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• • A—HUMAN NECESSITIES
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  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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  • C07H15/20—Carbocyclic rings
  • C07H15/24—Condensed ring systems having three or more rings
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  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
  • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
  • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
  • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J51/00—Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00

Definitions

• oxysterol binding protein i.e. , a cytosoiic component that bound to oxy sterols.
• LXRs liver X receptors
• fOSBPs cytoplasmic oxysterol receptors
• ORPs OSBP-related proteins
• OSBP oxysterol-binding protein
• OSBPl is an 89 kD protein comprising a sterol-binding domain and several other domains involved in protein-protein and protein-lipid interactions. These domains include a pleckstrin homology (PH) domain, which localizes proteins to phosphatidylinositol-containing membranes, and an FFAT domain, which is an endoplasmic reticulum (ER)-Iocalizing domain.
• PH pleckstrin homology
• FFAT domain which is an endoplasmic reticulum (ER)-Iocalizing domain.
• OSBPl does not exhibit enzymatic activity. Although an understanding of its cellular function remains incomplete, studies in the last few years have revealed that OSBPl is a sterol sensor that can exert control over key signaling pathways.
• ORPs OSBP-related proteins
• each ORP contains a sterol-binding domain, although beyond that there is considerable variation in ORP configuration.
• Most ORPs include a PH domain in addition to the sterol-binding domain.
• the sterol-binding domain of OSBP/ORPs is conserved among the family of twelve proteins, but it is not similar to sterol-binding domains of other proteins such as LXR, insig, or NPC-1.
• the ORPs are less well characterized than OSBPl, although they have been implicated in lipid metabolism, signaling, vesicular traffic, and non-vesicular sterol transport.
• ORP1 L leads to atherosclerotic lesions in mice. Additionally, atherosclerotic lesions were found to contain up-regulated ORP8, and ORP9 has been identified as a therapeutic target in raising high-density lipoprotein (HDL) levels. Knockdown of OSBP1 or ORPS led to enhanced levels of cholesterol efflux upon LXR agonism. Increased cholesterol efflux is a therapeutic approach in the treatment of atherosclerosis, suggesting that ORPs may be atherosclerosis drug targets. In yeast, there are 7 Osh proteins (ORP homologs).
• yeast can survive with any six of their seven Osh proteins deleted, but not all seven.
• Cephalostatin 1 (1), OSW-1 (2), ritterazine B (3), schweinfurthin A (4), and (-)- steliettin E (5) are natural products that are potently cytotoxic to selected human cancer cell lines with half-maximal growth inhibitory concentrations (Gl 50 ) in the low nanomolar range. These molecules also have highly similar cytotoxicity patterns against the sixty cultured human cancer cell lines evaluated by the National Cancer Institute (NCI-60), strongly suggesting that they share a cellular target or act by similar mechanisms.
• NCI-60 National Cancer Institute
• CRAMs Cerostatin and Related Antiproliferative Molecules
• WO 2010/068877 discloses that CRAMs and certain synthetic analogs thereof are high affinity ligands of oxysterol binding proteins (OSBPs) and OSBP-related proteins (ORPs), including OSBP1 and ORP4.
• OSBPs oxysterol binding proteins
• ORPs OSBP-related proteins
• the invention relates in part to a number of compounds structurally related to OSW-1 , a natural compound that binds OSBPs. Because OSBPs have been shown to be integral to atherosclerosis and Alzheimer's disease (AD), an aspect of the invention relates to the use of the OSW-1 analogs, or pharmaceutically acceptable salts, enantiomers, or stereoisomers thereof, in the treatment and/or prevention of atherosclerosis, Alzheimer's disease, and cancer, including p21 -deficient cancer. Another aspect of the invention relates to conjugates of OSW-1 analogs with monoclonal antibodies, including monoclonal antibodies targeted to cancer cells. A further aspect of the invention relates to methods of treating cancer using the conjugates of the invention. Additional aspects of the invention relate to pharmaceutical compositions containing the compounds and conjugates of the invention.
• FIG. 1A depicts the chemical structures of cephalostatin (1), OSW-1 (2), ritterazine B
• FIG. IB depicts selected OSW-1 analogs.
• FIG. 2 A is graph depicting selective growth inhibitory effects of compounds 1-5 and OSW-1 analogs 6-8 toward HCT-1 16 (human colorectal carcinoma) cells lacking p21.
• BFA brefeldin A.
• FIG. 2B is an image of a Coomassie-stained polyacrylamide gel electrophoresis (PAGE) chromatograph of HeLa cell lysates using 6b in the absence (left lane) and presence (right lane) of excess OSW-1. Arrow: band identified as OSBP1.
• PAGE polyacrylamide gel electrophoresis
• FIG. 2C is a scatter plot depicting 121 proteins identified and quantified by iTRAQ- based quantitative proteomics. Competition is plotted on the y-axis as logio fold change for 10 ⁇ OSW-1 over dimethyl sulfoxide (DMSO) control and p value representing statistical significance is plotted on x-axis. P-values are arbitrarily set to 1 for non-significant single peptide quantitations.
• VAP-A vesicle-associated membrane protein-associated protein A.
• FIG. 2D is a graph depicting competition binding experiments of [ H]-25-OHC (20 nM) with compounds l -4a in SI 00 lysate made from HEK-293T cells overexpressing OSB l -myc-his.
• CPM counts per minute.
• FIG. 2E is a graph depicting competition binding experiments of j 3 H]-25-OHC (20 nM) with compounds l-4a in S100 lysate made from HEK-293T cells overexpressing ORP4L-myc-his. CPM: counts per minute.
• FIG. 3 A is a series of four bar graphs depicting knockdown/dose-shift experiment in HCT-1 16 ⁇ 2 ⁇ cells stably transfected with either OSBPl -targeting shRNA (shOSBPl) or non-targeting shRNA (shNT). Bars represent s.d. of three or four individual experiments.
• FIG. 3C is a pair of Western blots depicting HeLa cell lysates expressing lacZ-
• FIG. 3D is a group of four graphs depicting effect of overexpression of lacZ, OSBPl , or ORP4L on the growth inhibitory activity (48 h) of 1, 2, 4a, and Taxol® in HeLa cells.
• FIG. 3E is a graph depicting correlation between binding affinity (Kj) to OSBP1 - myc-his and growth inhibition (GIso) in HCT-1 16 cells.
• FIG 3F is a graph depicting correlation between binding affinity (Kj) to ORP4L-mye- his and growth inhibition (GIso) in HCT-1 16 cells.
• FIG. 4A is a pair of graphs depicting effect of 25-hydroxycholesterol coadministration on growth inhibitory activity (48 h) of cephalostatin 1 (1) (left) or OSW-2 (2) (right) in HCT-1 16 cells.
• FIG. 4B is a bar graph depicting how removal of lipoproteins from the growth medium sensitizes CHO-7 cells to 25-OHC but not 1, 2, or 4a. Mean ⁇ s.d. of two individual experiments. Bref A, brefeldin A.
• FIG. 4C is a schematic depiction of the human OSBPl and ORP4L domains. OSBPl point mutations M446W and V582M are indicated.
• FIG. 4D is a table listing the binding coefficients (K D ) of 3 H-25-hydroxycholesterol for OSBP, ORP4L, and two OSBP point mutants: OSBP(M446W) and OSBP(V582M). It also lists the inhibition binding coefficient (Kj) values for cephalostatin 1, OSW-1 and schweinfurthin A inhibiting the binding of 20 nM 3 H-25-hydroxycholesteroi to OSBP, ORP4L, OSBP(M446W), and OSBP(V582M).
• K D binding coefficients
• 4E is a panel of 8 graphs depicting effect of overexpression of wild-type (WT) OSBPl or point mutants M446W (upper row) and V582M (lower row) on the growth inhibitory activity of 1, 2, 4a, and Taxol® in HeLa cells after 48 h.
• Relative GI50 values are averages from two individual experiments.
• FIG. 5 is a panel of 10 immunofluorescence photomicrographs depicting ORPphilin- induced OSBPl translocation in HCT-116 cells treated with the agents indicated in the lower left of each image and stained with fluorescently labeled antibodies for the antigen or antigens indicated in the upper left of each image.
• FIG. 6B is a bar graph depicting inhibition of reduction in OSBPl protein levels (at 24 h time point) in the presence of indicated concentrations of the proteasome inhibitors MG- 132 or lactacystin.
• FIG. 6C is a bar graph depicting schweinfurthin A (4a) and 25-OHC at indicated concentrations do not induce reduction in OSBPl levels after 24 h of treatment, but can block the reduction induced by OSW-1 .
• FIG. 7 is a graph depicting inhibition of sphingomyelin biosynthesis in CHO- l cells by indicated concentrations of indicated ORPphilins.
• Human OSBPl is an 807-amino acid protein with certain structural and functional characteristics described above. Nucleotide and amino acid sequences for human OSBPl are publicly available as, for example. GenBank Accession Nos. NM 002556 and NPJ)02547, respectively. Corresponding nucleotide and amino acid sequences are also available for a number of other species, including mouse and rat.
• OSBPl is known to be involved in 25-hydroxycholesterol (25-OHC)-induced increase of sphingomyelin synthesis through activation of ceramide transfer protein (CERT).
• 25-OHC 25-hydroxycholesterol
• CERT ceramide transfer protein
• Sphingomyelin is a phospholipid component of cell membranes that is synthesized from ceramide and phosphatidylcholine. Some inducers of apoptosis, such as Fas ligands, do so by activating sphingomyelinase causing sphingomyelin hydrolysis and generation of ceramide, which is known to cause apoptosis. Binding of 25-OHC to OSBP l causes, indirectly, an increase in transport of ceramide from the ER to the Golgi apparatus, where sphingomyelin synthase 1 is located. This leads to an increase of sphingomyelin synthesis.
• OSBPl and CERT work in concert to coordinate cholesterol and sphingomyelin levels in cells, which is important to maintain proper membrane structure.
• OSBPl knockout mouse was reported to be embryonic lethal. This suggests although OSBPl is required for animal development, cell lines can survive with OSBPl expression ablated.
• OSBPl A second activity of OSBPl was revealed when it was discovered that caveolae of the plasma membrane that were deprived of cholesterol or treated with 25-OHC caused differentia] phosphorylation states of ERKl/2 (extracellular signal-regulated kinase 1 and 2).
• OSBPl was found to be the sterol receptor mediating this event.
• OSBP l forms a ternary complex with two phosphatases, PP2A (a serine/threonine phosphatase) and HePTP (a tyrosine phosphatase), which then inactivates ERK l/2 through dephosphorylation.
• PP2A a serine/threonine phosphatase
• HePTP a tyrosine phosphatase
• ERKl /2 is a component of the MAP /ERK signaling pathway, which is responsible for linking growth factor signaling with cellular responses including cell division.
• OSB l can control ERKl/2 activation is notable since it shows that OSBPl can link sterol binding to key signaling pathways in the cell,
• OSBPl can control up-regulation of profilin-1 through activation of the JAK-STAT pathway in response to 25-OHC and 7-ketocho!esterol.
• Human ORP4 also known as OSBP2 is expressed in different isoforms as a consequence of alternative transcription start sites from the OSBP2 gene. Specifically, a long version (ORP4L) of 916 amino acids and a short version (ORP4S) of 461 amino acids have been characterized in humans. Nucleotide and amino acid sequences for human ORP4L are publicly available as, for example, GenBank Accession Nos. AF 125185 and AAD21618, respectively. Nucleotide and amino acid sequences for human ORP4S are publicly available as, for example, GenBank Accession Nos. NM_030758 and NP _1 10385, respectively.
• OSBP/ORP family As the OSBP/ORP family has been implicated in lipid transport and metabolism, vesicle trafficking and cell signaling, this protein family contains attractive targets for the treatment of a range of disorders, including but not necessarily limited to cancer, atherosclerosis, and Alzheimer's disease.
• ORPphilins As the OSBP/ORP family has been implicated in lipid transport and metabolism, vesicle trafficking and cell signaling, this protein family contains attractive targets for the treatment of a range of disorders, including but not necessarily limited to cancer, atherosclerosis, and Alzheimer's disease. ORPphilins
• OSBP 1 and ORP4L are targets of cephalo statin 1 (1), OSW-1 (2), ritterazine B (3), schweinfurthin A (4a), and (-)-stellettin E (5).
• CRAMs Cephalostatin and Related Antiproliferative Molecules
• ORPphilins CRAMs (Cephalostatin and Related Antiproliferative Molecules)
• Compound 1 induces apoptosis through an atypical mechanism.
• Compound 2 is 30-1 50 fold more cytotoxic toward glioblastoma and leukemia cells compared to non-transformed astrocytes and lymphocytes, respectively.
• Compounds 4a and 5 are selectively growth inhibitory toward tumor cells deficient for tumor suppressors neurofibromatosis 1 (NF l ) and p21 , respectively. Repression of p21 expression and loss of NF l are known to be important for the development of some cancers. Therefore, small molecules that selectively inhibit the growth of cancer cells with these genetic alterations are attractive candidates for cancer- specific therapeutics.
• NF l tumor suppressors neurofibromatosis 1
• p21 neurofibromatosis 1
• p21 neurofibromatosis 1
• Cancer is a well-known disease in mammals characterized by abnormal cell proliferation that is uncontrolled by external signals and, in many but not all instances, the capacity to invade tissues and metastasize to distant sites. Cancer causes significant morbidity and mortality through its disruption of normal cell and tissue function.
• a number of cancers are further characterized by somatic genetic mutations, loss of differentiation, and/or abnormal expression of any of a number of tumor suppressor genes including, for example, p53, p21 , and neurofibromatosis 1 (NFl). Loss of expression of p53 and/or p21 results in loss of control of cell replication and is frequently a characteristic of cancers that are resistant to conventional anti-cancer therapies.
• the p21 protein also known as p21/WAF l protein and as cyclin-dependent kinase (CDK) inhibitor 1 , also referred to herein simply as p21 , binds to and inhibits the activity of cyclin-CDK2 or -CDK4 complexes, and thus functions as a regulator of cell cycle progression at G ⁇ .
