Piperidinone derivatives as mdm2 inhibitors for the treatment of cancer

The present invention relates to a pharmaceutical combination comprising compound 2 ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (S) -1- (isopropylsulfonyl) ) -3-methylbutan-2-yl) -3-methyl-2-oxopiperidin-3-yl) acetic acid or a pharmaceutically acceptable salt, ester or amide thereof as defined in claim 1, together with a pharmaceutically acceptable excipient, diluent or carrier , which pharmaceutical combination is a solid dosage form, the combination is suitable for parenteral injection, or is a liquid dosage form for oral administration. The compound is an MDM2 inhibitor that is useful as a therapeutic agent, especially for the treatment of cancer. The present invention further relates to compound 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1- (isopropylsulfonyl) -3-methylbutane -2-yl) -3-methyl-2-oxopiperidin-3-yl) acetic acid or a pharmaceutically acceptable salt, ester or amide thereof for use in a method of treating various types of cancer, as defined in any one of claims 5 to 13. The present invention further relates to a medicament as defined in claims 14 to 16, as well as to esters, amides and prodrugs of the compounds as defined in claim 17, and to a pharmaceutical combination comprising an ester, amide or prodrug, such as as defined in claim 18.

BACKGROUND OF THE INVENTION p53 is a tumor suppressor and transcription factor that responds to cellular stress by activating the transcription of a number of genes involved in cell cycle arrest, apoptosis, senescence, and repair of DNA damage. In contrast to normal cells, which do not often need to activate p53, tumor cells are under constant cellular stress from a variety of sources, including hypoxia and activation of proapoptotic oncogenes. Therefore, there is a strong selective advantage of p53 pathway activation in tumors, and it has been suggested that elimination of p53 function could be a prerequisite for tumor survival. In support of this idea, three groups of researchers applied models to mice to show that the absence of p53 function is a constant condition for the maintenance of already established tumors. When the researchers restored p53 function to tumors in which p53 was inactivated, the tumors regressed.

P53 is deactivated by mutation and / or loss in 50% of solid tumors and 10% of liquid tumors. Other key parts of the p53 pathway are also genetically or epigenetically altered in the presence of cancer. The MDM2 oncoprotein inhibits p53 function and is activated by gene amplification with incidence rates for which

58366 Β1 reported to go up to 10%. MDM2, on the other hand, inhibits another tumor suppressor, pl4ARF. It has been hypothesized that changes downstream of p53 may be responsible for at least partial inactivation of p53 times in p53^m tumors (wild-type p53). In support of this concept, it appears that some p53^m tumors show decreased capacity for apoptosis, although their capacity to stop the cell cycle remains intact. One strategy for treating cancer involves the use of small molecules that bind MDM2 and neutralize its interaction with p53. MDM2 inhibits p53 activity by three mechanisms: 1) acting as EZ ubiquitin ligase in promoting p53 degradation; 2) binding to and blocking the p53 transcription-activation domain; and 3) transferring p53 from the nucleus to the cytiplasm. All three of these mechanisms would be blocked by neutralizing the MDM2-p53 interaction. More specifically, this therapeutic strategy could be applied to tumors that are p53^w, and studies with low molecular weight MDM2 inhibitors have yielded promising results in terms of reducing tumor growth in both vitro and in vivo. Furthermore, in patients with p53-deactivated tumors, stabilization of wild-type p53 in normal tissues by inhibition of MDM2 could allow selective protection of normal tissues from mitotic toxins.

The present invention relates to compounds capable of inhibiting the interaction between p53 and MDM2 and activating p53 downstream effector genes. As such, the compounds of the present invention would be useful in the treatment of cancer, bacterial infections, viral infections, ulcers and inflammation. More specifically, the compounds of the present invention are useful for the treatment of solid tumors such as: tumors of the breast, colon, lung and prostate; and liquid tumors such as lymphomas and leukemias. When used in the present application, MDM2 denotes the human protein MDM2, and p53 denotes the human protein p53. It should be noted that human MDM2 may also be referred to as HDM2 or hMDM2.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a pharmaceutical combination comprising compound 2 ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1) - (isopropylsulfonyl) -3-methylbutan-2-yl) -3-methyl-2-oxopiperidin-3-yl) acetic acid or a pharmaceutically acceptable salt, ester or amide thereof, as defined in claim 1, together with a pharmaceutically acceptable excipient , a diluent or carrier, which pharmaceutical combination is a solid dosage form, the combination is suitable for parenteral injection, or is a liquid dosage form for oral administration. The pharmaceutical combination of the present invention is defined in claim 1, and specific embodiments of the solid dosage form are defined in claims 2 to 4.

The present invention further relates to compound 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 (S) -1- (isopropylsulfonyl) -3 -methylbutan-2-yl) -3-methyl-2-oxopiperidin-3-yl) acetic acid or a pharmaceutically acceptable salt, ester or amide thereof for use in a method of treating various types of cancer, such as

58366 Β1 as defined in any one of claims 5 to 13. The present invention further relates to a medicament as defined in any one of claims 14 to 16, as well as to esters, amides and prodrugs of the compounds as defined in claim 17, and a pharmaceutical combination comprising an ester, amide or prodrug, as defined in claim 18.

DETAILED DESCRIPTION OF THE INVENTION Where the term alkyl is used, either alone or within other terms such as haloalkyl or alkylamino, it encompasses linear or branched radicals having the indicated number of carbon atoms. Examples of these radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, hexyl and the like.

The term alkoxy encompasses linear or branched oxy-containing radicals, each having alkyl moieties having the indicated number of carbon atoms. Examples of these radicals include methoxy, ethoxy, propoxy, butoxy and tert-butoxy.

The term aryl, alone or in combination, means a carbocyclic aromatic system containing one or two rings, wherein these rings may be fused to each other. The term aryl includes aromatic radicals such as phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl. More preferred aryl is phenyl.

The term carbonyl, whether used alone or with other terms, such as aminocarbonyl, means - (C = O) -.

The term N, N-dialkylamino encompasses a group wherein the amino group is independently substituted with two alkyl radicals having the indicated number of carbon atoms. Suitable N, N-dialkylamino radicals may be N, N-dimethylamino and N, N-diethylamino.

The term cycloalkyl includes saturated carbocyclic groups. Preferred cycloalkyl groups include cyclopentyl and cyclohexyl.

The term containing is intended to be of the open type, including said component but not excluding other elements.

The term therapeutically effective amount means an amount of a compound that ameliorates, soothes or eliminates one or more symptoms of a particular disease or condition, or prevents or delays the onset of one or more symptoms of a particular disease or condition.

58366 Β1 The term patient and subject can be used interchangeably to denote animals, such as dogs, cats, cows, horses, sheep, and humans. Mammals are especially interesting patients. The term patient encompasses both male and female individuals.

The term pharmaceutically acceptable means that the labeled substance, i. 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6 (4-chlorophenyl) -1 - ((S) -1- (isopropylsulfonyl) -3-methylbutan-2-yl) -3- methyl-2-oxopiperidin-3-yl) acetic acid or its salt, ester or amide, or formulation containing 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1 (isopropylsulfonyl) -3-methylbutan-2-yl) -3-methyl-2-oxopiperidin-3-yl) acetic acid or a salt, ester or amide thereof, or a particular excipient, suitable for use in a patient.

The terms treating, treating, treating and the like include both preventive (e.g., prophylactic) and palliative treatment.

The term excipient means any pharmaceutically acceptable additive, carrier, diluent, adjuvant or other ingredient, other than an active pharmaceutical ingredient (API), which is usually added to the composition and / or added during administration to a patient.

The compound of the present invention or a pharmaceutically acceptable salt, ester, amide or prodrug thereof is administered to a patient in a therapeutically effective amount. A compound or a pharmaceutically acceptable salt, ester or amide thereof for use in a method of treating various types of cancer in a subject in need thereof, as defined in claim 5, or a medicament comprising a compound described in claim 1 for use in a method of treating cancer , may be administered alone, or as part of a pharmaceutically acceptable combination or formulation. In addition, these compounds or combinations may all be administered simultaneously, such as by bolus injection, multiple times, as by a series of tablets, or delivered substantially evenly over a long period of time, such as by transdermal delivery. It should also be borne in mind that the dose of the compound may change over time.

In addition, the compound or a pharmaceutically acceptable salt, ester, amide or prodrug thereof may be administered singly or with other pharmaceutically active compounds. Other pharmaceutically active compounds may be used to treat the same disease or condition as the compound of the present invention, or a salt, ester, amide or prodrug thereof, or different diseases or conditions. If a patient is to receive, or is already receiving, multiple pharmaceutically active compounds, these compounds may be administered simultaneously or sequentially. For example, in the case of tablets, the active compounds may be present in a single tablet or in separate tablets, which may be administered simultaneously or sequentially, in any order. In addition, it should be borne in mind that combinations can take different forms. For example, one or more

58366 Β1 compounds can be delivered by tablet, while the other is given by injection or orally, as a syrup. All combinations, delivery procedures and administration sequences are included.

The term cancer refers to a physiological condition in a mammal characterized by unregulated cell growth. General classes of cancers include cancers, lymphomas, sarcomas and blastomas.

The compounds of the present invention can be used to treat cancer. Methods of treating cancer include administering to a patient in need thereof a therapeutically effective amount of 2 ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (S) -1- (isopropylsulfonyl) -3-methylbutan-2-yl) -3-methyl-2-oxopiperidin-3-yl) acetic acid or a pharmaceutically acceptable salt, ester or amide thereof.

The compounds of the present invention can be used to treat tumors. Methods of treating a tumor comprise administering to a patient in need thereof a therapeutically effective amount of 2 ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (S) -1- (isopropylsulfonyl) -3-methylbutan-2-yl) -3-methyl-2-oxopiperidin-3-yl) acetic acid or a pharmaceutically acceptable salt, ester or amide thereof.

Cancers that can be treated according to the present invention include, without limitation, cancers, such as cancer of the bladder, breast, colon, rectum, kidney, liver, lung (small cell lung cancer and non-small cell lung cancer), esophagus, bile bags, ovaries, pancreas, stomach, cervix, thyroid, prostate and skin (including squamous cell carcinoma); hematopoietic tumors of lymphoid lines (including leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, B cell lymphoma, T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy lymphoma); hematopoietic tumors of the myeloid lineage (including acute and chronic myelogenous leukemias, myelodysplastic syndrome, and promyelocytic leukemia); tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma, and other sarcomas, e.g., soft tissue and bone); tumors of the central and peripheral nervous system (including astrocyte, neuroblastoma, glioma, and schwannoma); and other tumors (including melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoacanthoma, thyroid follicular cell carcinoma, and Kaposi’s sarcoma). Other cancers that can be treated with a compound of the present invention include endometrial cancer, head and neck cancer, glioblastoma, malignant ascites and hematopoietic cancers.

Specific cancers that can be treated with the compounds of the present invention include soft tissue sarcomas, bone cancers such as osteosarcoma, breast tumors, bladder cancer, Li-Fraumeni syndrome, brain tumors, rhabdomyosarcoma, adrenocortical carcinoma, colorectal cancer. lung and acute myelogenous leukemia (AML).

