Smac mimetic

ES 2 565 337 T3

DESCRIPTION

SMAC Mimetic

Invention area

The present invention relates to the area of ​​SMAC mimetics and compositions for treating proliferative disorders including cancer.

Background of the invention

Apoptosis inhibitory proteins (lAPs) are naturally occurring intracellular proteins that suppress caspase-dependent apoptosis. SMAC, also known as DIABLO, is another intracellular protein that functions to antagonize, that is, inhibit the activity of IAPs. In normal healthy cells, SMAC and IAPs work together to keep cells healthy. However, in certain disease states, eg, cancer and other proliferative disorders, IAPs are not adequately antagonized and thus prevent apoptosis and cause or worsen abnormal proliferation and survival.

SMAC mimetics, also known as IAP antagonists, are synthetic small molecules that mimic the structure and IAP antagonist activity of the four N-terminal amino acids of SMAC. (SMAC mimetics are sometimes called IAP antagonists). When administered to animals suffering from proliferative disorders, SMAC mimetics antagonize IAPs, causing increased apoptosis among abnormally proliferating cells.

Examples of SMAC mimetic peptides are those disclosed in US 7,517,906; US 7,309,792; US 7,419,975; US 2005/0234042; US 2005/0261203; US 2006/0014700; US 2006/0025347; US 2006/0052311; US 2006/0128632; US 2006/0167066; US 2007/0042428; US 2007/032437; US 2008/0132485; WO 2005/069888; WO 2005/069894; WO 2006/010118; WO 2006/122408; WO 2006/017295; WO 2006/133147; WO 2006/128455; WO 2006/091972; WO 2006/020060; WO 2006/014361; WO 2006/097791; WO 2005/094818; WO 2008/045905; WO 2008/016893; WO 2007/136921; WO 2007/021825; WO 2007/130626; WO 2007/106192; and WO 2007/101347.

Summary of the Invention

This invention, in one aspect, is N- {1S- [2R- (6,6'-Difluoro-3 '- {4S-hydroxy-1- [2S- (2S-methylamino-propionylamino) butyryl] -pyrrolidin-2R -ylmethyl} -1 H, 1 'H- [2,2'] biindolyl-3-ylmethyl) -4S-hydroxy-pyrrolidine-1-carbonyl] -propyl} -2S-methylaminopropionamide and pharmaceutically acceptable salts thereof, in addition of various forms of said compound and salts thereof as described in greater detail later in the present patent.

This compound has the following structure:

<img file="ES2565337T3_D0001.tif" />

Wherein R5 is -CH2CH3. This compound is also referred to in the present patent as compound 15.

ES 2 565 337 T3

The invention, in related aspects, comprises a pharmaceutical composition comprising such a compound and its use in a method for the treatment of a proliferative disorder in a human or non-human mammalian subject in need thereof, which comprises internally administering to the subject a effective amount of said compound or a pharmaceutically acceptable salt thereof.

In other aspects, the invention comprises a compound for use in a method of treating a proliferative disorder in a mammal in need thereof, eg, a human, or a companion animal, a food-grade animal, or an animal. intended for sports activities, which comprises internally administering to the animal an effective amount of Compound 15 or a pharmaceutically acceptable salt thereof.

In another illustrative embodiment, the invention comprises a compound for use in a method of inducing apoptosis in a cell which comprises contacting the cell with Compound 15 or a pharmaceutically acceptable salt thereof. In this embodiment, the cell can be, for example, a cancer cell.

In illustrative embodiments, the invention comprises a compound for use in any or more of the foregoing methods, wherein said methods further comprise administering a second cancer-related therapy, such as, for example, chemotherapy, immunotherapy, photodynamic therapy, and combinations thereof.

In a further illustrative embodiment, the invention comprises a compound for use in a method of treating an autoimmune disease, wherein said condition is caused or aggravated by abnormal regulation of apoptosis, in a mammal in need thereof, which includes, for example, systemic lupus erythematosus, psoriasis, and idiopathic thrombocytopenic purpura (Werlhof's disease) which comprises internally administering to the animal an effective amount of Compound 15 or a pharmaceutically acceptable salt thereof.

Brief description of the Figures

Figure 1 shows the mean percentage of body weight loss in rats after 4 days of intravenous bolus dosing with SMAC mimetics, substantially as described in Example 4.

Figure 2 shows the mean tumor volume (2A) and body weight change (2B) that results from treatment of human xenografts in nude mice with SMAC mimetics, substantially as described in Example 5.

Detailed description of the invention

The compound of the invention is a SMAC mimetic that can be used in the treatment of proliferative disorders, for example: various benign tumors or malignant tumors (cancer), benign proliferative diseases (for example, psoriasis, benign prostatic hypertrophy, and restenosis) , or autoimmune diseases (eg, autoimmune proliferative glomerulonephritis, lymphoproliferative autoimmune responses). Cancer types that can potentially be treated with IAP antagonists include, but are not limited to, one or more of the following: lung adenocarcinoma, pancreatic cancer, colon cancer, ovarian cancer, breast cancer, mesothelioma, peripheral neuroma, bladder cancer, glioblastoma, melanoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, breast cancer, cervical cancer, chronic myeloproliferative disorders (e.g. chronic lymphocytic leukemia, chronic myelogenous leukemia), colon cancer, endocrine cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gestational trophoblastic tumors, hairy cell leukemia , Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, insular carcinoma, Kaposi's sarcoma, laryngeal cancer, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, lip cancer, oral cavity cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, melanoma, Merkel cell carcinoma, metastatic squamous neck cancer, multiple myeloma and other plasma cell neoplasms, mycosis fungoides and Sezary syndrome, myelodysplastic syndromes, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, small cell lung cancer, oropharyngeal cancer, bone cancer, including osteosarcoma and malignant fibrous histiocytoma of bone, epithelial ovarian cancer, germ cell tumors of the ovary, low malignant potential ovarian tumors, pancreatic cancer, paranasal sinus cancer, parathyroid cancer, cancer of the penis, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small bowel cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumors, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilm's tumor and other childhood kidney tumors.

ES 2 565 337 T3

Some embodiments of the invention include a compound for use in inducing apoptosis of cells, particularly pathologically proliferating cells. The methods can be performed in vitro or in vivo.

Methods may include administration of the compound of the invention alone, administration of a combination of IAP antagonists, or administration of the compound of the invention, with or without one or more additional IAP antagonists, and one or more agents. additional chemotherapeutics. The administration of multiple agents can be simultaneous or sequential. Useful chemotherapeutic agents include, but are not limited to, alkylating agents (eg, cyclophosphamide, mechlorethamine, chlorambucil, melphalan), anthracyclines (eg, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin disruptors) eg paclitaxel, docetaxel), epothilones (eg epothilone A, epothilone B, epothilone D), topoisomerase II inhibitors (eg etoposide, teniposide, tafluposide), nucleotide precursor analogs (eg, azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, thioguanine), peptide antibiotics (eg, bleomycin), platinum-based agents (eg, carboplatin cisplatin, oxaliplatin), retinoids (eg, trans-retinoic acid), and vinca alkaloids and derivatives (eg, vinblastine, vincristine, vindesine, vinorelbine). In some embodiments, chemotherapeutic agents include fludarabine, doxorubicin, paclitaxel, docetaxel, camptothecin, etoposide, topotecan, irinotecan, cisplatin, carboplatin, oxaliplatin, amsacrine, mitoxantrone, 5-fluoro-uracil, or gemcitabine.

In some embodiments of the invention, pharmaceutical compositions comprising the compound of the invention, alone or in combination with one or more other active pharmaceutical ingredients, are administered to a human or animal subject. The pharmaceutical compositions usually comprise at least one pharmaceutically acceptable excipient, eg, a carrier or diluent, and can be administered in a conventional manner by routes including the systemic, topical, or oral routes. Administration is usually by intravenous injection, either as a bolus or by infusion, but other routes of administration are not excluded. An intravenous formulation can be 1 mg / mL of Compound 15 in 0.05M PBS-citrate buffer, pH5. Specific modes of administration will depend on the indication and other factors, including the particular compound being administered. The amount of compound to be administered is that amount that is therapeutically effective. The dose to be administered will depend on the characteristics of the subject being treated, eg, the particular patient treated, age, weight, health, types of concomitant treatment, if any.

The frequency of treatments can easily be determined by one skilled in the art (eg, by the in-house physician).

Usually, the compound of the invention will be administered by intravenous injection, including, for example, infusion for about 1 to about 120 minutes, for example, about 30 minutes.

The pharmaceutical composition of the invention is a composition in which the active pharmaceutical ingredient, ie the compound of the invention, is sufficiently pure, and the composition is otherwise suitable, for internal administration to a human or other mammal. It can be prepared in a unit dose form, that is, a form suitable for a single administration to a subject. Thus, for example, a pharmaceutical composition in intravenous unit dose form may comprise a pre-filled vial or syringe, each comprising an effective amount or a convenient fraction of an effective amount, such that one of the contents of a vial or syringe at a given time. Such administration can be repeated up to 4 times per day over a period of time, if necessary to achieve a cumulative effective dose, eg tumor regression. A dosing regimen may be, for example, intravenous injections daily or twice a week, or, for example, weekly injections in cycles of three weeks with medication and one off for as long as the treatment is effective, for example, until the disease progresses or the drug is not tolerated. The effective dose administered in each injection is an amount that is effective and tolerated; it can be, for example, 0.01 to 30 mg / m²eg 0.2 to 10 mg / m², or, for example, 0.5 to 5 mg / m².

The compound of the invention can also be applied locally, such as in an isolated limb infusion. The compound of the invention can further be applied topically, for example, as in a cream, gel, lotion, or ointment, or in a patch of the depot or matrix type, or in an active transdermal delivery system.

An effective dose is one that over the course of therapy, which may be, for example, 1 or more weeks, multiple periods of 3 weeks on treatment / 1 week off, results in treatment of a proliferative disorder, i.e. , a decrease in the rate of disease progression, completion of progression, or regression or remission of the disease.

ES 2 565 337 T3

The pharmaceutical compositions to be used comprise a therapeutically effective amount as described above, or a pharmaceutically acceptable salt or other form thereof, together with one or more pharmaceutically acceptable excipients. The term "pharmaceutical composition" refers to a composition suitable for administration in medical use. It should be appreciated that determinations of appropriate dosage forms, dosage amounts, and routes of administration for a particular patient are at the level of those of ordinary skill in the medical and pharmaceutical arts.

Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the compound of the invention, which is preferably isotonic with the blood of the recipient. This aqueous preparation can be formulated according to known methods using suitable carriers or diluents that can include a buffer.

When the conjoint or combination therapy described in more detail below is practiced, administration of the compound and compositions of the present invention may occur concurrently with, subsequent to, or prior to, chemotherapy or radiation, provided that the chemotherapeutic agent or radiation sensitizes the system to the compound and compositions of the present invention.

The present invention is further directed to the use of the compound and compositions as a chemoenhancing agent with other treatment approaches. The term "chemoenhancing agent" refers to an agent that acts to increase the sensitivity of an organism, tissue, or cell to a chemical compound or treatment, mainly "chemotherapeutic agents" or "chemotherapeutic drugs" or a radiation treatment. Thus, the compound and compositions of the present invention can be used to inhibit tumor growth in vivo by administering them in combination with a biological or chemotherapeutic agent or by using them in combination with radiation. In these applications, administration of the compound and compositions of the present invention may occur prior to, and with sufficient time, to cause sensitization of the site to be treated. Alternatively, the compound and compositions of the present invention can be used concurrently with radiation and / or additional anticancer chemicals (infra). Such systems can avoid repeated administrations of the compound and compositions of the present invention, increasing convenience to the subject and physician, and may be particularly suitable for certain compositions of the present invention.

Biological and chemotherapeutic / anti-neoplastic agents and radiation induce apoptosis by activating apoptotic pathways, and, since the compound and compositions of the present invention release antagonists of apoptotic proteins (IAPs) and thus eliminate the blockade of apoptosis, the combination of chemotherapeutic / anti-neoplastic agents and radiation with the compound and compositions of the present invention would work additionally or synergistically to facilitate apoptosis.

A combination of the compound of the present invention and a biological or chemotherapeutic / anti-neoplastic agent and / or radiation therapy of any type that activates the extrinsic or intrinsic pathway, can provide a more effective approach to kill tumor cells. The compound of the present invention interacts with IAPs, such as XIAP, cIAP-1, cIAP-2, ML-IAP, etc., and removes the blockade of apoptosis mediated by IAP. Most chemotherapeutic / anti-neoplastic agents and / or radiation therapy destroy actively dividing cells by activating the intrinsic apoptotic pathway, leading to apoptosis and cell death. Biological antitumor agents such as TRAIL (TNF-related apoptosis inducing ligand) activate extrinsic apoptotic pathways. As described in more detail below, embodiments of the invention provide combinations of the compound of the present invention and a biological or chemotherapeutic / antineoplastic agent and / or radiation that provides synergistic action against unwanted cell proliferation. This synergistic action between the compound of the present invention and a biological or chemotherapeutic / anti-neoplastic agent and / or radiation therapy, can improve the efficacy of the biological or chemotherapeutic / anti-neoplastic agent and / or radiation therapies. This will allow an increase in the effectiveness of biological or chemotherapeutic / anti-neoplastic agents or radiation treatments, allowing a higher percentage of tumors that respond to therapy, an improvement in tumor response, and, potentially, a reduction in the dose of the biologic or chemotherapeutic / anti-neoplastic agent that is needed to treat a tumor, thereby providing the use of a more tolerable dose of the biological or chemotherapeutic / anti-neoplastic agent and / or radiation.

The patient is treated by administering the compound or a pharmaceutical composition of the present invention at the time the patient is undergoing concomitant or prior radiation or chemotherapy for the treatment of a neoproliferative pathology of a tumor such as, but not be limited to, bladder cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, gastric cancer, colon cancer, ovarian cancer, kidney cancer, hepatoma, melanoma, lymphoma, sarcoma, and combinations thereof. In another embodiment of the present invention, the compound or composition of the present invention can be administered in combination with a biological or chemotherapeutic agent and / or for use in combination with radiation therapy, immunotherapy, and / or photodynamic therapy, promoting apoptosis and increasing the effectiveness of chemotherapeutic therapy, radiotherapy, immunotherapy, and / or photodynamic therapy.

ES 2 565 337 T3

Embodiments of the invention also include a compound for use in a method of treating a patient suffering from cancer by the simultaneous or concurrent administration of a biological or chemotherapeutic agent. Such biological or chemotherapeutic agents include but are not limited to the chemotherapeutic agents described in Modern Pharmacology with Clinical Applications, Sixth Edition, Craig &amp; Stitzel, Chpt. 56, pp 639-656 (2004), incorporated herein by reference. The chemotherapeutic agent can be, but is not limited to, alkylating agents, antimetabolites, anti-tumor antibiotics, plant derived products such as taxanes, enzymes, hormonal agents, various agents such as cisplatin, monoclonal antibodies, glucocorticoids, mitotic inhibitors, inhibitors topoisomerase I, topoisomerase II inhibitors, immunomodulatory agents such as interferons, cell growth factors, cytokines, and non-steroidal anti-inflammatory compounds (NSAIDs), cell growth factors, and kinase inhibitors. Other classifications for chemotherapeutic agents include mitotic inhibitors, and anti-estrogenic agents.