• CDK cyclin-dependent kinase
• ORPphilins if lethal to cells lacking p21, may have an excellent therapeutic window in patients with late-stage, difficult-to-treat tumors. Since few small molecules have been reported to be highly selective towards isogenic cell lines lacking p21 , this characteristic indicates that ORPphilins are compounds with uncommon cellular targets and mechanisms of action that could render them useful in the treatment of cancer.
• Atherosclerosis results from high serum cholesterol levels (hypercholesterolemia) which leads to the accumulation of cholesterol in arterial walls.
• the plaques that characterize atherosclerosis inhibit blood flow and promote clot formation, and can ultimately cause death or severe disability via thrombotic or ischemic myocardial infarction (heart attack) and/or stroke.
• OSBPs may be useful in the treatment of atherosclerosis.
• mammalian oxysterol-binding protein-1 (OSBP1 ) binds oxygenated derivatives of cholesterol and mediates sterol and phospholipid synthesis.
• OSBP1 mammalian oxysterol-binding protein-1
• ORP8 mammalian oxysterol-binding protein-related protein 8 acts as a negative regulator of ABCA1 expression and macrophage cholesterol efflux, and thus may modulate the development of atherosclerosis. Yan et al. (2008) J Biol Chem 283:332-40.
• Alzheimer's disease is a common, chronic neurodegenerative disease that often leads to dementia and death, which is characterized by a progressive loss of memory and sometimes severe behavioral abnormalities, as well as an impairment of other cognitive functions. It ranks as the fourth leading cause of death in industrialized societies after heart disease, cancer, and stroke. The incidence of Alzheimer's disease is high, with an estimated 2.5 to 4 million patients affected in the United States and perhaps 1 7 to 25 million worldwide. Moreover, the number of sufferers is expected to grow as the population ages. A
• amyloid plaques insoluble deposits, known as amyloid plaques, in the brains of those affected.
• Autopsies have shown that amyloid plaques are found in the brains of virtually all Alzheimer's patients and that the degree of amyloid plaque deposition often correlates with the degree of dementia. While some opinion holds that amyloid plaques are a late-stage by-product of the disease process, the majority view is that amyloid plaques and/or soluble aggregates of amyloid peptides are more likely to be intimately, and perhaps causally, involved in Alzheimer's disease.
• Alzheimer's disease Because there is no cure for Alzheimer's disease, managing the disease usually involves medications to control symptoms, in combination with various non-drug strategies designed to ease the suffering of the person afflicted and that of his or her family and caregivers. Unfortunately, not all patients with Alzheimer's disease are responsive to the currently available therapies.
• OSBP1 has been shown to modulate processing and trafficking of the amyloid precursor protein. Laitinen et al. (2002) J Lipid Res 43:245-55. These results suggest that OSBP1 could play a role in linking cholesterol metabolism with intracellular amyloid production, and, more importantly, indicate that OSBP1 could provide an alternative target for a directed therapeutic for human maladies including Alzheimer's disease and
• Cephalostatins are a group of complex steroidal pyrazine alkaloids which were first isolated from the sea worm Cephalodiscus gilchrisli. They represent cytotoxins that are highly effective against the PS cell line (EDgo of 10 "7 - 10 "9 ⁇ g/mL) and are anti-tumor agents. They are natural marine products that occur rarely, however, and are available only in extremely small amounts. For example, only 139 mg of cephalostatin 1 and a total of 272 mg of other cephalostatins could be isolated from 166 kg of Cephalodiscus gilchrisli (tubular worms 5 mm long).
• cephalostatins such as cephalostatin 1 , cephalostatin 2, cephalostatin 3, cephalostatin 4, cephalostatin 5, cephalostatin 6, cephalostatin 7, 25'-epi ⁇ cephalostatin 7, 20-epi-cephalostatin 7, cephalostatin 8, cephalostatin 9, cephalostatin 10, cephalostatin 1 1 , cephalostatin 12, cephalostatin 13, cephalostatin 14, cephalostatin 15, cephalostatin 16, cephalostatin 17, cephalostatin 18, and cephalostatin 19) are intended to be encompassed.
• cephalostatin 1 induces apoptosis without formation of the apoptosome and without release of cytochrome c from the mitochondria, both events that occur with most cytotoxic anti-cancer small molecules.
• cephalostatin 1 leads to activation of caspase-4 and caspase-2 as well as release of Smac (second mitochondria-derived activator of caspase) from the mitochondria. This specific sequence of events is rarely observed in small molecule-induced apoptosis, suggesting that studying the mechanism of cephalostatin 1 may uncover new apoptotic signaling
• cephalostatin 1 may be useful in treating tumors that have become resistant to conventional cancer therapeutics.
• cephalostatin 1 also led to formation of the multi-protein complex known as the PIDDosome (comprising proteins P1DD, RAIDD, and caspase-2).
• the histone deacetylase inhibitor trichostatin A is the only other small molecule known to induce formation of the PIDDosome.
• Cephalostatin 1 was also shown to induce an ER stress response.
• cephalostatin analogues A few synthetically produced cephalostatin analogues, synthetic methods for preparing the cephalostatin analogues, pharmaceutical compositions containing the analogues, and methods for using the analogues as active agent in pharmaceutical uses, are described in U.S. Patent No. 5,708,164 (Winterfeldt, E. et al.); which is hereby incorporated by reference in its entirety, such as for the cephalostatin analogs, and their preparation, as described therein,
• OSW- 1 (2) is more abundant than cephalostatin 1 , which has enabled some in vivo studies.
• the GI5 0 of OSW-1 taken as an average against the NCI-60, is 0.78 nM.
• nude mice subcutaneous! y implanted with P388 tumor cells demonstrated a 59% increased life span upon a single intraperitoneal ( i.p.) treatment of 0,01 mg/kg OSW-1.
• OSW-1 also shows selectivity for malignant cell types over non-malignant cells.
• CLL chronic lymphocytic leukemia
• OSW-1 pancreatic cancers
• leukemias pancreatic cancers
• colon cancers malignant gliomas and other brain tumors
• ovarian cancers are disclosed in US Patent Application Publication No. 2005/0004044 (Huang, P. et al.); which is hereby incorporated by reference in its entirety, such as for the preparation of OSW-1 analogs.
• the family of natural products known as the schweinfurthins currently includes four compounds isolated from the African plant Macaranga schweinfurihii Pax.
• Schweinfurthins B and D also display significant activity in the NCI's 60-cell-line anticancer assay with mean GI50 values of less than 1 ⁇ .
• Their biological activity has attracted interest because some central nervous system (CNS), renal, and breast cancer cell lines are among the types most sensitive to these compounds. Inspection of the spectrum of activity shows no correlation with any currently used agents and suggests that these compounds may be acting at a previously unrecognized target or through a novel mechanism.
• (-)-Stellettin E (5) is a marine natural product also reported to have a cytotoxicity pattern similar to cephalostatin 1 (p not reported).
• (-)-Stellettin E is a light-sensitive compound only isolated in small quantities, prohibiting detailed cell-based studies and in vivo experiments.
• OSBP1 and its closest paralog ORP4L as high affinity receptors of cephalostatin 1 (1), OSW-1 (2), ritterazine B (3) and schweinfurthin A/B (4a/4b). These compounds are disclosed herein to alter several aspects of OSBPl cellular activity, revealing new activities of the largely mysterious OSBP/ORP proteins. For instance, 1 induces partial localization of OSBPl to the plasma membrane (FIG. 5E), 1 and 2 cause OSBPl degradation (FIG. 6), and all ORPphilins studied induce a reduction in the rate of sphingomyelin biosynthesis (FIG. 7).
• OSBPl and its paralogs Prior to the instant invention, the function of OSBPl and its paralogs, the ORPs, has not been well understood, due in part to a lack of reagents capable of altering the function of these proteins in cells. Small molecule perturbation of OSBP/ORPs by administration of 25- OHC or alteration of cellular cholesterol levels could affect many proteins beyond
• ORPphilins are likely more specific for OSB l and ORP4L than 25-OHC, since results disclosed herein show that neither NPC-1 or Insig— two cellular receptors of 25-OHC— re involved in the activity of these compounds. All of the ORPphilins appear to compete with 25-OHC for binding to OSBPl and ORP4L. The simplest explanation for this observation is that the ORPphilins displace 25-OHC from the highly conserved lipid-binding domain, which is present in the C- terminal end of all of the OSBP/ORP proteins.
• ORPphilins Convergence of the ORPphilins on the same protein targets validates the indication, provided by their similar pattern of growth inhibition in " NCl-60 cell line panel, that these compounds could have the same mechanism of action.
• findings disclosed herein reveal that the ORPphilins have individually distinct activities in targeting OSBPl and ORP4L.
• cephalostatin 1 (1) and OSW-1 (2) induce OSBPl degradation in cells, whereas schweinfurthin A (4a) does not.
• Immunofluorescence microscopy shows that the different ORPphilins affect OSBPl localization and Golgi integrity in different ways. Further, 1 and 2 bind OSBPl and ORP4L with only slight variations in affinity.
• ORPphilins and also the reason 4a falls off the ORP4L binding/cytotoxicity correlation.
• This large difference in schweinfurthin A's binding affinity for OSBPl and ORP4L suggests that it is feasible to discover small molecules that selectively interact with individual members of, or subsets of, the OSBP/ORP superfamily.
• the growth inhibitory activity of the ORPphilins was significantly suppressed by co-incubation of cells with sub-lethal doses of 25-OHC, a known high-affinity ligand of OSBPl and ORP4L.
• 25-OHC a known high-affinity ligand of OSBPl and ORP4L.
• 25-OHC competes with the ORPphilins for binding to OSBPl in cells came from observation that 25-OHC suppressed OSBPl degradation induced by OSW-1 (2). If OSBPl and ORP4L were decoy targets, displacement of the ORPphilins by 25-OHC would free more compound to interact with the actual efficacy target. This would be expected to sensitize cells to ORPphilins, not desensitize cells as observed.
• OSBPl over-expression of OSBPl (M446W) desensitized cells to 1, 2 and 4a, and over-expression of OSBPl (V582M) desensitized cells to 1.
• these mutants did not show altered binding to 1, 2, or 4a.
• One possible explanation for the desensitization of cells over- expressing these OSBPl mutants is that these mutations affect protein-protein interactions, and that the interaction of OSBPl and other proteins is involved in the antiproliferative activity of the ORPphilins. Mutants of the thyroid hormone ⁇ receptor provide a precedent for this possibility since known mutants in this protein suppress ligand-induced
• OSBPl has several known interacting partners and has been reported to function as a sterol-sensing scaffolding protein, capable of making multi-protein complexes in a ligand binding-dependent fashion. Therefore it is reasonable to speculate that the activity of the ORPphilins is reliant on OSBPl interacting with other proteins, and that mutations altering OSBPl protein-protein
• ORPphilins perturb the cellular functions of multiple members of the OSBP/ORP superfamily, beyond OSBPl and ORP4L, and the inhibition of these other OSBP/ORPs contribute to the antiproliferative activity of the ORPphilins. This would be a likely possibility if there was a critical cellular function performed redundantly by multiple OSBP/ORPs, which would mirror the situation in yeast.
• Yeast possess 7 Osh proteins, which are the yeast homologs of the OSBP/ORPs. Yeast reportedly can survive the loss of any six of the seven Osh proteins, but not loss of all seven. Furthermore, the
• OSBP/ORPs exhibit significant sequence homology in the lipid-binding domain, and most have been shown to bind 25-OHC. Given that the ORPphilins compete with 25-OHC for binding to OSBPl and ORP4L, other OSBP/ORPs may bind these compounds. The significant desensitization achieved by addition of 25-OHC (FIG. 4A) could be due to displacement of ORPphilins from other ORPs in addition to OSBP l and ORP4L.
• ORP4L mRNA levels have been reported to be increased in metastatic breast cancer tumors compared to localized tumors, signifying a possible role for ORP4L in the early steps of the metastatic cascade. Fournier et al. ( 1999) Cancer Ren. 59:3748-53. Therefore.
• ORPphilins will be useful reagents to further explore the role of ORP4L in cancer metastasis.
• cephalostatin 1 (1) and OSW-1 (2) induce a significant drop in OSBPl levels will be useful in studies of their anti-tumor activities, since this effect could be used to determine whether the compounds have reached specific tissues. Moreover, compounds 1 and 2 could be used to lower OSBPl protein levels in animal models of disease.
• OSW-1 analogs concern OSW-1 analogs, antibody conjugates formed with these analogs, pharmaceutical compositions containing the analogs or conjugates, and the use of the analogs, conjugates, and pharmaceutical compositions of the invention for the treatment of oxysterol binding protein (OSB l )-related diseases or conditions, including, in particular, cancer.
• An aspect of the invention is a compound of formula (I) or a pharmaceutically acceptable salt thereof:
• R', R 2 , R 3 , and R 6 are independently selected from the group consisting of hydrogen,
• Z is absent or is selected from the group consisting of O and NR 10 ;
• n is an integer 0-6;
• R is selected from the group consisting of substituted or unsubstituted: alky!, alkenyl, amino, alloc-protected amino, Fmoc-protected amino, aryl, 5- to 13-membered cycloaikyl or cycloalkenyl group, and 5- to 10-membered cyclic heteroaromatic group comprising 1-3 heteroatoms independently selected from the group consisting of N, O, and S;
• R 4 is selected from the group consisting of hydrogen and C1 -C6 alkyl
• R 5 is selected from the group consisting of hydrogen and acyl
• R 10 is selected from the group consisting of hydrogen and C1 -C6 alkyl
• R 1 , R 2 , R 3 , and R 4 are hydrogen and R s is acetyl, then R 6 is not hydrogen, /rora-methoxybenzoyl, or 3-phenyl-2-propenoyl;
• R 1 is 4-(alloc-aminomethyl)benzylaminocarbonyl, then at least one of R 2 , R 3 , R 4 , R 5 , and R 6 is not hydrogen; or (e) if R 2 or R 3 is 4-(alloc-aminomethyl)benzylaminocarbonyl, then R ⁇ is not acetyl or R 6 is not /?ara-methoxybenzoyl.
• the present invention relates to any one of the above- referenced compounds, wherein R 4 is hydrogen.
• the present invention relates to any one of the above-referenced compounds, wherein R 4 is a C 1 - C6 alkyl.
• the present invention relates to any one of the above-referenced compounds, wherein R 4 is methyl.
