoposide, Bax bak DKO cells were treated with etoposide in the presence of MBCQ (10 μΜ), or 3- MA (10 mM) as a positive control for 8 h. As shown in Figure 7A, the presence of MBCQ significantly reduced cell death of bax/bak DKO MEF cells. Furthermore, consistent with inhibition by MBCQ, the levels of LC3II were increased in etoposide treated cells but reduced in the presence of MBCQ (Figure 7B).

Example 4: MBCQ Selectively Reduces the Cellular Levels of PI3P

Since MBCQ inhibits autophagy induced by rapamycin and starvation, it was first determined if MBCQ affects the activity of mTOR. Western blotting assays demonstrated that MBCQ has no effect on the phosphorylation of mTOR and its targets, p70S6K and S6, in control or rapamycin treated cells. Nor does MBCQ have any effect on the

phosphorylation of GSK-3a/ , AKT. Since the phosphorylation of AKT is regulated by type I PtdIns3(PI3) kinase, this result also suggests that MBCQ has no effect on type I PI3 kinase. Thus, it was concluded that MBCQ has no effect for the mTOR pathway or type I PI3 kinase.

The effects of MBCQ on early endosomes using immunostaining of EEA1 as a marker, lysosomes using immunostaining of lamp2 as a marker or lysotracker, trans-Golgi using GalT-YFP as a marker was determined. No effect of MBCQ was detected in any of these experiments. Thus, it was concluded that MBCQ does not affect major intracellular organelles.

In addition, the effect of MBCQ on proteasomal degradation pathway using pEGFP- CL1, a GFP fusion with a short-lived peptide was determined (Bence, N.F., Sampat, R.M., and Kopito, R.R. (2001). Impairment of the ubiquitin-proteasome system by protein aggregation. Science 292, 1552-1555). It was found that MBCQ does not affect the levels of pEGFP-CLl, suggesting that MBCQ does not have a general effect on the proteasomal pathway (data not shown). In addition, the treatment of MBCQ has no effect on the general levels of polyubiquitination. Thus, it was concluded that MBCQ does not have a general effect the ubiquitin-proteasomal degradation pathway.

The levels of PtdIns3P (PI3P) are known to play a critical role in mediating autophagy (Levine, B., and Klionsky, D.J. (2004). Development by self-digestion:

molecular mechanisms and biological functions of autophagy. Dev Cell 6, 463-477). To ask if MBCQ has an effect on PI3P, H4 cells expressing FYVE-RFP were used. FYVE binds specifically to PI3P and is widely used as a marker for cellular levels for PI3P (Gaullier,

J.M., Simonsen, A., D'Arrigo, A., Bremnes, B., Stenmark, H., and Aasland, R. (1998).

FYVE fingers bind PtdIns(3)P. Nature 394, 432-433). Interestingly, the treatment of MBCQ rapidly and effectively reduced the levels of FYVE-RFP spots in both basal and rapamycin treated H4 cells while the levels of FYVE-RFP detected by western blotting were not changed (Figures 8).

To further determine the effect of MBCQ on the cellular levels of PtdIns3P, a lipid dot blot assay was used. The cellular Ptdlns species were extracted and applied onto polyvinylidene fluoride membrane. The levels of PtdIns3P was detected using GST-PX domain protein and anti-GST antibody. As shown in Figure 9, the treatment of MBCQ and

C43 selectively reduced the cellular levels of PtdIns3P in both basal and rapamycin treated cells. Taken together, it was concluded that MBCQ reduces the levels of PtdIns3P.

Example 5: MBCQ and its Active Derivatives Selectively Promotes the Degradation of Vps34 Complexes

Since the type III Ptdlns3 kinase complex, Vps34/Beclinl/pl50, is responsible for the phosphorylation of Ptdlns to produce PtdIns3P, MBCQ inhibitory activity on the kinase activity of the Vps34 complex was determined. 293T cells were transfected with HA-

Vps34/GFP-Beclinl. The Vps34 complex immunoprecipitated using anti-HA was incubated with Ptdlns in the presence of γ-32Ρ-ΑΤΡ. The phosphorylation product was analyzed by thin layer chromatography and followed by autoradiography. As shown in

Figure 10A, the phosphorylation of Ptdlns is inhibited by wortmannin but not by MBCQ.

Thus, it was concluded that MBCQ is not a direct inhibitor of Vps34 enzymatic activity.

On the other hand, it was noted that the levels of flag-tagged Beclinl and HA- Vps34 were considerably lower in MBCQ, C29 or C43 treated cells than that of C82, an inactive analog (Figure 10B). The treatment of MBCQ, C29 and C43, but not C82, also reduced the levels of GFP-pl50 and Atgl4L (Figures 10C-D). It was also found MBCQ and C43 could reduce the levels of endogenous Beclinl, Vps34 and Atgl4L (FigurelOE) in H4 cells and in 293T cells (FigurelOF) , while the known autophagy inhibitor 3-MA has no effect on endogenous Beclinl in H4 cells (Figure 10G).

To determine if MBCQ and C43 have similar effects on endogenous Beclinl, 293T cells were treated with MBCQ or C43 in the presence of CHX to inhibit protein synthesis. The levels of Beclinl were notably lower in the presence of MBCQ or C43 than with CHX alone after treatment for 6 h (Figure 10E). Thus, it can be concluded that both MBCQ and C43 may promote the degradation of endogenous Beclinl .

To explore the mechanism by which MBCQ and C43 reduce the levels of Vsp34 complexes, 293T cells were treated with C43 with proteasomal inhibitor MG132 or NH₄CI to inhibit lysosomal degradation. It was found that the presence of MG132 but not NH₄CI inhibited the reduction of GFP-Beclinl. This result suggests that the treatment of C43 promotes the degradation of Beclinl through the proteasomal pathway. It was therefore concluded that C43 inhibits autophagy by selectively promoting the degradation of type III PI3 kinase complexes including Vps34/Beclinl/pl50/Atgl4L/UVRAG.

