NZ724963B2 - Mdm2 inhibitors and therapeutic methods using the same - Google Patents

Abstract

Compounds of formula (I) which are inhibitors of MDM2 and MDM2-related proteins, and compositions containing the same, are disclosed. Methods of using the MDM2 inhibitors in the treatment of diseases and conditions wherein inhibition of an interaction between p53 and MDM2 provides a benefit, like cancers, also are disclosed. ncers, also are disclosed.

Description

MDM2 INHIBITORS AND THERAPEUTIC METHODS USING THE SAME FIELD OF THE ION The present invention relates to inhibitors of MDM2 and MDM2—related proteins and to therapeutic methods of treating conditions and diseases wherein inhibition of MDM2 and MDM2—related proteins provides a t.

BACKGROUND OF THE INVENTION The aggressive cancer cell phenotype is the result of a variety of genetic and epigenetic alterations g to deregulation of intracellular signaling ys (Ponder, Nature 4112336 (2001)). Cancer cells typically fail to execute an apoptotic program, and lack of appropriate apoptosis due to defects in the normal apoptosis machinery is considered a hallmark of cancer (Lowe et al., Carcinogenesis 212485 ). The inability of cancer cells to execute an apoptotic program due to s in the normal apoptotic machinery often is associated with an increase in resistance to chemotherapy, radiation, or immunotherapy—induced apoptosis. Primary or acquired ance of human cancer of different origins to current treatment protocols due to apoptosis defects is a major problem in t cancer therapy (Lowe et at. , Carcinogenesis 21:485 (2000); Nicholson, Nature 4072810 (2000)). Accordingly, current and future efforts ed to designing and developing new molecular target—specific anticancer therapies to improve survival and quality of life of cancer ts must include strategies that specifically target cancer cell resistance to apoptosis.

The p53 tumor suppressor plays a central role in controlling cell cycle progression, senescence, and apoptosis (Vogelstein et al., Nature 7 (2000); Goberdhan, Cancer Cell 7:505 (2005)). MDM2 and p53 are part of an auto-regulatory feed-back loop (Wu et al., Genes Dev. 7: 1 126 (1993)). MDM2 is transcriptionally activated by p53 and MDM2, in turn, ts p53 activity by at least three mechanisms (Wu et al., Genes Dev. 7: 1 126 (1993)). First, MDM2 protein directly binds to the p53 transactivation domain, and thereby inhibits p53-mediated transactivation. Second, MDM2 protein contains a nuclear export signal sequence, and upon binding to p53, induces the nuclear export of p53, preventing p53 from binding to the ed DNAs. Third, MDM2 protein is an E3 ubiquitin ligase and upon binding to p53 is able to promote p53 degradation.

Although high-affinity peptide-based inhibitors of MDM2 have been successfully designed in the past (Garcia-Echeverria et al., Med. Chem. 43:3205 (2000)), these inhibitors are not suitable therapeutic molecules because of their poor cell permeability and in vivo bioavailability. In the last few years, there have been s of discoveries of potent, non-peptide, small-molecule MDM2 inhibitors. See e.g., U.S. Patent Nos. 7,851,626;8,088,815; 7,759,383; 7,737,174; and 8,629,141; U.S. Pat. Appl. Publ.

Nos. 2012/0046306; 2010/0152190; 2011/0112052; 2012/0122947; Int. Pat. Appl. Publ. ; ; literature, Vassilev et al. Science 2004, 303, 844−48; Vu, et al. ACS Med. Chem. Lett., 2013, 4 (5), 466–69; Zhang, et al. ACS Med.

Chem. Lett., 2014, 5 (2), 124–27; Ding et. al., J. Med. Chem., 2013, 56 (14), 5979–83; Shu, et al. Org. Process Res. Dev., 2013, 17 (2), ; Zhao, et al. J. Med. Chem., 2013, 56 (13), 5553–61; Zhao, et al. J. Am. Chem. Soc., 2013, 135 (19), 7223–34; Sun et al. J.

Med. Chem., 2014, 57 (4), 1454–72; Turiso et al., J. Med. Chem., 2013, 56 (10), 0; and Rew et al. J. Med. Chem., 2012, 55 (11), 4). Despite these major advances, there is still a need to identify , non-peptide MDM2 inhibitors having suitable physiochemical and pharmacological properties that permit use of the inhibitors in therapeutic applications.

The present invention provides compounds designed to inhibit 53 interactions, and therefore activate the on of p53 and p53-related proteins for therapeutic applications.

SUMMARY OF THE INVENTION [0005a] According to a first aspect, the invention provides a compound having the structural formula: wherein is selected from the group consisting of , , , , and ; B is a C4-7 carbocyclic ring; R1 is H, C1-4alkyl, lkyl, heterocycloalkyl, ORa, or NRaRb; n is 0, 1, or 2; R2, R3, R4, R5, R7, R8, R9, and R10, independently, are selected from the group ting of H, F, Cl, CH3, and CF3; R6 is ; Ra is hydrogen or C1-4alkyl; Rb is hydrogen or C1-4alkyl; Rc and Rd are substituents on one carbon atom of ring B, wherein Rc is H, C1-3alkyl, C1-3alkyleneORa, ORa, or halo; Rd is H, C1-3alkyl, kyleneORa, ORa, or halo; Re is –C(=O)ORa, -C(=O)NRaRb, or –C(=O)NHSO2CH3, or a pharmaceutically acceptable salt thereof. [0005b] According to a second aspect, the invention provides a composition comprising (a) a compound of the invention and (b) an excipient and/or pharmaceutically acceptable carrier.

] According to a third , the invention provides a use of a compound according to the invention or a composition according to the invention for the manufacture of a medicament for the treatment of a disease or a condition for which a MDM2 inhibitor is indicated.

The present invention is directed to inhibitors of MDM2 and MDM2-related ns, to compositions comprising the tors, and to methods of using the inhibitors in a therapeutic treatment of conditions and diseases wherein inhibition of MDM2 and MDM2-related proteins activity provides a benefit.

The present invention therefore es compounds of structural formula (I) that not only demonstrate improvement in their chemical solution ity but also exhibited an unexpected improved anti-tumor activity, including achieving complete tumor regression in an animal model of human osteosarcoma.

More ularly, the present invention is directed to compounds having a structural a (I): 2b followed by page 3 wherein / \‘ R3 * \' (Ml * is ed from the group consisting of R2 R3 N * R5 R4 * N / \\“* /N 0“ L1a: I N / \“ I \ \ R3 * R3n>k R3 N * R2 and R2 ’ , ; B is a C4_7 carbocyclic ring; R1 is H, substituted or unsubstituted C1_4alkyl, tuted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, ORa, or NRR"; n is 0, l, or 2; R2, R3, R4, R5, R7, R3, R9, and R10, independently, are selected from the group consisting of H, F, Cl, CH3, and CF3; —< S>iRe —<>—Re R6 is or ; Ra is hydrogen or substituted or unsubstituted kyl; Rb is hydrogen or substituted or unsubstituted C1_4alkyl; RC and R01 are substituents on one carbon atom of ring B, wherein RC is H, C1_3alkyl, C1_3alkyleneORa, ORa, or halo; Rd is H, C1_3alkyl, C1_3a1kyleneORa, ORa, or halo; or Rc and Rd are taken together with the carbon to which they are attached to form a 4 to 6—membered spiro substituent, optionally containing an oxygen atom; and Re is —C(=O)ORa, -C(=O)NRaRb, or —C(=O)NHSOgCH3, or a pharmaceutically acceptable salt thereof.

In one embodiment, the present ion provides a method of treating a condition or disease by stering a therapeutically effective amount of a compound of structural formula (I) to an individual in need thereof. The e or condition of st is treatable by inhibition of MDM2 and MDM—2 related proteins, for example, a cancer or a hyperproliferative disorder.

The nds of structural formula (I) inhibit the interaction between p53 or p53— related proteins and MDM2 or MDM2-related proteins. Therefore, in another ment, methods are provided to induce senescence, cell cycle arrest, and/or sis in cells containing functional p53 or p53-related proteins comprising contacting the cells with a compound of structural formula (I).

Still another embodiment is to e methods of treating, rating, or preventing a hyperproliferative disease, 6.g. a cancer, for example, adrenal cortical , advanced cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone , bone metastasis, brain/CNS tumors in adults, brain/CNS tumors in children, breast cancer, breast cancer in men, cancer in children, cancer of unknown primary, Castleman disease, cervical cancer, rectum cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), gestational trophoblastic e, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and aryngeal cancer, leukemia — acute lymphocytic (ALL) in adults, leukemia — acute myeloid (AML), leukemia — chronic lymphocytic (CLL), leukemia — c myeloid (CML), ia — chronic myelomonocytic (CMML), leukemia in en, liver cancer, lung cancer — non—small cell, lung cancer - small cell, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and sal sinus cancer, nasopharyngeal cancer, neuroblastoma, non—Hodgkin lymphoma, non— Hodgkin lymphoma in children, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, myosarcoma, salivary gland cancer, sarcoma — adult soft tissue cancer, skin cancer — basal and squamous cell, skin cancer — melanoma, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, d cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, or Wilms Tumor, in a patient comprising administering to the patient a compound of structural a (I).

Another embodiment of the present invention is to provide a composition comprising (a) an MDM2 inhibitor of structural formula (I) and (b) an excipient and/or pharmaceutically acceptable carrier.

Another ment of the present ion is to utilize a composition sing a compound of structural formula (I) and a second therapeutically active agent in a method of treating an individual for a disease or condition wherein inhibition of MDM2 and MDM2—related proteins provides a benefit.

In another ment, methods of ting normal (e.g., non—hyperproliferative) cells in a mammal from the toxic side effects of chemotherapeutic agents and treatments are provided. This method comprises administering to the mammal or therapeutically—effective amount of one or more compound of structural formula (I).

In a further embodiment, the invention provides for use of a composition comprising an MDM2 inhibitor of structural formula (I) and an optional second therapeutic agent for the manufacture of a medicament for treating a disease or condition of interest, 6.g. a cancer.

Still another embodiment of the present invention is to provide a kit for human pharmaceutical use comprising (a) a container, (bl) a packaged composition comprising an MDM2 inhibitor of structural formula (I), and, ally, (b2) a packaged composition comprising a second therapeutic agent useful in the treatment of a disease or condition of interest, and (c) a e insert containing directions for use of the composition or compositions, administered simultaneously or tially, in the treatment of the disease or condition.

An MDM2 inhibitor of structural a (I) and the second eutic agent, e.g., an anticancer agent, can be administered together as a single—unit dose or separately as multi—unit doses, wherein the MDM2 inhibitor of structural formula (I) is administered before the second therapeutic agent or vice versa. It is oned that one or more dose of an MDM2 inhibitor of structural formula (1) and/or one or more dose of a second therapeutic agent can be administered.

In one embodiment, an MDM2 inhibitor of structural formula (I) and a second therapeutic agent are administered simultaneously. In related ments, an MDM2 inhibitor of structural formula (I) and a second therapeutic agent are administered from a single ition or from separate compositions. In a further embodiment, the MDM2 inhibitor of structural formula (I) and second therapeutic agent are administered tially. An MDM2 inhibitor of structural formula (I), as used in the present invention, can be administered in an amount of about 0.005 to about 500 milligrams per dose, about 0.05 to about 250 milligrams per dose, or about 0.5 to about 100 milligrams per dose.

These and other embodiments and features of the present invention will become apparent from the following detailed description of the preferred embodiments.

BRIEF DESCRIPTION OF DRAWINGS Figure 1A contains a graph of % purity vs. time (days) for compounds 06l, de No. 2, de No. 7, and de No. 8 in 1:1 CH3CN/H20.

Figure 1B contains a graph of % purity vs. time (days) for compounds AA—MI—06l, de No. 2, de No. 7, and de No. 8 in 1:1 MeOH/HZO.

Figure 1C contain graphs of % purity vs. time (days) for compounds AA-MI—06l, de No. 2, de No. 7, and de No. 8 in cell culture media.

Figure 2A contains a graph of mean tumor volume (mm3) vs. time (days) showing the efficacy of various tested compounds for tumor sion in the SJSA—l xenograft model.

Figure 2B contains a graph of mean tumor volume (mm3) vs. time (days) showing the efficacy of various tested compounds for tumor regression in the SJSA—l xenograft model.

Figure 3 ns a graph of mean tumor volume (mm3) vs. time (days) g the efficacy of various doses and dose schedules of de No. 8 for tumor regression in the SJSA-l xenograft model.

DETAILED PTION OF THE PREFERRED EMBODIMENTS Spiro-oxindole—based antagonists are a class of inhibitors of the p53—MDM2 interaction and are described in US. Patent Nos. 7,759,383, 7,737,174, and 8,629,141. Some spiro—oxindole MDM2 inhibitors quickly converted, in protic on, from one diastereomer to three other diastereomers (Zhao, et al. J Am Chem Soc. 2013, l35(19):7223—34). Efforts were made to improve the chemical stability of spiro—oxindole MDM2 inhibitors, such as those described in US. Patent No. 8,629,141. For example, compounds shown in Scheme 1 were shown to y isomerize from less potent diastereomers to more potent and ally more stable diastereomers as MDM2 inhibitors in US. Patent No. 8,629,141.

Less Potent Dlastereomers for MDM2 More Potent Dlastereomers for MDM2 Scheme 1. Conversion of less potent diastereomers to MDM2 to more potent diastereomers to MDM2 When the carboxamide substituent R is a benzoic acid, such as in AA—MI—061, the compound demonstrated high binding ty to MDM2, potent cell growth tion in SJSA—l cells and 90% tumor regression (at 100 mg/kg, once, daily dosing) in SCID mice bearing SJSA—l xenografts (Figure 2A). l other classes of spiro—oxindole compounds (US. Pat. Appl.

Publ. 2011, US 20110130398, Shu et al. Org. Process Res. Dev., 2013, 17 (2), 247—56, and Zhang et a1. ACS Med. Chem. Lett., 2014, 5 (2), 124-27) and pyrrolidines (Ding et. al J. Med.

Chem., 2013, 56 (14), 3, and US. Pat. Appl. Publ. US 20100152190, 2010) that contain the benzoic acid carboxamide substituent have shown high binding affinities to MDM2, good oral pharmacokinetics in animals, and strong antitumor activity in animal models of human cancer. These prompted us to explore replacements for the benzoic acid group for the design of new MDM2 tors (Scheme 11).