Specific examples of suitable biological and chemotherapeutic agents include, but are not limited to. cisplatin, carmustine (BCNU), 5-fluorouracil (5-FU), cytarabine (Ara-C), gemcitabine, methotrexate, daunorubicin, doxorubicin, dexamethasone, topotecan, etoposide, paclitaxel, vincristine, tamoxifen, TRAIL, and others-alpha Members, ie, other than TRAIL and TNF-alpha, of the TNF superfamily of molecules, interferon (in both its alpha and beta forms), thalidomide, thalidomide derivatives such as lenalidomide, melphalan, and PARP inhibitors. Other specific examples of suitable chemotherapeutic agents include nitrogen mustards such as cyclophosphamide, alkyl sulphonates, nitrosoureas, ethyleneimines, triacenes, folate antagonists, purine analogs, pyrimidine analogs, anthracyclines, bleomycins, mitomycins, dactinomycins, vincaloideslicamycin alkaloids. , epipodophyllotoxins, taxanes, glucocorticoids, L-asparaginase, estrogens, androgens, progestins, luteinizing hormones, octreotide acetate, hydroxyurea, procarbazine, mitotane, hexamethylmelamine, carboplatin, mitoxantrone, monoclonal antibodies, levamisole, interferons, interleukins, filgrastim, and sargramostim.

Another embodiment of the present invention refers to the compound or a composition of the present invention in combination with topoisomerase inhibitors to enhance their apoptosis inducing effect. Topoisomerase inhibitors inhibit DNA replication and repair, thereby promoting apoptosis, and are used as chemotherapeutic agents. Topoisomerase inhibitors promote DNA damage by inhibiting enzymes that are required in the DNA repair process. Therefore, the export of Smac from the mitochondria to the cell cytosol is caused by DNA damage caused by tpoisomerase inhibitors. Topoisomerase inhibitors of both the Type I class (camptothecin, topotecan, SN-38 (active metabolite of irinotecan) and the Type II class (etoposide) are expected to show potent synergy with the compounds herein Invention Additional examples of topoisomerase inhibitors that may be used include, but are not limited to, irinotecan, topotecan, etoposide, amsacrine, exatecan, gimatecan, etc. Other topoisomerase inhibitors include, for example, Aclacinomycin A, camptothecin, daunorubicin, doxorubicin, ellipticin, epirubicin, and mitaxantrone.

Another embodiment of the present invention refers to the compound or a composition of the present invention in combination with non-steroidal anti-inflammatory drugs (NSAIDs).

In another embodiment of the invention, the chemotherapeutic / anti-neoplastic agent for use in combination with the compound (s) of the present invention may be a platinum-containing compound. In one embodiment of the invention, the platinum-containing compound is cisplatin. Cisplatin may synergize with a compound of the present invention and enhance the inhibition of an IAP, such as, but not limited to, XIAP, cIAP-1, c-IAP-2, ML-IAP, etc. In another embodiment a platinum-containing compound is carboplatin. Carboplatin can synergize with a compound of the present invention and enhance the inhibition of an IAP, including, but not limited to, XIAP, cIAP-1, c-IAP-2, MLIAP, etc. In another embodiment a platinum-containing compound is oxaliplatin. Oxyplatin can synergize with a compound of the present invention and enhance the inhibition of an IAP, including, but not limited to, XIAP, cIAP-1, c-IAP-2, ML-iAp, etc.

Platinum chemotherapy drugs belong to a general group of DNA modifying agents. DNA modifying agents can be any highly reactive chemical agent that binds to various nucleophilic groups in nucleic acids and proteins and causes mutagenic, carcinogenic, or cytotoxic effects. DNA modifying agents work by different mechanisms, disruption of DNA function and cell death; damage to DNA / formation of cross-bridges or bonds between atoms in DNA; and induction of nucleotide mismatch leading to mutations, to achieve the same end result. Three non-limiting examples of platinum-containing DNA modifying agents are cisplatin, carboplatin, and oxaliplatin.

Still another embodiment of the present invention is the therapeutic combination or therapeutic use in combination of the compound or compositions of the present invention with TRAIL or TRAIL agonist antibodies, or other biological or chemical agents that bind to and activate the TRAIL receptor (s). . Many types of cancer cells are sensitive to TRAIL-induced apoptosis, while most normal cells appear to be resistant to this action of TRAIL. TRAIL-resistant cells can arise from a variety of

ES 2 565 337 T3 different mechanisms including receptor loss, presence of decoy receptors, overexpression of FLIP that competes for the binding of the proenzyme caspase-8 during the formation of DISC (death inducing signaling complex) and the inhibition of caspase-3 and / or caspase-9 activated by XIAP. Under conditions of resistance to TRAIL, a compound or composition of the present invention can increase the sensitivity of tumor cells to TRAIL, which leads to an increase in cell death, the clinical correlations of which are expected to be an increase in apoptotic activity. in TRAIL-resistant tumors, an improvement in clinical response, an increase in the duration of response, and finally, an increase in the survival rate of the patient.

In another embodiment of the invention, Compound 15 is administered in combination with a cytokine, eg, TNFq.

The compound and compositions of the present invention can also be used to augment radiation therapy (or radiotherapy), that is, the medical use of ionizing radiation as part of cancer treatment to control malignant cells. Although radiation therapy is often used as part of curative therapy, it is occasionally used as a palliative treatment, in which cure is not possible and symptomatic relief is the goal. Radiation therapy is commonly used to treat tumors.

It can be used as the primary therapy. It is also common to combine radiation therapy with surgery and / or chemotherapy. The tumors most commonly treated with radiation therapy are breast cancer, prostate cancer, kidney cancer, head and neck cancer, gynecologic tumors, bladder cancer, and lymphoma. Radiation therapy is commonly applied right to the localized area involved with the tumor. Radiation areas often also include lymph node drainage. It is possible but not usual to apply radiation therapy to the whole body, or to the entire surface of the skin. Radiation therapy is usually given daily up to 35-38 fractions (a daily dose is a fraction). These small frequent doses allow healthy cells time to regrow, repairing the damage done by radiation. Three main divisions of radiotherapy or teletherapy, brachytherapy or radiotherapy using closed or sealed sources of radioactive material, which are suitable examples of treatment protocol in the present invention. The differences refer to the position of the radiation source; the external is external to the body, whereas radiation therapy with a sealed or unsealed source delivers radioactive material internally. Sealed sources for brachytherapy are usually removed later, while unsealed sources are injected into the body.

Compound 15 is capable of forming pharmaceutically acceptable salts, including but not limited to acid addition and / or basic addition salts. Such salts are included in all aspects of the invention.

The present invention is intended to encompass Compound 15 synthesized in vitro using laboratory techniques, such as are well known to synthetic chemists; or synthesized using in vivo techniques, such as through metabolism, fermentation, digestion, and the like. It is further contemplated that the compound of the present invention can be synthesized using a combination of in vitro and in vivo techniques.

The present invention also includes isotopically enriched compounds, which are identical to Compound 15 but in that one or more atoms are replaced by an atom having an atomic mass and a mass number different from the atomic mass or mass number found. usually in nature. Examples of isotopes that can be included in the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁶OR, ¹⁷OR, ³¹P, ³²P, ³⁵Yes, ¹⁸F, and ³⁶Cl. Also included are substitutions with heavier isotopes such as deuterium, that is, ²H. Isotopically enriched compounds of the invention can generally be prepared by substituting a readily available isotopically-labeled reagent for a non-isotopically enriched reagent. For example, deuterium incorporation can be accomplished by substituting sodium d4-brohydride for sodium bohydride, or by replacing methyl iodide with methyl d3-iodide. Representative examples of specific deuterium analogs and their preparation are described in Example 1.

Compound 15 can exist in solvated forms in addition to unsolvated forms, including hydrated forms. In addition, Compound 15 can exist in various solid states including crystalline, semi-crystalline, and amorphous (non-crystalline) forms, and in the form of clathrates, prodrugs, biohydrolyzable esters, racemic mixtures, non-racemic mixtures, or purified stereoisomers including, but not limited to optically pure enantiomers and diastereomers. It is generally intended to include all of these and other such forms within the scope of the term Compound 15.

References to Compound 15 and the compound of the invention, and other similar phrases in this specification and in the claims, are intended to include not only the compound of formula (I), but also the pharmaceutically acceptable salts of Compound 15, in addition to various forms of said compound or salts thereof such as those described above and below.

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In further embodiments, the invention comprises compounds useful as intermediates in the synthesis of Compound 15, in addition to processes for the preparation of such intermediates and Compound 15. For example, in such embodiments, the invention comprises compounds shown in the Examples. below, such as Compounds 11, 12, 13, 14, and isotopically enriched compounds such as Compounds 19,20,28,29,31,32. One such embodiment is Compound 15 in which the 4-OH substituent in the pyrrolidine moiety is protected with a protecting group. An exemplary protecting group is an acetyl group, which is illustrated in Compounds 11-14, below. Other useful protecting groups will be apparent to those skilled in the art and include, for example, benzoyl, benzyl, trimethylsilyl, and triphenylmethyl groups. The protecting group is removed, for example, by contacting the protected intermediate with an acid or a base, as shown in Schemes XIII and XIV, below. Thus, the invention comprises the compound having the structure of Compound 15 in addition to protected versions of Compound 15 such as Compounds 13 and 14 in which the N-terminal ends are protected with carbamate moieties and / or hydroxyl groups. Free are protected as asters, where such compounds are referred to as protected Compound 15. The invention further comprises the step of deprotecting Protected Compound 15, by contacting Protected Compound 15 with an acid or base whereby the Protecting Group is removed to provide Compound 15. Isotopically enriched compounds of the invention include deuterated forms of the Compound 15 such as Compounds 20, 29, and 32. Protected forms of such compounds, eg, Compounds 19, 28, and 31, are also encompassed by the invention.

Examples

The following preparations and schemes illustrate the synthesis of the compounds of the present invention. Only the claimed compounds are part of the invention.

The abbreviations used throughout these diagrams and in the application in general are identified in the following table:

<td>ABBREVIATION</td><td>MEANING</td><td>ABBREVIATION</td><td>MEANING</td>

<td>ACN</td><td>Acetonitrile</td><td>NMP</td><td>N-methylpyrrolidinone</td>

<td>Ac2O</td><td>Acetic anhydride</td><td>PhCOCl</td><td>Benzoyl chloride</td>

<td>Cbz and Z</td><td>Benzyloxycarbonyl</td><td>DAY D</td><td>Di-isopropyl azo dicarboxylate</td>

<td>Boc and / or boc</td><td>tert-butyloxycarbonyl</td><td>DIBAL</td><td>Hydride Diisobutylaluminum</td>

<td>THF</td><td>Tetrahydrofuran</td><td>DMAP</td><td>4-dimethylamino pyridine</td>

<td>DCM</td><td>Dichloromethane</td><td>DMF</td><td>Dimethylformamide</td>

<td>DDQ</td><td>2,3-dichloro-5,6-dicyano-1,4-benzoquinone</td><td>DMSO</td><td>Dimethyl sulfoxide</td>

<td>mCPBA</td><td>3-chloroperbenzoic acid</td><td>TFA</td><td>Trifluoroacetic acid</td>

<td>Cbz-Cl</td><td>Benzyloxycarbonyl Chloride</td><td>TFAA</td><td>Trifluoroacetic anhydride</td>

<td>Hex</td><td>Hexanes</td><td>HOAc or AcOH</td><td>Acetic acid</td>

<td>HPLC</td><td>High performance liquid chromatography</td><td>DIPEA</td><td>Diisopropylethylamine</td>

<td>TLC</td><td>Thin layer chromatography</td><td>NMM</td><td>M-methylmorpholine</td>

<td>EtOAc</td><td>Ethyl acetate</td><td>NCS</td><td>M-chlorosuccinimide</td>

<td>Ph</td><td>Phenyl</td><td>TEA (EtaN)</td><td>Triethylamine</td>

<td>HEY YOU</td><td>2- (7-Aza-1 H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate</td><td>MsCl</td><td>Methane-sulfonylchloride</td>

ES 2 565 337 T3 (continued)

<td>ABBREVIATION</td><td>MEANING</td><td>ABBREVIATION</td><td>MEANING</td>

<td>I</td><td>Methyl*</td><td>Et</td><td>Ethyl</td>

<td>iPr</td><td>Iso-propyl</td><td>tBu or tert-Bu</td><td>Tert-butyl</td>

<td>cPr</td><td>Cyclopropyl</td><td>cHex</td><td>Cyclohexyl</td>

<td>(2R-EtOMe) and / or R-MeCHOMe</td><td>I</td><td>(2R-EtOH) and / or R-MeCHOH</td><td>o / tn</td>

<td>TBAF</td><td>Tetrabutylammonium fluoride</td><td>MsCI</td><td>Methanesulfonyl Chloride</td>

<td>Who</td><td>Methanesulfonyloxy</td><td>OTs</td><td>-BEAR₂-Ph-Me</td>

<td>TBDMSCI or TBSCI</td><td>Tert-Butyl-dimethyl-silyl chloride</td><td>OTBS</td><td>tert-butyl-dimethyl-silanyloxy</td>

<td>Ph₃P</td><td>Triphenylphosphine</td><td>Ac</td><td>0 . II -S — C — Me Acetyl ( ^ς )</td>

<td>n-Bu</td><td>Normal butyl</td><td>DMA</td><td>Dimethylamine</td>

<td>Swern [O]</td><td>Swern oxidation</td><td>HWE</td><td>Honer-WadsworthEmmons reaction</td>

<td>TBA-CI</td><td>Tetra-n-Butyl Ammonium Chloride</td><td>DMS</td><td>Dimethylsulfide</td>

<td>NP-HPLC</td><td>High performance liquid chromatography - normal phase</td><td>Meldrum acid</td><td>2,2-dimethyl-1,3-dioxane-4,6-dione</td>

<td>EDCI</td><td>N-3 (dimethylaminopropyl) -N'ethylcarbodiimide hydrochloride 1-Et¡l-3- (3- Dimethylaminopropyl) carbodiimideHCI</td><td>Imid.</td><td>Imidazole</td>

<td>Et2O</td><td>Ethylene oxide &lt;L&gt;)</td><td>HOBT, or HBT</td><td>Hydroxybenzotriazole</td>

<td>TES</td><td>Triethylsilane</td><td>RT</td><td>Room temperature</td>

<td>MeNO₂</td><td>Nitromethane</td><td>MeOH</td><td>Methanol</td>

<td>EtOH</td><td>Ethanol</td><td>NaOAc</td><td>Sodium acetate</td>

<td>DCE, or EDC</td><td>Dichloroethane, ethylene dichloride</td><td>CICO₂I</td><td>Ethyl Chloroformate</td>

<td>NaHMDS</td><td>sodium hexamethylsilylazide or sodium bis (trimethylsilyl) amide</td><td>TBSCI</td><td>Tert-Butyl-dimethyl-silanyl chloride</td>

<td>Boc-Chg-OH (Boc-Lcyclohexylglycin0061)</td><td>ΧΛΐγ 0</td><td>Cbz-N (Me) AlaOH ZN (Me) Ala-OH</td><td>0 Me</td>

ES 2 565 337 T3 (continued)

