OA16409A

OA16409A - Spiro-oxindole MDM2 antagonists.

Info

Publication number: OA16409A
Application number: OA1201300198
Authority: OA
Inventors: Shaomeng Wang; Yujun; SUN Wei ZHAO; Sanjeev Kumar; Lance Leopold; Laurent Debussche; Cedric Barriere; Jean-Christophe Carry; Kwame Amaning; Ming Guo
Original assignee: The Regents Of The University Of Michigan; Ascenta Licensing Corporation; Sanofi
Priority date: 2010-11-12
Filing date: 2011-11-11
Publication date: 2015-10-07

Abstract

Provided herein are compounds, compositions, and methods in the field of medicinal chemistry. The compounds and compositions provided herein relate to spirooxindoles which function as antagonists of the interaction between p53 and MDM2, and their use as therapeutics for the treatment of cancer and other diseases.

Description

SPIRO-OXINDOLE MDM2 ANTAGONISTS

BACKGROUND

The aggressive cancer cell phenotype is the resuit of a variety of genetic and epigenetic alterations leading to deregulation of intracellular signaling pathways (Ponder, Nature 411:336 (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 21:485 (2000)). The inability of cancer cells to execute an apoptotic program due to defects in the normal apoptotic machinery is often associated with an increase in résistance to chemotherapy, radiation, or immunotherapyinduced apoptosis. Prîmary or acquired résistance of human cancer of different origins to current treatment protocols due to apoptosis defects is a major problem in current cancer therapy (Lowe et al., Carcinogenesis 21:485 (2000); Nicholson, Nature 407:810 (2000)). Accordingly, current and future efforts towards designing and developing new molecular target-speclfic anticancer thérapies to improve survival and quality of life of cancer patients must include strategies that specîfically target cancer cell résistance to apoptosis.

The p53 tumor suppressor plays a central rôle in controlling cell cycle progression, senescence, and apoptosis (Vogelstein et al., Nature 408:307 (2000); Goberdhan, Cancer Cell 7:505 (2005)). MDM2 and p53 are part of an auto-regulatory feed-back loop (Wu et al., Genes Dev. 7:1126 (1993)). MDM2 is transcriptlonally activated by p53 and MDM2, in turn, inhibits p53 activity by at least three mechanisms (Wu et al., Genes Dev. 7:1126 (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 targeted DNAs. Third, MDM2 protein is an E3 ubiquitln ligase and upon binding to p53 is able to promote p53 dégradation.

Although high-affinity peptide-based inhibitors of MDM2 hâve been successfully designed in the past (Garcia-Echeverria ef al., Med. Chem. 43:3205 (2000)), these inhibitors are not suitable therapeutic molécules because of their poor cell permeability and in vivo bioavailability. Despite intensive efforts by the pharmaceutical industry, high throughput screening strategies hâve had very limited success in identifying potent, non-peptide small molécule inhibitors. Accordingly, there is a need for non-peptide, drug-like, small molécule inhibitors of the p53-MDM2 interaction.

The structural basis of the interaction p53 and MDM2 has been established by x-ray crystallography (Kussie et al., Science 274:948 (1996)).

Spiro-oxindole-based antagoniste of the p53-MDM2 interaction are described in U.S. Patent Nos. 7,759,383 B2 and 7,737,174 B2.

Skin cancer or melanoma is a commonly found type of cancer. Even though melanoma represents only a small fraction of the total number of cancer cases, it is responsible for many cancer deaths. According to statistics provided by the American Cancer Society, in contrast to many other types of cancers, the number of new cases of melanoma in the United States is still on the rise.

As with ail cancers, it is impérative to diagnose melanoma early. About 70% of melanomas are superfîcial spreading, meaning that they undergo a superficial, radial growth phase before they grow vertically and invade underlying tissue, a much more serious condition. Unfortunately, about 20% of cutaneous melanomas immediately start out with a vertical growth phase, which explains why these tumors are so dangerous. The 5-year survival rate for Stage 1 melanoma is very good. However, this drops off rapidly when cancer is allowed to progress and invade, first locally and then more distantly. Survival rate for Stage 2 disease is only 40-80%, Stage 3 10-70% and Stage 4 is almost invariably léthal within 5 years (<5-10% survives beyond 5 years) due to untreatable distant metastasis to especially lung and brain.

Melanoma originates from malignant transformation of mélanocytes, the pigment producing skin cells, via atypical and dysplastic premalignant intermediate stages to locally invasive and finally metastatic melanoma. A large number of genes hâve been implicated to play a rôle in these processes. Metastatic melanoma, the usual cause of death, is notoriously résistant to conventional therapy.

SUMMARY OF THE INVENTION

The présent disclosure contemplâtes that exposure of humans and animais to therapeutically effective amounts of drug(s) (e.g., small molécules) that increase the function(s) of p53 and p53-related proteins (e.g., p63, p73) inhibits the growth of p53 expressing cells. In some embodiments, the compounds provided herein inhibit the interaction between p53 or p53-related proteins and MDM2 or MDM2-related proteins (e.g., MDMX). Inhibiting the interaction between p53 or p53-related proteins and MDM2 or MDM2-related proteins inhibits the growth of cells. For example, inhibiting the interaction between p53 or p53-related proteins and MDM2 or MDM2-related proteins can inhibit cancer cells or supporting cells and/or renders such cells as a population more susceptible to the cell death-inducing activity of cancer therapeutic drugs or radiation thérapies. In 2 some embodiments, the inhibitors provided herein prolong the half-life of p53 by interfering with the p53-MDM2 interaction that would normally promote dégradation of p53. The compounds provided herein satisfy an unmet need for the treatment of multiple cancer types, either when administered as monotherapy to induce senescence, cell growth inhibition, apoptosis and/or cell cycle arrest in cancer ceils, 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 thérapies (combination thérapies), so as to render a greater proportion of the cancer cells or supportive cells susceptible to executing the apoptosis program compared to the corresponding proportion of cells in an animal treated only with the cancer therapeutic drug or radiation therapy alone.

In some embodiments, treatment of animais (including humans) with a therapeutically effective amount of one or more compounds provided herein and an anticancer agent produces a greater anti-tumor activity and clinical benefit in such animais compared to those treated with the compound or anticancer drugs/radiatîon alone. Put another way, because the compounds provided herein can lower the apoptotic threshold of cells that express p53 or p53-related protein, the proportion of cells that successfully execute the apoptosis program in response to the apoptosis inducing activity of anticancer drugs/radiation will be increased when used in combination with one or more of the compounds provided herein. Alternatively, the compounds provided herein 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. Since the doses for ail approved anticancer drugs and radiation treatments are known, the présent compounds, compositions, and methods provided herein can be used with one or more approved anticancer drugs and/or radiation treatment. Also, since the compounds provided herein may act at least in part by stimulating the proapoptotic and/or cell cycle-inhibiting activities of p53 and p53-related proteins, the exposure of cancer cells and supporting cells to therapeutically effective amounts of the compounds can be temporally linked to coïncide with the attempts of cells to execute the apoptosis program in response to the anticancer drug or radiation therapy. Thus, in some embodiments, administering the compounds or compositions provided herein in combination with other known anticancer drugs provide especially efficacious therapeutic practices.

In other embodiments, the inhibitors of the interaction between p53 or p53-related proteins and MDM2 and MDM2-related proteins provided herein may protect normal (e.g., nonhyperproliferative) 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 inhibitors provided herein may cause cell cycle arrest in cells comprising 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 différentiel protective effect may 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.

Applicants hâve found that certain spiro-oxindoles provided herein display an unexpected combination of drug-like properties. The unexpected combinations include, e.g., two or more of in vitro efficacy, in vivo efficacy, in vitro liver microsome stability, désirable absorption, distribution, metabolism, and excrétion (ADME) properties. For example, certain spiro-oxindoles provided herein are more résistant to metabolic dégradation e.g., as measured by in vitro liver microsomal stability and/or in vivo pharmacokinetics, and/or display improved in vivo efficacy as compared to known antagonists of the p53-MDM2 interaction.

Applicants hâve also found that metabolically cleavable groups can be used to increase the aqueous solubility of the parent molécule. Thus, in some embodiments, the spiro-oxindoles provided herein are useful prodrugs with improved aqueous solubility relative to the parent molécule.

In some embodiments, the compounds provided herein are spiro-oxindoles having Formulae l-XXXV (see below under “Compounds), or pharmaceutically acceptable salts, solvatés, or prodrugs thereof. In some embodiments, the compounds provided herein inhibit the interaction between p53 or p53-related proteins and MDM2 or MDM2-related proteins.

In some embodiments, the compounds provided herein contain a metabolically cleavable group. In particular, in some embodiments, the compounds provided herein contain a hydroxy group of a hydroxycycloalkyl side chain that can be used to attach a metabolically cleavable group. Suitable metabolically cleavable groups include, but are not limited to, amino acid esters or phosphate esters.

In some embodiments, the compounds provided herein can be used to induce senescence, cell cycle arrest and/or apoptosis in cells containing functional p53 or p53-related proteins. Also provided herein are methods of using the compounds provided herein for sensitizing cells to additional agent(s), such as inducers of senescence, apoptosis and/or cell cycle arrest. The compounds provided herein can also be used to provide chemoprotection of normal cells through the induction of cell cycle arrest prior to treatment with chemotherapeutic agents. In one embodiment, the methods of rendering a normal cell résistant to chemotherapeutic agents or treatments comprises contacting the cell with one or more compounds provided herein. In one embodiment, methods of protecting normal cells in an animal having a hyperproliferative disease from the toxic side effects of chemotherapeutic agents or treatments, comprises administering to the animal a compound provided herein. Provided herein are methods for the treatment, amelioration, or prévention 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 provided herein. Examples of such disorders and conditions caused by chemotherapy include, without limitation, mucositis, stomatitis, xerostomia, gastrointestinal disorders, and alopecia.

The compounds provided herein are useful for the treatment, amelioration, or prévention of disorders, such as those responsive to induction of apoptotic cell death, e.g., disorders characterized by dysrégulation of apoptosis, including hyperproliferative diseases such as cancer. In certain embodiments, the compounds can be used to treat, ameliorate, or prevent cancer that is characterized by résistance to cancer thérapies (e.g., those cancer cells which are chemoresistant, radiation résistant, hormone résistant, and the like). In other embodiments, the compounds can be used to treat hyperproliferative diseases characterized by expression of functional p53 or p53-related proteins. In other èmbodiments, the compounds provided herein 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, pharmaceutical compositions are provided. The pharmaceutical compositions can comprise one of more of the compounds provided herein and a pharmaceutically acceptable carrier.

In one embodiment, kits are provided. The kits can comprise one or more of the compounds provided herein, or a pharmaceutically acceptable sait thereof, and instructions for administering the compound to an animal. The kits may optionally contain other therapeutic agents, e.g., anticancer agents or apoptosis-modulating agents.

In one embodiment, methods of treating, preventing, or ameliorating a hyperproliferative disease, e.g., cancer, in a patient comprising pulsatile administration to the patient a therapeutically effective amount of one or more of the compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof are provided.

In one embodiment, methods of treating, preventing, or ameliorating a hyperproliferative disease, e.g., cancer, in a patient comprising pulsatile administration to the patient a therapeutically effective amount of one or more of the compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, in combination with one or more additional therapeutic, e.g., anticancer, agents.

In one embodiment, kits comprising one or more of the compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, and instructions for administering the compound(s) to a patient having a hyperproliferative disease by pulsatile dosing are provided. The kits can optionally contain one or more additional therapeutîc, e.g., anticancer, agents.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 is a reverse phase HPLC chromatogram of MI-519-64 after isolation by column chromatography on silica gel.

Fig. 2 is reverse phase HPLC chromatogram of MI-519-64 after treatment with acetonitrile/water for 12 h. Three isomers are présent. MI-519-64 and MI-519-6401 correspond to RP-HPLC peaks at 30.578 minutes, and 31.787 minutes, respectively. The isomer eluting at 29.162 minutes is referred to as MI-519-6402.

Fig. 3 is reverse phase HPLC chromatogram of MI-519-64 after treatment with acetonitrile/water for 3 days.

Fig. 4 is a line graph showing the binding affinities of MI-519-64, MI-519-6401, and MI-5196402 to human MDM2 protein, as determined using a fluorescence-polarization binding assay. The purity of each isomer used in this experiment (as determined by RP-HPLC) are as follows: MI-519-6402: 90% (with 10% of MI-519-64); MI-519-64: 93% (with 3% of MI519-64 and 4% of MI-519-6401); and MI-519-6401: >99%. The log IC_M values for MI-5196402, MI-519-64, and MI-519-6401 are 2.030 nM, 1.598 nM, and 0.8354 nM, respectively.

Fig. 5 is a line graph showing the binding affinities of MI-773 (TFA sait), MI-77301 (TFA sait), MI-77301 (free amine), and MI-77302 (TFA sait) to human MDM2 protein, as determined using a fluorescence-polarization binding assay.

Fig. 6 is a line graph showing the stability of MI-773 (TFA sait) at various time points in water/methanol = 1:1 with 0.1% of TFA, pH 2.1. The compound corresponding to peak 1 is MI-77302. The compound corresponding to peak 3 is MI-773. The compound corresponding to peak 4 is MI-77301.

Fig. 7 is a line graph showing the stability of MI-77301 (TFA sait) at various time points in water/methanol = 1:1 with 0.1% of TFA, pH 2.1. The compound corresponding to peak 1 is MI-77302. The compound corresponding to peak 3 is MI-773. The compound corresponding to peak 4 is MI-77301.

Fig. 8 is an illustration showing western blot analysis of p53 activation and apoptosis induced by MI-773 and MI-77301 in the SJSA-1 (osteosarcoma) cell line.

Fig. 9 is an illustration showing western blot analysis of p53 activation and apoptosis induced by MI-519-64 and MI-519-6401 in the SJSA-1 cell line.

Fig. 10 is a bar graph showing apoptosis induced by MI-773 and MI-77301 in the SJSA-1 cell line.

Fig. 11 is a bar graph showing cell death induced by MI-519-64 and MI-519-6401 in the SJSA-1 cell line.

Fig. 12 is a bar graph showing cell death induced by MI-519-64 and MI-519-6401 in the RS4;11 (human acute lymphoblastic leukemia (ALL)) cell line.

Fig. 13 is an illustration showing western blot analysis of in vivo activation of p53 and PARP cleavage induced by MI-519-64 and MI-519-6401 in SJSA-1 tumors in mice.

Fig. 14 is an illustration showing western blot analysis of in vivo activation of p53 and PARP cleavage induced by MI-519-64 and MI-519-6401 in RS4;11 tumors in mice.

Fig. 15 is an illustration showing western blot analysis of in vivo activation of p53 and PARP cleavage induced by MI-773 and MI-77301 in SJSA-1 tumors in mice.

Fig. 16 is an illustration showing three western blot analyses of p53 activation and apoptosis induced by MI-773 and MI-77301 in the RS4;11 cell line.

Fig. 17 is a line graph showing in vivo antitumor activity of MI-519-64, MI-519-6401, MI-773, and MI-77301 in the SJSA-1 osteosarcoma xenograft model in mice.

Fig. 18 is a line graph showing the animal weight following administration of MI-519-64, MI-519-6401, MI-773, and MI-77301 in mice.

Fig. 19 is a line graph showing in vivo antitumor activity of MI-519-6401 and MI-77301 in the 22Rv1 human prostate xenograft model in mice.

Fig. 20 is a line graph showing in vivo antitumor activity of MI-77301 in the SJSA-1 osteosarcoma xenograft model in mice (Cpd-B = MI-77301 ; treatment schedule = QD11 ).

Fig. 21 is a line graph showing the animal weight following administration of MI-77301 in mice (Cpd-B = MI-77301).

Fig. 22 is a line graph showing in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft model in mice (Cpd-B = MI-77301).

Fig. 23 is a line graph showing in vivo antitumor activity of MI-77301 in the SJSA-1 osteosarcoma xenograft model in mice (Cpd-B = MI-77301 ). ^r

Fig. 24 is a line graph showing the cell growth inhibition activity of MI-77301 in melanoma cell lines.

Fig. 25 is an illustration showing western blot analysis of p53 activation induced by MI77301 in SK-Mel-103 (human melanoma) cells.

Fig. 26 is an illustration showing western blot analysis of p53 activation induced by MI77301 in UACC-62 (p53 wt melanoma) and SK-Mel-19 (human melanoma)cells.

Fig. 27 is a bar graph showing apoptosis induced by MI-77301 in the UACC-62 cell line.

Fig. 28 is an illustration showing western blot analysis of in vivo activation of p53 induced by MI-773001 in SK-Mel-103 melanoma xenografts in mice.

Fig. 29 is a line graph showing in vivo antitumor activity of MI-77301 in the SK-Mel-103 melanoma xenograft model in mice.

Fig. 30 is reverse phase HPLC chromatogram of substantially pure CB061-lsomer B.

Fig. 31 is a line graph showing in vivo antitumor activity of MI-77301 in the HCT-116 human colorectal tumor xenograft model in mice.

Fig. 32 is a line graph showing in vivo antitumor activity of MI-77301 in the LNCAP human prostate tumor xenograft model in mice.

Fig. 33 is a line graph showing in vivo antitumor activity of MI-77301 in the RS4;11 human acute lymphoblastic leukemia xenograft model in mice.

Fig. 34 is a sériés of three ¹³C CPMAS NMR spectrograms showing MI-77301 (top), MI-773 (middle), and MI-77302 (bottom).

Fig. 35 is a reverse phase HPLC chromatogram of substantially pure MI-773 (eluent: MeOH/water with 0.1% TFA).

Fig. 36 is a reverse phase HPLC chromatogram of substantially pure MI-77301 (eluent: MeOH/water with 0.1% TFA).

Fig. 37 is a line graph showing the stability of MI-773 (TFA sait) at various time points in water/methanol = 1:1 with 0.1% of TEA, pH 10.8. The compound corresponding to peak 3 is MI-773. The compound corresponding to peak 4 is Ml 77301,

Fig. 38 is a line graph showing the stability of MI-773 (TFA sait) at various time points in water/methanol = 1:1, pH 3.9. The compound corresponding to peak 3 is MI-773. The compound corresponding to peak 4 is MI-77301.

Fig. 39 is reverse phase HPLC chromatogram of substantially pure C027 (eluent: acetonitrile/H₂O with 0.1% TFA).

Fig. 40 is reverse phase HPLC chromatogram of substantially pure C02701 (eluent: acetonitrile/H₂O with 0.1% TFA).

Fig. 41 is reverse phase HPLC chromatogram of C029 substantially free of other stereoisomers (eluent: acetonitrile/H₂O with 0.1% TFA).

Fig. 42 is reverse phase HPLC chromatogram of substantially pure C02901 (eluent: acetonitrile/H₂0 with 0.1% TFA).

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compounds that inhibit the interaction between p53 or p53-related proteins and MDM2 or MDM2-related proteins. By inhibiting the négative effect of MDM2 or MDM2-related proteins on p53 or p53-related proteins, these compounds sensitize cells to inducers of apoptosis and/or cell cycle arrest. In some embodiments, the compounds provided herein induce apoptosis and/or cell cycle arrest. 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. In some embodiments, the methods comprise contacting the cells with one or more compounds provided herein alone or in combination with additional agent(s), e.g., an inducer of apoptosis or a cell cycle disrupter.

Also provided herein are methods of treating, ameliorating, or preventing disorders, e.g., a hyperproliferative disease, e.g., cancer, in an patient, comprising admînistering to the patient one or more compounds provided herein and additional agent(s), e.g., an inducer of apoptosis. Such disorders include those characterized by a dysrégulation of apoptosis and those characterized by the prolifération of cells expressing functional p53 or p53-related proteins. In other embodiments, methods of protecting normal (e.g., non-hyperproliferative) cells in an animal from the toxic side effects of chemotherapeutic agents and treatments are provided. The methods comprise admînistering to the animal one or more compounds provided herein. Also provided herein are methods of treating preventing, or ameliorating a hyperproliferative disease, e.g., cancer, in a patient comprising admînistering to the patient a therapeutically effective amount of one or more of the compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, according to a pulsatile dosing regîmen.

Also provided herein are methods of treating preventing, or ameliorating a hyperproliferative disease, e.g., cancer, in a patient comprising admînistering to the patient a therapeutically effective amount of one or more of the compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, according to a pulsatile dosing regîmen in combination with one or more additional therapeutic, e.g., anticancer, agents.

Also provided herein are kits comprising one or more of the compounds provided herein, and instructions for admînistering the compound(s) to a patient having a hyperproliferative disease by pulsatile dosing. The kits can optionally contain one or more additional therapeutic, e.g., anticancer, agents

Définitions

The terms pulsatile administration, pulsatile dose administration, or pulsatile dosing as used herein, refer to intermittent (i.e., not continuous) administration of one or more of the compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, to a patient. Pulsatile dose administration regimens useful in the présent disclosure encompass any discontinuous administration regimen that provides a therapeutically effective amount of the compound(s) provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, to a patient in need thereof. Pulsatile dosing regimens can use équivalent, lower, or higher doses of compounds, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, than would be used in continuous dosing regimens. The compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, can be administered as a single agent under a pulsatile dosing regimen or can be administered under a pulsatile dosing regimen in combination with one or more additional anticancer agents (where the additional anticancer agents are administered either on a continuous or a pulsatile regimen). On the day that compounds, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, are scheduled to be administered to the patient, administration can occur in a single or in divided doses, e.g., once-a-day, twice-aday, three times a day, four times a day or more. In one embodiment, compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, are administered once (QD) or twice (BID) on the day It is schedule to be administered. In one embodiment the compounds provided, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, are administered orally to the patient according to a pulsatile dosing regimen. In one embodiment the compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, are administered intravenously to the patient according to a pulsatile dosing regimen.

The therapeutic utility of drug administration can be offset by the number and severity of adverse events a patient expériences. Pulsatile dosing of compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, can resuit in a réduction in the number and/or severity of clinical adverse events coupled with a maintenance or enhancement in clinical efficacy, as compared to continuous daily dosing. The clinical benefits of pulsatile dose administration of compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, can be more prominent when combined with the administration of other therapeutic agents to the patient.

In one embodiment, compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, are administered to a patient no more frequently than one day out of every two days (e.g., administration occurs on day 1, day 3, day 5, day 7, day 9, etc.), 10 one ont of every three days (e.g., administration occurs on day 1, day 4, day 7, day 10, etc.), one out of every four days, one out of every five days, one ouf of every six days, one out of every seven days, one out of every eight days, one out of every nine days, one out of every ten days, one out of every two weeks, one out of every three weeks, one out of every four weeks, one out of every five weeks, or longer. The pulsatile dosing regimen can continue for one, two, three or four weeks, one, two, three or four months, one, two, three or four years or longer.

In another embodiment, compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, are administered to a patient one day a week, e.g., a compound of Formulae l-XXXV, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof, is administered to a patient on one day followed by six consecutive days wherein the compound is not administered. In another embodiment, compounds having Formulae IXXXV, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, administered to a patient one day every two weeks. In another embodiment, compounds having Formulae IXXXV, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, are administered to a patient one day every three weeks. In another embodiment, compounds having Formulae l-XXXV, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, are administered to a patient one day every four weeks.

In another embodiment, compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, are administered to a patient on a least two consecutive days, e.g., at least three, four, five, six or seven consecutive days, followed by at least one day, at least two consecutive days, at least three consecutive days, at least four consecutive days, at least five consecutive days, at least six consecutive days, at least seven consecutive days, at least eight consecutive days, at least nine consecutive days, at least ten consecutive days, at least eleven consecutive days, at least twelve consecutive days, at least thirteen consecutive days, at least two consecutive weeks, at least three consecutive weeks, or at least four consecutive weeks or longer wherein the compound disclosed herein is not administered.

In one embodiment, compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, and one or more anticancer agents are administered to a patient on day 1 of an anticancer treatment cycle. Typically, the length of the treatment cycle is determined in accord with the approved dosing protocol(s) of the one or more anticancer agents that are to be administered to the patient in combination with the compounds having Formulae l-XXXV, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof. In one embodiment, the treatment cycle is about 14 days, about 21 days, or about 28 days. In a particular embodiment, the treatment cycle is 21 days. In one embodiment, the treatment cycle is repeated one or more times, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, or more times.

In another embodiment, compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, are administered to the patient on day 1, on days 1 and 2, or on days 1, 2, and 3 of a treatment cycle and one or more anticancer agents are administered starting on day 1 of the treatment cycle in accord with the recommended dosing schedule of the anticancer agent In one embodiment, the anticancer agent is a chemotherapeutic agent. In another embodiment, the anticancer agent is radiation therapy.

In another embodiment, compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, are administered via the sequential use of a combination of two or more pulsatile dosing schedules. The combination may comprise the same pulsatile dosing schedules or different pulsatile dosing schedules. The sequential use of a combination of two or more pulsatile dosing regimens may be repeated as many times as necessary to achieve or maintain a therapeutic response, e.g., from one to about fifty times, e.g., from one to about twenty times, e.g., from about one to about ten times. With every répétition any additional therapeutic agents may be the same or different from that used in the previous répétition.

In another embodiment, compounds provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof, are administered according to a pulsatile dosing schedule and/or sequential combination of two or more pulsatile dosing schedules followed by a waiting period. The term waiting period, as used herein, refers to a period of time between dosing schedules when a compound disclosed herein is not administered to the patient The waiting period may be one, two, three, four, five or six days, one, two or three weeks, one, two, three or four months, one, two, three or four years or longer. In certain embodiments, the waiting period may be one to thirty days, e.g., seven, fourteen, twenty one or thirty days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. After the waiting period, the same or a different pulsatile dosing schedule and/or sequential combination of one or more pulsatile dosing schedules of a compound disclosed herein can résumé. The pulsatile dosing/waiting period regimen may be repeated as many times as necessary to achieve or maintain a therapeutic response, e.g., from one to about fifty times, e.g., from one to about twenty times, e.g., from about one to about ten times. With every répétition any additional therapeutic agents may be the same or different from that used in the previous répétition.

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

The term “prodrug” as used herein, refers to a pharmacologically inactive dérivative of a parent drug” molécule that requires biotransformation (e.g., either spontaneous or enzymatic) within the target physiological System to release, or to convert (e.g., enzymatically, physiologically, mechanically, electromagnetically) the prodrug into the active drug. Prodrugs are designed to overcome problems associated with stability, water solubility, toxicity, lack of specificity, or limited bioavailability. Exemplary prodrugs comprise an active drug molécule itself and a chemical masking group (e.g., a group that reversibly suppresses the activity of the drug). Some prodrugs are variations or dérivatives of compounds that hâve groups cleavable under metabolic conditions. Prodrugs can be readily prepared from the parent compounds using methods known in the art, such as those described in A Textbook of Drug Design and Development, Krogsgaard-Larsen and H. Bundgaard (eds.), Gordon & Breach, 1991, particularly Chapter 5: Design and Applications of Prodrugs; Design of Prodrugs, H. Bundgaard (ed.), Elsevier, 1985; Prodrugs: Topical and Ocular Drug Delivery, K. B. Sloan (ed.), Marcel Dekker, 1998; Methods in Enzymology, K. Widder et al. (eds.), Vol. 42, Academie Press, 1985, particularly pp. 309-396; Burgeris Médicinal Chemistry and Drug Discovery, 5th Ed., M. Wolff (ed.), John Wiley & Sons, 1995, particularly Vol. 1 and pp. 172-178 and pp. 949-982; Pro-Drugs as Novel Delivery Systems, T. Higuchi and V. Stella (eds.), Am. Chem. Soc., 1975; and Bioreversible Carriers in Drug Design, E. B. Roche (ed.), Elsevier, 1987.

Exemplary prodrugs become pharmaceutically active in vivo or in vitro when they undergo solvolysis under physiological conditions or undergo enzymatic dégradation or other biochemical transformation (e.g., phosphorylation, hydrogénation, dehydrogenation, glycosylation). Prodrugs often offer advantages of water solubility, tissue compatibility, or delayed release in the mammalian organism. (See e.g., Bundgaard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam (1985); and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp. 352-401, Academie Press, San Diego, CA (1992)). Common prodrugs include acid dérivatives such as esters prepared by reaction of parent acids with a suitable alcohol (e.g., a lower alkanol) or esters prepared by reaction of parent alcohol with a suitable carboxylic acid, (e.g., an amino acid), amides prepared by reaction of the parent acid compound with an amine, basic groups reacted to form an acylated base dérivative (e.g., a lower alkylamide), or phosphorus-containing dérivatives, e.g., phosphate, phosphonate, and phosphoramidate esters, including cyclic phosphate, phosphonate, and phosphoramidate, see, e.g., US 2007/0249564 A1.

The term metabolically cleavable group” as used herein, refers to groups which can be cleaved from the parent molécule by metabolic processes and be substituted with hydrogen. Certain compounds containing metabolically cleavable groups may be prodrugs, Le., they are pharmacologically inactive. Certain other compounds containing metabolically cleavable groups may be antagonists of the interaction between p53 and MDM2. In such cases, these compounds may hâve more, less, or équivalent activity of the parent molécule. Examples of metabolically cleavable groups include those derived from amino acids (see, e.g., US 2006/0241017 A1; US 2006/0287244 A1; and WO 2005/046575 A2) or phosphoruscontaining compounds (see, e.g., U.S. 2007/0249564 A1) as illustrated in Scheme 1.

Scheme 1

parent amino acid drug

O metabolic --------- R-OH cleavage amino acid ester parent drug

R-OH +

O

CI'f'^0R OR”

O ^R-° or^m	metabolic	R-OH
cleavage

phosphate ester parent drug parent drug phosphite

The term pharmaceutically acceptable sait as used herein, refers to any sait (e.g., obtained by reaction with an acid or a base) of a compound provided herein that is physiologically tolerated in the target animal (e.g., a mammal) or human. Salts of the compounds of provided herein may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p*sulfonic, tartane, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the préparation of salts useful as intermediates in obtaining the compounds provided herein and their pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkalî métal (e.g., sodium) hydroxides, alkaline earth métal (e.g., magnésium) hydroxides, ammonia, and compounds of formula NW₄ ⁺, wherein W is Cm alkyl, and the like.

Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycérophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide, iodide, 2-hydroxyethanesulfonate, lactate, maleate, mesylate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds provided herein compounded with a suitable cation such as Na*. NH₄ ⁺, and NW₄ ⁺ (wherein W is a Cm alkyl group), and the like. For therapeutic use, salts of the compounds provided herein are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the préparation or purification of a pharmaceutically acceptable compound.

The term solvaté as used herein, refers to the physical association of a compound provided herein with one or more solvent molécules, whether organic or inorganic. This physical association often includes hydrogen bonding. In certain instances, the solvaté is capable of isolation, for example, when one or more solvaté molécules are incorporated in the crystal lattice of the crystalline solid. Solvaté encompasses both solution-phase and isolable solvatés. Exemplary solvatés include hydrates, ethanolates, and methanolates.

The term monovalent pharmaceutically acceptable cation as used herein refers to inorganic cations such as, but not limited to, alkaline métal ions, e.g., Na* and K*, as well as organic cations such as, but not limited to, ammonium and substituted ammonium ions, e.g., ΝΗΛ NHMe₃ ⁺, NH2Mez⁺, NHMe3* and NMe₄*.

The term divalent pharmaceutically acceptable cation as used herein refers to inorganic cations such as, but not limited to, alkaline earth métal cations, e.g., Ca²⁺ and Mg²*.

Examples of monovalent and divalent pharmaceutically acceptable cations are discussed, e.g., in Berge étal. J. Pharm. Sci., 66:1-19 (1997).

The term “therapeutically effective amount, as used herein, refers to that amount of the therapeutic agent (including the compounds, pharmaceutical compositions, and compositions of matter provided herein) sufficient to resuit in amelioration of one or more symptoms of a disorder, or prevent advancement of a disorder, or cause régression of the disorder. For example, with respect to the treatment of cancer, in one embodiment, a therapeutically effective amount can refer to the amount of a therapeutic agent that decreases the rate of tumor growth, decreases tumor mass, decreases the number of métastasés, increases time to tumor progression, increase tumor cell apoptosis, or increases survival time 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 at least 100%.

The terms sensitize” and “sensitizing, 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, prolifération, invasion, angîogenesis, necrosis, or apoptosis) of a second therapeutic agent. The sensitizing effect of a first agent on a target cell can be measured as the différence in the intended biological effect (e.g., promotion or retardation of an aspect of cellular function including, but not limited to, cell growth, prolifération, invasion, angîogenesis, 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 20%, 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.

The term dysrégulation of apoptosis, as used herein, refers to any aberration in the ability of (e.g., prédisposition) a cell to undergo cell death via apoptosis. Dysrégulation of apoptosis is associated with or induced by a variety of conditions, non-limiting examples of which include, autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, graft-versus-host disease, myasthenia gravis, or Sjôgren's syndrome), chronic inflammatory conditions (e.g., psoriasis, asthma or Crohn's disease), hyperproliferative disorders (e.g., tumors, B cell lymphomas, or T cell lymphomas), viral infections (e.g., herpes, papilloma, or HIV), and other conditions such as osteoarthritis and atherosclerosis. It should be noted that when the dysrégulation is induced by or associated with a viral infection, the viral infection may or may not be détectable at the time dysrégulation occurs or is observed. That is, virai-induced dysrégulation can occur even after the disappearance of symptoms of viral infection.

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 hâve at least 25% sequence homology with p53, hâve tumor suppressor activity, and are înhibited by 5 interaction with MDM2 or MDM2-related proteins. Examples of p53-related proteins include, but are not limited to, p63 and p73.

The term MDM2-related protein, as used herein, refers to proteins that hâve at least 25% 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 and MDM4.

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

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 15 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 prolifération învolving an încrease in cell number in a tissue or organ without significant 20 alteration in structure or function. Metaplasîa 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 results in an autoimmune disorder or a chronic inflammatory condition. As used herein, the term “autoimmune disorder” refers to any condition in which an organism produces antibodies or immune cells which recognize 25 the organism's own molécules, cells or tissues. Non-limiting examples of autoimmune disorders include autoimmune hemolytic anémia, autoimmune hepatitis, Berger’s disease or IgA nephropathy, celiac sprue, chronic fatigue syndrome, Crohn's disease, dermatomyositis, fibromyalgia, graft versus host disease, Grave’s disease, Hashimoto’s thyroiditis, idiopathic thrombocytopenia purpura, lichen planus, multiple sclerosis, myasthenia gravis, psoriasis, 30 rheumatic fever, rheumatic arthritis, scleroderma, SjÔgren’s syndrome, systemic lupus erythematosus, type 1 diabètes, ulcerative colitis, vitiligo, and the like.

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

The term melanoma as used herein refers to any form of cancer that begins in 35 mélanocytes. Melanoma includes, but is not limited to, the following subtypes: lentigo maligna, lentigo maligna melanoma, superficiel spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft-tissue melanoma, and metastatic melanoma. Melanoma, as used herein also includes metastatic melanoma.

The term normal cell, as used herein, refers to a cell that is not undergoing abnormal growth or division. Normal cells are non-cancerous and are not part of any hyperproliferative disease or disorder.

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

The terms “prevent, “preventing,” and “prévention,” as used herein, refer to a decrease in the occurrence of pathological cells (e.g., hyperproliferative or neoplastic cells) in an animal. The prévention may be complété, e.g., the total absence of pathological cells in a subject. The prévention may also be partial, such that the occurrence of pathological cells in a subject is less than that which would hâve occurred without treatment with one or more compounds provided herein.

The terms a and an refer to one or more.

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, DR1, DR2, DR3, DR4, DR5, DR6, FADD, and RIP. Other examples of apoptosis-modulating agents include, but are not limited to, TNFa, Fas ligand, antibodies to Fas/CD95 and other TNF family receptors, TRAIL (also known as Apo2 Ligand or Apo2L/TRAIL), antibodies to TRAIL-R1 or TRAIL-R2, Bcl-2, p53, BAX, BAD, Akt, CAD, PI3 kinase, PP1, 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 (e.g. ligand or receptor). Apoptosis-modulating agents include those which are inducers of apoptosis, such as TNF or a TNF-related ligand, particularly a TRAMP ligand, a Fas/CD95 ligand, a TNFR-1 ligand, or TRAIL.

The term pharmaceutically acceptable carrier or pharmaceutically acceptable vehicle encompasses any of the standard pharmaceutical carriers, solvents, surfactants, or vehicles. Suitable pharmaceutically acceptable vehicles include aqueous vehicles and nonaqueous vehicles. Standard pharmaceutical carriers and their formulations are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 19th ed. 1995.

The term '‘alkyl” as used herein by itself or part of another group refers to a straight-chain or branched saturated aliphatic hydrocarbon having from one to eighteen carbons or the number of carbons designated (e.g., C_rC_1B means 1 to 18 carbons). In one embodiment, the alkyl is a Ci-C^ alkyl. In another embodiment, the alkyl is a C₄-C₈ alkyl. In another embodiment, the alkyl is a C_rC₆ alkyl. In another embodiment, the alkyl is a C_rC₄ alkyl. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, isohexyl, n-heptyl, 4,4-dimethylpentyl, n-octyl, 2,2,4-trimethylpentyl, nonyl, decyl and the like.

The term optionally substituted alkyl as used herein by itself or part of another group means that the alkyl as defined above is either unsubstituted or substituted with one, two or three substituents independently selected from hydroxy (i.e., -OH), nitro (/.e., -NO₂), cyano (i.e., -CN), optionally substituted cycloalkyl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. In one embodiment, the optionally substituted alkyl is substituted with two substituents. In another embodiment, the optionally substituted alkyl is substituted with one substituent. In another embodiment, the substituents are selected from hydroxyl (i.e., a hydroxyalkyl), optionally substituted cycloalkyl (i.e., a (cycloalkyl)alkyl), or amino (i.e., an aminoalkyl). Exemplary optionally substituted alkyl groups include -CH₂OCH₃₁ -CH₂CH₂NH₂, CH₂CH₂NH(CH₃), -CH₂CH₂CN, -CH₂SO₂CH₃, hydroxymethyl, hydroxyethyl, hydroxypropyl, and the like.

The term alkylenyl” as used herein by itself or part of another group refers to a divalent alkyl radical containing one, two, three, four, or more joined methylene groups. Exemplary alkylenyl groups include -(CH₂)-, -(CH₂)₂-, -(CH₂)₃-, -(CH₂)₄-, and the like.

The term optionally substituted alkylenyl as used herein by itself or part of another group means the alkylenyl as defined above is either unsubstituted or substituted with one, two, three, or four substituents independently selected from the group consisting of optionally substituted Ci-C₈ alkyl, optionally substituted cycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl. In one embodiment, the optionally substituted Ci-C₆ alkyl is methyl. In one embodiment, the optionally substituted aryl is a phenyl optionally substituted with one or two halo groups. Exemplary optionally substituted alkylenyl groups include -CH(CH₃)-, -C(CH₃)₂-, -CH₂CH(CH₃)-, -CH₂CH(CH₃)CH₂-, -CH₂CH(Ph)CH₂-, -CH(CH₃)CH(CH₃)-, and the like.

The term haloalkyl as used herein by itself or part of another group refers to an alkyl as defined above having one to six halo substituents. In one embodiment, the haloalkyl has one, two or three halo substituents. Exemplary haloalkyl groups include trifluoromethyl, -CH₂CH₂F and the like.

The term hydroxyalkyl as used herein by itself or part of another group refers to an alkyl as defined above having one hydroxy substituent Exemplary hydroxyalkyl groups include hydroxymethyl, hydroxyethyl, hydroxypropyl, and the like.

The term dihydroxyalkyl as used herein by itself or part of another group refers to alkyl as defined above having two hydroxyl substituents. Exemplary dihydroxyalkyl groups include CH₂CH₂CCH₃(OH)CH₂OH, -CH₂CH₂CH(OH)CH(CH3)OH, -CH₂CH(CH₂OH)₂, CH₂CH₂CH(OH)C(CH3)₂OH -CH₂CH₂CCH₃(OH)CH(CH₃)OH, and the like, including stereoisomers thereof.

The term hydroxycycloalkyl as used herein by itself or part of another group refers to an optionally substituted cycloalkyl as defined below having a least one, e.g., one or two, hydroxy substituents. Exemplary hydroxycycloalkyl groups include:

and the like, including stereoisomers thereof.

The term optionally substituted (cycloalkyl)alkyl as used herein by itself or part of another group refers to an optionally substituted alkyl as defined above having an optionally substituted cycloalkyl (as defined below) substituent. Exemplary optionally substituted (cycloalkyl)alkyl groups include:

and the like, including stereoisomers thereof.

The term aralkyl as used herein by itself or part of another group refers to an optionally substituted alkyl as defined above having one, two or three optionally substituted aryl substituents. In one embodiment, the aralkyl has two optionally substituted aryl substituents. In another embodiment, the aralkyl has one optionally substituted aryl substituent. In another embodiment, the aralkyl is an aryl(Ci-C₄ alkyl). In another embodiment, the arylfCrC^ alkyl) has two optionally substîtuted aryl substituents. In another embodiment, the arylfCrC^ alkyl) has one optionally substîtuted aryl substituent. Exemplary aralkyl groups include, for example, benzyl, phenylethyl, (4-fluorophenyl)ethyl, phenylpropyl, diphenylmethyl (Le., Ph₂CH-), diphenylethyi (Ph₂CHCH₂-) and the like.

The term cycloalkyl as used herein by itself or part of another group refers to saturated and partially unsaturated (containing one or two double bonds) cyclic hydrocarbon groups containing one to three rings having from three to twelve carbon atoms (Le., C₃-C₁₂cycloalkyl) or the number of carbons desîgnated. In one embodiment, the cycloalkyl has one ring. In another embodiment, the cycloalkyl is a C₃-C₆ cycloalkyl. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin, adamantyl and the like.

The term “optionally substîtuted cycloalkyl” as used herein by itself or part of another group means the cycloalkyl as defined above is either unsubstituted or substîtuted with one, two or three substituents independently selected from halo, nitro, cyano, hydroxy, amino, optionally substîtuted alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, aralkyl, optionally substîtuted cycloalkyl, optionally substîtuted alkenyl, optionally substîtuted alkynyl, optionally substîtuted aryl, optionally substîtuted heteroaryl, optionally substîtuted heterocyclo, alkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. The term optionally substîtuted cycloalkyl also means the cycloalkyl as defined above may be fused to an optionally substîtuted aryl. Exemplary optionally substîtuted cycloalkyl groups include:

and the like.

The term alkenyl as used herein by itself or part of another group refers to an alkyl group as defined above containing one, two or three carbon-to-carbon double bonds. In one embodiment, the alkenyl has one carbon-to-carbon double bond. Exemplary alkenyl groups include -CH=CH₂, -CH₂CH=CH₂, -CH₂CH₂CH=CH₂, -CH₂CH₂CH=CHCH₃ and the like.