• the present invention relates to any one of the above- referenced compounds, wherein R 11 is hydrogen.
• the present invention relates to any one of the above-referenced compounds, wherein R 3 is acyl.
• the present invention relates to any one of the above- referenced compounds, wherein R 5 is acetyl (Ac).
• the present invention relates to any one of the above- referenced compounds, wherein R ! is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R 1 is trialkylsilyl. Alternatively, in certain embodiments he present invention relates to any one
• the present invention relates to any one of the above- referenced compounds, wherein R 2 is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R " is trialkylsilyl. Alternatively, in certain embodiments he present invention relates to any one
• the present invention relates to any one of the above- referenced compounds, wherein R 3 is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R J is trialkylsilyl. Alternatively, in certain embodiments, the present invention relates to any one
• the present invention relates to any one of the above- referenced compounds, wherein R 6 is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R 6 is trialkylsilyl. Alternatively, in certain embodiments, the present invention relates to any one
• the present invention relates to any one of the above-referenced compounds, wherein R 6 is /wo-methoxybenzoyl. In another example, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R 6 is 3-phenylpropanoyl.
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of R is alkyl. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is alkenyl. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is an amino group. In certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein one or more instances of R is an alloc-pro tected amino group. In certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein one or more instances of R is an Fmoc-protected amino group.
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of R is aryl. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is 5- to 13-membered cycloalkyl group. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is a 5- to 13-membered cycloalkenyl group.
• the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is a 5- to 10-membered cyclic heteroaromatic group comprising 1-3 heteroatoms independently selected from the group consisting of N, O, and S.
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of Z is oxygen (O).
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is O and R is alkyl.
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is O and R is 4-nitrophenyl.
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is O and n is zero (0).
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is O and n is one (1).
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is O and n is 2. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is 3. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is 4. Alternatively, in certain embodiments, the present invention relates to any one of the above -referenced compounds, wherein in one or more instances Z is O and n is 5. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is 0 and n is 6.
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of Z is NR 10 .
• the present invention relates to any one of the above-referenced compounds, wherein one or more instances of Z is Nil.
• the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is NR 10 and R 10 is a C1 -C6 alkyl.
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is Ni l and R is an amino group. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is Nil and R is an alloc- or Fmoc- N'- substituted amino group.
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is NH and n is 6. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is zero (0). In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is one (1). In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is 2.
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is NH and n is 3. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is 4. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is 5.
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is NH and R is a para- aminoalkylaryl group. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and R is an alloc- or Fmoc- /V'-substituted ra-aminomethylphenyl group.
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is NH, R is a /? r «-arninoalkylaryl group or an alloc- or Fmoc- N'- substituted /?ara-aminomethylphenyl group, and n is one (1).
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is NH, R is phenyl, and n is one (1).
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of Z is absent, such that there is a bond between the carbonyl carbon and the methylene group if n is non-zero, or between the carbonyl carbon and R if n is zero (0).
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is absent and R is an amino group. In certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is absent and R is an alloc- or Fmoc- N'-substituted amino group.
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is absent and n is 2. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is zero (0). In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is one (1). In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is 3. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is 4.
• the present invention relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is 5. In certain embodiments, the present invention relates to any one of the above -referenced compounds, wherein in one or more instances Z is absent and n is 6.
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is absent and R is a 1 -imidazolyl group. In certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein in one or more instances Z is absent, R is a 1 -imidazolyl group, and n is zero (0).
• the present invention relates to any one of the above- referenced compounds, wherein at least one of R 1 , R 2 , and R 3 is ter/-butyldimethylsilyl.
• the present invention relates to any one of the above- referenced compounds, wherein R 1 is fcr ⁇ butyldime hylsilyl.
• the present invention relates to any one of the above-referenced compounds, wherein R 2 is /eri-butyldimethylsilyl.
• the present invention relates to any one of the above-referenced compounds, wherein R is tert- butyldimethylsilyl.
• the present invention relates to any one of the above-referenced compounds, wherein each of R and R is tert- butyldimethylsilyl.
• the present invention relates to any one of the above-referenced compounds, wherein each of R and R " is tert- butyldimethylsilyl.
• the present invention relates to any one of the above-referenced compounds, wherein each of R " and R is tert- butyldimethylsilyl.
• the present invention relates to any one of the above-referenced compounds, wherein each of R " and R is tert- butyldimethylsilyl.
• An aspect of the invention is a compound selected from the group consisting of:
• An aspect of the invention is a pharmaceutical composition.
• the pharmaceutical composition includes any one of the above-referenced compounds and a pharmaceutically acceptable carrier.
• a pharmaceutical composition according to this aspect of the invention can be prepared by a method that includes the step of combining a therapeutically effective amount of any one of the above-referenced compounds and a pharmaceutically acceptable carrier.
• the pharmaceutical composition and method for its preparation can include more than one of the above-referenced compounds.
• the pharmaceutical composition and method for its preparation can of course optionally further include at least one additional pharmaceutically active agent other than a compound of the invention.
• An aspect of the invention concerns additional OSW- 1 analog compounds including distinct linker subunits containing functionality capable of bonding OSW- 1 analogs to other molecules at specific positions on the OSW-1 framework structure.
• the OSW-1 analogs according to this aspect of the invention may be particularly suited for forming conjugates with proteins, including, for example, antibodies.
• An aspect of the invention concerns a compound of formula ( ⁇ ) or a
• X is selected from the group consisting of O and NOR ;
• R l , R 2 , and R 3 are independently selected from the group consisting of hydrogen, R 20 ,
• Z is absent or is selected from the group consisting of O and NR . 1 1 0.
• n is an integer 0-6;
• R is selected from the group consisting of substituted or unsubstituted: alkyl, alkenyi, amino, alloc-protected amino, Fmoc-protected amino, aryl, 5- to 13-membered cycloalkyl or cycloalkenyl group, and 5- to 10-membered cyclic heteroaromatic group comprising 1 -3 heteroatoms independently selected from the group consisting of N, O, and S;
• R 4 is selected from the group consisting of hydrogen, C1 -C6 alkyl, and R 20 ;
• R 10 is selected from the group consisting of hydrogen and C 1 -C6 alkyl
• Z' is selected from the group consisting of 0 and l 10 ;
• n is an integer 1-6;
• the compound includes one and only one R 20 .
• the present invention relates to any one of the above- referenced compounds, wherein R 1 is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R is R' . Alternatively, in certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein R ! is trialkylsilyl. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R 1 is
• the present invention relates to any one of the above- referenced compounds, wherein R 2 is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R ⁇ is R ' Alternatively, in certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein is trialkylsilyl. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R 2 is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R ⁇ is R ' Alternatively, in certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein is trialkylsilyl. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R 2 is
• the present invention relates to any one of the above- referenced compounds, wherein R 3 is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one ol the above-referenced compounds, wherein R J is R: . Alternatively, in certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein R 3 is trialkylsilyl. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R 3 is
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of Z is oxygen (O).
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of Z is NR 10 .
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of Z is absent, such that there is a bond between the carbonyl carbon and the methylene group if n is non-zero, or between the carbonyl carbon and R if n is zero (0).
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances n is zero (0). Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances n is one (1 ). Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances n is 2. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances n is 3. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances n is 4.
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances n is 5. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances n is 6.
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of R is alkyl. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is alkenyl. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is an amino group. In certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein one or more instances of R is an alloc-protected amino group. In certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein one or more instances of R is an Fmoc-protected amino group.
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of R is aryl. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is 5- to 13-membered cycloalkyl group. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is a 5- to 13-membered cycloalkenyl group.
• the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is a 5- to 10-membered cyclic heteroaromatic group comprising 1-3 heteroatoms independently selected from the group consisting of N, O, and S.
• the present invention relates to any one of the above- referenced compounds, wherein R 4 is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R" is Cl-C6 alkyl. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R 4 is R 20 .
• the present invention relates to any one of the above- referenced compounds, wherein X is oxygen (O).
• the present invention relates to any one of the above- referenced compounds, wherein X is O and each of R 1 , R 2 , R 3 , and R 4 is selected from the group consisting of hy drogen and R 20 . Only one of R 1 , R 2 , R 3 , and R 4 is R 20
• the present invention relates to any one of the above-referenced compounds, wherein X is O, R 1 is R 20 , and each of R 2 , R 3 , and R 4 is hydrogen.
• the present invention relates to any one of the above- referenced compounds, wherein X is O, R 2 is R 20 , and each of R !
• the present invention relates to any one of the above-referenced compounds, wherein X is O, R 3 is R 20 , and each of R 1 , R 2 , and R 4 is hydrogen. As yet another example, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein X is O, R 4 is R 20 , and each of R ! , R 2 , and R is hydrogen.
• the present invention relates to any one of the above-referenced compounds, wherein X is NR 10 .
• the present invention relates to any one of the above-referenced compounds, wherein X is NR !0 and each of R 1 , R 2 , R 3 , and R 4 is hydrogen.
• the pre nt invention relates to any one of the above-
• present invention relates to any one of the above-
• the present invention relates to any one of ove-referenced compounds, wherein R 20 is
• the present invention relates to any one of the above-referen m r in R 20 is
• the present invention relates to any one of the above- referenced compounds, wherein m is one ( 1 ). Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein m is 2. Alternatively, in certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein m is 3. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein m is 4.
• the present invention relates to any one of the above- referenced compounds, wherein m is 5. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein m is 6.
• the present invention relates to any one of the above-referenced compounds, wherein Z r is oxygen (O).
• the present invention relates to any one of the above-referenced compounds, wherein ! is NR 10 2012/038562
• the present invention relates to any one of the
• the present invention relates to any one of the above-referenced compounds, wherein R is
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of R 10 is hydrogen. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R 10 is C1-C6 alkyl.
• An aspect of the invention concerns a compound of formula (III) or a
• X is selected from the group consisting of O and NOR " ;
• R' , R 2 , and R 3 are independently selected from the group consisting of hydrogen, R " ,
• Z is absent or is selected from the group consisting of O and NR 10 ;
• n is an integer 0-6;
• R is selected from the group consisting of substituted or unsubstituted: alkyl, alkenyl, amino, alloc-protected amino, Fmoc-protected amino, aryl, 5- to 13-membered cycloalkyl or cycloalkenyl group, and 5- to 10-memhered cyclic heteroaromatic group comprising 1 -3 heteroatoms independently selected from the group consisting of N, O, and S;
• R 4 is selected from the group consisting of hydrogen, C 1 -C6 alkyl, and R 20 ;
• R 10 is selected from the group consisting of hydrogen and C1-C6 alkyl
• R is selected from the group consisting of hydrogen, methyl, and C2-C6 alkyl;
• Z' is selected from the group consisting of O and NR 10 ;
• n is an integer 1-6;
• the compound includes one and only one R .
• the present invention relates to any one of the above- referenced compounds, wherein R 1 is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R is R . Alternatively, in certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein R 1 is trialkylsilyl. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R 1 is
• the present invention relates to any one of the above- referenced compounds, wherein R 2 is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R is R ⁇ . Alternatively, in certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein R 2 is trialkylsilyl. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R 2 is
• the present invention relates to any one of the above- referenced compounds, wherein R 3 is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R is R ⁇ . Alternatively, in certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein R J is trialkylsilyl. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R is
• the present invention relates to any one of the above- referenced compounds, wherein R 30 is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R 30 is methyl. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R is C2-C6 alkyl.
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of Z is oxygen (O).
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of Z is NR 10 .
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of Z is absent, such that there is a bond between the carbonyl carbon and the methylene group if n is non-zero, or between the carbonyl carbon and R if n is zero (0).
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances n is zero (0). Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances n is one (1). Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances n is 2. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances n is 3. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances n is 4.
• the present invention relates to any one of the above- referenced compounds, wherein in one or more instances n is 5. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein in one or more instances n is 6.
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of R is alkyl. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is alkenyl. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is an amino group. In certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein one or more instances of R is an alloc-protected amino group. In certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein one or more instances of R is an Fmoc-protected amino group.
• the present invention relates to any one of the above - referenced compounds, wherein one or more instances of R is aryl. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is 5- to 13-membered cycloalkyl group. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is a 5- to 13-membered cycloalkenyl group.
• the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R is a 5- to 10-membered cyclic heteroaromatic group comprising 1-3 heteroatoms independently selected from the group consisting of N. O, and S.
• the present invention relates to any one of the above- referenced compounds, wherein R 4 is hydrogen. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R 4 is C 1-C6 alkyl. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein R 4 is R 20 .
• the present invention relates to any one of the above- referenced compounds, wherein X is oxygen (O). In certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein X is O and each of R 1 , R 2 , R 3 , and R 4 is selected from the group consisting of hydrogen and R 20 . Only one of R 1 , R 2 , R 3 , and R 4 is R 20 . For example, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein X is O, R is R , and each of R , R " , and R is hydrogen.
• the present invention relates to any one of the above- referenced compounds, wherein X is O, R 2 is R 20 , and each of R 1 , R 3 , and R 4 is hydrogen.
• the present invention relates to any one of the above-referenced compounds, wherein X is O, R 3 is R 20 , and each of R 1 , R 2 , and R 4 is hydrogen.
• the present invention relates to any one of the above-referenced compounds, wherein X is O, R 4 is R 20 , and each of R 1 , R 2 , and R J is hydrogen.
• the present invention relates to any one of the above-referenced compounds, wherein X is NR 10 .
• the present invention relates to any one of the above-referenced compounds, wherein X is NR 10 and each of R ⁇ R 2 , R ⁇ and R 4 is hydrogen.
• the present invention relates to any one of the above-
• the resent invention relates to any one of the above-
• the present invention relates to any one of the above-ref renced compounds, wherein R 20 is
• the present invention relates to any one of the above-referenced compounds, wherein R 20 is
• the present invention relates to any one of the above- referenced compounds, wherein m is one (1). Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein m is 2. Alternatively, in certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein m is 3. Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein m is 4.
• the present invention relates to any one of the above- referenced compounds, wherein m is 5.
• the present invention relates to any one of the above-referenced compounds, wherein m is 6.