<td>ABBREVIATION</td><td>MEANING</td><td>ABBREVIATION</td><td>MEANING</td>

<td>Boc-N (Me) AlaOH</td><td>Me 0 And I saw..... 1 0 Me</td><td>Boc-Tle-OH</td><td>o 'A 0</td>

<td>Boc-Abu-OH</td><td>0 Et &gt; 1. Jk / 0 N H OR</td><td>Boc-Val-OH</td><td>A, O_r- OR</td>

<td>Boc-Ser-OH</td><td>.OH 0 í ^&gt; L, Jk Aa^ch0 N H 0</td><td>Cbz-Ser (tBu) OH</td><td>11 J Π 0</td>

<td>Boc-Ser (Me) -OH</td><td>.OMe / k Jk Xa / 0 N H OR</td><td>Cbz-Thr (tBu) OH</td><td>. ύ^λ 0'......^λα</td>

<td>Boc-Thr (tBu) OH</td><td>or ΛΑ / γ- or</td><td>Boc-Thr-OH</td><td> \ &gt;^0H0 &gt; v ..... ry or</td>

<td>Boc-Thr (Me) -OH</td><td>\ ^ OMe or ^{&gt; L}^X&lt;T o</td><td>PSI</td><td>Pounds per Square Inch (Gauge)</td>

<td>h</td><td>hour</td><td>NaOMe</td><td>Sodium methoxide</td>

Example 1 - Synthesis

ES 2 565 337 T3

Scheme I

<img file="ES2565337T3_D0002.tif" />

2

4- (tert-Butyl-dimethyl-silanyloxy) -pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester (2): A solution of Z-Hyp-OH (1,300 g, 1.13 mol), TEA (395 mL, 2.83 mol), and DBU (17.2 g, 1.13 mol) in DMF (1.25 L) was stirred in a cold water bath while a suspension of TBS-CI (188 g, 1.24 mol) in DMF (270 mL) was added slowly at 21-26 ° C [Note: moderately exothermic]. The resulting fine suspension was stirred for 22 h at room temperature. The reaction mixture was cooled to 2 ° C and quenched with water (1.54 L) at &lt;26 ° C [Note: the pH of the aqueous layer was 8.5-9.0]. MTBE (3 L) was added and the mixture was acidified to pH 3-4 with concentrated HCl (168 g) at 17-19 ° C. The organic layer was separated and washed with water (2 X 1.5 L). The organic layer was concentrated in vacuo and dried by further distillation of MTBE. Toluene (2 X 500 mL) was added and distilled to remove moisture, to provide 603 g of 2 as a yellow oil [Note: water content by KF analysis was 508 ppm]. Based on drying a small sample of 2 to its solid state, the contained weight of 2 was 412 g (96% yield, uncorrected for purity).¹H NMR (300 MHz, CDCI3): 57.34 (m, 3H), 7.29 (m, 2H), 5.24-5.11 (m, 2H), 4.52 (m, 1H), 4.43 (m, 1H), 3.64-3.42 ( m, 2H), 2.27-2.09 (m, 2H), 0.85 (s, 9H), 0.06 (s, 3H), 0.04 (s, 3H) ppm; ¹³C NMR (75 MHz, cfe-DMSO), rotomer mixture: 5178.7, 178.4, 159.3, 158.9, 141.9, 141.8, 133.4, 133.3, 132.8, 132.6, 132.3, 131.9, 75.3, 74.6, 71.0, 71.0, 62.8, 62.3 , 60.1, 59.7, 44.4, 43.4, 30.6, 30.6, 22.6, 22.6, 0.1, 0.0 ppm. Mass spectrum (ESI), m / z 379.5 [(M) +; calculated for CI9H29NO5YES: 379.5],

Scheme II

<img file="ES2565337T3_D0003.tif" />

Benzyl ester of 4- (tert-But¡ld¡met¡ls¡lan¡lox¡) -2- (6-fluoro-1H-¡ndol-3-carbon¡l) -p¡rrol¡d¡na- 1-carboxylic (3): Z-Hyp (OTBS) -OH (2.55.5 g, 145 mmol) was dissolved in toluene (265 mL). DMF (0.1 mL) and oxalyl chloride (22.4 g, 174 mmol) were added at room temperature. After 2-3 h, the bubbling stopped. After 4 h, the mixture was concentrated in vacuo (65 ° C bath, about 30 min) to provide 95 g of a light yellow solution which was confirmed to be acid chloride by analysis with ¹H NMR.

6-Fluoroindole (39.2 g, 290 mmol) was dissolved in anhydrous chlorobenzene (300 mL) and toluene (200 mL) and the solution was cooled to -4 ° C using an ice / acetone bath. A solution of 3M EtMgBr in diethyl ether (101 g, 294 mmol) was added over 31 minutes at &lt;2.5 ° C resulting in a light amber solution. After 30 min, the acid chloride / toluene solution (see above) was added over 45 min at &lt;2 ° C. The reaction mixture was kept cold for 1 hr and then allowed to warm slowly. After approximately 4 h (10.6 ° C), the reaction mixture was quenched with glacial HOAc (9.0 g, exothermic to 17.5 ° C) and then water (exothermic). Water (200 mL) and EtOAc (300 mL) were added and the organic layer was separated and washed with water (100 mL, slow separation). The organic layer was concentrated in vacuo to provide 227 g of 3 as an amber oil which was used without further purification. ¹H NMR (300 MHz, CDCI3), ~ 2: 1 mixture of rotomers: 59.38 (m, 0.7H), 8.58 (m, 0.3H), 8.35 (app. Dd, J = 5.2, 8.2 Hz, 0.3H), 8.03 (app. Dd, J = 5.2, 8.2 Hz, 0.7H), 7.74 (d, J = 2.9 Hz, 0.7H), 7.66 (d, J = 2.9 Hz, 0.3H), 7.38-7.32 (m, 5H ), 7.07 (m, 1H), 6.95 (m, 1H), 6.85 (m, 1H), 5.26-4.92 (m, 3H), 4.54 (m, 1H), 3.80 (app.dt, J = 5.2, 11.1 Hz, 1H), 3.61 (app. D, J = 11.1 Hz, 0.3H), 3.55 (app. D, J = 11.1 Hz, 0.7H), 2.25-2.07 (m, 2H), 0.88 (s, 9H) , 0.06 (s, 3H), 0.00 (s, 3H) ppm; ¹³C NMR (75 MHz, cfe-DMSO), rotomer mixture: 5193.4, 193.0, 159.3 (d, J_CF = 235.5 Hz), 153.9 (d, J_CF = 16.2 Hz), 136.7, 136.8 (d, J_CF = 34.0 Hz), 134.6, 128.3,

127.8, 127.2, 126.6, 122.4, 113.7 (d, J_CF = 13.5 Hz), 110.2 (d, J_CF = 20.2 Hz), 98.5 (d, J_CF = 25.4 Hz), 70.6, 69.8,

65.8, 65.8, 60.6, 60.3, 55.5, 55.0, 25.7, 25.6, 17.7, 17.7, -4.8, -4.9 ppm. Mass Spectrum (ESI), m / z 518.9 [[(MH) + Na] +; calculated for C27H32FN2O4SiNa: 518.6],

ES 2 565 337 T3

Scheme III

<img file="ES2565337T3_D0004.tif" />

4

2- (6-Fluoro-1H-indol-3-carbon¡l) -4-hydrox¡-p¡rrolld¡na-1-carboxylic acid benzyl ester (4): To a solution containing 3 (227 g) in THF (600 mL) was added 1 M TBAF in THF (160 mL) at room temperature. After 9 h, another 20 mL of the 1 M TBAF / THF solution was added. After approximately 48 h, the reaction mixture was concentrated in vacuo and then redissolved in EtOAc (600 ml_). The organic solution was washed with water (310 mL) and the product precipitated to form a thick suspension which was filtered (slowly). The solids were washed with EtOAc (165 mL portionwise) and dried to provide 43 g of 4. The combined filtrate was concentrated in vacuo to precipitate an additional 4.8 g of 4 after drying.¹H NMR (300 MHz, de-DMSO), mixed rotomers: Ó612.08 (br s, 1H), 8.43 (d, J = 10.5 Hz, 1H), 8.16 (ddd, J = 5.4, 8.7, 14.1 Hz, 1H), 7.36-7.31 (m, 2H), 7.27 (app. D, J = 10.2 Hz, 1H), 7.09-6.93 (m, 4H), 5.24 (dt, J = 8.1, 15.6 Hz, 1H), 5.14 (brs, 1H), 5.04 (app. d, J = 6.4 Hz, 1H), 4.90 (app. dd, J = 13.4, 28.4 Hz, 1H), 4.30 (brs, 1H), 3.58-3.43 (m, 2H ), 2.27 (m, 1H), 1.93 (m, 1H) ppm; ¹³C NMR (75 MHz, cfe-DMSO), rotomer mixture: O194.0, 193.6, 159.9 (d, J_CF = 235.2 Hz), 154.6 (d, J_CF = 9.6 Hz),

138.1. 137.5 (d, J_CF = 26.9 Hz), 136.0, 129.0, 128.5, 128.1 (d, J_CF = 40.0 Hz), 123.4, 123.3, 123.0, 122.9, 114.4 (d, Jcf = 11.7 Hz), 110.6 (d, J_CF = 23.7 Hz), 99.3 (d, J_CF = 25.2 Hz), 69.5, 68.8, 66.4, 66.3, 61.4, 61.1, 56.2, 55.7 ppm. Mass Spectrum (ESI), m / z 382.6 [(M) +; calculated for C21H19FN2O4: 382.3],

Scheme IV

<img file="ES2565337T3_D0005.tif" />

<img file="ES2565337T3_D0006.tif" />

2- (6-Fluoro-1H-indol-3-carbon¡l) -4- (4-nitro-benzo¡lox¡) -pyrrolld¡na-1-carboxylic acid benzyl ester ( 5): A solution containing 4 (51.1 g, 134 mmol), 4-nitrobenzoic acid (27.9 g, 167 mmol) and triphenylphosphine (48.9 g, 187 mmol) in anhydrous THF (700 mL) and DMF (175 mL) was cooled to 2 ° C. DIAD (37.4 mL, 194 mmol) was added over 1 hr at 2-3 ° C. After 1 hr, the solution was allowed to warm to room temperature. After approximately 16 h, the reaction mixture was concentrated in vacuo and MeOH (250 mL) was added and concentrated to form a thick suspension (322 g). Additional MeOH (250 mL) was added and the solution was concentrated in vacuo to provide a thick suspension (420 g) which was cooled in an ice bath. After approximately 1.5 h, the solid was collected on a vacuum filter and washed with chilled MeOH (190 mL). The product was air dried on the filter to provide 82.9 g (&gt; 100%) of 5 as a light yellow solid which was used directly in the next reaction. ¹H NMR (300 MHz, de-DMSO), mixed rotomers: 612.14 (brs, 1H), 8.47 (app. D, J = 6.6 Hz, 1H), 8.29-8.21 (m, 3H), 8.03 (dd, J = 2.7, 8.4 Hz, 2H), 7.43-7.33 (m, 2H), 7.28 (app. Dd, J = 2.1, 9.6 Hz, 1H), 7.20-7.08 (m, 4H), 5.55 (brs, 1H), 5.42 (dd, J = 8.4, 15.3 Hz, 1H), 5.13 (dd \ J = 12.6, 22.2 Hz, 1H), 5.04 (s, 1H), 3.99 (m, 1H), 3.73 (d, J = 12.3 Hz , 1H), 2.91 (m, 1H), 2.36 (m, 1H) ppm; ^theC NMR (75 MHz, cfe-DMSO), rotomer mixture: Ó192.9, 192.4, 164.2, 160.0 (d, J_CF = 235.5 Hz), 154.5 (d, J_CF = 12.0 Hz), 150.9, 137.5, 137.1 (d, J_CF = 12.6 Hz), 135.6, 135.1, 131.3, 128.9 (d, J_CF = 28.0 Hz), 128.5, 128.2, 128.1, 127.6, 124.2, 123.0, 113.5 (d, J_CF = 8.5 Hz), 110.9 (d, Jcf = 21.9 Hz), 99.1 (d, J_CF = 25.5 Hz), 75.2, 74.3, 66.7, 66.5, 62.4, 62.1, 53.6, 53.0, 38.6, 37.6 ppm. Mass Spectrum (ESI), m / z 531.8 [(M) +; calculated for C28H22FN3O7: 531.5],

ES 2 565 337 T3

Scheme V

<img file="ES2565337T3_D0007.tif" />

2- (6-Fluoro-1H-indol-3-carbon¡l) -4-hydrox¡-pyrroled¡na-1-carboxylic acid benzyl ester (6): A suspension of 5 (82.9 g) in THF (600 mL), MeOH (200 mL), and water (100 mL) was added 50% aqueous NaOH (16.0 g, 200 mmol) [Note: exothermic; temperature rise: 23.7 ° C to 25.9 ° C], After 2 h, glacial HOAc (5.3 g) was added to adjust the pH to 7-8 [Note: the orange solution changed to light yellow] and the reaction mixture was concentrated in vacuo. Water (500 mL) was added and the solvent was removed in vacuo to form a thick suspension. The solid was collected on a vacuum filter and washed with water (400 mL in portions). The solid was dried in a vacuum oven at 55 ° C to provide 42.6 g (83%, 2 steps) of 6 as an off-white solid.¹H NMR (300 MHz, de-DMSO): O8.38 (d, J = 11.1 Hz, 1H), 8.14 (ddd, J = 5.7, 8.7, 14.1 Hz, 1H), 7.35-7.29 (m, 2H), 7.25 (app. Dd, J =

2.1, 9.9 Hz, 1H), 7.10-6.95 (m, 4H), 5.16-4.98 (m, 2H), 4.90 (app. Q, J = 13.5, 25.8 Hz, 1H), 4.26 (m, 1H), 3.74 (app. ddd, J = 6.3, 11.1, 18.3 Hz, 1H), 3.22 (m, 1H), 2.59 (m, 1H), 1.73 (app. ddd, J = 6.6, 12.9, 25.2 Hz, 1H) ppm; ³C NMR (75 MHz, cfe-DMSO): O193.8, 193.3, 160.0 (d, J_CF = 235.2 Hz), 154.4 (d, J_CF = 14.5 Hz), 137.5 (d, J_CF = 26.0 Hz), 137.2 (d, Jcf = 12.3 Hz), 129.0, 128.5, 128.2 (d, J_CF = 35.4 Hz), 128.1, 127.4, 123.2, 123.1, 114.4 (d, J_CF = 11.4 Hz), 110.8 (d, Jcf = 23.7 Hz), 110.8 (d, J_CF = 23.7 Hz), 99.0 (d, J_CF = 25.8 Hz), 69.4, 68.6, 66.5, 66.4, 61.5, 61.2,

54.9, 54.6 ppm. Mass Spectrum (ESI), m / z 383.8 [(M + H) +; calculated for C21H20FN2O4: 383.3],

Scheme VI

<img file="ES2565337T3_D0008.tif" />

7

2- (6-Fluoro-1H-¡ndol-3-¡lmet¡l) -4-hydroxy-p¡rrol¡d¡na-1-carboxylic acid benzyl ester (7): To a suspension of 6 (10.1 g, 26 mmol) in anhydrous THF (200 mL) was added 2M L1BH4 in THF (26.2 mL, 52 mmol) over approximately 7 min [Note: exothermic; temperature rise: 21.5 ° C to 28.2 ° C], After 2.5 h, the light yellow solution was cooled to approximately 11 ° C and methanesulfonic acid (4.66 g, 48 mmol ) for about 4 min [Note: exothermic; temperature rise to 14.2 ° C],