The term optionally substîtuted alkenyl as used herein by itself or part of another group means the alkenyl as defined above is either unsubstituted or substîtuted with one, two or three substituents independently selected from halo, nitro, cyano, hydroxy, amino, optionally substîtuted alkyl, haloalkyl, hydroxyalkyl, aralkyl, optionally substîtuted cycloalkyl, optionally substîtuted alkenyl, optionally substîtuted alkynyl, optionally substîtuted aryl, optionally substîtuted heteroaryl, optionally substîtuted heterocyclo, alkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. Exemplary optionally substituted alkenyl groups include -CH=CHPh, -CH_zCH=CHPh and the like.

The term cycloalkenyl as used herein by îtself or part of another group refers to a cycloalkyl group as defined above containing one, two or three carbon-to-carbon double bonds. In one embodiment, the cycloalkenyl has one carbon-to-carbon double bond. Exemplary cycloalkenyl groups include cyclopentene, cyclohexene and the like.

The term optionally substituted cycloalkenyl as used herein by îtself or part of another group means the cycloalkenyl as defined above is either unsubstituted or substituted with one, two or three substituents independently selected from halo, nitro, cyano, hydroxy, amino, optionally substituted alkyl, haloalkyl, hydroxyalkyl, aralkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido.

The term alkynyl as used herein by îtself or part of another group refera to an alkyl group as defined above containing one to three carbon-to-carbon triple bonds. In one embodiment, the alkynyl has one carbon-to-carbon triple bond. Exemplary alkynyl groups include -C=CH, -CeCCH₃, -CH₂C=CH, -ΟΗ₂ΟΗ₂ΟξΟΗ and -CH₂CH₂C=CCH₃.

The term optionally substituted alkynyl as used herein by itself or part of another group means the alkynyl as defined above is either unsubstituted or substituted with one, two or three substituents independently selected from halo, nitro, cyano, hydroxy, amino, optionally substituted alkyl, haloalkyl, hydroxyalkyl, aralkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. Exemplary optionally substituted alkenyl groups include -C=CPh, -CH₂C^CPh and the like.

The term aryl as used herein by itself or part of another group refera to monocyclic and bicyclic aromatic ring Systems having from six to fourteen carbon atoms (/.e., Ο₆-Ο₁₄ aryl) such as phenyl (abbreviated as Ph), 1-naphthyl and 2-naphthyl and the like.

The term “optionally substituted aryl as used herein by itself or part of another group means the aryl as defined above is either unsubstituted or substituted with one to five substituents independently selected from halo, nitro, cyano, hydroxy, amino, optionally substituted alkyl, haloalkyl, hydroxyalkyl, aralkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. In one embodiment, the optionally substituted aryl is an optionally substituted phenyl. In one embodiment, the optionally substituted phenyl has four substituents. In another embodiment, the optionally substituted phenyl has three substituents. In another embodiment, the optionally substituted phenyl has two substituents. In another embodiment, the optionally substituted phenyl has one substituent. Exemplary substituted aryl groups include 2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2chlorophenyl, 2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3-fluorophenyl, 3chlorophenyl, 4-methylphenyl, 4-ethylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4chlorophenyl, 2,6-di-fluorophenyl, 2,6-di-chlorophenyl, 2-methyl, 3-methoxyphenyl, 2-ethyl, 3-methoxyphenyl, 3,4-di-methoxyphenyl, 3,5-di-fluorophenyl 3,5-di-methylphenyl and 3,5dimethoxy, 4-methylphenyl, 2-fluoro-3-chlorophenyl, 3-chloro-4-fluorophenyl and the like, The term optionally substituted aryl is meant to include groups having fused optionally substituted cycloalkyl and fused optionally substituted heterocyclo rings. Examples include:

and the like.

The term heteroaryl as used herein by itself or part of another group refers to monocyclîc and bicyclic aromatic ring Systems having from five to fourteen carbon atoms {i.e., C5-C14 heteroaryl) and one, two, three or four heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur. In one embodiment, the heteroaryl has three heteroatoms. In one embodiment, the heteroaryl has two heteroatoms. In one embodiment, the heteroaryl has one heteroatom. Exemplary heteroaryl groups include 1-pyrrolyl, 2pyrrolyl, 3-pyrrolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, purinyl, 2benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 2-benzthiazolyl, 4-benzthiazolyl, 5benzthiazolyl, 5-indolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 1-isoquinolyl, 5-isoquinolyl, 2quinoxalinyl, 5-quinoxalinyl, 2-quinolyl 3-quinolyl, 6-quinolyl and the like. The term heteroaryl is meant to include possible N-oxides. Exemplary N-oxides include pyridyl Noxide and the like.

The term optionally substituted heteroaryl as used herein by itself or part of another group means the heteroaryl as defined above is either unsubstituted or substituted with one to four substituents, typically one or two substituents, independently selected from halo, nitro, cyano, hydroxy, amino, optionally substituted alkyl, haloalkyl, hydroxyalkyl, aralkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. In one embodiment, the optionally substituted heteroaryl has one substituent In another embodiment, the substituent is an optionally substituted aryl, araîkyl, or optionally substituted alkyl. In another embodiment, the substituent is an optionally substituted phenyl. Any available carbon or nitrogen atom may be substituted. Exemplary optionally substituted heteroaryl groups include:

Ph Ph

and the like.

The term heterocyclo as used herein by itself or part of another group refers to saturated and partially unsaturated (containing one or two double bonds) cyclic groups containing one to three rings having from two to twelve carbon atoms (i.e., C₂-Ci₂ heterocyclo) and one or two oxygen, sulfur or nitrogen atoms. The heterocyclo can be optionally linked to the rest of the molécule through a carbon or nitrogen atom. Exemplary heterocyclo groups include:

and the like.

The term “optionally substituted heterocyclo as used herein by itself or part of another group means the heterocyclo as defined above is either unsubstituted or substituted with one to four substituents independently selected from halo, nitro, cyano, hydroxy, amino, optionally substituted alkyl, haloalkyl, hydroxyalkyl, aralkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, -COR^C, -SO2R^d, -N(R^e)COR^f, -N(R^e)SO2R⁹or -N(R^e)C=N(R^h)-amino, wherein R^c is hydrogen, optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R^d is optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R® is hydrogen, optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R^f is hydrogen, optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R⁹ is optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl; and R^h is hydrogen, -CN, optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl. Substitution may occur on any available carbon or nitrogen atom. Exemplary substituted heterocyclo groups include:

and the like. An optionally substituted heterocyclo may be fused to an aryl group to provide an optionally substituted aryl as described above.

The term alkoxy as used herein by itself or part of another group refers to a haloalkyl, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl or optionally substituted alkynyl attached to a terminal oxygen atom. Exemplary alkoxy groups include methoxy, tert-butoxy, -OCH₂CH=CH₂ and the like.

The term aryloxy as used herein by itself or part of another group refers to an optionally substituted aryl attached to a terminal oxygen atom. Exemplary aryloxy groups include phenoxy and the like.

The term aralkyloxy as used herein by itself or part of another group refers to an aralkyl attached to a terminal oxygen atom. Exemplary aralkyloxy groups include benzyloxy and the like.

The term alkylthio as used herein by itself or part of another group refers to a haloalkyl, aralkyl, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl or optionally substituted alkynyl attached to a terminal sulfur atom. Exemplary alkyl groups include -SCH₃ and the like.

The term halo or halogen as used herein by itself or part of another group refers to fluoro, chloro, bromo or iodo. In one embodiment, the halo is fluoro or chloro.

The term amino as used herein by itself or part of another group refers to a radical of formula -NR^aR^b wherein R^a and R^b are independently hydrogen, haloalkyl, aralkyl, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclo, optionally substituted aryl or optionally substituted heteroaryl; or R^a and R^b taken together with the nitrogen atom to which they are attached form a four to seven membered optionally substituted heterocyclo. Exemplary amino groups include -NH₂, -N(H)CH₃, -N(CH₃)₂, N(H)CH₂CH₃, N(CH₂CH₃), -N(H)CH₂Ph and the like.

The term carboxamido as used herein by itself or part of another group refers to a radical of formula -CO-amino. Exemplary carboxamido groups include -CONH₂, -CON(H)CH₃, CON(H)Ph, -CON(H)CH₂CH₂Ph, -CON(CH₃)2, CON(H)CHPh₂ and the like.

The term sulfonamido as used herein by itself or part of another group refers to a radical of formula -S0₂-amino. Exemplary sulfonamido groups include -SO₂NH₂, -SO₂N(H)CH₃, SO₂N(H)Ph and the like.

The term about, as used herein, includes the recited number ± 10%. Thus, about 10 means 9 to 11.

Certain of the compounds of the présent disclosure may exist as stereoisomers, Le., isomers that differ only in the spatial arrangement of atoms, încluding optîcal isomers and conformational isomers (or conformers). The disclosure includes ail stereoisomers, both as pure individual stereoisomer préparations and enriched préparations of each, and both the racemic mixtures of such stereoisomers as well as the individual diastereomers and enantiomers that may be separated according to methods 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 conventional analytical methods routinely used by those of skill in the art. In some embodiments, 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%, iess 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 or substantially pure stereoisomers.

Stereoisomerically enriched compounds that contain about 99% or more of a desired stereoisomer are referred to herein as pure or pure stereoisomers. The purity of any stereoisomerically enriched compound can be determined using conventional analytical methods such as, for example, normal phase HPLC, reverse phase HPLC, chiral HPLC, and ’Hand ¹³C NMR.

Compounds

In certain embodiments, compounds of Formula I are provided:

wherein:

R^1a, R^1b, R^1c, and R^1d are independently selected from the group consisting of hydrogen, halogen, hydroxy, amino, nitro, cyano, alkoxy, aryloxy, optionally substituted alkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted cycloalkenyl, optionally substituted aryl, optionally substituted heteroaryl, carboxamido, and sulfonamido;

R² is selected from the group consisting of optionally substituted aryl, aralkyl, and optionally substituted heteroaryl;

R³ is selected from the group consisting of optionally substituted alkyl, optionally substituted (cycloalkyl)alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted cycloalkenyl, optionally substituted aryl, and optionally substituted heteroaryl;

R⁴ is selected from the group consisting of hydrogen and optionally substituted alkyl;

R⁵ is selected from the group consisting of:

wherein:

each R^6a and R^eb is îndependently selected from the group consisting of hydrogen and optionally substituted Ci-C₆ alkyl;

R⁷ is selected from the group consisting of hydrogen, optionally substituted Ci-C₆ alkyl, and optionally substituted cycloalkyl;

R^8a and R^8b are each îndependently selected from the group consisting of hydrogen, optionally substituted C_rC₆ alkyl, and optionally substituted cycloalkyl; or

R^8a and R^ab taken together with the carbon that they are attached form a 3to 8-membered optionally substituted cycloalkyl;

W¹ is selected from the group consisting of -OR^9a and -NR^9bR^9c;

R^9a is hydrogen;

R^9b is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -SO₂R^9d, and -CONR^9eR^9f;

R^9c is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl; or

R^9b and R^9c taken together with the nitrogen atom to which they are attached form a 4- to 8-membered optionally substituted heterocyclo;

R^9d is selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl;

R^9e and R⁹ⁱ are each îndependently selected from the group consisting of hydrogen, optionally substituted alkyl, and optionally substituted cycloalkyl; or zx

R^9e and R^9f taken together with the nitrogen atom to which they are attached form a 4- to 8-membered optionally substituted heterocyclo;

W² is selected from the group consisting of -OR¹⁰ and -NR^11aR^11b;

with the proviso that when W¹ is -OR^9a and W² is -OR¹⁰ then at least one of R⁷, R^8a, and R^8b is other than hydrogen;

R¹⁰ is hydrogen; or one of R^9a and R¹⁰ is hydrogen and the other is a metabolically cleavable group;

R^11a is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -SO₂R^11c, and -CONR^11dR^11e;

R^11b is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl; or

R^11a and R^11b taken together with the nitrogen atom to which they are attached form a 4- to 8-membered optionally substituted heterocyclo;

R^11c is selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl;

R^11d and R^11e are each independently selected from the group consisting of hydrogen, optionally substituted alkyl, and optionally substituted cycloalkyl; or

R^11d and R¹¹® together with the nitrogen atom to which they are attached form a 4- to 8-membered optionally substituted heterocyclo;

n is 1,2, 3, 4, or 5;

each R^1Za, R^12b, R^12c and R^12d is independently selected from the group consisting of hydrogen and optionally substituted C^Ce alkyl;

R¹³ is selected from the group consisting of hydrogen and optionally substituted C_rC₆ alkyl;

R¹⁴ is selected from the group consisting of hydrogen, optionally substituted CtCs alkyl, and optionally substituted cycloalkyl;

Z is selected from the group consisting of -OR¹⁵ and -NR^16aR^16b; or

Z and R¹⁴ taken together form a carbonyl, i.e., a C=O, group.

R¹⁵ is selected from the group consisting of hydrogen and metabolically cleavable group;

R^16a is selected from the group consisting of -SO₂R^iec and -CONR^16dR^16e;

R^16b is selected from the group consisting of hydrogen and optionally substituted alkyl;

R^16c is selected from the group consisting of optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl;

R^10d and R¹⁶® are each independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl; or

R^16d and R¹⁶® taken together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclo;

is 1, 2, or 3;

p is 0, 1, 2, or 3;

each R^17a, R^17b, R^17c and R^17d is independently selected from the group consisting of hydrogen and optionally substituted Cj-Ce alkyl;

R¹⁸ is selected from the group consisting of hydrogen and optionally substituted Ci-C₆ alkyl;

R¹⁹ is selected from the group consisting of hydrogen, optionally substituted Ci-C₆ alkyl, and optionally substituted cycloalkyl;

R²⁰ is selected from the group consisting of hydrogen, optionally substituted Ci-C₆ alkyl, and optionally substituted cycloalkyl;

R²¹® and R^21b are each hydrogen; or one of R^21a and R^21b is hydrogen and the other is metabolically cleavable group;

q is 0,1,2, or 3;

r is 1, 2, or 3;

each R^22a, R^22b, R^22c, and R^22d is independently selected from the group consisting of hydrogen and optionally substituted C1-C6 alkyl;

R²³ is selected from the group consisting of hydrogen and optionally substituted CpCg alkyl;

R²⁴ is selected from the group consisting of -SO₂R^24a and -CONR^24bR^24c;

R^24a is selected from the group consisting of optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl;

R^24b and R^24c are each independently selected from the group consisting of hydrogen, optionally substituted cycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl; or

R^24b and R^24c taken together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclo;

s and t are each independently 1, 2, or 3;

X is selected from the group consisting of O, S, and NR*;

Y is selected from the group consisting of O, S, and NR;

R' is selected from the group consisting of hydrogen, optionally substituted alkyl, aralkyl, and optionally substituted cycloalkyl;

R is selected from the group consisting of hydrogen, optionally substituted alkyl, aralkyl, optionally substituted cycloalkyl, and -COR³¹;

R³¹ is selected from the group consisting of hydrogen and optionally substituted alkyl; and ~ represents a single or a double bond, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In certain embodiments, for the compounds of Formula I, ~ represents a single bond.

In certain embodiments, the compound of Formula I is a mixture of stereoisomers, e.g., a mixture of diastereomers and/or enantiomers, e.g., a racemic mixture. In another such embodiment, the compound is a mixture of diastereomers. In another such embodiment, the compound is a mixture of enantiomers. In particular embodiments, the compound is a single enantiomer.

In certain embodiments, R⁵ is selected from the group consisting of R5-1 and R5-2. In particular embodiments, R⁵ is R5-2 and Z is -OH. In particular embodiments, R⁵ is R5-2A and Z is OH.

In certain embodiments, compounds of Formula la are provided:

wherein R^1a, R^1b, R^1c, R^1d, R², R³, R⁴, R⁵, X, and Y hâve the meanings as described above for Formula I, or pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In certain embodiments, compounds of Formula Ib are provided:

wherein R^1a, R^1b, R^1c, R^1d, R², R³, R⁴, R⁵, X, and Y hâve the meanings as described above for Formula I, or tautomer thereof, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In certain embodiments, compounds of Formula ll-XVII are provided:

wherein R^1a, R^1b, R^1c, R^1d, R², R³, R⁴, R⁵, X, and Y hâve the meanings as described above for Formula I, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In some embodiments, compounds of Formula II are provided, wherein R^1a, R^1b, R^1c, R^1d, R², R³, R⁴, R⁵, X, and Y hâve the meanings as described above in connection with Formula I, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In some embodiments, compounds of Formula II are substantially free of one or more other stereoîsomers, i.e., compounds of Formulae lll-XVII. In some embodiments, compounds of

Formula II are substantially pure stereoisomers. In some embodiments, compounds of

Formula II are pure stereoisomers.

In some embodiments, compounds of Formula XIII are substantially free of one or more other stereoisomers. In some embodiments, compounds of Formula XIII are substantially pure stereoisomers. In some embodiments, compounds of Formula XIII are pure stereoisomers.

In some embodiments, compounds of Formula XIV are substantially free of one or more other stereoisomers. In some embodiments, compounds of Formula XIV are substantially pure stereoisomers. In some embodiments, compounds of Formula XIV are pure stereoisomers.

In some embodiments, compounds of Formula VI are provided, wherein R^1a, R^1b, R^1c, R^1d, R², R³, R⁴, R⁵, X, and Y hâve the meanings as described above in connection with Formula I, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In some embodiments, compounds of Formula VI are substantially free of one or more other stereoisomers, i.e., compounds of Formulae ll-V and VII-XVII. In some embodiments, compounds of Formula VI are substantially pure stereoisomers. In some embodiments, compounds of Formulae VI are pure stereoisomers.

In some embodiments, compounds of Formula X are provided, wherein R^1a, R^1b, R^1c, R^1d, R², R³, R⁴, R^s, X, and Y hâve the meanings as described above in connection with Formula I, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In some embodiments, compounds of Formula X are substantially free of one or more other stereoisomers, i.e., compounds of Formulae ll-IX and XI-XVII. In some embodiments, compounds of Formula X are substantially pure stereoisomers. In some embodiments, compounds of Formula X are pure stereoisomers.

In some embodiments, compounds of Formula XII are provided, wherein R^1a, R^1b, R^1c, R^1d, R², R³, R⁴, R⁵, X, and Y hâve the meanings as described above in connection with Formula I, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In some embodiments, compounds of Formula XII are substantially free of one or more other stereoisomers, i.e., compounds of Formulae ll-XI and XIII-XVII. In some embodiments, compounds of Formula XII are substantially pure stereoisomers. In some embodiments, compounds of XII are pure stereoisomers.

In some embodiments, compounds of Formula XII are unexpectedly more potent than compounds of Formulae ll-XI and XIII-XVII. For example, as demonstrated herein, compounds of Formula XII hâve lower IC₅₀ values than compounds of Formulae ll-XI and XIII-XVII against MDM2. In some embodiments, compounds of Formula XII are about 2-fold or more, e.g., about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 834 fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, or more, more potent than compounds of Formula H in fluorescence polarization-based MDM2 binding assays. In some embodiments, compounds of Formula XII are unexpectedly more efficacious than compounds of Formulae ll-XI and XIII-XVII in xenograft tumor models in mice and/or in other in vivo efficacy models.

In certain embodiments, compounds of Formulae l-XVII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided. In some embodiments, compounds of Formulae II, VI, X, and XII, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein:

a) R^1a, R^1b, R^1c, and R^1d are independently selected from the group consisting of hydrogen, fluoro, and chloro;

b) R^1a and R^1d are hydrogen; R^1b is selected from the group consisting of hydrogen and fluoro; and R^1c is selected from the group consisting of fluoro and chloro;

c) R² is optionally substituted phenyl;

d) R³ is selected from the group consisting of optionally substituted alkyl, optionally substituted (cycloalkyl)alkyl, and optionally substituted cycloalkyl;

e) R⁴ is hydrogen;

f) X is NH;

g) X is O;

h) X is S;

i) Y is O;

j) Y is S;

k) Yis NH;or

l) X and Y are NH;

or any combination thereof.

In certain embodiments, compounds of Formulae 11, VI, X, and XII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is R5-1 ; R^6a and R^6bare hydrogen; R⁷ is C1-C4 alkyl; R^8a and R^8b are hydrogen; W is -OR¹⁰, R⁹ and R¹⁰ are hydrogen; and n is 2.

In certain embodiments, compounds of Formulae II, VI, X, and XII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is R5-1; R^6a and R^6bare hydrogen; R⁷ is C_rC₄ alkyl; R^8a and R^8b are hydrogen; W is -NR^11aR^11b, R⁹is hydrogen; and n is 2.

In certain embodiments, the compounds of Formulae II, VI, X, and XII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R^s is R5-1; R^6a and R^6b are hydrogen; R⁷ is C1-C4 alkyl; R^ea and R^8b are hydrogen; W is -OR¹⁰, one of R⁹ and R¹⁰ is hydrogen and the other is a metabolically cleavable group; and n is 2.

In certain embodiments, compounds of Formulae II, VI, X, and XII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is R5-2; R^12a, R^12b, R^12c, and R^1Zd are each hydrogen; R¹³ is hydrogen; Z is -OR¹⁵ and R¹⁵ is hydrogen; o is 1 or 2; and p is 1 or 2.

In certain embodiments, compounds of Formulae II, VI, X, and XII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is R5-2; R^12a, R^1Zb, R^12c, and R^12d are each hydrogen; R¹³ is hydrogen; Z is -NR^16aR^16b; o is 1 or 2; and p is 1 or 2.

In certain embodiments, compounds of Formulae II, VI, X, and XII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is R5-2; R^12a, R^12b, R^12c, and R^12d are each hydrogen; R¹³ is hydrogen; Z is -OR¹⁵ and R¹⁵ a metabolically cleavable group; o is 1 or 2; and p is 1 or 2.

In certain embodiments, compounds of Formulae II, VI, X, and XII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is R5-3; R^17a, R^17b, R^17c, and R^17d are each hydrogen; R¹⁸, R¹⁹, and R²⁰ are hydrogen; R^21a and R^21b are hydrogen; and q and r are 1.

In certain embodiments, compounds of Formulae II, VI, X, and XII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is R5-3; R^17a, R^17b, R^17c, and R^17d are each hydrogen; R¹⁸, R¹⁹, and R²⁰ are hydrogen; one of R^21a and R^21b is hydrogen and the other is a metabolically cleavable group; and q and r are 1.

In certain embodiments, compounds of Formulae 11, VI, X, and XII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is R5-2A.

In certain embodiments, compounds of Formulae 11, VI, X, and XII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R² is selected from the group consisting of aralkyl and optionally substituted aryl having the Formula R2-1:

R2-1 and R^2Sa, R^25b, R^25c, R^25d, and R^25e are each independently selected from the group consisting of hydrogen, halogen, hydroxy, nitro, amino, cyano, alkoxy, optionally substituted alkyl, haloalkyl, optionally substituted aryl, and optionally substituted heteroaryl. In particular embodiments, R^25a is selected from the group consisting of hydrogen and fluoro; R^25b is chloro; R^25c is selected from the group consisting of hydrogen and fluoro; and R^25d and R^25e are hydrogen.

In certain embodiments, compounds of Formulae II, VI, X, and XII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is selected from the 5 group consisting of:

R5-9

	\
			—NR^16aR^16b
	-OR¹⁵	C	>14
R¹⁴
R5-10		R5-11

R¹⁴

	—NR^16aR^16b . R¹⁴ 4
n R5-13 ^R5'¹⁴

R5-12

o19 0R^£,d

R5-18

R5-17 including stereoisomers, e.g., enantiomers, thereof, wherein:

R⁷ is optionally substituted C_rC4 alkyl;

R^9a and R¹⁰ are each hydrogen; or one of R^9a and R¹⁰ is hydrogen and the other is a metabolically cleavable group;

R^9c is selected from the group consisting of hydrogen, optionally substîtuted alkyl, optionally substîtuted cycloalkyl, optionally substîtuted aryl, and optionally substituted heteroaryl; or

R⁹® and R^9f are each independently selected from the group consisting of hydrogen, optionally substituted alkyl, and optionally substituted cycloalkyl; or

R⁹® and R^9f taken together with the nitrogen atom to which they are attached form a 4- to 8-membered optionally substituted heterocyclo;

R^11a is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -SO₂R^11c, and -CONR^11dR¹¹®;

R^11d and R¹¹® are each independently selected from the group consisting of hydrogen, optionally substituted alkyl, and optionally substituted cycloalkyl; or

R^11d and R¹¹® taken together with the nitrogen atom to which they are attached form a 4- to 8-membered optionally substituted heterocyclo;

R¹⁴ is selected from the group consisting of hydrogen, CrC4 alkyl, or C₃-C₆cycloalkyl;

R¹⁵ is hydrogen or a metabolically cleavable group;

R^16a is selected from the group consisting of -SO₂R^16c and -CONR^iedR¹⁶®;

R^16d and R^1Be are each îndependently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl; or

R^16d and R^16e taken together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclo;

R^1S is selected from the group consisting of hydrogen, optionally substituted CrCe alkyl, and optionally substituted cycloalkyl;

R^21a and R^z1b are each hydrogen; or one of R^21a and R^21b is hydrogen and the other is metabolically cleavable group;

R²⁴ is selected from the group consisting of -SO₂R^24a and -CONR^24bR^24c;

R^24a is selected from the group consisting of optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl; and

R^24b and R^24c are each îndependently selected from the group consisting of hydrogen, optionally substituted cycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl, or

R^24b and R^24c taken together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclo.

In certain embodiments, R^s is selected from the group consisting of R5-5, R5-6, R5-10, R511, R5-12, R5-13, and R5-14.

In certain embodiments, R⁵ is selected from the group consisting of R5-10 and R5-12 and R¹⁴ is hydrogen or methyl and R¹⁵ is hydrogen.

In certain embodiments, compounds of Formulae II, VI, X, and XII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is selected from the group consisting of:

wherein:

R⁷ is selected from the group consisting of methyl, ethyl, propyl, isopropyl, and cyclopropyl; and

R^Sa and R^8b are each independently selected from the group consisting of hydrogen, methyl, ethyl, propyl, isopropyl, and cyclopropyl.

In certain embodiments, compounds of Formulae II, VI, X, and XII or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is selected from the

group consisting of:
OH j\^NHSO₂R^11c R⁷	OH / x/—NHSO₂R¹¹⁰ R⁷	OH /\^^]\,nhso₂r^11c R⁷] _R8a

f

11c

NHSO₂R

R^eb _R8a

I

and

11c

NHSO₂R^£ld

OH _Reb _R8a

R⁷ is selected from the group consisting of methyl, ethyl, propyl, isopropyl, and cyclopropyl;

R^8a and R^8b are each independently is selected from the group consisting of hydrogen, methyl, ethyl, propyl, isopropyl, and cyclopropyl;

R^9d is selected from the group consisting of methyl, trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl; and

R^11c is selected from the group consisting of methyl, trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl.

OH /jF,NHCONHR^11d

R⁷

NHCONHR¹¹⁰

11d

OH	OH	OH
z-F NHCONHR^11d	k,NHCONHR^11d / X	—F, NHCONHR^11d
R⁷ T	_R7	h₃ô P’R^0b
R^0a	_R8a	_R8a

NHCONHR⁹®

R⁷

NHCONHR⁹®

R⁸®

NHCONHR⁹®

OH

and

NHCONHR⁹®

_R8a

R^0a and R^eb are each independently is selected from the group consisting of hydrogen, methyl, ethyl, propyl, isopropyl, and cyclopropyl;

R^Se is selected from the group consisting of methyl, trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl; and

R^11d is selected from the group consisting of methyl, trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl.

In certain embodiments, compounds of Formulae II, VI, X, and XII, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is selected from the group consisting of:

wherein:

R¹⁴ is selected from the group consisting of methyl, ethyl, propyl, isopropyl, and cyclopropyl; and

R¹⁹ and R²⁰are each independently is selected from the group consisting of hydrogen, methyl, ethyl, propyl, isopropyl, and cyclopropyl.

In certain embodiments, compounds of Formulae II, VI, X, and XII, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is selected from the 10 group consisting of:

NHSO₂R^16c s

NHSO₂R^16c ^Ÿ^'R¹⁴

NHSO₂R^16c

vr¹⁴	—L ,
NHSO₂R^16c	NHSO₂R^16c	''NHSO₂R^16c
M—NHSO₂R^16c R¹⁴	L—\|^,,,NHSQ₂R^16c , R¹⁴	Y ^Xxx^^knhso₂r^16c
^ΖΌ· nhso₂r^16c	<Y-nhso₂r^16c K'·	^/xo , LJ'''Nhso₂r^16c R¹⁴
^^Y^ -'NHSOzR¹⁶⁰ ,		T Y’NHSO₂R^16c
r V-NHSO₂R^16c ,	*Y^\j'NHSO₂R^16c	**LG\-NHSO₂R^16c ' Y 4
\ \>'NHSO₂R^16cLV^V _R14	A \ >-NHSO₂R^16c , X__< Υ7Ύ⁴ *nhso₂r^16c
	and ' F^A-NHSOjR ^16c . k__/ W NHSO₂R^16c

16c wherein:

R^16c is selected from the group consisting of methyl, trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl.

NHCONHR^16d

NHC0NHR^ied

NHCONHR^16d

NHCONHR^16d ’

NHCONHR

16d

\hconhr^1m

L4“NHC0NHR^ied '

R¹⁴

A.

I—rNHCQNHR^16dr!4 '^^/NHCONHR^16d h, ^/γγ <^-NHCONHR^16d

NHCONHR^ied R¹⁴ <J-NHCONHR^ied ,

R¹⁴ ^*^S^2/^t '‘NHCONHR^16d , ^'^^/-‘NHCONHR¹⁶¹¹

Γ \->'NHCONHR^16d , ^*\^\-NHCONHR^16d

t. Λ.

Q_r ^N.^HC0NHR'“ ^NHCONHR''^^-nhconhr¹⁶⁰ t^^xY2S:^4^HCONHRl6d

NHCONHR^16d

wherein:

R^16d is selected from the group consisting of methyl, trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl.

In certain embodiments, compounds of Formulae II, VI, X, and XII, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof are provided, wherein R⁵ is:

In another embodiment, compounds of Formula XVIIIa are provided:

XVIIIa ιο wherein:

R^1b and R^1c are independently selected from the group consisting of hydrogen, fluoro, and chloro;

R³ is selected from the group consisting of optionally substituted alkyl, optionally substituted (cycloalkyl)alkyl, and, optionally substituted aryl, optionally substituted cycloalkyl;

R^26a, R^26b, and R^26c are independently selected from the group consisting of hydrogen, fluoro, and chloro; and

R²⁷ is selected from the group consisting of:

OR^0a

R⁷

R27-3

R¹⁴

R27-4

Z

I

R¹⁴

OR^21b

21a

»

R27-6

R27-7 »

R27-20 ' OR^21b .

R27-24

R27-22

K /

i pis' OR^21b

R27-27

R27-25

R27-26

R²⁰Î-OR^21a

R

A>or²¹⁰

R i-0R^21a

R²⁰

R¹⁴

R27-9

R27-19

R27-23

R27-18

R¹⁴

R27-21

R¹⁰'' 0_R21b

_ Z R¹⁴

R27-28

R27-29

R27-31

R27-32

R27-30 wherein:

R⁷ is optionally substituted 0_ν04 alkyl;

W² is selected from the group consisting of-OR¹⁰ and -NR^11aR^11b;

R^9a and R¹⁰ are each hydrogen; or one of R^9a and R^w is hydrogen and the other is a metabolically cleavable group;

R^11d and R^11e taken together with the nitrogen atom to which they are attached form a 4- to 8-membered optionally substituted heterocyclo;

R¹⁴ is selected from the group consisting of hydrogen, optionally substituted C1-C4 alkyl, and optionally substituted cycloalkyl;

Z is selected from the group consisting of -OR¹⁵ and -NR^16aR^16b;

R^1S is selected from the group consisting of hydrogen and metabolically cleavable group;

R^16a is selected from the group consisting of -SO₂R^16c and -CONR^16dR^16e;

R^16d and R^16b are each independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl; or

R¹⁹ is selected from the group consisting of hydrogen, optionally substituted CrCe alkyl, and optionally substituted cycloalkyl;

R²⁰ is selected from the group consisting of hydrogen, optionally substituted C_rC₆ alkyl, and optionally substituted cycloalkyl;

R^21a and R^21b are each hydrogen; or one of R^21a and R^21b is hydrogen and the other is metabolically cleavable group;

or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In another embodiment, compounds of Formula XVIIIb are provided:

r²Y
	H ^^N'r²⁷
_R26a	NH
R¹£
υυλ	?^R3
	*0 XVIIIb

wherein R^1b, R^1b, R³, R^26a, R^26b, R^26c, and R²⁷ hâve the meanings as described above for Formula XVIIIa, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In another embodiment, compounds of Formula XVIIIc are provided:

XVIIIc wherein R^1b, R^1b, R³, R^26a, R^26b, R^26c, and R²⁷ hâve the meanings as described above for Formula XVIIIa, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In certain embodiments, R²⁷ is selected from the group consisting of R27-2, R27-3, R27-5, R27-6, R27-8, R27-9, R27-11, R27-12, R27-14, R27-15, R27-16, R27-17, R27-19, R27-20, R27-21, R27-22, R27-24, R27-25, R27-27, R27-29, R27-30, R27-31, and R27-32. In certain embodiments, R²⁷ is selected from the group consisting of R27-2, R27-3, R27-5, and R27-6, R27-8, R27-9, R27-14, R27-15, R27-16, and R27-17. In certain embodiments, R²⁷ is a hydroxycycloalkyl group.

In certain embodiments, R^9a is hydrogen; W² is OH; Z is OH; R⁷ is CrC4 alkyl, e.g., methyl, ethyl, propyl, or isopropyl, or cyclopropyl; R¹⁴, R¹⁹, and R²⁰ are each independently hydrogen, C_rC₄ alkyl, e.g., methyl, ethyl, propyl, or isopropyl, or cyclopropyl; and R^21a and

R^21b are each hydrogen.

In certain embodiments, R^9a is hydrogen, R⁷ is hydrogen, CrC4 alkyl, or cyclopropyl; W² is NHR^11a; R^11a is CrC₄ alkyl, e.g., methyl, trifluoromethyl, ethyl, propyl, or isopropyl, or 5 cyclopropyl; R¹⁴ is hydrogen, Ci-C4 alkyl, e.g., methyl, ethyl, propyl, or isopropyl, or cyclopropyl; Z is -NHSO2R^16c or -NHC0NHR^16d; and R^16c and R^16d are each independently optionally substituted CrC4 alkyl, e.g., methyl, trifluoromethyl, ethyl, propyl, or isopropyl, or cyclopropyl.

In certain embodiments, compounds of Formulae XIX-XXXIV are provided:

XXIII

XXIV

XXV

XXVI

XXVIII XXIX

XXVII

XXX

XXXI

XXXII

XXXI 11

XXXIV wherein R^1b, R^1c, R³, R^Z6a, R^26b, R^26c, and R²⁷ hâve the meanings as described above in connection with Formula XVIIIa, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In certain embodiments, compounds of Formula XIX are provided, wherein R^1b, R^1c, R³, R^26a, R^26b, R^26c, and R²⁷ hâve the meanings as described above in connection with Formula XVIIIa, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In some embodiments, compounds of Formula XIX are substantially free of one or more other stereoisomers, i.e., compounds of Formulae XX-XXXIV. In some embodiments, compounds of Formula XIX are substantially pure stereoisomers. In some embodiments, compounds of Formula XIX are pure stereoisomers.

In certain embodiments, compounds of Formula XXIII are provided, wherein R^1b, R^1c, R³, R^26a, R^Z6b, R^26c, and R²⁷ hâve the meanings as described above in connection with Formula XVIIIa, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In some embodiments, compounds of Formula XXIII are substantially free of one or more other stereoisomers, i.e., compounds of Formulae XIX-XXII and XXIV-XXXIV. In some embodiments, compounds of Formula XXIII are substantially pure stereoisomers. In some embodiments, compounds of Formula XXIII are pure stereoisomers.

In certain embodiments, compounds of Formula XXVII are provided, wherein R^1b, R^1c, R³, R^26a, R^Z6b, R^26c, and R²⁷ hâve the meanings as described above in connection with Formula XVIIIa, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In some embodiments, compounds of Formula XXVII are substantially free of one or more other stereoisomers, i.e., compounds of Formulae XIX-XXVI and XXVIII-XXXIV. In some embodiments, compounds of Formula XXVII are substantially pure stereoisomers. In some embodiments, compounds of Formula XXVII are pure stereoisomers.

In certain embodiments, compounds of Formula XXIX are provided, wherein R^1b, R^1c, R³, R^26a, R^26b, R^26c, and R²⁷ hâve the meanings as described above in connection with Formula XVIIIa, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In some embodiments, compounds of Formula XXIX are substantially free of one or more other stereoisomers, i.e., compounds of Formulae XIX-XXVIII and XXX-XXIV. In some embodiments, compounds of Formula XXIX are substantially pure stereoisomers. In some embodiments, compounds of Formula XXIX are pure stereoisomers.

In certain embodiments, compounds of Formulae XIX, XXIII, XXVII, and XXIX are provided, wherein R²⁷ is selected from the group consisting of:

0R^9a

R⁷

R27-34 >

NR^11aR^11b

0R^9a ^n-T^^NR11aR11b

R⁷

R27-36

R¹⁴

OR¹⁵

R¹⁴

•OR¹⁵ »

R27-37

R27-38

R¹⁴

R27-40

Vx^-NHR¹⁶⁸r’¹⁴

R¹⁴ »

R27-41

R27-42

R27-39

16a

'NHR¹⁶®

R¹⁴

R27-44

R27-45 R 27 -46

R27-47

R27-48

R27-49 R27-50 R27-51

R27-52

y or¹⁵	i OR¹⁵ 'V'R .	y or¹⁵	y or¹⁵ * ^f'*_t F VS¹⁴
		R27-55	R27-56
R27-53	R27-54
/ NHR^16a M- .	i NHR^iea >/'-R» .	J NHR^10a	J NHR^1Sa and VV⁴
R27-57	R27-58	R27-59	R27-60

wherein:

R⁷is C1-C4 alkyl;

R^9a and R¹⁰ are hydrogen; or one of R^9a and R¹⁰ is hydrogen and the other is metabolically cleavable group;

R^11a and R^11b are each independently selected from the group consisting of hydrogen, optionally substituted C_rC₄ alkyl, optionally substituted cycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl;

or

R¹⁴ is selected from the group consisting of hydrogen and Ci-C₄ alkyl;

R¹⁵ is hydrogen or a metabolically cleavable group; and

R^16a is selected from the group consisting of -SC^R¹⁶⁰ and -CONR^16dR^16e;

R^16c is selected from the group consisting of optionally substituted Ci-C₄alkyl or cyclopropyl;

R^16d and R^16e are each independently selected from the group consisting of hydrogen, optionally substituted C_rC₄ alkyl or cyclopropyl; or

R^16d and R¹⁶® taken together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclo, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

OR^0a

W²

OR^0a

R27-63

I

OR^0a

X Ni²

R⁷ ~

R27-65

I

R27-66

R⁷ =

R27-68

OR^0a

wherein:

R⁷ is optionally substituted 0^04 alkyl;

W² is selected from the group consisting of-OR¹⁰ and -NR^11aR^11b;

R^0aand R¹⁰ are each hydrogen; or one of R^9a and R¹⁰ is hydrogen and the other is a metabolically cleavable group;

R^11a is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -S0₂R^11c, and -CONR^11dR^11e;

or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

OH

OH ^d“^0H

H₃C

OH

OH ch₃ ch₃ ^ν^2/’··ΌΗ , ^Λ'Χ> ° ^/.....Q-^0H

^/vQ\^{OH 1} ch₃	^/sQr^0H ^οη₃
^zV^oh	. ^zV'^oh

Λ.

V^0H ch₃

ΟΗ

In certain embodiments, compounds of Formulae XIX, XXIII, XXVII, and XXIX are provided, wherein R²⁷ is selected from the group consisting of;

wherein R^11a and R¹¹⁶ taken together with the nitrogen to which they are attached form a 5- or 6-membered optionally substituted heterocyclo.

In certain embodiments, compounds of Formulae XIX, XXIII, XXVII, and XXIX are provided, 10 wherein R²⁷ is selected from the group consisting of;

R¹⁴ is selected from the group consisting of hydrogen and Ci-C₄ alkyl; and

R¹⁵ is a metabolically cleavable group.

In certain embodiments, compounds of Formulae II, VI, X, XII, XIX, XXIII, XXVII, and XXIX are provided, wherein R¹⁵ is a metabolically cleavable group selected from the group consisting of:

O R^29a o ^^x^^vS)'^N'R^29b and s XR—OR^30aR28a¹’ R^2eb OR^30b wherein:

each R^zea and R^28b is îndependently selected from the group consisting of hydrogen, optionally substituted alkyl, and aralkyl;

R^Z9a and R^29b are each selected from the group consisting of hydrogen and optionally substituted alkyl;

vis 1, 2, 3, or4; and

R^30a and R^30b are each selected from the group consisting of hydrogen, optionally substituted alkyl, aralkyl, optionally substituted aryl, and monovalent pharmaceutically acceptable cation; or taken together R^30a and R^30b represent a divalent pharmaceutically acceptable cation or an optionally substituted alkylenyl.

In certain embodiments, R¹⁵ is the residue of a natural or unnatural amino acid. In other embodiments, R¹⁵ is the residue of glycine, îsoleucine alanine, leucine, asparagine, lysine, aspartic acid, méthionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, valine, proline, serine, tyrosine, arginine, and histidine.

In certain embodiments, compounds of Formulae II, VI, X, XII, XIX, XXIII, XXVII, and XXIX are provided, wherein R³ is CrC₁₀ alkyl.

In certain embodiments, compounds of Formulae II, VI, X, XII, XIX, XXIII, XXVII, and XXIX are provided, wherein R³ is selected from the group consisting of -CH2C(CH3)3, CH2C(CH3)2CH2CH3, -CH2C(CH3)2CH2CH2CH3, -CH2C(CH3)2CH2CH2CH2CH31 -CH2C(CH2CH3)2CH3, and -CH2C(CH3)2CH2CH(CH3)2. In certain embodiments, R³ is -CH2C(CH₃)₃.

In certain embodiments, compounds of Formulae II, VI, X, XII, XIX, XXIII, XXVII, and XXIX are provided, wherein R³ is optionally substituted aryl.

In certain embodiments, compounds of Formulae II, VI, X, XII, XIX, XXIII, XXVII, and XXIX are provided, wherein R³ is optionally substituted phenyl.