Example 6: MBCQ and its Active Derivatives Enhances Starvation-Induced Apoptosis

Since autophagy is activated under metabolic stress conditions to support cell survival, compounds were tested to determine if they promote cell death under starvation condition. Indeed, it was found that C43 reduced the survival of MDA-MB-231 cells under serum free condition (Figure 11A) and MCF-7 cells under glucose-free condition (Figure 1 IB). Western blot analysis confirmed that the treatment of C43 inhibited autophagy in MCF-7 cells under both basal and glucose-free condition.

In addition, it was found that C43 inhibited the proliferation of Bcap-37 cells, a breast cancer cell line, in the presence of 10% bovine serum (Figure 11C). Further, Mcap- 37 cells became highly sensitive to C43 under glucose free condition (Figure 1 ID). Western blot analysis of Bcap-37 cells cultured under control and glucose-free condition confirmed that the treatment of C43 inhibited autophagy under both basal and glucose-free conditions.

To explore the mechanism by which C43 induces the death of Bcap-37 cells, the DNA content was analyzed by FACS. It was found that the treatment of Bcap-37 cells under glucose-free condition induced a peak of sub-diploid DNA, consistent with apoptotic DNA fragmentation (Figure 1 IE). Furthermore, cleavage of PARP, a hallmark of caspase activation, was also detected in Bcap-37 cells treated with C43 under glucose-free condition for 6 h (Figure 1 IF). Another breast cancer cell line, BT549, also demonstrated a similar response towards the treatment of C43.

In contrast to the above cancer cell lines analyzed, the treatment of MDCK cells, which derived from the Madin-Darby canine kidney, with spautin under glucose -free condition did not induce apoptosis; only -25% growth suppression was observed when treated with 10 μΜ of spautin for 48 hrs (Figure 12A and 12B). Hs578Bst cells, established from normal tissue peripheral to the tumor and is myoepithelial in origin, also were not sensitive to spautin (Figure 12C and 12D). These results are consistent with the possibility that cancer cells may be under increased metabolic pressure and therefore more sensitive to the inhibition of autophagy than non-cancer cells.

Increased activation of autophagy under apoptotic deficient conditions has been shown to mediate cell death. To test this possibility, Bax-Bak double knockout (DKO) cells were tested with etoposide to induce by DNA damage response in the presence or absence of spautin and it was found that C43, MBCQ and 3-MA inhibits etoposide induced death of Bax-Bak DKO cell

Thus, it was concluded that a subset of cancer cells may be selectively sensitive to inhibition of autophagy.

Example 7: Effect of MBCQ Derivatives In Vivo

To begin to test the effect of MBCQ derivatives in vivo, the ability of MBCQ derivatives to inhibit autophagy in rapamycin injected mice was investigated. Mice were injected with rapamycin (10 mg/kg) alone as a positive control, or with C43 or MBCQ (40 mg/kg) intraperitoneally every hour for 4 h and then sacrificed at the fifth hour. The autophagy levels in liver were then analyzed by western blotting using anti-LC3 antibody. As shown in Figure 13 A, administration of C43 or MBCQ significantly reduced the levels of LC3II. Thus, it was determined that C43 and MBCQ are both active in vivo in inhibiting autophagy.

Since autophagy has been proposed to contribute to the tissue damage in pancreatitis, MBCQ derivatives were tested to see if they could reduce tissue damage induced by cerulein injection, a well-established animal model of pancreatitis (Hashimoto, D., Ohmuraya, M., Hirota, M., Yamamoto, A., Suyama, K., Ida, S., Okumura, Y.,

Takahashi, E., Kido, H., Araki, K., et al. (2008). Involvement of autophagy in trypsinogen activation within the pancreatic acinar cells. J Cell Biol 181, 1065-1072; and Ohmuraya, M., and Yamamura, K. (2008). Autophagy and acute pancreatitis: a novel autophagy theory for trypsinogen activation. Autophagy 4, 1060-1062). Rats were injected intraperitoneally with cerulein (50 ng/kg) alone or with C43 (40 mg/kg) hourly for 4 times. The rats were sacrificed at one hr after the last injection and the pancreas were isolated for western blotting analysis. As shown in Figure 13B, the injection of cerulein induced autophagy as reported; the co-injection of C43 significantly reduced the levels of autophagy induced by cerulein injection. Taken together, it was concluded that C43 is effective in reducing autophagy induced in cerulein induce pancreatitis.

Example 8: Preparation of Compounds

One general approach to the synthesis of compounds of formula I and II is depicted below in Scheme 1.

Scheme 1

[1] Step one is the formation of a quinazoline-4-ketone (or 8-aza-quinazoline-4- ketone).

In one approach, anthranilic acid methyl ester (or methyl 2-aminonicotinate) is mixed with formamide in a molar ratio of 1 :15-20 and heated at about 170-190 °C. After the reaction is complete, the mixture is cooled, leached, washed and dried. The resulting crude product is used in the next reaction without further processing.

[2] Step two is the formation of a 4-chloroquinazoline (or 8-aza-4- chloroquinazoline).

In one approach, the crude product from step one is mixed with phosphorus oxychloride in a molar ratio of 1 :8.7-10, then heated at about 100-115 °C. After the reaction is complete, approximately 10-12 hours, the mixture is cooled and excess phosphorus oxychloride is removed by rotary evaporation. An organic solvent, such as dichloromethane, is added to dissolve the solid, followed by pH adjustment of the resulting solution to about 7-8 by addition of ammonia. The resulting mixture is extracted with dichloromethane, dried and purified by column chromatography.

In another approach, the crude product from step one is mixed with thionyl dichloride in a molar ration of 1 : 15-20, with catalytic amount of anhydrous DMF (e.g. 0.5-1 mL), then heated at about 80-90 °C. After the reaction is complete, approximately 10-12 hours, the mixture is cooled and excessive thionyl dichloride was removed by rotary evaporator. An organic solvent, such as dichloromethane, is added to dissolve the solid, followed by pH adjustment of the resulting solution to about 7-8 by addition of ammonia. The resulting mixture is extracted with dichloromethane, dried and purified by column chromatography.