AA-Ml-061 Example No. 1 Example No. 2 R = Me, Example No. 7 R = Et, Example No. 8 MDM2 |C50 = 4.4 nM MDM2 |C50 = 5.4 nM MDM2 |C50 = 5.2 nM MDM2 IC50 < 5 nM SJSA—1 ICSD = 100 nM SJSA—1 |C50 = 480 nM SJSA—1 IC50 = 89 nM SJSA—1 ICSD < 70 nM 90% SJSA-1 tumor regression tumor growth inhibition complete and presisnt tumor regression Scheme 2. New MDM2 tors designed using replacements of the benzoic acid group.

In accordance with the t invention, the benzoic acid substituent of the carboxamide in AA—MI—06l was replaced with non—classical benzoic acid bioisosteres (J. Med.

Chem. 2012, 55, 3414), such as a bicyclo[l.1.1]pentane—l—carboxylic acid group or a bicyclo[2.2.2]octane— oxylic acid group, that produced nd No. l and Compound No. 2, respectively (Scheme 2). While, Compound No. 1 maintained a high binding affinity to MDM2 protein, it had a reduced potency in the cell growth inhibition activity in SJSA—l cells, compared to 06l. On the other hand, Compound No. 2, containing a bicyclo[2.2.2]octane— l—carboxylic acid group, maintained high binding affinity to MDM2 protein, and potent cell growth tion activity in SJSA—l cells, similar to the potency obtained for 06l. Compound No. 2, however, still showed only modest anti—tumor activity, merely inhibiting growth in mice bearing the SJSA—l xenograft tumors without achieving tumor regression (Figure 2B).

In an effort to improve the antitumor activity of nd No. 2 in animals, alkylation of the pyrrolidine nitrogen produced a series of compounds, including Compounds No. 7 and No. 8. Compounds No. 7 and No. 8 retained high binding affinity to MDM2 and was stable in solutions (Figure l). Unexpectedly, compounds No. 7 and No. 8 showed a much er antitumor activity than Compound No. 2 in mice bearing the SJSA—l xenograft .

Specifically, Compounds No. 7 and No. 8 demonstrated te and persistent tumor regression in mice bearing the SJSA-l xenograft tumors (Figure 2B).

Provided herein therefore are compounds of structural a (I) that inhibit the interaction between p53 or p53—related proteins and MDM2 or MDM2—related proteins. By inhibiting the negative effect of MDM2 or MDM2-related ns on p53 or p53—related proteins, the present compounds sensitize cells to inducers of apoptosis and/or cell cycle arrest.

In one embodiment, the present compounds induce apoptosis and/or cell cycle . Therefore, also provided herein are methods of sensitizing cells to inducers of apoptosis and/or cell cycle arrest and to methods of inducing apoptosis and/or cell cycle arrest in cells. The methods comprise contacting the cells with one or more compounds having a structural formula (I) either alone or in combination with additional agent(s), e.g., an inducer of apoptosis or a cell cycle disrupter.

The term "MDM2—related protein," as used herein, refers to proteins that have at least % sequence homology with MDM2, and interact with and inhibit p53 or p53—related proteins.

Examples of MDM2—related proteins include, but are not limited to, MDMX.

The term "functional p53," as used herein, refers to wild—type p53 expressed at normal, high, or low levels and mutant or allelic variants of p53 that retain(s) at least about 5% of the activity of wild—type p53, e.g., at least about 10%, about 20%, about 30%, about 40%, about 50%, or more of wild—type activity.

The term "p53—related protein," as used herein, refers to proteins that have at least 25% sequence homology with p53, have tumor suppressor activity, and are inhibited by interaction with MDM2 or elated proteins. Examples of p53—related ns include, but are not limited to, p63 and p73.

The term "disease" or "condition" denotes disturbances and/or anomalies that as a rule are regarded as being pathological conditions or functions, and that can manifest themselves in the form of particular signs, symptoms, and/or malfunctions. As demonstrated below, a compound of structural formula (I) is a potent inhibitor of an ction between p53 and p53— d proteins and MDM2 and MDM2—related ns and can be used in treating diseases and conditions wherein such tion provides a t.

The term "a e or condition wherein inhibition of MDM2 or elated proteins provides a benefit" pertains to a condition in which ting the interaction between p53 or p53—related proteins and MDM2 and MDM2-related proteins is important or necessary, e. g., for the onset, progress, expression of that disease or condition, or a disease or a ion which is known to be treated by MDM2 or MDM2-related protein inhibitor. Examples of such conditions include, but are not limited to, a cancer. One of ordinary skill in the art is readily able to determine r a compound treats a disease or condition mediated by a MDM2 or an MDM2—related protein, for any particular cell type, for example, by assays which conveniently can be used to assess the ty of particular nds.

The term "hyperproliferative disease," as used herein, refers to any condition in which a localized population of proliferating cells in an animal is not governed by the usual limitations of normal growth. Examples of hyperproliferative disorders include tumors, neoplasms, lymphomas, leukemias, and the like. A neoplasm is said to be benign if it does not undergo invasion or metastasis, and malignant if it does either of these. A "metastatic" cell means that the cell can invade neighboring body structures. Hyperplasia is a form of cell proliferation ing an increase in cell number in a tissue or organ without icant alteration in ure or function. Metaplasia is a form of controlled cell growth in which one type of fully differentiated cell substitutes for another type of differentiated cell.

The pathological growth of activated lymphoid cells often s in an autoimmune disorder or a chronic inflammatory condition. As used herein, the term "autoimmune disorder" refers to any condition in which an organism es antibodies or immune cells which recognize the sm's own molecules, cells or tissues. Non—limiting examples of mune disorders include autoimmune hemolytic anemia, autoimmune hepatitis, Berger's disease or IgA nephropathy, celiac sprue, chronic fatigue syndrome, Crohn's e, dermatomyositis, fibromyalgia, graft versus host disease, Grave's disease, Hashimoto‘s thyroiditis, idiopathic thrombocytopenia purpura, lichen planus, multiple sis, enia gravis, psoriasis, tic fever, rheumatic arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, type 1 diabetes, ulcerative colitis, vitiligo, and the like.

The term cence" as used , refers to the phenomenon whereby non—cancerous diploid cells lose the ability to divide, and characterized in part by telomeric dysfunction or shortening.

The terms "sensitize" and tizing," as used herein, refer to making, through the administration of a first therapeutic agent (e.g., a compound provided herein), an animal or a cell within an animal more susceptible, or more responsive, to the biological effects (e.g., promotion or retardation of an aspect of cellular function including, but not limited to, cell division, cell growth, proliferation, invasion, angiogenesis, necrosis, or apoptosis) of a second therapeutic agent. The sensitizing effect of a first agent on a target cell can be measured as the difference in the intended biological effect (6.g. or retardation of an aspect of cellular function , promotion including, but not limited to, cell growth, proliferation, invasion, angiogenesis, or apoptosis) observed upon the administration of a second agent with and without administration of the first agent. The response of the sensitized cell can be increased by at least about 10%, at least about %, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% over the response in the absence of the first agent. _10_ The term "dysregulation of apoptosis," as used herein, refers to any aberration in the ability of (e.g., predisposition) a cell to undergo cell death via apoptosis. Dysregulation of apoptosis is associated with or induced by a variety of conditions, non—limiting es of which include, autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, graft—versus—host disease, myasthenia gravis, or Sj6gren's syndrome), c inflammatory ions (e.g., psoriasis, asthma or Crohn's disease), hyperproliferative disorders (e.g., tumors, B cell lymphomas, or T cell lymphomas), viral infections (e.g., , papilloma, or HIV), and other conditions such as osteoarthritis and atherosclerosis. It should be noted that when the ulation is induced by or associated with a viral infection, the viral infection may or may not be detectable at the time ulation occurs or is observed. That is, Viral—induced dysregulation can occur even after the disappearance of symptoms of viral infection.

The term astic disease," as used herein, refers to any abnormal growth of cells being either benign (non—cancerous) or malignant (cancerous).

The term "normal cell," as used herein, refers to a cell that is not oing al growth or division. Normal cells are ncerous and are not part of any roliferative disease or disorder.

The term "anti—neoplastic agent," as used herein, refers to any compound that retards the proliferation, growth, or spread of a targeted (e.g., malignant) neoplasm.

The term "apoptosis-modulating agents," as used herein, refers to agents which are involved in modulating (e.g., inhibiting, decreasing, increasing, promoting) apoptosis. Examples of apoptosis—modulating agents include proteins which comprise a death domain such as, but not limited to, Fas/CD95, TRAMP, TNF RI, DRl, DR2, DR3, DR4, DR5, DR6, FADD, and RIP.

Other examples of apoptosis—modulating agents include, but are not limited to, TNFOL, Fas , antibodies to Fas/CD95 and other TNF family receptors, TRAIL (also known as Ap02 Ligand or ApoZL/TRAIL), antibodies to TRAIL—R1 or TRAIL—R2, Bel—2, p53, BAX, BAD, Akt, CAD, PI3 kinase, PPl, and caspase proteins. Modulating agents broadly include agonists and antagonists of TNF family receptors and TNF family ligands. Apoptosis—modulating agents may be soluble or membrane bound (6.g. ligand or or). Apoptosis—modulating agents include those which are inducers of apoptosis, such as TNF or a TNF—related ligand, particularly a TRAMP , a Fas/CD95 ligand, a TNFR—l ligand, or TRAIL. _11_ WO 61032 The term d eutic agent" refers to a therapeutic agent different from an MDM2 inhibitor of structural formula (I) and that is known to treat the disease or condition of st. For example when a cancer is the disease or condition of interest, the second therapeutic agent can be an anticancer agent.

The term "anticancer agent" as used herein, refers to any therapeutic agent (e.g., herapeutic compound and/or molecular eutic nd), antisense therapy, radiation therapy, or surgical intervention, used in the treatment of hyperproliferative diseases, such as cancer (6.g. in mammals, and particularly in humans).

As used herein, the terms "treat," "treating, II II treatment," and the like refer to eliminating, reducing, or ameliorating a disease or condition, and/or symptoms associated therewith. gh not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated. As used , the terms "treat, treating, treatment," and the like may include "prophylactic treatment," which refers to reducing the probability of redeveloping a disease or condition, or of a recurrence of a previously—controlled disease or condition, in a subject who does not have, but is at risk of or is susceptible to, redeveloping a e or condition or a recurrence of the disease or condition.

The term "treat" and synonyms contemplate administering a therapeutically effective amount of a compound of the invention to an individual in need of such treatment.

Within the meaning of the invention, "treatment" also includes relapse prophylaxis or phase prophylaxis, as well as the treatment of acute or chronic signs, ms and/or malfunctions. The ent can be orientated symptomatically, for example, to suppress ms. It can be ed over a short period, be oriented over a medium term, or can be a long—term treatment, for example within the context of a maintenance therapy.

The term "therapeutically effective amount" or "effective dose" as used herein refers to an amount of the active ingredient(s) that ) sufficient, when administered by a method of the invention, to efficaciously deliver the active ingredient(s) for the treatment of condition or disease of interest to an individual in need thereof. In the case of a cancer or other proliferation disorder, the therapeutically effective amount of the agent may reduce (i.e., retard to some extent and preferably stop) unwanted cellular proliferation; reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., retard to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., retard to some extent and preferably stop) tumor metastasis; _12_ inhibit, to some extent, tumor growth; reduce MDM2 and MDM2—related protein interactions with p53 and p53—related proteins; and/or relieve, to some extent, one or more of the symptoms ated with the cancer by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. To the extent the stered compound or composition prevents growth and/or kills existing cancer cells, it may be cytostatic and/or cytotoxic.

The term "container" means any receptacle and closure therefor suitable for storing, shipping, dispensing, and/or handling a pharmaceutical product.

The term "insert" means information accompanying a pharmaceutical product that provides a ption of how to administer the product, along with the safety and efficacy data required to allow the physician, pharmacist, and patient to make an informed decision regarding use of the product. The package insert generally is regarded as the " for a pharmaceutical product. rrent stration, administered in combination, simultaneous administration," and r phrases mean that two or more agents are administered concurrently to the subject being treated. By "concurrently," it is meant that each agent is administered either simultaneously or sequentially in any order at different points in time. However, if not administered simultaneously, it is meant that they are administered to an individual in a sequence and sufficiently close in time so as to provide the desired therapeutic effect and can act in concert. For example, an MDM2 inhibitor of structural formula (I) can be administered at the same time or sequentially in any order at ent points in time as a second therapeutic agent.

A t MDM2 inhibitor and the second eutic agent can be administered separately, in any appropriate form and by any suitable route. When a present MDM2 inhibitor and the second therapeutic agent are not administered concurrently, it is understood that they can be administered in any order to a t in need thereof. For example, a present MDM2 inhibitor can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 _13_ weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent treatment modality (e.g., radiotherapy), to an individual in need thereof. In various ments, an MDM2 inhibitor of structural formula (I) and the second therapeutic agent are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart. In one embodiment, the components of the combination therapies are administered at 1 minute to 24 hours apart.

The terms "pulsatile administration, pulsatile dose administration" or "pulsatile dosing" as used , refer to intermittent (i.e., not uous) administration of compounds of structural formula (I) to a patient. Pulsatile dose administration ns useful in the present disclosure encompass any tinuous administration regimen that provides a therapeutically effective amount of compounds of structural a (I) to a patient in need f. Pulsatile dosing regimens can use equivalent, lower, or higher doses of compounds of structural formula (I) than would be used in uous dosing ns. Advantages of pulsatile dose administration of compounds of structural formula (I) include, but are not limited to, improved safety, decreased toxicity, increased exposure, increased efficacy, and increased patient compliance. These advantages may be realized when compounds of structural formula (I) are administered as a single agent or are administered in combination with one or more additional anticancer agents. On the day that a compound of ural formula (I) is scheduled to be administered to the patient, stration can occur in a single or in divided doses, e.g., once—a— day, twice-a—day, three times a day, four times a day or more. In one embodiment, a compound having of structural formula (I) is administered once (QD) or twice (BID) on the day it is le to be stered.

The use of the terms "a", "an", "the", and similar referents in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated. Recitation of ranges of values herein merely are intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated , and each separate value is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to better _14_ illustrate the invention and is not a limitation on the scope of the invention unless otherwise d. No language in the ication should be construed as indicating any non—claimed element as essential to the ce of the invention.