In some embodiments, compounds of Formulae XIX, XXIII, XXVII, and XXIX are provided wherein R^1b, R^1c, R^26a, R^26b, and R^26c are each independently selected from the group consisting of hydrogen and halogen, e.g., chloro or fluoro, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In certain embodiments, compounds having Formulae VI, X, and XII are provided, wherein: R^1a, R^1b, R^1c, and R^1d are each independently selected from the group consisting of hydrogen, fluoro, and chloro:

R² is selected from the group consisting of aralkyl and:

wherein:

R^25a, R^25b, R^Z5c, R^25d, and R^25e are each independently selected from the group consisting of hydrogen, fluoro, and chloro;

R³ is selected from the group consisting of optionally substituted CrCa alkyl and optionally substituted aryl;

R⁴ is selected from the group consisting of hydrogen and optionally substituted C_rC_e alkyl;

R⁵ is selected from the group consisting of:

and

wherein:

R¹⁴ is selected from the group consisting of hydrogen and optionally substituted C_rC₄ alkyl;

X is selected from the group consisting of O, S, and NR’;

Y is selected from the group consisting of O, S, and NR;

R' is selected from the group consisting of hydrogen and optionally substituted C_rC₄ alkyl; and

R is selected from the group consisting of hydrogen, optionally substituted Ci-04 alkyl, and -COCH₃₁ wherein the compound is substantially free of one or more other stereoisomers, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In some embodiments, R⁴ is hydrogen. In some embodiments, X is NH. In some embodiments, Y is NH. In some embodiments, R³ is -CH₂C(CH₃)₃. In some embodiments, R⁵ is selected from the group consisting of:

OH

4-OH

Me

-OH

Me

In certain embodiments, compounds having Formula XII are provided, wherein:

R^1a, R^1b, R^1c, and R^1d are each independently selected from the group consisting of hydrogen, fluoro, and chloro;

R² is selected from the group consisting of benzyl and:

wherein:

R^25a, R^25b, R^25c, R^25d, and R²⁵® are each independently selected from the group consisting of hydrogen, fluoro, and chloro;

R³ is selected from the group consisting of optionally substituted Ci-Ce alkyl and phenyl;

R⁴ is selected from the group consisting of hydrogen and optionally substituted Ci-C₆ alkyl;

R⁵ is selected from the group consisting of:

-4-O H

R¹⁴

xl-OH

RÎ4

wherein:

R¹⁴ is selected from the group consisting of hydrogen and optionally substituted CrC.t alkyl;

X is selected from the group consisting of O, S, and NR;

Y is selected from the group consisting of O, S, and NR';

R is selected from the group consisting of hydrogen and optionally substituted C_rC₄ alkyl; and

R is selected from the group consisting of hydrogen, optionally substituted C1-C4 alkyl, and -COCH3, wherein the compound is substantially free of one or more other stereoisomers, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In some embodiments, R⁴ is hydrogen. In some embodiments, X is NH. In some embodiments, Y is NH. In some embodiments, R³ is -CH₂C(CH₃)₃. In some embodiments,

R⁵ is selected from the group consisting of:

	,ν_οΗ	Y LL_Oh
OH ''ΌΗ	Me	I Me
Υ'Ό·,	<x4-oh Me	^and Me

In certain embodiments, compounds having Formula XII are provided, wherein:

R^1a is hydrogen;

R^1b, R^1c, and R^1d are each independently selected from the group consisting of hydrogen, fluoro, and chloro;

R² is:

wherein:

R^25a, R^25b, R^Z5c, R^Z5d, and R^25e are each independently selected from the group consisting of hydrogen, fluoro, and chloro;

R³ is C₄-Ca alkyl;

R⁴ is hydrogen;

R⁵ is selected from the group consisting of:

A. ¹ k i	v . M-oh	A. I—L,_Oh
^0H ''OH	Me	1 Me
\^OH ΌΗ	Me	^and Me

; and

X and Y are NH, wherein the compound is substantially free of one or more other stereoisomers, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In some embodiments, R⁵ is selected from the group consisting of:

In some embodiments, compounds having Formula XXXV:

XXXV are provided, wherein:

R³ is C₄-C₈ alkyl; and

R^25a, R^25b, and R^25c are each independently selected from the group consisting of hydrogen, fluoro, and chloro, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

In some embodiments, compounds having Formula XXXV are substantially free of one or more other stereoisomers. In some embodiments, compounds having Formula XXXV are substantially pure stereoisomers. In some embodiments, compounds having Formula XXXV are pure stereoisomers.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable sait or solvaté thereof are provided. In some embodiments, compounds having the following structure:

H H or a pharmaceutically acceptable sait or solvaté thereof are provided.

In some embodiments, compounds having the following structure:

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable sait or solvaté thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable sait or solvaté thereof are provided. In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable sait or solvaté thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable sait or solvaté thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable sait or solvaté thereof are provided, werein the compound is substantially free of one or more other stereoisomers.

In certain embodiments, compounds having the following structure:

In certain embodiments, a compound having the following structure:

werein the compound is a substantially pure stereoisomer.

In certain embodiments, a compound having the following structure:

or a pharmaceutically acceptable sait or solvaté thereof is provided, werein the compound is a pure stereoisomer.

In certain embodiments, methods of preparing a compound having Formula XXXVII:

are provided, wherein:

R³² is selected from the group consisting of -OR³³ and -NR^34aR^34b;

R³³ is selected from the group consisting of hydrogen, alkyl, and aralkyl;

R^34a is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, aralkyl, optionally substituted aryl, and optionally substituted heteroaryl;

R^34b is selected from the group consisting of hydrogen and alkyl;

R² is selected from the group consisting of aralkyl and:

R²⁵⁹

R^25a, R^25b, R^ZSe, R^25d, and R^25e are each independently selected from the group consisting of hydrogen, fluoro, and chloro; and

R³ is selected from the group consisting of optionally substituted C^Ca alkyl and optionally substituted aryl.

In one embodiment, the method of preparing a compound having Formula XXXVII comprises allowing a compound having Formula XXXVI:

to isomerize to a compound having Formula XXXVII:

XXXVI

XXXVII

wherein R^1a, R^1b, R^1c, R^1d, R², R³, and R³² hâve the meanings as described above in connection with Formula XXXVII.

In one embodiment, the method of preparing a compound having Formula XXXVII comprises dissolving a compound having Formula XXXVI:

XXXVI in a solvent or a mixture of solvents, wherein R^1a, R^1b, R^1c, R^1d, R², R³, and R³² hâve the meanings as described above in connection with Formula XXXVII.

In one embodiment, the method of preparing a compound having Formula XXXVII comprises:

dissolving a compound having Formula XXXVI:

XXXVI in a solvent or a mixture of solvents; and

b) allowing the compound having Formula XXXVI to isomerize to a compound having Formula XXXVII, wherein R^1a, R^1b, R^1d, R^1d, R², R³, and R³² hâve the meanings as described above in connection with Formula XXXVII.

In one embodiment, the method of preparing a compound having Formula XXXVII:

comprises:

allowing the compound having Formula XXXVI:

XXXVI to isomerize to a compound having Formula XXXVII; and

b) isolating the compound having Formula XXXVII substantially free from the compound having Formula XXXVI, and one or more other stereoisomers, wherein R^1a, R^1b, R^1c, R^1d, R², R³, and R³² hâve the meanings as described above in connection with Formula XXXVII.

In one embodiment, the method of preparing a compound having Formula XXXVII:

comprises:

dissolving a compound having Formula XXXVI:

in a solvent or a mixture of solvents;

b) allowing the compound having Formula XXXVI to isomerize to a compound having Formula XXXVII; and

XXXVI

c) isolating the compound having Formula XXXVII substantially free from the compound having Formula XXXVI, and one or more other stereoisomers, wherein R^1a, R^1b, R^1e, R^1d, R², R³, and R³² hâve the meanings as described above in connection with Formula XXXVII.

In one embodiment, the solvent is selected from the group consisting of acetonitrile, methanol, ethyl acetate, and water, or a mixture thereof.

In one embodiment, the isomerization is carried out at a pH of less than 7, e.g., at a pH of about 6, about 5, about 4, about 3, about 2, or about 1. In one embodiment, the isomerization is carried out at a pH of about 7. In one embodiment, the isomerization is carried out at a pH of greater than 7, e.g., at a pH of about 8, about 9, about 10, about 11, about 12, or about 13.

In one embodiment, the isomerization is carried out in the presence of an acid, e.g., trifluoroacetic acid or acetic acid.

In one embodiment, the isomerization is carried out in the presence of a base, e.g., NaHCO₃.

In one embodiment, isomerization is carried out at a température of about 20°C to about 100°C, e.g., at a température of about 25°C to about 70°C, e.g., at a température of about 45°C to about 65°C. In one embodiment the isomerization is carried out at about room température, e.g., at about 25°C. In one embodiment the isomerization is carried out above room température, e.g., at about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, or about 100°C.

In one embodiment, the isomerization is carried about for a period of time between about 0.5 hours and about 2 weeks, e.g., for about 1 hour, about 3 hours, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 1 week. The period of time needed for isomerization to occur may dépend on a variety of factors including the chemical structure of Formula XXXVI, the solvent(s), the température, and/or the pH.

In one embodiment, R³² is -OR³³.

In one embodiment, R³² is -NR^34aR^34b.

In one embodiment, R^34b is hydrogen and R^34a is selected from the group consisting of alkyl, hydroxyalkyl, hydroxycycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl.

In one embodiment, R^34b is hydrogen and R^34a is selected from the group consisting of:

In one embodiment, the compound having Formula XXXVII is isolated as a substantially pure stereoisomer. In one embodiment, the compound having Formula XXXVII is isolated as a pure stereoisomer.

In one embodiment,

R³² is -NR^34aR^34b;

R^34a is:

R^34b is hydrogen;

R² is:

R³ is Ci-C₈ alkyl.

In one embodiment, a method of preparing MI-77301 comprising allowing MI-773 to isomerize to MI-77301 is provided.

In one embodiment, a method of preparing MI-77301 comprising:

allowing MI-773 to isomerize to MI-77301; and isolating MI-77301 substantially free from one or more other stereoisomers, is provided.

In one embodiment, a method of preparing MI-77301 comprising:

dissolving MI-773 in a solvent or a mixture of solvents;

allowing MI-773 to isomerize to MI-77301; and isolating MI-77301 substantially free from one or more other stereoisomers,

S.

is provided.

The compounds and processes provided herein will be better understood in connection with the following synthetic schemes which illustrate the methods by which the compounds provided herein may be prepared. Starting materials can be obtained from commercial 5 sources or prepared by well-established literature methods known to those of ordinary skill in the art. It will be readily apparent to one of ordinary skill in the art that the compounds defined above can be synthesized by substitution of the appropriate reagents and agents in the synthèses shown below.

Compounds of Formula la wherein Y is NH can be synthesized as described in Schemes 2 10 and 3.

Scheme 2

CAN 2.5 equiv

THF/H₂O or Pb(OAc)₄

Formula la (wherein Y is NH)

Scheme 3

tfa/ch₂ci₂


	R² k
NBS, THF/H₂O	R^1a NH R^1b k 3 R = p-OMeBn-, Bn-, Me-or other alkyl group
or DDQ, THF/HjO
JL JL ρΐο-χγ-χ
	RKJ
	Formula la (wherein Y is NH)

Compounds of Formula la can be separated by chiral resolution methods well known in the art, e.g., chiral column chromatography, to give compounds of Formulae ll-XVII. Suitable chiral columns for use in chiral resolutions include, for example, Daicel CHIRALCEL® OD-H, 5 Daicel CHIRAKPAK® AD-H and Regis Technologies ULMO chiral columns. Other chiral resolution methods are also possible. Compounds of Formulae ll-XVil can also be prepared by asymmetric synthetic methods. For example, compounds of Formula II, wherein Y is NH, can be synthesized by using a asymmetric 1,3-dipolar cycloaddition as the key step as previously described (See U.S. Patent Nos. 7,759,383 B2 and 7,737,174 B2, and Ding et 10 al., J. Am. Chem. Soc. 127:10130-10131 (2005)) (Scheme 4).

Scheme 4

A

B

F

Formula II (wherein Y = NH)

Reagents and conditions: a) CH₂CI₂-CH₃CN, KF-AI₂O₃, microwave, or methanol, piperidine reflux; b) 4A molecular sieves, toluene, 70°C; c) HNR⁴R⁶, r.t.; d) Pb(OAc)₄, CH₂CI₂-MeOH (1:1), 0“C, or ammonium cerium(IV) nitrate (CAN), CH₃CN, K₂CO_3l r.t.

Briefly, compound A reacts with aldéhyde B to give C. Compound C reacts with aldéhyde E and compound D to give F (a compound of Formula I wherein R is aralkyl). Treatment of F 5 with Pb(OAc)₄ or CAN gives the compound of Formula II wherein Y is NH,

Compounds of Formula XII can be prepared via isomerization of compounds of Formula II. Without intending to be bound by theory, the isomerization of a compound having Formula II to a compound having Formula XII (and other isomers, including compounds having Formula VI) may involve formation of the imine intermediate shown in Scheme 5. 10 Compounds of Formula XII may be less likely to isomerize, Le., they may be chemically more stable, than compounds of Formula II.

Scheme 5

(wherein Y = NH) isomerization

other isomers

X Formula XII / (wherein Y = NH)

imine intermediate

Methods

In some embodiments, compounds provided herein 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 signais. Therefore, it is contemplated that these compounds sensitize cells to induction of cell cycle arrest and/or apoptosis, including cells that are résistant to such inducing stimuli. By inhibiting the interaction between p53 or p53related proteins and MDM2 or MDM2-realted proteins, the compounds provided herein can be used to induce apoptosis in any disorder that can be treated, ameliorated, or prevented by the induction of apoptosis. In one embodiment, the inhibitors can be used to induce apoptosis in cells comprising functional p53 or p53-related proteins.

In another embodiment, the disclosure pertains to modulating apoptosis with compounds provided herein in combination with one or more additional apoptosis-modulating agents. Examples of apoptosis-modulating agents include, but are not limited to, Fas/CD95, TRAMP, TNF RI, DR1, DR2, DR3, DR4, DR5, DR6, FADD, RIP, TNFa, Fas ligand, TRAIL, antibodies 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 dégradation phase of apoptosis are also included. Examples of apoptosis-modulating agents include agents, the activity, presence, or change in concentration of which, can modulate apoptosis in a subject. Apoptosis-modulating agents include those which are inducers of apoptosis, such as TNF or a TNF-related ligand, particularly a TRAMP ligand, a Fas/CD95 ligand, a

TNFR-1 ligand, orTRAIL.

In some embodiments, the compounds, compositions, and methods provided herein, încluding the methods comprising pulsatile dose administration, are used to treat diseased cells, tissues, organs, or pathological conditions and/or disease states in an animal (e.g., a mammalian patient încluding, but not limited to, humans and veterinary animais). In this regard, various diseases and pathologies are amenable to treatment or prophylaxis using the présent methods and compositions. A non-limiting exemplary list of these diseases and conditions includes, but is not limited to, breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small 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, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourînary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, rénal cell carcinoma, endométrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperpiasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia (CLL) încluding B-CLL, acute myelogenous leukemia, chronic myeiogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, sarcoma such as liposarcoma, malignant fibrous histiocytoma, osteosarcoma, Ewing’s sarcoma, leiomyosarcoma, and rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, nonHodgkin's lymphoma, soft-tissue sarcomas such as lipoma, and malignant Schwannoma, ostéogénie sarcoma, primary macroglobulinemia, and retinoblastoma, and the like, T and B cell mediated autoimmune diseases; inflammatory diseases; infections; hyperproliferative diseases; AIDS; degenerative conditions, vascular diseases, and the like. In some embodiments, the cancer cells being treated are metastatic. In other embodiments, the cancer cells being treated are résistant to other anticancer agents.

In some embodiments, the compounds, compositions, and methods provided herein, încluding the methods comprising pulsatile dose administration, are used to treat, ameliorate, or prevent a cancer selected from the group consisting of melanoma, lung cancer, a sarcoma, colon cancer, prostate cancer, choriocarcinoma, breast cancer, retinoblastoma, stomach carcinoma, acute myeloid leukemia, a lymphoma, multiple myeloma, and a leukemia in a patient.

In some embodiments, the compounds, compositions, and methods provided herein, including the methods comprising pulsatile dose administration, are used to treat, ameliorate, or prevent melanoma in a patient.

In some embodiments, the compounds, compositions, and methods provided herein, including the methods comprising pulsatile dose administration, are used to treat, ameliorate, or prevent liposarcoma in a patient.

In some embodiments, the compounds, compositions, and methods provided herein, including the methods comprising pulsatile dose administration, are used to treat cancers that express functional or wild type p53 or p53-related proteins. In some embodiments, the compounds, compositions, and methods provided herein are used to treat cancers that express elevated levels of MDM2 or MDM2-related proteins.

In some embodiments, the methods, compounds, and compositions provided herein, including the methods comprising pulsatile dose administration, can be used to treat a patient having a sarcoma, including, for example, liposarcoma, malignant fibrous histiocytoma, osteosarcoma, and rhabdomyosarcoma. In some embodiments, the methods, compounds, and compositions provided herein, including the methods comprising pulsatile dose administration, can be used to treat a patient having a soft tissue tumor, including, for example, Ewing’s sarcoma, leiomyosarcoma, lipoma, and malignant Schwannomas. In some embodiments, the methods, compounds, and compositions provided herein, including the methods comprising pulsatile dose administration, can be used to treat a patient having lung, breast, liver, or colon cancer. In some embodiments, the methods, compounds, and compositions provided herein, including the methods comprising pulsatile dose administration, can be used to treat a patient having B-cell chronic lymphocytic leukemia and acute myeloid leukemia.

In some embodiments, infections suitable for treatment with the compounds, compositions, and methods provided herein include, but are not limited to, infections caused by viruses, bacteria, fungi, mycoplasma, prions, and the like.

In some embodiments, methods are provided, including the methods comprising pulsatile dose administration, for administering an effective amount of a compound or composition provided herein and at least one additional therapeutic agent (including, but not limited to, chemotherapeutic antineoplastics, apoptosis-modulating agents, antimicrobiais, antivirals, antifungals, and anti-inflammatory agents) and/or therapeutic technique (e.g., surgical intervention, and/or radiothérapies). In a particular embodiment, the additional therapeutic agent(s) is an anticancer agent.

A number of suitable therapeutic or anticancer agents are contemplated for use in the methods provided herein, including the methods comprising pulsatile dose administration.

Indeed, the methods provided 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., enzymes and antibodies); biological mimetics (e.g., gossypol or BH3 mimetics); agents that bind (e.g., 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., antibodies conjugated with anticancer drugs, toxins, defensins), toxins; radionuclides; biological response modifiers (e.g., interferons (e.g., IFN-α) and interleukine (e.g., IL-2)); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell différentiation (e.g., all-trans-retinoic acid); gene therapy reagents (e.g., antisense therapy reagents and nucléotides); 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 thérapies suitable for co-administration with the disclosed compounds are known to those skilled in the art.

In certain embodiments, anticancer agents comprise agents that induce or stimulate apoptosis. 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 damaging 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)-related factors (e.g., TNF family receptor proteins, TNF family ligands, TRAIL, antibodies to TRAILR1 or TRAIL-R2); kinase inhibitors (e.g., epidermal growth factor receptor (EGFR) kinase inhibitor. Additional anticancer agents include: vascular growth factor receptor (VGFR) kinase inhibitor, fibroblast growth factor receptor (FGFR) kinase inhibitor, platelet-derived growth factor receptor (PDGFR) kinase inhibitor, and Bcr-Abl kinase inhibitors (such as GLEEVEC)); antisense molécules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, and AVASTIN); anti-estrogens (e.g., raloxifene and tamoxifen); anti-androgens (e.g., flutamide, bicalutamide, finasteride, amînoglutethamide, kétoconazole, 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, DEXONE, HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxyphenbutazone, PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone, PRELONE, and TANDEARIL); and cancer chemotherapeutic drugs (e.g., irinotecan (CAMPTOSAR), CPT11, fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP-16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL); cellular signalîng molécules;

ceramides and cytokines; staurosporine, and the like.

In still other embodiments, the compositions and methods provided herein, including the methods comprising pulsatile dose administration, include one or more compounds provided herein and at least one anti-hyperproliferative or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g., herbs and other plant and/or animal derived compounds).

Alkylating agents suitable for use in the présent compositions and methods include, but are not limited to: 1) nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, 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 (methyl-CCNU); and streptozocin (streptozotocin)); and 5) triazenes (e.g., dacarbazine (DTIC; dimethyltriazenoimidazolecarboxamide).

In some embodiments, antimetabolites suitable for use in the présent compositions and methods include, but are not limited to: 1) folie acid analogs (e.g., methotrexate (amethopterin)); 2) pyrimidine analogs (e.g., fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorode-oxyuridine; FudR), and cytarabine (cytosine arabinoside)); and 3) purine analogs (e.g., mercaptopurîne (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2’-deoxycoformycin)).

In still further embodiments, chemotherapeutic agents suitable for use in the compositions and methods of the présent disclosure include, but are not limited to: 1) vinca alkaloids (e.g., Vinblastine (VLB), vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), 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 (cis-DDP) and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas (e.g., hydroxyurea); 9) methylhydrazine dérivatives (e.g., procarbazine (N-methylhydrazine; MIH)); 10) adrenocortical suppressants (e.g., mitotane (ο,ρ’-DDD) and aminoglutéthimide); 11) 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 propionate and fluoxymesterone); 16) antiandrogens (e.g., flutamide): and 17)gonadotropin-releasing hormone analogs (e.g., leuprolide).

Any oncolytic agent that is routinely used in a cancer therapy context finds use in the compositions and methods of the présent disclosure. For example, the U.S. Food and Drug Administration maintains a formulary of oncolytic agents approved for use in the United States. International counterpart agencies to the U.S.F.D.A. maintain similar formularies.

Table 1 provides a list of exemplary antineoplastic agents approved for use in the U.S. Those skilled in the art will appreciate that the product labels required on ail U.S. approved chemotherapeutics describe approved indications, dosing information, toxicity data, and the like, for the exemplary agents.

Table 1

Aldesleukin (des-alanyl-1, serine-125 human interleukin- 2)	Proleukin
Alemtuzumab (IgG 1k anti CD52 antibody)	Campath
Alitretinoin (9-cis-retinoic acid)	Panretin
Allopurinol (1,5-dihydro-4 H -pyrazolo[3,4-d]pyrimidin-4one monosodium sait)	Zyloprim
Altretamine (N,N,N^,,N'₁N,N_I- hexamethyl-1,3,5-triazine- 2, 4, 6-triamine)	Hexalen
Amifostine (ethanethiol, 2-[(3-aminopropyl)amino]-, dihydrogen phosphate (ester))	Ethyol
Anastrozole (1,3-Benzenediacetonitrile, a, a, a', a'tetramethyl-5“(1H-1,2,4-triazol-1-ylmethyl))	Arimidex
Arsenic trioxide	Trisenox
Asparaginase (L-asparagine amidohydrolase, type EC-2)	Elspar
BCG Live (lyophilized préparation of an attenuated strain of Mycobacterium bovis (Bacillus	TICE BCG

Calmette-Gukin [BCG], substrain Montreal)
bexarotene capsules (4-(1-(5,6,7,8-tetrahydro-3,5,5,8,8pentamethyl-2-napthalenyl) ethenyl] benzoic acid)	Targretin
bexarotene gel	Targretin
Bleomycin (cytotoxic glycopeptide antibiotics produced by Streptomyces verticillus·, bleomycin A₂ and bleomycin B₂)	Blenoxane
Capecitabine (5'-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]cytidine)	Xeloda
Carboplatin (platinum, diammine [1,1- cyclobutanedicarboxylato(2-)-0, O']-,(SP-4-2))	Paraplatin
Carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea)	BCNU, BiCNU
Carmustine with Polifeprosan 20 Implant	Gliadel Wafer
Celecoxib (as 4-[5-(4-methylphenyl)-3- (trifluoromethyl)- 1 H-pyrazol-1-yl] benzenesulfonamide)	Celebrex
Chlorambucil (4-[bis(2chlorethyl)amino]benzenebutanoic acid)	Leukeran
Cisplatin (PtCI₂H₆N₂)	Platinol
Cladribine (2-chloro-2'-deoxy-b-D-adenosine)	Leustatin, 2-CdA
Cyclophosphamide (2-[bis(2-chloroethyl)amino] tetrahydro-2H13,2-oxazaphosphorine 2-oxide monohydrate)	Cytoxan, Neosar
Cytarabine	Cytosar-U

( 1 -b-D-Arabinofuranosylcytosine, CgHi₃N₃O₅)
cytarabine liposomal	DepoCyt
Dacarbazine (5-(3,3-dimethyl-l-triazeno)-imidazole-4carboxamide (DTIC))	DTIC-Dome
Dactinomycin, actinomycin D (actinomycin produced by Streptomyces parvullus, CeïHaeNiîOie)	Cosmegen
Darbepoetin alfa (recombinant peptide)	Aranesp
daunorubicin liposomal ((8S-cis)-8-acetyl-10-[(3-amino-2,3,6trideoxy-â-L-lyxo-hexopyranosyl)oxy]7,8,9,10-tetrahydro-6,8,11-tri hydroxy-1methoxy-5,12-naphthacenedione hydrochloride)	DanuoXome
Daunorubicin HCl, daunomycin ((1 S ,3 S )-3-Acetyl-1,2,3,4,6,11-hexahydro3,5,12-trihydroxy-10-methoxy-6,11 -dioxo-1 naphthacenyl 3-amino-2,3,6-trideoxy-(alpha)L- lyxo -hexopyranoside hydrochloride)	Cerubidine
Denileukin diftitox (recombinant peptide)	Ontak
Dexrazoxane ((S)-4,4'-(1 -methyl-1,2-ethanediyl)bis-2,6- piperazinedione)	Zinecard
Docetaxel ((2R,3S)-N-carboxy-3-phenylisoserine, N-tertbutyl ester, 13-ester with 5b-20-epoxy12a,4,7b,10b,13a-hexahydroxytax-11-en-9one 4-acetate 2-benzoate, trihydrate)	Taxotere
Doxorubicin HCl (8S, 10S)-10-[(3-amino-2,3,6-trideoxy-a-Llyxo-hexopyranosyl)oxy] -8-glycolyl-7,8,9,10-	Adriamycin, Rubex

tetrahydro-6,8,11- trihydroxy-1-methoxy-5,12naphthacenedione hydrochloride)
doxorubicin	Adriamyctn PFS Intravenous injection
doxorubicin liposomal	Doxil
dromostanolone propionate (17b-Hydroxy-2a-methyl-5a-androstan-3-one propionate)	Dromostanolone
dromostanolone propionate	Masterone injection
Elliott's B Solution	Elliott's B Solution
Epirubicin ((8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-Larabino- hexopyranosyl)oxy]-7,8,9,10tetrahydro-6,8,11-trihydroxy-8(hydroxyacetyl)-l -methoxy-5,12naphthacenedione hydrochloride)	Ellence
Epoetin alfa (recombinant peptide)	Epogen
Estramustine (estra-1,3,5( 10)-triene-3,17-diol( 17(beta))-, 3[bis(2-chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium sait, monohydrate, or estradiol 3-[bis(2-chloroethyl)carbamate] 17(dihydrogen phosphate), disodium sait, monohydrate)	Emcyt
Etoposide phosphate (4'-Demethylepipodophyllotoxin 9-[4,6-O-(R)ethylidene-(beta)-D-glucopyranoside], 4'(dihydrogen phosphate))	Etopophos
etoposide, VP-16 (4'-demethylepipodophyllotoxin 9-(4,6-0-( R)ethylidene-(beta)-D-glucopyranoside])	Vepesid
Exemestane	Aromasin

(6-methylenandrosta-1,4-diene-3, 17-dione)
Filgrastim (r-metHuG-CSF)	Neupogen
floxuridine (intraarterial) (2'-deoxy-5-fluorouridine)	FUDR
Fludarabine (fluorinated nucléotide analog of the antiviral agent vidarabine, 9-b -Darabinofuranosyladenine (ara-A))	Fludara
Fluorouracil, 5-FU (5-fluoro-2,4(1H,3H)-pyrimidinedione)	Adrucil
Fulvestrant (7-alpha-[9-(4,4,5,5,5-penta fluoropentylsulphinyl) nonyl]estra-1,3,5-( 10)triene-3,17-beta-diol)	Faslodex
Gemcitabine (2’-deoxy-2', 2'-difluorocytidine monohydrochloride (b-isomer))	Gemzar
Gemtuzumab Ozogamicin (anti-CD33 hP67.6)	Mylotarg
Goserelin acetate	Zoladex Implant
Hydroxy u rea	Hydrea
Ibritumomab Tiuxetan (immunoconjugate resulting from a thiourea covalent bond between the monoclonal antibody Ibritumomab and the linker-chelator tiuxetan [N-[2-bis(carboxymethyl)amino]-3-(pisothiocyanatophenyl)- propyl]-[N-[2bis(carboxymethyl)amino]-2-(methyl) ethyljglycine)	Zevalin
Idarubicin (5, 12-Naphthacenedione, 9-acetyl-7-[(3amino-2,3₁6-trideoxy-(alpha)-L- lyxo hexopyranosyl)oxy]-7,8,9,10-tetrahydro- 6,9,11-trihydroxyhydrochloride, (7S- cis ))	Idamycin

Ifosfamide (3-(2-chloroethyl)-2-[(2chloroethyl)amino]tetrahydro-2H-1,3,2oxazaphosphorine 2-oxide)	IFEX
Imatinib Mesilate (4- [(4- Methyl-1 -piperazin yl )meth yl]- N- [4methyl-3-[[4-(3-pyridinyl)-2pyrimidinyl]amino]-phenyl]benzamide methanesulfonate)	Gleevec
Interferon alfa-2a (recombinant peptide)	Roferon-A
Interferon alfa-2b (recombinant peptide)	Intron A (Lyophïlized Betaseron)
Irinotecan HCl ((4S)-4,11-diethyl-4-hydroxy-9-[(4- piperidinopiperidino)carbonyloxy]-1 H-pyrano[3', 4': 6,7] indolizino[1,2-b] quinoline-3,14(4H, 12H) dione hydrochloride trïhydrate)	Camptosar
Letrozole (4,4'-( 1H-1,2,4 -T rïazol-1-ylmethylene) dibenzonitrile)	Femara
Leucovorîn (L-Glutamic acid, N[4[[(2amino-5-formyl- 1,4,5,6,7,8 hexahydro4oxo6- pteridinyl)methyl]amino]benzoyl], calcium sait (1:1))	Wellcovorin, Leucovorîn
Levamisole HCl ((-)-( 3)-2,3,5, 6-tetrahydro-6-phenylimidazo [2,1-b] thiazole monohydrochloride CnH^NïS-HCI)	Ergamisol
Lomustine (1 -(2-chloro-ethyl)-3-cyclohexyl-1 - nitrosourea)	CeeNU
Meclorethamine, nitrogen mustard	Mustargen

(2-chloro-N-(2-chloroethyl)-Nmethylethanamine hydrochloride)
Megestrol acetate 17a( acetyloxy)- 6- methylpregna- 4,6- diene- 3,20- dione	Megace
Melphalan, L-PAM (4-[bis(2-chloroethyl) amino]-L-phenylalanine)	Alkeran
Mercaptopurine, 6-MP (1,7-dihydro-6 H -purine-6-thione monohydrate)	Purinethol
Mesna (sodium 2-mercaptoethane sulfonate)	Mesnex
Methotrexate (N-[4-[[(2,4-diamino-6pteridinyl)methyl]methylamino]benzoyl]-Lglutamic acid)	Methotrexate
Methoxsalen (9-methoxy-7H-furo[3,2-g][1]-benzopyran-7one)	Uvadex
Mitomycin C	Mutamycin
mitomycin C	Mitozytrex
Mitotane (1,1-dichloro-2-(o-chlorophenyl)-2-(p- chlorophenyl) ethane)	Lysodren
Mitoxantrone (1,4-dihydroxy-5,8-bis[[2- [(2hydroxyethyl)amino]ethyl]amino]-9,10anthracenedione dihydrochloride)	Novantrone
Nandrolone phenpropionate	Durabolin-50
Nofetumomab	Verluma
Oprelvekîn (IL-11)	Neumega
Oxaliplatin (cis-[(1 R,2R)-1,2-cyclohexanediamine-N,N’] [oxalato(2-)-0,0'] platinum)	Eloxatin


Paclitaxel (5β, 20-Epoxy-1,2a, 4,7β, 10β, 13ahexahydroxytax-11-en-9-one 4,10-dîacetate 2- benzoate 13-ester with (2R, 3 S)- Nbenzoyl-3-phenylisoserine)	TAXOL
Pamidronate (phosphonic acid (3-amino-1hydroxypropylidene) bis-, disodium sait, pentahydrate, (APD))	Aredia
Pegademase ((monomethoxypolyethylene glycol succinimidyl) 11-17 -adenosine deaminase)	Adagen (Pegademase Bovine)
Pegaspargase (monomethoxypolyethylene glycol succinimidyl L-asparaginase)	Oncaspar
Pegfilgrastim (covalent conjugate of recombinant methionyl human G-CSF (Filgrastim) and monomethoxypolyethylene glycol)	Neulasta
Pentostatin	Nipent
Pipobroman	Vercyte
Plicamycin, Mithramycin (antibiotic produced by Streptomyces plicatus)	Mithracin
Porfimer sodium	Photofrin
Procarbazine (N-isopropyl-p-(2-methylhydrazino)-ptoluamide monohydrochloride)	Matulane
Quinacrine (6-chloro-9-( 1 -methyl-4-diethyl-amine) butylamino-2-methoxyacridine)	Atabrine

Rasburicase (recombinant peptide)	Elitek
Rituximab (recombinant anti-CD20 antibody)	Rituxan
Sargramostim (recombinant peptide)	Prokine
Streptozocin (streptozocin 2 -deoxy - 2 [[(methylnitrosoamino)carbonyl]amino] a(and b ) - D - glucopyranose and 220 mg citric acid anhydrous)	Zanosar
Talc (Mg₃Si₄O₁₀ (OH)₂)	Sclerosol
Tamoxifen ((Z)2-[4-( 1 _t2-diphenyl-1 -butenyl) phenoxy]-N, N-dimethylethanamine 2-hydroxy-1,2,3propanetricarboxylate (1:1))	Nolvadex
Temozolomide (3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]as-tetrazine-8-carboxamide)	Temodar
teniposide, VM-26 (4'-demethylepipodophyllotoxin 9-[4,6-0-(R)- 2- thenylidene-(beta)-D-glucopyranoside])	Vumon
T estolactone (13-hydroxy-3-oxo-13,17-secoandrosta-1,4dien-17-oic acid [dgr ]-lactone)	Teslac
Thioguanine, 6-TG (2-amino-1,7-dihydro-6 H - purine-6-thione)	Thioguanine
Thiotepa (Aziridine, Ι,Τ,Γ'-phosphinothioylidynetris-, or Tris (1-aziridinyl) phosphine sulfide)	Thioplex
Topotecan HCl ((S)-10-[(dimethylamino) methyl]-4-ethyl-4,9dihydroxy-1H-pyrano[3', 4': 6,7] indolizino [1,2-b] quinolîne-3,14-(4H, 12H)-dione	Hycamtin

monohydrochloride)
Toremifene (2-(p-[(Z)-4-chloro-1,2-d i pheny 1-1 -butenyl]phenoxy)-N,N-dimethylethylamine citrate (1:1))	Fareston
Tositumomab, 1131 Tositumomab (recombinant murine immunotherapeutic monoclonal lgG_2a lambda anti-CD20 antibody (I 131 is a radioimmunotherapeutic antibody))	Bexxar
Trastuzumab (recombinant monoclonal IgGi kappa anti- HER2 antibody)	Herceptin
Tretinoin, ATRA (all-trans retinoic acid)	Vesanoid
Uracil Mustard	Uracil Mustard Capsules
Valrubicin, N-trifluoroacetyladriamycin-14valerate ((2S-cis)-2- [1,2,3,4,6,11-hexahydro-2,5,12trihydroxy-7 methoxy-6,11-dioxo-[[4 2,3,6trideoxy-3- [(trifluoroacetyl)-amino-a-L-/yxohexopyranosyl]oxyl]-2-naphthacenyl]-2oxoethyl pentanoate)	Valstar
Vinblastine, Leurocristine (C₄₆H₅₆N₄O₁₀-H₂SO₄)	Velban
Vincristine (Ο₄6Η_5ΘΝ₄Οι₀·Η₂5Ο₄)	Oncovin
Vinorelbine (3’ ,4'-didehydro-4’-deoxy-C’norvincaleukoblastine [R-(R,R)-2,3dihydroxybutanedioate (1:2)(salt)])	Navelbine
Zolédronate, Zoledronic acid ((1-Hydroxy-2-imidazol-1-yl-phosphonoethyl)	Zometa

phosphonic acid monohydrate)

Anticancer agents further include compounds which hâve been identîfied to hâve anticancer activity. Examples include, but are not limited to, 3-AP, 12-0-tetradecanoylphorbol-13acetate, 17AAG, 852A, ABI-007, ABR-217620, ABT-751, ADI-PEG 20, AE-941, AG-013736, AGR0100, alanosine, AMG 706, antibody G250, antineoplastons, AP23573, apaziquone, APC8015, atiprimod, ATN-161, atrasenten, azacitidine, BB-10901, BCX-1777, bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib, bryostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cefixime, cetuximab, CG0070, cilengitide, clofarabine, combretastatin A4 phosphate, CP-675,206, CP-724,714, CpG 7909, curcumin, decitabine, DENSPM, doxercalciferol, E7070, E7389, ecteinascidin 743, efaproxiral, eflornithine, EKB569, enzastaurin, erlotinib, exisulind, fenretinide, flavopiridol, fludarabine, flutamide, fotemustine, FR901228, G17DT, galiximab, gefitinib, genistein, glufosfamide, GTI-2040, histrelin, HKI-272, homoharringtonine, HSPPC-96, hu14.18-interleukin-2 fusion protein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukîn-12, IPI-504, irofulven, ixabepilone, lapatinib, lenalidomide, lestaurtinib, leuprolide, LMB-9 immunotoxin, lonafarnîb, luniliximab, mafosfamide, MB07133, MDX-010, MLN2704, monoclonal antibody 3F8, monoclonal antibody J591, motexafin, MS-275, MVA-MUC1-IL2, nilutamide, nitrocamptothecin, nolatrexed dihydrochloride, nolvadex, NS-9, O6-benzylguanine, oblimersen sodium, ONYX015, oregovomab, OSI-774, panitumumab, paraplatin, PD-0325901, pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone, PS-341, PSC 833, PXD101, pyrazoloacridine, R115777, RAD001, ranpirnase, rebeccamycin analogue, rhuAngiostatin protein, rhuMab 2C4, rosiglitazone, rubitecan, S-1, S-8184, satraplatin, SB-, 15992, SGN-0010, SGN-40, sorafenib, SR31747A, ST1571, SU011248, suberoylanilide hydroxamic acid, suramin, talabostat, talampanel, tariquidar, temsirolimus, TGFa-PE38 immunotoxin, thalidomide, thymalfasin, tipifarnib, tirapazamine, TLK286, trabectedin, trimetrexate glucuronate, TroVax, UCN-1, valprotc 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 Goodman and Gilman’s Pharmaceutical Basis of Therapeutics tenth édition, Eds. Hardman étal., 2002.

In some embodiments, methods provided herein, including the methods comprising pulsatile dose administration, comprise administering one or more compounds provided herein 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 animal may receive photon radiotherapy, particle beam radiation therapy, other types of radiothérapies, and combinations thereof. In some embodiments, the radiation is delivered to the animal using a linear accelerator. In still other embodiments, the radiation is delivered using a gamma knife.

The source of radiation can be external or internai to the animal. External radiation therapy is most common and involves directing a beam of high-energy radiation to a tumor site through the skin using, for instance, a linear accelerator. While the beam of radiation is localized to the tumor site, it is nearly impossible to avoid exposure of normal, healthy tissue. However, external radiation is usually well tolerated by animais. Internai radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, particles, and the like, inside the body at or near the tumor site including the use of delivery Systems that specifically target cancer cells (e.g., using particles attached to cancer cell binding ligands). Such implants can be removed following treatment, or left in the body inactive. Types of internai radiation therapy include, but are not limited to, brachytherapy, interstitial irradiation, intracavity irradiation, radioimmunotherapy, and the like.

The animal may optionally receive radiosensitizers (e.g., metronidazole, misonidazole, intraarterial Budr, intravenous iododeoxyuridine (ludR), nitroimidazole, 5-substituted-4nitroimidazoles, 2H-isoindolediones, [[(2-bromoethyl)-amino]methyl]-nitro-1 H-imidazole-1ethanol, nitroaniline dérivatives, DNA-affinic hypoxia sélective cytotoxins, halogenated DNA ligand, 1,2,4 benzotriazine oxides, 2-nitroimidazole dérivatives, fluorine-containing nitroazole dérivatives, benzamide, nicotinamide, acridine-intercalator, 5-thiotretrazole dérivative, 3nitro-1,2,4-triazole, 4,5-dinitroimidazole dérivative, hydroxylated texaphrins, cisplatin, mitomycin, tiripazamine, nitrosourea, mercaptopurine, methotrexate, fluorouracil, bleomycin, vincristine, carboplatin, epirubicin, doxorubicin, cyclophosphamide, vindesine, etoposide, paclitaxel, heat (hyperthermia), and the like), radioprotectors (e.g., cysteamine, aminoalkyl dihydrogen phosphorothioates, amifostine (WR 2721), IL-1, IL-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 an animal, so long as the dose of radiation is tolerated by the animal without unacceptable négative 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 hâve sufficient energy to produce ionization, r.e., gain or loss of électrons (as described in, for example, U.S. 5,770,581 incorporated herein by reference in its entirety). The effects of radiation can be at least partially controlled by the clinician. In one embodiment, the dose of radiation is fractionated for maximal target cell exposure and reduced toxicity.

In one embodiment, the total dose of radiation 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 complété 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 1-8 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,

Gy, 4.2 Gy, or 4.5 Gy), or 1-2 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 animal to rest and the effects of the therapy to be realized. For example, radiation desirably is administered on consecutive days, and not administered on 2 days, for each week of treatment, thereby allowing 2 days of rest per week. However, radiation can be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5 days/week, 6 days/week, or ail 7 days/week, depending on the animal’s responsiveness and any potential side effects. Radiation therapy 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 therapeutic period comprising 6 weeks for treating, for instance, a solid tumor. Alternative^, radiation is administered in weeks 1-5 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 therapeutic agents may also be used as therapeutic agents in combination with the compounds provided herein. Any agent that can kill, inhibit, or otherwise attenuate the function of microbial organisms may be used, as well as any agent contemplated to hâve 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.

In some embodiments of the methods provided herein, one or more compounds provided herein and one or more therapeutic agents or anticancer agents are administered to an animal under one or more of the following conditions: at different periodicities, at different durations, at different concentrations, by different administration routes, etc. In some embodiments, the compound is administered prior to the therapeutic or anticancer agent, e.g., 0.5, 1,2, 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 therapeutic or anticancer agent. In some embodiments, the compound is administered after the therapeutic or anticancer agent, e.g., 0.5, 1, 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 administration of the anticancer agent. In some embodiments, the compound and the therapeutic or anticancer agent are administered concurrently but on different schedules, e.g., the compound is administered daily while the therapeutic or anticancer agent is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In other embodiments, the compound is administered once a week while the therapeutic or anticancer agent is administered daily, once a week, once every two weeks, once every three weeks, or once every four weeks.