58366 In a specific embodiment of the present invention relating to the treatment of cancer, wherein the cancer is identified as wild-type p53 (p53^w). In another specific embodiment, the cancer is identified as p53^m and a CDKN2A mutant. For example, a sample of a patient's cancer cells may be taken and analyzed to determine the status of that cancer cells with respect to p53 and / or CDKN2A. In one aspect, the patient having cancer is p53^w will be selected for treatment, in contrast to patients suffering from cancer that has mutated in relation to p53. In another aspect, a patient who has cancer that is also p53^m and has a mutated CDNK2A protein will be selected before a patient who does not have this trait. Taking cancer cells for analysis is well known to those skilled in the art. The expression r53^š means a protein encoded by genomic DNA sequence no. NC_000017 version 9 (7512445..7531642) (GenBank); protein encoded by cDNA sequence no. NM_000546 (GenBank); or a protein having GenBank sequence no. NP 000537.3. The term mutant for CDNK2A means a non-wild-type CDNK2A protein. The term CDKN2A wild-type denotes a protein encoded by genomic DNA sequence no. 9: 21957751-21984490 (Ensemble ID); protein encoded by cDNA sequence no. NM_000077 (GenBank) or NM_058195 GenBank) or; or a protein having GenBank sequence no. NP_000068 or NP_478102.

The compounds of the present invention may also be used to treat hyperproliferative disorders such as hyperplasia of the thyroid gland (especially Grave's disease) and cysts (such as hypervascularity of the ovarian stroma, characteristic of polycystic ovary syndrome (Stein-Levental syndrome)).

The compounds of the present invention may also be used to treat the following diseases or conditions: asthma, chronic obstructive pulmonary disease (COPD), emphysema, psoriasis, contact dermatitis, conjunctivitis, allergic rhinitis, systemic lupus erythematosus (SLE), ulcerative ulcers. , Crohn's disease, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, Alzheimer's disease, atherosclerosis and Huntington's disease.

The compounds of the present invention may also be used to treat inflammatory diseases, hypoxia, ulcers, viral infections, bacterial infections and bacterial sepsis.

Compound 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1- (isopropylsulfonyl) -3-methylbutan-2-yl) -3-methyl-2-oxopiperidin-3-yl) acetic acid or a pharmaceutically acceptable salt, ester, amide or prodrug thereof, as defined in the claims, may also be administered in combination with one or more pharmaceutically active compounds. funds. In one specific embodiment, the additional pharmaceutically active agent is an agent that can be used to treat cancer. For example, an additional pharmaceutically active agent may be selected from antineoplastic agents, antiangiogenic agents, chemotherapeutic agents, and peptidal cancer therapy agents. U

58366 In another embodiment, said antineoplastic agents are selected from antibiotic-like agents, alkylating agents, antimetabolic agents, hormonal agents, immune agents, interferon-like agents, kinase inhibitors, various agents, and combinations thereof. It is to be understood that additional pharmaceutically active compounds / agents may be conventional small organic chemical molecules, or may be macromolecules such as proteins, antibodies, peptibodies, DNA, RNA, or fragments of such macromolecules.

Examples of specific pharmaceutically active agents that can be used in the treatment of cancer and that can be used in combination with one or more compounds of the present invention include: methotrexate; tamoxifen; fluorouracil; 5-fluorouracil; hydroxyurea; mercaptopurine; cisplatin; carboplatin; daunorubicin; doxorubicin; etoposide; vinblastine; vincristine; pacitaxel; thioguanine; idarubicin; dactinomycin; imatinib; gemcitabine; altretamine; asparaginase; bleomycin; capecitabine; carmustine; bet. in aqueous NaCl solution; cyclophosphamine; cytarabine; decarazine; docetaxel; idarubicin; ifosfamide; irinotecan; fludarabine; mitosmicin; mitoxane; mitoxantrone; topotecan; vinorelbine; adriamycin; mitram; imiquimod; alemtuzmab; exemestane; bevacizumab; cetuximab; azacitidine; clofarabine; decitabine; desatinib; dexrazoxane; docetaxel; epirubicin; oxaliplatin; erlotinib; raloxifene; fulvestrant; letrozole; gefitinib; gemtuzumab; trastuzumab; gefitinib; ixabepilone; lapatinib; lenalidomide; aminolevulinic acid; temozolomide; nelarabine; sorafenib; nilotinib; pegaspargazu; pemetrexed; rituximab; dasatinib; thalidomide; bexarotene; temsirolimus; bortezomib; vorinostat; capecitabine; zoledronic acid; anastrozole; sunitinib; aprepitant and nelarabine, or a pharmaceutically acceptable salt thereof.

Additional pharmaceutically active agents that can be used in the treatment of cancer and that can be used in combination with one or more compounds of the present invention include: vascular endothelial growth factor inhibitors (VEGF), hepatocyte growth factor / disintegration factor inhibitors (HGF / SF), angiopoietin 1 and / or 2 inhibitors, apoptosis-inducing ligand-binding agonists (TRAIL), recombinant ligand human apo2 (TRAIL), insulin 1-like growth factor receptor (IGFR-1) inhibitors, cFMS inhibitors, HER 2 inhibitors, c-met inhibitors, aurora kinase inhibitors, CDK 4 and / or 6 inhibitors, and B-raf inhibitors.

Further additional pharmaceutically active agents that can be used in the treatment of cancer and that can be used in combination with one or more compounds of the present invention include drug and antibody conjugates (ADCs), wherein the antibody that binds to some protein, preferably it is on a cancer cell, conjugated via a linker to a chemical compound that is harmful to that cancer cell. Examples of chemical compounds that are harmful to cancer cells include maytansinoid derivatives and auristatin derivatives.

58366 Additional pharmaceutically active agents that may be used in the treatment of cancer and that may be used in combination with one or more compounds of the present invention include: epojetin alfa; darbepojetin alfa; panitumumab; pegfilgrastim; palifermin; filgrastim; denosumab; ancestry; AMG 102; AMG 319; AMG 386; AMG 479 (Ganitumab); AMG 511, AMG 900, AMG 655 (Konatumumab); AMG 745; AMG 951; and AMG 706 (Motezanib) or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention relates to the use of a compound of the present invention in combination with one or more pharmaceutical agents that are phosphathidylinositol 3-kinase (PI3K) protein inhibitors. Combinations of the compounds of the present invention together with protein inhibitors in the PI3K pathway have shown synergy in cancer cell growth assays, including enhanced apoptosis and cell killing. Examples of proteins in the PI3K pathway include PI3K, mTOR, and PKB (also known as Akt). The PI3K protein exists in several isoforms, including α, β, b, or γ. It has been hypothesized that a PI3K inhibitor that may be used in combination with a compound of the present invention may be selective for one or more isoforms. The term selective means that the compounds inhibit one or more isoforms more than other isoforms. Selectivity is a concept well known to those skilled in the art and can be measured by well-known activity in in vitro or cellular assays. Preferred selectivity means selectivity greater than 2-fold, more preferably greater than 10-fold, and even more preferred 100-fold higher selectivity for one or more isoforms over other isoforms. In one aspect, the PI3K inhibitors that can be used in combination with the compounds of the present invention are a PI3K inhibitor selective for the α isoform. In another aspect, the compound is a PI3K inhibitor selective for the b isoform.

Examples of PI3K inhibitors that can be used in combination with one or more compounds of the present invention include those described in the following documents: PCT published application no. WO2010 / 151791; PCT published application no. WO2010 / 151737; PCT published application no. WO2010 / 151735; PCT published application no. WO2010151740; PCT published application no. WO2008 / 118455; PCT published application no. WO2008 / 118454; PCT published application no. WO2008 / 118468; US application no. US20100331293; US application no. US20100331306; US application no. US20090023761; US application no. US20090030002; US application no. US20090137581; US application no.

US2009 / 0054405; US application no. US 2009/0163489; US application no. US 2010/0273764; US application no. US 2011/0092504; or PCT published application no. WO2010 / 108074.

58366 Preferred PI3K inhibitors for use in combination with the compounds of the present invention include:

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or a pharmaceutically acceptable salt thereof.

58366 Β1

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So where is X¹ fluorine or hydrogen; Υ¹ is a hydrogen or methyl group; and Z¹ is hydrogen or methyl.

Known compounds that inhibit both PI3K and mTOR (dual inhibitors). In another aspect, the present invention provides the use of dual PI3K and mTOR inhibitors for use in combination with a compound of the present invention.

MTOR is a protein in the PI3K pathway. Another aspect of the present invention involves the use of an mTOR inhibitor in combination with one or more compounds of the present invention. MTOR inhibitors that can be used in combination with the compounds of the present invention include those described in the following documents: PCT published application no. WO2010 / 132598 or PCT published application no. WO2010 / 096314.

PKB (Akt) is also a protein in the PI3K pathway. Another aspect of the present invention involves the use of an mTOR inhibitor in combination with one or more compounds of the present invention. PKB inhibitors that can be used in combination with the compounds of the present invention include those described in the following documents: U.S. Pat. 7,354,944; U.S. Pat. 7,700,636; U.S. Pat. 7,919,514; U.S. Pat. 7,514,566; U.S. Patent Application Publication No. US 2009/0270445 A1; U.S. Pat. 7,919,504; U.S. Pat. 7,897,619; or PCT published application no. WO 2010/083246 Al.

The compounds of the present invention may be used in combination with CDK4 and / or 6 inhibitors. CDK 4 and / or 6 inhibitors which may be used in combination with the compounds of the present invention

58366 Β1 of the invention include those described in the following documents: PCT published application no. WO 2009/085185 or U.S. Patent Application Publication no. US2011 / 0097305.

The compounds of the present invention may also be used in combination with pharmaceutically active agents for treating nausea. Examples of agents that can be used to treat nausea include: dronabinol; granisetron; metoclopramide; ondansetron; and prochlorperazine; or a pharmaceutically acceptable salt thereof.