After 16 h, the reaction mixture was cooled in an ice bath and carefully quenched with water (50 mL) [Note: the addition of water was exothermic and released a large amount of gas], Following the addition of water, the pH was adjusted to 1 with concentrated HCl (1.9 g). The reaction mixture was concentrated to remove THF and the aqueous solution was extracted with EtOAc (110 mL). The organic layer was separated and washed with water (2 X 50 mL) [Note: final pH approximately 5], The organic solution was concentrated in vacuo and azeotropically dried using anhydrous EtOAc to provide 10.2 g of 7 as a white foam [Note: 87.7 A% by HPLC analysis], ¹H NMR (300 MHz, cfe-DMSO), ~ 1: 1 mix of rotomers: 610.91 (app. D, J = 5.4 Hz, 1H), 7.69 (dd, J = 6.0, 8.4 Hz, 0.5H), 7.48- 7.30 (m, 4.5H), 7.13-7.07 (m, 3H), 6.85 (app. T, J = 8.4 Hz, 0.5H), 6.58 (app. T, J = 9.9 Hz, 0.5H), 5.195.10 (m, 3H), 4.25 (brs, 1H), 4.03-3.96 (m, 1H), 3.55 (dd, J = 5.1, 11.4 Hz, 1H), 3.29 (d, J = 11.4 Hz, 1H), 3.17- 2.98 (m, 2H), 1.79 (m, 2H) ppm. ¹³C NMR (300 MHz, c / 6-DMSO), rotomer mixture: Ó159.5 (d, J_CF = 232.1 Hz), 159.4 (d, Jcf = 232.3 Hz), 154.9, 137.7 (d, J_CF = 36.6 Hz), 136.7 (d, J_CF = 12.6 Hz), 136.6 (d, J_CF = 12.9 Hz), 129.1, 129.1,

128.7, 128.6 (d, J_CF = 26.3 Hz), 128.2, 125.0 (d, J_CF = 21.4 Hz), 124.5, 124.3, 120.1 (d, J_CF = 28.3 Hz), 120.0 (d, J_CF= 28.6 Hz), 112.4 (d, J_CF = 14.6 Hz), 107.4 (d, J_CF = 24.3 Hz), 107.3 (d, J_CF = 24.3 Hz), 69.9, 69.2, 67.1, 66.3, 58.7,

58.1, 56.1, 55.6, 38.3, 37.6, 31.2, 30.1 ppm. Mass Spectrum (ESI), m / z 368.6 [(M) +; calculated for C21H21FN2O3: 368.4],

ES 2 565 337 T3

Scheme Vil

<img file="ES2565337T3_D0009.tif" />

8

4-Acetox¡-2- (6-fluoro-1H-¡ndol-3-¡lmet¡l) -pyrrol¡din-1-carboxylic acid benzyl ester (8): To a solution containing 7 (4.7 g, 12.8 mmol) and DMAP (81 mg, 0.66 mmol) in DCM (100 mL) were added acetic anhydride (2.6 g, 25.5 mmol) at room temperature. After 16 h, the reaction mixture was quenched with MeOH (approximately 3 mL) and washed successively with 10% aqueous Na2CO3 (50 mL), dilute HCI (50 mL), and 10% aqueous Na2CO3 (50 mL). . The organic solution was concentrated in vacuo and filtered through a small column of silica gel (approximately 25 g) [eluent: DCM (200 mL) at 0.5% (v / v) MeOH / DCM (80 mL) to 2% MeOH / DCM (100 mL) to 5% MeOH / DCM (100 mL)]. The fractions containing the product were combined and concentrated to provide 3.28 g (63%) of 8 as a white foam [Note: 94.3 A% by HPLCj analysis.¹H NMR (300 MHz, CDCI3), ~ 1: 1 mixture of rotomers: 67.99 (m, 1H), 7.75-6.61 (m, 9H), 5.28 (m, 1H), 5.20 (m, 2H), 4.23 (m , 1H), 3.82 (dt, J = 5.4, 13.5 Hz, 1H), 3.60 (app. T, J = 13.2 Hz, 1H), 3.50 (d, J = 11.7 Hz, 0.5H), 3.31 (d, J = 12.9 Hz, 0.5H), 2.87 (dt, J = 5.1, 13.5 Hz, 1H), 2.13 (s, 3H), 2.01 (m, 2H) ppm; ¹³C NMR (75 MHz, CDCI3), ~ 1: 1 mixture of rotomers: 6170.8, 160.2 (JCF = 236.4 Hz), 155.2, 136.8, 136.6, 136.4, 128.9, 128.8, 128.5 (JCF = 24.3 Hz), 124.5 (Jcf = 21.4 Hz), 123.0, 123.0, 120.0 (Jcf = 27.1 Hz), 119.9 (Jcf = 26.0 Hz), 112.8 (Jcf = 10.5 Hz), 108.2 (Jcf = 24.3 Hz), 97.7 (Jcf = 25.7 Hz), 74.0 , 73.2, 67.9, 67.2, 58.5, 57.6, 53.4, 53.0, 35.4, 34.6, 30.8, 29.7, 21.5 ppm. Mass Spectrum (ESI), m / z410.6 [(M) +; calculated for C23H23FN2O4: 410.4],

Scheme VIII

<img file="ES2565337T3_D0010.tif" />

1. TFA, EtOAc

two. DDQ, EtOAc

<img file="ES2565337T3_D0011.tif" />

4-Acetox¡-2-3 '- (4-acetox¡-1-benz¡lox¡carbon¡lp¡rrol¡d¡n-2-¡lmet¡l) -6.6'-d¡fluoro acid benzyl ester -1H, 1'Hí2.2'1b¡¡ndol¡l-3-¡lmet¡lp¡rrol¡din-1-carboxylic (9): A solution containing 8 (2.9 g, 7.1 mmol) in EtOAc (approximately 5 mL) was cooled in an ice bath and pre-cooled TFA (20.3 mL) was added in one portion. The resulting yellow solution was stirred at 2-4 ° C. After 4.75 h, the cold reaction mixture was transferred (via cannula) with stirring to a mixture of pre-cooled EtOAc (30 mL), and 25% aqueous K2CO3 (80.7 g). The aqueous layer was separated and extracted with EtOAc (3 X 30 mL) and the combined organic extracts were washed with 10% aqueous Na2CO3 (30 g). The organic solution was concentrated in vacuo and azeotropically dried using anhydrous EtOAc to provide 2.95 g of indolylindoline diastereomers as a yellow foam that was used directly in the next reaction. Mass Spectrum (ESI), m / z 821.3 [(M) +; calculated for C46H46F2N4O8: 820.9],

To a solution containing the diastereomers (2.95 g) in EtOAc (30 mL) was added DDQ (885 mg, 3.9 mmol) in one portion [Note: exothermic; temperature rise: 26 ° C to 31.6 ° C], After 3 h, the dark brown / orange reaction mixture was filtered through Celite® which was subsequently rinsed with EtOAc (50 mL). [Note: a second reaction was performed on a 0.5 mmol scale for testing]. The filtrate was washed with 10% aqueous Na2CO3 (2 washes: 74 g, then 58 g). The organic layer was concentrated in vacuo to provide 2.14 g of 9 as a light brown solid.

The Celite® pad was further rinsed with THF (100 mL) which was concentrated in vacuo to provide another 1.12 g of 9 as a beige solid. The solids were dissolved in isopropyl acetate (¡PrAc, 50 mL). The PrAc solution was reduced to approximately 20 mL and the resulting suspension was heated to reflux, cooled to room temperature, and then placed in an ice bath. After 1 hr, the solid was collected

ES 2 565 337 T3 by vacuum filtration, washed with ¡PrAc (10 mL) and dried in a vacuum oven to provide 2.13 g (65%, 2 steps) of 9 as a beige solid [ Note: ~ 100 A% by HPLC analysis], ¹H NMR (300 MHz, CDCIs): 611.29 (brs, 2H), 7.57-7.36 (m, 14H), 6.90 (app. Dt, J = 2.1, 9.3 Hz, 2H), 5.39-5.30 (m, 6H), 4.28 (t, J = 9.0 Hz, 2H), 3.84-3.73 (m, 4H), 3.66 (d, J = 13.2 Hz, 2H), 3.40 (dd, J = 12.0, 14.4 Hz, 2H), 2.31 (s , 6H), 2.17 (m, 2H), 2.05 (m, 2H) ppm; ¹³C NMR (75 MHz, CDCI3): 6170.7, 161.9, 158.8, 156.3, 137.5, 137.3, 136.5, 128.9, 128.6, 128.5,

125.9, 118.8, 118.6, 108.8, 108.5, 108.3, 98.7, 98.3, 74.4, 68.0, 60.1, 53.5, 34.5, 28.9, 21.7 ppm. Mass Spectrum (ESI), m / z 818.2 [(M) +; calculated for C46H44F2N4O8: 818.8],

Scheme IX

<img file="ES2565337T3_D0012.tif" />

<img file="ES2565337T3_D0013.tif" />

5-í3 '- (4-acetox¡-p¡rrol¡d¡n-2-¡lmet¡l) -6.6'-d¡fluoro-1H.TH-í2.2'1b¡¡ndol¡l-3 -¡Lmethyl-p¡rrol¡d¡n-3-¡l acetic acid ester (10): A suspension containing 9 (35 g, 42.7 mmol) in 1: 1 EtOAc / MeOH (400 mL) was distributed in two Parr hydrogenators 500 mL (approximately 200 mL / each), and loaded with Pd on C 10% (wet, 5000 mg / each, Aldrich®). The reaction mixture was pressurized to 50 PSI H2 and stirred for 3 h. The reaction mixture was filtered through a pad of Celite® and the solids were washed with EtOAc. The clarified filtrate was concentrated in vacuo to provide 24 g of 10 as an off-white solid which was used directly in the next reaction.¹H NMR (300 MHz, CDCI3): 613.10 (br s, 2H) 7.45 (dd, J = 5.2, 8.9 Hz, 2H), 7.03 (dd, J = 2.3, 9.8 Hz, 2H), 6.85 (m, 2H) , 5.35 (m, 2H), 3.71 (m 2H), 3.18-3.35 (m, 4H), 2.90-3.14 (m, 4H), 2.56 (m, 2H), 2.00-2.10 (m, 2H), 2.04 ( s, 6H), 1.80-1.92 (m, 2H) ppm; ¹³C NMR (75 MHz, CDCI3): 6171.3, 161.7, 158.6,

136.1, 135.9, 130.5, 130.4, 125.4, 119.1, 118.9, 109.6, 108.0, 107.6, 97.6, 97.5, 75.1, 57.7, 51.6, 38.7, 32.8, 21.6 ppm. Mass Spectrum (ESI), m / z 550.9 [(M) +; calculated for C30H32F2N4O4: 550.6],

<img file="ES2565337T3_D0014.tif" />

5- (3'-4-acetox¡-1- (2-tert-butox¡carbon¡lam¡no-but¡r¡l) -pyrrol¡d¡n-2-¡lmet¡l1-6.6 ' -d¡fluoro-1H.1'H-í2.2'1bi¡ndol¡l-3-¡lmet¡ll-1 (2-tert-butox¡carbon¡lam¡no-but¡r¡l) -p ¡Rrolid¡n-3-¡l acetic acid ester (11): To a solution containing Boc-AbuOH (20.4 g, 100 mmol) and HATU (42.0 g, 110 mmol) in anhydrous NMP (150 mL) at 0 ° C NMM (16 mL, 150 mmol) was added followed by a solution of 10 (24 g, 42 mmol) in NMP (100 mL) The reaction mixture was slowly warmed to room temperature. After 16 h, the reaction mixture was diluted with MTBE (1000 mL) and the heterogeneous mixture was washed with water (500 mL). The layers were separated and the organic phase formed a heterogeneous suspension. MTBE (1000 mL) and EtOAc (500 mL) were added and the now homogeneous solution was washed successively with 1 N HCl (2 X 100 mL), saturated aqueous NaHCO3 (2 X 100 mL), brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was dissolved in 1: 1 DCM / MeOH (600 mL) and DCM (about 200 mL) was removed by distillation at 50 ° C [Note: a small amount of white precipitate was observed]. I know

ES 2 565 337 T3 added MeOH (200 mL) and additional solvent (approximately 200 mL) was removed at 50 ° C. The heterogeneous mixture was cooled to -5 ° C. After 16 h, the solid was collected by vacuum filtration and washed with cold MeOH. The solid was dried under high vacuum to provide 32 g of 11 as an off-white solid.¹H NMR (300 MHz, CDCI₃), mixture of rotomers: δ 11.22 (brs, 2H), 7.40 (dd, J = 5.1, 8.7 Hz, 2H), 7.31 (d, J = 9.3 Hz, 2H), 6.76 (dd, J = 8.40, 8.40, 2H) 6.26 (br s, 2H), 5.44 (m, 2H), 4.39 (dd, J = 7.5, 16.5 Hz, 2H), 4.24 (m, 2H), 4.15 (dd, J = 5.1, 12.9 Hz, 2H ), 3.79 (d, J = 12.9 Hz, 2H), 3.10-3.30 (m, 4H), 2.32 (d, J = 14.7 Hz, 2H), 2.24 (s, 6H), 1.90 (m, 2H), 1.74 (m, 2H), 1.56 (s, 18H), 0.99 (t, J = 7.5 Hz, 6H) ppm; ¹³C NMR (75 MHz, CDCI3): Ó172.2, 170.4, 161.4, 158.3, 155.8, 137.0, 136.9, 128.6, 125.5, 118.9, 118.7, 108.6, 108.4, 108.1, 98.3, 98.0, 80.8, 74.7, 60.4, 53.8 , 53.5,

34.1, 28.7, 28.6, 26.2, 21.5, 10.5 ppm. Mass Spectrum (ESI), m / z 920.5 [(M) +; calculated for C48H62F2N6O10: 921.0],

Scheme XI

<img file="ES2565337T3_D0015.tif" />

5- (3'-4-acetox¡-1- (2-am¡no-but¡r¡l) -pyrrol¡d¡n-2-¡lmet¡l1-6.6'-d¡fluoro-1H .TH-12.2Tb¡¡ndolyl-3-¡lmet¡ll-1- (2-am¡no-but¡r¡l) plrrolldln-3-ll acetic acid ester (12): A solution containing 11 (27.5 g, 30 mmol) in DCM (200 mL) was cooled to 0 ° C. TFA (50 mL) was added and the reaction mixture was monitored by LC / MS analysis until a complete conversion of 11 to 12 (approximately 3 h). The solvent was removed in vacuo and the dark green residue was dissolved in EtOAc (approximately 1 L). The EtOAc solution was carefully poured into a NaHCOs / ice / saturated aqueous water mixture to neutralize residual TFA. The organic phase was separated and washed twice with saturated aqueous NaHCO3 and then with brine. The combined aqueous washings were re-extracted with EtOAc (2 X 100 mL) and the combined organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated to provide 22 g of crude 12 as an off-white solid. ¹H NMR (300 MHz, CDCI3 + d4-MeOH), mixed rotomers: δ11.62 (br s, 2H), 7.48-7.62 (m, 4H), 6.89 (ddd, J = 2.4, 9.3, 9.3 Hz, 2H ), 5.48 (dd, J = 4.5, 4.8 Hz, 2H), 4.52 (dd, J = 9.3, 9.3 Hz, 2H), 4.06 (dd, J = 4.8, 12.3 Hz, 2H), 3.78 (d, J = 12.3 Hz, 2H), 3.54-3.70 (m, 4H), 3.30-3.40 (m, 2H), 2.33 (s, 6H), 2.02-2.16 (m, 2H), 1.70-1.96 (m, 4H), 1.09 (t, J = 7.2 Hz, 6H) ppm; ¹³C NMR (75 MHz, CDCI3 + d4-MeOH): δ173.5, 170.9, 161.8, 158.6, 137.2, 137.1, 128.2, 128.1, 125.6,