• the present invent relates to any one of the above-
• the present invention relates to any one of the above-referenced compounds, wherein Z' is oxygen (0). Alternatively, in certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein Z f is NR 10 .
• the present invention relates to any one of the above-referenced compounds, wherein R 20 is
• the present invention relates to any one of the
• the present invention relates to any one of the above- referenced compounds, wherein one or more instances of R 10 is hydrogen. In certain embodiments, the present invention relates to any one of the above-referenced compounds, wherein one or more instances of R i 0 is C1-C6 alkyl. In certain embodiments, the present invention relates to any one of the above- referenced compounds, wherein X is O, R 1 is
• the present invention relates to any one of the above- referenced compounds, wherein the compound is present as only a single stereoisomer.
• An aspect of the invention is a conjugate comprising a monoclonal antibody covarrily linked to any one of the above-referenced compounds.
• the compound is a compound of formula (II) or a pharmaceutically acceptable salt thereof, which includes an R 20 moiety as described herein.
• the compound is a compound of formula (III) or a pharmaceutically acceptable salt thereof, which includes an R moiety as described herein.
• the monoclonal antibody and the compound are covlusly linked through a cysteine thiol-maleimide Michael addition product, a valine- citrulline p-aminobenzyl linker, or a valine-citrulline p-aminobenzylcarbamate linker.
• linkage chemistry in general, is well known in the art and does not require further description here. See, for example, U.S. Pat. Nos. 7,659,241 to Senter et al., 7,745,394 to Doronina et ai., 7,829,531 to Senter et al., and 7,851 ,437 to Senter et al., the entire contents of which are incorporated herein by reference.
• the monoclonal antibody preferably retains its cognate antigen-binding specificity and avidity when it is part of the conjugate.
• the compound preferably retains its activity as an ORPphilin when it is part of the conjugate.
• Monoclonal antibodies and methods for their preparation are well known in the art.
• Monoclonal antibodies can include whole antibodies as well as antigen-binding fragments thereof, e.g.. Fab fragments and F(ab') 2 fragments of monoclonal antibodies.
• monoclonal antibodies also can include chimeric, humanized, and fully human antibodies.
• monoclonal antibodies can include bispecific antibodies.
• the monoclonal antibody binds specifically to an antigen expressed on a cancer cell.
• such antigens may be overexpressed by the cancer cell, characteristically expressed by the cancer cell, or uniquely expressed by the cancer cell. Antigens of these types have been termed cancer antigens or tumor antigens. Some of these antigens may be tumor-specific antigens or tumor- associated antigens.
• Tumor-specific antigens are antigens that are specifically present in tumor cells but not normal cells.
• tumor-specific antigens are viral antigens in tumors induced by DNA or R A viruses.
• Tuor-associated antigens are present in both tumor cells and normal cells but are present in a different quantity or a different form in tumor cells.
• antigens examples include oncofetal antigens (e.g., carcinoembryonic antigen (CEA)), differentiation antigens (e.g., T and T antigens), and oncogene products (e.g., HER/neu).
• CEA carcinoembryonic antigen
• T and T antigens differentiation antigens
• oncogene products e.g., HER/neu
• cancer antigen as used herein is a compound, such as a peptide or protein, associated with a tumor or cancer cell surface and which is capable of provoking an immune response when expressed on the surface of an antigen-presenting cell (APC) in the context of an MHC molecule.
• Cancer antigens can be prepared from cancer cells either by preparing crude extracts of cancer cells, for example, as described in Cohen et al. (1994) Cancer Res 54:1055, by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens. Cancer antigens include but are not limited to antigens that are
• antigens can be isolated or prepared recombinantly or by any other means known in the art.
• a cancer antigen is a human cancer antigen, i.e., a cancer antigen associated with or derived from a human cancer.
• Non-limiting examples of already-commercialized monoclonal antibodies that bind specifically to an antigen expressed on a human cancer cell include alemtuzumab (Campath; anti-CD52); bevacizumab (Avastin; anti-VEGF); cetuximab (Erbitux; anti-EGFR):
• gemtuzumab (Mylotarg; anti-CD33); ibritumomab (Zevalin; anti-CD20); panitumumab (Vectibix; anti-EGFR); rituximab (Rituxan; anti-CD20); tositumomab (Bexxar; anti-CD20); and trastuzumab (Herceptin; anti-ErbB2).
• ibritumomab Zevalin; anti-CD20
• panitumab Vectibix; anti-EGFR
• rituximab Rauxan; anti-CD20
• tositumomab Bexxar; anti-CD20
• trastuzumab Herceptin; anti-ErbB2
• Many additional monoclonal antibodies that bind specifically to an antigen expressed on a human cancer cell are under development, including those under regulatory review.
• the cancer cell expressing the antigen recognized by the monoclonal antibody is deficient for expression of p21.
• a cancer cell of this type is referred to herein as p21 -deficient.
• the level of p21 expression can be assessed using any suitable method, including, for example, binding by a p21 -specific monoclonal antibody either to whole cells or cell extracts, reverse transcriptase-polymerase chain reaction (RT-PC ) performed using appropriate sequence-specific oligonucleotide primers, Northern blotting, and the like.
• RT-PC reverse transcriptase-polymerase chain reaction
• Monoclonal anti-p21 antibodies are available from a variety of commercial suppliers.
• the nucleotide sequence of human p21/WAFI is publicly available as, for example, GenBank Accession No. S67388.
• a cell is said to be p21 -deficient when the expressed amount of p21 is objectively and measurably less than the expressed amount of p21 as measured in a suitable positive control cell.
• a cell is said to be p21 -deficient when the expressed amount of p21 is at least 5 percent less than the expressed amount of p21 as measured in a suitable positive control cell.
• a cell is said to be p21 - deficient when the expressed amount of p21 is at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 99, or 100 percent less than the expressed amount of p21 as measured in a suitable positive control cell.
• An aspect of the invention is a pharmaceutical composition.
• the pharmaceutical composition includes any one of the above-referenced conjugates and a pharmaceutically acceptable earner.
• a pharmaceutical composition according to this aspect of the invention can be prepared by a method that includes the step of combining a therapeutically effective amount of any one of the above-referenced conjugates and a pharmaceutically acceptable carrier.
• the pharmaceutical composition and method for its preparation can include more than one of the above-referenced conjugates.
• the pharmaceutical composition and method for its preparation can include more than one of the above-referenced conjugates.
• the pharmaceutical composition and method for its preparation can include more than one of the above-referenced conjugates.
• composition and method for its preparation can of course optionally further include at least one additional pharmaceutically active agent other than a conjugate of the invention.
• An aspect of the invention is a method of killing a mammalian cell.
• the method includes the step of contacting a mammalian cell with an effective amount of any one of the aforementioned compounds of the invention.
• the method relates to any one of the compounds according to formula (1) or a pharmaceutically acceptable salt thereof:
• R ! , R 2 , R 3 , and R 6 are independently selected from the group consisting of hydn
• Z is absent or is selected from the group consisting of O and NR 10 ;
• n is an integer 0-6;
• R is selected from the group consisting of substituted or unsubstituted: alkyi, alkenyl, amino, alloc-protected amino, Fmoc-protected amino, aryl, 5- to 13-membered cycloalkyl or cycloalkenyl group, and 5- to 10-membered cyclic heteroaromatic group comprising 1 -3 heteroatoms independently selected from the group consisting of N, O, and S;
• R 4 is selected from the group consisting of hydrogen and C1-C6 alkyi
• R 5 is selected from the group consisting of hydrogen and acyl
• R 10 is selected from the group consisting of hydrogen and C1 -C6 alkyi
• R 1 , R 2 , R 3 , and R 4 are hydrogen and R 5 is acetyl, then R 6 is not hydrogen, para-methoxy benzoyl, or 3-phenyl-2-propenoyl;
• R 1 is 4-(alloc-aminomethyl)benzylaminocarbonyl, then at least one of R 2 , R 3 , R 4 , R 5 , and R 6 is not hydrogen; or
• the method relates to any one of the above-referenced compounds, wherein R 4 is hydrogen. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein R 4 is a C1 -C6 alkyl. For example, in certain embodiments, the method relates to any one of the above -referenced compounds, wherein R 4 is methyl.
• the method relates lo any one of the above-referenced compounds, wherein R 3 is hydrogen.
• the method relates to any one of the above-referenced compounds, wherein R 5 is acyl.
• the method relates to any one of the above-referenced compounds, wherein R 5 is acetyl (Ac).
• the method relates to any one of the above-referenced compounds, wherein R 1 is hydrogen. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein R 1 is trialkylsilyl.
• the method relates to any one of the above-referenced
• the method relates to any one of the above-referenced compounds, wherein R 2 is hydrogen. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein R 2 is trialkylsilyl.
• the method relates to any one of the above-referenced
• the method relates to any one o the above-referenced compounds, wherein R 3 is hydrogen.
• the method relates to any one of the above-referenced compounds, wherein R J is trialkylsilyl.
• the method relates to any one of the above-referenced
• the method relates to any one of the above-referenced ompounds, wherein R 6 is hydrogen.
• the method 2 relates to any one of the above-referenced compounds, wherein R 6 is trialkylsilyl.
• the method relates to any one of the above-referenced
• the method relates to any one of the above-referenced compounds, wherein R 6 is para- methoxybenzoyl.
• the method relates to any one of the above-referenced compounds, wherein R 6 is 3-phenylpropanoyl.
• the method relates to any one of the above-referenced compounds, wherein one or more instances of R is alky!. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein one or more instances of R is alkenyl. In certain embodiments, the method relates to any one of the above- referenced compounds, wherein one or more instances of R is an amino group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein one or more instances of R is an alloc-protected amino group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein one or more instances of R is an Fmoc-protected amino group.
• the method relates to any one of the above-referenced compounds, wherein one or more instances of R is aryl. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein one or more instances of R is 5- to 13-membered cycloalkyl group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein one or more instances of R is a 5- to 13-membered cycloalkenyl group. In certain
• the method relates to any one of the above-reierenced compounds, wherein one or more instances of R is a 5- to 10-membered cyclic heteroaromatic group comprising 1 - 3 heteroatoms independently selected from the group consisting of N, 0, and S.
• the method relates to any one of the above-referenced compounds, wherein one or more instances of Z is oxygen (O),
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and R is alkyl.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and R is 4-nitrophenyl.
• the method relates to any one of the above -referenced compounds, wherein in one or more instances Z is O and n is zero (0). Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is one (1). Alternatively, in certain embodiments, in certain
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is 2. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is 3. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is 4.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is 5. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is 6.
• the method relates to any one of the above-referenced compounds, wherein one or more instances of Z is NR 10 .
• one or more instances of Z is NR 10 .
• the method relates to any one of the above-referenced compounds, wherein one or more instances of Z is NH. In another example, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NR l 0 and R )() is a Cl-C6 alkyl.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and R is an amino group, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and R is an alloc- or Fmoc- N'-substituted amino group.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is 6. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is zero (0). In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is one (1 ). In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is 2. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is 3.
• the method relates to any one of the above- referenced compounds, wherein in one or more instances Z is NH and n is 4. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is 5.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and R is a joara-aminoalkylaryl group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in. one or more instances Z is NH and R is an alloc- or Fmoc- TV -substituted para- aminomethylphenyl group.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH, R is a wra-aminoalkylaryl group or an alloc- or Fmoc- N'- substituted ara-aminomethylphenyl group, and n is one (1).
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH, R is phenyl, and n is one (1 ).
• the method relates to any one of the above-referenced compounds, wherein one or more instances of Z is absent, such that there is a bond between the carbonyl carbon and the methylene group if n is non-zero, or between the carbony! carbon and R if n is zero (0).
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and R is an amino group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and R is an alloc- or Fmoc- ⁇ '-substituted amino group.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is 2. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is zero (0). In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is one (1). In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is 3. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is 4.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is 5. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is 6.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and R is a 1-imidazolyl group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent, R is a 1-imidazolyl group, and n is zero (0).
• the method relates to any one of the above-referenced compounds, wherein at least one of R 1 , R 2 , and R 3 is 1 ⁇ 2r/-butyldimethylsilyl.
• the method relates to any one of the above-referenced compounds, wherein R 1 is ter -butyldimethylsilyl.
• the method relates to any one of the above-referenced compounds, wherein R 2 is tert- butyldimethylsilyl.
• the method relates to any one of the above-referenced compounds, wherein R is feri-butyldimethylsilyl.
• the method relates to any one of the above- referenced compounds, wherein each of R s and R 2 is tert-butyldimethylsilyl.
• the method relates to any one of the above-referenced compounds, wherein each of R 1 and R 3 is /eri-butyldimethylsilyl.
• the method relates to any one of the above-referenced compounds, wherein each of R 2 and R 3 is to -butyldimethylsilyl.
• the method relates to any one of the above-referenced compounds, wherein each of R ! , R 2 , and R 3 is ier/-butyldimethylsilyl.
• the method relates to a compound selected from the group consisting of:
• the mammalian cell is a cancer cell.
• the cancer cell is a human cancer cell, in one embodiment, the cancer cell is p21 -deficient.
• An aspect of the invention is a method of treating an OSBP-related disease or condition. The method includes the step of administering to a subject in need thereof an effective amount of a compound of the invention, thereby to increase apoptosis of OSBP- expressing cells associated with the OSBP-related disease or condition.
• an "OSBP-related disease or condition” refers to any disease or condition in which an oxysterol binding protein (OSBP) or OSBP-related protein (ORP) has been shown or can be shown to participate in the pathogenesis of the disease or condition.
• OSBP oxysterol binding protein
• ORP OSBP-related protein
• an OSBP-related disease or condition is cancer.
• an OSBP-related disease or condition is atherosclerosis.
• an OSBP-related disease or condition is Alzheimer's disease.
• treating refers to reducing or slowing the progression of at least one symptom or objective manifestation of a disease or condition in a subject having the disease or condition. In one embodiment, “treating” refers to eliminating a disease or condition in a subject having the disease or condition.
• a “subject” refers to a living mammal. In one embodiment a subject is a human.
• an “effective amount” is an amount that is sufficient to achieve a desired result.
• an effective amount to increase apoptosis of OSBP-expressing cells is an amount sufficient to increase apoptosis of OSBP-expressing cells.