In addition, the compounds of the present invention may be used in combination with other agents that can be used to treat cancer, such as acemanan; aclarubicin; aldesleukin; alitretinoin; amifostine; amrubicin; amsacrine; anagrelide; arglabin; arsenic trioxide; BAM 002 (Novels); bicalutamide; broxuridine; celmoleukin; cetrorelix; cladribine; clotrimazole; DA 3030 (Dong-A); daclizumab; denileukin diphthytox; deslorelin; dilazep; docosanol; doxercalciferol; doxyfluridine; bromocriptine; cytarabine; HIT diclofenac; interferon alpha; tretinoin; edelfosine; edrecolomab; eflornithine; emitefur; epirubicin; epoetin beta; etoposide phosphate; eksisulind; fadrozole; finasteride; fludarabine phosphate; formestan; fotemustine; gallium nitrate; gemtuzumab zogamycin; gimeracil / oteracil / tegafur combination; glycopene; goserelin; heptaplatin; human chorionic gonadotropin; human fetal alpha fetoprotein; ibandronic acid; interferon alpha; interferon alpha natural; interferon alfa-2; interferon alfa-2a; interferon alfa-2b; interferon alpha-NI; interferon alpha-n3; interferon alfacon-1; interferon alpha natural; interferon beta; interferon beta-la; interferon beta-1b; interferon gamma natural; interferon gammala; interferon gamma-lb; interleukin-1 beta; jobenguan; irzogladine; lanreotide; LC 9018 (lakult); leflunomide; lenograstim; lentin sulfate; letrozole; leukocyte alpha interferon; leuprorelin; levamisole + fluorouracil; liarozole; lobaplatin; lonidamine; lovastatin; mazoprokol; melarsoprol; metoclopramide; mifepristone; miltefosine; mirimostim; unpaired double-stranded RNA; mitoguazone; mitolactol; mitoxantrone; molgramostim; nafarelin; naloxone + pentazocine; nartograstim; nedaplatin; nilutamide; noskapin; a new protein that stimulates erythropoiesis; NSC 631570 octreotide; oprelvekin; osateron; paclitaxel; pamidronic acid; peginterferon alfa-2b; pentosan sodium polysulfate; pentostatin; picibanil; pirarubicin; rabbit antithymocyte polyclonal antibody; polyethylene glycol interferon alfa-2a; porfimer sodium; raltitrexed; razburikaza; rhenium Re 186 etidronate; Rll retinamide; romurtid; samarium (153 Sm) lexidronam; sargramostim; sisofiran; sobazoxan; sonermin; strontium-89 chloride; suramin; tazonermin; tazarotene; tegafur; temoporfin; teniposide; tetrachlorodecaxide; timalfasin; thyrotropin alfa; toremifene; tozitumomab-iodine 131; treosulfan; tretinoin; trilostane; trimetrexate; triptorelin; tumor necrosis factor alpha natural; ubenimex; bladder cancer vaccine; Maruyama vaccine; melanoma lysate vaccine; valrubicin; verteporfin; virulizin; zinostatin stimulant; abarelix; AE 941 (Aeterna); ambamustine; antisense oligonucleotide; bcl-2 (Genta); APC 8015 (Dendreon); dexaminoglutethimide; diazikvone; EL532 (Elan); EM 800 (Endorecherche); eniluracil; ethanidazole; fenretinide; filgrastim SDOl

58366 Β1 (Amgen); galocitabine; gastrin 17 immunogen; HLA-B7 gene therapy (Vical); granulocyte macrophage colony growth stimulating factor; histamine dihydrochloride; ibritumomab tiuxetan; ilomastat; IM 862 (Citran); interleukin-2; iproxyfen; LDI 200 (Milkhaus); leridistim; lintuzumab; CA 125 monoclonal antibody (MAb) (Biomira); MAb for Cancer (Japan Pharmaceutical Development); HER-2 and Fc MAb (Medarex); idiotypic MAb 105AD7 (CRC Technology); idiotypic CEA MAb (Trilex); LIM-l-iodine 131 MAb (Techniclone); polymorphic epithelial mucin-yttrium 90 MAb (Antisome); marimastat; menogaril; mitumomab; motexafin gadolinium; MH 6 (Galderma); nolatrexed; P 30 protein; pegvisomant; porphyromycin; prinomastat; RL 0903 (Shire); rubitekan; satraplatin; sodium phenylacetate; sparphosic acid; SRL 172 (SR Farm); SU 5416 (Pfizer); TA 077 (Tanabe); tetrathiomolybdate; taliblastine; thrombopoietin; tin ethyl ethiopurpurine; tirapazamine; cancer vaccine (Biomir); melanoma vaccine (New York University); melanoma vaccine (Sloan Kettering Institute); melanoma oncolysis vaccine (New York Medical College); vaccine for viral lysates of melanoma cells (Royal Newcastle Hospital); or lord. It should be borne in mind that the agents listed above may be used as pharmaceutically acceptable salts, where appropriate.

The compounds of the present invention may also be used in combination with radiotherapy, hormone therapy, surgery and immunotherapy, all of which are well known to those skilled in the art.

As one embodiment of the present invention contemplates treating a disease / condition with a combination of pharmaceutically active compounds that can be administered separately, the present invention further relates to a combination of separate pharmaceutical combinations in kit form. The kit contains two separate pharmaceutical combinations: a compound of the present invention and another pharmaceutical compound. The kit contains a container in which separate combinations are stored, such as a divided bottle or a divided foil bag. Additional examples of packaging include syringes, boxes and bags. It is common for the kit to contain instructions for using the separate components. The kit is particularly useful when it is desirable to administer the separate components in different dosage forms (e.g. oral and parenteral), when administered at different dosing intervals, or when it is desirable that the precise determination of the individual components in the combination be performed by the physician or veterinarian attributing the combination.

An example of such a kit is a so-called pocket pack. Pocket packs are well known in the packaging industry and are widely used in the packaging of pharmaceutical unit dosage forms (tablets, capsules and the like). Pocket packages usually consist of a board made of relatively solid material covered with plastic foil, for which it is desirable to be transparent. During the packaging process, pockets are formed in the plastic foil. These pockets fit tablets in size and shape

58366 Β1 or capsules to be packed. The tablets or capsules are then placed in the pockets, and a plate of relatively rigid material is glued to the plastic foil, on the side of the foil that is opposite to the direction of the pockets. This seals the tablets or capsules in pockets between the plastic foil and the tablet. Preferably, the durability of said panel is such that the tablets or capsules can be removed from the pocket pack by pressing on the pocket itself, thereby creating an opening in the panel in place of that pocket. The tablet or capsule can then be removed through said opening.

It may be desirable to provide a mnemonic on the kit, e.g. in the form of numbers immediately adjacent to the tablets or capsules, said numbers corresponding to the days of the therapy regimen in which the tablets or capsules so indicated are to be taken. Another example of such a mnemonic is a calendar printed on a board, e.g. first week, monday, tuesday, ... etc ... second week, monday, tuesday, ... etc. Other variations of the mnemonic will be obvious. The daily dose may mean a single tablet or capsule or several pills or capsules to be taken on a particular day. Also, the daily dose of a compound of the present invention may consist of one tablet or capsule, while the daily dose of another compound may consist of several tablets or capsules, or vice versa. It should be marked on the mnemonic, so that it helps in the correct application of active means.

In another specific embodiment according to the present invention, a package is provided designed to provide daily doses one by one, in the order in which they are to be used. It is desirable that such a dispenser has a mnemonic, in order to further facilitate adherence to the regimen. An example of such a mnemonic is a mechanical counter that shows the number of daily doses that have been issued. Another example of such a mnemonic is a battery-powered microchip memory connected to a liquid crystal display or an audible signal that, for example, reads the date the last daily dose was taken and / or reminds the patient when to take the next dose.

The compounds of the present invention and other pharmaceutically active compounds may, if desired, be administered to a patient either orally, rectally, parenterally (e.g., intravenously, intramuscularly, or subcutaneously), intracisternally, intravaginally, intraperitoneally, intravesically, topically (at example, powders, ointments or drops) or in the form of buccal or nasal spray. All methods used by those skilled in the art for the administration of a pharmaceutically active agent are included within the scope of the present invention. A specific aspect is a pharmaceutical combination as defined in claims 1 to 4, which is a solid dosage form, is a combination suitable for parenteral injection, or is a liquid dosage form for oral administration. A medicament for use in the treatment of cancer as defined in claims 14 to 16 or a compound for use in the treatment of certain cancers as defined in claims 5 to 13, as well as an ester, amide or prodrug as defined in patent

58366 The claim 17 and contained in the pharmaceutical combination as defined in claim 18 is not limited with respect to the dosage form.

Combinations suitable for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile solutions or dispersions for injection. Examples of suitable aqueous and non-aqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and organic esters suitable for injection, such as ethyl oleate. Adequate fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle dimensions in the case of dispersions, and by the use of surfactants.

These combinations may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersing agents. Contamination with microorganisms can be prevented by the addition of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of injected pharmaceutical combinations can be achieved by the use of agents that delay absorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert selected excipient (or carrier) such as disodium citrate or dicalcium phosphate or (a) fillers or extenders, such as, for example, starches, lactose, sucrose, mannitol and silicic acid; (b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, such as, for example, glycerol; (d) disintegrants, such as, for example, agargar, calcium carbonate, potato starch and tapioca starch, alginic acid, some complex silicates and sodium carbonate; (a) dissolving agents, such as, for example, paraffin; (f) absorption accelerators, such as, for example, quaternary ammonium compounds; (g) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (h) adsorbents, such as, for example, kaolin and bentonite; and (i) Lubricants, such as, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules and tablets, the dosage forms may also contain buffers.

Solid combinations of a similar type can be used as fillers in soft and hard gelatin filled capsules, using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

58366 Β1 Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and coatings, such as enteric coatings and the like, well known in the art. They may also contain clouding agents, and may be of such composition that they release the active compound or compounds in a particular part of the intestinal tract in a delayed manner. Examples of combinations used as carriers in such cases are polymeric substances and waxes. The active compounds may also be in microencapsulated form, if necessary, with one or more of the aforementioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubility enhancers, and emulsifiers, such as, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, more specifically cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and esters of fatty acids and sorbitan, or mixtures of such substances, and the like.

In addition to such inert diluents, the compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavors, and odorous substances. Suspensions, in addition to the active compound, may contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene and sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and gum tragacanth, or , and the like.

Combinations for rectal administration are preferably in the form of suppositories, which may be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol or suppository wax, which are solid at room temperature. , but liquid at body temperature, and therefore melt in the rectum or vaginal cavity and release the active ingredient.

Dosage forms for the topical administration of the compounds of the present invention include ointments, powders, sprays and inhalants. The active compound or suitable compounds are mixed under sterile conditions with a physiologically acceptable carrier, and any preservatives, buffers or propellants that may be required. Ophthalmic formulations, eye ointments, powders and solutions are also included within the scope of the present invention.

58366 The compounds of the present invention may be administered to a patient in doses ranging from about 0.1 to about 3000 mg per day. For a normal adult with a body weight of about 70 kg, a dose in the range of 0.01 to about 100 mg per kilogram of body weight is usually sufficient. The specific dose and dosage range that may be used depends on a number of factors, including the needs of the patient, the severity of the condition or disease being treated, and the pharmacological activity of the compound being administered. Determining dose ranges and optimal doses for a particular patient is a common skill in the art.

The compounds of the present invention may be administered in the form of pharmaceutically acceptable salts, esters, amides or prodrugs. The term salts refers to the inorganic and organic salts of the compounds of the present invention. Said salts may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in the form of the free base or acid with a suitable organic or inorganic base or acid, and isolating the salt formed in that reaction. Representative examples of salts include hydrobromides, hydrochlorides, sulfates, bisulfates, nitrates, acetates, oxalates, palmitates, stearates, laurates, borates, benzoates, lactates, phosphates, tosylates, citrates, maleates, fumarates, succinates, tartrates, naphthylates, mezlate lactobionates and laurylsulfonates, and the like. Salts may include cations based on alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations which include, but are not limited to, ammonium amethylamine, tetra , methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like. See, e.g., SM Berge et al., Pharmaceutical Salts, J. Pharm Sci, 66: 1-19 (1977).

Pharmaceutically acceptable esters of the compounds of this invention include C 1 -C 6 alkyl esters. Acceptable esters also include C₅-C₇ cycloalkyl esters as well as arylalkyl esters such as benzyl. C₃-C₄ alkyl esters are commonly used. Esters of the compounds of this invention may be prepared according to methods well known in the art.