In another approach, the crude product from step one is mixed with oxalyl chloride under argon and anhydrous DMF is added dropwise, to form a mixture with a molar ratio of 1 :1.5: 1.5 product of step one:oxalyl chloride:DMF, and then heated to about 85-95 °C. After about 7-10 hours the reaction is quenched with saturated disodium hydrogen phosphate. Then the reaction mixture is then extracted with an organic solvent, such as dichloromethane, by column chromatography.

[3] Step three is the formation of an N-substituted-4-amino-quinazoline (or 8-aza-N- substituted-4-amino-quinazoline).

Under argon, the product of step 2, HXC(R²)(R³)(CH₂)_nZ (as defined herein), and triethylamine are combined in a molar ratio of 1 : 1.25 : 1.68, in an organic solvent, such as tetrahydrofuran, and heated to about 75-80 °C. After about 12-18 hours, the organic solvent is removed by rotary evaporation. The resulting crude product is purified by column chromatograpy.

For additional illustration, the synthesis of compound A9, A30 and A36 are described in more detail below. As noted above, additional compounds can be prepared by varying the amine which is coupled with optionally substituted 4-chloroquinazoline (such as 9-3 shown below). Preparation of A9

9-3 A9

Scheme 2

To a suspension of AgN0₂ (448.5 mg, 2.92 mmol) in diethyl ether (5 niL) was added compound 9-1 (500 mg, 2.65 mmol) dropwise in an ice-salt bath under Argon. The mixture was warmed to RT and stirred overnight. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (EA: PE, 1 : 100) to give three compounds. By ^lH NMR it was difficult to judge which was the desired compound 9-2.

The mixture containing compound 9-2 (150 mg, 0.967 mmol, MC0449-41-2) and OH (81.4 mg, 1.451 mmol) in CH₃CN/H₂0 (1 mL/1 mL) was stirred for 2h at RT. Then selectfluor (514.0 mg, 1.451 mmol) was added in one portion. The mixture was stirred overnight at RT. The reaction mixture was poured into water (10 mL), extracted with ethyl acetate (2 x 20 mL). The combined organics were washed with brine (10 mL), dried over MgS04 , concentrated and purified by silica gel chromatography (PE) to afford compound 9-3 as a colorless oil (70 mg, yield: 42%).

To a solution of compound 9-3 (50 mg, 0.27 mmol) and (4-chlorophenyl)methan- amine (47 mg, 0.33 mmol) in isopropyl alcohol (5 mL) was added Et₃N (46 μί, 0.33 mmol). The solution was microwaved for 20 min at 150°C. TLC showed the reaction was completed. The mixture was concentrated and purified by flash chromatography to give A9 as a light yellow solid (52 mg, yield: 67 %, confirmed by 1H NMR, and LC-MS). The 1H NMR is shown in Figure 22. Preparation of A30

A30

Scheme 3

A solution of compound 9-3 (105 mg, 0.573 mmol), 30-9 (94 mg, 0.573 mmol) and NEt3 (0.22 mL, 1.64 mmol) in isopropanol (4 mL) was microwaved at 150 °C for 20 min. Concentration and purification by column chromatography gave A30 as a yellow solid (80 mg, yield: 45 %, confirmed by ^XH NMR). The 1H NMR is shown in Figure 23.

Preparation of Λ36

36-3 A36

Scheme 4

A solution of compound 36-1 (1.0 g, 5.3 mmol) and NaCN (520 mg, 10.6 mmol) in DMSO (10 mL) was stirred at 30 °C overnight. TLC showed the reaction was completed. The mixture was diluted with water (30 mL), and extracted with ethyl acetate (50 mL). The organic layer was washed by water (10 mL x 5) and NaHCC (sat., 20 mL), dried over anhydrous Na₂SC>4, and concentrated. The residue was purified by flash chromatography to give 36-2 as colorless oil (360 mg, yield: 50%). To a solution of compound 36-2 (346 mg, 2.56 mmol) in THF (10 mL) was added Raney Ni. Then the mixture was adjusted to PH=10 with concentrated aqueous ammonia and stirred at 30 °C overnight. TLC showed the reaction was completed. The mixture was filtered through Celite and the filtrate was concentrated to give 36-3 as a yellow oil (120 mg, yield: 34 %).

To a solution of compound 9-3 (50 mg, 0.27 mmol) and 36-3 (46 mg, 0.33 mmol) in isopropyl alcohol (5 mL) was added Et₃N (46 uL, 0.33 mmol). The solution was microwaved for 20 min at 150 °C. TLC showed the reaction was completed. Concentration and purification by flash chromatography gave A36 as a white solid (48.4 mg, yield: 63 %, confirmed by 1H NMR at 400 MHz in DMSO, and MS). The ^JH NMR is shown in Figure 24.

Example 9: Separating Autophagy-Inhibiting Activity from PDE5-Inhibiting Activity

The structural activity relationship (SAR) of MBCQ derivatives was investigated to determine if its activity in inhibiting autophagy may be separated from its PDE5 inhibitory activity. Among the MBCQ derivatives synthesized and analyzed for their autophagy inhibiting activity, as described above, some compounds exhibited autophagy inhibitory activity similar or above that of MBCQ and others had no anti-autophagy activity and thus can serve as negative controls.

Fourteen MBCQ derivatives were selected and screened for their activities on PDE5 (Wang, H., Yan, Z., Yang, S., Cai, J., Robinson, H., and Ke, H. (2008). Kinetic and structural studies of phosphodiesterase-8A and implication on the inhibitor selectivity. Biochemistry 47, 12760-12768). Among them, C43 (6-fluoro-N-(4- fiuorobenzyl)quinazolin-4-amine), an effective autophagy inhibitor with IC₅₀ of 0.87 μΜ which is comparable to that of MBCQ, was found to have much reduced inhibiting activity towards PDE5 and other PDEs. Thus, the PDE5 inhibiting activity of MBCQ can be chemically separated from that of autophagy inhibiting activity.