• a method that increases apoptosis of OSBP-expressing cells associated with an OSBP-related disease or condition reduces the number (or population) of cells associated with the OSBP-related disease or condition.
• Apoptosis refers to programmed cell death.
• biochemical events lead to characteristic changes in cell morphology and cell death. These changes include blebbing, loss of cell membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation.
• apoptosis produces cell fragments called apoptotic bodies that surrounding cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells and cause damage.
• Extracellular signals may include toxins, hormones, growth factors, nitric oxide, or cytokines, that must either cross the plasma membrane or transduce a signal across the plasma membrane to effect a response.
• a cell initiates intracellular apoptotic signaling in response to a stress, which may bring about cell suicide.
• the binding of nuclear receptors by glucocorticoids, heat, radiation, nutrient deprivation, viral infection, hypoxia, and increased intracellular calcium concentration, for example, by damage to the membrane, can all trigger the release of intracellular apoptotic signals by a damaged cell.
• a number of cellular components, such as poly ADP ribose polymerase, may also help regulate apoptosis.
• apoptotic signals Before the actual process of ceil death is precipitated by enzymes, apoptotic signals must cause regulatory proteins to initiate the apoptosis pathway. This step allows apoptotic signals to cause cell death, or the process to be stopped, should the cell no longer need to die.
• regulatory proteins Several proteins are involved, but two main methods of regulation have been identified: targeting mitochondria functionality, or directly transducing the signal via adaptor proteins to the apoptotic mechanisms.
• Another extrinsic pathway for initiation identified in several toxin studies is an increase in calcium concentration within a cell caused by drug activity, which also can cause apoptosis via a calcium-binding protease, calpain.
• Caspases are a family of cysteine proteases which can proteolyticaily degrade a host of intracellular proteins to carry out the cell death program.
• apoptosis There are a number of methods known in the art that are useful to measure apoptosis. These include caspase assays, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and DNA fragmentation assays, cell permeability assays, and annexin V assay, to name but a few. Any of these assays can be used to detect apoptosis of OSBP-expressing cells associated with an OSBP-related disease or condition. Using such assays, apoptosis is said to be increased when the amount or degree of apoptosis is objectively and measurably greater than suitable negative control amount or degree of apoptosis.
• TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling
• DNA fragmentation assays DNA fragmentation assays
• cell permeability assays cell permeability assays
• the apoptosis is increased if it is at least 5 percent greater than suitable negative control amount or degree of apoptosis. In alternative embodiments, apoptosis is increased if it is at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 100 percent greater than suitable negative control amount or degree of apoptosis.
• the invention relates to the any of the above-referenced methods, wherein the OSBP-related disease or condition is cancer, and the OSBP-expressing cells are cancer cells.
• the OSBP-related disease or condition is cancer
• the OSBP-expressing cells are cancer cells.
• ORPphilins may be highly selective for tumors that have lost p21 expression, an event that often occurs at later stages in the clinical progression of some types of tumors, ORPphilins might play a role as a new last line of defense in the treatment of cancer; as tumors become resistant to standard therapy through loss of p21 , these tumors could be become more sensitive to the ORPphilins. Therefore, in certain embodiments, the invention relates to any one of the above-referenced methods, wherein the cancer is a p21- deficient cancer, i.e., the cancer cells are p21 -deficient.
• Exemplary cancers that may be treated include cancers selected from the group consisting of Acute Lymphoblastic Leukemia (ALL); Acute Myeloid Leukemia (AML); Adrenocortical Carcinoma; AIDS-Related Cancers; AIDS-Related Lymphoma; Ana] Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Basal Cell
• Carcinoma Bladder Cancer; Bone Cancer; Brain Stem Glioma; Brain Tumor; Breast Cancer; Bronchial Adenomas/Carcinoids; Burkitt's Lymphoma; Carcinoid Tumor; Carcinoma of Unknown Primary Site; Central Nervous System Lymphoma, Primary; Cerebellar
• Chronic Lymphocytic Leukemia CLL
• Chronic Myelogenous Leukemia CML
• Chronic Myeloproliferative Disorders Colon Cancer; Colorectal Cancer; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma; Esophageal Cancer; Ewing's Family of Tumors; Ewing's Sarcoma; Extracranial Germ Cell Tumor; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Gallbladder Cancer; Gastric (Stomach) Cancer;
• Gastrointestinal Carcinoid Tumor Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma; Glioma, Childhood Brain Stem; Glioma, Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathway and Hypothalamic; Glioma, Hypothalamic and Visual Pathway; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma;
• Leukemia Adult Acute; Myeloid Leukemia, Childhood Acute; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Neuroblastoma; Non-Hodgkin's Lymphoma; Oral Cancer; Oropharyngeal Cancer; Osteosarcoma;
• Trophoblastic Tumor Gestational; Unusual Cancers of Childhood; Urethral Cancer; Uterine Cancer, Endometrial; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma; Vulvar Cancer; Waldenstrom's Macroglobulinemia; and Wilms' Tumor.
• the method relates to any one of the compounds according to formula (IJ or a pharmaceutically acceptable salt thereof:
• Z is absent or is selected from the group consisting of 0 and NR 10 ;
• n is an integer 0-6;
• R is selected from the group consisting of substituted or unsubstituted: alkyl, alkenyl, amino, alloc-protected amino, Fmoc-protected amino, aryl, 5- to 13-membered cycloalkyl or cycloalkenyl group, and 5- to 10-membered cyclic heteroaromatic group comprising 1 -3 heteroatoms independently selected from the group consisting of N. O, and S;
• R 4 is selected from the group consisting of hydrogen and C 1 -C6 alkyl
• R 5 is selected from the group consisting of hydrogen and acyl; and R 10 is selected from the group consisting of hydrogen and C I -C6 alkyl; provided that :
• R 1 is 4-(alloc-aminomethyl)benzylaminocarbonyl, then at least one of R 2 , R 3 , R 4 , R 5 , and R 6 is not hydrogen; or
• R or R is 4-(alloc-aminomethyl)benzylaminocarbonyl, then R is not acetyl or R 6 is not ara-methoxybenzoyl.
• the method relates to any one of the above-referenced compounds, wherein R 4 is hydrogen. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein R 4 is a C 1 -C6 a!kyl. For example, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein R 4 is methyl.
• the method relates to any one of the above-referenced compounds, wherein R 5 is hydrogen. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein R 5 is acyl. For example, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein R 5 is acetyl (Ac).
• the method relates to any one of the above-referenced compounds, wherein R 1 is hydrogen. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein R 1 is trialkylsilyl.
• the method relates to any one of the above-referenced
• the method relates to any one of the above-referenced compounds, wherein R 2 is hydrogen. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein R 2 is trialkylsilyl. Alternatively, in certain embodiments, the method relates to any one of the above-referenced
• the method relates to any one of the above-referenced compounds, wherein R 3 is hydrogen. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein R is trialkylsilyl.
• the method relates to any one of the above-referenced
• the method relates to any one of the above-referenced compounds, wherein R 6 is hydrogen. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein R 6 is trialkylsilyl.
• the method relates to any one of the above-referenced
• the method relates to any one of the above-referenced compounds, wherein R 6 is para- methoxy benzoyl. In another example, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein R 6 is 3-phenylpropanoyl.
• the method relates to any one of the above-referenced compounds, wherein one or more instances of R is alkyl. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein one or more instances of R is alkenyl. In certain embodiments, the method relates to any one of the above- referenced compounds, wherein one or more instances of R is an amino group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein one or more mstances of R is an alloc-protected amino group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein one or more instances of R is an Fmoc-protected amino group.
• the method relates to any one of the above-referenced compounds, wherein one or more instances of R is aryl. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein one or more instances of R is 5- to 13-membered cycloalkyl group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein one or more instances of R is a 5- to 13-membered cycloalkenyl group. In certain
• the method relates to any one of the above-referenced compounds, wherein one or more instances of R is a 5- to 10-membered cyclic heteroaromatic group comprising 1 - 3 heteroatoms independently selected from the group consisting of N, O, and S.
• the method relates to any one of the above-referenced compounds, wherein one or more instances of Z is oxygen (O).
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and R is alkyl.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and R is 4-nitrophenyl.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is zero (0). Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is one (1). Alternatively, in certain embodiments, in certain
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is 2. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, w r herein in one or more instances Z is O and n is 3. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is 4.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is 5. Alternatively, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is O and n is 6.
• the method relates to any one of the above-referenced compounds, wherein one or more instances of Z is NR 10 .
• one or more instances of Z is NR 10 .
• the method relates to any one of the above-referenced compounds, wherein one or more instances of Z is NH. In another example, in certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NR 10 and R i u is a C l -C6 alkyl.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is Ni l and R is an amino group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and R is an alloc- or Fmoc- N '-substituted amino group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is 6, In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is zero (0).
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is one (1 ). In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is 2. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and n is 3, In certain embodiments, the method relates to any one of the above- referenced compounds, wherein in one or more instances Z is NH and n is 4. In certain embodiments, the method relates to any one of the above- referenced compounds, wherein in one or more instances Z is NH and n is 5.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and R is rara-aminoalkylaryl group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH and R is an alloc- or Fmoc- jV'-substituted para- aminomethylphenyl group.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH, R is a /?ara-arninoalkylaryl group or an alloc- or Fmoc- N'-substituted jc/ra-aminomethylphenyl group, and n is one (1).
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is NH, R is phenyl, and n is one (1).
• the method relates to any one of the above-referenced compounds, wherein one or more instances of Z is absent, such that there is a bond between the carbonyl carbon and the methylene group if n is non-zero, or between the carbonyl carbon and R if n is zero (0).
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and R is an amino group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and R is an alloc- or Fmoc- N'-substituted amino group.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is 2. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is zero (0). In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is one (1). In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is 3. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is 4.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is 5. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and n is 6.
• the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent and R is a 1 -imidazolyi group. In certain embodiments, the method relates to any one of the above-referenced compounds, wherein in one or more instances Z is absent, R is a 1 -imidazolyl group, and n is zero (0).
• the method relates to any one of the above-referenced compounds, wherein at least one of R 1 , R 2 , and R 3 is ter/-butyldimethylsilyl.
• the method relates to any one of the above-referenced compounds, wherein R 1 is rt-butyldimethylsilyl.
• the method relates to any one of the above-referenced compounds, wherein K ⁇ is tert- butyldimethylsilyl.
• the method relates to any one of the above-referenced compounds, wherein R is /ert-butyldimethylsilyl.
• the method relates to any one of the above- referenced compounds, wherein each of R ; and R 2 is fert-butyldimethylsilyl.
• the method relates to any one of the above-referenced compounds, wherein each of R 1 and R 3 is /er/-butyldimethylsilyl .
• the method relates to any one of the above-referenced compounds, wherein each of R 2 and R 3 is /er/-butyldimethylsilyl.
• the method relates to any one of the above-referenced compounds, w r herein each of R 1 , R 2 , and R 3 is ⁇ butyldimethylsilyl. in certain embodiments, the method relates to a compound selected from the group consisting of:
• An aspect of the invention is a method of killing a cancer cell.
• the method includes the step of contacting a cancer cell with an effective amount of a conjugate of the invention.
• the cancer cell is p21 -deficient.
• An aspect of the invention is a method of treating a cancer.
• the method includes the step of administering to a subject in need thereof an effective amount of a conjugate of the invention.
• the cells of the cancer are p21 -deficient.
• Certain compounds of the invention which have acidic substituents may exist as salts with pharmaceutically acceptable bases.
• the present invention includes such salts.
• salts examples 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.
• 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.
• a compound of the invention When a compound of the invention contains more than one chiral center, it may exist in diastereoisonieric 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.
• One or more compounds of the invention can be administered alone to a subject or in the form of 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 subject as a simple mixture or in suitable formulated pharmaceutical compositions.
• 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.
• Suitable routes of administration include, for example, oral, eyedrop, rectal, transmucosal, topical, or intestinal administration; parenteral delivery, including intravenous, intramuscular, intraperitoneal, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intranasal, or intraocular injections.
• 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.
• compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable earners 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.
• 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.
• physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• 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
• PVP polyvinylpyrrolidone
• disintegrating agents 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.
• 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.
• compositions 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.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
• compositions may take the form of tablets or lozenges formulated in conventional manner.
• 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.,
• 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.
• 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.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
• a suitable vehicle e.g., sterile pyrogen-free water
• 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.
• 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).
• the compounds may be formulated with suitable polymeric or
• hydrophobic materials for example as an emulsion in an acceptable oil
• ion exchange resins for example as an emulsion in an acceptable oil
• sparingly soluble derivatives for example, as a sparingly soluble salt
• An example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water- miscible organic polymer, and an aqueous phase.
• the cosolvent system may be the VPD co- solvent system.
• VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
• the VPD co-solvent system (VPD:5W) consists of VPD diluted 1 : 1 with a 5% dextrose in water solution.
• This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
• the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
• identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. 12 038562
• 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.
• the compounds may be delivered using a sustained-release system, such as semi-permeable matrices of solid hydrophobic polymers containing the therapeutic agent.
• 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.
• compositions also may comprise suitable solid or gel phase carriers or excipients.
• suitable solid or gel phase 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.
• 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.
• pharmaceutically acceptable counterion is an ionic portion of a salt that is not toxic when released from the salt upon administration to a recipient.
• 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.
• inorganic acids such as hydrogen bisulfide, hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid
• organic acids such as para-toluenesulfonic, salicylic, tartaric, bitartaric, as
• Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bi sulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, deeanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacatc, fumarale, maleate, butyne-l ,4-dioate, hexyne-l ,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,
• 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; trielhylamme; mono-, bis-, or tris-(2-hydroxy-Iower alkyl amines), such as mono-, bis-, or tris-(2-hydroxyethyl)amine, 2- hydroxy-tert-butylamine, or tr is- (hydroxy methy I )methyl amine, alkyl-N- (hydroxy lower 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.
• 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.
• the therapeutically effective dose can be estimated initially from cellular assays.
• a dose can be formulated in cellular and animal models to achieve a circulating concentration range that includes the ICso as determined in cellular assays (i.e., the concentration of the test compound which achieves a half-maximal inhibition).