Pharmaceutically acceptable amides of the compounds of this invention include amides derived from ammonia, primary C1-C₈alkyl amines, and secondary C1-C₈ dialkyl amine. In the case of secondary amines, the amine may also be in the form of a 5- or 6-membered heterocycloalkyl group containing at least one nitrogen atom. Amides derived from ammonia, C1-C₃ primary alkyl amines and C1-C₂dialkyl secondary amines are commonly used. Amides of the compounds of this invention may be prepared according to methods well known to those skilled in the art.

The term prodrug represents compounds that are transformed in vivo to give a compound of the present invention. Transformation can occur through various mechanisms, such as hydrolysis in the blood.

58366 Β1

Discussion of the use of prodrugs is provided by T. Higuchi and W. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the ACS Symposium Series, and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

The compound of the present invention contains a carboxylic acid functional group, and the prodrug is selected from an ester formed by replacing the hydrogen atom of the acid group with a group selected from (C₂C₁₂) alkanoyloxymethyl, 1- (alkanoyloxy) ethyl having from 4 to 9 carbon atoms, 1-methyl-1 (alkanoyloxy) ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, l- (alkoxycarbonyloxy) ) ethyl having from 4 to 7 carbon atoms, 1-methyl-1 (alkoxycarbonyloxy) ethyl having from 5 to 8 carbon atoms, N- (alkoxycarbonyl) aminomethyl having from 3 to 9 carbon atoms, 1- (N- ( alkoxycarbonyl) aminomethyl having from 4 to 10 carbon atoms, 3phthalidyl, 4-Crotonolactonyl, gamma-butyrolacton-4-yl, di-N, N- (C1-C₂) alkylamino (C₂-C₃) alkyl (such as β-dimethylaminoethyl), carbamoyl- (C1-C6)₂) alkyl, N, N-di (C₁-C₂) alkylcarbamoyl- (C1-C₂) alkyl and piperidino-, pyrrolidino- or morpholino (C₂-₃) alkyl.

The compounds of the present invention may also exist in unsolvated and solvated form with pharmaceutically acceptable solvents such as water (hydrate), ethanol and the like. The present invention encompasses both solvated and unsolvated forms by its scope of protection.

It is also contemplated that the present invention encompasses compounds that have been synthesized in vitro using laboratory techniques, such as those well known to synthetic chemists; or synthesized by in vivo techniques, such as through metabolism, fermentation, digestion and the like. The scope of protection of the present invention also encompasses that the compounds of the present invention may be synthesized by a combination of in vitro and in vivo techniques.

The present invention also encompasses isotopically labeled compounds, which are identical to those listed herein, except that one or more atoms are substituted by an atom having an atomic mass or a mass number other than the atomic mass or mass number commonly encountered. in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁶0,¹⁷0,¹⁸Oh, ³¹P, ³²P, ³⁵S, ¹⁸F i ³⁶CI. In one aspect, the present invention relates to compounds in which one or more hydrogen atoms are substituted by deuterium atoms (²H).

Compounds of the present invention containing the aforementioned isotopes and / or other isotopes of other atoms are included within the scope of the present invention. Certain isotopically labeled compounds of the present invention, for example those incorporating radioactive

58366 Β1 isotopes such as ³H i ¹⁴C are useful in drug and / or substrate tissue redistribution tests. Tritium, ie.³X and 14-carbon, ie. ¹⁴C isotopes are particularly preferred for ease of preparation and detection. Further, substitution with heavier isotopes such as deuterium, i.²X may lead to certain therapeutic benefits resulting from greater metabolic stability, for example, a prolonged half-life in vivo or a reduction in the required dose, and may therefore be desirable in some circumstances. Isotopically labeled compounds of the present invention can, in general, be obtained by substituting an readily available isotopically labeled reagent with a non-isotopically labeled reagent.

The compounds of the present invention may exist in a variety of solid states, including crystalline states and amorphous states. Various crystalline states, also known as polymorphic, and amorphous states of the compounds of the present invention are encompassed by the scope of protection of the present invention.

In the synthesis of the compounds according to the present invention, it is possible that the use of certain leaving groups will be preferred. The term leaving group (LG) generally refers to groups that can be substituted by a nucleophile. Such departing groups are known in the art. Examples of leaving groups include, but are not limited to, halides (e.g., I, Br, F, Cl), sulfonates (e.g., mesylate, tosylate), sulfides (e.g., SCH₃), N-hydroxysuccinimide, N-hydroxybenzotriazole and the like. Examples of nucleophiles include, but are not limited to, amines, thiols, alcohols, Grignard reagents, anionic species (e.g., alkoxides, amides, carbanions), and the like.

The examples to be presented below illustrate specific embodiments of the present invention. These examples are intended as representative, and are not intended to limit in any way the scope of protection of the subject patent claims. Unless otherwise indicated, when a percentage in terms of a solid is used in the present application, that percentage means the percentage by weight relative to said solid combination. When a percentage in terms of a liquid is used, the stated percentage is the volume percentage in relation to the observed solution.

[0073] ¹H-NMR spectra were usually recorded on a Bruker Avance III 500 spectrometer (Bruker, Bilerica, MA), while working on ³N at a frequency of 500.13 MHz, equipped with a Bruker PABBI 5 mm probe with a gradient along the z axis; or on the Bruker Avance II 400 spectrometer during a frequency program³N of 400.23 MHz, equipped with a Bruker PABBO 5 mm probe with a gradient along the z axis. Samples are usually dissolved in 500 ml of DMSO-d6 or CD3OD for NMR analysis. Chemical shifts³N are given relative to the residual signals of the solvent DMSO-d6 at b 2.50 and CD₃OD to b 3.30.

58366 Β1 Significant maxima are presented in tabular form and usually include: number of protons, multiplicity (s, singlet; d, doublet; dd, doublet doublet; t, triplet; q, quartet; m, multiplet, br s, wide singlet ) and the coupling constant (s) in hertz.

Electron ionization (EI) mass spectra were typically recorded on an Agilent Technologies 6140 CJuadrupole LC / MS mass spectrometer. The results of mass spectrometry are given as the ratio of mass to charge, sometimes followed by the relative representation of each ion (in parentheses). The starting materials in the examples that follow are usually either available from commercial sources, such as Sigma-Aldrich, St. Louis, MO, or through procedures known from the literature.

The following abbreviations can be used here:

<td></td><td>eye</td>

<td>+ out or pos. ion</td><td>positive ion</td>

<td>Δ</td><td>heat</td>

<td>Ac</td><td>acetyl</td>

<td>Ac₂Oh</td><td>acetic anhydride</td>

<td>aq, vod.</td><td>water</td>

<td>AcOH</td><td>acetic acid</td>

<td>Bn</td><td>benzyl</td>

<td>Boc</td><td>tert-butyloxycarbonyl</td>

<td>BSA</td><td>bovine serum albumin</td>

<td>Bu</td><td>butyl</td>

<td>Bz</td><td>benzoyl</td>

<td>Assoc. or izr'to</td><td>calculated</td>

<td>Conc.</td><td>concentrated</td>

<td>CSA</td><td>camphor-10-sulfonic acid</td>

<td>d</td><td>days)</td>

<td>DBU</td><td>1,8-diazabicyclo [5.4.0] undecan-7-ene</td>

<td>DCE</td><td>dichloroethane</td>

<td>DCM</td><td>dichloromethane</td>

<td>DEA</td><td>diethylamine</td>

Jersey-Martin period; Dres-Martin reagent 1,1,1-triacetoxy-1,1-dihydro-1,2-

<td></td><td>benziodoxol-3- (1H) -one</td>

<td>DIEAili DIPEA</td><td>diisopropylethylamine</td>

<td>DMAP</td><td>4-dimethylaminopyridine</td>

<td>DME</td><td>1,2-dimethoxyethane</td>

<td>DMF</td><td>N, N-dimethylformamide</td>

58366 Β1

<td>DMSO</td><td>dimethyl sulfoxide</td>

<td>dr</td><td>diastereomeric ratio</td>

<td>DTT</td><td>dithiothreitol</td>

<td>DVB</td><td>divinylbenzene</td>

<td>EDC</td><td>N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide</td>

<td>eq</td><td>equivalent</td>

<td>ESI or ES</td><td>ionization electrospray</td>

<td>Et</td><td>ethyl</td>

<td>Et₂Oh</td><td>diethyl ether</td>

<td>Et₃N</td><td>triethylamine</td>

<td>EtOAc</td><td>ethyl acetate</td>

<td>EtOH</td><td>ethyl alcohol</td>

<td>g</td><td>gram (s)</td>

<td>h</td><td>hour (s)</td>

<td>HATU</td><td>O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetrarriethyluronium hexafluorophosphate</td>

<td>HBTU</td><td>O-Benzotriazole-N'N ^ N '^ N'-tetramethyl-uronium-hexafluorophosphate</td>

<td>Neh</td><td>hexanes</td>

<td>HMPA</td><td>hexamethylphosphoramide</td>

<td>HOAt</td><td>1-hydroxy-7-azabenzotriazole</td>

<td>HOBt</td><td>hydroxybenzotriazole</td>

<td>HPLC</td><td>high performance liquid chromatography</td>

<td>IPA or iPrOH</td><td>isopropyl alcohol</td>

<td>Jones reagent</td><td>a solution of chromium (IV) oxide and sulfuric acid in water</td>

<td>KHMDS</td><td>potassium hexamethyldisilazide</td>

<td>KOAc</td><td>potassium acetate</td>

<td>LCMS, LC-MS or LC / MS</td><td>liquid chromatography with mass spectrometry</td>

<td>LDA</td><td>lithium diisopropylamide</td>

<td>LHMDS or LiHMDS</td><td>lithium hexamethyldisilazide</td>

<td>L-Selectride®</td><td>lithium tri-sec-butyl borohydride (Sigma-Aldrich, St. Louis)</td>

<td>M</td><td>molar (mol L '¹)</td>

<td>m / z</td><td>mass divided by charge</td>

<td>mCPBA</td><td>m-chloroperoxybenzoic acid</td>

<td>Me</td><td>methyl</td>

<td>MeCN</td><td>acetonitrile</td>

<td>Mel</td><td>iodomethane</td>

58366 Β1

<td>MeON</td><td>methyl alcohol</td>

<td>mg</td><td>milligram (s)</td>

<td>min</td><td>minute (s)</td>

<td>Jr.</td><td>m ililitar (i)</td>

<td>M</td><td>mole (s)</td>

<td>MS</td><td>mass spectrometry</td>

<td>MsCI</td><td>methanesulfonyl chloride</td>

<td>MTBE or MtBE</td><td>methyl tert-butyl ether</td>

<td>m / z</td><td>mass-to-charge ratio</td>

<td>NaHMDS</td><td>sodium hexamethyldisilazide</td>

<td>NaOtBu</td><td>sodium tert-butoxide</td>

<td>NBS</td><td>N-bromosuccinimide</td>

<td>nBuLi</td><td>n-butyllithium</td>

<td>NMO</td><td>N-methylmorpholine-N-oxide</td>

<td>NMP</td><td>1-methyl-2-pyrrolidinone</td>

<td>NMR</td><td>nuclear magnetic resonance</td>

<td>N-Selectride®</td><td>sodium tri-sec-butyl borohydride (Sigma-Aldrich, St. Louis)</td>