In some embodiments, compositions provided herein comprise one or more of the compounds provided herein in an amount which is effective to achieve its intended purpose. While individual needs vary, détermination 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 équivalent amount of the pharmaceutically acceptable sait 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, ameliorate, or prevent such disorders. For intramuscular injection, the dose is generally about one-half of the oral dose. For example, a suitable intramuscular dose would be about 0.0025 to about 25 mg/kg, or from about 0.01 to about 5 mg/kg.

The unit oral dose may comprise from about 0.01 to about 1000 mg, for example, about 0.1 to about 100 mg of the compound. The unit dose may be administered one or more times daily as one or more tablets or capsules each containing from about 0.1 to about 10 mg, conveniently about 0.25 to 50 mg of the compound or its solvatés.

In a topical formulation, the compound may be présent at a concentration of about 0.01 to 100 mg per gram of carrier. In a one embodiment, the compound is présent at a concentration of about 0.07-1.0 mg/ml, for example, about 0.1-0.5 mg/ml, and in one embodiment, about 0.4 mg/ml.

In addition to administering the compound as a raw chemical, the compounds provided herein may be administered as part of a pharmaceutical préparation or composition. In some embodiments, the pharmaceutical préparation or composition can include one or more pharmaceutically acceptable carriers, excipients, and/or auxiliaries. In some embodiments, the one or more carriers, excipients, and auxiliaries facilitate processing of the compound into a préparation or composition which can be used pharmaceutically. The préparations, particularly those préparations which can be administered orally or topically and which can be used for one type of administration, such as tablets, dragees, slow release lozenges and capsules, mouth rinses and mouth washes, gels, liquid suspensions, haïr rinses, hair gels, shampoos and also préparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by intravenous infusion, injection, topically or orally, contain from about 0.01 to 99 percent, in one embodiment from about 0.25 to 75 percent of active compound(s), together with the one or more carriers, excipients, and/or auxiliaries.

The pharmaceutical compositions of provided herein may be administered to any patient which may expérience the bénéficiai effects of the compounds provided herein. 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 include veterinary animais (cows, sheep, pigs, horses, dogs, cats and the like).

The compounds and pharmaceutical compositions thereof may be administered by any means that achieve their intended purpose. For example, administration may be by patenterai, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal or topical routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dépendent upon the âge, health, and weight of the récipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

The pharmaceutical préparations provided herein are manufactured by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical préparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides, for example lactose or sucrose, mannitol or sorbitol, cellulose préparations 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 starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a sait thereof, such as sodium alginate. Auxiliaries can be suitable flow-regulating agents and lubricants. Suitable auxiliaries include, for example, silica, talc, stearic acid or salts thereof, such as magnésium stéarate or calcium stéarate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings which, if desired, are résistant to gastric juices. For this purpose, concentrated saccharide solutions may be used, which may optionally contain gum arable, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings résistant to gastric juices, solutions of suitable cellulose préparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose 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 préparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnésium stéarate and, optionally, stabilizers. In soft capsules, the active compounds are in one embodiment dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin. In addition, stabilizers may be added.

Possible pharmaceutical préparations 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 triglycérides, or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base materials include, for example, liquid triglycérides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parentéral 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 lipophilie solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycérides or polyethylene glycol-400. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. The topical compositions provided herein are formulated in one embodiment as oils, creams, lotions, ointments and the like by choice of appropriate carriers. Suitable carriers include vegetable or minerai oils, white petrolatum (white soft paraffin), branched chain fats or oils, animal fats and high molecular weight alcohol (greater than C12). The carriers may be those in which the active ingrédient is soluble. Emulsifiers, stabilizers, humectants and antioxidants may also be included as well as agents imparting color or fragrance, if desired. Additionally, transdermal pénétration enhancers can be employed in these topical formulations. Examples of such enhancers can be found in U.S. Pat Nos. 3,989,816 and 4,444,762.

Ointments may be formulated by mixing a solution of the active ingrédient in a vegetable oil such as almond oil with warm soft paraffin 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 may be conveniently prepared by dissolving the active ingrédient, in a suitable high molecular weight alcohol such as propylene glycol or polyethylene glycol.

In one embodiment, the présent disclosure relates to methods of treating a patient with a hyperproliferative disease, e.g., cancer, the methods comprising pulsatile administration to the patient one or more compounds or compositions provided herein, or pharmaceutically acceptable salts, solvatés, or prodrugs thereof.

The following examples are illustrative, but not limiting, of the compounds, compositions, 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.

In certain aspects, the following embodiments are provided:

Embodiment I: A method of treating, preventing, or ameliorating cancer in a patient, wherein the method comprises pulsatile administration to the patient a therapeutically effective amount of a compound having Formula XII:

or a pharmaceutically acceptable sait thereof, wherein:

R² is:

wherein:

R^0a, R^6b, R^6c, R^6d, and R^ee are each independently selected from the group consisting of hydrogen, fluoro, and chloro; R³ is optionally substituted C_rCa alkyl;

R⁴ is selected from the group consisting of hydrogen and optionally substituted

Ci-C₆ alkyl;

R⁵ is selected from the group consisting of:

wherein:

R⁷ is selected from the group consisting of hydrogen and optionally substituted

C_rC₄ alkyl;

X is selected from the group consisting of O, S, and NR ;

Y is selected from the group consisting of O, S, and NR’;

R is selected from the group consisting of hydrogen and optionally substituted C1-C4 alkyl, wherein the compound is substantially free of one or more other stereoisomers. Embodiment II: The method of Embodiment I, wherein R⁴ is hydrogen, or a pharmaceutically acceptable sait thereof.

Embodiment lll: The method of Embodiment I, wherein X is NH, or a pharmaceutically acceptable sait thereof.

Embodiment IV: The method of Embodiment I, wherein Y is NH, or a pharmaceutically acceptable sait thereof.

Embodiment V: The method of Embodiment I, wherein R³ is -CH₂C(CH3)3, or a pharmaceutically acceptable sait thereof.

Embodiment VI: The method of Embodiment I, wherein R⁵ is selected from the group consisting of:

or a pharmaceutically acceptable sait thereof. Embodiment VII. The method of Embodiment I, wherein:

R^1a is hydrogen;

R^1b, R^1c, and R^1d are each independently selected hydrogen, fluoro, and chloro;

R³ is C₄-C₈ alkyl;

R⁴ is hydrogen;

R⁵ is selected from the group consisting of:

from the group consisting of

X and Y are NH;

or a pharmaceutically acceptable sait thereof.

Embodiment VIII: The method of Embodiments VI or VII, wherein R⁵ is selected from the group consisting of:

'OH or a pharmaceutically acceptable sait thereof.

Embodiment IX: The method of Embodiment I, wherein the compound of Formula XII is selected from the group consisting of:

ιοο

or a pharmaceutically acceptable sait thereof.

Embodiment X: The method of Embodiment I, wherein the compound of Formula XII is:

or a pharmaceutically acceptable sait thereof.

Embodiment XI: The method of Embodiment I, wherein the compound of Formula XII is:

ΙΟΙ or a pharmaceutically acceptable sait thereof.

Embodiment XII: The method of any one of Embodiments ΙΧ-ΧΙ, wherein the compound is substantially free of other stereoisomers, or a pharmaceutically acceptable sait thereof.

Embodiment XIII: The method of Embodiment XII, wherein the compound is a substantially pure stereoisomer, or a pharmaceutically acceptable sait thereof.

Embodiment XIV: The method of any one of Embodiments l-XIII, wherein the compound is administered to the patient one day a week, one day every two weeks, one day every three weeks, or one day every four weeks.

Embodiment XV: The method of any one of Embodiments l-XIV, wherein cells of the hyperproliferative disease express functional p53.

Embodiment XVI: The method of any one of Embodiments l-XV, wherein the hyperproliferative disease is cancer.

Embodiment XVII.The method of Embodiment XVI, further comprising administering to the patient one or more anticancer agents.

Embodiment XVIII. The method of Embodiment XVII, wherein the anticancer agent is a chemotherapeutic agent.

Embodiment XIX: The method of Embodiment XVIII, wherein the anticancer agent is radiation therapy.

Embodiment XX: A kit comprising a compound having Formula XII:

or a pharmaceutically acceptable sait thereof, wherein:

R² is:

wherein:

_R6a, _R6b _R6c. _Red, _anc| _R6e _{are egch} j_n(j_ep_end_entiy selected from the group consisting of hydrogen, fluoro, and chloro;

102

R³ is optionally substituted C^Ca alkyl;

CrCe alkyl;

R⁵ is selected from the group consisting of:

wherein:

0,-04 alkyl;

X is selected from the group consisting of O, S, and NR ;

Y is selected from the group consisting of O, S, and NR;

R’ is selected from the group consisting of hydrogen and optionally substituted CrC₄ alkyl; and

R is selected from the group consisting of hydrogen and optionally substituted CrC₄ alkyl, wherein the compound is substantially free of one or more other stereoisomers, and instructions for pulsatile administration of the compound to a patient having a hyperproliferative disease.

Embodiment XXI: The kit of Embodiment XX, wherein the hyperproliferative disease is cancer.

EmbodimentXXII:The kit of Embodiment XXI, further comprising one or more anticancer agents.

Embodiment XXIII: The kit of Embodiment XXII, wherein the instructions direct co administration of the compound together with the one or more anticancer agents.

In certain aspects, the following particular embodiments are provided herein:

Embodiment XXIV: A method of treating, ameliorating, or preventing melanoma in a patient comprising admînistering to the patient a therapeutically effective amount of a compound having Formula XII:

wherein:

103

XII

R² is:

R^6b

wherein:

R^6a, R^6b, R^6c, R^6d, and R^6e are each independently selected from the group consisting of hydrogen, fluoro, and chloro;

R³ is optionally substituted CrC₈ alkyl;

Ci-C₈ alkyl;

R⁵ is selected from the group consisting of:

wherein:

R⁷ is selected from the group consisting of hydrogen and optionally substituted C_rC₄ alkyl;

X is selected from the group consisting of O, S, and NR';

Y is selected from the group consisting of O, S, and NR;

R' is selected from the group consisting of hydrogen and optionally substituted C_rC4 alkyl; and

R is selected from the group consisting of hydrogen and optionally substituted C1-C4 alkyl, wherein the compound is substantially free of one or more other stereoisomers, or a pharmaceutically acceptable sait thereof.

Embodiment XXV:The method of Embodiment XXIV, further comprising administering to the patient one or more additional anticancer agents.

Embodiment XXVI:

The method of Embodiment XXV, wherein the anticancer agent is

a chemotherapeutic agent.

Embodiment XXVII:

The method of Embodiment XXVI, wherein the anticancer agent is

radiation therapy.

Embodiment XXVIII: The method of any one of Embodiments XXIV-XXVII, wherein the melanoma is characterized by résistance to conventional cancer therapy.

104

Embodiment XXIX: The method of any one of Embodiments XXIV-XXVIII, wherein the melanoma expresses wild-type p53 protein.

Embodiment XXX:The method of any one of Embodiments XXIV-XXIX, wherein the compound of Formula XII is:

A kit comprising a compound having Formula XII:

Embodiment XXXI:

XII wherein:

R² is:

wherein:

R^6a, R^6b, R^6c, R^6d, and R⁶® are each independently selected from the group consisting of hydrogen, fluoro, and chloro;

R³ is optionally substituted C_rCa alkyl;

CrC₆ alkyl;

R⁵ is selected from the group consisting of:

105 wherein:

R⁷ is selected from the group consisting of hydrogen and optionally substituted C1-C4 alkyl;

X is selected from the group consisting of O, S, and NR;

Y is selected from the group consisting of O, S, and NR;

R is selected from the group consisting of hydrogen and optionally substituted 0,-0^ alkyl; and

R’ is selected from the group consisting of hydrogen and optionally substituted C1-C4 alkyl, wherein the compound is substantially free of one or more other stereoisomers, or a pharmaceutically acceptable sait thereof, and instructions for admînistering the compound to a patient having melanoma. Embodiment XXXII: The kit of Embodiment XXXI, further comprising one or more additional anticancer agents.

Embodiment XXXIII: The kit of Embodiment XXXI, wherein the instructions direct coadministration of the compound together with an additional anticancer agent.

In certain aspects, the following particular embodiments are provided herein: Embodiment XXXIV: A compound having Formulae XII:

XII wherein:

R² is:

wherein:

R^25a, R^2Sb, R^25c, R^25d, and R²⁵® are each independently selected from the group consisting of hydrogen, fluoro, and chloro;

106

R³ is optionally substituted CrCa alkyl;

Ci-C₆ alkyl;

R⁵ is selected from the group consisting of:

wherein:

R¹⁴ is selected from the group consisting of hydrogen and optionally substituted Ci-C₄ alkyl;

X is selected from the group consisting of O, S, and NR';

Y is selected from the group consisting of O, S, and NR”;

R is selected from the group consisting of hydrogen and optionally substituted C_rC₄alkyl; and

R” is selected from the group consisting of hydrogen and optionally substituted C_rC₄alkyl, wherein the compound is substantially free of one or more other stereoisomers, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

Embodiment XXXV: The compound of Embodiment XXXIV, wherein R⁴ is hydrogen, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

Embodiment XXXVI: The compound of Embodiment XXXIV, wherein X is NH, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

Embodiment XXXVII: The compound of Embodiment XXXIV, wherein Y is NH, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

Embodiment XXXVIII: The compound of Embodiment XXXIV, wherein R³ is ΟΗ₂Ο(ΟΗ₃)₃, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

Embodiment XXXIX: The compound of Embodiment XXXIV, wherein R⁵ is selected from the group consisting of:

—1		^/XU0H	L-I-oh
OH	Oh	Me	1 Me
'Xi	^ZO	Me	^Λ-Π ^and Ιχχί-ΟΗ Me

107 or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

Embodiment XL: The compound of Embodiment XXXVIII, wherein:

R^1a is hydrogen;

R^1b, R^1c, and R^1d are each independently selected from the group consisting of

hydrogen, fluoro, and chloro;
R³ is C₄-C_e alkyl; R⁴ is hydrogen; R⁵ is selected from the group consisting of:
	A^	VoH	A LL oh
	''OH	Me	1 Me
	O- ΌΗ	ru	and Q..oh
Me	Me ;and

XandYareNH;

or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

Embodiment XLI: The compound of Embodiments XXXIX or XL, wherein R^s is selected from the group consisting of:

or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

Embodiment XLII: The compound of Embodiment XXXIV selected from the group consisting of:

108

109

or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

Embodiment XLIII: A compound having the structure:

or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

Embodiment XLIV: A compound having the structure:

UO or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

Embodiment XLV:The compound of any one of Embodiments XLII-XLIV, wherein the compound is substantially free of other stereoisomers, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

Embodiment XLVI: The compound of Embodiment XLV, wherein the compound is a substantially pure stereoisomer, or a pharmaceutically acceptable sait, solvaté, or prodrug thereof.

Embodiment XLVII: A pharmaceutical composition comprising the compound of any one of Embodiments XXXIV-XLVI and a pharmaceutically acceptable carrier.

Embodiment XLVIII: A method of treating a patient comprising administering to the patient a therapeutïcally effective amount of the compound or a pharmaceutically acceptable sait thereof of any one of Embodiments XXXIV-XLVI, wherein the patient has a hyperproliferative disease.

Embodiment XLIX: A method of treating a patient comprising administering to the patient a therapeutïcally effective amount of the pharmaceutical composition of Embodiment XLVII, wherein the patient has a hyperproliferative disease.

Embodiment L: The method of Embodiments XLVIII or XLIX, wherein the hyperproliferative disease is cancer.

Embodiment Ll; The method of daims Embodiments XLVIII or XLIX, wherein cells of the hyperproliferative disease express functional p53.

Embodiment LII: The method of Embodiment L, further comprising administering to the patient one or more anticancer agents.

Embodiment LUI: The method of Embodiment LII, wherein the anticancer agent is a chemotherapeutic agent.

Embodiment LIV: The method of Embodiment LII, wherein the anticancer agent is radiation therapy.

Embodiment LV: A method of treating a patient, wherein the patient has a hyperproliferative disorder and is being treated with an anticancer agent, comprising administering to the patient a compound or pharmaceutically acceptable sait thereof of any one of Embodiments XXXIV-XLVI.

Embodiment LVI: The method of Embodiment LV, wherein the patient is experiencing sideeffects of the anticancer agent treatment selected from the group consisting of mucositis, stomatitis, xerostoma, alopecia, and gastrointestinal disorder.

Embodiment LVII: The method of Embodiment LVI, wherein cells of the hyperproliferative disorder express functional p53.

lll

Embodiment LVIII: A kit comprising a compound of any one of Embodiments XXXIVXLVI and instructions for administering the compound to a patient having a hyperproliferative disease.

Embodiment LIX: The kit of Embodiment LVIII, wherein the hyperproliferative disease is cancer.

Embodiment LX: The kit of Embodiment LIX, further comprising one or more anticancer agents.

Embodiment LXI: The kit of Embodiment LX, wherein the instructions direct coadministration of the compound together with the one or more anticancer agents.

EXAMPLE 1

Analytical Data for compounds

General Information

NMR spectra were recorded on a BRUKER AVANCE 250, BRUKER AVANCE 300, BRUKER AVANCE DRX-400, or BRUKER AVANCE DPX-500, or similar instrument Unless otherwise indicated all NMR chemical shifts reported herein are denoted by the delta (□) scale.

Liquid chromatography-mass spectrometry (denoted LC-MS) analysis was performed using method A, method B, or method C:

Method A: WATERS UPLC-SQD apparatus; lonization: electrospray in positive mode and/or négative mode (ES+/-); Chromatographie conditions: Column: ACQUITY BEH C18

1.7 pm - 2.1 x 50 mm; Solvents: A: H₂O (0.1 % formic acid) B: CH₃CN (0.1 % formic acid); Column température: 50 °C; Flow: 1 ml/min; Gradient (2 min): from 5 to 50 % of B in 0.8 min; 1.2 min: 100 % of B; 1.85 min: 100 % of B; 1.95 : 5 % of B; Rétention time = t_R (min).

Method B: WATERS ZQ apparatus; lonization: electrospray in positive mode and/or négative mode (ES+/-); Chromatographie conditions: Column: XBridge C18 2.5 pm - 3 x 50 mm; Solvents: A: H₂O (0.1 % formic acid) B: CH₃CN (0.1 % formic acid); Column température: 70°C; Flow: 0.9 ml/min; Gradient (7 min): from 5 to 100 % of B in 5.3 min; 5.5 min: 100 % of B; 6.3 min: 5 % of B; Rétention time = t_R (min).

Method C: WATERS UPLC-SQD apparatus; lonization: electrospray in positive mode and/or négative mode (ES+/-); Chromatographie conditions: Column: ACQUITY BEH C18

1.7 pm - 2.1 x 50 mm; Solvents: A: H₂O (0.1 % formic acid) B: CH₃CN (0.1 % formic acid); Column température: 50 °C; Flow: 0.8 ml/min; Gradient (2.5 min): from 5 to 100 % of B in

1.8 min; 2.4 min: 100 % of B; 2.45 min: 100 % of B; from 100 to 5 % of B in 0.05 min; Rétention time = t_R (min).

112

Purity analysis was performed using reverse-phase HPLC using a SunFire™ C18 5 pm 4.6 x150 mm column at a flow rate of 1 mL/min under the following conditions: Condition I: Gradient from 90% of solvent A (0.1% of TFA in water) and 10% of solvent B (0.1% of TFA in methanol) to 5% of solvent A and 95% of solvent B in 85 min; and Condition II: Gradient from 80% of solvent A (0.1% of TFA in water) and 20% of solvent B (0.1% of TFA in acetonitrile) to 50% of solvent A and 50% of solvent B in 30 min.

Low resolution ESl mass spectrum analysis was performed on Thermo-Scientific LCQ Fleet mass spectrometer or similar instrument.

The chemical names of the compounds provided in this example were determined with ADCLABS version 12.0.

C027 - TFA sait

¹H NMR (300 MHz, MeOH-d4): 7.50-7.36 (m, 1H), 7.24-7.10 (m, 2H), 6.88-6.76 (m, 3H), 5.12 (d, J = 10.17 Hz, 1H), 4.49 (d, J = 10.17 Hz, 1H), 4.23 (dd, J= 6.83, 2.09 Hz, 1H), 3.983.83 (m, 1H), 2.49-2.36 (m, 1H), 2.36-2.22 (m, 1H), 2.10-1.96 (m, 2H), 1.94-1.82 (m, 1H), 1.35-1.28 (m, 1H), 1.29 (s, 3H), 0.80 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 108.1, 166.0,

145.4, 136.9, 127.9, 126.1 (t, J_{C F} = 5.6 Hz), 125.4, 123.4 118.8 (d, J_C-f - 17-3 Hz), 112.0,

67.4, 64.5, 63.7, 61.6, 49.5, 45.6, 45.5, 42.4, 38.5, 30.9, 29.5, 27.6; ESI-MS calculated for CzaHas^CIFaNaOa [M+H]⁺: 532.2179, Found: 532.42.

C029 - TFA sait

¹H NMR (300 MHz, MeOH-d₄): 8.84 (d, J =6.80 Hz, 1H), 7.58 (t, J = 6.80 Hz, 1H), 7.39 (t, J = 7.11 Hz, 1H), 7.22 (t, J = 7.80 Hz, 1H), 6.88 (dd, J = 9.81, 7.80 Hz, 1H),6.78 (d, J= 10.13, 6.63 Hz, 1H), 5.11 (d, J = 10.37 Hz, 1H), 4.48 (d, J = 10.37 Hz. 1H), 4.21 (d, J = 10.37 Hz, 1H), 4.21 (dd, J = 7.32, 2.66 Hz, 1H), 3.95-3.75 (m, 1H), 2.46-2.22 (m, 2H), 2.12-1.96 (m, 2H), 1.94-1.80 (m, 1H), 1.34-1.28 (m, 1), 1.29 (s, 3H), 0.81 (s, 9H); ¹³C NMR (75 MHz,

113

MeOH-cL): 180.2, 169.2, 132.2, 128.7 (d, J_c*=2.2 Hz), 126.5 (d, J_C-f= 4.6 Hz), 124.7 (dd, Jc-f= 33.5, 19.2 Hz), 122.6 (d, J_C._F = 18.1 Hz), 101.5 (d, J_C-f = 23.0 Hz), 67.4, 64.4, 63.5, 61.9, 49.8, 45.6, 45.5, 42.4, 38.6, 30.9, 29.5, 27.6; ESI-MS calculated for C₂₈H₃₂ ³⁵CIF₃N₃O₃[M+Hf: 550.2084, Found: 550.33.

C031 - TFA sait

’H NMR (300 MHz, MeOH-d₄): 7.68-7.54 (m, 1H), 7.38-7.26 (m, 1H), 7.22-7.12 (m, 1H), 6.90-6.76 (m, 1H), 6.70-6.60 (m, 1H), 6.56-6.42 (m, 1H), 5.30-5.20 (m, 1H), 4.49 (d, J = 10.03 Hz. 1H), 4.25 (dd, J = 71.9, 2.39 Hz. 1H), 4.00-3.82 (m, 1H), 2.50-2.21 (m, 2H), 2.182.00 (m, 2H), 1.98-1.82 (m, 1H), 1.40-1.30 (m, 1H), 1.28 (s, 3H), 0.79 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 180.6, 165.1 (d, JC-f= 246.7 Hz), 166.1, 157.7 (d, JC-f= 247.9 Hz), 145.6 (d, JC-f= 12.0 Hz), 132.0, 128.6, 128.2 (d, JC-f= 10.2 Hz). 126.3 (d, JCF = 4.5 Hz), 125.0 (d, JC.F= 14.0 Hz), 122.4 (d, JC.F= 18.4Hz), 122.3, 109.8 (d, JC-f= 23.2 Hz), 99.9 (d, JC F= 27.8 Hz), 67.4, 64.5, 63.5, 61.5, 49.2, 45.6, 45.5, 42.3, 38.4, 30.9, 29.5, 27.5; ESI-MS calculated for C28H33³⁵CIF2N₃O₃ [M+Hf : 532.2179, Found: 532.42.

C034 - TFA sait

’H NMR (300 MHz, MeOH-d₄): 7.28-7.10 (m, 5H), 6.92-6.84 (m, 1H), 6.80-6.76 (m, 1H), 5.40-5.20 (m, 1 H), 5.08 (d, J = 10.96 Hz, 1H), 4.40-4.20 (m, 1H), 3.90-3.60 (m, 1 H), 2.502.30 (m, 1H), 2.30-2.15 (m, 1H), 2.15-2.00 (m, 2H), 1.90-1.75 (m, 1H), 1.57 (dd, J = 15.3, 3.71 Hz, 1H), 1.25 (s, 3H), 0.79 (s, 9H); ’³C NMR (75 MHz, MeOH-d4): 180.0, 165.9, 144.7, 136.7, 136.6, 135.8, 131.3, 130.1, 129.8, 128.1, 128.1, 126.8, 123.5, 112.0, 67.4, 64.3, 64.0,

62.2, 57.2, 45.7, 45.6, 42.7, 38.3, 31.0, 29.6, 27.5; ESI-MS calculated for C₂₈H₃₄ ³⁵CI₂N₃O₃[M+Hf: 530.1977, Found: 530.50.

C035 - TFA sait

114

¹H NMR (300 MHz, MeOH-d4): 7.40-7.00 (m, 5H), 6.80-6.40 (m, 1H), 5.60-5.00 (m, 2H), 4.60-4.20 (m, 1H), 4.00-3.80 (m, 1H), 2.60-2.40 (m, 1H), 2.40-2.20 (m, 1H), 2.20-2.00 (m, 2H), 2.00-1.80 (m, 1H), 1.70-1.50 (m, 1H), 1.28 (s, 3H), 0.83 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 180.0, 165.8, 160.0-145.0 (m, 2 χ C_sp2-F), 136.5, 135.9, 131.4, 130.0, 129.9, 128.0, 124.1 (d, J_C-f = 6.3 Hz), 119.1, 116.7 (d, J_C-f = 20.4 Hz), 101.4 (d, J_C-f = 23.0 Hz),

67.4, 64.2, 63.8, 62.5, 57.4, 45.6, 45.5, 42.7, 38.3, 31.0, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₃ ³⁵CIF₂N₃O₃ [M+H]⁺: 532.2179, Found: 532.42.

MI-519-73 - TFA sait

¹H NMR (300 MHz, MeOH-d4): 7.50-7.30 (m, 2H), 7.20-7.10 (m, 1H), 6.90-6.70 (m, 3H), 5.00-4.70 (m, 1H), 4.36 (d, J = 9.76 Hz, 1H), 4.05-3.96 (m, 1H), 3.70-3.50 (m, 1H), 1.94 (dd, J = 14.98, 7.30 Hz, 1H), 1.80-1.00 (m, 8H), 1.16 (s, 3H), 0.90-0.70 (m, 1H), 0.80 (s, 9H); ESI-MS calculated for C30H37³⁵CI2FN₃O₃ [M+H]⁺: 576.2196, Found: 576.58.

Ml-519-74 - TFA sait

¹H NMR (300 MHz, MeOH-d4): 7.50-7.30 (m, 2H), 7.25-7.10 (m, 1H), 6.85-6.70 (m, 3H), 5.00-4.70 (m, 1H), 4.32 (d, J = 9.69 Hz, 1H), 4.10-3.95 (m, 1H), 3.85-3.70 (m, 1H), 2.00-1.80 (m, 2H), 1.75-1.20 (m, 7H), 1.13 (s, 3H), 0.95-0.75 (m, 1H), 0.81 (s, 9H); ESI-MS calculated for C30H37³⁵CI2FN₃O₃ [M+Hf: 576.2196, Found: 576.58.

MI-7102-TFA sait

115

¹H NMR (300 MHz, MeOH-d4): 7.36-7.25 (m, 1H), 7.24-7.11 (m, 2H), 6.86 (d, J = 1.8 Hz, 1H), 6.80 (dd, J = 1.8, 8.1 Hz, 1H), 6.72 (d, 7 = 8.1 Hz, 1H), 4.82 (d, 7=9.6 Hz, 1H), 4.36 (d, 7 = 9.6 Hz, 1H), 4.04 (dd, 7 = 2.4, 7.4 Hz, 1H), 3.74-3.56 (m, 1H), 3.56-3.40 (m, 1H), 2.051.78 (m, 5H), 1.75-1.59 (m, 1H), 1.43-1.04 (m, 5H), 0.81 (s, 9H); ESI-MS calculated for C29H35CIF2N3O3 (M + H)⁺ requires 546.23, found 546.58; HPLC (Condition I) tR = 50.45 min (Purity 95.4%).

¹H NMR (300 MHz, MeOH-d₄): 8.38 (d, J = 7.7 Hz, 1H), 7.54 (t, J = 6.7 Hz, 1H), 7.40 (d, J = 7.1 Hz, 1H), 7.20 (t, J = 7.9 Hz, 1H), 6.93 (d, J = 6.1 Hz, 1H), 6.86 (d, J = 8.7 Hz, 1H), 4.45 (d, J = 10.3 Hz, 1H), 4.13 (dd, J = 2.8, 7.5 Hz, 1H), 3.77-3.55 (m, 1H), 3.55-3.42 (m, 1H), 2.09-1.71 (m, 4H), 1.70-1.56 (m, 1H), 1.4₅.1.₀2 (_m, 5_H). 0.82 <s, 9H); ESI-MS calculated for C₂₉H₃₄CI₂F₂N₃O₃ (M + H)* requires 580.19, found 580.67; HPLC (Condition I) t_R = 55.01 min (Purity 88.1%).

MI-7104-TFA sait

¹H NMR (300 MHz, MeOH-d₄); 7.49 (t, J = 7.2 Hz, 1H), 7.45-7.38 (m, 1H), 7.22 (t, J = 8.0 Hz, 1H), 6.85-6.68 (m, 2H), 4.80 (d, J = 9.8 Hz, 1H), 4.36 (d, J = 9.9 Hz, 1H), 4.01 (dd, J =

2.4, 7.6 Hz, 1 H), 3.74-3.57 (m, 1 H), 3.55-3.39 (m, 1 H), 2.04-1.77 (m, 4H), 1.74-1.59 (m, 1 H), 1.44-1.04 (m, 5H), 0.90 (d, J = 4.5 Hz, 1H), 0.82 (s, 9H); ESI-MS calculated for

116

C29H34CIF3N3O3 (Μ + Η)⁺ requires 564.22, found 564.58; HPLC (Condition I) t_R = 51.76 min (Purity 86.9%).

M1-7105- TFA sait

Ή NMR (300 MHz, MeOH-d₄): 7.49 (t, J = 7.2 Hz, 1H), 7.45-7.38 (m, 1H), 7.22 (t, J = 8.0

Hz, 1H), 6.85-6,68 (m, 2H), 4.80 (d, J = 9.8 Hz, 1H), 4.36 (d, J = 9.9 Hz, 1H), 4.01 (dd, J =

2.4, 7.6 Hz, 1 H), 3.74-3.57 (m, 1 H), 3.55-3.39 (m, 1 H), 2.04-1.77 (m, 4H), 1.74-1.59 (m, 1 H), 1.44-1.04 (m, 5H), 0.90 (d, J = 4.5 Hz, 1H), 0,82 (s, 9H); ESI-MS calculated for C₂₉H₃₅CIF2N₃O3 (M + H)⁺ requires 546.23, found 546,58; HPLC (Condition I) t_R = 49.20 min (Purity 99.4%).

MI-7106-TFA sait

’H NMR (300 MHz, MeOH-d₄): 8.36 (d, J = 7.0 Hz, 1 H), 7.59 (d, J = 8.1 Hz, 1 H), 7.41-7.11 (m, 4H), 7.04 (d, J= 7.6 Hz, 1H), 6.78 (d, J= 1.8 Hz, 1H), 5.19 (d, J = 11.3 Hz, 1H), 4.44 (J = 8.1 Hz. 1H), 4.07 (d, J = 11.3 Hz, 1H), 3.74-3.53 (m, 1H), 3.53-3.37 (m, ÏH), 2.08-,.83 (m, 3_h), 1.83-1.^e9 (m, 1H), 1.61-1.44 (m. 1H), 1.44-1.08 (m, 4H), 1.07-0.72 (m, 1H), 0.88 (s, 9H); ESI-MS calculated for CjgHaeCIzNaOa (M + H)⁺ requires 544.21, found 544.67; HPLC (Condition I) t_R = 51.41 min (Purity 93.0%).

MI-7108-TFA sait

’H NMR (300 MHz, MeOH-d₄/DMSO-d₀): 10.15 (s, 1H), 7.76 (d, J = 8.2 Hz, 1H), 7.22 (s,

1H), 7.17-7.00 (m, 3H), 6.94 (d, J = 7.1 Hz, 1H), 6.81 (d, J = 6.0 Hz, 1H), 4.42 (d, J = 8.3 Hz.

117

H), 4.09 (d, J = 3.0 Hz, 1H), 3.79 (d, J = 8.3 Hz, 1H), 3.73-3.49 (m, 2H), 3.35 (d, J = 9.5 Hz,

1H), 2.10-1.84 (m, 4H), 1.52-1.11 (m, 5H), 0.87 (s, 9H); 13C NMR (75 MHz, MeOHdVDMSO-de): 177.1, 172.4, 153.6 (d, J_C-_F = 242.7 Hz), 138.7, 138.5 (d, J_C._F = 2.4 Hz), 133.2, 129.0, 127.544, 127.541 (d, J_C._F = 6.7 Hz), 126.8, 126.5, 119.7 (d, J_C._F = 19.2 Hz), 111.3,

110.4 (d, J_C._F = 24.1 Hz), 68.4, 66.6, 65.7, 64.0, 58.6, 46.8, 42.2, 33.26, 33.20, 30.4, 30.2, 29.7, 29.5; ESI-MS calculated for C₂₉H3₅CI₂FN₃O3 (M + H)+ requires 562.20, found 562.67; HPLC (Condition I) t_R = 55.08 min (Purity 96.1%); HPLC (Condition II) t_R = 21.44 min (Purity 92.7%).

MI-7109 - TFA sait

¹H NMR (300 MHz, MeOH-d4): 7.47 (t, J = 6.7 Hz, 1H), 7.42-7.33 (m, 1H), 7.18 (t, J = 7.7 Hz, 1H), 6.87 (d, J = 1.8 Hz, 1 H), 6.78 (dd, J = 1.8, 8.1 Hz, 1H), 6.70 (d, J = 8.1 Hz, ÏH), 4.40 (d, J =9.7 Hz, 1H), 4.11 (dd, J = 2.5, 7.6 Hz, 1H), 2.77-2.65 (m, 1H), 1.99 (dd, J =7.6, 15.3 Hz, 1H), ¹ 24 (dd, J = 2.5, 15.3 Hz, 1H), 0.92-0.62 (m, 2H), 0.81 (s, 9H), 0.56-0.30 (m, 2H); ESI-MS calculated for CZ6H₂₉CI₂FN₃O₂ (M + H)⁺ requires 504.1_e, found 504.58; HPLC (Condition I) t_R = 53.99 min (Purity 94.4%).

B059 - TFA sait

’H NMR (300 MHz, CD₃OD): 7.45-7.34 (m, 1H), 7.26-7.12 (m, 1H), 7.04-6.93 (m, 1H), 6.90 (d, J= 1.80 Hz, 1H), 6.65 (dd, J = 8.08, 1.80 Hz, 1H), 4.41 (d, J= 9.25 Hz, 1H), 3.96 (quint, J = 8.13 Hz, 1H), 2.51-2.07 (m, 2H), 2.40-2.20 (m, 2H), 1.88 (dd, J = 14.20, 9.91 Hz, 1H), 1.32 (s, 3H), 1.20-0.80 (m, 1H), 0.88 (s, 9H); ¹³C NMR (75 MHz, CD₃OD): 181.3, 172.9 (d, Jc-_F = 266.9 Hz), 168.6, 162.7, 145.3, 135.8, 131.7,130.7 (d, J_C._F = 38.6 Hz), 126.2 (d, J_C-_F = 4.5 Hz), 126.1, 123.6, 122.9, 122.7, 111.4, 78.4, 67.7, 63.4, 46.0, 45.8, 44.3, 38.0, 31.4,

30.2, 27.6; ESI-MS calculated for C₂₈H₃₁ ³⁵CI₂FN₃O₃ [M+Hf: 546.1727, Found: 546.50.

MI-519-77 - TFA sait

U8

¹H NMR (300 MHz, CD₃OD): 7.50-7,40 (m, 1H), 7.40 (m, 1H), 7.20-7.10 (m, 1H), 6.85 (d, J = 1.40 Hz, 1H), 6.84-6.72 (m, 2H), 5.00-4.80 (m, 1H), 4.45 (d, J = 10.10 Hz, 1H), 4.02 (t, J =

6.61 Hz, 1H), 3.90 (quintet, J = 8.07 Hz, 1H), 2.50-2.25 (m, 2H), 2.10-1.82 (m, 3H), 1.811.31 (m, 8H), 1.30 (s, 3H), 1.10-0.91 (m, 1H), 0.91-0.81 (m 1H); ESI-MS calculated for C₂₉H₃₃ ³⁵CI₂FN₃O₃ [M+Hf: 560.1883, Found: 560.50.

MI-519-78 - TFA sait

H ¹H NMR (300 MHz, CD3OD): 7.45-7.31 (m, 2H), 7.20-7.11 (m, 1H), 6.86-6.82 (m, 1H), 6.816.78 (m, 2H), 4.90-4.80 (m, 1H), 4.45 (d, J = 10.33 Hz, 1H), 4.10-3.95 (m, 1H), 3.70-3.60 (m, 1H), 3.50-3.40 (m, 1H), 2.10-1.05 (m, 17 H), 1.05-0.95 (m, 1H), 0.95-0.80 (m, 1H); ESI-MS calculated for C3oH35³⁵CI2FN₃0₃ [M+H]⁺: 574.2040, Found: 574.58.

MI-519-80 - TFA sait

¹H NMR (300 MHz, CD₃OD): 7.80-7.72 (m, 1H), 7.50-7.38 (m, 2H), 6.87 (d, J = 1.81 Hz, 1H), 6.71 (dd, J = 8.16, 1.81 Hz, 1H), 6.52-6.40 (m, 1H), 4.96-4.80 (m, 1H), 4.62 (d, J = 8.69

Hz, 1H), 4.10-3.95 (m, 1H), 3.70-3.55 (m, 1H), 3.50-3.45 (m, 1H), 2.00-1.80 (m, 3H), 1.80119

1.60 (m, 1H), 1.40-1.00 (m, 5H), 0.95-0.85 (m, 1H), 0.80 (s, 9H); ESI-MS calculated for C₂₉H₃₅ ³⁵CI₃N₃O₃ [M+H]⁺: 578.1744, Found: 578.75.

C02701 - TFA sait (2’S,3'R,4'S,5'R)-6-Chloro-4'-(2,3-difluoro-phenyl)-2^,-(2,2-dimethyl-propyl)-2-oxo-1,2dihydro-spiro[indole-3,3'-pyrrolidine]-5'-carboxylic acid (3-hydroxy-3-methylcyclobutyl)-amide, trifluoroacetate

1H), 7.20-7.05 (m, 3H), 6.80-6.75 (m, 1H), 5.40-5.10 (m, 1H), 4.61 (d, J = 11.39 Hz, 1H), 4.50 (d, J =7.66 Hz, 1H), 3.95-3.80 (m, 1H), 2.45-2.30 (m, 1H), 2.30-2.15 (m, 1H), 2.05-1.80 (m, 2H), 1.80-1.60 (m, 1H), 1.27 (s, 3H), 1.20-1.08 (m. 1H), 0.86 (s, 9H); ¹³C NMR (75 MHz, MeOH-d₄): 177.8, 167.0, 160.0-148.0 (m, 2 x C_sp2-F), 145.2, 137.2, 126.8, 126.5-126.0 (m), 125.0, 124.1, 123.5, 122.1 (d, J_C-f = 9.74 Hz), 119.1 (d, J_C-f = 17.1 Hz), 112.1, 67.3, 64.5,

64.2, 62.6, 48.5, 45.6, 45.5, 43.3, 38.3, 31.0, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₃ ³⁵CIF₂N₃O₃ [M+H]⁺: 532.2179, Found: 532.50.

LC-MS: t_R (min) = 0.86; [M+H]⁺: m/z 532; [M-H]’: m/z 530 (method A).

¹H NMR (400 MHz, DMSO-d_e + TFA): 0.80 (s, 9 H); 1.01 (d broad, J=15.2 Hz, 1 H); 1.20 (s, 3 H); 1.60 (m, 1 H); 1.85 to 1.98 (m, 2 H); 2.08 (m, 1 H); 2.25 (m, 1 H); 3.72 (m, 1 H); 4.49 (m, 2 H); 5.30 (d, J=12.1 Hz, 1 H); 6.78 (d, J=2.0 Hz, 1 H); 7.16 (dd, J=2.0 and 8.3 Hz, 1 H); 7.23 (m, 1 H); 7.42 (m, 1 H); 7.48 (m, 1 H); 7.74 (d, J=8.3 Hz, 1 H).

C02901 - TFA sait (2'S,3^,R,4'S,5’R)-4'-(3-Chloro-2-fluoro-phenyl)-2’-(2,2-dimethyl-propyl)-5,6-difluoro-2oxo-1,2-dihydro-spiro[indole-3,3'-pyrrolidine]-5'-carboxylic acid (3-hydroxy-3-methylcyclobutyl)-amide, trifluoroacetate

120 ¹H NMR (300 MHz, MeOH-d4): 7.80-7.65 (m, 1H), 7.60-7.50 (m, 1H), 7.40-7.30 (m, 1H), 7.20-7.10 (m, 1H), 6.80-6.65 (m, 1H), 5.50-5.10 (m, 1H), 4.60 (d, J = 11.39 Hz, 1H), 4.50 (d, J = 6.96 Hz, 1 H), 3.95-3.80 (m, 1H), 2.50-2.30 (m, 1H), 2.30-2.20 (m, 1H), 2.10-1.80 (m, 2H), 1.80-1.65 (m, 1H), 1.27 (s, 3H), 1.20-1.05 (m, 1H), 0.87 (s, 9H); ¹³C NMR (75 MHz, MeOH-di): 177.8, 167.0, 160.0-145.0 (m, 3* Csp2-F), 132.6, 128.6, 126.6, 122.5 (d, J_C-f = 18.9 Hz), 121.3 (d, J_C-f = 13.0 Hz), 118.8, 115.4 (d, J_C._F= 21.7 Hz), 115.1, 101.8 (d, J_C-f = 23.3 Hz), 67.3, 64.6, 64.3, 62.5, 48.7, 45.6, 45.5, 43.4, 38.3, 31.0, 29.5, 27.5; ESI-MS calculated for C_2aH₃₂ ³⁵CIF₃N₃O₃ [M+H]*: 550.2084, Found: 550.33.