Table 1. Summary of Determination of % Inhibition of PDE5 activity

A41 -9.1 4.2 -18.1 6.5 -8.0 5.2

A64 7.59 7.14 1.97 1.12 -3.35 8.68

A68 0.92 7.99 -2.71 9.86 -14.46 3.61

A69 -1.96 3.92 -2.84 6.29 1.78 11.76

A70 -8.55 6.00 -2.88 2.79 -11.26 7.73

A72 -2.07 7.16 -0.23 0.77 -0.15 9.36

Sildenafil 101.7 2.0 98.8 2.0 96.5 3.9

Zaprinast 101.6 3.0 88.4 2.6 38.0 3.5

MBCQ 98.8 4.7 91.0 10.4 54.4 11.1

Consistent with this conclusion, there were a number of other known PDE5 inhibitors in the bioactive library that were screened, including MY-5445, dipyridamole, IBMX and sildenafil, but not recovered as autophagy inhibitors. To further confirm this conclusion, H4-LC3-GFP cells were treated with rapamycin and other PDE5 inhibitors including MY-5445 (30 μΜ), dipyridamole (80 μΜ), IBMX (100 μΜ) or sildenafil (10 μΜ) using MBCQ as a positive control. None of the PDE5 inhibitors tested, including the most potent PDE5 inhibitor, sildenafil (Viagra) which has an EC5₀ of 2.5 nM for PDE5, has any activity on autophagy. From these data, it was concluded that the autophagy inhibitory activity of MBCQ is not related to its PDE5 inhibitory activity.

Example 10: Identification of a Deubiquitinating Protease Complex for Vps34

Complex I

Ubiquitination represents an essential key step in mediating proteasomal degradation. Experiments were therefore run to determine if ubiquitination of Beclinl is increased in cells treated with C43. As depicted in Figure 16, it was found that C43 promoted the ubiquitination of Beclin 1.

It was therefore hypothesized that C43 targets a deubiquitinating protease complex (DUB) which normally functions to negatively regulate the ubiquitination of Vps34 complex I. This follows the common finding that a small molecule is more likely to be an inhibitor than an activator. To directly test this hypothesis, a collection of 127 siRNAs targeting Human Deubiquitinating Enzymes from Dharmacon library SMART pools were screened for DUBs that when knockeddown lead to inhibition of autophagy using LC3- GFP-H4 cells as an assay.

siPL l was used for validation of transfection effiency, and siVps34 was included in as a positive control. Seventy-two hours post-transfection, cells were treated with DMSO, rapamycin (200nM) to induce autophagy, or rapamycin (200 nM) and spautin (10 μΜ), respectively in duplicate for additional 8h. Cells were counterstained with Hoechst 33342 (0.5 μΜ) and fixed in 3.8% PFA. The fluorescent images were acquired and quantified using a CellWoRx High Content Cell Analysis System.

The screen identified USP10, USP13, USP3, USP16 and USP18 as five genes that when knockdown led to a decrease in the levels of autophagy under the basal condition as well as in the presence of rapamycin by at least 1.5 standard deviation from the plate median. The effects of knockdown of these five USPs on the protein expression levels in the Vps34 complexes in H4 cells were analyzed. It was found that knockdown of any of the five USPs reduced the levels of endogenous Vps34, Beclinl, Atgl4L and UVRAG (Figure 17). Furthermore, knockdown of any of the five USPs also led to reductions in the protein levels of the other four USPs (Figure 18). Interestingly, the treatment of C43 also reduced the levels of these five USPs (Figure 18). Treatment of spautin also can reduce the levels of USP13 and USP10 in 293T cells and Bcap-37 cells, but have little effect on the levels of USP44, an unrelated USP.

These results suggest that the stabilities of USP3, USP10, USP13, USP 16 and USP 18 are co-dependent upon each other which might happen if they exist in a large complex. To test this posibility, GFP-USP10 and Myc-USP13 plasmids were transfected into 293T cells and examined by GFP-USP10 interaction and Myc-USP13

immunoprecipitation. It was found found that GFP-USP10 and Myc-USP13 could indeed interact and importantly, the interaction was inhibited in spautin-treated cells (Figure 19). Thus, it was concluded that spautin disrupts the USP 10 and USP 13 interaction which might be needed for appropriately targeting this deubiquitinating protease complex to regulate the ubiquitination status of Vps34 complexes.

Example 11, Regulation of p53 via Vsp34 Complexes and Deuubiquitination

Since USP 10 is known as the DUB of p53, the effects of knocking down these USPs on p53 was also investigated. It was found that the knockdown of any one of the five USPs could lead to the reduction of p53 (Figure 20). These data suggest that USP3, USP10, USP 13, USP 16 and USP 18 are all regulators of p53.

To test the possibility that inhibition of USP 10 by spautin- 1 might affect the levels of p53, H4 cells were transfected with MDM2 siRNAs for 72 hours and treated with rapamycin (0.25 μΜ), or spautin- 1. The treatment with spautin- 1 led to a reduction in the levels of p53 which were inhibited with knockdown of MDM2, the major E3 ubiquitin ligase for p53 (Figure 21 A). On the other hand, knockdown of Mdm2 had no effect on spautin-1 induced reduction of USPIO, USP13, Vps34 or Beclinl . Consistent with an effect of spautin-1 in promoting the degradation of p53 through the proteasomal pathway, the reduction in p53 levels was blocked in the presence of MG132 but not NH₄C1 (Figure 21B). Furthermore, the knockdown of USPIO, USP13, Beclinl or Vps34 all led to the reduction of p53 levels (Figure 21C-F). These data demonstrate that the cellular levels of p53 are coordinately regulated with that of Vps34 complexes via deubiquitinating enzymes such as USPIO and USP13.