118.7, 118.6, 108.6, 108.3, 108.0, 98.6, 98.1, 74.6, 60.1, 53.5, 33.5, 28.0, 21.4, 9.7 ppm. Mass Spectrum (ESI), m / z 721.4 [(M) +; calculated for C38H46F2N6O6: 720.8],

Scheme XII

<img file="ES2565337T3_D0016.tif" />

<img file="ES2565337T3_D0017.tif" />

5- (3 '- (4-acetox¡-1-i2- (2-methyl- (tert-butox¡carbon¡l) -am¡no-prop¡on¡lam¡no) -but¡r¡ l1-p¡rrol¡d¡n-2-¡lmet¡ll-6.6'-difluoro1H.TH-í2.2'1b¡¡ndol¡l-3-¡lmet¡l) -1-í2- (2- methyl- (tert-butox¡carbon¡l) -am¡no-prop¡on¡lam¡no) -but¡r¡l1-pyrrol¡d¡n-3-¡l acetic acid ester (13) : To a solution containing Boc-N (Me) Ala-OH (14.6 g, 72 mmol) and HATU (30.4 g, 80 mmol) in anhydrous NMP (150 mL) at 0 ° C was added NMM ( 12 mL, 105 mmol) followed by the addition of 12 (30 mmol) in NMP (200 mL). The resulting mixture was allowed to warm to room temperature. After 16 h, the reaction mixture was diluted with diethyl ether (1 L) and washed successively with water (1 L), 1N HCl (2 X 100 mL), aqueous NaHCO3

ES 2 565 337 T3 saturated (2 X 100 mL), brine, dried over anhydrous Na2SO4, filtered, concentrated to provide 33.5 g of crude 13 .

The crude 13 was dissolved in EtOH (50 mL) and then slowly added to water (1000 mL) with vigorous stirring at 50 ° C resulting in the precipitation of a white solid. The heterogeneous mixture was cooled to 5 ° C. After 16 h, the solid was collected by vacuum filtration and washed with water. The wet solid was dried under high vacuum conditions at 50 ° C to provide 29.9 g of 13 as an off-white solid.¹H NMR (300 MHz, CDCI₃): O11.57 (br s, 2H), 7.40-7.60 (m, 4H), 6.89 (m, 2H), 5.50 (m, 2H), 4.75 (m, 2H), 4.67 (q, J = 6.9 Hz , 2H), 4.50 (t, J = 9.6 Hz, 2H), 4.20 (dd, J = 3.9, 12.3 Hz, 2H) 3.85 (d, J = 12.3 Hz, 2H), 3.57 (br d, J = 13.5 Hz , 2H), 3.34 (dd, J = 12.0, 13.8 Hz, 2H), 2.89 (s, 6H), 2.34 (s, 6H), 2.1 (m, 2H), 1.95 (dt, J = 6.0, 13.8 Hz, 2H), 1.79 (dt, J = 7.2, 14.1 Hz, 2H), 1.52 (s, 18H), 1.39 (d, J = 7.2 Hz, 6H), 1.03 (t, J = 7.2 Hz, 6H) ppm; ¹³C NMR (75 MHz, CDCI3): Ó174.0, 172.1, 171.9, 170.5, 161.8, 158.7, 137.5, 137.3, 128.4, 125.8, 118.7, 118.6, 108.8, 108.4, 108.1, 98.8, 98.5, 81.0, 74.6, 60.1 , 54.0, 52.0, 33.7, 30.5, 28.6, 28.1, 25.9, 21.6, 14.0, 9.9 ppm. Mass Spectrum (ESI), m / z 1091.7 [(M) +; calculated for C56H76F2N8O10: 1091.2],

Scheme XIII

<img file="ES2565337T3_D0018.tif" />

5- (3 '- (4-acetox¡-1-í2- (2-meth¡lam¡no-prop¡on¡lam¡no) -but¡r¡l1-p¡rrol¡d¡n-2- ¡Lmet¡ll-6.6'-d¡fluoro-1H, 1'H-í2.2'1b¡¡ndol¡lmet¡l) 1-í2- (2-meth¡lam¡no-prop¡on¡lam¡ no) -butıryl-pırrolıdın-3-α acetic acid ester (14): A solution containing 13 (28.5 g, 26 mmol) in DCM (150 mL) was cooled to 0 [deg.] C. TFA (50 mL) was added. After 30 min, the reaction mixture was warmed to room temperature and monitored until LC / MS analysis revealed complete conversion from 13 to 14 (about 4 h). The solvent was removed in vacuo and the dark green residue was dissolved in EtOAc (500 mL) and carefully poured into an aqueous NaHCO / ice mixture. The aqueous phase was separated and re-extracted with EtOAc (2 x 250 mL). The combined extracts were washed several times with saturated aqueous NaHCO3, then brine, dried over anhydrous Na2SO4, filtered, and concentrated to provide 24 g of 14 as a light yellow solid. ¹H NMR (300 MHz, CDCI3): Ó1.66 (brs, 2H), 8.16 (d, J = 8.4 Hz, 2H), 7.52 (dd, J = 2.1, 9.6 Hz, 2H), 7.43 (dd, J = 5.4, ​​8.4 Hz, 2H), 6.83 (ddd, J =

2.1, 9.0, 9.0 Hz, 2H), 5.41 (dd, J = 4.2, 4.5 Hz, 2H), 4.64 (dd, J = 7.8, 14.1 Hz, 2H), 4.36 (br d, J = 9.3, 9.6 Hz, 2H), 4.13 (dd, J = 4.8, 12.6 Hz, 2H), 3.81 (d, J = 12.0 Hz, 2H), 3.44 (d, J = 13.2 Hz, 2H), 3.0-3.18 (m, 4H), 2.50 (s, 6H), 2.30 (s, 6H), 2.15 (d, J = 14.4 Hz, 2H), 1.90-2.08 (m, 2H), 1.76-1.90 (m, 2H), 1.33 (d, J = 7.2 Hz, 6H), 1.08 (t, J = 7.2 Hz, 6H) ppm; ¹³C NMR (75 MHz, CDCI₃): Ó175.3, 172.6, 170.4, 161.8, 137.5, 137.3, 128.4, 128.3, 125.9, 118.6,

118.5, 108.5, 108.1, 107.8, 98.7, 98.3, 74.5, 60.9, 59.9, 53.9, 51.3, 35.8, 33.6, 27.6, 26.2, 21.5, 20.2, 10.1 ppm. Mass Spectrum (ESI), m / z 891.6 [(M) +; calculated for C46H60F2N8O8: 891.0],

Scheme XIV

<img file="ES2565337T3_D0019.tif" />

N- (1S-í2R- (6.6'-D¡fluoro-3 '- (4S-h¡drox¡-1-¡2S- (2S-meth¡lam¡no-prop¡on¡lam¡no) -but¡ r¡l1-p¡rrol¡d¡n-2R-¡lmet¡ll-1H, 1'Hí2.2'1b¡¡ndol¡l-3-¡lmet¡l) -4S-h¡drox¡-p ¡Rrol¡d¡na-1-carbon¡l1-prop¡ll-2S-met¡lamino-prop¡onam¡da (15): To a solution that

ES 2 565 337 T3 contained 14 (24 g) in MeOH (200 mL) 1 M NaOH (80 mL) was added at 0 ° C. The reaction mixture was degassed and kept under a nitrogen atmosphere wrapped with an aluminum foil. The ice bath was removed. After 60 min, the MeOH was removed in vacuo and the residue was diluted with water (200 mL) and extracted with EtOAc (500 mL). The aqueous phase was separated and re-extracted with EtOAc (2 X 150 mL). The combined organic extracts were washed with brine and dried over anhydrous Na2SO4, filtered, and concentrated to provide 22.5 g of crude as a yellow / light brown solid.

The crude (22.5 g) was dissolved in MeOH (50 mL) and EtOAc (200 mL). The volume was reduced (50%) by distillation under reduced pressure at 60 ° C using a rotary evaporator. MTBE (300 mL) was added and the cloudy solution was heated to 60 ° C. After 30 min, the solution was cooled to room temperature and then kept at -5 ° C.

After 16 h, the solid was collected by vacuum filtration and washed with cold 25% EtOAc / MTBE and dried under high vacuum at room temperature to provide 16.6 g of 15 as an off-white solid. An additional 5.5 g of 15 was recovered from the filtrate by removing the solvent and drying under vacuum.¹H NMR (300 MHz, CDCIs): Ó11.74 (s, 2H), 8.27 (d, J = 8.7 Hz, 2H), 7.71 (dd, J = 5.4, 8.4 Hz, 2H), 7.55 (dd, J = 2.4, 9.6 Hz, 2H), 6.88 (ddd, J = 2.4, 9.3, 9.3 Hz, 2H), 4.62-4.78 (m, 4H), 4.43 (dd, J = 9.3, 9.9 Hz, 2H), 4.03 (dd, J = 4.8, 11.4 Hz, 2H ), 3.80 (d, J = 11.4 Hz, 2H), 3.66 (dd, J = 2.7, 14.4 Hz, 2H), 3.53 (dd, J = 11.4, 14.4 Hz, 2H), 3.11 (q, J = 6.9 Hz , 2H), 2.56 (s, 6H), 2.45 (m, 2H), 2.19 (d, J = 14.4 Hz, 2H), 1.76-2.10 (m, 6H), 1.59 (br s, 2H), 1.39 (d , J = 6.9 Hz, 6H), 1.22-1.38 (m, 2H), 1.07 (t, J = 7.2 Hz, 6H) ppm; ¹³C NMR (75 MHz, cfe-DMSO): O175.2, 172.8, 161.6, 158.5, 137.3,

137.2, 128.4, 128.3, 126.4, 120.8, 120.6, 109.4, 108.7, 108.4, 98.4, 98.0, 70.8, 60.2, 59.9, 56.6, 51.8, 36.4, 35.3, 28.3, 25.6, 20.0, 10.6 ppm. Mass Spectrum (ESI), m / z 807.5 [(M) +; calculated for C42H56F2N8O6: 806.9],

Scheme XV

<img file="ES2565337T3_D0020.tif" />

<img file="ES2565337T3_D0021.tif" />

N-tert-butoxycarbonyl-N-yds-methylanine (17): To a solution of Boc-Ala-OH (16, 3.5 g, 18.5 mmol) in anhydrous THF (50 mL) was added NaH (2, 1 g, 60% in mineral oil, 51.0 mmol) at 0 ° C. After 45 min, the reaction mixture was warmed to room temperature and then heated to 45 ° C for an additional 20 min. The reaction mixture was cooled to 0 ° C and O3-iodomethane (10.0 g, 69.0 mmol) was added. The resulting mixture was stirred at room temperature. After 16 h, the reaction mixture was quenched with water, and extracted with EtOAc. The organic phase was discarded and the aqueous solution was acidified to pH 3 with 1N HCl and extracted with EtOAc. The organic phase was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by reverse phase HPLC (Dynamax 2 C18 column; 0.1% HOAc containing 10-100% ACN / water over 30 min; 40 mL / min) to provide 17 (3.6 g, 94 %) as a white solid after lyophilization. ¹H NMR (300 MHz, d₄-MeOH), mixed rotomers: 64.80 (brs, 1H), 4.67 (q, J = 6.9 Hz, 0.5H), 4.38 (q, J = 6.9 Hz, 0.5H), 1.36-1.52 (m, 12H) ppm ; Mass Spectrum (ESI), m / z 207.0 [(M + H) +; calculated for C9H15D3NO4: 207.2],

Scheme XVI

<img file="ES2565337T3_D0022.tif" />

ES 2 565 337 T3

5- (3 '- (4-acetox¡-1-i2- (2-cÍ3-methyl- (tert-butox¡carbon¡l) -am¡no-prop¡on¡lam¡no) -but¡ r¡l1-p¡rrol¡d¡n-2-¡lmet¡ll-6.6'-d¡fluoro1H.1'H-í2.2'1b¡¡ndol-3-¡lmet¡l) -1-í2 - (2-cÍ3-methyl- (tert-butox¡carbon¡l) -am¡no-prop¡on¡lam¡no) -but¡r¡l1-p¡rrol¡d¡n-3-¡ l Acetic acid ester (18): To a solution containing Boc-N (O3-Me) Ala-OH (17, 1.00 g, 4.83 mmol) and HATU (2.00 g, 5.30 mmol) in anhydrous NMP (20 mL) a 0 ° C NMM (0.8 mL, 7.20 mmol) was added followed by addition of 12 (crude, 1.73 g, 2.40 mmol) in NMP (20 mL). The resulting mixture was allowed to warm to room temperature. After 16 h, the reaction mixture was diluted with diethyl ether (200 mL) and washed successively with water (200 mL), 1N HCl (2 X 100 mL), saturated aqueous NaHCO3 (2 X 100 mL), brine, dried over saturated aqueous Na2SO4, filtered, and concentrated. The residue was purified by reverse phase HPLC (Dynamax 2 C18 column; 0.1% HOAc containing 10-100% ACN / water for 30 min; 40 mL / min). The product-containing fractions were combined, frozen, and lyophilized to provide 1.1 g of 18 (42%) as an off-white solid. ¹H NMR (300 MHz, CDCI₃), mixture of rotomers: Ó11.56 (br s, 2H), 7.56 (dd, J = 5.4, 8.7 Hz, 2H), 7.52 (m, 2H), 7.10 (brs, 2H), 6.89 (ddd, J = 2.1, 9.0, 9.0 Hz , 2H), 5.47 (t, J = 4.8 Hz, 2H), 4.75 (brs, 2H), 4.67 (q, J = 6.9 Hz, 2H), 4.50 (t, J = 9.3 Hz, 2H), 4.18 (dd , J = 4.2, 11.7 Hz, 2H) 3.85 (d, J = 12.6 Hz, 2H), 3.57 (dd, J = 2.1, 14.4 Hz, 2H), 3.34 (dd, J = 12.0, 14.4 Hz, 2H), 2.34 (s, 6H), 2.29 (brs, 2H), 2.10 (m, 2H), 1.97 (m, 2H), 1.79 (m, 2H), 1.51 (s, 18H), 1.39 (d, J = 6.9 Hz , 6H), 1.03 (t, J = 7.5 Hz, 6H) ppm. Mass Spectrum (ESI), m / z 1097.7 [(M) +; calculated for CseHyoDeFzNsOiz: 1097.3],