Research has established that targeting the p53—MDM2 interaction using small molecule tors is a viable cancer therapeutic strategy. The prior discovery of MDM2 inhibitors and early data have demonstrated that non-peptide, small molecule inhibitors of MDM2—p53 interactions have great therapeutic potential for the treatment of diseases and ions in which MDM2 and MDM2—related proteins have a role.

The present invention is directed to a new class of potent and specific inhibitors of MDM2—p53 interactions. The t compounds function as potent antagonists of MDM2—p53 interactions. The MDM2 inhibitors of the present invention therefore are useful in the treatment of a variety of diseases and conditions, including cancers, in subjects in need of such treatment.

Also provided are methods of treating a subject having unwanted roliferative cells comprising administering a therapeutically ive amount of a present compound to a subject in need of such treatment. Also provided are methods of preventing the proliferation of unwanted proliferating cells, such as cancers, in a subject comprising the step of administering a therapeutically effective amount of a nd of structural formula (I) to a subject at risk of developing a condition characterized by unwanted proliferating cells.

The present invention is directed to MDM2 inhibitors having a structural formula (I): wherein _15_ 2015/026098 R4 -* \ R5 (Ml / \‘ R3 * \' * is selected from the group consisting of R2 R3 N * R5 R4 N/ /N fl.*N / \“ |\‘\'* \ \ R3 * R3n>k|\\\‘* R3 N * R2 and R2 3 3 ; B is a C4_7 carbocyclic ring; R1 is H, substituted or unsubstituted C1_4alkyl, substituted or unsubstituted cycloalkyl, tuted or tituted heterocycloalkyl, ORa, or NRaRb; n is 0, l, or 2; R2, R3, R4, R5, R7, R8, R9, and R10, independently, are selected from the group consisting of H, F, Cl, CH3, and CF3; —< S>iRe —<>—Re R6 is or ; Ra is hydrogen or substituted or unsubstituted C1_4alkyl; Rb is hydrogen or substituted or unsubstituted C1_4a1kyl; RC and R01 are substituents on one carbon atom of ring B, wherein Rc is H, C1_3alkyl, C1_3alkyleneORa, ORa, or halo; Rd is H, C1_3alkyl, C1_3a1ky1eneORa, ORa, or halo; or RC and Rd are taken together with the carbon to which they are attached to form a 4 to 6—membered spiro substituent, optionally ning an oxygen atom; and Re is ORa, —C(=O)NRaRb, or —C(=O)NHSOZCH3, or a pharmaceutically acceptable salt thereof.

The compounds of structural formula (I) inhibit MDM2—p53 interactions and are useful in the ent of a variety of diseases and conditions. In particular, the compounds of structural formula (I) are used in methods of treating a disease or condition wherein inhibition of —16- MDM2 and MDM2—related protein provides a benefit, for example, cancers and proliferative diseases. The method comprises administering a therapeutically effective amount of a compound of structural formula (I) to an individual in need thereof. The present methods also encompass administering a second therapeutic agent to the individual in addition to the compound of structural formula (I). The second therapeutic agent is selected from drugs known as useful in treating the disease or condition afflicting the individual in need thereof, e.g., an anticancer agent known as useful in treating a particular cancer.

As used herein, the term "alkyl" refers to straight chained and branched saturated C1_10 hydrocarbon groups but not limited to methyl, ethyl, n—propyl, yl, n-butyl, , including sec—butyl, t—butyl, n—pentyl, 2—methylbutyl, 3—methylbutyl, 2,2—dimethylpropyl, n—hexyl, 2—methylpentyl, ylpentyl, 4—methylpentyl, 2,2-dimethylbutyl, 2,3—dimethylbutyl, 3,3—dimethylbutyl, and 2—ethybutyl. The term Cm—n means the alkyl group has "m" to "n" carbon atoms. The term "alkylene" refers to an alkyl group having a substituent. An alkyl, e.g., methyl, or ne, e.g., —CH2—, group can be substituted with one or more, and typically one to three, of ndently selected halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, or amino groups, for e.

As used herein, the term "halo" is defined as fluoro, , bromo, and iodo.

The term "hydroxy" is defined as —OH.

The term "alkoxy" is d as —OR, n R is alkyl.

The term "amino" is defined as —NH2, and the term amino" is defined as —NR2, wherein at least one R is alkyl and the second R is alkyl or en.

The term "carbamoyl" is defined as —C(=O)NR2.

The term "carboxy" is defined as -C(=O)OH or a salt thereof.

The term "nitro" is defined as —N02.

The term "cyano" is d as —CN.

The term "trifluoromethyl" is defined as —CF3.

The term "trifluoromethoxy" is defined as —OCF3. _17_ | I As used herein, groups such as is an abbreviation for CH3_ As used herein, the term "ary " refers to a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, yl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. Aryl also refers to bicyclic and tricyclic carbon rings, where one ring is ic and the others are saturated, lly unsaturated, or aromatic, for e, dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl). Unless otherwise indicated, an aryl group can be unsubstituted or tuted with one or more, and in particular one to four, groups independently selected from, for example, halo, alkyl, alkenyl, —OCF3, —N02, —CN, —NC, —OH, , amino, alkylamino, —C02H, —COgalkyl, —OCOalkyl, aryl, and heteroaryl.

As used herein, the term "heterocyclic" refers to a heteroaryl and heterocycloalkyl ring systems.

As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, , or sulfur atom in an aromatic ring. Each ring of a heteroaryl group can contain one or two 0 atoms, one or two S atoms, and/or one to four N atoms, ed that the total number of atoms in each ring is four or less and each ring contains at least one carbon atom. In certain embodiments, the heteroaryl group has from 5 to 20, from 5 to 15, or from 5 to 10 ring atoms. Examples of monocyclic heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, yl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, triazinyl, and triazolyl. es of bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzimidazolyl, benzoisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzothiophenyl, benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridinyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolinyl, isothiazolyl, yridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl, opyridyl, quinolinyl, quinoxalinyl, quiazolinyl, thiadiazolopyrimidyl, and thienopyridyl. Unless otherwise indicated, a aryl group can be tituted or substituted with one or more, and in particular one to four, substituents selected from, for example, halo, alkyl, alkenyl, —OCF3, —N02, —CN, — NC, —OH, alkoxy, amino, alkylamino, —C02H, —C02all<yl, —OCOalkyl, aryl, and heteroaryl. _ 18 _ As used herein, the term "cycloalkyl" means a monocyclic or bicyclic, saturated or partially unsaturated, ring system containing three to eight carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, exyl, cycloheptyl, and cyclooctyl, optionally substituted with one or more, and lly one to three, of ndently selected halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, or amino groups, for example.

As used herein, the term "heterocycloalkyl" means a monocyclic or a bicyclic, saturated or partially unsaturated, ring system containing 4 to 12 total atoms, of which one to five of the atoms are ndently selected from nitrogen, oxygen, and sulfur and the remaining atoms are carbon. Nonlimiting examples of heterocycloalkyl groups are azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, dihydropyrrolyl, morpholinyl, thiomorpholinyl, opyridinyl, oxacycloheptyl, dioxacycloheptyl, thiacycloheptyl, diazacycloheptyl, each optionally substituted with one or more, and typically one to three, of independently selected halo, C1_6 alkyl, C1_6 alkoxy, cyano, amino, carbamoyl, nitro, carboxy, C37 l, C37 alkynyl, or the like on an atom of the ring.

R4 2k <1 Rm .3 [0108] In some preferred embodiments, * is R2 or In other ments, B ism or <>.

In various embodiments, n is 0 or 1 and R1 is H or CH3. In various embodiments, —(CH2)n—R1 is H, CH3, or CH2CH3.

In various embodiments, R2 is H. In other embodiments, R3 is halo, and preferably chloro. In still another embodiments, R4 is H, R5 is H, or both R4 and R5 are H.

In some preferred ments, R7 is halo, and more preferably is fluoro.

In some embodiments, each of R8, R9, and R10 are H.

In various embodiments, Ra and Rh, individually, are H, CH3, or CH2CH3.

In other embodiments, RC and Rd, individually, are H, halo, OH, CH3, CH2CH3, or CHZOH. In some embodiments, Rc and Rd are F and F, H and H, OH and CH3, CH3 and CH3, CH3 and OH, H and OH, CH2CH3 and CH2CH3, and CHon and CHQOH. _19_ In other embodiments, Rc and Rd are taken together with ring B to form a spiro moiety, for example In other embodiments, RC and RC1 taken with ring B form: 235%,M, Mii,o.zjw%.

In some embodiments, Re is —C(=O)OH, NH2, or —C(=O)NHSOZCH3. o 0 *w @4 In various ments, R6 is OH, NH * <9 6 O H *—< H0 (I? HN—?=O * ,or HN—S=O Additionally, salts of the present compounds also are included in the present invention and can be used in the methods disclosed herein. The present invention further includes all possible stereoisomers and geometric isomers of the compounds of structural formula (I). The t invention includes both racemic nds and optically active isomers. When a compound of structural formula (I) is desired as a single enantiomer, it can be ed either by resolution of the final product or by specific synthesis from either isomerically pure starting material or use of a chiral auxiliary reagent, for example, see Z. Ma et al., Tetrahedron.- Asymmetry, 8(6), pages 883—888 (1997). Resolution of the final product, an intermediate, or a _20_ starting material can be ed by any suitable method known in the art. Additionally, in situations where tautomers of the compounds of structural a (I) are possible, the present invention is intended to include all tautomeric forms of the nds.

Certain of the nds of the present disclosure may exist as isomers, i.e., isomers that differ only in the spatial arrangement of atoms, including optical isomers and conformational isomers (or conformers). The disclosure includes all stereoisomers, both as pure individual stereoisomer ations and enriched preparations of each, and both the racemic mixtures of such stereoisomers as well as the individual diastereomers and enantiomers that may be separated according to s that are well known to those of skill in the art.

The term "substantially free of" as used herein means that the compound comprises less than about 25% of other stereoisomers, e.g., diastereomers and/or enantiomers, as established using tional analytical methods routinely used by those of skill in the art. In one embodiment, the amount of other stereoisomers is less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.5%.

Stereoisomerically enriched compounds that contain about 95% or more of a desired stereoisomer, for example, about 96% or more, about 97% or more, about 98% or more, or about 99% or more are referred to herein as "substantially pure stereoisomers." Stereoisomerically enriched compounds that n about 99% or more of a desired stereoisomer are referred to herein as "pure" stereoisomers." The purity of any Stereoisomerically ed compound can be determined using conventional analytical methods such as, for example, normal phase HPLC, reverse phase HPLC, chiral HPLC, and 1H and 13C NMR.

Compounds of the invention can exist as salts. Pharmaceutically acceptable salts of the compounds of the invention often are preferred in the methods of the invention. As used herein, the term "pharmaceutically acceptable salts" refers to salts or zwitterionic forms of the compounds of structural formula (I). Salts of compounds of a (I) can be prepared during the final isolation and purification of the compounds or separately by reacting the compound _21_ with an acid having a suitable cation, such as, but not limited to, alkali and alkaline earth metal ions, e.g., Na+, K+, Ca2+, and Mg2+as well as organic cations such as, but not limited to, ammonium and substituted ammonium ions, e.g., NH4+, NHMe3+, NH2M62+, NHMe3+ and NMe4+. Examples of monovalent and divalent pharmaceutically acceptable cations are discussed, 6.57., in Berge et al. J. Pharm. Sci, 6621—19 (1997).

The pharmaceutically acceptable salts of compounds of structural a (I) can be acid addition salts formed with pharmaceutically acceptable acids. Examples of acids which can be employed to form pharmaceutically acceptable salts e nic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and c acids such as oxalic, maleic, succinic, and citric. iting examples of salts of compounds of the invention include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2—hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, rate, rsulfonate, digluconate, glycerolphsphate, hemisulfate, heptanoate, ate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2— naphthalenesulfonate, oxalate, pamoate, ate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzene nate, and p—toluenesulfonate salts. In on, available amino groups present in the compounds of the invention can be quatemized with methyl, ethyl, propyl, and butyl chlorides, es, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, , myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. In light of the foregoing, any reference to compounds of the present invention appearing herein is intended to include nds of structural formula (I) as well as pharmaceutically acceptable salts thereof.

Specific compounds of the present invention include, but are not limited to, compounds having the structure set forth below. _22_ The t invention provides MDM2 inhibitors, as exemplified by compounds of structural formula (I), for the treatment of a variety of diseases and conditions wherein inhibition of MDM2 and MDM—2 related proteins has a beneficial effect. In one embodiment, the present invention s to a method of treating an individual suffering from a disease or ion n inhibition of the MDM2 and MDM2—related proteins provides a benefit comprising administering a therapeutically effective amount of a compound of structural formula (I) to an individual in need thereof. _23_ The present methods contemplate that exposure of animals or patients suffering from cancer to therapeutically effective amounts of drug(s) (e.g., small molecules) that increase the function(s) of p53 and p53—related proteins (e.g., p63, p73) inhibits the growth of cancer cells or supporting cells. The present MDM2 inhibitors provided herein inhibit the interaction between p53 or p53—related proteins and MDM2 or MDM2—related proteins (6. g., MDMX). Inhibiting the interaction between p53 or p53—related proteins and MDM2 or MDM2—related proteins inhibits the growth of cancer cells or supporting cells and/or renders such cells as a population more susceptible to the cell death—inducing ty of cancer therapeutic drugs or radiation therapies.

In one embodiment, the MDM2 inhibitors provided herein g the half—life of p53 by interfering with the p53—MDM2 interaction that would normally promote degradation of p53.

The compounds provided herein satisfy an unmet need for the treatment of multiple cancer types, either when administered as erapy to induce senescence, cell growth inhibition, apoptosis and/or cell cycle arrest in cancer cells, or when administered in a temporal relationship with additional agent(s), such as other cell death—inducing or cell cycle disrupting cancer therapeutic drugs or radiation therapies (combination therapies), so as to render a r proportion of the cancer cells or tive cells tible to executing the apoptosis program compared to the ponding proportion of cells in an animal or a patient treated only with the cancer therapeutic drug or radiation therapy alone.