The levels of p53 in BECN+/- mice were examined. As shown in Figure 21G, the levels of Beclinl in newborn BECN+/- mice were approximately half of that in wt mice. Consistent with a coordinated regulation of Vps34 complex components, the levels of Vps34, Atgl4L and UVRAG were also significantly reduced in BECN+/- tissues.

Interestingly, the levels of USPIO and p53 in the heart and lung of newborn BECN+/- mice were correspondingly reduced. The levels of USP13 could not be examined currently due to a lack of antibody that can recognize murine USP13. The reduced levels of p53 and class III Ptdlns3 kinase activity in BECN+/- mice provide a molecular mechanism to explain the increased rate of tumorigenesis after monoallelic loss of Beclinl.

Example 12, Spautin-1 kills cancer cells under non-proliferating condition

Treatment with spautin-1 for 5 days induced cell death of certain cancer cell lines, such as MDA-MB-231 cells, a model for metastasis, (Figure 26A). The possibility that spautin-1 kills non-proliferating MDA-MD-231 cells was tested by plating the cells in high density so they are confluent in 24 hrs, and treating with spautin-1 (Figure 1B-D).

Interestingly, after treatment with spautin-1 for 48-60 h, MDA-MB-231 cells and SUM159 cells, as well as two ovarian cancer cell lines (OVCA 3 and ES-2), which all carry mutant p53, died; while SKOV3 ovarian cancer cells, which are null for p 3, were more resistant. Thus, spautin-1 kills selected cancer cells under non-proliferating conditions and cancer cells expressing mutant p53 may be more sensitive to spautin-1 than null p53 expressing cells. To further confirm this conclusion, multiple cancer lines were treated with spautin-1 for 6 days. Although spautin-1 had no strong effect on cancer cell proliferation in the first 3 days, most of spautin-1 -treated cells died when assayed on day 6 (Figure IE). H1299 cells expressing a mutant p53 allele (175H) were more sensitive to spautin-1 than control H1299 cells. INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. published patent applications cited herein are hereby incorporated by reference.

EQUIVALENTS

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or

modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

Claims

We claim:

1. A method of producing induced pluripotent stem cells, comprising the steps of: a) administering to non-pluripotent cells a compound represented by formula I:

I

or a pharmaceutically acceptable salt, biologically active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, wherein n is 0, 1, 2, 3 or 4;

Y is -C( ¹)= or -N=;

R is -H, lower alkyl, -CH₃, lower fluoroalkyl, -CH₂F, -CHF₂, -CF₃, -N0₂, - OH, -NH₂, -NH(lower alkyl), -N(lower alkyl)₂, or lower alkynyl;

R¹ is independently selected for each occurrence from the group consisting of -H, -F, -CI, -Br, -I, -N0₂, -OH, -NH₂, -NH(lower alkyl), -N(lower alkyl)₂, -CH₃, -CF₃, -C(=0)(lower alkyl), -CN, -0(lower alkyl), -0(lower fluoroalkyl), -S(=0)(lower alkyl), -S(=0)₂(lower alkyl), and -C(=0)0(lower alkyl);

X is -0-, -S-, -N(H)-, -N(lower alkyl)-, -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-. -CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂- or -CH₂CH₂CH₂CH₂CH₂CH₂-; and

Z is phenyl, pyridyl, vinyl, morphinyl, phenanthrolinyl, naphthyl, furyl or benzo[d]thiazolyl; and optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH , -OCF₃, lower fiuoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4-methylene dioxy, and -C(=0)0(lower alkyl);

, wherein J is CI, OCHF₂, OCH₂CH₃, OCH₂CF₃,

0(CH₂)₂CH₃, OCH(CH₃)₂, 0(CH₂)₃CH₃, or O(cyclopentyl); and b) culturing said cells under conditions and for a period of time such that at least a portion of said non-pluripotent cells become pluripotent.

2. A method of claim 1, wherein Y is -CiR¹)^.

3. A method of claim 1, wherein n is Y is -N=.

4. A method any one of claims 1-3, wherein R is -H.

5. A method of any one of claims 1-4, wherein at least one R¹ is -NH₂, -CI, -N0₂, -I, or -OMe.

6. A method of any one of claims 1-5, wherein at one R¹ is -NH₂, -CI, -N0₂, -I, or -OMe; and at least two R¹ are -H.

7. A method of any one of claims 1-6, wherein R² is -CH₃.

8. A method of any one of claims 1-6, wherein R² is -H.

9. A method of any one of claims 1-8, wherein R³ is -CH₃.

10. A method of any one of claims 1-8, wherein R³ is -H.

11. A method of any one of claims 1-10, wherein X is -0-, -S-, -N(H)-, -N(lower alkyl)- or -CH₂-.

12. A method of any one of claims 1-10, wherein X is -N(H)- or -N(lower alkyl)-.

13. A method of any one of claims 1-10, wherein X is -N(H)-.

14. A method of any one of claims 1-13, wherein Z is phenyl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4-methylene dioxy, and -C(=0)0(lower alkyl).