• ICso as determined in cellular assays
• a "therapeutically effective dose” refers to that amount of the compound that results in amelioration of symptoms in a subject.
• 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 ED 50 (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 ED 5 o.
• the data obtained from these cell culture assays and animal studies can be used in
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 5 o 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).
• MEC minimal effective concentration
• the MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% inhibition of protein kinase using the assays described herein. 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.
• the effective local concentration of the drug may not be related to plasma concentration.
• 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
• compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
• the pack may for example comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
• the compounds of the present invention in the form of particles of very small size, for example as obtained by fluid energy milling.
• Capsules containing an active compound can be prepared. In the preparation of capsules, 10 parts by weight of active compound and 240 parts by weight of lactose can be de-aggregated and blended. The mixture can be filled into hard gelatin capsules, each capsule containing a unit dose or part of a unit dose of active compound.
• Tablets can be prepared, for example, from the active compound (10 parts by weight), lactose (190 parts by weight), maize starch (22 parts by weight), polyvinylpyrrolidone (10 parts by weight) and magnesium sterate (3 parts by weight).
• the active compound, the lactose and some of the starch can be de-aggregated, blended and the resulting mixture can be granulated with a solution of the polyvinylpyrrolidone in ethanol.
• the dry granulate can be blended with the magnesium stearate and the rest of the starch.
• the mixture is then compressed in a tabletting machine to give tablets each containing a unit dose or a part of a unit dose of active compound.
• the tablets can be enteric coated in a conventional manner using a solution of 20% cellulose acetate phthalate and 3% diethyl phthalate in ethanol :dichloromethane (1 : 1 ).
• Suppositories containing an active compound can be prepared.
• 100 parts by weight of active compound can be incorporated in 1300 parts by weight of triglyceride suppository base and the mixture formed into
• suppositories each containing a therapeutically effective amount of active ingredient.
• alkenyl as used herein, means a straight or branched chain hydrocarbon containing from 2 to 10 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens.
• Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5- hexenyl, 2-heptenyl, 2 -methyl- 1 -heptenyl, and 3-decenyl.
• alkoxy means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
• Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.
• Representative examples of alkoxycarbonyl include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, and tert-butoxycarbonyl.
• alkoxysulfonyl as used herein, means an alkoxy group, as defined herein, appended to the parent molecular moiety through a sulfonyl group, as defined herein.
• alkoxysulfonyl include, but are not limited to, methoxysulfonyl, ethoxysulfonyl and propoxysulfonyl.
• arylalkoxy and “heteroalkoxy” as used herein, means an ary! group or heteroaryl group, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein.
• Representative examples of arylalkoxy include, but are not limited to, 2-chlorophenylmethoxy, 3-trifluoromelhylethoxy, and 2,3-methylmethoxy.
• arylalkyl as used herein, means an alkoxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein,
• alkoxyalkyl include, but are not limited to, tert-butoxymethyl, 2- ethoxyethyl, 2-methoxyethyl, and methoxymethyl.
• alkyl means a straight or branched chain hydrocarbon containing from 1 to
• 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, and n-hexyl.
• alkylene is art-recognized, and as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound, which may be aliphatic or alicyclic, or a combination thereof, and which may be saturated, partially unsaturated, or fully unsaturated.
• linear saturated C ⁇ .i alkylene groups include, but are not limited to, -(CH 2 ) literal- where n is an integer from 1 to 10, for example, -CH 2 - (methylene), -CH 2 CH 2 - (ethylene), -CH 2 CH 2 CH 2 - (propylene), -CH2CH2CH2CH2-
• branched saturated Cs-io alkylene groups include, but are not limited to, -CH(CH 3 )-, -CH(CH 3 )CH 2 -, -CH(CH 3 )CH 2 CH 2 -, -CH(CH 3 )CH 2 CH 2 CH 2 -, - CH 2 CH(CH 3 )CH2-, -CH 2 CH(CH 3 )CH 2 CH 2 -, -CH(CH 2 CH 3 )- !
• .so alkyiene groups include, but are not limited to,
• alicyclic saturated Cj-io alkyiene groups include, but are not limited to, cyclopentylene (e.g., cyclopent-1 ,3- ylene), and cyclohexylene (e.g., cyclohex-l ,4-ylene).
• alicyclic partially unsaturated Cj-io alkyiene groups include, but are not limited to, cyclopentenylene (e.g., 4- cyclopenten-l ,3-ylene), and cyclohexenylene (e.g., 2-cyclohexen-l,4-ylene, 3-cyclohexen- 1 ,2-ylene, and 2,5-cyclohexadien-l,4-ylene).
• cyclopentenylene e.g., 4- cyclopenten-l ,3-ylene
• cyclohexenylene e.g., 2-cyclohexen-l,4-ylene, 3-cyclohexen- 1 ,2-ylene, and 2,5-cyclohexadien-l,4-ylene.
• alkylcarbonyl as used herein, means an alkyl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein.
• alkylcarbonyl include, but are not limited to, acetyl, 1 -oxopropyl, 2,2-dimethyl-l -oxopropyl, 1-oxobutyl, and 1-oxopentyl.
• alkylcarbonyioxy and "aryicarbonyloxy” as used herein, means an alkylcarbonyl or arylcarbonyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
• Representative examples of alkylcarbonyioxy include, but are not limited to, acetyloxy, ethylcarbonyloxy, and tert-butylcarbonyloxy.
• Representative examples of aryicarbonyloxy include, but are not limited to phenylcarbonyloxy.
• alkylsulfonyl as used herein, means an alkyl group, as defined herein, appended to the parent molecular moiety through a sulfonyl group, as defined herein.
• alkylsulfonyl include, but are not limited to, methylsulfonyl and efhylsulfonyl.
• alkylthio as used herein, means an alkyl group, as defined herein, appended to the parent molecular moiety through a sulfur atom.
• Representative examples of alkylthio include, but are not limited, methylthio, ethylthio, tert-butylthio, and hexylthio.
• arylthio alkenylfhio
• arylakylthio for example, are likewise defined.
• alkynyl as used herein, means a straight or branched chain hydrocarbon group containing from 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond.
• Representative examples of alkynyl include, but are not limited, to acetyienyl, 1 - propynyl, 2-propynyl, 3-butynyl, 2-pentynyI, and 1 -butynyl.
• amino refers to radicals of both unsubstituted and substituted amines appended to the parent molecular moiety through a nitrogen atom.
• the two groups are each independently hydrogen, alkyl, alkylcarbonyl, alkylsulfonyl,
• arylcarbonyi or formyl.
• Representative examples include, but are not limited to
• aromatic refers to a planar or polycyclic structure characterized by a cyclically conjugated molecular moiety containing 4n+2 electrons, wherein n is the absolute value of an integer.
• Aromatic molecules containing fused, or joined, rings also are referred to as bicylic aromatic rings.
• bicyclic aromatic rings containing heteroatoms in a hydrocarbon ring structure are referred to as bicyciic heteroaryl rings.
• aryl means a phenyl group or a naphthyl group.
• the aryl groups of the present invention can be optionally substituted with one, two, three, four, or five substituents independently selected from the group consisting of deuterium, tritium, alkenyl, alkoxy, alkoxycarbonyl, alkoxysulfonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, alkylsulfonyl, alkylthio, alkynyl, amido, amino, carboxy, cyano, formyl, halo, haloalkoxy, haloalkyl, hydroxyl, hydroxyalkyl, mercapto, nitro, silyl and silyloxy.
• arylalkyl or “aralkyl” as used herein, means an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
• Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3- phenylpropyi, and 2-naphth-2-ylethyl,
• arylalkoxy or "arylalkyloxy” as used herein, means an arylalkyl group, as delined herein, appended to the parent molecular moiety through an oxygen atom.
• heteroarylalkoxy as used herein, means a heteroaryl alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
• arylcarbonyi as used herein, means an aryl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein.
• arylcarbonyi include, but are not limited to, benzoyl and naphihoy!.
• aryloxy means an aryl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
• heteroaryloxy means a heteroaryl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
• carboxy as used herein, means a -C0 2 H group.
• cycioalkyl as used herein, means monocyclic or multicyclic (e.g., bicyclic, tricyclic, etc.) hydrocarbons containing from 3 to 12 carbon atoms that is completely saturated or has one or more unsaturated bonds but does not amount to an aromatic group.
• Examples of a cycioalkyl group include cyclopropyl, cyciobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl.
• cycloalkoxy as used herein, means a cycioalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
• cyano as used herein, means a -CM group.
• halo or halogen means -CI, -Br, -I or -F.
• haioalkoxy means at least one halogen, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein.
• haioalkoxy include, but are not limited to, chloromethoxy, 2- fluoroethoxy, trifluoromethoxy, and pentafluoroethoxy.
• haloalkyi means at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
• Representative examples of haloalkyi include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl.
• heterocyclyl include non-aromatic, ring systems, including, but not limited to, monocyclic, bicyclic and tricyclic rings, which can be completely saturated or which can contain one or more units of unsaturation, for the avoidance of doubt, the degree of unsaturation does not result in an aromatic ring system) and have 3 to 12 atoms including at least one heteroatom, such as nitrogen, oxygen, or sulfur.
• heterocyclic rings azepines, azetidinyl,
• heterocyclyl groups of the invention are substituted with 0, 1, 2, or 3 substituents independently selected from deuterium, tritium, alkenyl, alkoxy, alkoxycarbonyl, aikoxysulfonyl, alkyl,
• alkylcarbonyl alkylcarbonyloxy, alkylsulfonyl, alkylthio, alkynyl, amido, amino, carboxy, cyano, formyl, halo, haioalkoxy, haloalkyi, hydroxyl, hydroxyalkyl, mercapto, nitro, silyi and silyloxy.
• heteroaryl as used herein, include aromatic ring systems, including, but not limited to, monocyclic, bicyclic and tricyclic rings, and have 3 to 12 atoms including at least one heteroatom, such as nitrogen, oxygen, or sulfur.
• heteroaryl include: azaindolyl, benzo(b)thienyl, benzimidazolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzoxadiazolyl, furanyl, imidazolyi, imidazopyridinyl, indolyl, indolinyl, indazolyl, isoindolinyl, isoxazolyl, isothiazolyl, isoquinolinyl, oxadiazolyl, oxazolyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl, pyrazolo[3,4-d]pyrimi
• heteroaryl groups of the invention may be substituted with 0, 1, 2, or 3 substituents independently selected from deuterium, tritium, alkenyl, alkoxy, alkoxycarbonyl, alkoxysulfonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, alk lsulfony], alkylthio, alkynyl, amido, amino, carboxy, cyano, formyf, halo, haloalkoxy, haloalkyl, hydroxyl, hydroxyalkyl, mercapto, nitro, sily! and silyloxy.
• substituents independently selected from deuterium, tritium, alkenyl, alkoxy, alkoxycarbonyl, alkoxysulfonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, alk lsulfony], alkylthio, alkynyl, amido, amino, carboxy, cyano,
• heteroarylalkyl or “heteroaralkyl” as used herein, means a heteroaryl, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
• Representative examples of heteroarylalkyl include, but are not limited to, pyridin-3- ylmethyl and 2-(thien-2-yl)ethyI.
• hydroxy as used herein, means an -OH group.
• hydroxyalkyl as used herein, means at least one hydroxy group, as defined herein, is appended to the parent molecular moiety through an alkyl group, as defined herein.
• Representative examples of hydroxyalkyl include, but are not limited to, hydroxymethyl, 2- hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypentyl, and 2-ethyl-4-hydroxyheptyl.
• mercapto as used herein, means a -SH group.
• nitro as used herein, means a -N0 2 group.
• prenyl dimethylallyl, geranyl, neryl, farnesyl and geranylfarnesyl, as well as partially saturated equivalents thereof.
• prenylaryl or "prenylheteroaryl” means a prenyl, as defined herein, appended to the parent molecular moiety through an aryl or heteroaryl, as defined herein.
• Representative examples of haloalkoxy include geranylphenyi and geranylpyridinyl.
• silyl as used herein includes hydrocarbyl derivatives of the silyl (H3S1-) group (i.e., (hydrocarbyl ⁇ Si-), wherein a hydrocarbyl groups are univalent groups formed by removing a hydrogen atom from a hydrocarbon, e.g., ethyl, phenyl.
• the hydrocarbyl groups can be combinations of differing groups which can be varied in order to provide a number of silyl groups, such as trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS), tert- butyldimethylsilyi (TBS/TBDMS), triisopropylsilyl (TIPS), and [2- (trimethylsilyl)ethoxy]methyl (SEM).
• TMS trimethylsilyl
• TDPS tert-butyldiphenylsilyl
• TIPS triisopropylsilyl
• SEM [2- (trimethylsilyl)ethoxy]methyl
• silyloxy as used herein means a silyl group, as defined herein, is appended to the parent molecule through an oxygen atom.
• Cephalostatin 1 (1) ( ⁇ 2 mg) was supplied via total synthesis in accordance with published method Former et al. (2009) J Am Chem Soc
• OSW-1 (2) was obtained through isolation from nature by Y. Mimaki and coworkers and also through total synthesis.
• OSW-1 analogs 6-9 (FIG. IB) were prepared as detailed in WO 2010/068877, the entire contents of which is incorporated herein by reference.
• Schweinfurthin A (4a) ( ⁇ 3 mg) and ritterazine B (3) (0.2 mg) were obtained from the NCI.
• mice anti-actin polyclonal was obtained from Sigma; mouse anti-tubulin monoclonal (DSHB E7) was obtained from Developmental Studies Hybridoma Bank; rabbit anti-c-Myc (N-262) polyclonal was obtained from Santa Cruz Biotechnologies; mouse anti-c- Myc (9E10) monoclonal was obtained from Santa Cruz Biotechnologies; rabbit anti- COL4A3BP/CERT (ab72536) polyclonal was obtained from Abeam: mouse anti-P230 monoclonal was obtained from BD Biosciences.
• HCT-1 16 wild-type and HCT- 1 16 p21 "' ' cell lines were supplied by the Bert Vogelstein lab (Johns Hopkins).
• CHO-7 cells were a gift from Michael Brown and Joseph Goldstein (UT-Southwestern).
• Ml 2 cells were a gift from Daniel Ory (Washington University in St. Louis). All other cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). Plasmids, Vectors, and OSBP1 shR As.