Scheme XVII

<img file="ES2565337T3_D0023.tif" />

5- (3 '- (4-acetox-1-i2- (2-d-meth¡lam¡no-prop¡on¡lam¡no) -but¡r¡l1-pyrrole¡d¡n-2-¡ lmet¡ll-6 6'-d¡fluoro-1H, 1'H-i2.2'1b¡¡ndol¡l-3methyl) -1-i2- (2-cÍ3-methylamno-prop¡on ¡Lam¡no) -but¡r¡l1-p¡rrol¡d¡n-3-¡l acetic acid ester (19): A solution containing 18 (1.10 g, 1.00 mmol) in DCM (15 mL) was cooled to 0 [deg.] C. TFA (5 mL) was added. After 30 min, the reaction mixture was warmed to room temperature and monitored until LC / MS analysis revealed a complete conversion from 18 to 19 (approximately 4 hrs). The solvent was removed in vacuo and the dark green residue was dissolved in EtOAc (100 mL) and carefully poured into an aqueous NaHCOs / ice mixture. The aqueous phase was separated and re-extracted with EtOAc (2 X 50 mL). The combined organic extracts were washed several times with saturated aqueous NaHCO3, then brine, dried over anhydrous Na2SO4, filtered, and concentrated to provide crude 19 which was used without further purification. Mass Spectrum (ESI), m / z 897.5 [(M) +; calculated for C46H54D6F2N8O8: 897.0],

Scheme XVIII

<img file="ES2565337T3_D0024.tif" />

N- (1S-í2R- (6.6'-D¡fluoro-3 '- (4S-h¡drox¡-1-¡2S- (2S-cÍ3-met¡lam¡no-prop¡on¡lam¡no) - but¡r¡l1-p¡rrol¡d¡n-2R-¡lmet¡ll-1H.1'Hí2.2'1b¡¡ndol¡l-3-¡lmet¡l) -4S-h¡drox¡ -p¡rrol¡d¡na-1-carbon¡l1-prop¡ll-2S-cÍ3-meth¡lam¡no-prop¡onam¡da (20): To a solution containing 19 crude (approximately 1.00 mmol) in MeOH (20 mL) was added 1 M NaOH (2 mL) at room temperature.After 35 min, the MeOH was removed in vacuo and the residue was diluted with water (50 mL) and extracted with EtOAc (2 X 50 mL). The combined organic extracts were washed with brine and dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by reverse phase HPLC (Dynamax 2 C18 column; 0.1% HOAc containing 10-100% ACN / water for 30 min; 40 mL / min). The fractions that contained

ES 2 565 337 T3 the product was combined, frozen, and lyophilized to provide 0.6 g of 20 (75%) as a white flocculating solid. ¹H NMR (300 MHz, CD₃CN), mixture of rotomers: δ 11.86 (s, 2H), 7.91 (d, J = 7.8 Hz, 2H), 7.71 (dd, J = 5.4, 8.7 Hz, 2H), 7.45 (dd, J = 2.4, 9.9 Hz, 2H), 6.83 (m, 2H), 4.56 (m, 2H), 4.47 (m, 2H), 4.20 (m, 2H), 3.84 (dd, J = 4.2, 11.1 Hz, 2H), 3.66 (d , J = 11.1 Hz, 2H) 3.45 (m, 4H), 2.93 (q, J = 6.9 Hz, 2H), 1.60-1.89 (m, 8H), 1.19 (d, J = 6.9 Hz, 6H), 0.94 ( t, J = 7.2 Hz, 6H) ppm; NMR (75 MHz, CD₃CN + d₄-MeOH), mixture of rotomers:

6175.2, 173.0, 162.4, 159.3, 137.8, 137.6, 128.8, 128.7, 126.8, 110.8, 120.7, 109.5, 108.7, 108.4, 98.5, 98.1, 71.6,

60.5, 60.1, 56.8, 52.6, 36.6, 28.6, 26.0, 22.7, 19.0, 10.1 ppm. Mass Spectrum (ESI), m / z 813.4 [(M) +; calculated for C42H50D6F2N8O6: 813.0],

Scheme XIX

<img file="ES2565337T3_D0025.tif" />

2- (6-Fluoro-1H-¡ndol-3-¡l-d2-methyl) -4-hydrox¡-p¡rrol¡dlna-1-carboxylic acid benzyl ester (21): A suspension of 6 (3.0 g, 7.85 mmol) in anhydrous THF (50 mL) was cooled to 0 ° C. D4-NaBH4 (0.66 g, 15.7 mmol) was added in one portion followed by the addition of BF3-etherate (1.1 mL, 8.60 mmol). After approximately 10 min, the ice bath was removed and the reaction mixture was heated to reflux.

After 3 h, the reaction mixture was cooled in an ice bath and carefully quenched with saturated aqueous NH4Cl (50 mL). The biphasic mixture was diluted with EtOAc and the organic layer was separated and washed with water (2 X 50 mL) and then with brine. The EtOAc layer was dried over Na2SO₄ anhydrous, filtered and concentrated to provide 3.2 g of crude 21 (&gt; amount) which was used without further purification. Mass Spectrum (ESI), m / z 371.2 [(M + H) +; calculated for C2iH₂oD2FN₂0₃: 371.4],

Scheme XX

<img file="ES2565337T3_D0026.tif" />

22

4-Acetox¡-2- (6-fluoro-1H-lndol-3-¡l-cy? -Metal) -pyrrol¡d¡na-1-carboxylic acid benzyl ester (22): To a solution containing crude 21 (approximately 7.85 mmol), Et3N (1.2 g, 12.0 mmol), and DMAP (50 mg, cat.) In DCM (30 mL) was added acetic anhydride (0, 74 mL, 7.85 mmol) at room temperature. After 3 h, the reaction mixture was quenched with NaHCO₃ aqueous saturated (50 mL) and then diluted with DCM. The DCM layer was separated and washed successively with dilute HCl (50 mL), water (50 mL), and brine (50 mL). The organic solution was dried over Na2SO₄ anhydrous, filtered, and connected. The crude product was purified by silica gel flash chromatography [30-40% EtOAc in hexane] to provide 2.0 g (62%, 2 steps) of 22 as a white foam.¹H NMR (300 MHz, CDCI3), ~ 1: 1 mixture of rotomers: 68.41 (br s, 1H), 7.80-6.50 (m, 9H), 5.25 (m, 1H), 5.21 (m, 2H), 4.27 ( m, 1H), 3.82 (dt, J = 5.1, 13.2 Hz, 1H), 3.61 (dd, J = 11.4, 11.7 Hz, 1H), 2.13 (s, 3H), 2.00 (m, 2H) ppm; ¹³C NMR (75 MHz, CDCh), ~ 1: 1 mix of rotomers: 6170.8 160.2 (J_CF = 236.2 Hz), 155.2, 136.9, 136.6,

136.5, 129.0, 128.9, 128.6 (J_CF = 24.4 Hz), 124.5 (J_CF = 22.1 Hz), 123.1, 120.1 (J_CF = 27.2 Hz), 119.9 (J_CF = 27.2 Hz),

112.8, 108.2 (J_CF = 23.5 Hz), 97.7 (J_CF = 25.7 Hz), 74.1, 73.3, 68.0, 67.2, 58.5, 57.6, 53.4, 53.1, 35.4, 34.6, 21.5 ppm. Mass Spectrum (ESI), m / z 413.1 [(M) +; calculated for C2₃H2iD2FN2O4: 412.4],

ES 2 565 337 T3

Scheme XXI

<img file="ES2565337T3_D0027.tif" />

<img file="ES2565337T3_D0028.tif" />

4-Acetox¡-2-3 '- (4-acetox¡-1-benz¡lox¡carbonyl-p¡rrol¡d¡n-2-¡l-cit? -Meth¡l) -6.6 '-d¡fluoro-1H.THí2,2'1b¡¡ndolyl-3-¡l-cy? -met¡l1-p¡rrol¡d¡na-1-carboxylic (231: Indole 22 ( 2.0 g, 4.80 mmol) in TFA (10 mL) precooled (-5 ° C) The resulting yellow solution was allowed to warm slowly to room temperature over 2 h. The reaction mixture was concentrated in vacuo to remove TFA and the crude mixture of indolylindoline diastereomers was used directly in the next reaction. Mass Spectrum (ESI), m / z 825.4 [(M) +; calculated for C46H42D4F2N4O8: 824.9],

To a solution containing diastereomers of indolylindoline in EtOAc (100 mL) was added DDQ (0.58 g, 2.5 mmol) in one part. After 15 min, the dark brown / orange reaction mixture was quenched with saturated aqueous NaHCO3. The layers were separated and the organic phase was washed successively with saturated aqueous NaHCO3 (3 x 50 mL) and brine, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was dissolved in DCM (10 mL) and the solution was then diluted with MeOH (50 mL). Slow removal of DCM in vacuo provided a precipitate which was collected by vacuum filtration, washed with cold MeOH, and dried to provide 1.7 g of 23 (86%, 2 steps).¹H NMR (300 MHz, CDCI₃): 511.30 (br s, 2H), 7.60-7.30 (m, 14H), 6.90 (app. Dt, J = 2.4, 9.3 Hz, 2H), 5.40 (m, 2H), 5.36 (d, J = 3.6 Hz , 4H), 4.28 (d, J = 8.1 Hz, 2H), 3.79 (m, 4H), 2.31 (s, 6H), 2.06 (m, 4H) ppm; Mass Spectrum (ESI), m / z 823.3 [(M) +; calculated for C46H40D4F2N4O8: 822.9],

Scheme XXII

<img file="ES2565337T3_D0029.tif" />

<img file="ES2565337T3_D0030.tif" />

5-í3 '- (4-acetoxy-p¡rrol¡d¡n-2-¡l-cit? -Me¡l) -6.6'-d¡fluoro-1H.TH-í2.2Tb¡¡ndol¡l -3-¡l-cy? -Me¡l1-p¡rrol¡d¡n-3-¡l ester of acetic acid (241: A suspension containing 23 (0.40 g, 0.48 mmol) in 1 : 1 EtOAc / MeOH (40 mL) was placed in a 500 mL Parr flask and charged with 10% Pd on C (wet, approximately 200 mg) .The reaction mixture was pressurized to 50 PSI H2 and stirred for 3 h. The reaction mixture was filtered through a pad of Celite® and the solids were washed with EtOAc. The cleared filtrate was concentrated in vacuo to provide crude 24 as an off-white solid that was used directly in the next reaction. Mass Spectrum (ESI), m / z 555.2 [(M) +; calculated for C30H28D4F2N4O4: 554.6],

ES 2 565 337 T3

Scheme XXIII

<img file="ES2565337T3_D0031.tif" />

<img file="ES2565337T3_D0032.tif" />

5- (3'-4-acetox¡-1- (2-tert-butox¡carbon¡lam¡no-but¡ril) -pyrrol¡d¡n-2-¡l-cy? -Met¡l1 -6.6'-d¡fluoro-1H.TH-í2.2'1b¡¡ndol¡l-3-¡l-c? D¡met¡l-1- (2-tert-butox¡carbon¡lam¡no -but¡ril) -p¡rrol¡d¡n-3-¡l acetic acid ester (251: A solution containing Boc-Abu-OH (224 mg, 1.1 mmol) and HATU (442 mg, 1, 2 mmol) in anhydrous NMP (10 mL) at 0 ° C, NMM (0.2 mL, 1.7 mmol) was added followed by a solution of 24 (0.48 mmol) in NMP (10 mL). Reaction slowly warmed to room temperature. After 16 h, the reaction mixture was diluted with diethyl ether (100 mL) and washed successively with water (5 X 50 mL), 1N HCl (50 mL), saturated aqueous NaHCO3 (50 mL), and brine, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by reverse phase HPLC (Dynamax 2 C18 column; 0.1% HOAc containing 10-100% ACN / water for 30 min; 40 mL / min). The product containing fractions were combined, concentrated, and lyophilized to provide 310 mg of 25 (70%, 2 steps) as a white flocculating solid. ¹H NMR (300 MHz, CDCI3), mixed rotomers: Ó11.17 (brs, 2H), 7.39 (dd, J = 5.4, 8.4 Hz, 2H), 7.29 (d, J = 9.3 Hz, 2H), 6.75 ( dd, J = 8.40, 8.40, 2H), 6.40 (brs, 2H), 5.44 (m, 2H), 4.40 (dd, J = 7.8, 16.5 Hz, 2H), 4.22 (d, J = 7.8 Hz, 2H) , 4.15 (dd, J = 5.1, 12.9 Hz, 2H), 3.80 (d, J = 12.9 Hz, 2H), 2.23 (s, 6H), 1.90 (m, 2H), 1.74 (m, 2H), 1.57 ( s, 18H), 0.99 (t, J = 7.2 Hz, 6H) ppm; ¹³C NMR (75 MHz, CDCI3), mixed rotomers: 6172.1, 170.4, 161.4, 158.2, 155.8, 137.0, 136.9, 128.6, 125.5, 118.9, 118.8,

108.6, 108.4, 108.1, 98.3, 98.0, 80.8, 74.7, 60.3, 53.8, 53.6, 34.1, 28.7, 28.6 (br), 26.2, 21.5, 10.5 ppm. Mass Spectrum (ESI), m / z 925.4 [(M) +; calculated for C48H58D4F2N6O10: 925.0],

Scheme XXiV

<img file="ES2565337T3_D0033.tif" />

5- (3'-4-acetox¡-1- (2-am¡no-butryl) -pyrrol¡d¡n-2-¡l-c? -Me¡l1-6.6'-d¡fluoro- 1H.1'H-i2.2'1b¡¡ndol¡l-3-¡l-c? -Met¡ll-1- (2-am¡nobutihlí-pyrrolidin-3-yl ester of acetic acid (26) : A solution containing 25 (310 mg, 0.34 mmol) in DCM (20 mL) was cooled to 0 ° C. TFA (5 mL) was added and the reaction was monitored by LC / MS analysis until complete conversion from 25 to 26 (approximately 3 h). The solvent was removed in vacuo and the dark green residue was dissolved in EtOAc (50 mL). The EtOAc solution was poured into a NaHCOs / ice / saturated aqueous water mixture to neutralize residual TFA. The organic phase was separated and washed twice with saturated aqueous NaHCO3 and then once with brine. The combined aqueous washes were re-extracted with EtOAc (2 X 20 mL) and the organic extracts were dried over Na2SO4, filtered, and concentrated to provide crude 26 (250 mg) as an off-white solid. Mass Spectrum (ESI), m / z 725.3 [(M) +; calculated for C38H42D₄F₂N₆OR₆: 724.8],