15. A method of producing induced pluripotent stem cells, comprising the steps of: a) administering to non-pluripotent cells a compound represented by formula II:

II

or a pharmaceutically acceptable salt, biologically active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, wherein n is 0, 1, or 2;

Y is -C( ¹)= or -N=;

R is -H, lower alkyl, -CH₃, lower fluoroalkyl, -CH₂F, -CHF₂, or -CF₃;

R¹ is independently selected for each occurrence from the group consisting of -H, -CH₃, -F, -CI, -Br, -I, and -N0₂;

R² and R³ are independently selected from the group consisting of -H, -CH_3, -CH₂CH₃, -CH₂CH₂CH₃, and -CH(CH₃)₂;

R⁴, R⁵ and R⁸ are independently selected from the group consisting of -H, - CH₃, -CF₃, -OCH₃, -OCF₃, -F, -CI, -B, and -I; and

R⁶ and R⁷ are independently selected from the group consisting of -H, -CH₃, -CF₃, -OCH₃, -OCF₃, -F, -CI, -Br, -I, and -C(=0)0(lower alkyl); or R⁶ and R⁷ taken together are -OCH₂0-;

provided that the compound is not

or

b) culturing said cells under conditions and for a period of time such that at least a portion of said non-pluripotent cells become pluripotent. 16. The method of claim 15, wherein Y is - R¹)^

17. The method of claim 15, wherein Y is -N=.

18. The method of any one of claims 15-17, wherein n is R is -H, -CH₃, -CH₂F, -CHF₂ or -CF₃.

19. The method of any one of claims 15-17, wherein R is H.

20. The method of any one of claims 15-17, wherein R is -CH₂F, -CHF₂ or -CF₃.

21. The method of any one of claims 15-18, wherein R¹ is -F, -CI, -Br or -I.

22. The method of any one of claims 15-18, wherein R¹ is -CH₃.

23. The method of any one of claims 15-18, wherein R¹ is -N0₂.

24. The method of any one of claims 15-23, wherein R² is -H.

25. The method of any one of claims 15-23, wherein R² is -CH₃.

26. The method of any one of claims 15-25, wherein R³ is -H.

27. The method of any one of claims 15-25, wherein R³ is -CH3.

28. The method of any one of claims 15-27, wherein R⁴ is -H.

29. The method of any one of claims 15-27, wherein R⁴ is -F, -CI, -CH₃ or -OCH₃.

30. The method of any one of claims 15-29, wherein R⁵ is -H.

31. The method of any one of claims 15-29, wherein R⁵ is -F, -CI, -CH₃ or -CF₃.

32. The method of any one of claims 15-31, wherein R⁶ is -H.

33. The method of any one of claims 15-31, wherein R⁶ is -F, -CI or -Br.

34. The method of any one of claims 15-31, wherein R⁶ is -CH₃ or -CF₃.

35. The method of any one of claims 15-31, wherein R⁶ is -OCH₃, -OCF₃, or - C(=0)0(lower alkyl).

36. The method of any one of claims 15-31 , wherein R⁶ and R⁷ taken together are -OCH₂O-.

37. The method of any one of claims 15-36, wherein R⁷ is -H.

38. The method of any one of claims 15-37, wherein R⁸ is -H.

39. A method of producing induced pluripotent stem cells, comprising the steps of: a) administering to non-pluripotent cells a compound, or a pharmaceutically acceptable salt thereof, selected from the group consisting of

PCT/US2011/064432

b) culturing said cells under conditions and for a period of time such that at least a portion of said non-pluripotent cells become pluripotent.

A method of producing induced pluripotent stem cells, comprising the steps of: a) administering to non-pluripotent cells a compound represented by formula III:

III

n is 0, 1, 2, 3 or 4;

Y is -C( ¹)= or -N=;

R¹ is independently selected for each occurrence from the group consisting of -H, -F, -CI, -Br, -I, -N0₂, -OH, -NH₂, -NH(lower alkyl), -N(lower alkyl)₂, -CH₃, -CF₃, -C(=0)(lower alkyl), -CN, -0(lower alkyl), -0(lower fluoroalkyl), -S(=0)(lower alkyl), -S(=0)₂(lower alkyl) and -C(=0)0(lower alkyl);

X is -0-, -S-, -N(H)-, -N(lower alkyl)-, -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-.

Z is phenyl, pyridyl, vinyl, morphinyl, phenanthrolinyl, naphthyl, furyl or benzo[d]thiazolyl; and optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH , -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4-methylene dioxy, and -C(=0)0(lower alkyl); and

A method of claim 40, wherein Y is -C(R¹)=.

A method of claim 40, wherein n is Y is -N=.

A method any one of claims 40-42, wherein R is -H.

44. A method of any one of claims 40-43, wherein at least one R¹ is -NH₂, -CI, -N0₂, -I, or -OMe.

45. A method of any one of claims 40-44, wherein at one R¹ is -NH₂, -CI, -N0₂, -I, or -OMe; and at least two R¹ are -H.

46. A method of any one of claims 40-45, wherein R² is -CH₃.

47. A method of any one of claims 40-45, wherein R² is -H.

48. A method of any one of claims 40-47, wherein R³ is -CH₃.

49. A method of any one of claims 40-47, wherein R³ is -H.

50. A method of any one of claims 40-49, wherein X is -0-, -S-, -N(H)-, -N(lower alkyl)- or -CH₂-.

51. A method of any one of claims 40-49, wherein X is -N(H)- or -N(lower alkyl)-.

52. A method of any one of claims 40-49, wherein X is -N(H)-.

53. A method of any one of claims 40-52, wherein Z is phenyl optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4-methylene dioxy, and - C(=0)0(lower alkyl).

54. A method of producing induced pluripotent stem cells, comprising the steps of: a) administering to non-pluripotent cells a compound represented by formula IV:

IV

Y is -0φ )= or -N=;

R is independently selected for each occurrence from the group consisting of -H, lower alkyl, -CH₃, lower fluoroalkyl, -CH₂F, -CHF₂, and -CF₃; R is independently selected for each occurrence from the group consisting of -H, -CH₃, -F, -CI, -Br, -I, and -N0₂;

R² and R³ are independently selected from the group consisting of -H, -CH₃, -CH2CH3, -CH2CH2CH3, and -CH(CH₃)₂;

R⁴, R⁵ and R⁸ are independently selected from the group consisting of -H, - CH₃, -CF₃, -OCH3, -OCF3, -F, -CI, -Br, and -I; and