LC-MS: t_R (min) = 0.87; [M+H]⁺: m/z 550; [M-H]‘: m/z 548 (method A).

¹H NMR (400 MHz; DMSO-d₆ + TFA): 0.80 (s, 9 H); 1.01 (d, J=15.2 Hz, 1 H); 1.20 (s, 3 H); 1.62 (m, 1 H); 1.85 to 1.98 (m, 2 H); 2.08 (m, 1 H); 2.26 (m, 1 H); 3.73 (m, 1 H); 4.52 (m, 2 H); 5.28 (d, J=12.1 Hz, 1 H); 6.79 (dd, J=6.7 and 10.1 Hz, 1 H); 7.25 (t, J=7.8 Hz, 1 H); 7.50 (m, 1 H); 7.60 (m, 1 H); 8.08 (m, 1 H).

C03001 - TFA sait

methyl-cyclobutyl)-amide, trifluoroacetate

¹H NMR (300 MHz, MeOH-d4): 7.70 (d, J = 7.30 Hz, 1H), 7.60-7.50 (m, 1H), 7.45-7.35 (m, 1H), 7.25-7.15 (m, 1H), 6.88 (d, J = 6.00 Hz, 1H), 5.21 (d, J = 11.35 Hz, 1H), 4.61 (d, J = 11.37 Hz, 1H), 4.53 (d, J = 8.19 Hz, 1H), 3.95 -3.80 (m, 1H). 2.50-2.35 (m, 1H), 2.35-2.15 (m, 1H), 2.00-1.80 (m, 2H), 1.80-1.60 (m, 1H), 1.29 (s, 3H), 1.25-1,05 (m, 1H), 0.89 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 177.3, 166.7, 157.6 (d, J_C-f= 249.5 Hz), 155.7 (d, J_C-f= 243.5 Hz), 140.4 (d, J_C-f= 2.8 Hz), 132.5, 128.4, 126.4 (d, J_C._F = 4.9 Hz), 125.0 (d, J_CF= 7.4 Hz),

123.4 (d, J_C._F= 19.5 Hz), 122.3 (d, J_C._F = 18.9 Hz), 121.0 (d, J_C-f= 13.0 Hz), 114.5 (d, J_C-f = 25.1 Hz), 104.8, 67.1, 64.6, 64.2, 62.4, 47.3, 45.4, 45.3, 43.2, 38.2, 30.8, 29.2, 27.3; ESI-MS calculated for C₂₈H₃₂ ³⁵CI₂F₂N₃O₃[M+H]⁺: 566.1789, Found: 566.50.

LC-MS: t_R (min) = 0.93; [M+H]⁺: m/z 566 (method A).

¹H NMR (400 MHz, DMSO-d_e + TFA): 0.81 (s, 9 H); 1.02 (d broad, J=15.2 Hz, 1 H); 1.20 (s, 3 H); 1.62 (m, 1 H); 1.87 to 1.99 (m, 2 H); 2.09 (m, 1 H); 2.27 (m, 1 H); 3.75 (m, 1 H); 4.55

121 (m, 2 H); 5.30 (d, J=12.1 Hz, 1 H); 6.89 (d, J=6.3 Hz, 1 H); 7.25 (t, J=7.8 Hz, 1 H); 7.50 (m,

H); 7.61 (m, 1 H); 8.04 (d, J=8.9 Hz, 1 H)

C031 - TFA sait

LC-MS: t_R (min) = 0.84; [M+Hf: m/z 532; [M-H]’: m/z 530 (method A).

’H NMR (400 MHz, DMSO-d_e + TFA): 0.83 (s, 9 H); 1.11 (d broad, J=15.2 Hz, 1 H); 1.22 (s, 3 H); 1.83 (m, 1 H); 2.00 to 2.36 (m, 4 H); 3.82 (m, 1 H); 4.20 (dd, J=2.9 and 7.7 Hz, 1 H);

4.36 (d, J=10.5 Hz, 1 H); 5.00 (d, J=10.5 Hz, 1 H); 6.53 to 6.74 (m, 2 H); 6.94 (dd, J=5.6 and 8.8 Hz, 1 H); 7.21 (t, J=8.0 Hz, 1 H); 7.41 (t, J=8.0 Hz, 1 H); 7.71 (t, J=8.0 Hz, 1 H).

Cl ’H NMR (300 MHz, MeOH-d₄): 7.58 (d, J = 8.07 Hz, 1H), 7.30-7.10 (m, 4H), 7.02 (d, J = 7.67 Hz, 1H), 6.77 (d, J = 1.54 Hz, 1H), 5.40-5.20 (m, 1H), 4.44 (d, J = 7.09 Hz, 1H), 4.10 (d, J = 11.25 Hz, 1H), 3.95-3.80 (m, 1H), 2.45-2.30 (m, 1H), 2.30-2.15 (m, 1H), 2.05-1.85 (m, 2H), 1.80-1.70 (m, 1H), 1.27 (s, 3H), 1.20-1.10 (m,1H), 0.86 (s, 9H); ’³C NMR (75 MHz,

MeOH-d₄): 177.8, 167.3, 145.3, 137.1, 135.8, 134.4, 131.4, 130.4, 129.5, 128.3, 126.3,

124.2, 124.1, 112.2, 67.3, 64.9, 64.2, 62.8, 57.2, 45.7, 45.6, 43.4, 38.3, 31.0, 29.5, 27.5;

ESI-MS calculated for C₂₈H₃4³⁵CI₂N₃O₃ [M+Hf: 530.1977, Found: 530.58.

LC-MS: t_R (min) = 0.84; [M+Hf: m/z 530; [M-H]’: m/z 528 (method A).

’H NMR (400 MHz, DMSO-d₆ + TFA): 0.80 (s, 9 H); 1.02 (d broad, J=15.5 Hz, 1 H); 1.20 (s, 3 H); 1.68 (m, 1 H); 1.82 to 2.00 (m, 2 H); 2.09 (m, 1 H); 2.27 (m, 1 H); 3.73 (m, 1 H); 4.07 (d, J=11.9 Hz, 1 H); 4.32 (dd, J=2.2 and 8.4 Hz, 1 H); 5.29 (d, J=11.9 Hz, 1 H); 6.74 (d, J=2.0 Hz, 1 H); 6.98 (d, J=7.8 Hz, 1 H); 7.15 to 7.35 (m, 4 H); 7.78 (t, J=8.3 Hz, 1 H).

C03701 - TFA sait

122 (2'S,3^,R,4'R,5'R)-6-Chloro-4'-(3-chloro-phenyl)-2’-(2_>2-dimethyl-propyl)-5-fluoro-2oxo-1,2-dihydro-spiro[indole-3,3'-pyrrolidine]-5'-carboxylic acid (3-hydroxy-3-methylcyclobutyl)-amide, trifluoroacetate

’H NMR (300 MHz, MeOH-d₄): 9.00-8.80 (m, 1 H), 7.73 (d, J = 8.42 Hz, 1H), 7.40-7.20 (m, 3H), 7.15-7.05 (m, 1H), 6.89 (d, J = 6.00 Hz, 1 H), 5.32 (d, J = 11.34 Hz, 1H), 4.52 (d, J = 7.91 Hz, 1H), 4.20 (d, J = 11.28 Hz, 1H), 4.00-3.80 (m, 1H), 2.50-2.35 (m, 1H), 2.35-2.20 (m, 1H), 2.20-1.90 (m, 2H), 1.90-1.80 (m, 1H), 1.31 (s, 3H), 1.30-1.15 (m, 1H), 0.91 (s, 9H); ’³C NMR (75 MHz, MeOH-d₄): 178.8, 168.2, 157.3 (d, J_C-f = 255.8 Hz), 142.1 (d, J_C._F= 2.6 Hz), 137.1, 135.5, 132.8, 131.7, 130.7, 129.6, 127.2 (d, J_C-f= 7.2 Hz), 124.7 (d, J_{C F} = 19 3 Hz), 115.5 (d, J_C._F = 24.9 Hz), 114.8, 68.6, 66.6, 65.3, 64.0, 58.2, 47.0, 46.8, 44.7, 39.5,

32.2, 30.8, 28.8; ESI-MS calculated for C_2eH₃₃ ³⁵CI2FN3O3 [M+H]⁺: 548.1883, Found: 548.42. LC-MS: tR (min) = 0.87; [M+Hf: m/z 548; [M-H]‘: m/z 546 (method A).

¹H NMR (400 MHz, DMSO-d₀): 0.70 to 2.23 (m, 18 H); 3.68 to 5.10 (m, 4 H); 6.78 to 8.03 (m, 6 H).

C04801 - TFA sait (2’S,3'R,4’S,5'R)-6-Chloro-4'-(2,3-difluoro-phenyl)-2'-(2,2-dimethyl-propyl)-5-fluoro-2oxo-1 ^-dihydro-spirolindole-S.S'-pyrrolidinel-S’-carboxylic acid (3-hydroxy-3-methylcyclobutyl)-amide, trifluoroacetate

’H NMR (300 MHz, MeOH-d₄): 9.00-8.80 (m, 1H), 7.70 (d, J = 8.35 Hz, 1H), 7.50-7.35 (m, 1H), 7.30-7.10 (m, 2H), 6.88 (d, J = 6.88 Hz, 1 H), 5.30 (d, J = 11.32 Hz, 1H), 4.66 (d, J = 11.33 Hz, 1H), 4.56 (d, J = 7.43 Hz, 1H), 4.00-3.80 (m, 1H), 2.50-2.35 (m, 1H), 2.35-2.20 (m, 1H), 2.10-1.90 (m, 2H), 1.80-1.70 (m, 1H), 1.30 (s, 3H), 1.16 (d, J = 15.34 Hz, 1H), 0.90 (s, 9H); ¹³C NMR (75 MHz, MeOH-d₄): 177.5, 166.9, 160-145 (m, 2 * C_sp2-F), 155.9 (d, J_CF =

243.4 Hz), 140.7 (d, J_C-_F = 2.69 Hz, 1H), 126.5-126.1 (m), 125.6 (d, J_{C F}= 7.6 Hz), 125.0 (d,

123

7_C._F = 3.4 Hz), 123.6 (d, 7_C._F = 19.5 Hz), 122.0 (d, 7_C._F = 9.8 Hz), 119.1 (d, 7_C._F=17.1 Hz), 114.7 (d, J_C._F = 25.0 Hz), 113.4, 67.3, 64.7, 64.3, 62.5, 48.2, 45.6, 45.6, 43.4, 38.3, 31.0, 29.5, 27.5; ESI-MS calculated for 0_2βΗ₃₂ ³⁵0ΙΕ₃Ν₃03 [M+Hf: 550.2084, Found: 550.42. LC-MS: t_R (min) = 0.89; [M+Hf: m/z 550; [M-H]’: m/z 548 (method A) ¹H NMR (400 MHz, DMSO-d₆ + TFA): 0.81 (s, 9 H); 1.02 (d broad, J=15.5 Hz, 1 H); 1.20 (s, 3 H); 1.62 (m, 1 H); 1.87 to 1.98 (m, 2 H); 2.09 (m, 1 H); 2.26 (m, 1 H); 3.73 (m, 1 H); 4.56 (m, 2 H); 5.29 (d, J=12.4 Hz, 1 H); 6.89 (d, J=6.2 Hz, 1 H); 7.20 to 7.49 (m, 3 H); 8.06 (d, J=9.3 Hz, 1 H).

MI-710201 - TFA sait (2'S_l3^,R,4'S,5^,R)-6-Chloro-4^,-(2,3-difluoro-phenyl)-2^,-(2,2-dimethyl-propyl)-2-oxo-1,2dihydro-spiro[indole-3,3'-pyrrolidine]-5'-carboxylic acid (4-hydroxy-cyclohexyl)-amîde, trifluoroacetate

¹H NMR (300 MHz, MeOH-d4): 7.57 (d, J = 8.0 Hz, 1H), 7.50-7.36 (m, 1H), 7.27-7.07 (m, 3H), 6.79 (s, 1H), 5.11 (d, J = 11.1 Hz, 1H), 4.55 (d, J = 11.0 Hz, 1H), 4.39 (d, J = 7.7 Hz, 1H), 3.71-3.52 (m, 1H), 3.52-3.37 (m, 1H), 3.21 (dd, J = 7.4, 14.5 Hz, 1H), 1.92 (d, J = 9.6 Hz, 1H), 1.86-1.70 (m, 2H), 1.58 (d, 7= 11.8 Hz. 1H), 1.43-1.18 (m, 4H), 1.12 (d, 7= 15.5 Hz, 1H), 0.99 (d, 7= 13.0 Hz, 1H), 0.88 (s, 9H); ESI-MS calculated for C29H35CIF2N3O3 (M + H)⁺ requires 546.23, found 546.58; HPLC (Condition I) tR = 52.15 min (Purity 98.8%). LC-MS: t_R (min) = 0.84; [M+Hf: m/z 546; [M-H]': m/z 544 (method A).

¹H NMR (400 MHz; DMSO-d₆): mixture of isomers: 0.80 (s, 9 H); 0.84 to 1.30 (m, 5 H); 1.41 to 1.87 (m, 5 H); 3.43 to 3.54 (m, 2 H); 4.03 (m broad, 1 H); 4.36 (d broad, J=10.3 Hz, 2 H); 4.83 (m broad, 1 H); 6.72 (d, J=2.0 Hz, 1 H); 7.10 (dd, J=2.0 and 8.3 Hz, 1 H); 7.14 (m, 1 H); 7.24 (m, 1 H); 7.40 (m, 1 H); 7.58 (d, J=8.3 Hz, 1 H); 7.97 (m broad, 1 H); 10.56 (m broad, 1 H).

Ml-710401 - TFA sait (2’S,3’R,4'S,5'R)-4'-(3-Chloro-2-fluoro-phenyl)-2’-(2,2-dimethyl-propyl)-5,6-difluoro-2oxo-1,2-dihydro-spiro[indole-3,3’-pyrrolidine]-5’-carboxylic acid (4-hydroxycyclohexyl)-amide, trifluoroacetate

124

’H NMR (300 MHz, MeOH-d₄): 7.67 (t, J = 8.6 Hz, 1H), 7.57 (t, J = 6.8 Hz, 1H), 7.38 (t, J = 6.8 Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H), 6.71 (dd, J = 6.6, 10.1 Hz, 1H), 5.01 (d, J = 10.8 Hz. 1H), 4.52 (d, J = 10.8 Hz, 1H), 4.40-4.21 (m, 1H), 3.74-3.56 (m, 1H), 3.56-3.40 (m, 1H), 2.08-1.87 (m, 2H), 1.87-1.68 (m, 2H), 1.68-1.53 (m, 1H), 1.45-1.18 (m, 3H), 1.17-0.97 (m, 2H), 0.89 (s, 9H); ESI-MS calculated for C₂₉H3₄CIF₃N3O₃ (M + Hf requires 564.22, found 564.58; HPLC (Condition I) t_R = 52.15 min (Purity 98.8%).

LC-MS: t_R (min) = 0.84; [M+H]⁺: m/z 564; [M-H]': m/z 562 (method A) ’H NMR (400 MHz; DMSO-d₆ + TFA): 0.81 (s, 9 H); 0.81 to 1.98 (m, 10 H); 3.35 (m, 1 H); 3.52 (m, 1 H); 4.53 (d, J=12.2 Hz, 1 H); 4.59 (dd, J=2.3 and 8.7 Hz, 1 H); 5.33 (d, J=12.2 Hz, 1 H); 6.78 (dd, J=6.8 and 10.3 Hz, 1 H); 7.21 (t, J=9.0 Hz, 1 H); 7.48 (m, 1 H); 7.66 (m, 1 H); 8.00 (m, 1 H).

MI-710501 - TFA sait (2^,S,3’R₁4'S,5'R)-4'-(3-Chloro-2-fluoro-phenyl)-2’-(2,2-dimethyl-propyl)-6-fluoro-2oxo-1,2-dihydro-spiro[Îndole-3,3'-pyrrolidine]-5’-carboxylic acid (4-hydroxycyclohexyl)-amide, trifluoroacetate

’H NMR (300 MHz, MeOH-d₄): 7.66-7.53 (m, 1H), 7.44-7.33 (m, 1H), 7.22-7.09 (m, 1H), 6.93-6.79 (m, 1H), 6.59-6.51 (m, 1H), 5.40-5.31 (m, 1H), 4.63-4.48 (m, 1H), 4.41-4.30 (m, 1H), 2.41-2.20 (m, 2H), 2.15-1.97 (m, 2H), 1.95-1.85 (m, 1H), 1.85-1.71 (m, 1H), 1.71-1.47 (m, 3H), 1.19-1.07 (m, 1H), 0.88 (s, 9H); ESI-MS calculated for C₂gH3₄CIF₃N3O₃ (M + H)⁺requires 546.23, found 546.58; HPLC (Condition I) t_R= 52.05 min (Purity 98.8%); HPLC (Condition II) t_R = 19.26 min (Purity 100%).

LC-MS: t_R (min) = 0.80; (M+Hf: m/z 546; [M-H]': m/z 544 (method A).

’H NMR (400 MHz; DMSO-d₆ + TFA): 0.81 (s, 9 H); 0.81 to 1.99 (m, 10 H); 3.35 (m, 1 H); 3.52 (m, 1 H); 4.47 (d, J=12.0 Hz, 1 H); 4.53 (d broad, J=8.3 Hz, 1 H); 5.37 (d, J=12.0 Hz, 1

125

H); 6.58 (dd, J=2.6 and 9.1 Hz, 1 H); 6.91 (m, 1 H); 7.21 (t, J=9.0 Hz, 1 H); 7.45 (m, 1 H);

7.62 to 7.72 (m, 2 H).

MI-710601 - TFA sait (2’S,3'R,4'R,5^,R)-6-Chloro-4'-(3-chloro-phenyl)-2'-(2,2-dimethyl-propyl)-2-oxo-1,2dihydro-spiro[indole-3,3'-pyrrolidine]-5'-carboxylic acid (4-hydroxy-cyclohexyl)-amide, trifluoroacetate

LC-MS: t_R (min) = 0.82; [M+H]⁺: m/z 544; [M-H]’: m/z 542 (method A).

’H NMR (400 MHz; DMSO-d₆ + TFA): 0.80 (s, 9 H); 0.83 to 1.96 (m, 10 H); 3.34 (m, 1 H); 3.50 (m, 1 H); 4.02 (d, J=12.1 Hz, 1 H); 4.48 (d broad, J=8.5 Hz, 1 H); 5.32 (d, J=12.1 Hz, 1 H); 6.75 (dd, J=1.9 and 8.3 Hz, 1 H); 6.98 (d, J=7.8 Hz, 1 H); 7.18 to 7.32 (m, 4 H); 7.74 (d, J=8.3 Hz, 1 H).

MI-710801 (2^,S_r3'R,4^,R,5'R)-6-Chloro-4'-(3-chloro-phenyl)-2’-(2,2-dimethyl-propyl)-5-fluoro-2oxo-1 ^-dihydro-spiroOndole-S.S'-pyrrolidinel-S’-carboxylic acid (4-hydroxycyclohexyl)-amide

LC-MS: t_R (min) = 0.85; [M+H]⁺: m/z 562; [M-H]': m/z 560 (method A).

¹H NMR (400 MHz; DMSO-d₆): 0.78 (m, 1 H); 0.81 (s, 9 H); 1.08 to 1.33 (m, 5 H); 1.70 to 1.90 (m, 4 H); 3.25 (m, 1 H); 3.35 to 3.58 (m, 3 H); 3.92 (d, J=9.2 Hz, 1 H); 4.43 (t, J=9.2 Hz, 1 H); 4.49 (d, J=4.8 Hz, 1 H); 6.79 (d, J=6.4 Hz, 1 H); 6.92 (d, J=7.8 Hz, 1 H); 7.09 to 7.21 (m, 3 H); 7.75 (d, J=8.5 Hz, 1 H); 7.83 (d, J=9.3 Hz, 1 H); 10.38 (s broad, 1 H).

MI-710901 - TFA sait (2’S,3'R,4^,S,5’R)-6-Chloro-4’-(3-chloro-2-fluoro-phenyl)-2'-(2,2-dimethyl-propyl)-2oxo-1 ^-dihydro-spirotindole-S.S'-pyrrolidineJ-S'-carboxylic acid cyclopropylamide, trifluoroacetate

126

cf₃co₂h ’H NMR (300 MHz, MeOH-d₄): 7.61 (d, J = 8.1 Hz, 1 H), 7.53 (t, J = 6.7 Hz, 1 H), 7.40 (t, J =

7.0 Hz, 1H), 7.21-7.08 (m, 2H), 6.79 (d, J= 1.6 Hz, 1H), 5.14 (d, J = 11.3 Hz, 1H), 4.60 (d, J = 11.3 Hz, 1 H), 4.48 (d, J = 7.0 Hz, 1 H), 2.78-2.58 (m, 1 H), 1.86 (dd, J = 8.4, 15.4 Hz, 1 H), 1.13 (d, J= 15.4 Hz, 1H), 0.88 (s, 9H), 0.78-0.60 (m, 2H), 0.47-0.16 (m, 2H); ¹³C NMR (75 MHz, MeOH-d₄): 177.9, 169.5, 157.8 (d, J_{C F} = 249.4 Hz), 145.2, 137.2, 132.6, 128.7, 126.8 (d, J_{C F} = 1-6 Hz), 126.6 (d, J_C-f - 4.9 Hz), 124.2, 123.6, 122.5 (d, J_C._F = 18.8 Hz), 121.8 (d,

Jc-f =13.1 Hz), 64.7, 64.4, 62.9, 43.5, 31.0, 29.6, 23.9, 6.7, 6.5; ESI-MS calculated for CzeHzgC^FNgOi (M + H)⁺ requires 504,16, found 504.58; HPLC (Condition I) t_R = 58.22 min (Purity 99.6%).

LC-MS: t_R (min) = 0.98; [M+H]⁺: m/z 504; [M-H]': m/z 502 (method A) ¹H NMR (400 MHz; DMSO-d_e + TFA): 0.15 (m, 1 H); 0.35 (m, 1 H); 0.54 to 0.70 (m, 2 H);

0.81 (s, 9 H);1.01 (d, J=15.2 Hz, 1 H); 1.91 (dd, J=8.4 and 15.2 Hz, 1 H); 2.66 (m, 1 H); 4.52 (m, 2 H); 5.27 (d, J=12.0 Hz. 1 H); 6.79 (d, J=2.0 Hz, 1 H); 7.18 (dd, J=2.0 and 8.3 Hz, 1 H);

7.23 (d, J=8.1 Hz, 1 H); 7.48 (m, 1 H); 7.59 (m, 1 H); 7.74 (d, J=8.3 Hz, 1 H).

C08301 - TFA sait

CF₃CO₂H ¹H NMR (300 MHz, MeOH-d₄): 7.64-7.54 (m, 1H), 7.20-7.46 (m, 1H), 7.46-7.38 (m, 1H),

7.28-7.14 (m, 2H), 5.26 (d, J= 11.34 Hz, 1H), 4.65 (d, J = 11.43 Hz, 1H), 4.55 (dd, J =8.28,

1.59 Hz. 1H), 3.95-3.80 (m, 1H), 2.46-2.33 (m, 1H), 2.30-2.20 (m, 1H), 2.40-1.84 (m, 2H),

1.76-1.64 (m,1H), 1.29 (s, 3H), 1.17 (dd, J = 15.40, 1.5 Hz, 1H), 0.88 (s, 9H); ¹³C NMR (75

MHz, MeOH-d₄): 177.4, 166.9, 157.8 (d, J_C-f = 249.9 Hz), 144.3 (d, J_C._F = 248.0 Hz), 132.8,

132.5 (d, J_c-F = 12.3 Hz), 128.6, 126.7 (d, J_{C F} = 4.87 Hz), 126.3 (d, J_C-f = 3.32 Hz), 125.6,

123.9 (d, J_C._F = 14.3 Hz), 122.6 (d, J_C-f = 18.9 Hz), 122.1, 121.3 (d, J_C-f =13.1 Hz), 67.2,

64.7, 64.5, 62.6, 48.9, 45.6, 45.5, 43.4, 38.3, 31.0, 29.5, 27.5; ESI-MS calculated for C2₈H₃2³⁵Cl2F₂N₃O₃ [M+H]⁺: 566.18, Found: 566.50.

127

C08601-TFA sait

¹H NMR (300 MHz, MeOH-d4): 8.83 (d, J = 6.98 Hz, 1H), 7.54-7.38 (m, 2H), 7.30-7.14 (m, 3H), 5.26 (d, J =11.37 Hz, 1H), 4.64 (d, J = 11.37 Hz, 1H), 4.56 (dd, J = 8.35, 1.27 Hz, 1H), 3.95-3.80 (m, 1H), 2.48-2.34 (m, 1H), 2.32-2.20 (m, 1H), 2.08-1.86 (m, 2H), 1.80-1.64 (m, 1 H), 1.30 (s, 3H), 1.18 (d, J = 14.37 Hz, 1H), 0.90 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 177.4, 166.9, 133.9, 132.4, 130.1, 126.35 (dd, J_C-f = 9.43, 2.72 Hz), 125.5, 125.0 (d, J_C-f = 3.72 Hz), 124.0 (d, J_C-f = 14.4 Hz), 122.1 (d, J_C._F = 3.13 Hz), 121.9 (d, J_C._F = 9.77 Hz), 119.3 (d, J_C-f = 17.1 Hz), 119.9, 115.1, 67.3, 64.8, 64.7, 64.5, 62.6, 45.6, 45.5, 43.4, 38.4, 31.0, 29.5, 27.5; ESI-MS calculated for C_2aH₃₂ ³⁵CIF₃N₃O₃ [M+Hf: 550.20, Found: 550.35.

C09101-TFA sait

¹H NMR (300 MHz, MeOH-d4): 8.43 (d, J = 7.73 Hz, 1H), 7.66-7.54 (m, 2H), 7.39 (t, J= 7.31 Hz, 1H), 7.24-7.10 (m, 2H), 6.82 (d, J =1.43 Hz, 1H), 5.26 (d, J =12.22 Hz, 1H), 4.61 (d, J = 11.38 Hz, 1H), 4.53 (d, J = 7.73 Hz, 1H), 3.57-3.54 (m, 1H), 1.93 (dd, J = 15.41, 8.26 Hz, 1H), 1.80-1.10 (m, 9H), 1.16 (s, 3H), 0.89 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 177.6, 166.6, 157.7 (d, J_C._F = 249.6 Hz), 144.9, 137.0, 132.4, 128.6, 126.5, 126.4 (d, J_C._F = 4.9 Hz), 124.0, 123.3, 122.3 (d, J_C-f= 19.2 Hz), 121.3 (d, J_C._F= 13.0 Hz), 118.7, 114.9, 112.0, 68.7, 64.4, 64.0, 62.8, 50.2, 48.9, 43.2, 37.9, 37.8, 30.9, 30.8, 29.4, 28.5, 28.3; ESI-MS calculated for C₃₀H₃7³⁵CI₂FN₃O₃ [M+H]⁺: 576.21, Found: 576.58.

C09601-TFA sait

CF₃CO₂I-I

128 ’H NMR (300 MHz, MeOH-d₄): 8.84 (d, J = 6.87 Hz, 1H), 7.62 (d, J= 8.10 Hz, 1H), 7.19 (dd, J = 8.10, 1.79 Hz, 1H), 7.15 (dt, J = 8.42, 1.87 Hz, 1H), 7.01 (s, 1H), 6.94 (d, J= 9.62, 1.30 Hz, 1 H), 6.83 (d, J = 1.69 Hz, 1 H), 5.21 (d, J ~ 11.25 Hz, 1 H), 4.45 (dd, J = 8.21, 1.56 Hz, 1H), 4.15 (d, J = 11.25 Hz, 1H), 4.00-3.82 (m, 1H), 2.50-2.36 (m,1H), 2.36-2.22 (m, 1H), 2.10-1.98 (m, 1H) 1.91 (dd, J = 15.44, 8.30 Hz, 1H), 1.84-1.72 (m, 1H), 1.31 (s, 3H), 1.16 (dd, J = 15.44,1.47 Hz, 1H), 0.90 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 177.7,167.0, 164.0 (d, JC-f= 249.6 Hz), 145.3, 137.3, 136.6 (d, JC-f= 20.6 H), 136.6, 126.4, 126.1 (d, JC-p= 2.91 Hz), 124.3, 123.8, 117.9 (d, JC-f = 25.0 Hz), 115.4 (d, J = 23.0 Hz), 112.3, 67.3, 64.8, 64.3, 62.7, 56.7, 45.7, 45.6, 43.3, 38.4, 31.0, 29.6, 27.6; ESI-MS calculated for C28H33^3SCI2FN₃O3 [M+H]⁺: 548.18 , Found: 548.67.

C09701-TFA sait

’H NMR (300 MHz, MeOH-d₄): 8.21 (d, J ~ 6.81 Hz, 1H), 7.68-7.54 (m, 2H), 7.39 (td, J = 7.60, 1.36 Hz, 1H), 7.18 (t, J = 8.09 Hz, 1H), 7.13 (dd, J = 7.97, 1.92 Hz, 1H), 6.79 (d, J = 1.66 Hz, 1H), 5.30 (d, J = 11.51 Hz, 1H), 4.56 (d, J = 11.84 Hz, 1H), 4.52 (d, J - 8.26 Hz, 1H), 3.86-3.70 (m, 1H), 2.00-1.80 (m, 2H), 1.65-1.05 (m, 8H), 1.04 (s, 3H), 0.87 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 177.8, 167.2, 145.1, 137.2, 132.7, 128.8, 126.7 (d, JC.F = 3.6 Hz), 126.6, 124.2, 123.4, 122.6 (d, JC-f = 18.7 Hz), 121.7 (d, JC-f = 12.5 Hz), 112.1, 69.7, 64.6, 64.2, 62.9, 49.2, 43.4, 36.8, 36.4, 31.0, 29.5, 28.3, 28.3; ESI-MS calculated for C3oH37³⁵CI2FN₃0₃ [M+H]⁺: 576.21, Found: 576.67.

C11701-TFA sait

’H NMR (300 MHz, MeOH-d₄): 8.83 (d, J = 6.61 Hz, 1H), 7.64 (dd, J = 6.06, 2.50 Hz, 1H), 7.59 (d, J= 8.11 Hz, 1H), 7.36-7.22 (m, 1H), 7.11 (dd, J = 8.09, 1.76 Hz, 1H), 7.00-6.82 (m, 1H), 1.72 (d, J =1.72 Hz, 1H), 5.13 (d, J = 11.21 Hz, 1H), 4.58 (d, J = 11.21 Hz, 1H), 4.51 (dd, J = 8.23, 1.66 Hz, 1H), 3.98-3.80 (m, 1H), 2.44-2.32 (m, 1H), 2.32-2.20 (m, 1H), 2.101.94 (m, 1H), 1.88 (dd, J= 15.38, 8.24 Hz, 1H), 1.80-1.68 (m, 1H), 1.29 (s, 3H), 1.14 (dd, J

129 = 15,38, 1.45 Hz, 1H), 0.89 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 177.9, 167.1, 161.1 (d, Jc-f = 243.6 Hz), 145.2, 137.3, 136.9, 132.1 (d, JCF = 9.0 Hz), 131.2 (d, JC-f = 3.60 Hz), 130.0 (d, JC.F = 2.22 Hz), 127.0, 123.8 (d, JC-f = 57.7 Hz), 121.6 (d, JC-f = 14.8 Hz), 118.5 (d, Jc.f = 25.4 Hz), 112.1, 67.4, 64.5, 64.4, 62.7, 48.4, 45.7, 45.5, 43.3, 38.4, 31.0, 29.6, 27.6; ESI-MS calculated for CZ8H33³⁵CI2FN₃O₃ [M+H]*: 548.19, Found: 548.67.

C29701-TFA sait

• CF₃CO₂H ¹H NMR (MeOH-d₄): 7.66 (d, J = 8.47 Hz, 1H), 7.56 (t, J = 6.87 Hz, 1H), 7.43 (td, J = 7.60,

1.47 Hz, 1H), 7.22 (d, J = 7.79 Hz, 1H), 7.16 (dd, J = 8.17, 1.89 Hz, 1H), 6,81 (d, J = 1.80 Hz, 1H), 5.17 (d, J- 11.92 Hz, 1H), 4.68-4.58 (m, 2H), 3.91-3.78 (m, 1H), 3.78-3.66 (m, 2H), 2.44-2.32 (m, 1H), 2.26-2.10 (m, 2H), 1.98-1.88 (m, 1H), 1.64-1.52 (m, 1H), 1.43 (t, J=7.15 Hz, 1H), 1.33-1.25 (m, 1H), 1.29 (s, 3H), 0.81 (s, 3H); ESI-MS: Calculated for C₃₀H₃₇CI₂FN₃O₃ [M+H]* = 576.22, Found: 576.92.

C30201-TFA sait

¹H NMR (MeOH-d₄): 7.68 (dd, J = 8.16, 1.47 Hz, 1H), 7.48 (t, J= 7.09 Hz, 1H), 7.39 (td, J= 7.30, 1.29 Hz, 1H), 7.20-7.08 (m, 2H), 6.79 (d, J= 1.79 Hz, 1H), 5.19 (d, J = 11.56 Hz, 1H), 4.83 (d, J - 11.56 Hz, 1H), 4.51 (t, J = 3.96 Hz, 1H), 3.94-3.78 (m,

1H), 3.12 (s, 3H), 2.50-2.36 (m, 1H), 2.32-2.20 (m, 1H), 2.10-1.92 (m, 2H), 1.73 (dd, J = 10.67, 9.23 Hz, 1H), 1.40-1.25 (m, 1H), 1.29 (s, 3H). 0.75 (s, 9H); ESI-MS: Calculated for C_ZgH₃₅Cl_zFN₃O₃ [M+H]* = 562.20, Found: 562.58.

EXAMPLE 2

Fluorescence-polarization MDM2 binding assay

The binding affinity of the MDM2 inhibitors was determined using an optimized, sensitive and quantitative fluorescence polarization-based (FP-based) binding assay using a

130 recombinant human His-tagged MDM2 protein (residues 1-118) and a fluorescently tagged p53-based peptide.

The design of the fluorescence probe was based upon a previously reported high-affrnity p53 -based peptidomimetic compound (5-FAM-pAla~pAla-Phe-Met-Aib-pTyr-(6-CI-LTrp)Glu-Ac3c-Leu-Asn-NH₂ (SEQ ID NO: 1)) (Garcia-Echeverria étal., J. Med. Chem. 43: 32053208 (2000)). This tagged peptide is called PMDM6-F. The K<j value of PMDM6-F with the recombinant MDM2 protein was determined from the saturation curve. MDM2 protein was serially double diluted in a Dynex 96-well, black, round-bottom plate, and the PMDM6-F peptide was added at 1 nM concentration. The assay was performed in the buffer: 100 mM potassium phosphate, pH 7.5; 100 pg/mL bovine gamma globulin; 0.02% sodium azide, 0.01% Triton X-100) and the polarization values were measured after 3 h of incubation using an ULTRA READER (Tecan U.S. Inc., Research Triangle Park, NC). The IC50 value was obtaîned by fitting the mP values in a sigmoidal dose-response curve (variable slope) with a non-linear régression, and was determined to be 1.40 nM ± 0.25. The value was caiculated using the équation: Ka value = IC₅₀ - L0/2. L0 is the total concentration of the fluorescent ligand; LO/2 is the total concentration of the fluorescent ligand divided by 2. Since PMDM6-F was used at a final concentration of 1 nM, LO/2 was 0.5 nM.

Dose-dependent, compétitive binding experiments were performed with serial dilutions of a tested compound in DMSO. A 5 pL 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 phosphate, pH 7.5; 100 pg/mL bovine gamma globulin; 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 pL. For each assay, the controls included the MDM2 protein and PMDM6-F (équivalent to 0% inhibition), PMDM6-F peptide alone (équivalent to 100% inhibition). The polarization values were measured after 3 h of incubation. The ICso values, /.e. the inhibitor concentration at which 50% of bound peptide is displaced, were determined from a plot using nonlinear least-squares analysis. Curve fitting was performed using GRAPHPAD PRISM software (GraphPad Software, Inc., San Diego, CA).

In the alternative, fluorescence polarization values were measured using the Infinité M-1000 plate reader (Tecan U.S., Research Triangle Park, NC) in Microfluor 2 96-well, black, roundbottom plates (Thermo Scientific). In the saturation experiments, 1 nM of PMDM6-F and increasing concentrations of proteins were added to each well to a final volume of 125 Cl in the assay buffer (100 mM potassium phosphate, pH 7.5, 100 Dg/ml bovine y-globulin, 0.02% sodium azide (Invitrogen), with 0.01% Triton X-100 and 4% DMSO). Plates were mixed and incubated at room température for 30 minutes with gentle shaking to assure equilibrium. The polarization values in millipolarization units (mP) were measured at an

131 excitation wavelength of 485 nm and an émission wavelength of 530 nm. Equilibrium dissociation constants (K_d) were then calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 5.0 software (Graphpad Software, San Diego, CA).

K) values of tested compounds were determined in a dose-dependent compétitive binding experiment. Mixtures of 5 DI of the tested compound in different concentrations in DMSO and 120 Cl of preincubated protein/fluorescent probe complex with fixed concentrations in the assay buffer (100 mM potassium phosphate, pH 7.5, 100 Dg/ml bovine y-globulin, 0.02% sodium azide, with 0.01% Triton X-100) were added into assay plates and incubated at room température for 30 minutes with gentle shaking. Final concentrations of the protein and fluorescent probe in the compétitive assays were 10nM and 1nM, respectively, and final DMSO concentration is 4%. Négative controls containing protein/fluorescent probe complex only (équivalent to 0% inhibition), and positive controls containing free fluorescent probe only (équivalent to 100% inhibition), were included in each assay plate. FP values were measured as described above. ICa, values were determined by nonlinear régression fitting of the sigmoidal dose-dependent FP decreases as a function of total compound concentrations using Graphpad Prism 5.0 software (Graphpad Software, San Diego, CA). Kj values of tested compounds to the MDM2 protein were calculated using the measured IC₅₀values, the Kd value of the fluorescent probe to the protein, and the concentrations of the protein and fluorescent probe in the compétitive assays (Nikolovska-Coleska et al., Anal. Biochem. 332:261-73 (2004)).

Compounds shown in Table 2A as the free base were tested either as the free base or as the CF₃CO₂H (TFA) or HCl sait. In general, comparable assay responses are expected between the free base and sait form of a compound (See, e.g., Ml-77301 (free base) and MI-77301 (TFA sait)).

Binding Kinetics of Different Isomers in Binding Media

Aliquots of freshly prepared DMSO stock solutions of compounds were diluted in FP binding assay buffer to préparé the aqueous compound incubation solutions in which compound isomerization was taking place. Final compound concentration in the incubation solution was 25μΜ, and 5% of DMSO was présent to enhance the solubility. These solutions were stored at room température for the whole time range of the experiment. 80 μΙ_ of aliquots of compound solutions were mixed with 20 μΙ_ of freshly prepared MDM2/PMDM6-F mixture in the assay plates at different time points. Final concentrations of the protein, fluorescent probe, and DMSO are same as those in the compétitive assays described above. Négative and positive controls were included in each assay plate as well. Following 15 minutes of

132 incubation at room température with gentle shaking, mP values were measured and IC₅₀values were determined as described above (Table 2B). Due to the plate préparation and incubation time required before measurement, it should be noticed that ail IC50 values presented below are values actually obtained 20 minutes after the labeled incubation time.

EXAMPLE 3

Fluorescence-polarization MDM2 binding assay

The binding affinity of the MDM2 inhibitors was optionally determined using a fluorescence polarization-based (FP-based) binding assay using a recombinant human MDM2 protein ÎO (residues 5-109) and PMDM6-F as follows:

MDM2 protein was serially diluted with a step of 1.8 in a Costar 96-well, black, non binding surface reference 3686 plate, and the PMDM6-F peptide was added at 5 nM concentration. The assay was performed in the buffer: 100 mM potassium phosphate, pH 7.5; 100 pg/mL bovine gamma globulin, 0.01% Triton X-100) and the anisotropy values were measured at 15 equilibrium using a Fusion reader (Packard). The fraction of ligand bound, F_Sb, was calculated using the following équation F_SB = (Aobs -AF)/[(Ab-Aobs)Q + Aobs -AF] (ref) where Aobs= anisotropy observed, Ab= anisotropy when ail p53 is bound, AF = anisotropy when p53 is free, Q= ratio Fluorescence intensity Bound /fluorescence intensity Free (Biochemistry 43:16056-16066 (2004)). KD was determined, using the Langmuir équation 20 applied to fluorescence polarization, to be 1.8 nM.

Dose-dependent, compétitive binding experiments were performed with serial dilutions of a tested compound in DMSO. A 5 pL sample of the tested compound and PMDM6-F peptide (5 nM) and MDM2 protein (6 or 8 nM) in the assay buffer (100 mM potassium phosphate, pH 7.5; 100 pg/mL bovine gamma globulin, 0.01% Triton X-100), were added in Costar 9625 well, black, non binding surface reference 3686 to produce a final volume of 125 pL. For each assay, the contrais included the MDM2 protein and PMDM6-F (équivalent to 0% inhibition), PMDM6-F peptide alone (équivalent to 100% inhibition). The polarization values were measured at equilibrium. The ICso values, Le. the inhibitor concentration at which 50% of bound peptide is displaced, were determined from a plot using the 4-parameter logistic 30 model (Ratkowsky and Reedy, Biométries 42(3):575-82 (1986). The adjustment was obtained by non-linear régression using the Marquardt algorithm in Xlfit software (Table 3).

Table 3

Compound	IC₅₀ (biot MDM2)	IC₅₀ (without Tag)
MI-710201	308.32 nM	272.83 nM
MI-710501	309.08 nM	260.40 nM

133

MI-710601	157.86 nM	137.94 nM
MI-710801	289.62 nM	256.02 nM
MI-710901	224.96 nM	184.74 nM
C02701	149.49 nM	111.52 nM
C02901	159.97 nM	121.90 nM
C03001	120.23 nM	89.59 nM
C031	3339.44 nM	2742.86 nM
C03401	107.66 nM	85.61 nM
C03701	270.09 nM	220.41 nM
C04801	473.01 nM	388.72 nM

EXAMPLE 4

Cell growth assay

Isogenic HCT-116 colon cancer cell Unes were a kind gift from Prof. Bert Vogelstein (Johns Hopkins, Baltimore, MD) and were maintained in McCoy’s 5A medium containing 10% FBS. Ail other cell lines were obtained from ATCC (Manassas, VA) and were maintained in RPMI1640 medium containing 10% FBS.