In one ment, treatment of patients with a therapeutically effective amount of one or more compounds of structural formula (I) and one or more anticancer agents es a r anti—tumor activity and clinical benefit in such patients ed to those treated with the compound or anticancer drugs/radiation alone. Alternately stated, because the present compounds lower the apoptotic threshold of cells that express p53 or p53-related n, the proportion of cells that sfully execute the sis program in se to the apoptosis ng activity of anticancer drugs/radiation will be increased when used in combination with one or more of the present compounds. Compounds of structural formula (I) therefore can be used to allow administration of a lower, and therefore less toxic and more tolerable, dose of an anticancer drug and/or radiation to produce the same tumor response/clinical benefit as the conventional dose of the anticancer drug/radiation alone. Because the doses for approved anticancer drugs and radiation treatments are known, the compounds, compositions, and methods provided herein can be used with one or more approved ncer drugs and/or radiation treatment. Also, because compounds of structural formula (I) can act, at least in part, by _24_ 2015/026098 stimulating the pro—apoptotic and/or cell cycle—inhibiting activities of p53 and p53—related ns, the re of cancer cells and supporting cells to therapeutically ive amounts of these compounds can be temporally linked to coincide with the attempts of cells to execute the apoptosis program in response to the anticancer drug or radiation therapy. Thus, in one ment, administering the compounds or pharmaceutical compositions provided herein in combination with other known ncer drugs provides especially efficacious therapeutic practices.

In one embodiment, the inhibitors of the interaction between p53 or p53—related proteins and MDM2 and MDM2—related proteins of structural formula (I) can protect normal (e.g., non—hyperproliferative) cells from the toxic effects of certain chemotherapeutic agents and radiation, possibly through the ability of the inhibitors to induce cell cycle arrest of normal cells.

For example, the MDM2 inhibitors provided herein may cause cell cycle arrest in cells sing wild—type or functional p53 (and/or wild-type or functional p53-related proteins) while having no or less effect on cancer cells comprising mutated, deleted, or otherwise non— or less functional p53 (and/or mutated, deleted, or otherwise non—or less functional p53—related proteins). This differential protective effect can allow for more effective treatment of cancer by allowing the use of higher doses or longer treatments of chemotherapeutic agents or treatments without increasing the toxic side effects of such treatment when administered in combination with inhibitors provided herein.

Also provided herein are methods of using compounds of structural formula (I) for izing cells to onal s), such as inducers of ence, apoptosis, and/or cell cycle arrest. Compounds of structural formula (I) also can be used to provide chemoprotection of normal cells through the induction of cell cycle arrest prior to treatment with chemotherapeutic agents. In one embodiment, methods of rendering a normal cell resistant to chemotherapeutic agents or treatments comprises contacting the cell with one or more compounds of structural formula (I) are provided. In another embodiment, methods of protecting normal cells in an animal having a hyperproliferative disease from the toxic side s of chemotherapeutic agents or treatments, comprises administering to the animal a compound of structural a (I) are ed. Also ed herein are methods for the treatment, amelioration, or prevention of disorders, side effects, or conditions caused by the administration of chemotherapeutic agents to normal cells comprising administering to an animal undergoing chemotherapy a compound of structural formula (I). Examples of such disorders and _25_ 2015/026098 conditions caused by chemotherapy include, without limitation, tis, stomatitis, xerostomia, gastrointestinal disorders, and alopecia.

Compounds of structural formula (I) are useful for the treatment, amelioration, or prevention of disorders, such as those sive to induction of apoptotic cell death, e.g., disorders characterized by dysregulation of apoptosis, including hyperproliferative diseases such as cancer. In one embodiment, these compounds can be used to treat, or ameliorate cancer that is characterized by resistance to cancer therapies (e.g., those cancer cells which are chemoresistant, radiation ant, e resistant, and the like). In another embodiment, the present compounds can be used to treat hyperproliferative diseases characterized by expression of functional p53 or lated ns. In another embodiment, the present compounds can be used to protect normal (e.g., non—hyperproliferative) cells from the toxic side effects of chemotherapeutic agents and treatments by the induction of cell cycle arrest in those cells.

In one embodiment, compounds of structural formula (I) induce cell cycle arrest and/or apoptosis and also potentiate the induction of cell cycle arrest and/or apoptosis either alone or in response to additional apoptosis induction signals. Therefore, it is plated that the present compounds ize cells to induction of cell cycle arrest and/or apoptosis, including cells that are resistant to such inducing stimuli. By inhibiting the interaction between p53 or p53—related proteins and MDM2 or MDM2—realted proteins, the present compounds can be used to induce apoptosis in any disorder that can be treated, ameliorated, or prevented by the induction of apoptosis. In one embodiment, compounds of structural formula (I) can be used to induce apoptosis in cells sing functional p53 or p53-related proteins.

The compounds of structural formula (I), in combination with one or more additional apoptosis—modulating agents, e.g., anticancer agents, to modulate apoptosis. Examples of apoptosis—modulating agents include, but are not limited to, Fas/CD95, TRAMP, TNF RI, DRl, DR2, DR3, DR4, DR5, DR6, FADD, RIP, TNFOL, Fas ligand, TRAIL, dies to TRAIL—R1 or TRAIL-R2, Bcl-2, p53, BAX, BAD, Akt, CAD, PI3 kinase, PP1, and caspase proteins. Other agents involved in the initiation, decision and degradation phase of apoptosis also are included.

Examples of apoptosis-modulating agents include , the activity, presence, or change in concentration of which, can modulate sis in a subject. Apoptosis—modulating agents include those which are rs of apoptosis, such as TNF or a TNF—related ligand, particularly a TRAMP ligand, a Fas/CD95 ligand, a TNFR—l ligand, or TRAIL. —26- The compounds, compositions, and methods herein are used to treat diseased cells, tissues, organs, or pathological ions and/or disease states in an animal (e.g., a mammalian patient including, but not limited to, humans and veterinary animals). In this regard, various diseases and pathologies are le to treatment or prophylaxis using the present methods and compositions. A nonlimiting exemplary list of these diseases and conditions includes, but is not limited to, breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, ma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head—neck cancer, glioma, glioblastoma, liver cancer, r cancer, all cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small—cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, h carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, geal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, al hyperplasia, leukemia, acute lymphocytic leukemia, c lymphocytic leukemia (CLL) including B—CLL, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell ia, neuroblastoma, sarcoma such as liposarcoma malignant s histiocytoma, osteosarcoma, Ewing’s sarcoma, leiomyosarcoma, and rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft— tissue sarcomas such as , and malignant Schwannoma, osteogenic sarcoma, primary macroglobulinemia, and blastoma, and the like, T and B cell mediated autoimmune es; inflammatory diseases; infections; hyperproliferative diseases; AIDS; degenerative conditions, vascular diseases, and the like. In one embodiment, the cancer cells being treated are metastatic. In another embodiment, the cancer cells being treated are resistant to other anticancer agents.

The compounds, compositions, and s herein are used to treat s that express functional or Wild type p53 or p53—related ns. In one embodiment, the compounds, itions, and methods provided herein are used to treat cancers that express elevated levels of MDM2 or MDMZ-related proteins.

The nds, compositions, and methods herein can be used to treat a patient having a sarcoma, including, for example, liposarcoma, malignant fibrous histiocytoma, _27_ osteosarcoma, and rhabdomyosarcoma. In another ment, the compounds, compositions, and methods ed herein can be used to treat a patient having a soft tissue tumor, ing, for example, Ewing’s sarcoma, leiomyosarcoma, lipoma, and malignant Schwannomas. In another embodiment, the nds, compositions, and methods provided herein can be used to treat a t having lung, breast, liver, or colon cancer. In another embodiment, the compounds, compositions, and methods provided herein can be used to treat a patient having B— cell chronic lymphocytic leukemia and acute myeloid leukemia.

The compounds, compositions, and methods provided here also can be used to treat a patient having melanoma, lung cancer, sarcoma, colon cancer, prostate cancer, choriocarcinoma, breast cancer, blastoma, stomach carcinoma, acute myeloid leukemia, lymphoma, multiple myeloma, or leukemia.

The compounds, compositions, and methods provided here further can be used to treat a patient having liposarcoma or melanoma.

Infections suitable for treatment using the compounds, compositions, and methods herein e, but are not limited to, infections caused by viruses, bacteria, fungi, mycoplasma, prions, and the like.

The t compounds of structural formula (I), or a pharmaceutical composition comprising a compound of structural formula (I), are useful in ng a hyperproliferative disease such as cancer.

The methods ed for administering an effective amount of a compound of structural formula (I) in combination with at least one second therapeutic agent (including, but not limited to, chemotherapeutic antineoplastics, apoptosis—modulating agents, crobials, antivirals, antifungals, and anti—inflammatory agents) and/or therapeutic technique (e.g., surgical intervention and/or radiotherapies). In preferred embodiments, the second therapeutic agent(s) is an anticancer agent.

A number of second suitable therapeutic or ncer agents are contemplated for use in the present methods. Indeed, the s ed herein can include but are not limited to, administration of numerous therapeutic agents such as: agents that induce apoptosis; polynucleotides (e.g., anti—sense, ribozymes, siRNA); polypeptides (e.g., s and antibodies); biological mimetics (e.g., gossypol or BH3 mimetics); agents that bind (e.g., —28- oligomerize or complex) with a Bcl—2 family protein such as Bax; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., dies conjugated with anticancer drugs, toxins, defensins), toxins; uclides; biological se modifiers (e.g., interferons (e.g., ) and interleukins (e.g., IL—2)); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all-trans—retinoic acid); gene therapy reagents (e.g., antisense therapy ts and nucleotides); tumor vaccines; angiogenesis inhibitors; proteosome inhibitors: NF-KB modulators; anti—CDK compounds; HDAC inhibitors; and the like. Numerous other examples of therapeutic agents, such as chemotherapeutic compounds and anticancer therapies suitable for co—administration with the disclosed nds, are known to those skilled in the art.

Anticancer agents comprise agents that induce or stimulate sis. Agents that induce or stimulate apoptosis include, for example, agents that interact with or modify DNA, such as by intercalating, cross—linking, alkylating, or otherwise ng or chemically modifying DNA. Agents that induce apoptosis include, but are not limited to, radiation (e.g., X— rays, gamma rays, UV); tumor necrosis factor (TNF)-re1ated factors (e.g., TNF family receptor proteins, TNF family ligands, TRAIL, antibodies to TRAIL-R1 or TRAIL—R2); kinase inhibitors (e.g., epidermal growth factor receptor (EGFR) kinase inhibitor. Additional ncer agents include: ar growth factor receptor (VGFR) kinase inhibitor, fibroblast growth factor or (FGFR) kinase inhibitor, platelet—derived growth factor receptor (PDGFR) kinase inhibitor, and Bcr—Abl kinase inhibitors (such as GLEEVEC)); antisense molecules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, and AVASTIN); anti—estrogens (e.g., raloxifene and tamoxifen); anti-androgens (e.g., flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole, and corticosteroids); cyclooxygenase 2 (COX—2) inhibitors (e.g., celecoxib, meloxicam, NS-398, and non-steroidal anti—inflammatory drugs (NSAIDs)); anti—inflammatory drugs (e.g., butazolidin, DECADRON, DELTASONE, dexamethasone, dexamethasone intensol, , HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, nbutazone, PEDIAPRED, butazone, PLAQUENIL, prednisolone, prednisone, E, and TANDEARIL); and cancer chemotherapeutic drugs (6. g., irinotecan (CAMPTOSAR), CPT-l 1, fludarabine (FLUDARA), azine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP—l6, cisplatin, carboplatin, oxaliplatin, S—FU, doxorubicin, _29_ gemcitabine, omib, gefitinib, bevacizumab, TAXOTERE or TAXOL); cellular signaling molecules; ceramides and cytokines; staurosporine, and the like.

The itions and methods herein include one or more compounds of structural formula (I) and at least one antihyperproliferative or ncer agent, e.g., alkylating agents, tabolites, and natural products (e.g., herbs and other plant and/or animal derived compounds). ting agents suitable for use in the present compositions and methods e, but are not limited to: l) nitrogen mustards (e.g., mechlorethamine, hosphamide, ifosfamide, melphalan (L—sarcolysin); and chlorambucil); 2) ethylenimines and methylmelamines (e.g., hexamethylmelamine and thiotepa); 3) alkyl sulfonates (e.g., busulfan); 4) nitrosoureas (e.g., carmustine (BCNU); lomustine (CCNU); semustine l-CCNU); and streptozocin (streptozotocin)); and 5) triazenes (e.g., dacarbazine (DTIC; dimethyltriazenoimid— azolecarboxamide).

Antimetabolites suitable for use in the present compositions and methods e, but are not limited to: l) folic acid analogs (e.g., methotrexate (amethopterin)); 2) pyrimidine analogs (e.g., fluorouracil (S—fluorouracil; S—FU), floxuridine (fluorode—oxyuridine; FudR), and bine (cytosine arabinoside)); and 3) purine analogs (e.g., mercaptopurine (6— topurine; 6—MP), thioguanine (6—thioguanine; TG), and pentostatin (2’— deoxycoformycin)).

Chemotherapeutic agents suitable for use in the present compositions and methods include, but are not limited to: l) vinca alkaloids (e.g., Vinblastine (VLB), Vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; mycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g., L—asparaginase); 5) biological response modifiers (e.g., interferon—alfa); 6) platinum coordinating complexes (e.g., cisplatin DP) and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas (e.g., hydroxyurea); 9) methylhydrazine tives (e.g., procarbazine (N—methylhydrazine; MIH)); ) adrenocortical suppressants (e.g., ne (o,p’—DDD) and aminoglutethimide); ll) adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13) estrogens (e.g., diethylstilbestrol and ethinyl estradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g., testosterone _30_ propionate and fluoxymesterone); 16) antiandrogens (e.g., flutamide): and 17) gonadotropin— releasing hormone analogs (e.g., leuprolide).

Any anticancer agent routinely used in a cancer therapy context finds use in the compositions and methods of the present invention. Table 1 provides a list of exemplary antineoplastic agents. Those skilled in the art appreciate that the "product labels" ed on all US. approved chemotherapeutics describe approved indications, dosing information, toxicity data, and the like, for the exemplary agents.