ES 2 565 337 T3

Scheme XXV

<img file="ES2565337T3_D0034.tif" />

<img file="ES2565337T3_D0035.tif" />

5- (3 '- (4-acetox¡-1-i2- (2-methyl- (tert-butox¡carbon¡l) -am¡no-prop¡on¡lam¡no) -but¡r¡ l1-p¡rrol¡d¡n-2-¡ld? -met¡ll-6.6'-d¡fl uoro1H.1'H-í2.2Jb¡¡ndol¡ld? -met¡l) -1-í2 - (2-methyl- (tert-butox¡carbon¡l) -am¡no-prop¡on¡lam¡no) -but¡r¡l1-p¡rrol¡d¡n-3-¡laster of Acetic acid (27): To a solution containing Boc-N (Me) Ala-OH (83 mg, 0.41 mmol) and HATU (172 mg, 0.45 mmol) in anhydrous NMP (5 mL) at 0 ° C NMM (0.1 mL, 0.85 mmol) was added followed by the addition of crude 26 (123 mg, 0.17 mmol) in NMP (5 mL). The resulting mixture was allowed to warm to room temperature. After 16 h, the reaction mixture was diluted with diethyl ether (100 mL) and washed successively with water (50 mL), 1N HCl (2 X 50 mL), saturated aqueous NaHCC&gt; 3 (2 X 50 mL), and brine, dried over saturated aqueous Na2SC&gt; 4, filtered, and concentrated. The crude product was purified by reverse phase HPLC (Dynamax 2 C18 column; 0.1% HOAc containing 10-100% ACN / water for 30 min; 40 mL / min). The fractions containing the product were combined, concentrated, and lyophilized to provide 170 mg of 27 (91%, 2 steps) as an off-white flocculating solid. ¹H NMR (300 MHz, CDCI3), mixed rotomers: 611.51 (br s, 2H), 7.40-7.60 (m, 4H), 6.86 (m, 2H), 5.46 (m, 2H), 4.74 (br s, 2H ), 4.65 (q, J = 6.9 Hz, 2H), 4.45 (d, J = 8.7 Hz, 2H), 4.17 (dd, J = 4.8, 12.3 Hz, 2H) 3.82 (d, J = 12.3 Hz, 2H) , 2.87 (s, 6H), 2.28 (s, 6H), 2.05 (m, 2H), 1.92 (m, 2H), 1.78 (m, 2H), 1.48 (s, 18H), 1.37 (d, J = 7.2 Hz, 6H), 1.01 (t, J = 7.2 Hz, 6H) ppm; ¹³C NMR (75 MHz, CDCI3), mixed rotomers: Ó173.3,

170.2, 170.1, 170.5, 168.6, 159.9, 135.5, 135.4, 126.5, 126.4, 123.8, 116.8, 116.7, 106.8, 106.4, 106.1, 96.8, 96.5, 79.1, 72.6, 57.9, 52.1, 50.1, 31.7, 28.5, 26.6, 25.5 (br), 23.9, 19.6, 19.0, 12.1, 8.0 ppm. Mass Spectrum (ESI), m / z 1095.5 [(M) +; calculated for C56H72D4F2N8O12: 1095.3],

Scheme XXVI

<img file="ES2565337T3_D0036.tif" />

5- (3 '- (4-acetox¡-1- [2- (2-meth¡lam¡no-prop¡on¡lam¡no) -but¡r¡l1-p¡rrol¡d¡n-2 -¡L-d2-met¡ll-6.6'-d¡fluoro-1H.1'H- [2.2'1b¡¡ndol¡l-3-¡ld2-methyl) -1- [2- (2 -meth¡lam¡no-prop¡on¡lam¡no) -but¡r¡l1-p¡rrol¡d¡n-3-¡l acetic acid ester (28): A solution containing 27 (170 mg 0.15 mmol) in DCM (15 mL) was cooled to 0 [deg.] C. TFA (5 mL) was added. After 30 min, the reaction mixture was allowed to warm to room temperature and monitored until LC / MS analysis revealed complete conversion from 27 to 28 (about 4 h). The solvent was removed in vacuo and the dark green residue was dissolved in EtOAc (100 mL) and carefully poured into an aqueous NaHCOs / ice mixture. The aqueous phase was separated and re-extracted with EtOAc (2 X 20 mL). The combined organic extracts were washed several times with saturated aqueous NaHCO3, then brine, dried over anhydrous Na2SO4, filtered, and concentrated to provide crude 28 as a light yellow solid. Mass Spectrum (ESI), m / z 895.3 [(M) +; calculated for C46H56D4F2N8O8: 895.0],

ES 2 565 337 T3

Scheme XXVII

<img file="ES2565337T3_D0037.tif" />

N- {1S- [2 / R- (6,6'-D¡fluoro-3 '- {4S-h¡drox¡-1- [2S- (2S-meth¡lam¡no-prop¡on¡lam ¡No) -but¡r¡l] -p¡rrol¡d¡n-2R-¡l-d2-methyl} -1H, 1'H [2,2 '] b¡¡ndolil-3-¡ l-d2-methyl) -4S-hydrox¡-p¡rrol¡d¡na-1-carbonyl] -prop¡l} -2S-meth¡laminoprop¡onam¡da (29): To a solution containing crude 28 (0.15 mmol) in MeOH (20 mL) 1 M NaOH (5 mL) was added at 0 ° C. The reaction mixture was degassed and kept under a nitrogen atmosphere wrapped in aluminum foil. The ice bath was removed. After 60 min, the MeOH was removed in vacuo and the residue was diluted with water (20 mL) and extracted with EtOAc (50 mL). The aqueous phase was separated and re-extracted with EtOAc (2 x 50 mL). The combined organic extracts were washed with brine and dried over Na2SO4, filtered, and concentrated. The crude product was purified by reverse phase HPLC (Dynamax 2 C18; 0.1% HOAc containing 10-100% ACN / water for 30 min; 40 mL / min). The fractions containing the product were combined, concentrated, and lyophilized to provide 110 mg of 29 (90%, 2 steps) as a white flocculating solid. ¹H NMR (300 MHz, CDCIs + c / 4-MeOH), mixed rotomers: 611.58 (s, 2H), 7.80 (dd, J = 5.4, 8.7 Hz, 2H), 7.45 (dd, J = 2.4, 9.9 Hz , 2H), 6.87 (ddd, J = 2.4, 9.2, 9.2 Hz, 2H), 4.66 (dd, J = 5.7, 7.8 Hz, 2H), 4.60 (br s, 2H), 4.47 (d, J = 7.2 Hz , 2H), 4.00 (dd, J = 4.8, 11.4 Hz, 2H), 3.76 (d, J = 11.4 Hz, 2H), 3.43 (q, J = 6.9 Hz, 2H), 2.55 (s, 6H), 2.19 (d, J = 14.4 Hz, 2H), 1.78-2.02 (m, 8H), 1.46 (d, J = 7.2 Hz, 6H), 1.09 (t, J = 7.2 Hz, 6H) ppm; ¹³C NMR (75 MHz, CDCI3 + d4-MeOH), mixed rotomers: O173.6, 171.8, 161.7, 158.6, 137.1, 136.9, 128.1, 128.0, 125.9, 119.8, 119.7, 108.3, 108.2,

107.8, 97.8, 97.5, 70.9, 69.4, 59.0, 56.1, 52.0, 36.3, 35.7, 25.5, 18.5, 9.8 ppm. Mass Spectrum (ESI), m / z 811.4 [(M) +; calculated for C42H52D4F2N8O6: 810.9],

Scheme XXVIII

<img file="ES2565337T3_D0038.tif" />

OAC OAc

5- (3 '- {4-acetox¡-1- [2- (2-d3-methyl- (tert-butoxycarbon¡l) -am¡no-prop¡on¡lam¡no) -but ¡L] -pyrrol¡d¡n-2-¡l-d2-methyl} -6,6'-d¡fluoro1H, 1'H- [2,2 '] b¡¡ndol¡l- 3-¡l-d2-methyl) -1- [2- (2-d3-methyl- (tert-butoxycarbon¡l) -am¡no-prop¡on¡lam¡no) -but ¡R¡l] -p¡rrol¡d¡n-3-¡l ester of acetic acid (30): A solution containing Boc-N (d3-Me) Ala-OH (17.83 mg, 0.41 mmol) and HATU (172 mg, 0.45 mmol) in anhydrous NMP (5 mL) at 0 ° C was NMM (0.1 mL, 0.85 mmol) was added followed by the addition of crude 26 (123 mg, 0.17 mmol) in NMP (5 mL). The resulting mixture was allowed to warm to room temperature. After 16 h, the reaction mixture was diluted with diethyl ether (100 mL) and washed successively with water (50 mL), 1N HCl (2 X 50 mL), saturated aqueous NaHCO3 (2 X 50 mL), and brine. , dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by reverse phase HPLC (Dynamax 2 C18 column; 0.1% HOAc containing 10-100% ACN / water for 30 min; 40 mL / min). The fractions containing the product were combined, concentrated and lyophilized to provide 160 mg of 30 (85%, 2 steps) as a white flocculating solid. ¹H NMR (300 MHz, CDCI3), rotomer mixture: 611.51 (br s, 2H), 7.40-7.60 (m, 4H), 6.87 (ddd, J = 2.1, 9.0, 9.0 Hz, 2H), 5.47 (t, J = 4.8 Hz, 2H), 4.74 (brs, 2H), 4.65 (q, J = 7.2 Hz, 2H), 4.46 (d, J = 8.1 Hz, 2H), 4.18 (dd, J = 3.9, 11.7 Hz, 2H) 3.83 (d, J = 12.3 Hz, 2H), 2.30 (s, 6H), 2.24 (m, 2H), 2.05 (m, 2H), 1.93 (m, 2H), 1.79 (m, 2H), 1.49 (s, 18H), 1.38 (d, J = 6.9 Hz, 6H), 1.02 (t, J = 7.2 Hz, 6H) ppm; ¹³C NMR (75 MHz, CDCI3), mixed rotomers: O175.6, 172.2, 172.1, 170.6, 161.8, 158.7, 137.5, 137.3, 128.5, 128.4, 125.8, 118.7, 118.6,

108.7, 108.4, 108.0, 98.8, 98.4, 81.1, 74.6, 66.1, 59.9, 54.0, 52.1, 33.7, 28.6, 27.5 (br), 25.8, 21.6, 20.9, 14.0, 9.9 ppm; Mass Spectrum (ESI), m / z 1101.5 [(M) +; calculated for C56H66D10F2N8O12: 1101.3],

ES 2 565 337 T3

Scheme XXIX

<img file="ES2565337T3_D0039.tif" />

OAc

5- (3 '- (4-acetox¡-1-i2- (2-cÍ3-meth¡lam¡no-prop¡on¡lam¡no) -but¡r¡l1-p¡rrol¡d¡n- 2-¡l-cl? -Met¡ll-6.6'-d¡fluoro-1H.TH-Í2.2'1b¡ndol¡l3-¡ld? -Met¡l) -1-¡2- (2- d3-meth¡lam¡no-prop¡on¡lam¡no) -but¡r¡l1-p¡rrol¡d¡n-3-¡l acetic acid ester (31): A solution containing 30 (160 g , 0.14 mmol) in DCM (15 mL) was cooled to 0 [deg.] C. TFA (5 mL) was added. After 30 min, the reaction mixture was warmed to room temperature and monitored until LC / MS analysis revealed a complete conversion of 30 to 31 (approximately 4 h). The solvent was removed in vacuo and the dark green residue was dissolved in EtOAc (100 mL) and carefully poured into an aqueous NaHCOs / ice mixture. The aqueous phase was separated and re-extracted with EtOAc (2 X 50 mL). The combined organic extracts were washed several times with saturated aqueous NaHCO3, then brine, dried over anhydrous Na ?SO4, filtered, and concentrated to provide crude 31 as a yellow solid. Mass Spectrum (ESI), m / z 901.5 [(M) +; calculated for C46H50D10F2N8O8: 901.1],

Scheme XXX

<img file="ES2565337T3_D0040.tif" />

<img file="ES2565337T3_D0041.tif" />

N-í1S-í2R- (6.6'-D¡fluoro-3'-í4S-h¡drox¡-1-í2S- (2S-met¡lam¡no-prop¡on¡lam¡no) -but¡r¡ l1-p¡rrol¡d¡n-2R-¡l-cl? -met¡ll-1H.THí2.2'1bi¡ndol¡l-3-¡l-cl? -met¡l) -4S-h ¡Drox¡-p¡rrol¡d¡na-1-carbon¡l1-prop¡ll-2S-met¡la¡n¡noprop¡onam¡da (32):

To a solution containing crude 31 (0.14 mmol) in MeOH (20 mL) was added 1 M NaOH (5 mL) at 0 ° C. The reaction mixture was degassed and kept under a nitrogen atmosphere wrapped in aluminum foil. The ice bath was removed. After 60 min, the MeOH was removed in vacuo and the residue was diluted with water (20 mL) and extracted with EtOAc (50 mL). The aqueous phase was separated and re-extracted with EtOAc (2 x 50 mL). The combined organic extracts were washed with brine and dried over Na2SO4, filtered, and concentrated. The crude product was purified by reverse phase HPLC (Dynamax 2 C18; 0.1% HOAc containing 10-100% ACN / water for 30 min; 40 mL / min). The fractions containing the product were combined, concentrated, and lyophilized to provide 100 mg of 32 (87%, 2 steps) as a white flocculating solid.¹H NMR (300 MHz, CDCI3 + d4-MeOH), mixed rotomers: δ 11.62 (s, 2H), 7.79 (dd, J = 5.4, 8.4 Hz, 2H), 7.47 (dd, J = 2.4, 10.2 Hz, 2H), 6.87 (ddd, J = 2.4, 9.2, 9.2 Hz, 2H), 4.68 (dd, J = 5.4, 7.5 Hz, 2H), 4.58 (m, 2H), 4.45 (d, J = 6.6 Hz, 2H ), 3.99 (dd, J = 4.8, 11.1 Hz, 2H), 3.75 (d, J = 11.1 Hz, 2H), 3.19 (q, J = 6.9 Hz, 2H), 2.15 (brd, J = 12 Hz, 2H ), 1.78-2.02 (m, 8H), 1.39 (d, J = 6.6 Hz, 6H), 1.07 (t, J = 7.5 Hz, 6H) ppm; ¹³C NMR (75 MHz, CDCI3 + d4-MeOH), mixed rotomers: O175.4, 172.0, 161.8, 158.7, 137.1, 137.0, 128.2, 128.0, 126.0, 119.9, 119.7, 108.4, 108.3,

107.9, 98.0, 97.6, 71.0, 60.0, 59.6, 56.2, 51.6, 36.4, 25.8, 19.5, 9.8 ppm; Mass Spectrum (ESI), m / z 817.4 [(M) +; calculated for C42H46D10F2N8O6: 817.0],

Examples 2, 3, 4, and 5

Compounds tested in Examples 2, 3, 4, and 5 are shown in Table 1.

ES 2 565 337 T3

Table 1

<td colspan="3">OH R_or OR^{/ N} Λ '~ &lt;\ / 7 R5^R Oh</td>

<td>Compound</td><td>R5</td><td>R</td>

<td> 15</td><td>-CH2CH3</td><td>6-F</td>

<td> 2</td><td>-CH (CH3) CH3</td><td>6-F</td>

<td> 3</td><td>-R-CH (OH) CH3</td><td>6-F</td>

<td> 4</td><td>-S-CH (OH) CH3</td><td>6-F</td>

<td> 5</td><td>-R-CH (OCH3) CH3</td><td>6-F</td>

Example 2A. ClAP degradation assay

The concentration that induces the degradation of clAP-1 and clAP-2 by 50% (IC50) was determined for various compounds by monitoring the disappearance of the Green Fluorescent Protein (GFP) signal in A375 cells. Briefly, A375 cell lines expressing GFP-labeled clAP-1 and clAP-2 were generated by transfection of a HA2xEGFP-pcDNA3 vector containing a coding region for clAP-1 (A375Gc1) or clAP-2 (A375Gc2). . 2x10⁴ of A375Gc1 or A375Gc2 cells were grown in 96-well plates and treated with various concentrations of the test compounds for 2 h. After incubation, cells were harvested by trypsinization and suspended in 150 µl of DMEM-10% FBS. A total of 10⁴ cells were analyzed using a FACScan (Becton Dickinson). Fluorescence was monitored with GFP using a 488 nm filter and emission was measured with a 530 nm filter. IC50 was defined as the drug concentration at which 50% of the GFP signal was inhibited.