R⁶ and R⁷ are independently selected from the group consisting of -H, -CH₃,

-CF₃, -OCH₃, -OCF₃, -F, -CI, -Br, -I, and -C(=0)0(lower alkyl); or R⁶ and

R⁷ taken together are -OCH₂0-; and

55. The method of claim 54, wherein Y is -Q ¹)^

56. The method of claim 54, wherein Y is -N=.

57. The method of any one of claims 54-56, wherein n is R is -H, -CH₃, -CH₂F, -CHF₂ or -CF₃.

58. The method of any one of claims 54-56, wherein R is H.

59. The method of any one of claims 54- -56 wherein R is -CH₂F, -CHF₂ or■

60. The method of any one of claims 54- -57 wherein R¹ is -F, -CI, -Br or -I.

61. The method of any one of claims 54- -57 wherein R is -CH₃.

62. The method of any one of claims 54- -57 wherein R is -NO₂.

63. The method of any one of claims 54- -62 wherein R² is -H.

64. The method of any one of claims 54- -62 wherein R is -CH₃.

65. The method of any one of claims 54- -64 wherein R is -H.

66. The method of any one of claims 54- -64 wherein R is -CH₃.

67. The method of any one of claims 54- -66 wherein R is -H.

68. The method of any one of claims 54- -66 wherein R is -F, -CI, -CH₃ or -OCH₃. 69. The method of any one of claims 54- -68 wherein R is -H.

70. The method of any one of claims 54- -68 wherein R is -F, -CI, -CH₃ or -CF₃ 71. The method of any one of claims 54- -70 wherein R is -H.

72. The method of any one of claims 54- -70 wherein R is -F, -CI or -Br.

73. The method of any one of claims 54- -70 wherein R is -CH₃ or -CF?

74. The method of any one of claims 54- -70 wherein R is -OCH₃, -OCF₃, or

C(=0)0(lower alkyl).

75. The method of any one of claims 54-70, wherein R⁶ and R⁷ taken together are

76. The method of any one of claims 54-70, wherein R⁷ is -H.

77. The method of any one of claims 54-76, wherein R⁸ is -H.

78. A method of producing induced pluripotent stem cells, comprising the steps of:

a) administering to non-pluripotent cells

, or a

pharmaceutically acceptable salt, biologically active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer thereof; and

79. The method of any preceding claim, wherein the non-pluripotent cells are somatic cells.

80. The method of claim 79, wherein the somatic cells are fibroblast cells.

81. The method of any preceding claim, wherein the non-pluripotent cells express elevated levels of c-Myc, Klf4, Sox2, Oct4, or combinations thereof.

82. The method of any preceding claim, wherein the non-pluripotent cells express elevated levels of Oct4 and Sox2.

83. The method of any preceding claim, wherein the non-pluripotent cells are human cells.

84. The method of any preceding claim, wherein the non-pluripotent cells are mouse cells.

85. A compound represented by formula III :

III

Y is -C( ¹)= or -N=; R is independently selected for each occurrence from the group consisting of -H, lower alkyl, -CH₃, lower fluoroalkyl, -CH₂F, -CHF₂, -CF₃, -N0₂, -OH, -NH2, -NH(lower alkyl), -N(lower alkyl)₂, and lower alkynyl; R¹ is independently selected for each occurrence from the group consisting of -H, -F, -CI, -Br, -I, -N0₂, -OH, -NH₂, -NH(lower alkyl), -N(lower alkyl)₂, -CH₃, -CF₃, -C(=0)(lower alkyl), -CN, -0(lower alkyl), -0(lower fluoroalkyl), -S(=0)(lower alkyl), -S(=0)₂(lower alkyl) and -C(=0)0(lower alkyl);

-CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH2- or -CH₂CH₂CH₂CH₂CH₂CH₂-; and

Z is phenyl, pyridyl, vinyl, morphinyl, phenanthrolinyl, naphthyl, furyl or benzo[d]thiazolyl; and optionally substituted with one or more substitutents selected from the group consisting of -CH₃, lower alkyl, fluoroalkyl, -0CH₃, -0CF₃, lower fluoroalkoxy, -F, -CI, -Br, -I, -N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4-methylene dioxy, and -C(=0)0(lower alkyl).

86. The compound of claim 85, wherein Y is -C(R¹)=.

87. The compound of claim 85, wherein n is Y is -N=.

88. The compound any one of claims 85-87, wherein R is -H.

89. The compound of any one of claims 85-88, wherein at least one R¹ is -NH₂, -CI, - N0₂, -I, or -OMe.

90. The compound of any one of claims 85-89, wherein at one R¹ is -NH₂, -CI, -NO2, -I, or -OMe; and at least two R¹ are -H.

91. The compound of any one of claims 85-90, wherein R² is -CH₃.

92. The compound of any one of claims 85-90, wherein R² is -H.

93. The compound of any one of claims 85-92, wherein R³ is -CH₃.

94. The compound of any one of claims 85-92, wherein R³ is -H.

95. The compound of any one of claims 85-94, wherein X is -0-, -S-, -N(H)-, -N(lower alkyl)- or -CH₂-.

96. The compound of any one of claims 85-94, wherein X is -N(H)- or -N(lower alkyl)-.

97. The compound of any one of claims 85-94, wherein X is -N(H)-.

98. The compound of any one of claims 85-97, wherein Z is phenyl optionally

substituted with one or more substitutents selected from the group consisting of

-CH , lower alkyl, fluoroalkyl, -OCH₃, -OCF₃, lower fluoroalkoxy, -F, -CI, -Br, -I,

-N0₂, lower alkoxy, -NH(lower alkyl), -N(lower alkyl)₂, -CF₃, 3,4-methylene dioxy, and -C(=0)0(lower alkyl).

99. A compound represented by formula IV:

Y is -QR¹^ or -N=;

R is independently selected for each occurrence from the group consisting of -H, lower alkyl, -CH , lower fluoroalkyl, -CH₂F, -CHF₂, and -CF₃;

R² and R³ are independently selected from the group consisting of -H, -CH , -CH₂CH₃, -CH₂CH₂CH₃, and -CH(CH₃)₂;

R⁴, R⁵ and R⁸ are independently selected from the group consisting of -H, - CH₃, -CF₃, -OCH₃, -OCF₃, -F, -CI, -Br, and -I; and

R⁶ and R⁷ are independently selected from the group consisting of -H, -CH₃, -CF₃, -OCH₃, -OCF₃, -F, -CI, -Br, -I, and -C(=0)0(lower alkyl); or R⁶ and R⁷ taken together are -OCH₂0-.