Cells were seeded in 96-well fiat bottom cell culture plates at a density of 2-3x10³ 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-(2methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium sait (Dojindo Molecular Technologies Inc., Gaithersburg, Maryland). 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 compounds that inhibited cell growth by 50% (ICso) was calculated by comparing absorbance in the untreated cells and the cells treated with the compounds using the GraphPad Prism software (GraphPad Software, La Jolla, CA 92037, USA). The results of this assay are presented in Tables 2A and 2C. Under conditions used in this assay, it is possible that a compound having Formula II isomerizes to a compound having Formula XII and other isomers (e.g., MI-773 isomerizes to MI-77301; MI-519-64 isomerizes to MI-519-6401). The results of this assay for MI-77301 in a variety of melanoma cell lines (Fernandez, Y., et al., Cancer Res. 65:6294-6304 and référencés cited therein) is presented in Fig. 24.

EXAMPLE 5

134

Cell death assay

Cell death assays were performed using trypan blue staining. Cells were treated in the presence and absence of indicated compounds. Both the floating and adhèrent cells were stained with trypan blue. Cells that stained blue or the morphologically unhealthy cells were 5 scored as dead cells. At least 100 cells were counted in each of three separate areas under microscope. As shown in Figs. 11 and 12, MDM2 inhibitors provided herein induce cell death in SJSA-1 and RS4;11 cancer cells with wild-type p53.

135

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Table 2B

	IC_go values (nM) to human MDM2 protein in FP assay
Time (hours)	MI-773	MI-77301	MI-519-64	MI-519-6401
0.1	79.9	11.3	77.2	6.5
3.0	74.3	11.2	54.0	7.0
7.0	65.2	12.3	46.2	8.8
24.0	57.7	15.1	43.4	8.9
32.0	49.2	10.6	42.7	8.3
48.0	46.6	14.5	34.8	8.8
72.0	33.3	12.8	27.9	9.6

Table 2C

Example	RS4;11 (p53 wt) IC_S0 (μΜ)
MI-519-6401	0.13 ±0.02’
MI-773 (TFA sait)	0.13¹
MI-77301 (free amine)	0.102; 0.081¹
MI-77301 (TFA sait)	0.059 ± 0.025¹
MI-7102	0.46
MI-710201	0.54
Ml-710301	0.16
MI-7104	0.52

150

MI-710401	0.66
MI-7105	0.37
MI-710601	0.13

¹ Four day treatment.

EXAMPLE 6

Western blotting

For Western blot analysis, cells were lyzed in ice-cold RIPA buffer: 20 mM Tris-HCI (pH 7.5), 150 mM NaCI, 1 mM EDTA, 1 mM EGTA, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1mM sodium orthovanadate and 1 pg/ml leupeptin. The proteins in the whole cell lysâtes were detected by Western blot analysis using the following antibodies: anti-p53 (clone DO-1), anti-MDM2 (clone SMP-14), anti-p21 (clone SX118), anti-p-actin (clone AC-40) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; HRP conjugated). As shown in Figs. 8, 9, and 13-16, MDM2 inhibitors provided herein are active in this assay.

PARP cleavage was used as a biochemical marker of apoptosis. During apoptosis, caspases cleave Poly (ADP-rîbose) polymerase (PARP). Rabbit anti-PARP (Cell Signaling Cat # 9542), used in the experiment, detects cleavage of full length (116kD) Poly (ADPribose) polymerase (PARP) and larger (89kD) cleaved fragment of PARP. A total of 2 χ 10⁶adhèrent cells were treated in the presence or absence of MI-77301 and incubated at 37°C for 19 hr. Cells were harvested using 0.05% trypsin-EDTA (Invitrogen), washed in PBS and lysed on ice for 15min using RIPA buffer (Sigma), supplemented with protease inhibitor cocktail (Roche). Clarîfied cell lysate was obtained by centrifuging the lysed cells at 13000 ^xg at 4°C for 15 min. Protein in the cell lysate was estimated using commercially available Bio-Rad protein assay dye. A total of 25 pg protein was loaded on a 4-20% SDS-PAGE gel, electrophoresed, and transferred to a PVDF membrane for 3 hours at 40 V. Membrane was blocked in TBST (20 mM Tris, 0.5 M NaCI, 0.1% Tween-20, pH 7.5) containing 5 % dry milk (Bio-Rad) for 1 hr at room température. Primary antibody diluted in TBST, containing 5% dry milk, was applied to the membrane overnight in cold room at 4 °C on an orbital shaker. Membrane was washed in TBST, incubated for 1 hr at room température with either an antirabbit secondary antibody (Immunopure goat anti-rabbit antibody, Thermo Scientific) or an anti-mouse antibody (Pierce goat anti-mouse antibody, Thermo Scientific), diluted 1:2000 in TBST. Membrane was washed in TBST and developed using SuperSignal West Pico

157 reagent (Thermo Scïentific). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody conjugated to HRP (Santa Cruz) was used as a loading control for proteins. Western blot analyses for MI-77301 are provided in Figs. 25, 26, and 28.

EXAMPLE 7

In vivo efficacy studies using SJSA-1 and 22Rv1 xenograft models

SJSA-1 (osteosarcoma) tumor cells were harvested with Trypsin (0.05%)-EDTA (0.53mM) (GIBCO™, Invitrogen Corp.), growth medium added and cells placed on ice. A cell sample was mixed 1:1 with Trypan Blue (GIBCO™, Invitrogen Corp.) and counted on a hemocytometer to détermine the number of live/dead cells. Cells were washed once with 1X PBS (GIBCO™, 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, Invitrogen Corp.) for a final Matrigel protein concentration of 5 mg/ml. SJSA-1 tumors were inoculated into C.B-17 SCID mice at 5 x 10⁶ cells in 0.1ml with Matrigel. Cells were injected s.c. into the flank région 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) = (AxB^z)/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 stopped, tumor volume (Figs. 17, 20, 22, and 23) and body weight (Figs. 18 and 21) was measured at least once a week. Mice were kept for an additional 60 days for further observation of tumor growth and toxicity. As shown in Fig. 22, a single 200 mg/kg dose of MI-77301 (QD1 treatment) shows comparable efficacy to a continuous dosing regimen (QD7 treatment).

Before treatment began, tumors were allowed to grow to 60-140 mm³ in volume, at which point the blood vessel supplies to the tumor should hâve been established. Mice with tumors within acceptable size range were randomized into treatment groups of 8 mice for experimental compounds and 10 mice for the Control group. Experimental compounds were given orally, once per day for 2-3 weeks, The Control group received vehicle alone (10% PEG 400:3% Cremophor:87% PBS). Other suitable vehicles for in vivo administration of the compounds provided herein include, without limitation, 98% PEG 200:2% polysorbate 80; 98% PEG 200:2% TPGS; and 0.5% polysorbate 80:0.6% methyl cellulose:98.9% water.

Using similar protocole, the antitumor activity of MI-519-6401 and MI-77301 was evaluated in the 22Rv1 prostate cancer model in mice (Fig. 19), and the antitumor activity of MI-77301 was evaluated in the HCT-116 human colorectal tumor model (Fig. 31), the LNCAP human prostate tumor model (Fig. 32), and the RS4;11 human ALL model (Fig. 33).

152.

EXAMPLE 8

Synthesis of MI-519-64 and MI-519-65

Step 1 : benzyl 3-oxocyclobutanecarboxylate (2)

Referring to Scheme 6A, BnBr was added to the mixture of compound 1 and K₂CO₃ in acetonitrile 150 mL. The mixture was stirred at room température over 24 h and the solid was filtered. The solvent was removed and the residue was purified by column chromatography to give compound 2.

Step 2: benzyl 3-hydroxy-3-methylcyclobutanecarboxylates (3 and 4)

MeMgCI in THF was added dropwise to the solution of compound 2 in diethyl ether at 78°C and the mixture was stirred at the same température for half an hour. After TLC monitoring showed the disappearance of the starting material, the reaction was quenched by adding aqueous NH₄CI solution. The aqueous phase was extracted with ethyl acetate three times and the combined organic phase was washed with brine and dried (Na₂SO₄). The solid was filtered and the solvent was removed. The residue was purified by column chromatography to give compounds 3 and 4 (5:1 based on TLC analysis).

Step 3: benzyl 3-(tert-butyldimethylsilyloxy)-3-methylcyclobutane carboxylates (5 and 6)

To the mixture of compounds 3 and 4 in DMF (10 mL) was added immidazole and TBSCI, and the resulting mixture was stirred at 80°C for 30 h. After cooling to room température, water was added and the aqueous phase was extracted with ethyl acetate three times. The combined organic phase was washed with brine and dried (Na₂SO₄). The solid was filtered and the solvent was removed. The residue was purified by column chromatography to get compounds 5 and 6.

Step 4: 3-(tert-butyldimethylsilyloxy)-3-methylcyclobutanecarboxylic acids (7 and 8)

To the mixture of compounds 5 and 6 in îsopropanol was added Pd/C. The resulting mixture was stirred under 1 atm hydrogen for 1 h. TLC showed the disappearance of the starting material and the solid was filtered. The solvent was removed to give compounds 7 and 8.

Step 5: benzyl-3-(tert-butyldimethylsilyloxy)-3-methylcyclobutylcarbamates 9 and 10

To a 0°C stirring solution of compounds 7 and 8 and Et₃N in acetone was added CICOOEt dropwise. The resulting mixture was stirred at 0°C for 30 min. A solution NaN₃ in water was added, and the resulting mixture was stirred at 0°C for an additional 20 min. Water was added, and the aqueous phase was extracted with ethyl acetate three times. The combined organic phase was washed with brine and dried (Na₂SO₄). The solvent was removed and the residue was dissolved in toluene. Benzyl alcohol and NaHCO₃ were added. The

5 resulting mixture was stirred at 80°C for 2 h. Ali the solvent was removed and the residue was purified by column chromatography to obtain two isomers 9 and 10 in a 5:1 ratio.

Step 6: 3-(tert-butyldimethylsilyloxy)-3-methylcyclobutanamine (11)

To a mixture of the major isomer 9 and NaHCO₃ in isopropanol was added Pd/C and the 5 resulting mixture was stirred under 1 atm hydrogen for 1 h. The solid was filtered and the solvent was removed to give compound 11.

Step 7: 3-(tert-butyldimethylsilyloxy)-3-methylcyclobutanamine (12)

To a mixture of the minor isomer 10 and NaHCO₃ in isopropanol was added Pd/C and the resulting mixture was stirred under 1 atm hydrogen for 1h. The solid was filtered and the 10 solvent was removed to give compound 12.

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Step 8: MI-519-64

Referring to Scheme 6B, to a solution of compound 11 in THF was added compound 13 and the resulting solution was stirred overnight. The solvent was removed and the residue thus obtained was dissolved in CH3CN/H2O (1:1). CAN was added and the reaction mixture was stirred for 30 min. Water was added and the aqueous phase was extracted with ethyl acetate three times. The combined organic layers were dried (Na₂SO₄), filtered, and concentrated. The residue was purified by column chromatography on siiica gel to give compound 14. Compound 14 was dissolved in methanol, 12M HCl in water was added, and the reaction mixture was stirred for 1 h at room température. The solvent was removed and the residue was purified by HPLC to give MI-519-64 as the TFA sait. ¹H NMR (300 MHz, CD₃OD) δ 7.54-7.52 (m, 1H), 7.42-7.38 (m, 1H), 7.23-7.18 (m, 1H), 6.88-6.75 (m, 3H), 5.04 (d, J= 9.9 Hz, 1H), 4.45 (d, J= 9.9 Hz, 1H), 4.19-4.16 (m, 1H), 3.92-3.89 (m, 1H), 2.42-2.11 (m, 2H), 2.10-1.87 (m, 3H), 1.32-1.24 (m, 4H), 0.82 (s, 9H); MS (ESI) m/z 548 [M+H]⁺.

Step 9: MI-519-65

To a solution of compound 12 in THF was added compound 13 and the resulting solution was stirred overnight. The solvent was removed and the residue was dissolved in CH₃CN/H₂O (1:1). CAN was added and the reaction mixture was stirred for 30 min. Water was added and the aqueous phase was extracted with ethyl acetate three times. The combined organic layers were dried (Na₂SO₄), filtered, and concentrated. The residue was purified by column chromatography on siiica gel to give compound 15. Compound 15 was dissolved in methanol, 12M HCI in water was added, and the reaction mixture was stirred for 1 h at room température. The solvent was removed and the residue was purified by HPLC to give MI-519-65 as the TFA sait. ¹H NMR (300 MHz, CD₃OD) □ 7.50 (m, 1H), 7.44-7.38 (m, 1H), 7.24-7.20 (m, 1H), 6.89-6.88 (m, 1H), 6.80 (m, 1H), 6.71 (m, 1H), 4.91-4.88 (m, 1H), 4.40-4.36 (m, 2H), 4.10-4.06 (m, 1H), 2.41-2.33 (m, 2H), 2.07-1.87 (m, 3H), 1.25-1.21 (m, 4H), 0.82 (s, 9H); MS (ESI) m/z 548 [M+H]*.

EXAMPLE 9

Synthesis of MI-519-6401

Scheme 7

MI-519-64 (100 mg) purified by flash chromatography on silica gel was placed in a 50 mL round-bottom-flask equipped with magnetic stirring bar. Acetonitrile (20 mL) was added to fully dissolve the compound and deionized water (7 to 10 mL) was added. NaHCO₃saturated aqueous solution (ca. 0.5 mL) was then added to adjust the pH value between 7 and 8. This solution was allowed to stir at room température for at least 12 h. TFA (0.1 mL) and another 10 mL of deionized water were added to the solution and the solution was purified by semi-preparative RP-HPLC immediately using acetonitrile and water as the eluents to give MI-519-6401 as the TFA sait. ¹H NMR (300 MHz, MeOH-d4): 7.62-7.53 (m, 2H), 7.45-7.35 (m, 1H), 7.20-7.10 (m, 2H), 6.80-6.85 (m, 1H), 5.11 (d, J = 11.07 Hz, 1H), 4.57 (d, J =11.11 Hz, 1H), 4.40 (d, J = 7.39 Hz, 1H), 4.00-3.80 (m, 1H), 2.50-2.35 (m, 1H), 2.35-2.20 (m, 1H), 2.10-1.90 (m, 1H), 1.90-1.60 (m, 2H), 1.30 (s, 3H), 1.20-1.05 (m, 1H), 0.88 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 177.8, 168.0, 157.6 (d, J_C-f - 249 Hz). 144.9, 136.8, 132.2, 128.5, 126.5, 126.3 (d, J_C-f = 4.76), 123.9, 123.7, 122.3 (d, J_C-f= 18.97 Hz), 122.0 (d, Jc-f =13.1 Hz), 111.8, 67.1, 64.6, 64.5, 62.9, 49.0, 45.5, 45.4, 43.3, 38.0, 30.8, 29.5, 27.3; ESI-MS calculated for C₂₈H₃₃ ³⁵CI₂FN₃O₃ [M+H]⁺: 548.1883, Found: 548.25.

Analytical RP-HPLC spectra are presented in Figs. 1-3. Referring to Fig. 3, MI-519-6401 corresponds to the RP-HPLC peak at 31.787 minutes.

In an alternate procedure, MI-519-64 (100 mg) purified by flash chromatography on silica gel was placed in a 50 mL round-bottom-flask equipped with magnetic stirring bar. Methanol (20 mL) was added to fully dissolve the compound and deionized water (10 to 20 mL) was added. NaHCO₃ saturated aqueous solution (ca. 0.5 mL) was then added to adjust the pH value between 7 and 8. This solution was allowed to stir at room température for at least 12 h. TFA (0.1 mL) and another 10 mL of deionized water were added to the solution and the solution was purified by semi-preparative RP-HPLC immediately using acetonitrile and water as the eluents to give MI-519-6401 as the TFA sait.

C02701, C02901, C03001, C03401, C03701, C03801, C04801, C08301, C08601, and C11701 of EXAMPLE 1 were prepared using procedures similar to that used to préparé MI-519-6401.

EXAMPLE 10

156

Synthesis of MI-773

Scheme 8

CAN

CH₃CN/H₂0/acetone 2:1:1 ice bath. 5 min

MI-773

Step 1

To a stirred solution of oxindole 1 (4.19 g, 25 mmol) in methanol (50 mL) was added aldéhyde 2 (3.96 g, 25 mmol) and piperidine (2.45 mL, 25 mmol). The reaction mixture was stirred at room température for 3 h and the yellow precipitate was collected, washed successively with methanol, hexanes, and ethyl ether and dried to give compound 3 (6.25 g, 81% yield).

Step 2

To a solution of compound 3 (6.25 g, 21 mmol) in toluene (75 ml) was added compound 4 (5.43 g, 21 mmol), compound 5 (2.15 g, 21 mmol) and 4Â molecular sieves (4 g). The reaction mixture was heated at reflux overnight and filtrated. The filtrate was evaporated and the residue was purified by silica gel flash column chromatography (n-hexane/ethyl acetate = 9:1 to 5:1 ) to give compound 6 (8.78 g, 65% yield).

Step 3

The solution of compound 6 (965 mg, 1.5 mmol) and amine 7 (346 mg, 3 mmol) in 5 mL of THF was stirred at room température for 2 days and the solvent was removed under reduced pressure. The residue was purified by silica gel flash column chromatography (nhexane/ethyl acetate = 1:1 to 1:4) to give compound 8 (819 mg, 72% yield).

15θ

Step 4

To an ice-bath cooled solution of compound 8 (800 mg, 1.05 mmol) in CH3CN (8 ml), H₂O (4 ml) and acetone (4 ml) was added CAN (ammonium cérium) (1.15 g, 2.1 mmol). Progress of the reaction was monitored by TLC. When ail the starting material disappeared (around 5 min), 100 mg of NaHCO₃ powder was added and the reaction mixture was diluted with 50 mL of ethyl acetate. The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue was purified by silica gel flash column chromatography (methylene chloride/methanol/triethylamine = 200:1:1 to 200:10:1) to give (2'R,3S,4'S,57?)6-chloro-4'-(3-chloro-2-fluorophenyl)-N-((trans-4-hydroxycyclohexyl)-2^,-neopentyl-2oxospiro[indoline-3,3’-pyrrolidine]-5’-carboxamide (MI-773) (402 mg, 68% yield, Purity (HPLC): > 95% (Fig. 35)). The absolute stereochemical configuration of MI-773 was determined by x-ray analysis.

MI-773 was dissolved in DCM, TFA was added, and the solvent was removed by évaporation. The residue was further purified by chromatography on a C18 reverse phase semi-preparative HPLC column with solvent A (0.1% of TFA In water) and solvent B (0.1% of TFA in methanol) as eluents (gradient: 45% of solvent A and 55% of solvent B to 30% of solvent A and 70% of solvent B in 30 min) to give MI-773 as the TFA sait. NMR for MI-773 (TFA sait): ¹H NMR (300 MHz, CD₃OD) δ 7.47 (t, J - 7.0 Hz, 1H), 7.34 (t, J = 7.4 Hz, 1H), 7.14 (t, J= 7.9 Hz, 1H), 6.83 (s, 1H), 6.80 (s, 2H), 4.39 (d, J = 10.0 Hz, 1H), 4.15-4.05 (m, 1H), 3.72-3.53 (m, 1H), 3.53-3.85 (m, 2H), 2.10-1.75 (m, 4H), 1.62 (d, J = 12.2 Hz, 1H), 1.45-1.05 (m, 5H), 0.78 (s, 9H).

Stability of MI-773 (TFA sait): MI-773 (TFA sait) was dissolved in a water/méthanol mixture: 1) water/methanol = 1:1 with 0.1% of TFA, pH 2.1; 2) water/methanol = 1:1 with 0.1% of TEA, pH 10.8; or 3) water/methanol = 1:1, pH 3.9. The solution was allowed to stand at room température. The purity was tested using a C18 reverse phase analytical HPLC column at the time points of 0, 12 h, 24 h, 48 h, and 72 h. The results showed transformation of MI-773 (corresponding to peak 3) to ΜΙ-77302 (corresponding to peak 1 ), MI-77301 (corresponding to peak 4) and another compound (corresponding to peak 2) having the same molecular weight (Figs. 6, 37, and 38). The purity of an identical sample solution stored at 4°C was also tested at 0 and 36 h. The results showed comparably slow transformation of MI-773 at 4°C.

EXAMPLE 11

Synthesis of MI-77301

Scheme 9

160 c

OH

MeOH/HjO

Ml-773

Cl

MI-77301

MI-77302

MI-773 (as the TFA sait) was dissolved in MeOH/H₂0 (1:1 v/v ratio) and allowed to stand at room température for 1-4 days. The solution was purified by chromatography on a C18 reverse phase semi-preparative HPLC column with solvent A (0.1% of TFA in water) and solvent B (0.1% of TFA in methanol) as eluents (gradient: 45% of solvent A and 55% of solvent B to 30% of solvent A and 70% of solvent B in 30 min). MI-77301 was isolated as the TFA sait. ¹H NMR (300 MHz, MeOH-d4): 8.35 (d, J = 7.8 Hz, 1H), 7.54-7.62 (m, 2H), 7.37-7.43 (m, 1H), 7.12-7.20 (m, 2H), 6.80 (d, J = 1.5 Hz, 1H), 5.20 (d, J = 11.4 Hz, 1H), 4.58 (d, J = 11.4 Hz. 1 H), 4.51 (d, J = 7.2 Hz, 1H), 3.50-3.75 (m, 1H), 3.30-3.50 (m, 1H), 1.82-2.00 (m, 3H), 1.76 (d, J = 10.5 Hz, 1H), 1.52 (d, J = 12.3 Hz, 1H), 1.05-1.42 (m, 4H), 0.88-1.00 (m, 1H), 0.88 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 177.7, 166.9, 157.6 (d, JC-F = 248.0 Hz), 145.0, 137.0, 132.4, 128.6, 126.6, 126.4 (d, JC-F = 4.9), 124.0, 123.4, 122.3 (d, JC-F = 18.8 Hz), 121.5 (d, JC-F = 12.8 Hz), 111.9, 69.9, 64.4, 64.0, 62.8, 49.7, 34.3, 34.2, 30.9, 30.82, 30.77, 29.4; ESI-MS calculated for C29H35CI2FN3O3 (M + H)+ requires 562.20, found 562.33; [a]_D ²⁵ = -27.2° (c = 0.005 g/mL in MeOH); Purity (HPLC): > 95% (See Fig. 36).

In an alternative procedure, MI-773 (77 mg) was dissolved in 15 mL MeOH/H₂O (v/v =1:1). After 3 days, the needle crystals that had formed were collected, washed with cold MeOH/H₂O (v/v =1:1) and dried in vacuum to give MI-77301 as the free amine (20 mg; >95% purity as determined by HPLC). ’H NMR (300 MHz, MeOH-d₄): 7.49-7.55 (m, 1H), 7.25-7.31 (m, 1 H), 7.10-7.16 (m. 1H), 6.82 (d, J = 1.8 Hz, 1H), 6.50-6.71 (m, 1H), 6.49 (d, J = 8.4 Hz, 1H), 4.32 (d, J = 9.0 Hz, 1H), 4.09 (d, J = 8.7 Hz, 1H), 3.57-3.69 (m, 1H), 3.49 (d, J = 9.2 Hz, 1H), 3.46-3.57 (m, 1H), 1.83-2.07 (m, 3H), 1.68-1.80 (m, 1H), 1.54 (dd, J = 9.0, 14.3 Hz, 1H), 1.12-1.45 (m, 5H), 0.80 (s, 9H). The absolute stereochemical configuration of MI-77301 was determined by X-ray analysis.

Stability of MI-77301 (TFA sait): MI-77301 (TFA sait) was dissolved in a water/methanol mixture (water/methanol =1:1 with 0.1% of TFA). The solution was allowed to stand at room température. The purity was tested using a C18 reverse phase analytical HPLC column at the time points of 0, 12 h , 48 h, and 72 h. The results showed slow transformation of MI77301 (corresponding to peak 4) to MI-77302 (corresponding to peak 1) and two other compounds (corresponding to peaks 2, and 3) having the same molecular weight (Fig. 7). The absolute stereochemistry of MI-77302 was determined by x-ray analysis.

161

MI-710201, Ml-710401, MI-710501, MI-710601, MI-710801, and MI-710901 of EXAMPLE 1 were prepared using procedures similar to that used to préparé MI-77301.

¹³C CPMAS NMR spectroscopy (400 MHz) of MI-77301 (top), MI-773 (middle), and Ml77302 (bottom) is presented in Fig. 34. Chemical shift différences are observed in the 5 carbonyl région (170-185 ppm).

In an alternative procedure, MI-77301 was prepared as described in Scheme 9A.

Scheme 9A ci

toluene. 115°C 75% yield (250 g scale)

OH

2.1 eq. CAN

MeTHF/ H₂O 90% (crude) yield (35 g scale)

Cl

1. Isomerization In AcOIPr ! cal/ AcOH

2. crystalllzatlon In AcOiPr

3. desolvation / drying

55% yield (15 g scale)

Cl

chemical purity > 97%

EXAMPLE 12

Synthesis of:

162 (2'S,3R,4'R,5'S)-6-chloro-4'-(3-chloro-2-fluorophenyl)-N-((trans)-4hydroxycyclohexyl)-2'-neopentyl-2-oxospiro[indoline-3,3^,-pyrrolidine]-5'carboxamide; and (2‘R,3S,4'R_I5'S)-6-chloro-4^,-(3-chloro-2-fluorophenyl)-N-((trans)-4hydroxycyclohexyO^'-neopentyl^-oxospiroIindoline-S.S'-pyrrolidineJ-S'carboxamide

(5S,6R)-5,6-diphenylmorpholin-2-one was prepared according to J. Org. Chem. 2005, 70, 6653. mp: 139°C (Kofler); LC-MS: t_R (min) = 0.96; [M+Hf: m/z 254 (method C). (3R.3^,R.4'S.6'S.8^,R,8a'S)-6-chloro-8^l-(3-chloro-2-fluoropheny|)-6^l-neopentyl-3’,4'-diphenvl 3'.4',8^t.8a'-tetrahvdrospiro[indoline-3,7'-Dvrrolor2,1-cl[1.4]oxazinel -1',2(6’H)-dione

To a suspension of 496 mg (1.61 mmol) of (E)-6-chloro-3-(3-chloro-2fluorobenzylidene)indolin-2-one in toluene under argon, were added 408 mg (1.61 mmol) of (5S,6R)-5,6-diphenylmorpholin-2-one and 213 pL (1.61 mmol) of 3,3-dimethylbutyraldehyde. The reaction mixture was heated at reflux température for 6 hours, upon which it was cooled down to room température and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography on a 70 g silica cartridge (15-40 pm silica gel; eluting solvent: cyclohexane/ ethyl acetate 90/10 v/v; flow: 50 mL/min). 0.58 g of (3R,3'R₁4^,S,6^,S,8’R,8a'S)-6-chloro-8'-(3-chloro-2-fluorophenyl)-6^,-neopentyl-3^,,4'-diphenyl S'.^.S'.ea'-tetrahydrospirotindoline-SJ’-pyrrolo^.l-cHI ,4]oxazine] -T,2(6’H)-dione was obtained as an amorphous yellow solid. LC-MS: t_R (min) = 1.81; [M+Hf: m/z 643;[M-H]': m/z 641 (WATERS UPLC-SQD apparatus; lonization: electrospray in positive mode and/or négative mode (ES+/-); Chromatographie conditions: Column: ACQUITY BEH C18 1.7 pm 2.1 x 50 mm; Solvents: A: H2O (0.1 % formic acid) B: CH₃CN (0.1 % formic acid); Column température: 50 °C; Flow: 0.8 ml/min; Gradient (2.5 min): from 5 to 100 % of B in 1.8 min; 2.4 min: 100 % of B; 2.45 min: 100 % of B; from 100 to 5 % of B in 0.05 min; Rétention time = t_R (min); referred to herein as Method C); ¹H NMR (400 MHz, DMSO-de): 0.39 (s, 9 H); 1.30 (dd, J=4.0 and 15.2 Hz, 1 H); 1.93 (dd, J=6.3 and 15.2 Hz, 1 H); 3.49 (dd, J=4.0 and 6.3 Hz, 1 H); 4.47 (d, J=11.2 Hz, 1 H); 5.04 (d, J=4.2 Hz, 1 H); 5.08 (d, J=11.2 Hz, 1 H); 6.53 (d, J-8.1 Hz, 1 H); 6.66 to 6.76 (m, 3 H); 6.96 (m, 2 H); 7.10 to 7.26 (m, 9 H); 7.34 (t broad, J=7.9 Hz, 1 H); 7.62 (t broad, J=7.9 Hz, 1 H); 10.71 (s broad, 1 H).

163 (2^,S.3R.4^,R.5^,S)-6-chloro-4^,-(3-chloro-2-fluorophenyl)-1^,-((1S.2R)-2-hvdroxv-1.2diphenvlethvl)-N-((frans)-4-hvdroxvcvclohexvl)-2'-neopentvl-2-oxospiroίindoline-3.3^,pyrrolidinel-S’-carboxamide

To a mixture of 112 mg (0.97 mmol) of trans-4-aminocyclohexanol in 2 mL of tetrahydrofuran under argon, was added 0.57 g (0.89 mmol) of (3R,3'R,4'S,6'S,8’R,8a'S)-6chloro-8'-(3-chloro-2-fluorophenyl)-6'-neopentyl-3',4'-diphenyl 3',4',8',8a’tetrahydrospiro[indoline-3,7'-pyrrolo[2,1-c][1,4]oxazine] -T,2(6’H)-dtone in 8 mL of tetrahydrofuran. The resulting mixture was heated at 60°C for 17 hours, upon which 136 pL (0.97 mmol) of triethylamine was added and heating was pursued.

After 24 hours, 13.6 mg (0.12 mmol) of trans-4-aminocyclohexanol and 2.5 mL of tetrahydrofuran were added. After 41 hours, the reaction mixture was cooled down to room température and concentrated to dryness under reduced pressure. The residue was diluted with a mixture of 20 mL of ethyl acetate and 6 mL of water and decanted. The aqueous phase was extracted with 6 mL of ethyl acetate. The combined organic phases were dried over magnésium sulfate and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography on a 70 g siiica cartridge (15-40 pm siiica gel; eluting solvent: cyclohexane/ ethyl acetate 50/50 v/v followed by 40/60 v/v; flow: 50 mL/min). 0.55 g of (2'S,3R,4'R,5'S)-6-chloro-4'-(3-chloro-2-fluorophenyl)-1 '-((1 S,2R)-2-hydroxy-1,2diphenylethyl)-N-((trans)-4-hydroxycyclohexyl)-2'-neopentyl-2-oxospiro[indoline-3,3' pyrrolidine]-5'-carboxamide was obtained as a white meringue, mp: 190°C (Kofler); LC-MS: t_R (min) = 1.56; [M+H]⁺: m/z 758; [M-H]‘: m/z 756 (method C); ¹H NMR (400 MHz , DMSOd₆): 0.51 (s, 9 H); 0.62 (d, J=15.2 Hz, 1 H); 0.81 (m, 1 H); 1.02 to 1.35 (m, 4 H); 1.60 (m, 1 H); 1.79 (m, 2 H); 2.36 (dd, J=9.3 and 15.2 Hz, 1 H); 3.24 to 3.34 (m partially hidden, 2 H);

3.48 (m, 1 H); 3.69 (d, J=10.9 Hz, 1 H); 3.94 (d, J=7.9 Hz, 1 H); 4.13 (d, J=10.9 Hz, 1 H); 4.45 (d, J=4.4 Hz, 1 H); 5.05 (dd, J=4.4 and 7.9 Hz, 1 H); 5.14 (d, J=4.4 Hz, 1 H); 6.18 (d, J=8.2 Hz, 1 H); 6.50 (d, J=2.0 Hz, 1 H); 6.59 (t broad, J=7.9 Hz, 1 H); 6.67 (dd, J=2.0 and 8.2 Hz, 1 H); 6,73 (t broad, J=7.9 Hz, 1 H); 7.20 (t, J=7.8 Hz, 1 H); 7.24 (t broad, J=7.9 Hz, 1 H); 7.28 (t, J=7.8 Hz, 2 H); 7.35 (m, 2 H); 7.39 to 7.45 (m, 3 H); 7.49 (d, J=8.2 Hz, 1 H); 7.57 (d, J=7.8 Hz, 2 H); 10.32 (s broad, 1 H).

(2^,S.3R₁4'R,5'S)-6-chloro-4^,-(3-chloro-2-fluorophenvl)-N-((frans)-4-hydroxvcvclohexvl)-2^,-neopentvl-2-oxospiro[indoline-3,3^,-pyrrolidine1-5^,-carboxamide

A mixture of 542 mg (0.71 mmol) of (2'S,3R,4'R,5’S)-6-chloro-4'-(3-chloro-2-fluorophenyl)-1'((1S,2R)-2-hydroxy-1,2-diphenylethyl)-N-((frans)-4-hydroxycyclohexyl)-2'-neopentyl-2oxospirolindolîne-S.S'-pyrrolidinel-S'-carboxamide in 10 mL of éthanol was cooled to 0°C and

16¼

989 mg (1.79 mmol) of ceric ammonium nitrate was added slowly via spatula in 15 min. The reaction mixture was stirred at 0°C for 1 hour, upon which it was treated with 4 mL of toluene, 2 mL of éthanol, 5 mL of saturated brine and 3 mL of ethyl acetate, and decanted. The organic phase was separated and the aqueous phase was extracted with 2x5 mL of ethyl acetate. The organic phases were combined and washed with 3 mL of 5% sodium carbonate. After décantation, the aqueous phase was diluted with water and reextracted with 10 mL of ethyl acetate. The organic phases were combined and successively washed with 2 mL of 11% sodium disulfite and 2 mL of saturated brine. It was then dried over magnésium sulfate and concentrated to dryness under reduced pressure. 222 mg of the residue was purified by flash chromatography on a 40 g silica cartridge (15 pm silica gel; eluting solvent: dichloromethane/ acetone 75/25 v/v followed by 65/35 v/v; flow: 30 mL/min). 0.183 g of an off-white meringue was obtained, taken up twice in diisopropyl oxide and dried at 25°C under reduced pressure. 157 mg of (2’S,3R,4^,Rl5’S)-6-chloro-4'-(3-chloro-2fluorophenyl)-N-((frans)-4-hydroxy-cyclohexyl)-2’-neopentyl-2-oxospiro[indoline-3,3'pyrrolidineJ-S'-carboxamide were obtained as a white amorphous solid. mp: 176°C (Kofler); LC-MS: tR (min) =1.13 (91%) and 1.03 (9%); [M+H]⁺: m/z 562; [M-H]’: m/z 560 (method C); ¹H NMR (400 MHz, DMSO-d6): 0.76 (s, 9 H); 0.85 (dd, J=1.8 and 14.3 Hz, 1 H); 0.97 to 1.28 (m, 4 H); 1.38 to 1.50 (m, 2 H); 1.67 (m, 1 H); 1.80 (m, 2 H); 2.56 (t, J=12.1 Hz, 1 H);

3.23 to 3.38 (m partially hidden, 2 H); 3.45 (m, 1 H); 3.93 (d, J=9.6 Hz, 1 H); 4.22 (dd, J=9.6 and 12.1 Hz, 1 H); 4.46 (d, J=4.4 Hz, 1 H); 6.62 (d, J=8.1 Hz, 1 H); 6.70 to 6.75 (m, 2 H); 7.13 (t broad, J=7.9 Hz, 1 H); 7.31 (t broad, J=7.9 Hz, 1 H); 7.64 (t broad, J=7.9 Hz, 1 H); 7.92 (d, J=7.7 Hz, 1 H); 10.51 (s broad, 1 H).

(2^,R.3S.4^,R.5^,S)-6-chloro-4'-(3-chloro-2-fluorophenyl)-N-((trans)-4-hvdroxvcvclohexvl)-2^,-neopentyl-2-oxospiro[indoline-3.3^,-pyrrolidinel-5^,-carboxamide

The rest of the crude compound (206 mg) was dissolved and stirred into 10 mL of ethyl acetate, treated with 41 pL (0.07 mmol) of glacial acetic acid and the reaction mixture was heated at 60°C for 3 hours, upon which it was cooled down to room température and stirred for 16 hours. It was then washed with 5 mL of saturated sodium hydrogencarbonate and 3 mL of water. The organic phase was dried over magnésium sulfate and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography on a 30 g silica cartridge (15-40 pm silica gel; eluting solvent: dichloromethane/ acetone 75/25 v/v; flow: 20 mL/min). The isolated product was taken up twice in diisopropyl oxide. The solid was filtered and dried at 25°C under reduced pressure. 106 mg of (2’R,3S,4'R,5’S)-6-chloro4'-(3-chloro-2-fluorophenyl)-N-((frans)-4-hydroxycyclohexyl)-2'-neopentyl-2oxospiro[indoline-3,3' pyrrolidine]-5’-carboxamide were obtained as a pinkish amorphous

16&

solid. mp: 193°C (Kofler); LC-MS: t_R (min) = 1.03; [M+H]⁺: m/z 562; [M-H]’: m/z 560 (method C); ¹H NMR (400 MHz. DMSO-d₆): 0.72 (dd, J=1.5 and 14.2 Hz, 1 H); 0.80 (s, 9 H); 1.08 to 1.30 (m, 5 H); 1.68 to 1.91 (m, 4 H); 3.28 to 3.49 (m, 3 H); 3.58 (m, 1 H); 4.29 (d, J=9.0 Hz, 1 H); 4.39 (t, J=9.0 Hz, 1 H); 4.50 (d, J=4.4 Hz, 1 H); 6.68 (d, J=2.0 Hz, 1 H); 7.05 (dd, J=2.0 and 8.1 Hz, 1 H); 7.12 (t broad, J=7.9 Hz. 1 H); 7.34 (t broad, J=7.9 Hz, 1 H);

7.48 to 7.57 (m, 2 H); 7.72 (d, J=8.1 Hz, 1 H); 10.40 (s broad, 1 H); 0_D = +18.4° +/- 0.9 (c = 1.525 mg/ O.ômLMeOH).

EXAMPLE 13

Synthesis of (2’8,3Ή,4'5,5^,β)-Γ-3θθΙγΙ-6-οήΙθΓθ-4'-(3-οΚΙθΓθ-2-ίΙυοΓθ-ρήθηγΙ)-2'(2,2-dimethyl-propyl)-2-oxo-1,2-00170^-^^0(^016-3^^^0110^6)-5^ carboxylic acid (frans-4-hydroxy-cyclohexyl)-amide

Acetic acid 4-fl(2^,S,3'R.4'S,5'R)-1,1'-diacetvl-6-chloro-4'-(3-chloro-2-fluoro-phenvn-2'f2,2-dimethvl-propvn-2-oxo-1,2-dihvdro-spirofindole-3.3^l-pyrrolidinel-5’-carbonyllaminol-cyclohexvl ester

To a solution of 281 mg (0.50 mmol) of (2'S,3^,R,4'S,5'R)-6-chloro-4^,-(3-chloro-2-fluorophenyl)-2'-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro [indole-3,3'-pyrrolidine]-5'-carboxylic acid (frans-4-hydroxy-cyclohexyl)-amide in 5.0 mL of pyridine under argon, was added 178 pL (2.50 mmol) of acetyl chloride. The resulting mixture was stirred at room température for 4 days, upon which it was poured into a mixture of water and ethyl acetate. The organic phase was separated and the aqueous phase was extracted twice with ethyl acetate. The combined organic extracts were washed with brine, dried with magnésium sulfate and then concentrated to dryness under reduced pressure. The residue was purified by flash chromatography on a 50 g silica cartridge (15-40 pm silica gel; eluting solvent: dichloromethane/ methanol 98/2 v/v; flow: 40 mL/min) followed by a second purification by flash chromatography on a 30 g silica cartridge (15-40 pm silica gel; eluting solvent: dichloromethane/ methanol 98/2 v/v; flow: 20 mL/min). 123 mg of acetic acid 4{[(2^,S,3^,R,4‘S,5’R)-1,T-diacetyl-6-chloro-4^,-(3-chloro-2-fluoro-phenyl)-2'-(2,2-dimethyl166 propyl)-2-oxo-1,2-dihydro-spiro [indole-3,3'-pyrrolidin]e-5'-carbonyl]-amino}-cyclohexyl ester were obtained as a white powder. LC-MS: t_R (min) = 1.19; [M+Hf: m/z 688; [M-H]’: m/z 686 (method A).

(2'S,3’R.4’S.5'RÎ-T-Acetvl-6-chloro-4‘-(3-chloro-2-fluoro-phenyl)-2'-(2,2-dimethvl· ΡΓθρνΠ-2-οχο-1 ^-dihvdro-spirofindole-S.S'-pvrrolidinel-S'-carboxvlic acid (trans-4hvdroxv-cyclohexvD-amide

To a solution of 117 mg (0.17 mmol) of acetic acid 4-{[(2’S,3'R,4'S,5^,R)-1l1‘-diacetyl-6chloro-4'-(3-chloro-2-fluoro-phenyl)-2’-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole3,3'-pyrrolidine]-5'-carbonyl]-amino}-cyclohexyl ester in 10.0 mL of methanol under argon, was added 10 mL (81 mmol) of a saturated potassium carbonate solution. The resulting mixture was stirred at room température for 1 hour, upon which the methanol was evaporated under reduced pressure. The remaining aqueous phase was extracted 3 times with 20 mL of dichloromethane. The combined organic extracts were washed with 20 mL of brine, dried with magnésium sulfate and then concentrated to dryness under reduced pressure. 92 mg of (2^,S,3'R,4^,S,5^,R)-T-acetyl-6-chloro-4‘-(3-chloro-2-fluoro-phenyl)-2'-(2,2dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3'-pyrrolidine]-5'-carboxylic acid (trans-4hydroxy-cyclohexylfamide were obtained as a white powder. mp: 220°C (Kofler); LC-MS: tR (min) = 0.97; [M+Hf: m/z 604; [M-H]’: m/z 602 (method A); ¹H NMR (60°C, CHLOROFORM-d, 400 MHz): 0.68 (s, 9 H); 0.75 to 2.62 (m partially hidden, 14 H); 3.51 (m, 1 H); 3.65 (m, 1 H); 4.28 (m broad, 1 H); 4.48 (dd, J=3.4 and 5.9 Hz, 1 H); 4.98 (d, J=10.3 Hz, 1 H); 6.65 (d, J=1.5 Hz, 1 H); 6.99 (t, J=7.8 Hz, 1 H); 7.05 to 7.48 (m, 5 H).