<td>PBS</td><td>phosphate buffered saline solution</td>

<td>PMB</td><td>paramethoxybenzyl</td>

<td>Pr</td><td>propyl</td>

<td>ppm</td><td>parts in a million</td>

<td>rac</td><td>racemic</td>

<td>RP-HPLC or RPHPLC</td><td>reversed-phase high pressure liquid chromatography</td>

<td>ST or st</td><td>room temperature</td>

<td>for S. or sat'd or satd</td><td>saturated</td>

<td>SFC</td><td>chromatography with supercritical fluid</td>

<td>TBAF</td><td>tetrabutylammonium fluoride</td>

<td>TBDMS</td><td>tert-butyldimethylsilyl</td>

<td>TBDMS-CI</td><td>tert-butyldimethylsilyl chloride</td>

<td>TBDPS</td><td>tert-butyldiphenylsilyl</td>

<td>TIME</td><td>(2,2,6,6-tetramethylpiperidin-1-yl) oxidanyl</td>

<td>terc or t</td><td>tertiary</td>

<td>TFA</td><td>trifluoroacetic acid</td>

<td>THF</td><td>tetrahydrofuran</td>

<td>TIPS</td><td>triisopropylsil il</td>

<td>TLC</td><td>thin layer chromatography</td>

58366 Β1

<td>TMS</td><td>trimethylsilyl or trimethylsilane</td>

<td>TPAP</td><td>tetrapropylammonium perrutenate</td>

<td>t_R</td><td>retention time</td>

<td>tBuOH</td><td>tert-butyl alcohol</td>

<td>v / v</td><td>volume by volume</td>

EXAMPLES In the following, reference examples and an example of how the compounds of the present invention may be prepared will be presented. Reference examples are not covered by the scope of the present invention.

REFERENCE EXAMPLE 1 [0077]

<img file="RS58366B1_D0007.tif" />

2 - ((3R, 5R, 6S) -1 - ((S) -1-tert-butoxy-1-oxobutan-2-yl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -2 -oxopiperidin-3yl) acetic acid

Step A. 2- (3-Chlorophenyl) -1- (4-chlorophenyl) ethanone [0078]

<img file="RS58366B1_D0008.tif" />

To a solution of 2- (3-chlorophenyl] acetic acid (10 g, 58.6 mmol) in THF (58 mL) was slowly added 117 mL of a 1 M solution of sodium bis- (trimethylsilyl) amide in THF over 1 h After stirring the mixture at -78 [deg.] C. for 40 minutes, a solution of methyl 4-chlorobenzoate (10 g, 58.6 mmol) in THF (35 mL) was added.

58366 Β1 in a period of 10 min. The reaction mixture was stirred at -78 ° C for 3 h, after which it was allowed to cool to 25 ° C and stirred for an additional 2 h until the reaction was complete. The reaction was quenched by the addition of saturated aqueous NH₄CI, and most of the THF was removed under reduced pressure. The residue was extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with saturated NaCl solution, dried over Na₂SO₄, filtered, and the filtrate was concentrated. The product was recrystallized from ether / propane to give the title compound as a white solid.

Step B. Methyl 4- (3-chlorophenyl) -5- (4-chlorophenyl) -5-oxopentanoate [0080]

<img file="RS58366B1_D0009.tif" />

co₂r To a solution of 52.1 g (197 mmol) of 2- (3-chlorophenyl) -1- (4-chlorophenyl) ethanone (Reference Example 1, step A) and methyl acrylate (19.5 ml, 216 mmol) to 360 ml of THF was slowly added 20 ml of a 1 M solution of potassium tert-butoxide in THF at 0 ° C for 20 minutes (reaction temperature was maintained &lt;10 ° C). The reaction mixture was allowed to cool to room temperature. After stirring for 1 h, the reaction mixture was concentrated under reduced pressure, diluted with water and extracted with ethyl acetate. The combined organic layers were washed with saturated NaCl solution, dried over Na₂SO₄, filtered, and the filtrate was concentrated. Purification of the residue by gas chromatography on silica gel (eluent: 15% EtOAc / hexanes) gave the title compound as a colorless liquid. R is CH₃.

StepC. (4S, 5S) -methyl 4- (3-chlorophenyl) -5- (4-chlorophenyl) -5-hydroxypentanoate and (4R, 5R) -methyl 4- (3-chlorophenyl) -5 (4-chlorophenyl) -5 -hydroxy pentanoate [0082]

<img file="RS58366B1_D0010.tif" />

<img file="RS58366B1_D0011.tif" />

58366 Β1 To a solution of 75.1 g (213 mmol) of methyl 4- (3-chlorophenyl) -5- (4-chlorophenyl) -5-oxopentanoate (reference example 1, step B) in MeOH (0.71 L, c = 0.3 M) at 0 ° C sodium borohydride (8058 mg, 213 mmol) was added in several small portions. After the reaction mixture was stirred at 0 ° C for 30 min, the reaction was quenched with ice-cold H₂Oh, the reaction mixture was concentrated under reduced pressure and extracted with EtOAc. The combined organic layers were washed (saturated aqueous NaCl solution), dried over Na₂SO₄, filtered, and the filtrate was concentrated. Purification of the residue by gas pressure chromatography on silica gel (eluent: 20 to 30% EtOAc / hexanes, gradient elution) gave a mixture of the title compounds as a colorless liquid.

Step D. (4S, 5R) -Methyl 5-azido-4- (3-chlorophenyl) -5- (4-chlorophenyl) pentanoate and (4R, 5S) -methyl 5-azido-4- (3-chlorophenyl) -5- (4-chlorophenyl) pentanoate [0084]

<img file="RS58366B1_D0012.tif" />

<img file="RS58366B1_D0013.tif" />

To a solution of 63.1 g (179 mmol) of (4S, 5S) -methyl 4- (3-chlorophenyl) -5- (4-chlorophenyl) -5-hydroxypentanoate and (4R, 5R) -methyl 4- ( 3-Chlorophenyl) -5- (4-chlorophenyl) -5-hydroxypentanoate (Reference Example 1, Step C) and triethylamine (49.8 mL, 357 mmol) in DCM (600 mL, 0.3 M) was added methanesulfonyl chloride (18 mL, 232 mmol) at 0 ° C dropwise over 10 minutes. The reaction mixture was stirred at 0 ° C for 40 minutes, while TLC was used to monitor the completion of the reaction. The reaction was then quenched with ice-cold water, extracted (3 h DCM) and washed with sat. vod. NaCl solution. The combined organic layers were dried (Na₂SO₄) and concentrated under reduced pressure.

The crude mesylate synthesized in this way was dissolved in DMF (350 mL, 0.5 M) and sodium azide (58 g, 893 mmol) was added in several portions. The mixture was heated to 100 ° C and, after stirring at 100 ° C for 30 minutes, cooled to room temperature, diluted with water and extracted with EtOAc. The combined organic layers were washed (saturated aqueous NaCl solution), dried over Na₂SO₄, filtered, and the filtrate was concentrated. Purification of the residue by gas chromatography on silica gel (eluent: 5 to 20% EtOAc / hexanes, gradient elution) gave the title compound as a colorless liquid.

Step E. (5R, 6S) -5- (3-Chlorophenyl) -6- (4-chlorophenyl) piperidin-2-one

58366 Β1 [0087]

<img file="RS58366B1_D0014.tif" />

To a solution of 45.9 g (121 mmol) of methyl 5-azido-4- (3-chlorophenyl) -5- (4-chlorophenyl) pentanoate (reference example 1, step D) in a THF / H mixture₂O (4: 1, 375 mL) was added 152 mL of a 1 M solution of trimethylphosphine in THF (152 mmol). After stirring at 25 ° C for 1 h, most of the THF was removed under reduced pressure. The residue was basified (ice-cold 2 M LiOH) and the product was extracted with methylene chloride. The combined organic layers were washed with saturated NaCl solution, dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure to give a white solid.

This solid was dissolved in a mixture of MeOH / saturated aqueous NaHCO₃ (4: 1, 2.4 L, c = 0.05 M) and the reaction was heated to reflex for 3 h. Excess organic solvent was removed under reduced pressure, the residue was diluted with water and extracted (2 x 10% MeOH / DCM). The combined organic layers were washed with saturated NaCl solution, dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure to give trans-5- (3-chlorophenyl) -6- (4-chlorophenyl) piperidin-2-one as a mixture of stereoisomers. The individual stereoisomers were separated by chiral HPLC (flow rate: 18 ml / min on a Chiralcel® OD-H 20 mm, ID h 250 mm, 5 mic columns) (Daicel Inc., Fort Lee, NJ) using 40% isopropyl alcohol / hexene as eluent) to give the title compound (t_R = 8.2 min) as a white solid.

[a]_D = + 158 (T = 23.4 ° C, c = 1.12, MeOH); ^:1 H NMR (400 MHz, CHLOROFORM-d) b ppm 7.21 (2 H, d, J = 8.2 Hz), 7.09-7.19 (3 ​​H, m), 7.04-7.01 (1H, m), 6.97 (2H, d, J = 8.2 Hz), 6.80-6.77 (1H, m), 5.83 (1H, s, br), 4.51 (1H, d, J = 9.8 Hz), 2.94-2.77 (1H, m), 2.74-2.60 (2H, m), 2.34-2 .20 (1H, m), 2.17-2.08 (1H, m); MS (ESI) 320.0 [M + H]⁺.

The enantiomer of the title compound, (5S, 6R) -5 (3-chlorophenyl) -6- (4-chlorophenyl) piperidin-2-one: t was also obtained by the process described above._R = 12.4 min; [a]_D = -156 (T = 23.4 ° C, c = 1.13, MeOH).

Step F. Tert-butyl (2S) -2 - ((2S, 3R) -3- (3-chlorophenyl) -2- (4-chlorophenyl) -6-oxo-1-piperidinyl) butanoate and tert-butyl ) -2 - ((2S, 3R) -3- (3-chlorophenyl) -2- (4-chlorophenyl) -6-oxo-1-piperidinyl) butanoate

58366 Β1 [0091]

<img file="RS58366B1_D0015.tif" />

<img file="RS58366B1_D0016.tif" />

To a solution of 13.5 g (42.2 mmol) (5R, 6S) -5,6-bis (4-chlorophenyl) piperidin-2-one (Reference Example 1, step E) in 140 ml of DMF 4.22 g (105 mmol) of a dispersion of 60% sodium hydride in mineral oil at 0 ° C were added. After stirring for 20 minutes, tert-butyl 2-bromobutanoate (28.2 g, 126 mmol) was added at 0 ° C, and the resulting solution was stirred at 25 ° C for 1.5 h until the reaction was complete. Then sat. vod. NH solution₄CI after which the mixture was extracted with ethyl acetate. The combined organic layers were washed with water and saturated NaCl solution, dried over Na₂SO₄, filtered, tejefiltrate concentrated under reduced pressure. Purification of the residue by gas chromatography on silica gel (eluent: 20 to 50% EtOAc / hexanes, gradient elution) gave tert-butyl (2S) -2 - (2S, 3R) -3- (3-chlorophenyl) -2 - (4-chlorophenyl) -6oxo-1-piperidinyl) butanoate as a less represented isomer with faster elution:¹1 H NMR (400 MHz, CHLOROFORM-d) b ppm 7.22 (2H, d, J = 8.2 Hz), 7.20-7.10 (2H, m), 7.08 (2H, t, J = 8.2 Hz) 6.99-6.96 (1H, m), 6.77-6.73 (1H, m), 4.48 (1H, d, J = 9, 4 Hz), 3.24 (1H, t, J = 7.0 Hz), 3.04-2.94 (1H, m), 2.72-2.58 (2H, m), 2 , 25-2.00 (3 H, m), 1.93-1.82 (1 H, m), 1.45 (9 H, s), 0.98 (3 H, t, J = 7, 4 Hz); MS (ESI) 462.1 [M + H]⁺.