• the pcDNATM3.1/myc-His vector and the pcDNATM3.1/myc-His lacZ control vector were purchased from Invitrogen.
• OSBP1 cDNA was purchased from Open Biosystems.
• ORP4L cDNA was cloned from HeLa total RNA extract using standard molecular biology techniques.
• the OSBP1 and ORP4L cDNAs were cloned into the pcDNATM3.1/myc-His vector (Invitrogen). 0SBP1(M446W),
• OSBPl(V582M), ORP4L(M550W), and ORP4L(V686M) mutants were prepared with the QuikChange® II XL Site-directed Mutagenesis Kit (Stratagene) as instructed by the supplier.
• OSBP1 shRNAs in the pSIREN-RetroQ vector (BD Biosciences) were generously provided by Giulio Romeo (Joslin Diabetes Center).
• Cell viability assay Cells were plated into 96-well plates in 75 ⁇ of media. After 24 h, a time-zero plate was produced by adding 25 of media and 20 ⁇ , of CellTiter- Blue® (Promega) to the wells, followed by incubation at 37 °C for 90 min. Fluorescence (544 nm excitation; 590 nm emission) was detected using a SPECTRAmax® Gemini XS to establish cell viability at time of dosing. Then, compounds were serially diluted in media and delivered to the cells as 4X solutions in 25 ⁇ of media. At either 48 h or 72 h, CellTiter- Blue® was added and the fluorescence was recorded as described above. Growth relative to 8562 untreated cells was calculated and this data was fitted to a four-parameter dose-response curve using Graphpad Prism 5.
• LipofectamineTM LTX with PLUSTM reagent (Invitrogen) as instructed, and after 8 h of transfection, the cells were re-plated for the cell viability assay, performed as described above. Expression levels were evaluated by immunoblotting at the time of dosing.
• HCT- 1 36 or A549 adenocarcinoma human alveolar basal epithelial cells were plated on coverslips in 6-well plates. After 72 h, the culture media was removed and replaced with media containing either compound or vehicle control. At the indicated time-points, the cells were fixed for 10 min using 3.7%
• the coverslips were washed with BSA/PBS and incubated for 60 min at RT with Alexa Fluor®-conjugated secondary antibodies (Invitrogen) in BSA/PBS, with the final 30 min including incubation with Iloescht 33258.
• the coverslips were then washed sequentially with BSA/PBS, PBS, and water and finally mounted on microscope slides using Aqua Poly/Mount (Polysciences Inc.). Imaging data was acquired using Zeiss LSM 510 META scanning confocal microscope.
• Mass spectra were obtained from the Harvard University Mass Spectrometry Laboratory using an Agilent 6210 TOF LC/MS instrument.
• High pressure liquid chromatography (HPLC) purification was performed on an Agilent 1 100. All compounds used for biological experimentation were HPLC purified. The yields reported are following silica gel purification, prior to HPLC purification.
• Semi-preparative ITPLC was performed using a diode-array detector with the indicated columns and solvent conditions.
• OSF3P l (M446W), OSBP ] (V582M), ORP4L, ORP4L(M550W), ORP4L(V686M), and lacZ were cloned into the pcDNA3.1 vector (Invitrogen), which encodes a C -terminal myc-his tag.
• pcDNA3.1 vector Invitrogen
• Binding Buffer 50 mM HEPES pH 7.4, 50 mM KC1, 5 mM DTT, 1 x HALTTM Protease Inhibitor with EDTA (ThermoScientific).
• OSBPl , ORP4L and LacZ cD A were cloned into the pcDNA3.1 vector (Invitrogen) with standard molecular biology methods and verified via sequencing.
• OSBPl (M446W) and OSBP 1 (V582M) mutants were prepared with the QuikChange® II XL Site-directed
• C/D Charcoal/Dextran
• the 20% PVA solution was prepared in a 50 mL conical tube, adding water to the solid PVA.
• the PVA solution was heated in an 80 °C water bath, with frequent sonication and vortexing, until homogeneity was achieved ( ⁇ 1 h).
• the 20% PVA solution was then kept at room temperature (rt).
• the cells were washed once with rt PBS, then 2 mL of the M- Per®/HALTTM with EDTA (ThermoScientific) cell-lysis reagent was added per plate. The plates were rocked for 5 min at 250 rprn at rt. All subsequent steps were performed at 4 °C or with tubes on ice. The lysate was transferred to ultracentrifuge tubes and centrifuged for 1 h at 100,000 g at 4 °C in either a 70 Ti or a 70.1 Ti rotor (Beckman Coulter) in a Beckman Coulter ultracentrifuge.
• 3 H-25-OHC was added to the large stock of binding assay lysate to a final concentration of 20 nM.
• This H-25-OHC-containing binding lysate was then dispensed using a multi-channel pipettor into the 96-well plates. Then, 1.5 pL of a serial dilution of the inhibitors in either 100% ethanol (25-OHC) or DMSO (the ORPphilins) was added to each well.
• the binding lysate used in each well of the 96-well plate consisted of 60 pL of 0.2 mg/mL SI 00 lysate, 7.5 pL 20% PVA, and 5.6 pL 2M KC1.
• 0.4 pL of buffer was then added (total volume per well: 73.5 pL).
• 0.1 1 pL of 13.2 pM 3 H-25-OHC and 0,29 pL binding buffer was added for each well (total volume per well: 73.5 pL). " New serial dilutions were made for each experiment.
• Binding assays were incubated, standing, for 16 h at 4 °C. Then 30 ⁇ . of the rt C/D solution was multichannel pipetted into new "V shaped 96-well plates, and 60 ⁇ , of the binding lysate mixture was then added. The plates were briefly, lightly vortexed (-10 sec), and then rocked at 250 rpm for 30 min at rt. The plates were then centrifuged at 1900 g for 15 min at 4 °C in Microplate Carriers (Sorvall cat. #75006449S) in a Sorvall Legend RT centrifuge.
• OSBP1 and ORP4L Are Receptors of ORPphilins
• the growth inhibitory activities of compounds l-4a were also significantly enhanced in HCT-1 16 ⁇ 2 ⁇ ⁇ cells versus HCT- 1 16 wt cells (FIG. 2A).
• the high degree of ⁇ 2 ⁇ selectivity of compounds 1 -5 was a unique characteristic of these compounds since many other antiproliferative small molecules tested did not exhibit this selectivity (FIG. 2A).
• the ⁇ 2 ⁇ ' " selectivity of compounds 1-5 provided evidence beyond the NCI-60 COMPARE analysis that these compounds were mechanistically related.
• this ⁇ 2 ⁇ _ selectivity could be used as a defining feature for this class of compounds, allowing analogs of 1-5 to be identified that shared the same characteristic mechanism of action.
• OSW-1 (2) Affinity purification was used to identify putati ve targets of compounds l-4a.
• OSW-1 (2) analogs were prepared with the goal of maintaining the potency and the ⁇ 2 ⁇ ⁇ selectivity of 2; these same analogs were also used to prepare an OSW-1 affinity matrix (FIG. IB).
• FIG. IB OSW-1 affinity matrix
• Alloc-protected amine 6a prepared through formation of a carbamate with the C3-hydroxyl of the xylosyl saccharide, maintained the antiproliferative potency of 2 and, critically, also exhibited ⁇ 2 ⁇ 7" selectivity commensurate with that of 2 (FIG. 2A).
• OSBPl and OSBP-paralog ORP4L showed the most significant competition by soluble 2 (FIG. 2C). Additionally, VAP-A (vesicle- associated membrane protein-associated protein A) showed a >50% competition by 2. VAP- A is known to interact with both OSBP1 and ORP4L. Furthermore, OSBP1 and ORP4L are believed to physically interact. Wyles et al. (2007) Exp Cell Res 313: 3426-37. Therefore, any of these three proteins could have been the direct binding partner of affinity reagent 6b, with the other two proteins being co-purified. However, since OSBP1 and ORP4L are high- affinity receptors of oxysterols, and 2 comprises an oxysterol moiety, OSBP1 and ORP4L were chosen for investigation as putative targets for compounds 1-4.
• short hairpin RNA (shRNA) knockdown of OSBPl expression was performed and then its effect on the growth inhibitory activity of 1, 2, and 4a measured.
• Cells were treated with compounds for 72 h and GI50S were determined as described in connection with FIG. 2. While reduction of OSBPl levels by 85% (FIG. 3B) did not affect HCT-1 16 p21 " ⁇ cell growth, it did sensitize cells to 1 , 2, and 4a by 4-, 6-, and 9- fold, respectively (FIG. 3A).
• ORPphilins antiproliferative activity of the ORPphilins is further supported by the activity of OSW-1 analog 9, which bears a tert-butyldimethylsilyl group on the C3 carbinol (FIG. IB).
• 25-OHC (I I) is a high-affinity ligand of OSBPl and ORP4L that is not cytotoxic at concentrations up to 10 ⁇ .
• Example 1 established that the ORPphilins compete with 25- OHC for binding to OSBP l and ORP4L (FIG. 2D,E). Therefore, if OSB l and ORP4L mediate the biological activities of these compounds, co-administration of sub-lethal doses of 25-OHC with the ORPphilins should inhibit binding of the ORPphilins to OSBP and ORP4L, thereby desensitizing cells to the cytotoxic effects of the ORPphilins.
• NPC1 and Insig are also cellular receptors of 25- OHC.
• NPC1 is involved in the egress of lipoprotein-derived cholesterol from lysosomes.
• Insig is a chief regulator of SREBP-dependent cholesterol homeostasis, and 25-OHC exerts much of its ability to inhibit cholesterol biosynthesis through its binding to Insig, The possibility that suppression of the antiproliferative activity of the ORPphilins by 25-OHC could be due to displacement of these compounds from NPC1 or Insig was considered.
• OSBP1 and ORP4L are targets of the ORPphilins
• dominant drug-resistant alleles of these proteins were sought.
• 15 OSBP1 residues inside and outside the region of the protein previously reported to mediate sterol binding were mutated (FIG. 4C)
• the growth inhibition induced by the ORPphilins was determined in HeLa cells overexpressing either wild-type or mutant OSBPl-myc-his proteins. Expression of wt OSBPl -myc-his and mutant OSBPl-myc-his were nearly equivalent as measured by western blot.
• OSBPl(M446W)-myc-his caused an 18-fold reduction in the antiproliferative activity of cephalostatin 1 (1) at the GI 50 level compared to expression of wt OSBPl-myc-his (FIG. 4E1).
• the same mutant caused a 3.9-fold reduction in the antiproliferative activity of OSW-1 (2) (FIG. 4E2) and a 2.7-fold reduction in the activity of schweinfurthin A (4a) (FIG. 4E3),
• a second OSBP1 mutant, V582M reduced the antiproliferative activity of 1 by 9,4-fold when compared to wt OSBPl -myc-his, yet this mutant did not affect the
• the binding affinity of 1, 2 and 4a to the 0SBP1 (M446W) and 0SBP1(V582M) mutants were then determined using [ 3 H]-25-OHC competition binding.
• the binding affinity of 25-OHC for 0SBP1(M446W) was identical to wt.
• the 0SBP1(V582M) mutant had a slightly higher 25-OHC 3 ⁇ 4 as compared to wt.
• the binding affinity of 1, 2 and 4a for these two OSBP1 mutants were unchanged compared to wt OSBPl-myc-his (FIG. 4D). This suggests that these mutations provide resistance to the ORPphilins not by affecting compound binding but by altering the cellular function of OSBPl .
• ORP4L Based on the sequence alignment of OSBPl and ORP4L, mutations in ORP4L were then made at positions equivalent to OSBPl (M446W) and OSB l (V582M). Unlike expression of the two aforementioned OSBPl mutants, expression of ORP4L(M550W) and ORP4L(V686M) did not reduce cell sensitivity to the ORPphilins when compared to wt ORP4L-myc-his.
• OSBPl translocates from cytoplasmic sites to the trans- Golgi network (TON) upon addition of 25-OHC to cells.
• TON trans- Golgi network
• HCT-1 16 cells were treated with ORPphilins for short time periods (4 h) and the effects on OSBPl localization monitored using immunofluorescence microscopy.
• OSBPl green
• protein P230 red
• HCT-1 16 cells were visualized in HCT-1 16 cells using indirect immunofluorescence microscopy.
• Cells were treated with compound for 4 h, fixed with 3.7% formaldehyde/PBS, washed, and permeabilized using 0.5% Triton X-100 in PBS. Proteins were labelled using specific primary antibodies against OSBPl and P230 overnight at 4 °C. After washing, cells were treated with Alexa Fluor 488 and Alexa Fluor 546 secondary antibodies for 1 h at RT.
• schweinfurthin A (4a) treatment caused OSBPl to accumulate at perinuclear positions with a large degree of co-localization with the trans-Go ⁇ ⁇ marker p230, suggesting that 4a causes OSBPl to translocate to the trans-Go ⁇ g ⁇ network (FIG. 5C).
• OSW-1 (2) caused OSBPl to localize to a perinuclear position that did not co-localize with p230 (FIG. 5D). Instead, OSW-1 treatment caused the p230 marker to appear as small, dispersed vesicles, which is indicative of trans-Go ⁇ gi dismemberment. Similar to 2.
• Cephalostatin 1 and OSW-1 Cause a Reduction in Cellular OSBPl Levels
• cephalostatin 1 (1) and OSW- 1 (2) were treated with 1 and 2 caused a decrease in OSBPl levels.
• Treatment of HCT-1 16 cells with vehicle (D SO) or 25-OHC (10 ⁇ ) had no effect on OSBPl levels, as assessed by western blotting (FIG. 6A).
• treatment with 1 or 2 induced a dramatic, time-dependent reduction of OSBPl levels (FIG. 6A).
• 50 nM of 1 or 10 nM of 2 caused an approximately 75% reduction in OSBPl protein levels at 24 h in HCT-1 16 cells (FIG. 6A), with a similar effect recorded in A549 cells.
• OSBPl levels were determined using both an antibody recognizing the far N -terminus of OSBP l (FIG. 6) and also an antibody recognizing the C-terminus of OSBP l . Both antibodies showed the OSBPl reduction induced by 1 and 2, indicating degradation of the entire protein, as opposed to proteolysis or modification to one terminal region of OSBPl .