The results of the clAP-1 and -2 degradation assay are shown in Table 2.

Table 2

<td>Compound</td><td>GFP-clAP-1 IC_5n (nM)</td><td>SD</td><td>GFP-clAP-2 IC_5n (nM)</td><td>SD</td><td>ClAP-2 / clAP-1 ICsn ratio</td>

<td> 15</td><td>27 (n = 56)</td><td> 15</td><td>174 (n = 61)</td><td> 100</td><td> 6,4</td>

<td> 2</td><td>4 (n = 3)</td><td> 0,6</td><td>7 (n = 3)</td><td> 0,8</td><td> 1,8</td>

<td> 3</td><td>328 (n = 3)</td><td> 83</td><td>674 (n = 3)</td><td> 69</td><td> 2,1</td>

<td> 4</td><td>464 (n = 12)</td><td> 112</td><td>604 (n = 12)</td><td> 192</td><td> 1,3</td>

<td> 5</td><td>10 (n = 41)</td><td> 2</td><td>37 (n = 38)</td><td> 19</td><td> 3,7</td>

These data show that Compound 15 has greater relative potency in degrading clAP-1 relative to clAP-2 compared to Compounds 2, 3, 4, and 5.

Example 2B. Caspase-3 derepression assay

MDA-MB-231 tumor cells were seeded with exponential growth by trypsinization, and harvested by centrifugation in a benchtop centrifuge at 1000xg for 10 minutes at room temperature. The cell pellet was washed once by resuspending in a 5 mL hypotonic lysis buffer.

ES 2 565 337 T3 (20 mM HEPES, pH7.5, 10 mM KCI, 1.5 mM MgCU, 1.0 mM EDTA, 1.0 mM DTT) and was collected by centrifugation. The pellet was then resuspended in 1 volume of hypotonic lysis buffer supplemented with a complete protease inhibitor tablet (Roche) and allowed to swell on ice for 30 minutes. The cells were disrupted by approximately 50 passages through a 27 gauge needle. The lysis was monitored by light microscopy. The lysate was centrifuged at 12000xg for 10 minutes at 4 ° C to remove the membrane fraction, non-lysed cells and debris. The soluble fraction was collected to determine protein concentration and for further analysis. The hypotonic lysate (25 pg protein), 50 pg / mL cytochrome c and 10 mM dATP were combined in a microcentrifuge tube to a final volume of 9 ml in a hypotonic lysis buffer by addition of the test compound and incubated for 30 minutes at room temperature. After incubation, a 50 μl hypotonic lysis buffer containing a caspase-3 substrate zDEVD-R110 (2) based on 5 μΜ of rhodamine-110 (2) pro-fluorescent was added, and the intensity was monitored. fluorescence over time. Activation of the lysate by the addition of cytochrome c and dATP results in apoptosome formation and subsequent activation of caspases-9 and -3. Endogenous XIAP inhibits much of this activity, and addition of test compound to activated lysate results in more caspase activity than is generated by activated lysate alone as measured by increasing fluorescence intensity after cleavage. of zDEVDR110 (2) by caspase-3. IC50 values ​​were calculated using GraphPad Prism representing the increase in fluorescence intensity vs. different concentrations of the compounds tested and the results are shown in Table 3.

Table 3

<td>Compound</td><td>ICsode derepression (nm) of XIAP Caspase-3</td><td>SD</td>

<td> 15</td><td>24.3 (n = 3)</td><td> 2,0</td>

<td> 2</td><td>13.4 (n = 3)</td><td> 4,3</td>

<td> 3</td><td>0.2 (n = 3)</td><td> 0</td>

<td> 4</td><td>0.35 (n = 3)</td><td> 0,2</td>

<td> 5</td><td>0.36 (n = 3)</td><td> 0,05</td>

These data show that Compound 15 has a lower potency to antagonize XIAP function compared to Compounds 2, 3, 4, and 5.

Example 3- Cytotoxicity

Cytotoxicity data on SKOV-3 ovarian tumor cells was generated substantially as follows. The MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay is an example of an assay that has been used to measure cell growth as described above (Hansen , *. B., Nielsen, SE, and Berg, K. (1989) J. Immunol. Methods 119, 203-210) and have been incorporated herein by reference in their entirety. Briefly, SK-OV-3 cells were seeded in 96-well plates in McCoy's medium containing 10% fetal bovine serum albumin (5,000 per well) and incubated overnight at 37 ° C. The next day, test compounds were added in various concentrations (0.003-10 mM) and the plates were incubated at 37 ° C for an additional 72 hrs. This incubation time was optimal to measure the inhibitory effects of different analogs. Fifty microliters of a 5mg / mL MTT reagent were added to each well and the plates were incubated at 37 ° C for 3 hours. At the end of the incubation period, 50 microliters of DMSO was added to each well to dissolve the cells and the optical density of Iso wells (OD) was measured using a dfe microplate reader (Victor2 1420, Wallac, Finland ) at 535 nm. Cell survival (CS) was calculated using the following equation:

CS = (OD treated well / mean OD control wells) X100%

The CC50, defined as the drug concentration resulting in 50% CS, was obtained by calculating the point where the dose response curve crosses the 50% CS point using GraphPad Prism. These results suggest that clAP-1 binding Smac mimetics can be used in cancer treatment either as monotherapy or in combination with chemotherapeutics. The results of the SKOV-3 cytotoxicity assays for the compounds tested in this assay are shown in Table 4.

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Table 4

<td>Compound</td><td>CCspnM</td><td>Std Dev (nM)</td>

<td> 15</td><td>0.14 (n = 76)</td><td> 0,02</td>

<td> 2</td><td>0.5 (n = 6)</td><td> 0,1</td>

<td> 3</td><td>13 (n = 4)</td><td> 4</td>

<td> 4</td><td>2 (n = 23)</td><td> 0,3</td>

<td> 5</td><td>0.13 (n = 76)</td><td> 0,1</td>

These data indicate that Compound 15 has potency equivalent to Compound 5 and is more potent than Compounds 2, 3, and 4.

Example 4 - Tonicity

Additional body weight loss (BWL), mortality and toxicity data were generated substantially as follows. Sprague-Dawley rats were dosed daily (QDx4, IV bolus slowly) with Compounds 15, 4 and 5. Body weights were taken on day 4 and shown as a percentage change from day 1. Compounds 4 and 5 were administered at a rate of 0.3 mg / Kg, 1 mg / Kg, or 3 mg / Kg; Compound 15 was administered at a rate of 1, 5, or 10 mg / Kg.

The results of the BWL assay are shown in Fig 1.

Mortality. Compounds 4 and 5 were not tolerated at doses of 3 mg / Kg, and caused the death of the animals at these doses. No mortality was observed with Compound 15 at a rate of 5 mg / Kg (mortality was observed at doses of 10 mg / Kg.)

Clinical results. No clinical signs were observed at doses of 1 mg / kg / day with Compound 15 after 4 days of administration. Animals treated with Compound 15 at a dose of 5 mg / kg / day showed similar clinical signs to Compounds 4 and 5 at a dose of 1 mg / kg / day such as lethargy, increased / irregularity of the respiratory rhythm and an increase in the rhythm. cardiac. Rats treated with 1 mg / kg of Compound 5 showed additional clinical observations including dehydration, poor appearance, cromorrinorrhea, alopecia (head), and excessive itching from days 2 to 4.

Body weight. In doses of 1 mg / kg, the animals that received Compounds 4 and 5 suffered weight loss while the animals that received Compound 15 in doses of 1 mg / kg / day gained weight. At doses of 5 mg / kg / day with Compound 15, a treatment-related mean weight loss of approximately 8% was observed from day 1 to day 4. Treatment-related mean body weight loss of approximately 4% and 6% was observed in animals treated with Compounds 4 and 5, respectively, at doses of 1 mg / kg / day. Pathology. Evaluation of anatomical pathology after treatment with Compounds 4 and 5 at doses of 1 mg / kg / day resulted in the following observations. Marked to severe bone marrow hypocellularity occurred in the erythroid series, mild to moderate hypercellularity in the myeloid series, and mild to moderate megakaryocyte hypertrophy and hyperplasia in the tibia and sternum when Compounds 4 and 5 were administered in doses of 1 mg / kg / day. For Compounds 4 and 5 the lungs exhibited mild to moderate dose-related diffuse pneumocyte hypertrophy / hyperplasia of type 2 due to increased alveolar macrophages, hypertrophied bronchiolar epithelium, proliferation of perivascular mononuclear cells, and hypertrophied visceral pleural cells. In contrast, evaluation of anatomical pathology following treatment with Compound 15 at the same dose (1 mg / kg / day) identified minimal to mild hypocellularity of erythroid cells, minimal to mild hypocellularity of myeloid cells, and minimal pneumocytic hypertrophy. type 2 in the lungs.

The data described above indicates that Compound 15 is approximately 5 times better tolerated in rats compared to Compounds 4 and 5 based on each dose.

Example 5 - Reduction in tumor volume and change in body weight

MDA-MB-231 xenograft data was generated substantially as follows. MDA-MB-231 human breast tumor cells were injected into the mammary fat pad of female nude mice and dosing was started twelve days later at a tumor volume of approximately 148 mm³. No tumor burden was associated with this model based on lack of weight loss or animal morbidity in the

ES 2 565 337 T3 control groups. 1x10 were injected⁷ cells subcutaneously in mice in the adipose mammary panicle, with cells suspended in 200pl of 1: 1 HBSS: solution with Matrigel plug; the injected cells were within nine passages of the original group. A previous study of tumor volumes was recorded beginning approximately one week before the estimated start date. When the tumors reached approximately 150 mm³ animals are grouped by tumor volume into treatment and control groups and dosing is started (Day 0); mice are individually tagged and tracked throughout the experiment. The animals were dosed by weight (0.01 mL per gram; 10 ml / Kg).

Starting on Day 0, the animals were observed daily and weighed twice a week using a digital scale (Ohaus SP601); data including individual weights and mean of weights in grams (Mean Pe ± SD), mean percentage of weight change versus Day 0 (% vDü) and mean percentage of weight change versus previous measurements (% vD-x) they were recorded for each group and represented at the end of the study. Starting on Day 0, tumor dimensions were measured twice a week by digital caliper (Fowler Ultra-Cal IV) and data including estimated mean and individual tumor volumes (Mean VT ± SEM) were recorded for each group. ; tumor volume was calculated using the formula: VT = width² x length x 0.52. Individual mice achieving the designated endpoint of the study (an estimated tumor volume of approximately 1 cm) were assigned³) a value of time until the end of study effect (TTE) corresponding to that day; the tumor growth delay (TGD) study was concluded once all mice reached the final effect or sixty days after the start of the study. Upon completion of the study, the TGD and% TGD were calculated using the mean TTE value (MTTE) for each treatment group versus control (C) using the formulas: TGD (days) = TC and% TGD = TC / C x100, in where TC is the difference between the MTTE- treatment group and the MTTE-Control group. Animals with tumors that do not reach the designated final effect volume at the end of the study are considered long-term survivors (LTS) and a TTE value corresponding to the final study day was assigned; tumor-free animals are not included in TGD calculations. A log-rank test is used to statistically determine differences in overall survival between each treated group compared to controls. Individual mice exhibiting a tumor volume of &lt;50% at the Day 0 measurement, for two consecutive measurements over a seven-day period, were considered to be partially responsive (PR). If the PR persists until the end of the study, the percentage of tumor regression (% TR) was determined using the formula:% TR = 1-Tf / Ti x100; A mean value was calculated if multiple PR mice appeared in one group. Individual mice were classified as lacking palpable tumors (&lt;4x4 mm² for two measurements over a seven-day period) with a complete response criterion (CR); a CR that persisted until study completion was considered a tumor-free survivor (TFS); TFS animals are excluded from TGD calculations and statistical analysis. MTTE values ​​for statistical differences between treatment and control groups are compared using a log-rank test.

Compound 15 was administered by ip injection alone in doses of 20, 40 or 60 mg / Kg on a q3dx5 schedule (every three days for 5 cycles). The 22-day TC values ​​were calculated for these groups, where all of them were statistically significant compared to the control (p = 0.005, p &lt;0.0001, op = 0.0001). In the 20 mg / kg group, 6/10 mice were considered long-term survivors and partial tumor regression was indicated in three mice. In the 40 mg / kg group, 9/10 mice were considered long-term survivors and partial tumor regression was indicated in three mice. In the 60 mg / kg group, 8/10 mice were considered long-term survivors and partial tumor regression was indicated in seven mice.

Compound 5 was administered by ip injection alone at a dose of 15 mg / Kg under a q3dx5 scheme. A 21-day TC value was calculated for this group which was found to be statistically significant compared to the control (p = 0.002). In this group 3/10 mice were considered long term survivors and partial tumor regression was indicated in five mice. The efficacy of this dose level produced half the number of long-term survivors according to a dose of 20 mg / Kg of Compound 15. The results of the MDA-MB-231 xenograft assay are shown in Figures 2A and 2B. Compound 15 in doses of 20 mg / Kg presented an anti-tumor activity comparable to that of Compound 5 in doses of 15 mg / Kg. Subsequent studies have shown that the minimum effective dose of Compound 15 in this model is less than 1 mg / Kg. Weight loss was greater in mice administered Compound 5 at a dose of 15 mg / Kg compared to mice dosed with Compound 15 at a dose of 20 mg / Kg. Thus, Compound 15 has comparable efficacy with less toxicity relative to Compound 5 and therefore exhibits an improvement in therapeutic index.

The compound of Formula 1 is particularly well tolerated and quite suitable for use in a pharmaceutical composition, as well as in a method for the treatment of a proliferative disorder or an autoimmune disorder. In particular, the pharmaceutical composition of the invention for the treatment of a proliferative disorder, which comprises an effective amount of Compound 15 in addition to a pharmaceutically acceptable excipient, can improve the therapeutic index by reducing toxicities. Reduced toxicities include, for example, one of, or any combination of one or more of:

• reduction in body weight loss, • reduction in the incidence of mortality,

ES 2 565 337 T3 • reduction of hypocellularity of the bone marrow erythroid series, • reduction of hypercellularity of the myloid series, • reduction of megakaryocyte hypertrophy and hyperplasia, • reduction of diffuse hypertrophy / hyperplasia of pneumocytes of Type 2 • decreased lethargy, • more regular breathing, • less increased heart rate

The reduction of toxicities detailed above are those observed in the animals tested.

A similar, additional or different reduction in toxicities will be observed in humans. The reductions are relative, for example, in relation to the degree to which toxicities would be observed after internal administration of a pharmaceutical composition in which the active pharmaceutical ingredient is an analog of Compound 15, for example, one or more of the analogs. where R5 is -CH2CH3, -CH (CH3) CH3, -R-CH (OH) CH3, -S-CH (OH) CH3, and R-CH (OCH3) CH3, for example, at the same dose or a comparable potency dose.