16T

EXAMPLE 14

Synthesis of (2'S_l3^,R,4'S₁5’R)-6-chloro-4'-(3-chloro-2-fluoro-phenyl)-2^,-(2,2» dimethyl-propyl^-oxo-l^-dihydro-spiroljndole-S.S'-pyrroliclinel-S’-carboxylic acid (frans-4-hydroxy-cyclohexylmethyl)-amîde

(2^,R_<3¹S.4'S,5'R)-6-Chloro-4^l-(3-chloro-2-fluoro-phenvl)-2'-(2.2-dimethvl-propyl)-T((1 R.2S)-2-hydroxy-1,2-diphenvl-ethyl)-2-oxo-1 .Z-dihydro-spirofindole-S.S'pyrrolidinel-5'-carboxvlic acid (trans-4-hvdroxv-cyclohexylmethvO-amide

To a solution of 0.28 g (1.71 mmol) of 4-aminomethyl-cyclohexanol hydrochloride in 12.0 mL of tetrahydrofuran, was added 0.46 mL (3.26 mmol) of triethylamine. The resulting mixture was stirred at room température for 30 minutes and 1 g (1.55 mmol) of:

was added progressively via spatula, followed by 2 mL of tetrahydrofuran. The reaction mixture was heated at reflux température for 7 hours and then stirred at room température for 2.5 days, upon which it was diluted with 10 mL of water and 15 mL of ethyl acetate. The organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic extracts were dried with magnésium sulfate and then concentrated to dryness under reduced pressure. The residue was purified by flash chromatography on a 70 g silica cartridge (15-40 pm silica gel; eluting solvent: dichloromethane, then dichloromethane/ methanol 98/2 v/v; flow: 50 mL/min). 0.40 g of (ZR.S'S^'S.S'RJ-e-chloro-^S-chloro^-fluorophenyl)-2'-(2,2-dimethyl-propyl)-1^,-((1R,2S)-2-hydroxy-1,2-diphenyl-ethyl)-2-oxo-1,2dihydro-spiro[indole-3,3'-pyrrolidine]-5’-carboxylic acid ( trans-4-h ydroxycyclohexylmethyl)-amide were obtained as a white solid. LC-MS: t_R (min) = 1.16; [M+Hf: m/z 772; [M-H]’: m/z 770 (method A); Ή NMR (CHLOROFORM-d, 400 MHz): 0.74 (s large, 9 H); 0.83 (m, 2 H); 1.13 (m, 2 H); 1.20 (m, 1 H); 1.25 (d, J=15.6

16S

Hz, 1 H); 1.35 (d, J=4.9 Hz, 1 H); 1,42 (m, 1 H); 1.55 (m partially hidden, 1 H); 1.84 to 2.00 (m, 2 H); 2.29 (d, J=2.9 Hz, 1 H); 2.67 (dd, J=9.3 and 15.6 Hz, 1 H); 2.86 (m, 1 H); 3.38 (m, 1 H); 3.48 (m, 2 H); 4.11 (m, 2 H); 4.36 (d, J=8.3 Hz, 1 H); 5.20 (dd, J=2.9 and 8.3 Hz, 1 H); 5.64 (d, J=8.1 Hz, 1 H); 6.28 (m, 1 H); 6.44 (t, J=7.8 Hz, 1 H); 6.53 (d, J=2.2 Hz, 1 H); 6.62 (dd, J=2.2 and 8.1 Hz, 1 H); 6.67 (t, J=7.8 Hz, 1 H); 7.09 (t, J=7.8 Hz, 1 H); 7.25 (s, 1 H); 7.30 to 7.45 (m, 5 H); 7.54 to 7.67 (m, 5 H).

(2'S,3^lR.4'S,5'R)-6-Chloro-4'-(3-chloro-2-fluoro-Dhenvl)-2'-(2,2-dimethyl-proDvl) -2oxo-1 ^-dihydro-spirofindole-S.S-pyrrolidinel-S'-carboxylic acid_ (/rafis-4-hvdroxvcvclohexylmethvD-amide

In a three-neck 50 mL flask were successively introduced 0.70 g (0.91 mmol) of (2'R,3’S_l4'S,5^,R)-6-chloro-4'-(3-chloro-2-fluoro-phenyl)-2'-(2,2-dimethyl-propyl)-r-((1R,2S)-

2-hydroxy-1,2-diphenyl-ethyl)-2-oxo-1,2-dihydro-spiro[indole-3,3'-pyrrolidine]-5'-carboxylic acid (trans-4-hydroxy-cyclohexylmethyl)-amide, 7.0 mL of acetonitrile, 3,5 mL of distilled water and 3.5 mL of acetone. The resulting mixture was stirred and cooled to 0°C and 0.99 g (1.81 mmol) of cérium ammonium nitrate was added in small portions. The reaction mixture was stirred at 0°C for 20 minutes, upon which 89 mg (1.06 mmol) of sodium hydrogencarbonate were added and stirring was maintained for 5 minutes. The mixture was diluted with 60 mL of ethyl acetate and decanted. The organic phase was separated and the aqueous phase was extracted twice with 10 mL of ethyl acetate. The combined organic extracts were washed with 20 mL of water, dried with magnésium sulfate and then concentrated to dryness under reduced pressure. The residue was purified by flash chromatography on a 30 g silica cartridge (15-40 pm silica gel; eluting solvent:

dichloromethane; flow: 30 mL/min) followed by a second purification by flash chromatography on a 15 g silica cartridge (15-40 pm silica gel; eluting solvent:

dichloromethane/ methanol/ 28% ammonia 97/2/1 v/v/v; flow: 30 mL/min). 87 mg of (2’S,3^,R,4'S,5’R)-6-chloro-4'-(3-chloro-2-fluoro-phenyl)-2^,-(2,2-dimethyl-propyl)-2-oxo-1,2dihydro-spiro[indole-3,3’-pyrrolidine]-5'-carboxylic acid (trans-4-hydroxy-cyclohexylmethyl)amide were obtained as a white solid. mp: 192°C (Kofler); LC-MS: t_R (min) = 0.84; [M+H]⁺: m/z 576; [M-H]': m/z 574 (method A); ¹H NMR (CHLOROFORM-d, 400 MHz): mixture of isomers: 0.90 (s, 9 H); 0.99 to 2.09 (m partially hidden, 11 H); 3.05 to 3.27 (m, 2 H); 3.59 (m, 2 H); 4.39 (d, J=8.8 Hz, 1 H); 4.59 (m, 1 H); 6.76 (s large, 1 H); 6.99 (t, J=7.8 Hz, 1 H); 7.06 to 7.56 (m, 6 H); 7.82 (t broad, J=6.1 Hz, 1 H).

EXAMPLE 15

1«

Synthesis of (2'S,3'R,4'S,5'R)-6-Chloro-4^,-(3-chloro-2-fluoro-phenyl)-2^,-(2,2dimethyl-propyl)-T-methyl-2-oxo-1,2-(111^^0^^0(^016-3^^^0^106)-5^ carboxylic acid (frans-4-hydroxy-cyclohexyl)-amide

To a suspension of 0.50 g (0.89 mmol) of (2^,S,3’R,4’S,5'R)-6-chloro-4'-(3-chloro-2-fluorophenyl)-2'-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro [indole-3,3’-pyrrolidine]-5’-carboxylic acid (trans-4-hydroxy-cyclohexyl)-amide in 18.0 mL of acetonitrile under argon, were added 0.89 mL (0.97 mmol) of a 36.5% solution of formaldéhyde in water, followed by 65 mg (0.98 mmol) of sodium cyanoborohydride. The resulting solution was stirred at room température for 2 hours, upon which it was poured into 50 mL of ethyl acetate. The aqueous phase was separated and the organic phase was washed with a saturated solution of sodium hydrogencarbonate. The latter aqueous phase was reextracted with ethyl acetate. The combined organic extracts were washed with brine, dried with magnésium sulfate and then concentrated to dryness under reduced pressure. The residue (0.52 g) was purified by chiral HPLC chromatography on a Kromasil C18 column (1100 g batch 4680, 10 pm, 7.65 x 35 cm), eluting solvent: acetonitrile/ water 40/60 v/v + 0.1 % trifluoroacetic acid; flow: 250 mL/min. The collected solution was treated with sodium hydrogencarbonate up to pH 8 and then extracted 3 times with 200 mL of ethyl acetate. The combined organic extracts were washed twice with 100 mL of water, dried with magnésium sulfate and then concentrated to dryness under reduced pressure. The residue was then dried in a dessicator under reduced pressure for 16 hours. 0.17 g of ^’S.S'R^'S.S'RJ-S-chloro^’TS-chloro^-fluoro-phenyl)^’(2,2-dimethyl-propyl)-T-methyl-2-oxo-1,2-dihydro-spiro[indole-3l3'-pyrrolidine]-5^,-carboxylic acid (4-hydroxy-cyclohexyl)-amide was obtained as a white solid. mp: 188°C (Kofler); LCMS: tR (min) = 0.85-0.97 (mixture of isomers); [M+Hf: m/z 576; [M-Hf: m/z 574 (method A); ¹H NMR (400 MHz, CHLOROFORM-d): 0.73 (d, J=15.6 Hz, 1 H); 0.79 (s, 9 H); 1.15 to

1.48 (m, 4 H); 1.85 to 2.12 (m, 5 H); 2.75 (s, 3 H); 3.60 to 3.77 (m, 3 H); 4.17 (d, J=9.8 Hz, 1 H); 4.33 (d broad, J=9.8 Hz, 1 H); 6.69 (d, J=1.5 Hz, 1 H); 6.99 (t, J=7.8 Hz, 1 H); 7.05 (dd, J=1.5 and 8.3 Hz, 1 H); 7.10 to 7.25 (m, 3 H); 7.37 (m broad, 1 H); 7.56 (t, J=7.8 Hz, 1 H). Similar methodology was used to prépare C29701 and C30201.

170

EXAMPLE 16

Isomerization Studies

General Information

Expérimente involving moisture and/or air sensitive components were performed in ovendried glassware under an atmosphère of nitrogen. Commercial solvents and reagents were used without further purification with the following exception: THF was freshly distilled from sodium wire.

Flash chromatography was performed using silica gel (type H) from TM chemicals, Inc. Columns were typically packed as slurry and equilibrated with hexane prior to use. Analytical thin layer chromatography (TLC) was performed using Merck 60 F254 precoated silica gel plate (0.2 mm thickness). Subjected to elution, plates were visualized using UV radiation. Further visualization was possible by staining with basic solution of potassium permanganate or acidic solution of phosphomolybdic acid, followed by heating with heating gun.

Compounds were purified by HPLC using a Waters Sunfire C18 reverse phase semipreparative HPLC column (19 mm * 150 mm) using solvent A (water, 0.1% of TFA) and solvent B (CH3CN, 0.1% of TFA or MeOH, 0.1% of TFA) as eluents with a flow rate of 10 mL/min on a Waters Delta 600 instrument. Analytical reverse phase HPLC was conducted using Waters 2795 Séparation module.

Proton nuclear magnetic résonance (’H NMR) and carbon nuclear magnetic résonance (¹³C NMR) spectroscopy were performed on a Bruker Advance 300 NMR spectrometer. Chemical shifts of ’H NMR spectra are reported as δ in units of parts per million (ppm) downfield from SiMe4 (δ 0.0) or relative to the signal of chloroform-d (δ = 7.26, singlet), methanol-d4 (δ = 3.31, quintuplet) and DMSO-de (δ = 2.50, quintuplet). Multiplicities were given as: s (singlet); d (doublet); t (triplet); q (quartet); dd (doublet of doublets); ddd (doublet of doublets of doublets); dt (doublet of triplets); m (multiplets) and etc. The number of protons for a given résonance is indicated by nH. Coupling constants are reported as J values in Hz. Carbon nuclear magnetic résonance spectra (¹³C NMR) are reported as δ in units of parts per million (ppm) downfield from SiMe4 (δ 0.0) or relative to the signal of chloroform-d (δ = 77.23, triplet), MeOH-d₄ (δ = 49.20, septuplet) and DMSO-d_e (δ = 39,52, septuplet).

Low resolution ESl mass spectrum analysis was performed on Thermo-Scientific LCQ Fleet mass spectrometer.

Compounds 1-10 were prepared according to Scheme 10 and Table 4 using methods previous described (See, e.g., Ding, K. et al., J. Am. Chem. Soc. 727:10130-10131 (2005);

172

Ding, K. et al., J. Med. Chem. 49:3432-3435 (2006); Yu, S. et al., J. Med. Chem. 52:79707973 (2009); Shangary, S., Proc. Natl. Acad. Sci. 705:3933-3938 (2008); US 7,759,383) as a mixture of isomers. Isomer A was identified as the prédominant isomer following CAN oxidation in most cases. Applicants hâve found that dissolving the mixture of isomers obtained from CAN oxidation in a solvent or a mixture of solvents and allowing the reaction mixture to mature for a period of time under various conditions provides a mixture of isomers having Isomer B as the prédominant isomer. In some cases, Isomers C and D were isolated in pure or substantially pure form. Likewise, compounds 11 and 12 were prepared according to Scheme 11 and Table 5.

The procedure for isomerization used in this study was as follows: approximately 30 mg product obtained from CAN oxidation (pre-purified by flash column chromatography) was placed in a round bottom flask equipped with magnetic stirring bar. Acetonitrile (2.4 mL) was added to dissolve the product. To the acetonitrile solution was added water (2.0 mL) and 0.5 mL NaHCO₃ (saturated) solution to give a pH of approximately 8. The reaction mixture was allowed to stir at room température for approximately 3 days. The percentage of isomers was determined using analytical HPLC. Further purification was performed on semi-preparative or préparative reverse phase HPLC using MeOH (0.1% TFA) and water (0.1% TFA) as mobile phase.

Isomerization of Isomer A to Isomer B can also be carrîed out under acidic conditions, e.g., MeCN-H₂O, CF₃CO₂H (pH <1), room température, 3 days; ethyl acetate, acetic acid, 60°C, 3 h, or neutral conditions e.g., MeOH or MeOH-H₂O.

172

Scheme 10

1.0 equlv

1.1 equiv 1.1 equlv

HNR^bR^c

1,3 equlv THF, rt, 12 h

Oxidation

CAN 2.1 equiv

MeCN:H₂O (1:1) rt. 1 h

Isomer A

Isomer B

Isomer izatlon

Isomers A-D sat. NaHCOj MeCN:H₂O (1:1) rt, 3 days

173

Table 4

C P d	R 1	R 2	-NR^bR^c	Yi el d^a(% )	Isomer Ratio after Oxidati on A:B:(C+ D)^b	Isomer Ratio after Isomerizat ion A:B:(C+D)⁶
1	I-	H	-NMe₂	50 C	95:2:3	6:71:24
2	h	H	-NHMe	61	87:5:8	3:71:26
3	h	^F	-NMe₂	57	75:19:6	35:46:19
4	H	F	NHMe	84	67:2:31	9:79:12
5	H	F	H OH	86	88:2:10	1:80:19
6	H	F	, r H	79	89:10:1	12:61:27
7	H	F	-NH₂	56	0:44:56	0:96:4
8	F	H	-NMe_z	60	56:25:19	10:71:19 2:88:8
9	F	H	-NHMe	87	19:52:27	1:74:25
1 0 e	F	H	OH	81	30:32:38	3:58:37

^a Yield after oxidation;⁶ Ratio was determined by HPLC analysis;^c Yield after HPLC séparation; ^ύ Ratio determined by HPLC analysis after allowing oxidation product to stand in MeOH for two hours;⁹ Compound 10 Isomer A and Compound 10 Isomer B are referred to as MI-219 and MI-21901, respectively, in Table 2A .

17¼

Scheme 11

CAN 2.1 equiv

MeCN-H₂O1:1 rt, 1 hour

74%

lund 11 C and D

25%

Compound 12 Isomer A

Compound 12 Isomer B

Compound 12 Isomers C and D tsomerizalion

Isomers A-D sat. NaHCO₃MeCN:H₂O (1:1) rt. 3 days

Table 5

C	Yield	Isomer Ratio	Isomer Ratio
P	(%)	after Oxidation	after Equilibration
d	a:b:(c+d)^b	a:b:(c+d)¹’

175

1 1	74^a	89:6:3	44:50:6
1 2	25^e	—	41:34:25*

^e Yield after oxidation;^b Ratio was determined by HPLC analysis;^c Hydrolysis yield; ^dStarting with pure Compound 12-lsomer A.

Analytical Data

Compound 1 - Isomer A (TFA sait): ESl: Calculated for C₂5H₃o³⁵CI₂N₃0₂ [M+H]⁺= 474.17, Found: 474.50.

Compound 1 - Isomer B (TFA sait): ESl: Calculated for C₂₅H₃o³⁵CI₂N₃0₂ [M+H]⁺= 474.17, Found: 474.68.

Compound 1 - Isomer D (TFA sait): ¹H NMR (300 MHz, CD3OD): 7.72 (d, J = 8.08 Hz, 1H), 7.28-7.22 (m, 1H), 7.20-7.12 (m, 2H), 7.12-7.08 (m, 1H), 7.04 (d, J = 7.65 Hz, 1H), 6.81 (d, J = 1.79 Hz, 1H), 5.54 (d, J - 10.87 Hz, 1H), 4.37 (dd, J = 6.54, 4.52 Hz, 1H), 4.15 (d, J = 10.85 Hz, 1H), 2.98 (s, 3H), 2.81 (s, 3H), 1.70 (dd, J = 15.24, 6.67 Hz, 1H), 1.20 (dd, J = 15.26, 4.43 Hz, 1H), 0.91 (s, 9H); ESl: Calculated for C25H30³⁵CI2N₃O₂ [M+H]⁺= 474.17, Found: 474.50.

Compound 2 - Isomer A (TFA sait): ¹H NMR (300 MHz, CD3OD): 7.28-7.16 (m, 3H), 7.167.06 (m, 1H), 6.92-6.82 (m, 2H), 6.80-6.76 (m, 1H), 4.92 (d, J =10.23, 4.20-4.10 (m, 2H), 2.73 (s, 3H), 1.99 (s, J = 15.32, 6.75 Hz, 1H), 1.47 (dd, J = 15.54, 3.49 Hz, 1H), 0.80 (s, 9H); ESl: Calculated for C24H28³⁵CI2N₃O₂ [M+H]⁺= 460.16, Found: 460.52.

Compound 2 - Isomer B (TFA sait): ¹H NMR (300 MHz, CD3OD): 7.63 (d, J = 8.06 Hz, 1H), 7.30-7.14 (m, 4H), 7.12-7.00 (m, 1H), 6.82-6.76 (m, 1H), 5.29 (d, J= 11.24 Hz, 1H), 4.47 (d, J = 6.68 Hz, 1H), 4.16 (d, J = 11.22 Hz, 1H), 2.73 (s, 3H), 1.92 (dd, J= 15.40, 8.39 Hz, 1H), 1.17 (d, J = 16.85 Hz, 1H), 0.90 (s, 9H); ¹³C (75 MHz, CD3OD): 177.78, 168.54, 145.38, 137.07, 135.77, 134.60, 131.40, 130.29, 129.48, 128.29, 126.32, 124.24, 124.16, 112.18, 65.11, 64.18, 62.85, 56.95, 43.42, 30.97, 29.66, 26.98; ESl: Calculated for C₂₄H₂₈ ³⁵CI₂N₃O₂[M+H]⁺= 460.16, Found: 460.48.

Compound 2 - Isomer D (TFA sait): ¹H NMR (300 MHz, CD3OD): 7.57 (d, J = 8.12 Hz, 1 H), 7.24-7.08 (m, 3H), 7.04-6.98 (m, 1 H), 6.92 (d, J = 7.62 Hz, 1H), 6.76 (d, J = 1.78 Hz, 1H), 5.02 (d, J = 12.47 Hz, 1H), 4.42 (dd, J = 7.06, 4.40 Hz, 1H), 4.13 (d, J = 12.47 Hz, 1H), 2.70 (s, 3H), 1.74 (dd, J = 15.35, 7.14 Hz, 1H). 1.17 (dd, J = 15.37, 4.38 Hz, 1H), 0.88 (s, 9H); ¹³C (75 MHz, CD3OD): 176.72, 167.32, 144.88, 137.13, 135.62, 133.98, 131.17, 130.24, 129.73, 128.41, 127.88, 123.91, 123.53, 112.57, 65.42, 64.33, 62.97, 59.03, 44.86, 31.01,29.57, 27.03; ESl: Calculated for C₂₄H₂₈ ³⁵CI₂N₃O2 [M+H]⁺= 460.16, Found: 460.50.

17ff

Compound 3 - Isomer A (TFA sait): ¹H NMR (300 MHz, CD3OD): 7.80-7.70 (m, 1H), 7.507.40 (m, 1H), 7.32-7.22 (m, 1H), 6.92-6.88 (m, 1H), 6.74 (dd, J =8.15, 1.75 Hz, 1H), 6.48 (d, J = 8.11 Hz, 1H), 5.58 (d, J = 7.94 Hz, 1H), 4.38 (d, J = 7.94 Hz, 1H), 4.26 (d, J= 7.55 Hz, 1H), 2.99 (s, 3H), 2.84 (s, 3H), 2.06 (dd, J= 15.42, 7.72 Hz, 1H), 1.13 (d, J = 15.37 Hz, 1H), 0.89 (s, 9H); ¹³C (75 MHz, CD3OD): 180.07, 166.93, 157.51 (d, Jc-f = 243.98 Hz), 145.59, 137.11, 132.54, 128.77, 127.66, 126.74 (d, J_C._F = 4.55 Hz), 125.89 (d, J_C._F =13.40 Hz), 123.68, 122.85 (d, J_C._F = 10.19 Hz), 123.10 (d, J_C._F = 18.37 Hz), 112.07, 63.03, 62.05, 61.76, 42.09, 37.70, 36.89, 30.77, 29.34; ESl: Calculated for C₂₅H₂₉ ³⁵CI₂FN₃O₂ [M+H]⁺= 492.16, Found: 492.44.

Compound 3 - Isomer B (TFA sait): ¹H NMR (300 MHz, CD₃OD): 7.68-7.57 (m, 2H), 7.45-

7.35 (m, 1H), 7.22-7.10 (m, 2H), 6.84-6.77 (m 1H), 5.68 (d, J = 10.24 Hz, 1H), 4.64 (d, J = 10.24 Hz, 1H), 4.48 (dd, J = 8.20, 1.70 Hz, 1H), 2.98 (s, 3H), 2.80 (s, 3H), 1.89 (dd, J = 15.48, 8.26 Hz. 1 H), 1.14 (dd, J = 15.48, 1.67 Hz, 1H), 0.89 (s, 9H); ¹³C (75 MHz, CD3OD): 177.96, 168.37, 152.99 (d, JC.F = 253.32 Hz), 145.26, 137.39, 132.82, 128.82, 126.95, 126.70 (d, JC F = 4.79 Hz), 124.28, 122.09 (d, JC.F = 13.13 Hz), 118.90, 115.11, 112.24, 64.69, 59.97, 42.64, 37.96, 37.03, 31.03, 29.62; ESl: Calculated for C25H29³⁵CI2FN₃O₂[M+H]*= 492.16, Found: 492.46.

Compound 3 - Isomer C (TFA sait): ¹H NMR (300 MHz, CD3OD) (TFA sait): 7.63 (d, J = 8.13 Hz, 1H), 7.52-7.38 (m, 2H), 7.24-7.12(m, 2H), 6.83 (d, J = 1.80 Hz, 1H), 5.47 (d, J = 11.16 Hz, 1H), 4.42 (d, J = 11.31 Hz, 1H), 4.35 (t, J = 5.66 Hz, 1H), 2.95 (s, 3H), 2.93 (s, 3H), 1.91 (dd, J= 15.39, 5.80 Hz, 1H), 1.71 (dd, J= 15.24, 5.43 Hz, 1H), 0.84 (s, 9H); ESl: Calculated for C25H29³⁵Cl2FN3O₂ [M+H]⁺= 492.16, Found: 494.20.

Compound 3 - Isomer D (TFA sait): ¹H NMR (300 MHz, CD3OD) (TFA sait): 7.60 (d, J = 8.07 Hz, 1H), 7.40-7.30 (m, 1H), 7.22-7.12 (m, 1H), 7.07 (dd, J = 8.16, 1.87 Hz, 1H), 7.046.95 (m, 1H), 6.80 (d, J = 1.87 Hz, 1H), 5.58 (d, J = 9.80 Hz, 1H), 4.51 (d, J = 9.84 Hz, 1H), 4.43 (dd, J = 6.69, 4.15 Hz. 1H), 2.99 (s, 3H), 2.80 (s, 3H), 1.68 (dd, J = 15.41, 6.81 Hz, 1 H), 1.42 (dd, J = 15.41, 4.26 Hz, 1H), 0.90 (s, 9H); ESl: Calculated for C25H2g³⁵CI2FN₃O2 [M+H]⁺= 492.16, Found: 492.28.

Compound 4 - Isomer A (TFA sait): ¹H NMR (300 MHz, CD3OD): 7.66-7.54 (m, 1H), 7.387.28 (m, 1H), 7.22-7.10 (m, 1H), 6.87 (s, 1H), 6.82-6.72 (m, 2H), 5.21 (d, J = 10.00 Hz, 1H), 4.50 (d, J = 9.93 Hz, 1 H), 4.30-4.24 (m, 1H), 2.75 (s, 3H), 2.07 (dd, J= 15.35, 7.35 Hz, 1H), 1.8 (d, J = 14.57 Hz, 1H), 0.80 (s, 9H); ¹³C (75 MHz, CD3OD): 180.31, 167.24, 157.81 (d, Jq. _F = 247.78 Hz), 145.44, 136.94, 132.11, 128.66, 127.94, 126.47 (d, J_C._F = 4.45 Hz), 125.29, 125.01 (d, J_C-f= 13.96 Hz), 123.37, 122.64 (d, J_C._F = 18.03 Hz), 122.04, 64.35, 63.69, 61.74, 49.51, 42.34, 30.92, 29.55, 27.10; ESl: Calculated for C₂₄H₂₇ ³⁵CI₂FN₃O₂ [M+H]⁺= 478.15, Found: 478.92.

177

Compound 4 - Isomer B (TFA sait): ’H NMR (300 MHz, CD₃OD): 7.65-7.55 (m, 2H), 7.407.34 (m, 1H), 7.20-7.10 (m, 2H), 6.84-6.80 (m, 1H), 5.26 (d, J = 11.15 Hz, 1H), 4.64 (d, J = 11.19 Hz, 1H), 4.45 (d, J = 7.86 Hz, 1H), 2.74 (s, 3H), 1.87 (dd, J = 15.19, 8.58 Hz, 1H), 1.11 (d, J= 15.21 Hz, 1H), 0.90 (s, 9H); ’³C (75 MHz, CD3OD): 177.75, 168.20, 159.53 (d, Jc-f - 248.25 Hz), 145.22, 137.24, 132.59, 128.58, 126.63 (d, Jc* - 5.25 Hz), 124.16, 123.43, 126.66, 122.59 (d, JC-f - 4.05 Hz), 121.54 (d, JC.F = 12.75 Hz), 112.13, 64.49, 64.40, 62.73, 55.34, 43.33, 31.01, 29.58, 27.06; ESI: Calculated for C24H27³⁵CI2FN₃O₂ [M+H]⁺= 478.15, Found: 478.25.

Compound 4 - Isomer C (TFA sait): ’H NMR (300 MHz, CD₃OD): 7.62 (d, J = 8.17 Hz, 1H), 7.46-7.36 (m, 2H), 7.22 (dd, J = 8.13, 1.89 Hz, 1 H), 7.15 (t, J = 8.08 Hz, 1H), 6.80 (d, J = 1.85 Hz, 1H), 4.84 (d, J = 12.32, 1H), 4.35 (d, J = 12.32 Hz, 1H), 4.24 (t, J = 5.62 Hz, 1H), 2.67 (s, 3H), 1.87 (dd, J = 5.44, 2.53 Hz, 2H), 0.77 (s, 9H); ESI: Calculated for C_Z4H₂₇ ³⁵CI₂FN₃O₂ [M+H]⁺= 478.15, Found: 478.52.

Compound 4 - Isomer D (TFA sait): ’H NMR (300 MHz, CD₃OD): 7.53 (d, J = 8.11 Hz, 1 H), 7.36-7.26 (m, 1H), 7.11 (dd, J= 8.08, 1.88 Hz, 1H), 6.98-6.90 (m, 2H), 6.77 (d, J = 1.85 Hz, 1 H), 5.03 (d, J = 11.95 Hz, 1 H), 4.51 (d, J = 11.87 Hz, 1 H), 4.50-4.42 (m, 1 H), 2.72 (s, 3H), 1.72 (d, J = 15.36, 7.30 Hz, 1H), 1.16 (dd, J = 15.37, 4.09 Hz, 1H), 0.89 (s, 9H); ESI: Calculated for C₂₄H₂₇ ³⁵CI₂FN₃O₂ [M+H]⁺= 478.15, Found: 478.38.

Compound 5 - Isomer A (TFA sait): ’H NMR (300 MHz, CD₃OD): 7.63-7.53 (m, 1H), 7.407.30 (m, 1H), 7.23-7.13 (m, 1H), 6.87 (d, J = 1.36 Hz, 1H), 6.86-6.74 (m, 2H), 5.19 (d, J =

10.22 Hz, 1H), 4.49 (d, J = 10.22 Hz,1H), 4.25 (dd, J ~ 7.35, 2.71 Hz, 1H), 3.56-3.43 (m, 1H), 3.42-3.30 (m, 4H), 2.07 (dd, J = 15.43, 7.44 Hz,1 H), 1.72-1.58 (m, 1H), 1.56-1.40 (m, 1H), 1.30 (dd, J = 15.43, 2.62 Hz, 1H), 0.80 (s, 9H); ’³C (75 MHz, CD3OD): 180.26, 166.78, 157.81 (d, JC-f = 247.93 Hz), 145.43, 136.90, 132.16, 128.82, 127.96, 126.48 (d, JC-f = 4.49 Hz), 125.43, 124.94 (d, JC.F = 14.00 Hz), 123.37, 122.63 (d, JC.F = 18.23 Hz), 112.00, 71.01, 67.25, 64.38, 63.80, 61.69, 50.06, 42.36, 38.30, 33.80, 30.94, 29.55; ESI: Calculated for C27H33³⁵CI2FN₃O₄ [M+H]⁺= 552.18, Found: 552.92.

Compound 5 - Isomer B (TFA sait): ’H NMR (300 MHz, CD₃OD): 7.60 (d, J = 8.15 Hz, 1 H), 7.55 (t, J= 7.12 Hz, 1H), 7.38 (t, J = 7.57 Hz, 1H), 7.20-7.10 (m, 2H), 6.78 (d, J= 1.70 Hz, 1 H), 5.27 (d, J = 11.40 Hz, 1 H), 4.62 (d, J = 11.40 Hz, 1 H), 4.52 (dd, J = 8.33, 1.29 Hz, 1 H), 3.45-3.25 (m, 5H), 1.90 (dd, J= 15.46, 8.38 Hz, 1H), 1.64-1.48 (m, 1H), 1.46-1.32 (m, 1H), 1.14 (dd, J = 15.46, 1.34 Hz, 1H), 0.87 (s, 9H); ¹³C (75 MHz, CD3OD): 177.77, 167.67, 157.90 (d, Jc-F - 251.2 Hz), 145.21, 137.26, 132.67, 128.64, 126.89 (d, JC.F = 1.88 Hz), 126.65 (d, Jc-f = 4.89 Hz), 124.17, 123.43, 122.60 (d, JC.F = 19.0 Hz), 121.52 (d, JC-f = 13.0 Hz), 112.14, 79.99, 67.25, 64.45, 62.873, 48.8, 43.39, 38.26, 33.78, 31.02, 29.57; ESI: Calculated for C27H33^3SCI2FN₃O₄ [M+H]⁺= 552.18, Found: 552.42;

17ff

Compound 5 - Isomer C (TFA sait): Ή NMR (300 MHz, CD₃OD): 8.37 (s, broad, NH), 7.62 (d, J = 8.11 Hz, 1H), 7.50-7.34 (m, 2H), 7.36-7.10 (m, 2H), 6.83 (d, J = 1.83 Hz, 1H), 4.32 (d, J = 12.09 Hz, 1H), 4.24-4.14 (m, 1H), 3.50-3.10 (m, 5H), 1.86-1.72 (m, 2H), 1.58-1.44 (m, 1H), 1.44-1.26 (m, 1H), 0.80 (s, 9H); ESI: Calculated for C₂₇H₃₃ ³⁵CI₂FN₃O₄ [M+H]⁺= 552.18, Found: 552.42.

Compound 5 - Isomer D (TFA sait): ¹H NMR (300 MHz, CD3OD): 7.49 (d, J = 8.02 Hz, 1H), 7.36-7.24 (m, 1H), 7.11 (dd, J = 8.09, 1.89 Hz, 1H), 7.06-6.96 (m, 1H), 6.96-6.88 (m, 1H), 6.80 (d, J = 1.88 Hz, 1 H), 4.89 (d, J = 11.57 Hz, 1 H), 4.46 (d, J = 11.57 Hz, 1 H), 4.35-4.25 (m, 1H), 3.56-3.40 (m, 1H), 3.40-3.20 (m, 4H), 1.70-1.40 (m, 3H), 1.10 (dd, J = 14.59, 3.06 Hz, 1H), 0.91 (s, 9H); ESI: Calculated for C27H33³⁵CI2FN₃O₄ [M+H]⁺= 552.18, Found: 552.40. Compound 6 - Isomer A (TFA sait): ¹H NMR (300 MHz, CD3OD): 7.59 (t, J = 6.93 Hz, 1H), 7.41 (td, J = 7.55, 0.97 Hz, 1 H), 7.22 (t, J = 7.90 Hz, 1 H), 6.90 (d, J = 1.79 Hz, 1 H), 6.77 (dd, J =8.14, 1.79 Hz, 1H), 6.48 (d, J = 8.15 Hz, 1H), 5.21 (d, J=8.22 Hz, 1H), 4.50 (d, J = 8.19 Hz, 1 H), 4.17 (dd, J = 7.63, 2.23 Hz, 1H), 4.10-3.80 (m, 4H), 3.75-3.45 (m, 4H), 3.40-3.00 (m, 4H), 2.08-1.96 (m, 1H), 1.19 (dd, J = 15.32, 2.23 Hz, 1H), 0.81 (s, 9H); ¹³C (75 MHz, CD3OD): 180.28, 168.91, 157.75 (d, J_C-f = 248.38 Hz), 145.50, 137.13, 132.29, 128.63, 127.67, 126.63 (d, J_{C F} = 4.50 Hz), 125.74 (d, J_C-f = 13.69 Hz), 124.72, 123.44, 122.83 (d, Jc-_F = 18-25 Hz), 112.20, 65.16, 64.25, 63.84, 62.22, 58.01, 53.84, 50.05, 42.39, 35.72, 30.95, 29.52; ESI: Calculated for C₂₉H₃₆ ³⁵CI₂FN₄O₃ [M+H]⁺= 577.21, Found: 577.48.

Compound 6 - Isomer B (TFA sait): ¹H NMR (300 MHz. CD3OD): 7.66-7.48 (m, 2H), 7.427.32 (m, 1H), 7.20-7.10 (m, 2H), 6.78 (d, J = 1.68 Hz, 1H), 5.38 (d, J = 11.48 Hz, 1H), 4.65 (d, J = 11.48 Hz, 1H), 4.52 (d, J = 7.59 Hz, 1H), 4.00-3.70 (m, 4H), 3.70-3.60 (m, 2H), 3.503.40 (m, 2H), 3.40-3.10 (m, 4H), 1.97 (dd, J = 15.40, 8.62 Hz, 1H), 1.12 (d, J = 15.40 Hz, 1H), 0.88 (s, 9H); ¹³C (75 MHz, CD3OD): 177.73, 169.20, 145.19, 137.15, 132.54, 128.69, 126.84, 126.64 (d, J_{C F} = 4.80 Hz), 124.11, 123.72, 122.57 (d, J_C._F = 13.19 Hz), 121,77 (d, J_C._F = 13.19 Hz), 112.06, 65.08, 64.59, 64.32, 62.69, 57.41, 53.70, 48.47, 43.55, 35.61, 31.06, 29.56; ESI: Calculated for C₂₉H_3e ³⁵CI₂FN₄O₃ [M+H]⁺= 577.21, Found: 577.48.

Compound 7 - Isomer B (TFA sait): ’H NMR (300 MHz, CD₃OD): 7.54-7.44 (m, 2H), 7.367.26 (m, 1 H), 7.14-7.00 (m, 2H), 6.70 (d, J = 1.76 Hz, 1 H), 5.22 (d, J = 11.36 Hz, 1 H), 4.50 (d, J = 11.36 Hz, 1H), 4.41 (d, J = 8.23, 1.85 Hz, 1H), 1.81 (dd, J =15.46, 8.31 Hz, 1H), 1.06 (dd, J = 15.46, 1.90 Hz, 1H), 0.78 (s, 9H); ESI: Calculated for C₂₃H_Z5 ³⁵CI₂FN₃O₂ [M+H]⁺= 464.13, Found: 464.60.

Compound 7 - Isomer D (TFA sait): ’H NMR (300 MHz, CD₃OD): 7.64 (d, J = 8.01 Hz, 1H), 7.54-7.40 (m, 2H), 7.28-7.12 (m, 2H), 6.83 (d, J = 1.68 Hz, 1H), 4.96 (d, J = 12.33 Hz, 1H),

4.36 (d, J = 12.33 Hz, 1H), 4.28 (t, J = 5.60 Hz, 1H), 1.93-1.86 (m, 2H), 0.80 (s, 9H); ESI: Calculated for C_Z3H₂₅ ³⁵CI_ZFN₃O_Z [M+H]⁺= 464.13, Found: 464.42.

173

Compound 8 - Isomer B (TFA sait): ¹H NMR (300 MHz, CD₃OD): 7.66 (d, J = 8.49 HZ, 1 H),

7.35-7.12 (m, 4H), 6.86 (d, J = 6.02 Hz, 1H), 5.67 (d, J = 10.03 Hz, 1H), 4.41 (dd, J = 8.20,

1.79 Hz, 1H), 4.13 (d, J= 10.03 Hz, 1H), 2.97 (s, 3H), 2.74 (s, 3H), 1.91 (dd, J= 15.46, 8.20 Hz, 1H) 1.17 (dd, J = 15.46, 1.82 Hz, 1H), 0.91 (s, 9H); ¹³C (75 MHz, CD3OD):177.80, 168.54, 156.04 (d, JC-f = 243.74 Hz), 141.04, 136,12, 135.16, 131.81, 130.66, 129.73, 128.52, 125.50 (d, JC-f =7.37 Hz), 123.70 (d, JC-f = 19.58 Hz), 114.43 (d, JC F =25.50 Hz), 113.62, 65.81, 64.45, 60.58, 57.31, 42.59, 38.04, 37.00, 31.04, 29.63; ESI: Calculated for C25H29³⁵CI2FN₃O₂ [M+H]*= 492.16, Found: 492.50.

Compound 8 - Isomer D (TFA sait): ¹H NMR (300 MHz, CD₃OD): 7.73 (d, J = 8.75 Hz, 1H), 7.30-7.18 (m, 1H), 7.18-7.12 (m, 1H), 7.12-7.06 (m, 1H), 6.89 (d, J = 6.12 Hz, 1H), 5.52 (d, J = 10.56 Hz, 1H), 4.37 (dd, J = 6.56, 4.48 Hz, 1H), 4.18 (d, J= 10.62 Hz, 1H), 2.99 (s, 3H),

2.80 (s, 3H), 1.69 (dd, J= 15.35, 6.58 Hz, 1H), 1.21 (dd, J= 15.35, 4.37 Hz, 1H), 0.93 (s, 9H); ESI: Calculated for C₂₅H₂₉ ³⁵CI₂FN₃O_Z [M+H]*= 492.16, Found: 492.62.

Compound 9 - Isomer A (TFA sait): ¹H NMR (300 MHz, CD3OD): 7.25-7.20 (m, 3H), 7.107.00 (m, 1H), 6.85 (d, J = 6.00 Hz, 1H), 5.13 (d, J = 11.10 Hz, 1H), 4.30-4.10 (m, 2H), 2.72 (s, 3H), 2.02 (dd, J = 15.30, 7.20 Hz, 1H), 1.52 (dd, J = 15.30, 3.90 Hz, 1H), 0.79 (s, 9H); ESI: Calculated for C24H27^3SCI2FN₃O₂ [M+H]*= 478.15, Found: 478.46.

Compound 9 - Isomer B (TFA sait):

¹H NMR (300 MHz, CD₃OD): 7.68 (d, J = 8.47 Hz, 1H), 7.32-7.16 (m, 3H), 7.04 (d, J = 7.63 Hz, 1 H), 6.85 (d, J = 6.01 Hz, 1 H), 5.23 (d, J = 11.21 Hz, 1 H), 4.46 (dd, J = 8.23, 1.76 Hz, 1H), 4.13 (d, 11.21 Hz, 1H), 2.71 (S, 3H), 1.90 (dd, J= 15.46, 8.23 Hz, 1H), 1.17 (dd, J =

15.48, 1.75 Hz, 1H), 0.89 (s, 9H) ¹³C (75 MHz, CD3OD): 177.57, 168.31, 159.98 (d, JCF = 244.73 Hz), 140.90, 135.98, 134.31, 131.59, 130.56, 129.45, 128.47, 125.84 (d, JC-f = 7.48 Hz), 123.61 (d, JCF = 19.45 Hz), 114.33 (d, JC.F = 25.29 Hz), 113.57, 65.62, 64.15, 62.93, 57.04, 63.42, 31.01, 29.60, 27.02; ESI: Calculated for C24H27³⁵CI2FN₃O₂ [M+H]⁺= 478.15, Found: 478.46.

Compound 9 - Isomer C (TFA sait):

¹H NMR (300 MHz, CD₃OD): 7.66 (d, J = 8.70 Hz, 1H), 7.32 (d, J = 8.20 Hz, 1 H), 7.23 (t, J = 7.89 Hz, 1 H), 7.09 (t, J = 1.70 Hz, 1 H), 6.93 (d, J = 7.66 Hz, 1 H), 6.89 (,J = 6.13 Hz, 1 H),

4.22 (t, J = 5.73 Hz, 1 H), 3.91 (d, J = 12.36 Hz,1 H), 2.70 (s, 3H), 1.90-1.84 (m, 2H), 0.83 (s, 9H); ESI: Calculated for C₂₄H₂₇ ³⁵CI₂FN₃O₂ [M+H]⁺= 478.15, Found: 478.58.

Compound 9 - Isomer D (TFA sait): ¹H NMR (300 MHz, CD3OD): 7.51 (d, J = 8.79 Hz, 1 H), 7.24-7.12 (m, 2H), 7.08 (s, 1H), 7.25 (d, J =7.13 Hz, 1H), 6.85 (d, J = 6.13 Hz, 1H), 4.80 (d, J = 11.25 Hz, 1H), 4.25-4.15 (m, 1H), 4.09 (d, J = 11.25 Hz, 1H), 2.76 (s, 3H), 1.44 (dd, J = 15.26, 7.62 Hz, 1H), 1.08 (dd, J = 15.26, 3.19 Hz, 1H), 0.92 (s, 9H); ESI: Calculated for C24H27³⁵CI2FN₃O_Z [M+H]*= 478.15, Found: 478.56.