• Sphingomyelin is biosynthesized from ceramide at the Golgi apparatus. Ceramide is primarily transported from the ER to the Golgi by ceramide transport protein (CERT), and the transport activity of CERT is both OSBPI- and VAP-A dependent. In CHO-K l cells, 25- OHC has been shown to induce an increase in sphingomyelin biosynthesis in an OSBPI - dependent fashion. To determine whether the ORPphilins affect sphingomyelin synthesis, pulse-labeling with [ 3 H]-serine was performed in CHO-Kl and HCT-1 16 cells and the amount of tritium incorporated into sphingomyelin and other lipids then measured.
• CERT ceramide transport protein
• ORPphilins caused a significant reduction in the biosynthesis of SM (FIG. 7).
• Treatment of CHO-Kl cells with a GI 90 dose of 25-OHC (15 ⁇ ) caused a robust stimulation of SM biosynthesis, which is contrary to the reduction that was observed with 1.
• Inhibition of SM biosynthesis did not occur upon exposure of cells to taxol or the ER stress-inducing small molecule thapsigargin, demonstrating that 1, 2 and 4a do not inhibit SM biosynthesis due to induction of general cytotoxicity or ER stress.
• CERT levels akin to the effects of 1 and 2 on OSBP1 levels.
• Treatment of HCT-1 16 cells or A549 cells with high dose (GI 90 ) 1 or 2 did not affect CERT protein levels.
• the mono-Fmoc- 1,6-diaminohexane solution was added slowly drop-wise through a large-gauge needle to the triphosgene solution, over 15 min. With washing of the reaction flask, a total of 2.3 mL of CH2G 2 /CH3CN was used to transfer the mono-Fmoc- 1 ,6-diaminohexane. Upon addition of the mono-Fmoc- 1 ,6-diaminohexane to the triphosgene, the solution became close to homogeneous. After 10 min stirring, argon was bubbled for 5 min directly into the reaction to remove excess phosgene.
• OSW- 1 (9.4 mg, 0.01 mmol), under argon, was dissolved in 0.5 mL of CH 2 CI 2 . 0.3 mL (-0.01 mol, ⁇ 1 eq.) of the isocyanate solution was added to the OSW-1 solution. After - 10 h, 0.15 mL more of the isocyanate solution was added (-0.005 mmol, -0.5 eq.). After a further 16 h, the reaction had progressed considerably by TLC analysis.
• OSW-1 was also recovered.
• 100, 101 , and 102 were fully purified using semi -preparative reverse-phase HPLC with a Zorbax C-18SB column (I.D. 9.4 mm, 3.9 mL/min) using isocratic elution with 90% MeOH/ H2O.
• 100 (elution time 15.3 min) U 2012/038562 was obtained as a white solid (1.2 mg, 9%, 25% borsm).
• 101 (elution time 17.4 min) was obtained as a white solid (1.4 mg, 11%, 29% borsm).
• 102 (elution time 20.8 min) was obtained as a white solid (1.8 mg, 14%, 38% borsm).
• OSW-1 analogs 7 and 8 were prepared as previously described. ubo et al. ( 1992) PhylQchemisiry 3 1(1 1 ):3969-73. HPLC purification of 7: Zorbax C 18SB column, isocratic 82% MeOH/H 2 0. HPLC purification of 8: Eclipse C8 column, isocratic 60% CH 3 CN/H 2 0.
• Methyl ester 105 was isolated from the reaction of mono-Fmoc-l 5 6-diaminohexane isocyanate and OSW-1 ; this is the same reaction that produced 100-102. 105 is a minor product of this reaction discovered when HPLC purification was used to purify the remaining products of this reaction. Presumably, 105 arises from attack of MeOH on an activated ester form of OSW-1.
• the activated ester of OSW-1 could be the OSW-1 acid chloride, formed from OSW-1 reacting with phosgene in the reaction. Apparently this activated OSW-1 ester then reacted with MeOH upon pTLC purification. This is a trace product of the reaction (yield ⁇ 10%).
• the reaction was purified by pTLC, loaded on 2 plates and eluted with 8% MeOH7CH 2 Cl 2 .
• Several products were isolated and identified via ⁇ NMR and LRMS. -1-2 mg of >80% 115, 123, and 124 were produced (-10% yield), with less than 1 mg of 127 produced ( ⁇ 10% yield).
• the carbonate products showed some instability on TLC, suggesting a significant portion was degraded during purification.
• carbonate 123 and 124 are contaminated with each other, which could be due lo incomplete purification, but there are also suggestions that the compounds can interconvert in solution, transferring the ⁇ -nitrophenyl carbony] group between the 3" and 4" xylosyl hydroxyls.
• Carbonate 124 ⁇ NMR (500 MHz, D 3 CN). Significantly impure with 123.
• Carbonate 123 ( ⁇ 1 mg, 0.001 mmol) was dissolved in 0.2 mL CH2CI 2 .
• a stock solution of mono- Alloc- 1 ,6-hexanediamine was prepared in CH 2 G2 and added (0.6 mg, 0.003 mmol) to the 123 solution.
• a catalytic amount of DMAP solid was added ( ⁇ 0.5 mg) to the reaction, which was sealed and stirred overnight.
• the yellow solution was concentrated and purified using pTLC (1/2 plate) using 8%
• Method 2 Using Carbonate 124 as substrate: Performed identically as Method 1.
• 4"-xylosyl carbamate 126 (-0.4 mg, -35%) was obtained, in addition to some cyclic carbonate 122.
• This outcome suggests that either 1) DMAP initially catalyzes the formation of cyclic carbonate 122, which then proceeds to form the carbamate product; or 2) DMAP catalyzes the acyl transfer from the 3-xylose hydroxy] to the 4-xylose, which then proceeds to form the carbamate product.
• Method 1 Mono- Alloc- 1 ,6-hexanediamine hydrochloride (Sigma-AIdrich) (23.5 mg, 0.1 18 mmol) was azeotroped from benzene and subjected to a high vacuum to ensure complete removal of solvent. It was then dissolved in CH2CI2 0.5 mL to give a homogenous, weakly yellow solution. DIPEA (22 pL, 0.126 mmol) was added and the solution stirred for — 15 min. Meanwhile, 32 mg of triphosgene (98%, Sigma- Al lrich) was charged to reaction flask in the atmosphere, placed under an argon atmosphere, and dissolved in 0.5 mL CL ⁇ CL.
• Method 2 115 (3-(4-Nitrophenyl)-OSW-l carbonate) (-1 mg, 0.001 mmol), under argon, was dissolved in 0.2 mL of CH2CI2.
• a reagent stock solution of mono-Alloc-1 ,6- hexanediamine was prepared by dissolving 4.0 mg in 0.5 mL of CH2CI2, 75 pL of the reagent solution (0.6 mg, 0.003 mmol) was added. The reaction was sealed, and quickly became yellow. After 16 h, little desired formed, added more mono- Alloc- 1 ,6- hexanediamine (1 mg, 0.005 mmol) as a solid to the reaction.
• reaction was dissolved in CHCI3, and extracted with aq. NH 4 C1 twice, followed by washing the organic layer with 3 ⁇ 40 and brine, and drying over Na 2 S0 4 .
• the reaction was pTLC purified with 5% MeOH CF Ch, to yield slightly impure 103
• OSW-1 carbamate 103 (6.0 mg, 0.0054 mmol) was dissolved in 0.20 mL of a 4% KOH/EtOH solution. The lightly yellow colored solution was stirred for 40 min. The reaction was carefully neutralized to neutral pH with 10% HC1, then diluted with CHCI3. Extracting with with H 2 0 gave a cloudy, poorly separated extraction, to which aq. sat.
• Bis-carbamate 109 was obtained as a white solid (6.7 mg, 19%, 24% borsm). Both carbamate 6c and 6a were purified to analytical purity via HPLC (Eclipse C8 column, isocratic 83% MeOH/H 2 0) for biological experimentation.
• OSW- 1 (5.6 mg, 0.006 mmol) was dissolved in 0.1 mL of CH 2 C1 2 while under argon.
• 15 mg of Alloc-(N)-diaminomethyl benzene isocyanate was azeotroped from benzene followed by CHCI3.
• 0.3 mL of CH 2 C1 2 was then added to the isocyanate to produce a cloudy, brownish reagent stock solution.
• 0.2 mL of the isocyanate solution (10 mg, 0.04 mmol) was added to OSW- 1 via syringe at ambient temperature. The reaction was slightly cloudy and yellow in color.
• OSW-1 carbamate 106 (4.0 mg, 0.0036 mmol) was dissolved in 0.25 mL of a 4% KOH/EtOH solution. The lightly yellow colored solution was stirred for 70 min. The reaction was carefully neutralized to neutral pH with 10% HO, then diluted with CHCI3. Extracting with with H 2 0 gave a cloudy, poorly separated extraction. Aq. sat. NaHCC>3 was then added. The organic layer was separated, the aqueous fraction back-extracted with CHCI3, and the combined organic phases dried over N 2 S0 4 . 20 was purified with silica gel chromatography, using a 5-15% eOH/CH 2 C!2 gradient eluenl. 20 was obtained as white solid (2.3 mg, 68%). .
• OSW-1 carbamate 6c (5.5 mg, 0.005 mmol) was dissolved in 0.30 mL of a 4% OH/EtOH solution. The lightly yellow colored solution was stirred for 70 min. The reaction was carefully neutralized to neutral pH with 10% HQ, then diluted with CHC! 3 . Extracting with with H 2 0 gave a cloudy, poorly separated extraction. Aq. sat. NaHC0 3 was then added.
• the organic layer was separated, the aqueous fraction back-extracted with CHCI 3 , and the combined organic phases dried over Na 2 S04- 107 was purified with silica gel chromatography, using a 5-1.2% MeOH/CF C ⁇ gradient eluent, which only produced a portion of the desired compound pure (1.1 mg, 23%), as a white solid.
• the remaining 107 was in mixed fractions with impurities (estimated total amount of product from the reaction was 50-70%).
• OSW-1 carbamate 101 (1.3 mg, 0.0011 mmol) was dissolved, under argon, in 0.2 mL THF. Piperidine (8 0.08 mmol) was added. The reaction was monitored by TLC, and more piperidine was added until deprotection was complete (total piperidine ⁇ 40 ⁇ , requiring ⁇ 1 hr of time). The piperidine was removed by azeotroping the reaction with toluene twice, followed by three times with CIICI3. The crude NMR should the desired product, with the expected dibenzylfulvene by-product. The product was poorly soluble in several solvents, so purification was very difficult. The product was used crude.
• OSW-1 Amine 111 LRMS (+TOF): calculated for C 54 I1 ⁇ 2Oi 6 [M+2H + j: 1016.5664; found: 1016.3.
• efhanolamine in DMSO solution was added and incubated for 4 h at ambient temperature.
• the resin was then centrifuged to remove the 5% ethanolaminc solution and washed three times with 0.5 mL DMSO.
• the resin was stored dry at -20 °C until use.
• OSW-1 (5 mg, 0.005 mmol) under argon was taken into a nitrogen glove bag. Copper (I) chloride (-1 mg, 0.01 mmol) was added to the OSW-1, and under argon, the reaction was dissolved in DMF (0.1 mL). Alloc-(iV)-diaminomethyl benzene isocyanate (7 mg) was charged into a dry vial and dissolved in DMF (0.29 mL) to make a reagent stock solution.
• the reaction was purified via pTLC with 5% MeOH/CH 2 C! 2 (1/2 plate). Further purification with semi-preparative reverse-phase IIPLC with a Zorbax C- 18SB column (LD.9.4 mm, 3.9 mL/min) using isocratic elution with 95% MeOH/ H2O was performed, yielding 120 (1.1 mg, 17%, 35% borsm) and 110 (1.1 mg, 17%, 35% borsm) as white solids.
• Fmoc-p-alanine acid chloride (3.56 mg, 0.01 1 mmol) was dried in vacuo for 30 minutes.
• a solution of OSW-1 (10 mg, 0.01 1 mmol) and triethylamine ( 1.16 mg, 0.01 1 mmol) in 4 mL CH 2 Cl2 was cooled to 0 °C. 9 was dissolved in 0.5 mL CH 2 CI 2 and injected into OSW-1 solution. TLC in 7% MeGH/CLLCb showed the reaction was not done and another 0.8 equivalents of 9 was added.
• OSW-1 25 mg, 0.029 mmol
• Benzylisocyanate 4 mg, 0.034 mmol
• the pyridine solution was added to the flask containing OSW-1 via syringe and stirred at rt for 20h.
• TLC in 100% EtOAc showed multiple spots.
• Solvent was removed in vacuo.
• Product was purified via column
• OSW-3 (2.0 mg, 0.0023 mmol) was dissolved in 0.5 mL MeOH. Hydrogen gas was bubbled through the reaction, then the atmosphere of the reaction was flushed with argon. 10% Palladium on carbon ( ⁇ 0.2 mg, -0.0002 mmol) was added, and the reaction was placed under a hydrogen atmosphere. The reaction was stirred for 45 min. The reaction was filtered through a pad of Celite to remove the Pd C, with MeOH washing. The reaction was condensed, and purification with a silica gel pipette column was performed using a 4-12% MeOH/CHC gradient. 133 ( ⁇ 1 .0 mg, ⁇ 50% yield) was obtained as a white solid.
• OSW-1 and OSW- 1 analogs presented in Examples 9 - 35 were characterized in vitro in direct and competition binding assays, respectively, with tritiated 25-OHC and either OSB l-myc-his or ORP4L-myc-his. Methods are described above in the General Methods section. Results are shown in Table 1. Items for which no data is available are reported as "-
• OSW- 1 and OSW-1 analogs presented in Examples 9 - 35 were characterized in vitro in growth inhibition assays with wild-type (p21 + + ) and p21 -deficient ( ⁇ 2 /_ ) HCT- 1 16 cells. Methods are described above in the General Methods section. Results are shown in Table 2. items for which no data is available are reported as
• OSW-1 and OSW-1 analogs presented in Examples 9 - 35 were characterized in vitro in growth inhibition assays with wild-type A549 cells. Methods are essentially as described above in the General Methods section in connection with assays using HCT-1 16 cells.

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