Further elution gave tert-butyl (2R) -2 - ((2S, 3R) -3- (3-chlorophenyl) -2- (4-chlorophenyl) -6-oxo-piperidinyl) butanoate as slower, more abundant isomer.

^gN NMR (400 MHz, CHLOROFORM-d) b ppm 7.24 (2 H, d, J = 8.2 Hz), 7.18-7.10 (2 H, m), 7.01 (2 H, d, J = 8.2 Hz), 7.02-6.98 (1H, m), 6.82-6.78 (1H, m), 5.83 (1H, s), 4, 54 (1H, d, J = 9.8 Hz), 3.09 (1H, dd, J = 8.2, 4.3 Hz), 3.05-2.99 (1H, m), 2.70-2.64 (2H, m), 2.28-2.18 (2H, m), 2.08-2.02 (1H, m), 1.48 (9H, s) ), 0.57 (3H, t, J = 7.4 Hz); MS (ESI) 462.1 [M + H]⁺.

StepG. Tert-butyl (2S) -2- (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -2-oxopiperidin-1-yl) butanoate i. tert-butyl (2S) -2 - ((3R, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -2-oxopiperidin-1-yl) butanoate [0094]

58366 Β1

<img file="RS58366B1_D0017.tif" />

<img file="RS58366B1_D0018.tif" />

To a solution of 1.45 g (3.14 mmol) of tert-butyl (2S) -2 - ((2S, 3R) -3- (3-chlorophenyl) -2- (4-chlorophenyl) -6-oxo -lpiperidinylbutanoate (reference example 1, step F) and allyl bromide (0.326 mL, 3.76 mmol) in 12.5 mL of THF was added 3.3 mL of a 1 M solution of lithium bis in trimethylsilylamide in THF (3, 3 mmol), dropwise, at -78 [deg.] C. After stirring at -78 [deg.] C. for 3 h, the reaction was quenched with saturated aqueous NH₄CI and extracted with ethyl acetate. The combined organic layers were washed with saturated NaCl solution, dried over Na₂SO4, filtered, and the filtrate was concentrated under reduced pressure. Purification of the residue by gas pressure chromatography on silica gel (50 g SiO₂, eluent: 5 to 20% EtOAc / hexanes, gradient elution) tert-butyl (2S) -2 - ((3R, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4) was obtained -chlorophenyl) -2-oxopiperidin-1-yl) butanoate as a more represented isomer with faster elution.

^gN NMR (400 MHz, CHLOROFORM-d) b ppm 7.27-7.24 (2 H, m), 7.21-7.12 (2 H, m), 7.11-7.00 (3 H , m), 6.93-6.87 (1H, m), 5.90-5.77 (1H, m), 5.19-5.09 (2H, m), 4.64 ( 1 H, d, J = 8.6 Hz), 3.21-3.10 (2 H, m), 2.80-2.71 (1 H, m), 2.70-2.63 (1 H, m), 2.56-2.48 (1H, m), 2.30-2.15 (2H, m), 2.07-1.99 (1H, m), 1,601.48 (1H, m), 1.47 (9H, s), 0.61 (3H, t, J = 7.6 Hz); MS (ESI) 446.0 [M + H]⁺.

Further elution gave tert-butyl (2S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -2-oxopiperidin-1-yl) butanoate as a less represented isomer with a slower elution.

^gN NMR (400 MHz, CHLOROFORM-d) b ppm 7.23 (2 H, d, J = 8.2 Hz), 7.19-7.07 (2 H, m), 7.01-6.95 (3H, m), 6.77-6.72 (1H, m), 5.95-5.77 (1H, m), 5.16-4.99 (2H, m), 4 , 51 (1H, d, J = 10.6 Hz), 3.13-3.04 (1H, m), 2.94 (1H, dd, J = 7.8, 4.3 Hz) , 2.87-2.77 (1H, m), 2.68-2.58 (1H, m), 2.39-2.27 (2H, m), 2.16-1.95 (2H, m), 1.54-1.50 (1H, m), 1.51 (9H, s), 0.55 (3H, t, J = 7.4 Hz); MS (ESI) 446.0 [M + H]⁺.

Step H. 2 - ((3R, 5R, 6S) -1 - ((S) -1-tert-butoxy-1-oxobutan-2-yl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) ) -2-oxopiperidin-3-yl) acetic acid [0096]

58366 Β1

<img file="RS58366B1_D0019.tif" />

To a rapidly stirred solution of 842 mg (1.67 mmol) of tert-butyl (2S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6) (4 -chlorophenyl) -2-oxopiperidin-1-yl) butanoate (reference example 1, step G) in a mixture of 7 ml of water, 5 ml of acetonitrile and 5 ml of SSC was added sodium periodate (1.43 g, 6.70 mmol), followed by ruthenium (111) chloride hydrate (37.8 mg, 0.168 mmol). After stirring vigorously for 18 h, the reaction mixture was acidified (10% citric acid) and diluted with EtOAc. The reaction mixture was filtered through celite and the filtrate was extracted with EtOAc. The combined organic layers were washed with saturated NaCl solution, dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase preparative HPLC (GeminiTM Prep C18 5 μιη column, Phenomenex, Torrance, CA; eluent: 60 to 80% acetonitrile + 0.1% TFA in water + 0.1% TFA, gradient elution) to obtained the title compound as a white solid.

³1 H NMR (400 MHz, CHLOROFORM-d) b ppm 7.35 (2 H, d, J = 8.6 Hz), 7.27-7.24 (3 H, m), 7.22-7.16 (1H, m), 7.18 (2H, d, J = 8.6 Hz), 4.85 (1H, d, J = 5.1 Hz), 3.36 (1H, dd, J = 8.6, 3.5 Hz), 3.18-3.14 (1 H, m), 2,922.80 (2 H, m), 2.79-2.72 (1 H, m), 2.32-2.18 (2H, m), 2.15-2.06 (1H, m), 1.63-1.50 (1H, m), 1.44 (9H, s) ), 0.67 (3H, t, J = 7.4 Hz); MS (ESI) 520.2 [M + H]⁺, 518.0 [Μ - Η] '.

The following Reference Example 2 was prepared as described in Reference Example 1, with the substitution of tert-butyl 2-bromopentanoate in step F with an appropriate amount of ethyl 2-bromobutanoate, ethyl 2-bromo-3-methylpentanoate, ethyl 2-bromopentanoate and ethyl 2-bromo-2-cyclopropyl acetate, respectively.

REFERENCE EXAMPLE 2 [0099]

<img file="RS58366B1_D0020.tif" />

58366 Β1

2 - ((3R, 5R, 6S) -5- (3-Chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-ethoxy-1-oxobutan-2-yl) -2-oxopiperidine -3-yl) acetic acid [0100] ^gN NMR (400 MHz, CHLOROFORM-d) b ppm 7.42-7.33 (3 H, m), 7.32-7.28 (3 H, m), 7.27-7.24 (2 H , m), 4.91 (1H, d, J = 3.5 Hz), 4.23-4.10 (2H, m), 3.54 (1H, dd, J = 8.6, 3.5 Hz), 3.22-3.16 (1H, m), 2.84-2.73 (3H, m), 2.38-2.30 (2H, m), 2, 05-1.97 (1H, m), 1.60-1.50 (1H, m), 1.27 (3H, t, J = 7.4 Hz), 0.70 (3H, t, J = 7.4 Hz); MS (ESI) 491.8 [M + H]⁺, 489.9 [Μ - Η] '.

REFERENCE EXAMPLE 3 [0101]

<img file="RS58366B1_D0021.tif" />

2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxo-1- (pentan-3-yl) piperidin-3-yl) acetic acid

Step A. (5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (2,4-dimethoxybenzyl) piperidin-2-one.

[0102]

<img file="RS58366B1_D0022.tif" />

58366 Β1 To a cloudy solution of (2,4-dimethoxyphenyl) methanol (97.00 g, 577 mmol) and pyridine (93 mL, 1153 mmol) in anhydrous Et₂O (1153 mL) was added thionyl chloride (116 mL, 1586 mmol) dropwise over 1 h at 0 ° C under nitrogen with mechanical stirring. After 1 h, the reaction mixture was poured into 2 L of ice water, and the layers were separated. The aqueous layer was extracted with Et₂O (2x11) and the organic layers were collected, washed with ice water (1.2 L), cold mixture sat. vod. NaCl solution / sat. vod. NaHCO₃(5: 1) (1.2 L), dried₄), filtered, and most of the ether was removed in vacuo at 12 ° C. Benzene (300 ml) was added and the mixture was concentrated to 12 ° C until 100 ml of benzene remained to give a solution of 1- (chloromethyl) -2,4-dimethoxybenzene. 80g (250 mmol) (5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) piperidin-2-one (Reference Example 1, Step E) was added portionwise over 20 minutes to the NaH mixture. (19.98 g, 500 mmol) in anhydrous DMF (400 mL) at 0 ° C under a nitrogen atmosphere. After this addition, the ice bath was removed and the mixture was stirred at rt for 1 h before cooling the solution to 0 ° C. A solution of 1- (chloromethyl) -2,4-dimethoxybenzene (107 g, 575 mmol) in benzene was added to the cooled solution, and the reaction mixture was allowed to cool to rt. After 16 h, the reaction mixture was poured into ice water (2 L) and extracted with EtOAc (3x11). The organic layers were combined, washed with water (3x11), sat. vod. NaCl solution (11), dried₄), filtered and concentrated in vacuo to give a thick yellow oil. Purification on Combiflash XL (gas pressure column chromatography, Teledyne Isco, Lincoln, NE) with four 330 g series connected columns and one 1.5 kg column and eluting with 35-40-45-50-55% EtOAc / hexanes, gave a very pale yellow oil. The oil was dissolved in benzene and the solvent was removed in vacuo and the residue dried in vacuo for 2 days to give the title compound as a white foam (105.8 g, 90%).

Step B. (5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (2,4-dimethoxybenzyl) -3-methylpiperidin-2-one.