180

Compound 10 - Isomer A (TFA sait) (MI-219-TFA sait): ’H NMR (300 MHz, CD₃OD): 7.32-

7.22 (m, 3H), 7.18-7.10 (m, 1H), 7.06 (d, J = 8.74 Hz, 1H), 6.88 (d, J = 6.08 Hz, 1H), 4.97 (d, J = 10.79 Hz, 1H), 4.22 (d, J = 10.79 Hz, 1 H), 4.26-4.18 (m, 1H), 3.50-3.20 (m, 5H), 2.03 (dd, J = 15.35, 6.56 Hz, 1H), 1.68-1.52 (m, 2H), 1.50-1.40 (m, 1H), 0.84 (s, 9H); ’³C (75 MHz, CD₃OD): 179.64, 166.47, 140.40 (d, J_P._C = 2.94 Hz), 136.54, 136.06, 131.64, 130.16, 130.02, 128.14, 127.27 (d, J_P._C = 40.14 Hz). 123.05 (d, J_P._C = 19.44 Hz), 115.94 (d, J_P._C =

25.54 Hz), 113.09, 70.91, 67.24, 64.19, 64.10, 62.73, 57.52, 42.66, 38.14, 33.85, 30.99, 29.57; ESI: Calculated for C₂₇H₃₃ ³⁵CI₂FN₃O₄ [M+Hf= 552.18, Found: 552.75; HPLC Purity = 82%.

Compound 10 - Isomer B, (TFA sait) (MI-21901 - TFA sait): Ή NMR (300 MHz, CD₃OD): 7.68 (d, J = 8.47 Hz, 1H), 7.32-7.17 (m, 3H), 7.12-7.02 (m, 1H), 6.85 (d, J = 6.00 Hz, 1H),

5.23 (d, J= 11.28 Hz, 1H), 4.46 (dd, J =8.41, 1.91 Hz, 1H), 4.10 (d, J = 11.28 Hz. 1H), 3.50-

3.20 (m, 5H), 1.90 (dd, J = 15.18, 8.62 Hz. 1H), 1.65-1.47 (m, 1H), 1,47-1.33 (m,1H), 1.18 (d, J = 15.18 Hz,1 H), 0.89 (s, 9H); ’³C (75 MHz, CD3OD): 177.56, 167.81, 155.99 (d, JP.C = 243.27 Hz), 140.87, 135.98, 134.28, 131.63, 130.60, 129.51, 128.60, 125.82 (d, JP.C = 7.39 Hz), 123.58 (d, JP.C = 19.68 Hz), 114.30 (d, JP.C = 25.32 Hz), 113.55, 70.86, 67.23, 65.53, 64.16, 63.07, 57.35, 43.43, 38.15, 33.78, 31.01, 29.57; ESI: Calculated for C27H33³⁵CI2FN₃O₄[M+Hf= 552.18, Found: 552.38; HPLC Purity = 95%.

Compound 10 - Isomer D (TFA sait): ’H NMR (300 MHz, CD₃OD): 7.60-7.53 (m, 1H), 7.307.10 (m, 2H), 7.08-7.02 (m, 1H), 7.00-6.92 (m. 1H), 6.84 (d, J = 6.13 Hz, 1H), 4.96 (d, J = 12.25 Hz, 1H), 4.39 (dd, J = 7.22, 4.31 Hz, 1 H), 4.10 (d, J = 12.27 Hz, 1H), 3.50-3.20 (m, 5H), 1.68 (dd, J = 15.29, 7.23 Hz, 1H), 1.62-1.48 (m, 1H), 1.48-1.32 (m, 1H), 1.17 (dd, J = 15.29, 4.16 Hz, 1H), 0.90 (s, 9H); ESI: Calculated for C₂₇H₃₃ ³⁵CI₂FN₃O₄ [M+Hf= 552.18, Found: 552.40.

Compound 11 - Isomer A (TFA sait): ’H NMR (300 MHz, CD₃OD): 7.50-7.40 (m, 2H), 7.28-

7.18 (m, 1H), 6.91 (d, J= 1.81 Hz, 1H), 6.78(dd, J= 8.13, 1.87 Hz, 1H), 6.46 (d, J = 8.14 Hz, 1H), 5.30 (d, J = 8.52 Hz, 1H), 4.43 (d, J = 8.52 Hz, 1H), 4,40-4.20 (m, 2H), 4.08 (dd, J = 7.47, 2.43 Hz, 1H), 2.00 (dd, J= 15.32, 7.53 Hz, 1H), 1.22 (t, J= 7.12 Hz, 3H), 1.19 (dd, J = 15.32, 2.55 Hz, 1H), 0.81 (s, 9H); ¹³C (75 MHz, CD₃CI, note free amine): 181.33, 171.91,

156.54 (d, Jc-f = 248.02 Hz), 142.85, 134.15, 129.91, 128.37 (d, J_C-f = 14.61 Hz), 127.19 (d, J_C._F = 3.19 Hz), 125.94, 124.94, 124.63 (d, J_{C F} = 4.50 Hz), 122.10, 121.69 (d, J_C._F = 18.50 Hz), 110.72, 67.16, 65.70, 63.19, 61.74, 51.17, 43.45, 30.33, 29.97, 14.32; ESI: Calculated for C₂₅H₂e³⁵CI₂FN₂O₃ [M+Hf = 493.15, Found: 493.44.

Compound 11 - Isomer B (TFA sait): ’H NMR (300 MHz, CD₃OD): 7.59 (d, J = 7.41 Hz, 1H), 7.50-7.42 (m, 1H), 7.40-7.32 (m, 1H), 7.17-7.07 (m, 2H), 6.78 (d, J= 1.65 Hz, 1H), 5.61 (d, J = 12.25 Hz, 1H), 4.56 (d, J = 12.25 Hz, 1H), 4.47 (dd, J = 8.50, 1.50 Hz, 1H), 4.25 (dq,

182

J = 10.77, 7.12 Hz, 1H), 4.13 (dq, J = 10.77, 7.12 Hz, 1H), 1.93 (dd, J = 15.39, 8.67 Hz, 1H), 1.34 (dd, J = 15.39, 1.57 Hz, 1H), 1.10 (t, J =7.12 Hz, 3H), 0.87 (s, 9H); ESl: Calculated for C₂₅H_2e ³⁵CI₂FN₂O₃ [M+H]⁺= 493.15, Found: 493.44.

Compound 12 - Isomer A (TFA sait): ¹H NMR (300 MHz, CD₃OD): 7.56-7.46 (m, 1H), 7.46-

7.36 (m, 1H), 7.26-7.18 (m, 1H), 6.91 (d, J =1.61 Hz, 1H), 6.76 (dd, J = 8.10, 1.50 Hz, 1H), 6.47 (d, J = 8.14 Hz, 1H), 5.29 (d, J = 8.67 Hz, 1H), 4.43 (d, J = 8.67 Hz, 1H), 4.11 (d, J = 7.47, 2.13 Hz, 1H), 2.02 (dd, J = 15.36, 7.50 Hz, 1H), 1.16 (dd, J= 15.36, 2.24 Hz, 1H), 0.81 (s, 9H); ESl: Calculated for C₂₃H₂₄ ³⁵CI₂FN₂O₃ [M+H]*= 465.11, Found: 465.42.

Compound 12 - Isomer B (TFA sait): ¹H NMR (300 MHz, CD3OD): 7.58-7.42 (m, 2H), 7.367.26 (m, 1H), 7.14-7.02 (m, 1H), 6.75 (s, 1H), 5.43 (d, J = 12.00 Hz, 1H), 4.58 (d, J = 11.97 Hz, 1H), 4.35 (d, J = 8.53 Hz, 1H), 1.87 (dd, J= 15.08, 9.31 Hz, 1H), 1.09 (d, J = 15.31 Hz, 1H), 0.85 (s, 9H); ESl: Calculated for C23H24³⁵CI2FN₂O₃ [M+Hf= 465.11, Found: 465.38.

Compound 12 - Isomer D (TFA sait): ¹H NMR (300 MHz, CD3OD): 7.49 (d, J = 8.10 Hz, 1H), 7.36-7.26 (m, 1H), 7.13 (dd, J = 8.10, 1.85 Hz, 1H), 7.08-7.00 (m, 1H), 6.96-6.86 (m, 1H), 6.78 (d, J= 1.80 Hz, 1H), 5.32 (d, J = 12.69 Hz, 1H), 4.56 (d, J= 12.69 Hz, 1H), 4.42 (dd, J = 7.27, 3.88 Hz, 1H), 1.69 (dd, J= 15.44, 7.64 Hz, 1 H), 1.13 (dd, J= 15.44, 3.76 Hz, 1H), 0.90 (s, 9H); ESl: Calculated for C23H24³⁵CI2FN₂O₃ [M+H]*= 465.11, Found: 465.54.

EXAMPLE 17

Synthesis of CB061-lsomer B (TFA sait)

Scheme 12

182

Cl
o
	- °xY · d N Ph X H	3 MS4A
ciXA-h	PhMe, reflux 12 h
1.0 equiv	1.1 equiv 1.1 equiv

OH

1.3 equiv THF. rt, 12 h

H

Oxidalion

CAN 2.1 equiv MeCN:H₂O (1:1) ri. 1 h

CB061 Isomer A

CB061 Isomer B

CB061 Isomers C and D

Isomerization ________ CB061

Isomers A-D sat. NaHCO₃

MeCN:H₂O (1:1) ri, 3 days

CB061 was prepared according to Scheme 12 using methodology described in EXAMPLE 16 to give an A:B:(C+D) isomer ratio after oxidation of 15:67:18, an A:B:(C+D) isomer ratio after isomerization of 2:74:24, and substantially pure CB061-Isomer B (as the TFA sait) 5 after chromatography (Fig. 30).

Analytical data for CB061-lsomer B (TFA sait): ¹H NMR (300 MHz, CD3OD): 7.66 (d, J = 8.45 Hz, 1H), 7.30-7.16 (m, 3H), 7.10-7.03 (m, 1H), 6.85 (d, J = 5.98 Hz, 1H), 5.22 (d, J = 11.37 Hz, 1H), 4.46 (dd, J = 8.31, 1.68 Hz, 1H), 4.09 (d, J = 11.37 Hz, 1H), 3.70-3.50 (m, 1H), 3.50-3.39 (m, 1H), 2.00-1.84 (m, 3H), 1.82-1.70 (m, 1H), 1.58-1.46 (m, 1H), 1.40-1.12 10 (m, 4H), 1.08-0.90 (m, 1H), 0.88 (s, 9H); ¹³C NMR (75 MHz, CD3OD): 177.42, 166.80,

155.82 (d, J_c-F = 243.61 Hz), 140.67 (d, J_C-f = 2.77 Hz), 135.73, 134.18, 131.36, 130.33, 129.44, 128.37, 125.77 (d, J_C-f = 7.38 Hz), 123.38 (d, J_C._F = 19.54 Hz), 114.06 (d, J_C._F =

25.18 Hz), 113.39, 69.97, 65.12, 64.06, 62.94, 57.41, 49.88, 43.37, 34.38, 34.29, 30.98, 30.82, 29.39; ESI: Calculated for C₂₉H3₅CI₂FN₃O₃ [M+H]⁺= 562.20, Found: 562.36. The

183 absolute stereochemistry of CB061 - Isomer B was determined by single crystal x-ray crystallography.

EXAMPLE 18

Synthesis of CB087 - Isomer B (TFA sait)

Scheme 13

Oxidation

Isomerizalion

TFA(pH<1)

MeCN-H₂O 3 deys

CAN 2.1 equiv MeCN :H₂0 (1:1) ri, 1 h

compound A 85:15 mixture of Isomers

CB087 Isomer B

CB087

Isomers A, C, and D

CB087 Isomers A-0

CB087 was prepared according to Scheme 13 using methodology described in EXAMPLE to give an (A+C+D):B isomer ratio after oxidation of 65:35, an (A+C+D):B isomer ratio after isomerization of 18:82 (in acetonitrile-water in the presence of TFA), and substantially 10 pure CB087-lsomer B (as the TFA sait) after chromatography. In this study, intermediate A was isolated as an 85:15 mixture of isomers based on ¹H NMR analysis. This mixture was used in the oxidation step. The major isomer was characterized by ¹H NMR and ESI. Also, little isomerization was observed in MeOH or in MeOH-water at a pH of 8 after 3 days at room température.

Analytical data for compound A major ïsomer: ¹H NMR (300 MHz, CD₃OD): 7.40 (s, 6H),

7.10 (s, 9H), 7.20 (t, J = 7.12 Hz, 1H), 6.74 (t, J = 7.86 Hz, 1H), 6.48 (s, 1H), 6.44 (d, J =

8.20 Hz, 1H), 6.08 (t, J = 7.02 Hz, 1H), 5.51 (d, J =8.22 Hz, 1H), 5.35 (d, J = 4.67 Hz, 1H),

4.55 (d, J = 4.45 Hz, 1H), 4.18 (s, 1H), 4.15-3.90 (m, 3H), 3.70-3.55 (m, 1 H), 2.30-2.00 (m, 4H), 1.60-1.30 (m, 4H); ¹³C (75 MHz, CD₃OD): 179.68, 175.39, 155.86 (d, J_C-p =247.63 Hz), 18<+

142.81, 134.49, 134.21, 132.34, 132.09, 129.46, 129.28 (d. J_C._P = 12.91 Hz), 128.45, 128.09, 127.78, 127.16, 126.90, 126.67, 125.20, 124.90, 124.08 (d, J_C-p= 3.27 Hz), 121.32 (d, J_C-p= 8.18 Hz), 121.17, 110.01, 76.10, 75.35, 69.39, 67.82, 66.89, 61.60, 49.60, 48.14, 33.60, 33.48, 30.12, 29.82; ESI: Calculated for C44H41CI2FN3O4 [M+H]⁺= 764.25, Found: 5 764.26.

Analytical data for CB087-lsomer B (TFA sait): ¹H NMR (300 MHz, CD₃OD): 8.34 (d, J =

7.70 Hz, 1H), 7.70 (d, J = 8.11 Hz, 1H), 7.70 (t, J = 7.02 Hz, 1H), 7.42 (t, J = 7.61 Hz. 1H),

7.40-7.30 (m, 5H), 7.22 (d, J = 7.90 Hz, 1H), 7.16 (dd, J= 8.17, 1.85 Hz, 1 H), 6.61 (d, J = 1.74 Hz, 1 H), 5.58 (s, 1H). 5.34 (d, J = 10.97 Hz, 1H), 4.83 (d, J = 11.04 Hz, 1H), 3.78-3.60 10 (m, 1H), 3.50-3.40 (m, 1H), 2.00-1.86 (m, 2H), 1.86-1.78 (m, 1H), 1.70-1.60 (m, 1H), 1.42-

1.20 (m, 3H), 1.02 (qd, J = 12.68, 3.30 Hz, 1H); ¹³C (75 MHz, CD₃0D): 177.29, 167.33,

154.20 (d, J_c-F = 284.04 Hz), 144.70, 136.97, 132.50, 131.26, 130.24, 130.03, 128.83,

128.70, 126.61, 126.54, 123.98, 122.91, 122.35 (d, J_C-f= 18.95 Hz), 121.70 (d, J_C-f= 12.66 Hz), 111.78, 70.44, 69.95, 64.89, 62.47, 49.98, 49.87, 34.33, 34.28, 30.91, 30.80; ESI: 15 Calculated for C₃₀H₂₉CI₂FN₃O₃ [M+H]*= 568.16, Found: 568.54.

185

EXAMPLE 19

Synthesis of CB083 - Isomer B (TFA sait)

Scheme 14

MS 4A

1.1 equiv 1.1 equiv

PhMe, reflux 12 h

H mixture ot isomers

1.3 equiv

THF. rt. 12 h

65%

compound A tsomer Ratio: 84:16

CAN 2.1 equlv MeCN :H₂O (1:1) rt, 1 h

Oxidation:

CB083

CB083 Isomers A. C, and D

Isomer B

Isomerization

TFA (pH < 1) MeCN-HîO.

days

CB083 Isomers A-D

CB083 was prepared according to Scheme 14 using methodology described in EXAMPLE 16 to give an (A+C+D):B isomer ratio after oxidation of 41:59, an (A+C+D):B isomer ratio after isomerization of 10:90 (in acetonitrile-water in the presence of TFA), and substantially pure CB083-lsomer B (as the TFA sait) after chromatography. In this study, intermediate A was isolated as an 84:16 mixture of isomers, and the major isomer was used in the oxidation step. Also, little isomerization was observed in MeOH or in MeOH-water at a pH of 8 after 3 days at room température.

Analytical data for compound A major isomer: ¹H NMR (300 MHz, CD₃OD): 8.42 (d, J = 7.49 Hz, NH, 1H), 7.40-7.25 (m, 5H), 7.25-7.10 (m, 9H), 7.20-6.98 (m, 3H), 6.94 (dd, J = 8.06, 1.88 Hz, 1H), 6.82 (d, J = 8.07 Hz, 1H), 6.63-6.55 (m, 2H), 6.53 (d, J = 1.84 Hz, 1H),

5.18 (d, J = 5.70 Hz, 1H), 4.32 (s, 1H), 3.97 (d, J = 5.70 Hz, 1H), 3.70 (d, J = 7.57 Hz, 1H), 3.70-3.55 (m, 2H), 3.25-3.10 (m, 1H), 2.64 (dd, J = 14.03, 6.91 Hz, 1H), 2.45 (dd, J = 13.97, 9.24 Hz, 1H), 2.15-1.90 (m, 4H), 1.55-1.30 (m, 4H); ¹³C (75 MHz, CD₃OD): 181.03, 176.38, 144.79, 144.55, 139.09, 138.54, 135.17, 134.94, 133.16, 130.13, 130.07, 129.77,

186

129.26, 129.11, 129.03, 128.95, 128.80, 128.18, 127.66, 127.35, 122.68, 111.27, 79.40, 76.05, 72.19, 70.6, 70.56, 62.05, 53.09, 49.63, 37.84, 34.88, 34.77, 31.34, 31.07; ESI: Calculated for C₄₅H₄₅CIN₃O₄ [M+H]⁺= 726.31, Found: 726.44.

Analytical data for CB083-lsomer B (TFA sait): ¹H NMR (300 MHz, DMSO-d6): 10.28 (s, NH, 1H), 8.51 (d, J = 6.67 Hz, NH, 1H), 7.59 (d, J = 8.03 Hz, 1H), 7.25-7.00 (m, 9H), 6.606.50 (m, 3H), 4.82 (s, 1H), 4.42 (d, J= 9.65 Hz, 1H), 3.64-3.50 (m, 1H), 3.46-3.32 (m, 1H), 3.30-3.16 (m, 1H), 2.85 (dd, J = 13.83, 4.77 Hz, 1H), 2.28 (dd, J = 13.83, 10.20 Hz, 1H), 1.90-1.66 (m, 4H), 1.38-1.10 (m, 4H); ¹³C (75 MHz, DMSO-d6): 178.36, 166.47, 143.95, 136.55, 133.32, 131.90, 128.69, 128.46, 128.09, 127.88, 127.18, 126.83, 126.52, 123.21, 120.72, 109.97, 70.38, 67.78, 63.18, 62.17, 52.83, 47.76, 34.54, 33.57, 29.82, 29.78; ESI: Calculated for C31H₃₃CIN₃O₃ [M+H]*= 530.22, Found: 530.40.

EXAMPLE 20

Synthesis of Synthesis of CB084 - Isomer B (TFA sait)

CB084 was prepared according to Scheme 15 using methodology described in EXAMPLE 16 to give an A:B:(C+D) isomer ratio after oxidation of 97:1:2, an A:B:(C+D) isomer ratio after isomerization of 46:52:2 in acetonitrile-water in the presence of TFA after 3 days, an A:B:(C+D) isomer ratio after isomerization of 9:49:42 in acetonitrile-water at pH 8 after 3 days, and substantially pure CB084-lsomer B (as the TFA sait) after chromatography.

Analytical data for CB084-lsomer A (free amine): ¹H NMR (300 MHz, DMSO-d6): 10.02 (s, NH, 1H), 7.86 (d, J = 7.60 Hz, NH, 1H), 7.31 (d, J =7.91 Hz, 1H), 7.05 (d, J = 7.91 Hz, 1H), 6.97 (s, 3H), 6.54 (s, 1H), 6.46 (d, J = 3.65 Hz, 2H), 4.50 (d, J = 4.08 Hz, NH, 1H), 3.60-3.40 (m, 2H), 3.40-3.30 (m, 1H), 3.25-3.10 (m, 1H), 2.80-2.70 (m, 2H), 2.60-2.40 (m, 1H), 1.901.60 (m, 4H), 1.34 (dd, J= 13.86, 10.33 Hz, 1H), 1.30-1.05 (m, 4H), 0.90-0.70 (m, 1H), 0.71 (s, 9H); ¹³C (75 MHz, DMSO-d6): 180.50, 169.86, 143.83, 138.61, 131.88, 129.53, 127.91, 127.64, 125.83, 125.55, 120.61, 108.90, 68.15, 68.08, 68.03, 61.27, 55.94, 47.20, 43.56, 34.36, 33.83, 33.77, 30.30, 30.02, 29.80, 29.67; ESI: Calculated for C30H39³⁵CIN3O₃ [M+H]⁺=

524.27, Found: 524.55.

Scheme 15

18716409

MS4A

PhMe, reflux 12 h

mixture of 2 isomers

OH

CB084 Isomer A

CB084 Isomer B

CB084 Isomers C, and D

Isomerization

MeCN:H₂0 (1:1) sat. NaHCOj 3 days; or

MeCN:H₂O (1:1) TFA 3 days

CB084 Isomers A-D

Analytical data for CB084-lsomer B (TFA sait): ¹H NMR (300 MHz, CD₃OD): 8.54 (d, J =

7.42 Hz, NH, 1H), 7.48 (d, J = 8.06 Hz, 1H), 7.23 (d, J = 8.06 Hz, 1H), 7.20-7.08 (m, 3H), 6.90 (s, 1H), 6.67 (d, J = 6.94 Hz, 1H), 4.49 (d, J = 11.08 Hz, 1H), 4.17 (dd, J = 7.91, 1.68 Hz, 1H), 3.75-3.60 (m, 1H), 3.60-3.45 (m, 1H), 3.40-3.30 (m, 1H), 2.79 (dd, J = 14.10, 6.05 Hz, 1H), 2.36 (dd, J= 14.10, 10.21 Hz, 1H), 2.10-1.80 (m, 4H), 1.49 (dd, J = 15.58, 8.30 Hz, 1H), 1.45-1.20 (m, 4H). 1.00-0.80 (m, 1H), 0.87 (s, 9H); ’³C (75 MHz, DMSO-d6): 175.40, 144.42, 136.34, 133.82, 128.42, 127.93, 127.02, 126.60, 123.34, 121.24, 110.53, 67.72,

63.74, 63.35, 61.30, 51.75, 47.89, 42.56, 33.47, 33.44, 32.92, 29.78, 29.66, 29.52, 28.85; ESI: Calculated for C₃oH3₉ ³⁵CIN₃03 [M+H]⁺= 524.27, Found: 524.44.

EXAMPLE 21

Synthesis of C144

Scheme 16

180

Q
J/ t	₃ MS4A
PhMe. reflux 12 h
1.0 equiv 1.1 equiv 1.1 equiv

BnNHj

2.0 equiv. THF, d, 12 h

C144 was prepared using methodology described above. The absolute stereochemistry of C144 determined by single crystal x-ray crystallography.

Analytical data for C144: ¹H NMR (300 MHz, CD3CI): 7.70-7.40 (m, 5H), 7.50-7.00 (m, 9H), 7.00-6.80 (m, 5H), 6.80-6.60 (m, 2H), 6.43 (d, J = 7.68 Hz, 1H), 6.11 (t, J = 5.79 Hz, 1H), 5.99 (d, J = 7.56 Hz, 1H), 5.27 (d, J = 6.91 Hz, 1 H), 4.48 (dd, J = 14.79, 6.31 Hz, 1H), 4.34 (d, J =6.91 Hz, 1H), 4.21 (dd, J = 14.79, 5.41 Hz, 1H), 4.13 (d. J= 11.24 Hz, 1H), 3.80 (d, J = 11.24 Hz, 1H), 3.51 (d, J = 9.46 Hz, 1H), 2.83 (dd, J = 15.40, 9.46 Hz. 1H), 1.32 (d, J = 15.40 Hz, 1H), 0.76 (s, 9H); ¹³C (75 MHz, CD3CI): 178.42, 173.63, 142.12, 139.27, 137.97, 134.54, 133.23, 132.49, 131.78, 129.21, 128.88, 128.74, 128.48, 128.21, 127.92, 127.86, 127.77, 127.52, 127.29, 124.63, 122.27, 109.35, 75.06, 72.14, 70.40, 66.08, 60.65, 60.51, 43.62, 43.56, 30.11,29.91; ESI: Calculated for C₄₄H₄₆N₃O₃ [M+H]⁺= 664.35, Found: 664.36.

EXAMPLE 22

Cellular Activity of Ml-77301

The activity of MI-773001 in a variety of tumor cell lines is presented in Table 6. The cytotoxic concentration is the first concentration where cell death corresponding to the cytotoxic index is observed. Cytotoxicity was quantified by blue trypan exclusion at 96 h except for CCF-STTG1 at 192 hours (medium 20-50% cell death and high >50% cell death). The anti-proliferative IC₅o was determined by ATP assay.

Table 6

tum or

origin

p53

BM

Cyto tox

Cytot ox*

IC50 (nM)

cell line					(μΜ)
H12 99	lung		-	-	-	>10,0 00
U2O S	bone	WT/W T	-	-	-	>10,0 00
SJS A1	bone	WT/W T	MDM2 amplifi ed	high	1	145
HCT 116	colon	WT/W T	-	-	-	229
RKO	colon	WT/W T	-	-	-	480
22R V1	prostat e	WT/M UT	-	medi um	3	399
LnC ap	prostat e	WT/W T	-	high	3	50
JAR	placent a	WT/W T	MDM2 amplifi ed	high	3	169
CCF STT G1	CNS	WT/W T	MDM2 amplifi ed	medi um	1	162
Cap an2	pancre as	-/-	-	-	-	>10,0 00
MCF 7	breast	WT/W T	-	-	-	201
Y79	retinoblasto me	WT/W T	Rb mutate d	medi um	1	430
Weri -RB1	retinoblasto me	WT/W T	Rb mutate d	high	0.3	not deter mine d
SNU	gastric	WT/W	KRAS	high	10	77

I90

-1		T	mut
MKN 45	gastric	WT/W T	Met amplifi ed	-	-	>10,0 00
Hs7 46T	gastric		Met amplifi ed	-	-	10,00 0
SNU -5	gastric		Met amplifi ed	-	-	>10,0 00
MOL M13	AML	WT/W T	FLT3- ITD	high	0.1	14.7
MV4 ;11	AML	WT/W T	FLT3- ITD	medi um	0.3	45.4
RS4; 11	ALL	WT/W T	-	high	0.1	28
LY3	ABC- DLBCL	WT/W T	-	medi um	0.3	280
Ly10	ABC- DLBCL	WT/W T	-	medi um/h igh	0.3	90
Ly7	GCB- DLBCL	WT/M UT	-	-	-	>10,0 00
DoH H2	GCB- DLBCL	WT/W T	BCL2 overex pressi on	high	0.3	39.3
Rec 1	MOL	WT/W T	-	high	10	738
SR	lympho ma	WT/W T	-	medi um	0.3	14.9
H92 9	MM	WT/W T	-	high	1	114
Ku8 12	CML- BC	WT/W T	-	medi um	3	>10,0 00
EHE	CLL	WT/W	-	medi	0.3	70

191

B

T

um

EXAMPLE 23

Cell Growth Inhibition and Cytoxic Effects on 22Rv1 Cell Lines

MI-519-6401 and MI-77301 were evaluated for their cell growth inhibition and cytotoxic effects on a prostate cancer cell line 22Rv1 (from The DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, reference DSMZ ACC438). For growth inhibition assays, cells were incubated in the presence of MI-519-6401 or MI-77301 for 96 h in 96-well format. Cell seeding conditions were adapted to get significant cell growth în this assay format. Growth inhibition assays were performed using the Celltiter-GIo Luminescent kit (Promega). The IC50 values (concentration where the growth inhibition percentage is equal to half of the maximum inhibitory effect of the tested compound) were calculated and ranged between 100 nM and 500 nM in this prostate cancer cell line for both compounds.

For cytotoxicity assays, cells were incubated with MI-519-6401 or MI-77301 for 96 h in 6well format. Cell seeding conditions were adapted to get significant cell growth in this assay format. Cytotoxic effects were measured using trypan blue staining. Both the floating and adhèrent cells were stained with trypan blue. Quantification was performed by Vi-CELL® Cell Viability Analyzer (Beckmann-Coulter) which détermines both cell concentration and percentage of viable cells. For both compounds at concentrations which were close to IC₉₀concentrations (concentration where the growth inhibition percentage is equal to 90 percent of the maximum inhibitory effect of the tested compound), the percentages of viable cells were sîgnîfîcantly decreased compared to untreated cells and were at best between 50 and 70 % in the 22Rv1 cell line.

EXAMPLE 24

Apoptosis Assay

Apoptosis was determined using Annexin-V-FLUOS/propidium iodide staining kit (Roche Applied Science) by modifications of the manufacturées instructions. Early stage apoptotic cells display translocation of phosphatidylserine from inner to outer surface of plasma membrane which can be detected by Annexin V fluorescein staining. Propidium iodide (PI) staining détermines the late stage apoptotic cells or necrotic cells. A total of 0.25 * 106 cells were plated overnight in a 6-well tissue culture. Next day, adhèrent cells were treated in the presence or absence of MI-77301 and incubated at 37°C for 2.5 days. Cells were harvested using 0.05% trypsin-EDTA (Invitrogen) by pooling the floating and the adhèrent cell populations, and were washed with PBS. Next, cells were stained with Annexin-V-FLUOS and PI in the incubation buffer at room température for 10 minutes in the dark. Modifications were made by using 0.5 pl each of Annexin-V-FLUOS and PI (instead of 2 μΙ each,

191 mentîoned in the instruction manuai) for staining. These modifications were found to decrease the background noise, especially of fluorescein, in the assay. Cells were acquired and analyzed in a flow cytometer. Annexin V+/PI- cells were scored as early apoptotic cells and Annexin V+/PI+ cells as late apoptotic cells. The results of this assay are presented in Fig. 27.

EXAMPLE 25

In vivo effîcacy in the SK-Mel-103 xenograft model in mice

Drug préparation

MI-77301 was dissolved in 10% PEG400 (polyethylene glycol, mol wt 400, Sigma # P3265) 3% Cremophor EL (Sigma # C5135) and 87% 1X PBS (GIBCO™, Invitrogen Corp.) at the desired concentration (prepared fresh each day and dosed orally within 1 hour). The pH of the drug solutions were checked before use and required to be between pH 3.0 and 9.0 for PO (oral gavage) and between pH 4.5 and 9.0 for IV (intravenous) administration. The pH of a solution was adjusted with 0.5N NaOH when necessary.

Cell culture

Human melanoma cells SK-MEL-103 were maintained at 37°C, 95% air, 5% carbon dioxide in HyQ® RPMI-1640 medium (with 2.05 mM L-glutamîne, 0.1 pM stérile filtered, Hyclone®, QB Perbio) supplemented with 10% FBS and penicillin/ streptomycin and passaged twice weekly.

Xenograft tumor cell injection

Tumor cells for xenografts were harvested with Trypsin (0.05%)-EDTA (0.53mM) (GIBCO™, Invitrogen Corp.), growth medium added and cells placed on ice. A cell sample was mixed 1:1 with Trypan Blue (GIBCO™, Invitrogen Corp.) and counted on a hemocytometer to détermine the number of live/dead cells. Cells were washed once with 1X PBS (GIBCO™, Invitrogen Corp.) and resuspended in PBS. Cells in 0.1 ml were injected subcutaneously (s.c.) into the flank région of each mouse using a 27 gauge needle. For Matrigel injections, after washing in PBS, cells were resuspended in an ice cold mixture of 1:1 PBS and Matrigel (BD Biosciences, Invitrogen Corp.) for a final Matrigel protein concentration of 5 mg/ml. SKMEL-103 tumors were inoculated into SCID mice (UM breeding strain:236 C.B-17 SCID, Charles River) at 5 x 10® cells in 0.1 ml with Matrigel. Treatment was started on day 4-5 after tumor injection.

Xenograft tumor growth and weight monitoring

The size of tumors growing in the mice was measured in two dimensions using calipers. Tumor volume (mm³) = (AxB^z)/2 where A and B are the tumor length and width (in mm), respectively. During treatment, tumor volume and body weight were measured three times a week. After the treatment was stopped, tumor volume and body weight was measured at

193 least once a week and mice were kept for additional 60 days for further observation of tumor growth and toxicity.

Assessment of toxicity and end point

Tumors were not allowed to exceed 10% of the animal’s total body weight. If an animal had two or more tumors the total weight of ail tumors were not allowed to exceed 10% of the animal's total body weight. At the end of the experimental period or when tumor size approached 10% of the total body weight, the animal was euthanized. Animais that showed profound morbidity or a weight loss of over 20% of body weight were euthanized.

The efficacy of MI-77301 at 100 mg/kg PO in this assay is presented in Fig. 29.

Having now fully described the methods, compounds, and compositions of matter provided herein, it will be understood by those of skill in the art that the same can be performed within a wide and équivalent range of conditions, formulations, and other parameters without affecting the scope of the methods, compounds, and compositions provided herein or any embodiment thereof. Ail patents, patent applications and publications cited herein are fully incorporated by reference herein in their entirety.

Claims

What Is Claimed Is:

1. A compound having Formula XII:

XII wherein:

R^1a, R^1b, R^1c, and R^1d are each independently selected from the group consisting of hydrogen, fluoro, and chloro;

R² is selected from the group consisting of aralkyl and:

wherein:

R^25a, R^25b, R^25c, R^25d, and R²⁵® are each independently selected from the group consisting of hydrogen, fluoro, and chloro;

R³ is selected from the group consisting of optionally substituted C_rC₈ alkyl and optionally substituted aryl;

R⁴ is selected from the group consisting of hydrogen and optionally substituted

Ci-C₈ alkyl;

R⁵ is selected from the group consisting of:

wherein:

R¹⁴ is selected from the group consisting of hydrogen and optionally substituted C1-C4 alkyl;

195

X is selected from the group consisting of O, S, and NR ;

Y is selected from the group consisting of O, S, and NR;

R is selected from the group consisting of hydrogen and optionally substituted C1-C4 alkyl; and

R is selected from the group consisting of hydrogen, optionally substituted C_rC₄ alkyl, and -COCH₃, wherein the compound is substantially free of one or more other stereoisomers, or a pharmaceutically acceptable sait thereof.
2. The compound of claim 1, wherein:

R^z is:

R³ is optionally substituted C^Ce alkyl;

R⁵ is selected from the group consisting of:

R’ is selected from the group consisting of hydrogen and optionally substituted C_rC₄ alkyl, or a pharmaceutically acceptable sait thereof.
3. The compound of claim 2, wherein R⁴ is hydrogen, or a pharmaceutically acceptable sait thereof.
4. The compound of claim 2, wherein X is NH, or a pharmaceutically acceptable sait thereof.
5. The compound of claim 2, wherein Y is NH, or a pharmaceutically acceptable sait thereof.
6 The compound of claim 2, wherein R³ is -CH₂C(CH₃)₃, or a pharmaceutically acceptable sait thereof.

196
7.

The compound of claim 2, wherein R⁵ is selected from the group consisting of:

or a pharmaceutically acceptable sait thereof.

The compound of claim 2, wherein: is hydrogen;

R^1b, R^1c, and R^1d are each îndependently selected hydrogen, fluoro, and chloro;

R³ is C₄-C₈ alkyl;

R⁴ is hydrogen;

R⁵ is selected from the group consisting of:
8.

R^1a from the group consisting of

I 'OH

OH

I

Me

Me

X and Y are NH; or a pharmaceutically acceptable sait thereof,
9. The compound of daims 7 or 8, wherein R⁵ is selected from the group consisting of:

'OH or a pharmaceutically acceptable sait thereof.

19*
10. A compound having Formula XXXV:

XXXV wherein:

R^1b and R^1c are independently selected from the group consisting of hydrogen, fluoro, 5 and chloro;

R³ is C₄-C₈ alkyl; and

R^25a, R^Z5b, and R^25c are each independently selected from the group consisting of hydrogen, fluoro, and chloro, wherein the compound is substantially free of one or more other stereoisomers,

10 or a pharmaceutically acceptable sait thereof.

198
11. The compound of claim 2 selected from the group consisting of:

wherein the compound is substantially free of one or more other stereoisomers, or a pharmaceutically acceptable sait thereof.

200
12. The compound of claim 1 selected from the group consisting of:

wherein the compound is substantially free of one or more other stereoisomers, or a

5 pharmaceutically acceptable sait thereof.

20Î
13.

A compound selected from the group consisting of:

wherein the compound is substantially free of one or more other stereoîsomers, or a pharmaceutically acceptable sait thereof.
14. A compound having the structure:

wherein the compound is substantially free of one or more other stereoîsomers, or a pharmaceutically acceptable sait thereof.
15. A compound having the structure:

201 pharmaceutically acceptable sait thereof.
16. The compound of any one of daims 1-15, wherein the compound is a substantially pure stereoisomer, or a pharmaceutically acceptable sait thereof.
17. The compound of claim 16, wherein the compound is a pure stereoisomer, or a pharmaceutically acceptable sait thereof.
18. A pharmaceutical composition comprising the compound of any one of daims 117, or a pharmaceutically acceptable sait thereof, and a pharmaceutically acceptable carrier.
19. A pharmaceutical composition comprising the compound of any one of daims 117, or a pharmaceutically acceptable sait thereof, for use in treating a hyperproliferative disease.
20. A compound of any one of daims 1-17, or a pharmaceutically acceptable sait thereof, for use in treatment of a hyperproliferative disease.
21. Use of a compound of any one of daims 1-17, or a pharmaceutically acceptable sait thereof, for the manufacture of a médicament for treatment of hyperproliferative disease.
22. The pharmaceutical composition of claim 19, wherein the hyperproliferative disease is cancer.
23. The compound of daim 20, or a pharmaceutically acceptable sait thereof, wherein the hyperproliferative disease is cancer.
24. The use of claim 21, wherein the hyperproliferative disease is cancer.

203
25. The pharmaceutîcal composition of claim 22, wherein the cancer is selected from the group consisting of melanoma, lung cancer, sarcoma, colon cancer, prostate cancer, choriocarcinoma, breast cancer, retinoblastoma, stomach carcinoma, acute myeloid leukemia, lymphoma, multiple myeloma, and leukemia.
26. The compound of claim 23, wherein the cancer is selected from the group consisting of melanoma, lung cancer, sarcoma, colon cancer, prostate cancer, choriocarcinoma, breast cancer, retinoblastoma, stomach carcinoma, acute myeloid leukemia, lymphoma, multiple myeloma, and leukemia.
27. The use of claim 24, wherein the cancer is selected from the group consisting of melanoma, lung cancer, sarcoma, colon cancer, prostate cancer, choriocarcinoma, breast cancer, retinoblastoma, stomach carcinoma, acute myeloid leukemia, lymphoma, multiple myeloma, and leukemia.
28. The pharmaceutîcal composition of claim 25, wherein the cancer is selected from the group consisting of liposarcoma and melanoma.
29. The compound of claim 26, wherein the cancer is selected from the group consisting of liposarcoma and melanoma.
30. The use of claim 27, wherein the cancer is selected from the group consisting of liposarcoma and melanoma.
31. A kit comprising the compound of any one of daims 1-17, or a pharmaceutically acceptable sait thereof, and instructions for administering the compound, or a pharmaceutically acceptable sait thereof, to a patient having cancer.
32. A kit comprising the pharmaceutîcal composition of claim 18 and instructions for administering the pharmaceutîcal composition to a patient having cancer.
33. The kit of daims 31 or 32, wherein the cancer is selected from the group consisting of melanoma, lung cancer, sarcoma, colon cancer, prostate cancer, choriocarcinoma,

204 breast cancer, retinoblastoma, stomach carcinoma, acute myeloid leukemia, lymphoma, multiple myeloma, and leukemia.
34. The kit of claims 31 or 32, further comprising one or more anticancer agents.
35. The kit of claims 31 or 32, wherein the instructions direct co-administration of the compound, or pharmaceutically acceptable sait thereof, or pharmaceutical composition together with the one or more anticancer agents.
36. The kit of claims 31 or 32, wherein the instructions direct pulsatile dose administration of the compound, or pharmaceutically acceptable sait thereof, or pharmaceutical composition to the patient.
37. A method of preparing a compound having Formula XXXVII:

the method comprising allowing a compound having Formula XXXVI:

to isomerize to a compound having Formula XXXVII, wherein:

R³² is selected from the group consisting of-OR³³and -NR^34aR^34b;

R³³ is selected from the group consisting of hydrogen, alkyl, and aralkyl;

R^34a is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, aralkyl, optionally substituted aryl, and optionally substituted heteroaryl;

R^34b is selected from the group consisting of hydrogen and alkyl;

R^1a, R^1b, R^1c, and R^1d are each independently selected from the group consisting of hydrogen, fluoro, and chloro;

205

R² is selected from the group consisting of aralkyl and:

_R25b

R²⁵®

R²⁵®, R^25b, R^25c, R^25d, and R²⁵® are each independently selected from the group consisting of hydrogen, fluoro, and chloro; and

R³ is selected from the group consisting of optionally substituted Cj-Ca alkyl and optionally substituted aryl.
38. The method of claim 37, wherein the solvent is selected from the group consisting of acetonitrile, methanol, ethyl acetate, and water, or a mixture thereof.
39. The method of claims 37 or 38, wherein the isomerization is carried out at a pH of less than 7.
40. The method of claims 37 or 38, wherein the isomerization is carried out at a pH of 7.
41. The method of claims 37 or 38, wherein the isomerization is carried out at a pH of greater than 7.
42. The method of any one of claims 37-39, wherein the isomerization is carried out in the presence of an acid selected from the group consisting of trifluoroacetic acid and acetic acid.
43. The method of any one of claims 37, 38, or 41, wherein the isomerization is carried out in the presence of NaHCO₃.
44. The method of any one of claims 37-43, wherein the isomerization is carried out at a température of about 20°C to about 100°C.
45. The method of any one of claims 37-44, wherein R³² is -OR³³.
46. The method of any one of claims 37-44, wherein R³² is -NR^34aR^34b.

206
47. The method of claim 46, wherein R^34b is hydrogen and R^34a is selected from the group consisting of alkyl, hydroxyalkyl, hydroxycycloalkyl, optionally substituted aryJ, and optionally substituted heteroaryl.
48.

The method of claim 47, wherein R^34a is selected from the group consisting of:
49. The method of any one of claims 37-48, wherein the compound of Formula XXXVII is isolated as a substantially pure stereoisomer.
50. The method of claim 49, wherein the compound having Formula XXXVII is isolated as a pure stereoisomer.
51. The method of any one of claims 37-50, wherein: R³² is -NR^34aR^34b;

R^34a is:

R^33b is hydrogen;

R^z is:

; and

R³ is Ci-C_a alkyl.
52. The method of claim 37 further comprising isolating the co

Formula XXXVII substantially free from the compound having Formula XXXVI. il * I )/