100. The compound of claim 99, wherein Y is -C(R¹)=.

101. The compound of claim 99, wherein Y is -N=.

102. The compound of any one of claims 99-101, wherein n is R is -H, -CH₃, -CH₂F, -CHF₂ or -CF₃.

103. The compound of any one of claims 99-101, wherein R is H.

104. The compound of any one of claims 99- 101, wherein R is - CH₂F, - CHF₂ or -CF₃.

105. The compound of any one of claims 99- 102, wherein R¹ is -F, -CI, -Br or -I.

106. The compound of any one of claims 99- 102, wherein R¹ is -CH₃.

107. The compound of any one of claims 99- 102, wherein R¹ is -N0₂.

108. The compound of any one of claims 99- 107, wherein R² is -H.

109. The compound of any one of claims 99- 107, wherein R² is -CH₃.

110. The compound of any one of claims 99- 109, wherein R³ is -H.

111. The compound of any one of claims 99- 109, wherein R³ is -CH₃.

112. The compound of any one of claims 99- 111, wherein R⁴ is -H.

113. The compound of any one of claims 99- 111, wherein R⁴ is -F, -CI, -CH₃ or - OCH₃

114. The compound of any one of claims 99- 113, wherein R⁵ is -H.

115. The compound of any one of claims 99- 113, wherein R⁵ is -F, -CI, -CH₃ or - CF₃.

116. The compound of any one of claims 99- 115, wherein R⁶ is -H.

117. The compound of any one of claims 99- 115, wherein R⁶ is -F, -CI or -Br.

118. The compound of any one of claims 99- 115, wherein R⁶ is -CH₃ or -CF₃.

119. The compound of any one of claims 99- 115, wherein R⁶ is -OCH₃, -OCF₃, or -

C(=0)0(lower alkyl).

120. The compound of any one of claims 99- 115, wherein R⁶ and R⁷ taken together are

-OCH₂0-.

121. The compound of any one of claims 99- 120, wherein R⁷ is -H.

active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer thereof.

124. A pharmaceutical composition comprising a compound according to any one of claims 85-123, or a pharmaceutically acceptable salt, biologically active metabolite, solvate, hydrate, prodrug, enantiomer or stereoisomer thereof; and a

pharmaceutically acceptable diluent or carrier.

125. A method of treating cancer, pancreatitis, neurodegeneration, an inflammatory disease, an infectious disease, or an infection caused by an intracellular pathogen, comprising the step of administering to a subject in need thereof a therapeutically effective amount of one or more compounds of any one of claims 85-123, or a pharmaceutical composition of claim 124.

126. The method of claim 125, wherein the method is for treating cancer.

127. The method of claim 126, wherein said cancer is selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer.

128. The method of claim 125, wherein the method is for treating pancreatitis.

129. The method of claim 125, wherein the method is for treating neurodegeneration.

130. The method of claim 125, wherein the method is for treating a neurodegenerative condition is selected from the group consisting of vascular dementia, presenile dementia, neurodegeneration in Down syndrome, and HIV-related dementia.

131. The method of claim 125, wherein the method is for treating neurodegeneration; and the method enhances cognition or inhibits cognitive decline in said subject having said neurodegenerative condition.

132. The method of claim 125, wherein the method is for treating an infection caused by an intracellular pathogen.

133. The method of claim 132, wherein the infection is caused by a bacteria or virus.

134. The method of any one of claims 125-133, further comprising administering at least one additional therapeutic agent.

135. The method of claim 134, wherein the at least one additional therapeutic agent is administered simultaneously with said one or more compounds of any one of claims 85-123, or a pharmaceutical composition of claim 124.

136. The method of claim 134, wherein the at least one additional therapeutic agent is administered sequentially with said one or more compounds of any one of claims 85-123, or a pharmaceutical composition of claim 124.

137. The method of claim 135 or claim 136, wherein the at least one additional

therapeutic agent is an anti-angiogenesis agent.

138. The method of claim 137, wherein said anti-angiogenesis agents is selected from the group consisting of bevacizumab (Avastin®), carboxyamidotriazole, TNP-470, CM101, IFN-a, IL-12, platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonists, angiostatic steroids with heparin, Cartilage-Derived

Angiogenesis Inhibitory Factor, matrix metalloproteinase inhibitors, angiostatin, endostatin, 2-methoxyestradiol, tecogalan, thrombospondin, prolactin, νβ3 inhibitors and linomide.

139. The method of claim 135 or claim 136, wherein the at least one additional

therapeutic agent is an anti-cancer compound which convert glycolysis dependent cancer to cells incapable of glycolysis.

140. The method of claim 139, wherein the anti-cancer compound which converts

glycolysis dependent cancer to cells incapable of glycolysis is selected from the group consisting of busulfan, cisp latin, carboplatin, chlorambucil,

cyclophosphamide, ifosfamide, dacarbazine (DTIC), mechlorethamine (nitrogen mustard), melphalan, carmustine (BCNU), lomustine (CC U), dactinomycin, daunorubicin, doxorubicin (Adriamycin), idarubicin, mitoxantrone, prednisone, dexamethasone, tamoxifen, fulvestrant, anastrozole, letrozole, megestrol acetate, bicalutamide, flutamide, leuprolide, goserelin, gleevac, Iressa, Tarceva, Herceptin, Avastin, L-asparaginase and tretinoin.

141. A method of inactivating a deubiquitinating protease complex comprising the step of contacting the deubiquitinating protease complex with one or more compounds of any one of claims 85-123; wherein the deubiquitinating protease complex comprises USP3,USP10, USP13, USP16 and USP18.