Table l Aldesleukin (des-alanyl- l , serine- 125 human interleukin—2) Alemtuzumab Campath (IgGlK anti CD52 antibody) Alitretinoin Panretin (9-cis-retinoic acid) Allopurinol Zyloprim (1,5-dihydro-4 H olo[3,4-d]pyrimidin—4-one monosodium salt) Altretamine Hexalen (N,N,N',N',N",N",- thyl-l,3,5-triazine-2, 4, 6-triamine) Amifostine Ethyol (ethanethiol, 2-[(3-aminopropyl)amino]—, dihydrogen phosphate (ester)) Anastrozole Arimidex (l,3-Benzenediacetonitrile, a, a, a', a'-tetramethyl( l H- l riazol- l -ylmethyl)) Arsenic de Trisenox Asparaginase Elspar (L-asparagine amidohydrolase, type EC-2) BCG Live (lyophilized ation of an attenuated strain of Mycobacterium bovis (Bacillus TICE BCG Calmette-Gukin [BCG], substrain al) bexarotene capsules Targretin (4-[l-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethylnapthalenyl) l] benzoic acid) bexarotene gel Targretin Bleomycin (cytotoxic glycopeptide antibiotics produced by Streptomyces illus; bleomycin A2 ane and bleomycin B2) _31_ oxy-5 -fluoro-N-[(pentyloxy)carbonyl]—cytidine) Carboplatin Paraplatin (platinum, diammine [1,1-cyclobutanedicarboxylato(2-)-0, O']—,(SP2)) Carmustine BCNU, BiCNU (1 , 3 -bis(2-chloroethyl)-1 sourea) Carmustine with Polifeprosan 20 Implant Gliadel Wafer Celecoxib (as 4-[5—(4-methylphenyl)—3- (trifluoromethyl)-1H-pyrazolyl] Celebrex benzenesulfonamide) Chlorambucil (4- [bis(2chlorethyl)amino]benzenebutanoic acid) Cisplatin (PtCleéNz) Cladribine Leustatin, 2-CdA oro-2'-deoxy-b-D—adenosine) Cyclophosphamide Cytoxan, Neosar (2-[bis(2-ch10roethy1)amino] tetrahydro-ZH-13,2-0xazaphosphorine 2-oxide monohydrate) Cytarabine Cytosar—U (1 -b-D-Arabinofuranosylcytosine, C9H13N305) cytarabine mal DepoCyt Dacarbazine DTIC—Dome (5-(3,3—dimethyl-l-triazeno)-imidazolecarboxamide (DTIC)) Dactinomycin, actinomycin D (actinomycin produced by Streptomyces parvullus, C62H86N12016) Cosmegen Darbepoetin alfa Aranesp (recombinant peptide) daunombicin liposomal ((8 S -cis)-8 -acetyl[(3 -2,3,6-tride0xy-a-L-lyx0-hex0pyranosyl)oxy]-7,8,9,10- DanuoXome tetrahydro-6,8, l l-trihydroxy-l-methoxy-5,12-naphthacenedione hloride) Daunorubicin HCl, daunomycin ((1 S ,3 S )—3-Acetyl-1,2,3,4,6,1 1—hexahydro-3,5,12-trihydroxy—10-methoxy-6,1 l-dioxo- l — Cerubidine naphthacenyl 3 -amino-2,3,6-tride0xy-(alpha)—L- lyxo —heX0pyranoside hydrochloride) Denileukin diftitox Ontak (recombinant peptide) Dexrazoxane Zinecard ((S)-4,4'—(1-methyl-1,2-ethanediyl)bis-2,6-piperazinedi0ne) ((2R,3S)-N-carboxy-3 -phenylisoserine, N-tert-butyl ester, 13-ester with 5bepoxy- 12a,4,7b,10b,13a—hexahydroxytax- 11-enone 4-acetate 2-benzoate, trihydrate) Doxorubicin HCl (8 S ,1OS)[(3—arnino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy] -8 -glycolyl-7, 8 ,9,10- Adriamycin, Rubex tetrahydro-6,8, l 1- trihydroxymethoxy-5, 12-naphthacenedione hydrochloride) nycin PFS doxorubicm. .

Intravenous IIIJCCUOII. . . doxorubicin liposomal drornostanolone propionate tanolone (17b-Hydroxy-2a-n1ethyl—5a-androstanone propionate) dromostanolone propionate Masterone injection Elliott's B Solution Elliott's B Solution Epirubicin is)[(3-an1ino-2,3,6-trideoxy-a-L-arabino- hexopyranosyl)oxy]—7,8,9, 10— tetrahydro-6,8, 1 l-trihydroxy-S- (hydroxyacety1)methoxy-5, 12-naphthacenedione hydrochloride) Epoetin alfa (recombinant peptide) ustine (estra-l,3,5(10)-triene-3,l7-diol(l7(beta))-, 3-[bis(2-chloroethyl)carbarnate] 17- (dihydrogen phosphate), disodium salt, monohydrate, or estradiol 3-[bis(2— chloroethyl)carban1ate] hydrogen phosphate), disodium salt, monohydrate) Etoposide phosphate methylepipodophyllotoxin 9-[4,6-0—(R)-ethylidene-(beta)-D-glucopyranoside], 4'- Etopophos (dihydrogen phosphate» etoposide, VP-16 Vepesid (4'-demethylepipodophyllotoxin (R)—ethylidene-(beta)—D-glucopyranoside]) Exemestane Aromasin (6-methylenand1'osta-1,4-diene-3 , 17-dione) Filgrastim Neupogen (r-metHuG-CSF) floxuridine (intraarterial) FUDR (2'—deoxy-5 -fluorouridine) Fludarabine (fluorinated nucleotide analog of the antiviral agent Vidarabine, 9-b -D- Fludara arabinofuranosyladenine (ara-A)) Fluorouracil, 5-FU (5-fluoro-2,4(1H,3H)-pyrimidinedione) 2015/026098 Fulvestrant (7-alpha—[9-(4,4,5,5,5-penta fluoropentylsulphinyl) nonyl]estra—1,3,5-(10)- triene—3, 17- Faslodex beta-diol) Gemcitabine Gemzar (2'-deoxy-2', 2'-difluorocytidine monohydrochloride (b-isomer)) Gemtuzumab Ozogamicin Mylotarg (anti-CD33 hP67.6) lin acetate Zoladex Implant Ibritumomab Tiuxetan (immunoconjugate resulting from a thiourea covalent bond between the monoclonal antibody Ibritumomab and the linker-chelator tiuxetan [N-[2-bis(carboxymethyl)amino] Zevalin (p-isothiocyanatophenyl)- propyl] —[N- (carboxymethyl)amino] -2—(methyl) — ethyl] e) Idarubicin (5, hthacenedione, 9-acetyl[(3-amino-2,3,6-trideoxy-(alpha)-L- lyxo - Idamycin hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,9,1 1-trihydroxyhydrochloride, (7S- cis )) Ifosfamide (3-(2-chloroethy1)—2-[(2-chloroethyl)amino]tetrahydro-ZH-1,3,2-oxazaphosphorine 2- IFEX oxide) ib Mesilate (4-[(4-Methylpiperazinyl)methyl]-N-[4-methyl-3 - [ [4-(3 inyl)—2— Gleevec dinyl]amino] -phenyl]benzamide methanesulfonate) Interferon alfa-2a (recombinant peptide) Interferon alfa-2b Intron A (Lyophllized (recombinant peptide) Betaseron) Irinotecan HCl ((4S)-4,11-diethylhydroxy[(4— piperi-dinopiperidino)carbonyloxy]—1H-pyrano[3', 4': Camptosar 6,7] zino[l,2-b] quinoline-3,l4(4H, 12H) dione hydrochloride trihydrate) Letrozole Femara (4,4'-(1H-1,2,4 —Triazol—1—ylmethylene) dibenzonitrile) Leucovorin Wellcovorin, (L-Glutamic acid, N[4[[(2amino—5-formyl1,4,5,6,7,8 hexahydro4oxo6— Leucovorin pteridinyl)methyl]amino]benzoyl], calcium salt (1:1)) Levamisole HCl ((-)—( S)—2,3,5, 6—tetrahydrophenylimidazo [2,1-b] thiazole monohydrochloride Ergamisol CllHnNZSOHCl) Lomustine CeeNU (1 -(2-chlor0-ethyl)—3 -cyclohexyl- 1 —nitrosourea) Meclorethamine, nitrogen mustard Mustargen (2-chloro-N-(2-chlor0ethyl)-N-rnethylethanamine hloride) Megestrol acetate Megace 170.( oxy)— 6- methylpregna- 4,6- diene- 3,20- dione lan, L-PAM (4- -chlor0ethyl) amino] -L-phenylalanine) Mercaptopurine, 6-MP (1,7-dihydro-6 H -purinethione monohydrate) Mesna Mesnex (sodium 2-mercaptoethane sulfonate).

Methotrexate Methotrexate (N-[4-[[(2,4-diaminopteridinyl)methyl]methylamino]benzoyl]-L-glutan1ic acid) Methoxsalen Uvadex (9-methoxy-7H-furo[3,2-g] [ 1]—benzopyranone) Mitomycin C Mutamycin mitomycin C Mitozytrex Mitotane Lysodren (1 , 1-dichloro(0-chlorophenyl)(p-chlorophenyl) ethane) Mitoxantrone (1 ,4-dihydroxy-5,8 -bis [ [2— [(2-hydroxyethyl)an1ino] ethyl]amino] -9, 1 O-anthracenedione Novantrone dihydrochloride) Nandrolone phenpropionate Durabolin-SO Nofetumomab Verluma Oprelvekin Neumega (IL-11) Oxaliplatin Eloxatin (cis-[(1R,2R)-1,2—cyclohexanediamine-N,N’] [oxalato(2-)-0,0’] platinum) Paclitaxel (5J3, xy-1,2a, 4,78, 108, 13a—hexahydroxytaXenone 4,10-diacetate 2— benzoate 13-ester with (2R, 3 S)- N-benzoyl—3-phenylisoserine) Pamidronate (phosphonic acid (3-arninohydr0xypropylidene) bis-, disodium salt, ydrate, (APD)) Pegademase Adagen (Pegademase methoxypolyethylene glycol succinimidyl) 11 adenosine deaminase) Bovine) Pegaspargase Oncaspar 2015/026098 (monomethoxypolyethylene glycol succinimidyl L-asparaginase) — Pegfilgrastim (covalent conjugate of recombinant methionyl human G-CSF (Filgrastim) and Neulasta monomethoxypolyethylene glycol) Plicamycin, mycin Mithracin (antibiotic produced by Streptomyces plicatus) Procarbazine Matulane (N-isopropyl-p—(2-methy]hydrazino)-p-toluamide monohydrochloride) Quinacrine Atabrine' oro( l —methyl—4-diethyl—amine) mino—2-methoxyacridine) Rasburicase (recombinant peptide) Rituximab (recombinant anti-CD20 antibody) mostim (recombinant peptide) ozocin (streptozocin 2 —deoxy [[(methylnitrosoamino)carbonyl]amino] - a(and b ) - D - Zanosar glucopyranose and 220 mg citric acid anhydrous) Talc Sclerosol (Mg3Si4010 (OH)2) Tamoxifen ((Z)2-[4-(1,2-diphenylbutenyl) phenoxy] —N, N-dimethylethanamine 2-hydroxy-1,2,3- Nolvadex propanetricarboxylate ( 1 : 1)) Temozolomide (3,4-dihydromethyloxoimidazo[5,1-d]—as-tetrazinecarboxamide) teniposide, VM—26 (4'-demethylepipodophyllotoxin 9-[4,6(R) thenylidene-(beta)-D-glucopyranoside]) Testolactone (13-hydroxyoxo-l3,17—secoandrosta-1,4-dienoic acid [dgr ]-lactone) anine, 6—TG Thioguanine (2-amino-1,7-dihydro-6 H - purine—6-thione) Thiotepa Thioplex (Aziridine, 1,1',1"-phosphinothioylidynetris—, or Tris (l-aziridinyl) phosphine sulfide) Topotecan HCl Hycamtin ((S)[(dimethylamino) methyl] ethyl-4,9-dihydroxy-1H-pyrano[3', 4': 6,7] indolizino [1,2-b] quinoline—3,14-(4H,12H)-di0ne monohydrochloride) Toremifene (2-(p-[(Z)chloro-1,2-diphenyl-1—butenyl]—phenoxy)—N,N-dimethylethylamine citrate (1:1)) Tositumomab, I 131 momab (recombinant murine immunotherapeutic monoclonal Inga lambda anti—CD20 antibody (1 Bexxar 131 is a radioimmunotherapeutic antibody)) Trastuzumab Herceptin (recombinant monoclonal IgG1 kappa ER2 antibody) Tretinoin, ATRA Vesanoid (all-trans retinoic acid) Uracil Mustard Uracil Mustard Capsules Valrubicin, N-trifluoroacetyladriamycinva1erate ((2S-cis) [1,2,3,4,6,11-hexahydro-2,5,12-trihydroxy-7 methoxy-6,11-dioxo-[[4 2,3,6- trideoxy [(trifluoroacetyl)-amino-0.—L-lyxo-hexopyranosyl]oxyl] naphthacenyl] oxoethyl pentanoate) Vinblastine, Leurocristine (C46H56N4010°stO4) Vincristine Oncovin (C46H56N4010°HZSO4) Vinorelbine (3' ,4'-didehydro-4'—deoxy-C'-norVincaleul<oblastine [R-(R*,R*)-2,3- ine dihydroxybutanedioate (1 :2)(salt)]) Zoledronate, Zoledronic acid ((1 xyimidazol- 1 —yl-phosphonoethyl) phosphonic acid monohydrate) Anticancer agents r include nds which have been identified to have anticancer activity. es include, but are not limited to, 3—AP, l2—O—tetradecanoylphorbol— 13-acetate, 17AAG, 852A, ABI—007, AER—217620, ABT-751, ADI—PEG 20, AE-941, AG— 013736, O, alanosine, AMG 706, antibody G250, antineoplastons, AP23573, apaziquone, APC8015, atiprimod, ATN—16l, atrasenten, azacitidine, BB—10901, BCX—l777, bevacizumab, BGOOOOl, tamide, BMS 247550, bortezomib, bryostatin—l, buserelin, calcitriol, CCI—779, CDB—29l4, cefixime, cetuximab, CG0070, cilengitide, clofarabine, combretastatin A4 phosphate, CP-675,206, ,714, CpG 7909, curcumin, decitabine, _37_ DENSPM, doxercalciferol, E7070, E7389, ecteinascidin 743, efaproxiral, eflomithine, EKB—569, enzastaurin, erlotinib, exisulind, fenretinide, flavopiridol, fludarabine, flutamide, fotemustine, FR901228, G17DT, galiximab, gefitinib, genistein, glufosfamide, GTI—2040, histrelin, HKI—272, homoharringtonine, HSPPC—96, hul4.18—interleukin—2 fusion protein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukin—12, IPI—504, irofulven, ixabepilone, nib, lenalidomide, lestaurtinib, leuprolide, LMB-9 immunotoxin, mib, luniliximab, mafosfamide, MB07133, O, MLN2704, monoclonal antibody 3F8, monoclonal antibody J591, motexafin, MS— 275, Cl—ILZ, mide, amptothecin, nolatrexed dihydrochloride, nolvadex, NS—9, O6—benzylguanine, oblimersen sodium, 15, oregovomab, OSI—774, panitumumab, paraplatin, 5901, pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone, PS-341, PSC 833, PXD101, pyrazoloacridine, R115777, RAD001, ranpirnase, rebeccamycin analogue, rhuAngiostatin protein, rhuMab 2C4, rosiglitazone, rubitecan, S—l, S— 8184, satraplatin, SB—, 15992, SGN—OOlO, , sorafenib, SR31747A, ST1571, SU011248, suberoylanilide hydroxamic acid, suramin, talabostat, talampanel, tariquidar, temsirolimus, TGFa—PE38 immunotoxin, thalidomide, thymalfasin, tipifamib, zamine, TLK286, trabectedin, trimetrexate glucuronate, TroVax, UCN—l, valproic acid, vinflunine, VNP40101M, volociximab, vorinostat, VX—680, ZD1839, ZD6474, zileuton, and zosuquidar trihydrochloride.