[0104]

<img file="RS58366B1_D0023.tif" />

58366 Β1 (5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (2,4-dimethoxybenzyl) piperidin-2-one solution (reference example 3, step A) (140.34 g, 298 mmol) in anhydrous THF (994 mL) was evacuated by bubbling argon through solution for 20 minutes while the solution was cooled to -78 ° C. Iodomethane (23.32 mL, 373 mmol) was added followed by LHMDS (328 mL, 328 mmol) over 15 minutes. The reaction mixture was stirred for 15 minutes at -78 ° C after which the ice bath was removed and the mixture was stirred at rt for 12 hours. The reaction was quenched by the addition of sat. vod. NH₄CI these layers separated. The aqueous layer was extracted with Et₂O (2 x 500 ml) and the organic layers were collected, washed with sat. vod. NaCl solution, dried (MgSO 4)₄), filtered and concentrated in vacuo to give an orange oil. Purification (wet moiety with low DCM) on Combiflash Companion XL (gas column chromatography, Teledyne Isco, Lincoln, NE) with SiO₂with a 1.5 kg column and eluting with 4 I of each of the following mixtures: 15-20-25-30-35% EtOAc / hexanes the title compound was obtained as a very thick yellow oil and in the form of a mixture of C-3 diastereomers in a ratio of 3 , 7: 1.

Step C. (5R, 6S) -3-Allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (2,4-dimethoxybenzyl) -3-methylpiperidin-2-one.

[0106]

<img file="RS58366B1_D0024.tif" />

From a solution of (5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (2,4-dimethoxybenzyl) -3-methylpiperidin-2-one (Reference Example 3, step B, a mixture of C-3 diastereomers) (117.0 g, 242 mmol) in anhydrous THF (966 mL) was removed by gas bubbling argon through the solution for 20 minutes. Allyl bromide (105 mL, 1208 mmol) was added followed by LHMDS (725 mL, 725 mmol) over 20 minutes. The reaction mixture was heated at 40 ° C under argon for 5 hours. The reaction mixture was cooled to rt, and the reaction was quenched by the addition of sat. vod. NH₄CI (500 mL), after which the layers were separated. The aqueous layer was extracted with EtOAc (2x11) and the organic layers were combined, washed with sat. vod. NaCl solution (11), dried₄), filtered and concentrated in vacuo to give a red oil (180 g). By purification on a Biotage system

58366 Β1 (Charlotte, NC) with SiO₂ by a 1.5 kg column and eluting with a 10-30% EtOAc / hexanes mixture gave the title compound as a very pale yellow oil, in a mixture of (3S) :( 3R) diastereomers in a ratio of 3.7: 1.

Step D. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methylpiperidin-2-one [0108]

<img file="RS58366B1_D0025.tif" />

Solution (5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (2,4-dimethoxybenzyl) -3-methylpiperidin-2one (Reference Example 3, step C, a mixture of diastereomers) (105.87 g, 202 mmol) in TFA (778 mL, 1.01Ε + 04 mmol) was heated at 50 ° C for 2 h before concentrating the reaction mixture in vacuo. The residue was azeotroped with hexanes to remove all TFA. The deep purple oil, which contained some residues, was collected with a minimal amount of DCM, filtered and well evaporated with DCM. The filtrate was concentrated in vacuo to give a dark purple oil. Purification (wet application with a minimum amount of DCM) on a Biotage Isolera (Biotage, Charlotte, NC) system with a 1.5 kg column and eluting with 25-40% EtOAc / hexanes gave the title compound as a white solid.^gN NMR (500 MHz, CDCl 3)₃b ppm 1.30 (s, ZN), 2.06 (m, 2H), 2.52 (dd, J = 13.7 and 7.1 Hz, 1H), 2.60 (dd, J =

13.7 and 7.8 Hz, 1H), 3.06 (m, 1H), 4.50 (d, J = 10.7 Hz, 1H), 5.17 (m, 2H), 5.81 br s, 1H), 5.86 (m, 1H), 6.77 (d, J = 7.6 Hz, 1H), 6.96 (d, J = 8.3 Hz, 2H), 7.00 (s, 1H), 7.12 (t, J = 7.7 Hz, 1H), 7.17 (m, 1H), 7.20 (d, J = 8.3 Hz, 2H), [q]²²_d + 182.2 ° (c 1.55, CHCl 3)₃).

Step E. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1- (pentan-3-yl) piperidin-2-one [ 0110]

<img file="RS58366B1_D0026.tif" />

58366 Β1 In suspension 1.81 g (4.8 mmol) (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methylpiperidin-2one ( reference example 3, step D) in 3-bromopentane (17.6 ml) was added 967 mg (60% by weight in mineral oil, 24.2 mmol) of sodium hydride. The resulting milky white slurry was heated at 120 ° C for 20 hours, then more 3-bromopentane (5.1 mL) was added. After an additional 24 h at 120 ° C, the reaction mixture was cooled to room temperature and quenched with saturated aqueous ammonium chloride. The mixture was extracted with ethyl acetate (3 h) and the combined organic layers were dried over Na₂SO₄, then filtered and the filtrate was concentrated. Purification of the residue by gas pressure chromatography on silica gel (2 to 26% EtOAc / hexanes, gradient elution) gave the title compound as a white solid.

Step F. Synthesis of 2- (3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxo-1- (pentan-3-yl) piperidine- 3yl) acetic acid In a solution of 725 mg (1.63 mmol) (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1 - (pentan-3yl) piperidin-2-one (reference example 2, step A) in a mixture of acetonitrile (4 ml), carbon tetrachloride (4 ml) and water (5.9 ml) was added 1.40 g (6, 53 mmol) of sodium periodate, followed by 44 mg (0.20 mmol) of ruthenium (II) chloride hydrate. The dark brown biphasic mixture was stirred vigorously at room temperature for 21 h, after which it was acidified by the addition of 1 N HCl. The mixture was diluted with EtOAc and filtered through a pad of Celite® (JT Baker, Phillipsberg, NJ, diatomaceous earth). After filtration, the layers were separated, and the aqueous layer was extracted with EtOAc (1 h). The combined organic layers were washed with saturated aqueous sodium chloride (1 h), then dried over Na.₂SO₄, filtered, and the filtrate was concentrated. Purification of the residue by gas pressure chromatography on silica gel (0 to 25% MeOH / DCM, gradient elution) gave the title compound as a white solid.

^gN NMR (400 MHz, CDCl 3)₃b ppm 7.06-7.27 (5H, m), 6.90-7.01 (2H, m), 6.68 (d, 1H, J = 7.8 Hz), 4, 34 (1H, d, J = 10.4 Hz), 3.00-3.15 (2H, m), 2.63-2.79 (2H, m), 2.15-2.27 (1H, m), 1.85-2.03 (3H, m), 1.51 (s, 3H), 1.38-1.51 (2H, m), 0.95 H, t, J = 7.4 Hz), 0.50 (3 H, t, J = 7.4 Hz). Mass spectrum (ESI) m / z = 462 (M + 1).

REFERENCE EXAMPLE4

2 - ((3R, 5R, 6S) -5- (3-Chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1- ((S) -1-morpholinobutan-2-yl) -2-oxopiperidine -3yl) acetic acid [0113]

58366 Β1

<img file="RS58366B1_D0027.tif" />

Step Α. (S) -Methyl 2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1-yl) butanoate [ 0114]

<img file="RS58366B1_D0028.tif" />

To a solution of (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methylpiperidin-2-one (Reference Example 3, Step D) 4.00 g, 10.7 mmol) in 45 mL of DMF was added a dispersion of 60% sodium hydride in mineral oil (1.71 g, 42.7 mmol) at 0 ° C. After stirring for 20 minutes, methyl 2-bromobutanoate (6.15 mL, 53.4 mmol) was added at 0 ° C, and the resulting solution was stirred at 25 ° C for 12 h until the reaction was complete. Then sat. vod. NH solution₄CI after which the mixture was extracted with ethyl acetate. The combined organic layers were washed with water and sat. vod. NaCl solution, dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure. Purification of the residue by silica gel chromatography under gas pressure (eluent: 0 to 100% MTBE / hexanes, gradient elution), followed by separation of the individual stereoisomers by chiral SFC (flow rate: 65 ml / min on a ChiralPak® AD-H column (Diacel Inc ., Fort Lee, NJ) using a 3: 1 mixture of heptanes: IPA (0.1% DEA) / CO₂ as eluent) the title compound was obtained as the isomer with a faster elution.

³1 H NMR (400 MHz, CHLOROFORM-d) b ppm 7.23 (2 H, d, J = 8.4 Hz), 7.06-7.17 (2 H, m), 7.00 (3 H, t, J =

1.8 Hz), 6.77 (1 H, d, J = 7.6 Hz), 5.79 - 5.94 (1 H, m), 5.20 (1 H, d, J = 4.7 Hz) ), 5.17 (1H, s), 4.56 (1H, d, J =)

10.8 Hz), 3.73 (3 H, s), 3.25 - 3.37 (1 H, m), 3.18 (1 H, dd, J = 7.6 Hz, 4.9 Hz), 2.61 (2H, d, J = 7.4 Hz), 2.20 - 2.34 (1H, m), 2.09 - 2.19 (1H, m), 1.99 (1 H, d, J = 3.1 Hz), 1.57 -1.72 (1 H, m), 1.24 (3 H, s), 0.61 (3 H, t, J = 7.5) Hz); Mass spectrum (ESI) m / z = 474.1 [M + H]⁺.

Further elution gave:

58366 N1 (R) -methyl 2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1-yl) butanoate

<img file="RS58366B1_D0029.tif" />

as an isomer with a slower elution.

^gN NMR (400 MHz, CHLOROFORM-d) b ppm 7.22 (2 H, d, J = 8.0 Hz), 6.99-7.19 (4 H, m), 6.95 (1 H, t, J =

1.8 Hz), 6.71 (1H, d, J = 7.6 Hz), 5.81 - 5.95 (1H, m), 5.19 (1H, d, J = 2, 7 Hz), 5.16 (1 H, d, J = 1.0 Hz), 4.48 (1 H, d, J = 10.6 Hz), 3.67 (3 H, s), 3, 24 - 3.32 (1 H, m), 3.20 (1 H, dd, J = 7.8 Hz, 6.1 Hz), 2.61 - 2.72 (1 H, m), 2, 49 - 2.60 (1H, m), 1.91 - 2.21 (4H, m), 1.27 (3H, s), 1.00 (3H, t, J = 7.5) Hz); MS (ESI) m / z = 474.1 [M + H]⁺.

Step B. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((R) -1-hydroxybutan-2-yl) -3-methylpiperidine -2-on [0116]

<img file="RS58366B1_D0030.tif" />

U (S) -methyl 2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-lyl solution) (R) -methyl 2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1-yl) butanoate (1.73 g, 3.64 mmol) (mixture of stereoisomers from Reference Example 4, step A) in 27 ml Et₂A solution of lithium tetrahydroborate in THF (0.238 mL, 7.28 mmol) at 0 ° C was added to 9 mL of THF. The resulting solution was stirred at 25 ° C for 2 h. The reaction was quenched (10% citric acid), extracted (2 h EtOAc) and washed (1 h sat. Aqueous NaCl solution). The combined organic layers were washed with sat. vod. NaCl solution, dried over Na₂SO4, filtered, and the filtrate was concentrated under reduced pressure. The rest is

58366 Β1 purified by chromatography on silica gel under gas pressure (eluent: 0-60% EtOAc in hexanes) to give the title compound as an isomer with faster elution.