For a more detailed description of anticancer agents and other therapeutic agents, those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician’s Desk Reference and to n and Gilman s aceutical Basis of Therapeutics" tenth n, Eds. n et al., 2002.

The methods provided herein comprise administering one or more compounds of structural formula (I) in combination with radiation therapy. The methods provided herein are not limited by the types, amounts, or delivery and administration systems used to deliver the therapeutic dose of radiation to an animal. For example, the mammal can receive photon herapy, particle beam radiation therapy, other types of radiotherapies, and combinations thereof. In one embodiment, the radiation is delivered to the animal using a linear accelerator.

In another embodiment, the radiation is red using a gamma knife.

The source of radiation can be external or internal to the mammal. External radiation therapy is most common and involves directing a beam of high—energy ion to a tumor site through the skin using, for instance, a linear accelerator. While the beam of radiation is localized _38_ to the tumor site, it is nearly impossible to avoid exposure of normal, healthy tissue. However, external radiation is usually well tolerated by . Internal radiation therapy involves ting a radiation—emitting source, such as beads, wires, s, capsules, particles, and the like, inside the body at or near the tumor site including the use of delivery systems that ically target cancer cells (6.g. , using particles attached to cancer cell binding ligands).

Such implants can be removed following treatment, or left in the body inactive. Types of internal ion therapy include, but are not limited to, brachytherapy, interstitial ation, intracavity irradiation, radioimmunotherapy, and the like.

The mammal optionally can receive radiosensitizers (e.g. , metronidazole, misonidazole, intra—arterial Budr, intravenous iododeoxyuridine (ludR), nitroimidazole, 5— substituted—4—nitroimidazoles, 2H—isoindolediones, [[(2—bromoethyl)-amino]methyl]—nitro—1H— imidazole— 1—ethanol, nitroaniline derivatives, DNA—affinic hypoxia selective cytotoxins, halogenated DNA ligand, 1,2,4 benzotriazine oxides, 2—nitroimidazole derivatives, fluorinecontaining nitroazole derivatives, benzamide, nicotinamide, acridine—intercalator, tretrazole derivative, 3—nitro—1,2,4—triazole, 4,5—dinitroimidazole derivative, ylated texaphrins, cisplatin, mitomycin, tiripazamine, nitrosourea, mercaptopurine, methotrexate, fluorouracil, bleomycin, stine, carboplatin, epirubicin, doxorubicin, cyclophosphamide, vindesine, etoposide, axel, heat (hyperthermia), and the like), radioprotectors (e.g., cysteamine, aminoalkyl dihydrogen phosphorothioates, amifostine (WR 2721), lL—l, lL—6, and the like).

Radiosensitizers enhance the killing of tumor cells. Radioprotectors protect healthy tissue from the harmful effects of radiation.

Any type of radiation can be administered to the , is long as the dose of radiation is ted by the mammal without unacceptable negative side—effects. Suitable types of radiotherapy include, for example, ionizing (electromagnetic) radiotherapy (e.g., X—rays or gamma rays) or particle beam radiation therapy (e.g., high linear energy radiation). Ionizing radiation is defined as radiation comprising particles or photons that have ient energy to produce ionization, i.e., gain or loss of electrons (as described in, for example, U.S. 5,770,581 incorporated herein by reference in its ty). The effects of radiation can be at least partially controlled by the ian. In one embodiment, the dose of radiation is fractionated for maximal target cell exposure and d toxicity. _39_ 2015/026098 In one embodiment, the total dose of ion administered to an animal is about .01 Gray (Gy) to about 100 Gy. In another embodiment, about 10 Gy to about 65 Gy (e.g., about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60 Gy) are administered over the course of treatment. While in some embodiments a complete dose of radiation can be administered over the course of one day, the total dose is ideally fractionated and administered over several days. Desirably, radiotherapy is administered over the course of at least about 3 days, e.g., at least 5, 7, 10, 14, 17,21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about l-8 weeks). Accordingly, a daily dose of radiation will comprise approximately 1—5 Gy (e.g., about 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy, 2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or 4.5 Gy), or l—2 Gy (e.g., 1.5—2 Gy). The daily dose of radiation should be sufficient to induce destruction of the targeted cells. If stretched over a period, in one embodiment, radiation is not administered every day, thereby allowing the mammal to rest and the effects of the therapy to be ed. For example, radiation desirably is stered on 5 consecutive days, and not stered on 2 days, for each week of ent, thereby allowing 2 days of rest per week.

However, ion can be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5 days/week, 6 days/week, or all 7 days/week, depending on the responsiveness of the mammal and any potential side effects. Radiation y can be initiated at any time in the therapeutic period. In one embodiment, radiation is initiated in week 1 or week 2, and is administered for the remaining duration of the therapeutic period. For example, radiation is administered in weeks 1—6 or in weeks 2—6 of a eutic period comprising 6 weeks for treating, for instance, a solid tumor. Alternatively, radiation is administered in weeks l—5 or weeks 2—5 of a therapeutic period comprising 5 weeks. These exemplary radiotherapy administration schedules are not intended, however, to limit the methods provided herein.

Antimicrobial eutic agents may also be used as eutic agents in combination with the compounds of structural formula (I). Any agent that can kill, inhibit, or otherwise attenuate the function of ial organisms may be used, as well as any agent contemplated to have such activities. Antimicrobial agents include, but are not limited to, natural and synthetic antibiotics, antibodies, inhibitory proteins (e.g., defensins), antisense nucleic acids, membrane disruptive agents and the like, used alone or in combination. Indeed, any type of antibiotic may be used including, but not limited to, antibacterial agents, antiviral agents, antifungal agents, and the like. _40_ 2015/026098 In the present methods, one or more compounds of ural formula (I) are administered to a mammal in need f. In another embodiment of the methods, one or more compound and one or more second therapeutic agents, i.e., as anticancer agents, are administered to a mammal in need thereof under one or more of the following ions: for example, at different periodicities, at different durations, at different concentrations, by different administration routes. In one embodiment, the compound of structural formula (I) is administered prior to the therapeutic or anticancer agent, e.g., 0.5, l, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks prior to the administration of the second therapeutic or anticancer agent. In another ment, the compound of structural formula (I) is administered after the second therapeutic or anticancer agent, e.g., 0.5, l, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks after the stration of the anticancer agent. In another embodiment, the compound of structural formula (I) and the second therapeutic or anticancer agent are administered concurrently, but on ent schedules, e.g., the nd is administered daily while the second therapeutic or anticancer agent is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In another embodiment, a present compound is administered once a week and the second therapeutic or anticancer agent is administered daily, once a week, once every two weeks, once every three weeks, or once every four weeks.

In one embodiment, a method of treating, or ameliorating cancer in a patient comprises a pulsatile administration of a therapeutically effective amount of a compound of ural formula (I) to the patient.

Toxicity and therapeutic efficacy of the nds of structural formula (I) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the maximum tolerated dose (MTD) of a compound, which defines as the highest dose that causes no ty in animals. The dose ratio between the maximum tolerated dose and therapeutic effects (e.g. inhibiting of tumor growth) is the therapeutic index. The dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized. Determination of a eutically effective amount is well within the capability of those skilled in the art, especially in light of the ed disclosure provided herein.

A therapeutically ive amount of a compound of structural formula (I) required for use in therapy varies with the nature of the condition being treated, the length of time that _41_ 2015/026098 activity is desired, and the age and the condition of the patient, and ultimately is determined by the ant physician. Dosage amounts and intervals can be ed individually to provide plasma levels of the MDM2 inhibitor that are sufficient to in the desired therapeutic effects. The desired dose conveniently can be administered in a single dose, or as multiple doses administered at appropriate intervals, for example as one, two, three, four or more subdoses per day. Multiple doses often are desired, or required. For example, a present MDM2 inhibitor can be administered at a ncy of: four doses delivered as one dose per day at ay intervals (q4d x 4); four doses delivered as one dose per day at three—day intervals (q3d x 4); one dose delivered per day at five—day intervals (qd x 5); one dose per week for three weeks (qwk3); five daily doses, with two days rest, and another five daily doses (5/2/5); or, any dose regimen determined to be appropriate for the circumstance.

The pharmaceutical compositions provided herein comprise one or more compounds of structural formula (I) in an amount effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typically, the compounds may be administered to mammals, e.g. humans, orally at a dose of 0.0025 to 50 mg/kg, or an equivalent amount of the pharmaceutically acceptable salt thereof, per day of the body weight of the mammal being treated for disorders responsive to induction of apoptosis. In one embodiment, about 0.01 to about 25 mg/kg is orally administered to treat or ameliorate disorders. For intramuscular ion, the dose is generally about one—half of the oral dose. For example, a suitable intramuscular dose is about 0.0025 to about 25 mg/kg, or from about 0.01 to about 5 mg/kg.

The unit oral dose can comprise from about 1 to about 2000 mg, for example, about 100 to about 1000 mg of a present compound. The unit dose can be administered one or more times daily as one or more tablets or capsules each containing from about 5 to about 500 mg, conveniently about 50 to 250 mg of the compound or its salts.

In a topical formulation, the compound can be present at a concentration of about 0.01 to 100 mg per gram of carrier. In a one ment, the compound is present at a concentration of about 5—100 mg/ml.

In on to administering the compound as a heat chemical, compounds of ural a (I) can be stered as a component of a pharmaceutical preparation or composition.

The pharmaceutical ition comprises one or more pharmaceutically acceptable carriers, _42_ excipients, and/or auxiliaries. The one or more carriers, excipients, and auxiliaries facilitate processing of a compound of structural formula (I) into a preparation which can be used pharmaceutically. The compositions, particularly compositions that can be administered orally or topically that can be used for one type of administration, such as tablets, dragees, slow e lozenges and capsules, mouth rinses and mouth washes, gels, liquid suspensions, hair rinses, hair gels, shampoos, and also preparations that can be administered rectally, such as suppositories, as well as suitable solutions for stration by intravenous infusion, injection, topically or orally, contain from about 0.01 to 99 percent, or from about 0.25 to 75 percent, of a nd of ural formula (I), together with the one or more carriers, excipients, and/or aries.

The pharmaceutical compositions provided herein can be administered to any patient which may experience the beneficial effects of compounds of structural formula (I). Foremost among such patients are mammals, e.g., humans, although the methods and compositions provided herein are not intended to be so limited. Other patients e veterinary animals (cows, sheep, pigs, horses, dogs, cats and the like).

Compounds of structural formula (I) and pharmaceutical compositions thereof can be administered by any means that achieve their intended purpose. A compound of structural formula (I) can be administered by any suitable route, for e by oral, buccal, inhalation, sublingual, rectal, vaginal, intracisternal or intrathecal through lumbar re, transurethral, nasal, percutaneous, i.e., transdermal, or parenteral (including intravenous, intramuscular, aneous, intracoronary, intradermal, intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a particular site) administration. Parenteral administration can be lished using a needle and syringe or using a high pressure technique. Alternatively, or concurrently, administration can be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the ent, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

The present pharmaceutical compositions and preparations are manufactured by conventional mixing, granulating, dragee—making, dissolving, or lyophilizing processes.

Pharmaceutical compositions for oral use can be obtained by ing a present nd with solid excipients, optionally ng the ing mixture and processing the mixture of _43_ granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee COI'CS.

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

Other pharmaceutical preparations that can be used orally include push-fit es made of gelatin, as well as soft, sealed capsules made of gelatin and a cizer such as glycerol or sorbitol. The push—fit capsules can contain the active compounds in the form of es which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin. In addition, stabilizers may be added. le pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of one or more of the active compounds with a suppository base. Suitable suppository bases are, for example, natural or synthetic cerides, or paraffin arbons. In on, it is also possible to use gelatin rectal capsules which _44_ 2015/026098 consist of a combination of the active compounds with a base. Possible base materials include, for example, liquid triglycerides, hylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water—soluble form, for example, water-soluble salts and alkaline solutions.

In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides or polyethylene glycol—400. Aqueous injection suspensions may contain substances which increase the Viscosity of the sion including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. ally, the suspension may also contain stabilizers.

Topical compositions are formulated in one embodiment as oils, creams, lotions, ointments, and the like by choice of appropriate carriers. Suitable carriers include vegetable or mineral oils, white petrolatum (white soft paraffin), branched chain fats or oils, animal fats and high molecular weight alcohol (greater than C12). The carriers can be those in which the active ingredient is soluble. Emulsifiers, stabilizers, humectants and antioxidants also can be included as well as agents imparting color or fragrance, if desired. Additionally, ermal penetration enhancers can be employed in these topical formulations. Examples of such ers can be found in US. Pat. Nos. 3,989,816 and 4,444,762, are orated herein by reference. nts may be formulated by mixing a solution of the active ingredient in a vegetable oil such as almond oil with warm soft in and allowing the mixture to cool. A typical example of such an ointment is one which includes about 30% almond oil and about 70% white soft paraffin by weight. Lotions conveniently are prepared by ving the active ingredient, in a suitable high molecular weight alcohol such as propylene glycol or polyethylene glycol.

The following examples are illustrative, but not limiting, of the compounds, itions, and methods provided herein. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapy and which are obvious to those skilled in the art are within the spirit and scope of the methods, compounds, and compositions provided herein.

Further ed are kits comprising a compound of structural a (I) and, optionally, a second therapeutic agent useful in the treatment of diseases and conditions wherein _45_ inhibition of MDM2 and MDM2—related ns provides a benefit, packaged tely or together, and an insert having instructions for using these active agents.

In many embodiments, a compound of structural formula (I) is stered in conjunction with a second therapeutic agent useful in the treatment of a disease or condition wherein tion of MDM2 and MDM2—related proteins provides a benefit. The second therapeutic agent is different from the compound of structural formula (I). A compound of structural formula (I) and the second therapeutic agent can be administered simultaneously or sequentially to achieve the desired effect. In addition, the compound of structural a (I) and second eutic agent can be administered from a single composition or two separate compositions.

The second therapeutic agent is administered in an amount to provide its desired therapeutic effect. The effective dosage range for each second eutic agent is known in the art, and the second eutic agent is administered to an individual in need thereof within such established ranges.

A compound of structural formula (I) and the second therapeutic agent can be administered together as a single—unit dose or separately as multi—unit doses, wherein the compound of structural formula (I) is administered before the second therapeutic agent or vice versa. One or more dose of the compound of structural formula (I) and/or one or more dose of the second therapeutic agent can be administered. The nds of structural formula (I) therefore can be used in conjunction with one or more second therapeutic agents, for example, but not limited to, anticancer agents.

As an additional embodiment, the present invention includes kits which comprise one or more nds or compositions packaged in a manner that facilitates their use to practice methods of the invention. In one simple embodiment, the kit includes a compound or composition described herein as useful for practice of a method (e.g., a composition comprising a compound of structural a (I) and an al second therapeutic agent), packaged in a container, such as a sealed bottle or vessel, with a label affixed to the container or included in the kit that describes use of the compound or ition to practice the method of the invention.

Preferably, the compound or composition is packaged in a unit dosage form. The kit further can include a device suitable for administering the composition according to the intended route of administration. —46- As discussed below, MDM2 inhibitors possessed properties that hindered their development as therapeutic . In accordance with an important feature of the present invention, compounds of structural formula (I) were synthesized and ted as inhibitors of MDM2 and MDM2—related proteins. For example, compounds of the present invention typically have a binding affinity (IC50) to MDM2 of less than 50 nM, less than 25 nM, less than 10 nM, and less than 5 nM.

SYNTHESIS OF COMPOUNDS Compounds of the present invention were prepared as follows. The following synthetic schemes are representative of the ons used to size nds of ural formula (1). Modifications and alternate schemes to prepare MDM2 inhibitors of the invention are readily within the capabilities of persons skilled in the art by substitution of the appropriate ts and agents in the syntheses shown below.

Solvents and reagents were obtained commercially and used without further purification. Chemical shifts (8) of NMR a are reported as 8 values (ppm) downfield relative to an internal standard, with multiplicities ed in the usual manner.

Unless otherwise stated all temperatures are in degrees Celsius.

In the synthetic methods, the examples, and throughout the specification, the abbreviations have the following meanings [Bis(dimethylamino)methylene] — lH— 1,2,3— triazolo[4,5—b]pyridinium 3—oxid hexafluorophosphate DIEA N,N—diisopropylethylamine 2015/026098 Final compounds are in trifluoroacetate salt form.

Compounds of structural formula (I) can also be prepared by asymmetric synthetic methods, as described in U.S. Patent Nos. 7,759,383 and 7,737,174 (each incorporated herein by reference), and Ding et a1., J. Am. Chem. Soc. 127: 10130-10131 (2005)). In the case of an asymmetric synthesis, compounds of structural a (I) can be separated by chiral resolution methods well known in the art, e. g., chiral column chromatography. Suitable chiral columns for use in chiral resolutions include, for example, Daicel CEL® OD—H, Daicel CHIRAKPAK® AD—H, and Regis Technologies ULMO chiral columns. Other chiral resolution methods are also possible. —48- 1.) DCM,HATU, IOH.HZO, NaOH, DIEA, 10min 21:1 THFZMeOHZHZO F 0w“ , ' 2. H2N.6 HO RT OMe CI DMAP, overnight de No. 2 HATU (616 mg, 1.62 mmol), DIEA (0.550 mL, 3.24 mmol) were added to a suspension of acid I (500 mg, 1.08 mmol) in DCM (15 mL) and stirred. After 10 minutes, methyl 4—aminobicyclo[2.2.2]octane—l—carboxylate (396 mg, 2.16 mmol) and DMAP (132 mg, 1.08 mmol) were added to the reaction. After overnight, the solvent was removed in vacuo and the crude was purified by column chromatography to give 549 mg of intermediate II. 20 (110 mg 2.62 mmol) and sodium hydroxide (105 mg, 2.62 mmol) were added to a solution of intermediate II (549 mg, 0.873 mmol) dissolved in a mixture of THF (3 mL), H20 (3 mL), and MeOH (3 mL). After the hydrolysis was complete, as determined by TLC, the on was quenched with TFA (3 mL) and stirred. After 5 minutes, the solution was trated in vacuo (not to dryness) and the resulting oil was redissolved in CH3CN and H20 (1:1) and the solution was purified by ative HPLC. The purified fractions were combined, concentrated in vacuo, re—dissolved in H20, frozen and lyophilized to give de No. 2 (TFA salt) as a white powder. 1H-NMR (300MHz, CDgOD) 5 ppm 7.63 (t, J = 6.84 Hz, 1H), 7.45 (d, J = 6.76 Hz, 1H), 7.35 (t, J = 7.21 Hz, 1H), 7.18—7.04 (m, 2H), 6.77 (dd, J = 1.26 Hz, 1H), 4.68 (d, J = 10.61 Hz, 1H), 2.73-2.48 (m, 1H), .98 (m, 1H), 1.98-1.43 (m, 18H), 1.27-1.02 (m, 2H); ESI—MS m/z 614.92 (M+H)+. de No. 1 Chemical Formula: C29H28C|2FN3O4 Exact Mass: 571.14 Molecular Weight: 572.45 _49_ 2015/026098 de No. 1 was obtained using the same synthetic strategy described for de No. 2. 1H— NMR (300MHz, CD30D) 5 ppm 7.61 (t, J = 6.55 Hz, 1H), 7.49 (dd, J = 2.34, 8.20 Hz, 1H), 7.39 (t, J = 6.90 Hz, 1H), 7.15 (t, J = 8.53 Hz, 1H), 7.10 (dd, J = 1.94, , 1H), 6.78 (d, J = 1.88 Hz, 1H), 4.98 (d, J = 10.87 Hz, 1H), 4.78 (d, J = 10.92 Hz, 1H), 2.84-2.71 (m, 1H), 2.26 (s, 6H), 2.14 (d, J = 13.90 Hz, 1H), 2.02-1.67 (m, 5H), 1.60-1.38 (m, 1H), 1.31-1.10 (m, 2H); ESI—MS m/z 572.25 (M+H)+. de No. 3 Chemical Formula. C32H32CIZF3N304 Exact Mass: 649.17 Molecular Weight: 650.52 de No. 3 was obtained using the same synthetic strategy described for de No. 2. 1H— NMR (300MHz, CD30D) 5 ppm 7.71 (s, 1H), 7.63 (t, J = 6.61 Hz, 1H), 7.50 (dd, J = 2.08, 8.18 Hz, 1H), 7.36 (t, J = 7.54 Hz, 1H), 7.18-7.05 (m, 2H), 6.79 (d, J 21.83 Hz, 1H) 4.96 (d, J = 10.48 Hz, 1H), 4.71 (d, J = 10.51 Hz, 1H), 2.78 (d, J 214.25 Hz, 1H), 2.59—1.91 (m, 6H), 1.91-1.70 (m, 12H), 1.53-1.33 (m, 1H); ESI—MS rn/z 650.92 (M+H)+. _50_ de No.4 Chemical Formula: C33H37C|2FN4O5S Exact Mass: 690.18 Molecular Weight: 691.64 de No. 4: CD1 (49 mg, 0.303 mmol), DIEA (88 uL, 0.505 mmol), and DMAP (cat.) were added to a solution of de No. 2 (62 mg, 0.101 mmol) in chloroethane (10 mL) and the reaction was heated to 40°C. After 20 minutes, methanesulfonamide (96 mg, 1.01 mmol) was added and the reaction was refluxed. After overnight, the sovent was removed in vacuo and the crude was purified by prepartive HPLC to give de No. 4 (TFA salt) as a white solid. 1H-NMR (300MHz, CD3OD) éppm 7.64 (t, J = 7.23 Hz, 1H), 7.45 (dd, J = 1.93, 8.22 Hz, 1H), 7.36 (t, J = 7.23 Hz, 1H), 7.18-7.04 (m, 2H), 6.77l (d, J = 1.66 Hz, 1H), 4.69 (d, J = 10.70 Hz, 1H), 3.19 (s, 3H), 2.75-2.52 (m, 1H), 2.21-1.99 (m, 1H), 1.99-1.44 (m, 17H), 1.41-1.27 (1n, 1H), 1.27-1.03 (m, 2H); ESI—MS m/z 691.42 (M+H)+. de No.5 Chemical a: 033H36C|2FN3O5 Exact Mass: 643.20 Molecular Weight: 644.56 _51_ de No. 5 was obtained using the same tic strategy described for de No. 2. 1H-NMR (300MHz, CD3OD) 6 ppm 7.69-7.60 (m, 2H), 7.48 (, dd, J = 2.09, 8.23 Hz, 1H), 7.40 (t, J = 6.93 Hz, 1H), 7.16 (t, J = 8.05 Hz, 1H), 7.09 (dd, J = 1.91, 8.21 Hz, 1H), 6.79 (d, J = 1.87 Hz, 1H), 5.07 (d, J = 11.01 Hz, 1H), 4.72 (d, J = 11.08 Hz, 1H), 2.60 (d, J = 12.07 Hz, 1H), 2.30 (dt, J = 4.11, 13.45 Hz, 1H), .93 (m, 2H), 1.92—1.52 (m, 16H), 1.25 (s, 3H); ESI—MS m/z 644.25 (M+H)+. de No. 6 Chemical Formula: C33H36C|2FN3O5 Exact Mass: 643.20 Molecular Weight: 644.56 de No. 6 was obtained using the same tic strategy described for de No. 2. 1H—NMR (300MHz, CD3OD) 5 ppm 7.70 (s, 1H), 7.62 (t, J = 7.05 Hz, 1H), 7.52 (dd, J = 2.08, 8.21 Hz, 1H), 7.38 (t, J = 7.41 Hz, 1H), 7.15 (d, J = 7.93 Hz, 1H), 7.10 (dd, J = 1.76, 8.19 Hz, 1H), 6.79 (d, J = 1.83 Hz, 1H), 4.99 (d, J = 11.35 Hz, 1H), 4.70 (d, J: 11.00 Hz, 1H), 2.76—2.59 (m, 1H), 2.22—1.91 (m, 3H), 1.89—1.19 (m, 16H), 1.03 (s, 3H); ESI—MS m/z 644.75 (M+H)+.

ACOH, paraformaldehyde NaBH(OAc)3, overnight de No. 2 de No. 7 Paraformaldehyde (15 mg, 0.506 mmol) was added to a solution of compound de No. 2 (20 mg, 0.028 mmol) dissolved in AcOH (1 mL). After 15 s sodium triacetoxyborohydride (59 mg, 0.28 mmol) was added and after reacting overnight the reaction was quenched with saturated ammonium chloride solution and extracted with ethyl e. The ethyl acetate solvent was removed in vacuo and the resulting oil was re—dissolved in a solution of _52_ WO 61032 acetonitrile and water (1:1 with 0.1% TFA) and purified by preparative HPLC. The pure de No. 7 fractions were combined, concentrate in vacuo, re—dissolved in water (with minimum amount of acetonitrile), frozen and 1yophi1ized to give de No. 7 (TFA salt) as a white powder. 1H-NMR (300MHz, CD30D) 6 ppm 7.94 (s, 1H), 7.61-7.52 (m, 2H), 7.40 (t, J = 7.32 Hz, 1H), 7.19-7.08 (m, 2H), 6.78 (d, J = 1.56 Hz, 1H), 4.99 (d, J = 11.86 Hz, 1H), 4.63 (d, J = 12.06 Hz, 1H), 3.27 (s, 3H), 2.61-2.48 (m, 1H), 2.32-2.14 (m, 2H), 1.88-1.40 (m, 18H), 1.37—1.12 (m, 1H); ESI—MS m/z 628.83 (M+H)+. de No. 8 Chemical Formula: C34H38C|2FN3O4 Exact Mass: 641.22 Molecular Weight: 642.59 de No. 8 was obtained using the same synthetic strategy described for de No. 7. 1H-NMR (300MHz, CD30D) 6 ppm 7.63 (t, J = 7.04 Hz, 1H), 7.56—7.48 (m, 2H), 7.42 (t, J = 7.39 Hz, 1H), 7.18 (t, J = 7.96 Hz, 1H), 7.10 (d, J = 8.06 Hz, 1H), 6.79 (s, 1H), 5.08-4.96 (m, 1H), 4.57 (d, J = 11.85 Hz, 1H), .99 (m, 1H), 3.87-3.69 (m, 1H), .54 (m, 1H), 2.36- 2.13 (m, 2H), 1.94-1.45 (m, 18H), 1.39 (t, J = 6.65 Hz, 3H), 1.32-1.14 (m, 1H); ESI—MS m/z 642.50 (M+H)+. _53_ de No. 9 Chemical Formula: C33H37C|2FN4O3 Exact Mass: 626.22 Molecular Weight: 627.58 de No. 9 was obtained using the same synthetic gy described for de No. 4 (ammonium hydroxide solution was added instead of methanesulfonamide). 1H—NMR (300MHz, CD30D) 5 ppm; ESI-MS m/z 627.58 (M+H)+.

To demonstrate the ability of the present MDM2 inhibitors to bind to MDM2 ns, competitive FP binding assays were performed. Stability tests, cell growth assays, pharmacokinetic studies, and in vivo efficacy studies in SJSA—l xenograft models using the present MDM2 inhibitors also were performed.

Fluorescence-polarization MDM2 binding assay The binding affinity of the MDM2 tors disclosed herein was determined using a cence polarization—based (FP—based) binding assay using a recombinant human His— tagged MDM2 protein (residues l—118) and a cently tagged p53—based peptide.

The design of the fluorescence probe was based upon a previously reported high— affinity p53 -based peptidomimetic compound called PMDM6—F (Garcia-Echevern’a et al., J.

Med. Chem. 43: 3205—3208 (2000)). The Kd value of F with the recombinant MDM2 protein was determined from the saturation curve. MDM2 protein was serially double diluted in a DyneX l, black, round—bottom plate, and the PMDM6—F peptide was added at lnM concentration. The assay was performed in the buffer: 100 mM potassium phosphate, pH 7.5; 100 ug/mL bovine gamma globulin; 0.02% sodium azide, 0.01% Triton X—100) and the polarization values were ed after 3 h of incubation using an ULTRA READER (Tecan US. Inc., Research Triangle Park, NC). The IC50 value was obtained by fitting the mP values in _54_ a sigmoidal dose—response curve (variable slope) with a non—linear sion, and was determined to be 1.40 nM 4.- 0.25. The Kd value was calculated using the equation: Kd value = IC50 — L0/2. L0/2 is the concentration of the free ligand (PMDM6—F). Since PMDM6—F was used at a final concentration of lnM, L0/2 was 0.5 nM.

Dose—dependent, competitive g experiments were performed with serial dilutions of a tested compound in DMSO. A 5 uL sample of the tested compound and pre—incubated MDM2 protein (10 nM) and PMDM6—F peptide (1 nM) in the assay buffer (100 mM potassium ate, pH 7.5; 100 ug/mL bovine gamma in; 0.02% sodium azide, 0.01% Triton X— 100), were added in a Dynex 96—well, black, round—bottom plate to produce a final volume of 125 uL. For each assay, the controls included the MDM2 protein and PMDM6—F (equivalent to 0% inhibition), PMDM6—F e alone (equivalent to 100% inhibition). The polarization values were measured after 3 h of incubation. The IC50 values, i.e. the inhibitor concentration at which 50% of bound peptide is displaced, were determined from a plot using ear least— squares analysis. Curve fitting was med using GRAPHPAD PRISM software (GraphPad Software, Inc., San Diego, CA). The results of this assay are summarized in Table 2.

Cell growth assay Isogenic HCT—1 16 colon cancer cell lines were a kind gift from Prof. Bert Vogelstein (Johns Hopkins, Baltimore, MD) and were maintained in McCoy's 5A medium containing 10% PBS. The SJSA—l cell lines were obtained from ATCC, (Manassas, VA) and were maintained in RPMI—l640 medium containing 10% PBS.

Cells were seeded in 96—well flat bottom cell culture plates at a density of 2—3><103 cells/well with compounds and incubated for 4 days. The rate of cell growth inhibition after treatment with increasing concentrations of the tested compounds was determined by WST—8 (2— (2—methoxy—4—nitrophenyl)—3—(4-nitrophenyl)—5-(2,4—disu1fopheny1)—2H—tetrazolium monosodium salt (Dojindo Molecular Technologies Inc., Gaithersburg, nd). WST—8 was added at a final concentration of 10% to each well, and then the plates were incubated at 37°C for 2—3 hrs.

The absorbance of the samples was measured at 450 nm using a TECAN ULTRA Reader. The concentration of the nds that inhibited cell growth by 50% (IC50) was calculated by comparing absorbance in the untreated cells and the cells treated with the compounds using the ad Prism software (GraphPad Software, La Jolla, CA 92037, USA). The results of this assay are presented in Table 2. _55_ Table 2.

Cell Growth Inhibition MDM2 Compound Chemical (FP g assay) IC50 (HM) ID Structure HCTl16 HCTl16 IC50(nM) Ki(nM) p53 WT p53 deleted .U‘ 4; /\ 9‘N /\ ._. 9° 00 A._. >1 \o >1 U.) /\ 4; . UI /\ _56_ In vivo efficac studies usin S SA-xeno raft models SJSA-l (osteosarcoma) tumor cells were harvested with Trypsin (0.05%)—EDTA (0.53 mM) (GIBCOTM, Invitrogen Corp), growth medium was added, and the cells were placed on ice.

A cell sample was mixed 1:1 with Trypan Blue (GIBCOTM, Invitrogen Corp.) and counted on a hemocytometer to determine the number of live/dead cells. Cells were washed once with 1X PBS (GIBCOTM, Invitrogen Corp.) and resuspended in PBS. For Matrigel injections, after washing in PBS, cells are resuspended in an ice cold mixture of 1:1 PBS and Matrigel (BD Biosciences, ogen Corp.) for a final Matrigel protein tration of 5 mg/ml. SJSA-l tumors were inoculated into C.B-17 SCID mice at 5 x 106 cells in 0.1m1 with Matrigel. Cells were injected s.c. into the flank region of each mouse using a 27 gauge needle.

The size of tumors growing in the mice was measured in two dimensions using calipers. Tumor volume (mm3) = (AxB2)/2 where A and B are the tumor length and width (in mm), respectively. During treatment, tumor volume and body weight was measured three times a week. After the treatment was d, tumor volume and body weight was measured at least once a week. Mice were kept for an additional 60 days for further observation of tumor growth and toxicity. The anti-tumor activity of compounds No. 1, No. 7 and No. 8 are shown in Fig. 2.

The umor activity of compound No. 8 (administered via oral gavage) at different doses and according to different dosing schedules, including weekly for 3 weeks ks), every other day for 3 weeks, daily for 3 days out of a week for 3 weeks (qd1—3/w*3wks), and daily for 2 weeks (qd*l4d), is shown in Fig. 3.

Suitable es for in viva administration of the compounds provided herein include, without limitation, 10% PEG 400:3% Cremophor:87% PBS; 98% PEG 200:2% polysorbate 80; 98% PEG 200:2% TPGS; and 0.5% polysorbate 80:0.6% methyl cellulose:98.9% water. _57_ WO 61032 Stability of Compounds in Solution The stability of the compounds were determined in 1:1 MeOH2HgO, 1:1 CH3CNzHgO, and cell culture medium using ultra mance liquid chromatography.

The following Tables 3, 4, and 5 summarize additional test results showing the microsomal stability, oral pharmakinetics, and cell growth inhibition for compounds de N0. 2, de No. 7, and de No. 8.

Table 3. Microsomal stability of representative compounds in mouse, rat, dog and human microsomes Tl/2 (min) Compound Mouse Rat Dog Human de No. 2 >60 >60 >60 >60 de No. 7 >60 >60 >60 >60 de No. 8 >60 >60 >60 >60 Table 4. y of oral Pharmacokinetic data in Sprague—Dawley Rats Dose Compound route Cmax(ng/mL) Tmax (h) AUCO-l mL) AUCO-oo(ng-h/mL) t 1,2 (h) F(AUCO—oo) (mglkg) de N0. 2 25 oral 8234 i278 3.33 i 1.15 73603 i5022 74319i5260 4.29 i0.371 35.0:2.48 de N0. 7 25 oral 4391 i2826 4.00 i0.0 35205 i 15223 35426 $15489 3.891L 1.02 48.6:213 de N0. 8 25 oral 5453 i894 4.00 i0.0 39083i8473 39528i8521 4.61 i 1.35 40.3i8.69 Table 5. tion of cell growth by representative compounds. Cells were treated for 4 days and cell growth was determined using WST assay.

Compound ID Cell Lines Tumor Type p53 Status Compound Compound Compound No. 2 No. 7 No. 8 SS.” (HM) 70mm MW) RS4;11 Wild-type 62:26 (nM) 56118 (nM) 3815 (nM) _58_ LNCaP Wild-type 36:19 (nM) 30:15 (nM) 18:13 (nM) HCT116 Wild-type 137 i 31 (nM) 117 i 33 (nM 104 i 36 (nM) HCT116 p53-i- 14 i 2 (11M) 18 i 8 (11M) 8 i 1 (11M) 1 Wild—type 677 i 252 (nM) 713 i 165 (nM) 462 i 36 (nM) The present invention encompasses compounds of structural formula (I) and pharmaceutical compositions comprising a compound of structural formula (I) and a pharmaceutically acceptable carrier.

The present invention also encompasses a method of treating a patient comprising administering to the patient a therapeutically ive amount of the compound of structural formula (I), wherein the patient has a hyperproliferative e, wherein cells of the hyperproliferative disease, such as a cancer, express functional p53, further comprise administering to the patient one or more anticancer agents e.g., a chemotherapeutic agent or radiation therapy.

The present invention is bed in connection with red embodiments.

However, it should be iated that the invention is not limited to the disclosed embodiments.

It is understood that, given the description of the embodiments of the invention , various modifications can be made by a person skilled in the art. Such modifications are encompassed by the claims below. _59_ WHAT IS D: 1. A compound having the structural formula: wherein is selected from the group consisting of , , , , and ; B is a C4-7 carbocyclic ring; R1 is H, C1-4alkyl, cycloalkyl, heterocycloalkyl, ORa, or NRaRb; n is 0, 1, or 2; R2, R3, R4, R5, R7, R8, R9, and R10, independently, are selected from the group consisting of H, F, Cl, CH3, and CF3; R6 is ; Ra is hydrogen or C1-4alkyl; Rb is hydrogen or kyl; Rc and Rd are substituents on one carbon atom of ring B, wherein Rc is H, C1-3alkyl, C1-3alkyleneORa, ORa, or halo; Rd is H, C1-3alkyl, C1-3alkyleneORa, ORa, or halo; Re is –C(=O)ORa, -C(=O)NRaRb, or –C(=O)NHSO2CH3, or a pharmaceutically acceptable salt thereof. 2. The compound of claim 1 wherein is or 3. The compound of claim 1 or claim 2 wherein B is or 4. The compound of any one of the preceding claims where n is 0 or 1.

. The compound of claim 4 wherein R1 is H or CH3. 6. The compound of any one of the preceding claims n –(CH2)n-R1 is H, CH3, or CH2CH3. 7. The nd of any one of the preceding claims wherein R2 is H. 8. The nd of any one of the preceding claims wherein R3 is chloro. 9. The compound of any one of the preceding claims wherein at least one of R4 or R5 is H, or both R4 and R5 are H.

. The compound of any one of the preceding claims wherein R7 is fluoro. 11. The compound of any one of the preceding claims wherein each of R8, R9, and R10 is H. 12. The compound of any one of the preceding claims wherein Ra and Rb, individually, are H, CH3, or CH2CH3. 13. The compound of any one of the preceding claims wherein Rc and Rd, individually, are H, halo, OH, CH3, CH2CH3, or CH2OH. 14. The compound of any one of the ing claims n Rc and Rd are F and F, H and H, OH and CH3, OH and H, CH3 and CH3, CH3 and OH, H and OH, CH2CH3 and CH2CH3, or CH2OH and CH2OH.

. The compound of any one of claims 1 to 14 wherein Rc and Rd taken together with ring B to form: , , , , , , , , , , , , or . 16. The compound of any one of the preceding claims wherein Re is – C(=O)OH, -C(=O)NH2, or –C(=O)NHSO2CH3. 17. The nd of any one of the preceding claims wherein R6 is , , or . 18. A compound according to claim 1, selected from the group consisting and . 19. A compound ing to claim 1, having the structural formula: . A composition comprising (a) a compound of any one of claims 1 to 19 and (b) an excipient and/or pharmaceutically acceptable carrier. 21. Use of a compound according to any one of claims 1 to 19 or a composition according to claim 20 for the manufacture of a ment for the treatment of a disease or a condition for which a MDM2 inhibitor is indicated. 22. The use of claim 21 wherein the disease or condition is a hyperproliferative disease. 23. The use of claim 22 wherein the hyperproliferative disease is a cancer. 24. A compound according to any one of claims 1 to 19 substantially as herein bed with reference to any one or more of the es or figures.

. A composition according to claim 20 substantially as herein described with reference to any one or more of the examples or figures. 26. A use according to any one of claims 21 to 23 substantially as herein described with nce to any one or more of the examples or figures.

WO 61032 figure “1A Figure la Figure ‘3 8 Stability of AA-fl‘livcomgmunds Stabifity of AA—Mifiommunds liiy N ANN? 1-compounds in 1:1 CchszO in 15:1 MeOHngO in can cuttura mefiia my. -‘!‘- Ms-MI-fi‘fi + Cigar] Ex More 105 —V— AAANIALW +(1ng Ex 9m. 5 ~ «v ‘ 1- ngl F". 0.? 4&- 655d Six No. ‘5 +flip-r3 EEK-”$0. ‘3‘ -9— Bed Ex No- 33 {1‘ E ‘1 t‘« 3 Days 98313 Days Chemical stability comparison of AA—MI—06l, de EX No. 2, de EX No. 7 and de EX No. 8 in Figure 1A. 1:1 CH3CN to H20; Figure 1B. 1:1 MeOH to H20; and Figure 1C. cell culture media figure 2?: Figure 2B I Treatment 8.58M TumorGrth A m MEMSO- I I I I I I I I I I I I I I -.- vgmmmmi Exam 0 Vehicle Control "E 2090 '3‘“ CI"! E“ ”0'23“?“ka ‘19 G) ‘6- de Ex No.?,1&fimgikg qD AA~M1~061 I E ‘ 3 use. 1590 :2 35(3— § L. . 5 I Treatmentqe €600- §1im IIIIIIIIIIIIII — h,“ I: 400— *5 r: 500 g 200— E E \ 01 . . .5%?‘I‘T“-“f“"“fil 9 L. r. ,, '10 is 20 25 m ‘15 20 25 so as 40 45 Days Days post implantation Efficacy in the SJSA—l xenograft model in mice of Figure 2A. AA—MI—06l achieving 90% tumor regression; and Figure 2B. Compound No. 2 showing l tumor growth tion, and Compounds No. 7 and No. 8 demonstrating complete (100%) tumor regression.

WO 61032 Figure 3 835%»? “$133330? Gr‘m’éh QQ 'Vfihiflk {stdml g“ {333% E 4% SE38 (Tint 8 (3mm gikgmw‘fiwm 2‘: I as“ am mwmm E 3: m '6 ammmmwwks 3 Qifix} (am 3 {IQMgfltgmmxy ether day E 35333 ~S\. Cyfi Qifififimgfimmva :3» «that-w? any h 3333 , {‘3‘ Cyd Siimxsigfig)g&i~$ew Writs g 2.515% 4;» Cf": :rfargmzskgmxsimm :8 351337 3&3} {2 . g $339 53 '33 §§ R3 3’3 3‘3 Qays past 3mg)!" {Karim Efficacy in the SJSA—l xenograft model in mice of Compound No. 8. Compound No. 8 demonstrated tumor regression with different dosing schedules (e.g., daily at 100 mg/kg, every other day at 200 mg/kg, and days 1—3 per week).