³1 H NMR (400 MHz, CHLOROFORM-d) b ppm 0.99 (t, J = 7.4 Hz, 3 H), 1.29 (s, 3 H), 1.79 - 2.03 (m, 4 H), 2.62 (d, J = 7.4 Hz, 2 H), 2.80 - 2.85 (m, 1H), 3.05 - 3.16 (m, 1H), 3, 40 - 3.49 (m, 2H), 4.33 (d, J = 10.4 Hz, 1H), 5.13 - 5.22 (m, 2H), 5.79 - 5.95 (m, 1H), 6.7 (d, J = 7.6 Hz, 1H), 6.85 - 6.97 (m, 3H), 7.08 - 7.15 (m, 1H) ), 7.17 - 7.19 (m, 1H), 7.23 (d, J = 8.6 Hz, 2H); Mass spectrum (ESI) m / z = 446 (M + 1).

Further elution gave:

(3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-hydroxybutan-2-yl) -3-methylpiperidin-2- he

<img file="RS58366B1_D0031.tif" />

as an isomer with a slower elution.

³1 H NMR (400 MHz, CHLOROFORM-d) b ppm 0.68 (t, J = 7.5 Hz, 3 H), 1.27 (s, 3 H), 1.38 -1.52 (m, 1 H), 1.90 - 2.08 (m, 4 H), 2.61 (d, J = 7.4 Hz, 2 H), 3.10 - 3.25 (m, 2 H), 3, 59 - 3.68 (m, 2H), 4.46 (d, J = 10.2 Hz, 1H), 5.18 (dd, J = 13.7, 1.8 Hz, 2H), 5.79 - 5.93 (m, 1H), 6.72 (d, J = 7.6 Hz, 1H), 6.93 - 7.04 (m, 2H), 7.09 7, 13 (m, 1H), 7.15 - 7.20 (m, 1H), 7.24 (d, J = 8.6 Hz, 2H); Mass spectrum (ESI) m / z = 446 (M + 1).

StepC. (S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1-yl) butanal ]

<img file="RS58366B1_D0032.tif" />

To a solution of 218 mg (0.49 mmol) (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-hydroxybutane- 2-yl) -3-methylpiperidin-2-one (reference example 4, step B) in a mixture of water (13.20 μΙ, 0.733 mmol) and DCM (4883 μΙ) was added 1,1-tris acetoxy) -1,1-dihydro-1,2-benzodioxol-3- (1H) one

Martin periodin) (311 mg, 0.733 mmol) at room temperature. The reaction was monitored by LCMS, a

58366 Β1 several smaller portions of additional periodinan were added until the reaction was complete. The reaction was stopped (2 ml of 1M Na₂S₂Oh₃), extracted (2 h DCM), after which the combined organic layers were washed with sat. NaHCO solution₃ (2 h), sat. NaCl solution, dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure. Purification of the residue by gas pressure chromatography on silica gel (20 to 35% EtOAc / hexanes, gradient elution) gave the title compound.

Step D. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1- ((S) -1-morpholinobutan-2-yl) piperidine- 2on.

[0120]

<img file="RS58366B1_D0033.tif" />

To a solution of 100 mg (0.225 mol) (S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl- Morpholine (200 ml, 2,297 mmol), acetic acid (1,288 μΙ, 0,023 mmol) and sodium triacetoxyborohydride (95 mg, 2) oxopiperidin-1-yl) butanal (reference example 4, step C) in DCE (2420 ml) 0.450 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction was stopped. sodium bicarbonate solution and extracted with DCM (2x10 ml). The combined organic layers were washed with sat. NaCl solution, dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure to give the crude title compound as an oil.

Step E. 2- (3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1- ((S) -1-morpholinobutan-2-yl) - 2-oxopiperidin-3-yl) acetaldehyde [0122]

58366 Β1

<img file="RS58366B1_D0034.tif" />

In a round balloon filled with (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1- ((S) -1-morpholinobutane-2- yl) THF (2 mL) was added to piperidin-2-one (Reference Example 4, Step D) (125 mg, 0.242 mmol). Approximately 1 ml of water was added dropwise until the solution became and remained cloudy with gentle stirring. T-BuOH (0.350 mL) was added dropwise until the solution became homogeneous. NMO (42.6 mg, 0.364 mmol), apotome and osmium tetroxide, 4% by weight in water (1 drop from a glass Pasteur pipette) were added. The reaction mixture was stirred at room temperature for 16 hours. Another drop of osmium tetroxide, 4 wt. % in water. After 5 hours, two more drops of osmium tetroxide, 4 wt. % in water, and the reaction mixture was stirred at room temperature for an additional 16 hours. Sodium periodate (145 mg, 0.679 mmol) was added and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with ethyl acetate (10 mL) and water (10 mL) and filtered. The aqueous filtrate layer was extracted with additional ethyl acetate (10 mL), after which the combined organic layers were washed with sat. vod. NaCl solution, dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the title compound.

Step F. 2- (3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1- ((S) -1-morpholinobutan-2-yl) - 2-Oxopiperidin-3-yl) acetic acid [0124]

<img file="RS58366B1_D0035.tif" />

58366 Β1 U Solution 2 - ((3Κ, 5Κ, 65) -5- (3-θίήΙθΓηίΙ) -6- (4-θίθηϊΙ) -3-θΐϊΙηθΐϊΙ-1 - ((5) -1-ηηοΓίοΙϊηοόυΐ3η-2- ϊΙ) -2-Oxopiperidin-3-yl) acetaldehyde (Reference Example 4, Step E) (125 mg, 0.242 mmol) in acetone (2 mL) was added 3 mL of a CrO mixture₃ in water (2 ml) and concentrated H₂SO₄ (1 ml). The reaction mixture was stirred at room temperature for 2 hours and then diluted with water (10 ml) and ethyl acetate (10 ml), after which the layers were separated. The aqueous layer was extracted with additional ethyl acetate (10 mL). The combined organic layers were concentrated under reduced pressure. The residue was purified by reverse phase preparative HPLC (column: Gemini-NX C18 5um; Phenomonex, Torrance, CA; eluent: 0 to 100% MeCN + 0.1% TFA in water + 0.1% TFA) to give the title unification.

1 NMR (400 MHz, CHLOROFORM-d) b ppm 0.57 (t, J = 7.53 Hz, 1 H) 1.26 (s, 1 H) 1.39 (s, 3 H) 1.54 - 1.70 (m, 1H) 1.72 -1.89 (m, 1H) 2.02-2.27 (m, 3H) 2.49 (br. S. 2H) 2.69 ( No. s, 2 H) 2.82 (m, 2 H) 3.02 (No. s, 2 H) 3.13 - 3.30 (m, 2 H) 3.74 - 3.93 (m, 4 H) 4.47 - 4.72 (m, 1 H) 6.75 (d, J = 7.82 Hz, 1 H) 6.96 (t, J = 1.86 Hz, 1 H) 7, 01 (br.s, 1H) 7.04 - 7.17 (m, 3H) 7.22 (d, J = 8.41 Hz, 2H); Mass spectrum (ESI) m / z = 533 [M + H]⁺.

REFERENCE EXAMPLE 5

2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1- (S) -1- (methylsulfonamido) -butan-2-yl) -2-Oxopiperidin-3-yl) acetic acid [0126]

<img file="RS58366B1_D0036.tif" />

Step A. (3R, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (S) -1 - ((4-methoxybenzyl) amino) butane- 2-yl) -3-methylpiperidin-2-one

58366 Β1

<img file="RS58366B1_D0037.tif" />

To a solution of (S) -2 - ((5S, 5S, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1-yl) butanal (300 mg, 0.675 mmol; reference example 4, step C) and (4-methoxyphenyl) methanamine (131 μΙ, 1.01 mmol) in DCE (4.5 mL) was added sodium triacetoxyborohydride (429 mg, 2 , 03 mmol) at 0 ° C in several portions. After the mixture was stirred at 25 ° C for 18 h, the reaction was quenched by the addition of ice-cold saturated aqueous NaHCO₃ and extracted (2 x DCM). The combined organic layers were washed (1 h sat. With aqueous NaCl solution) and concentrated under reduced pressure to provide the title compound as a yellow film. The product was used in the next step without further purification.

Step B. (S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1-yl) butane-lamonium 2,2,2-trifluoroacetate [0129]

<img file="RS58366B1_D0038.tif" />

To a solution of (3R, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (S) -1- (4-methoxybenzylamino) butan-2-yl ) -3-methylpiperidin-2-one (370 mg, 0.654 mmol; Reference Example 5, Step A) in acetonitrile (8.0 mL) and water (1.6 mL) was added cerium ammonium nitrate (2.87 g, 5.23 mmol) at 25 ° C. After stirring for 2 days, the reaction was quenched (sat. Aqueous NaCl), the mixture extracted (3 h EtOAc) and washed (1 h sat. Aqueous NaCl). The combined organic layers were dried (Na₂SO₄) and concentrated under reduced pressure. Purification by RP-HPLC (35 to 70% MeCN / H₂O (0.1% TFA), gradient elution) gave the title compound as a pale yellow powder.

58366 Β1

StepC. N- (S) -2 - ((3S, 5R, 6S) -3-Allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1-yl) butyl) methanesulfonamide [0131]

<img file="RS58366B1_D0039.tif" />

(S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1-yl) butane-lamonium 2,2,2-trifluoroacetate (74 mg, 0.14 mmol; reference example 5, step B) was dissolved in DCM at 0 ° C and 2 N lithium hydroxide was added thereto (0.34 ml, 0.68 mmol), after which the resulting solution was stirred for 5 min at 0 ° C. The solution was extracted (2 h DCM), washed (sat. Aqueous NaCl solution), dried (Na₂SO₄) and concentrated under reduced pressure to give the free amine. To a solution of this free amine in DMF (0.34 mL) was added methanesulfonyl chloride (53 μΙ, 0.68 mmol) and pyridine (66 μΙ, 0.820 mmol), respectively, at 0 ° C. After stirring at 25 ° C overnight, the reaction mixture was acidified (10% citric acid) and extracted (2 h EtOAc), then washed (sat. Aqueous NaCl solution). The combined organic layers were dried (Na₂SO₄) and concentrated under reduced pressure. Purification by RP-HPLC (45 to 80% MeCN / H₂O (0.1% TFA), gradient elution) gave the title compound as a white powder.

Step D. 2- (3R, 5R, 6S) -5- (3-Chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1- (S) -1- (methylsulfonamido) butane-2- yl) -2-oxopiperidin-3-yl) acetic acid In a rapidly stirred solution of N - ((S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1-yl) butyl) methanesulfonamide (34 mg, 0.064 mmol, reference example 4, step C) in a mixture of water (0.55 ml), acetonitrile (0.37 ml) and CCI₄ Sodium periodate (55 mg, 0.26 mmol) and ruthenium (III) chloride hydrate (1.5 mg, 6.5 pmol) were added (0.37 mL). After stirring vigorously for 20 h, the reaction mixture was acidified (10% citric acid) and diluted with EtOAc. The insoluble material was separated by filtration through a Celite® cake (JT Baker, Phillipsberg, NJ, diatomaceous earth). The filtrate was extracted (2 h EtOAc) and washed (sat. Aqueous NaCl solution). The combined organic layers were dried (Na₂SO₄) and concentrated under reduced

58366 Β1 by pressing. Purification by RP-HPLC (40 to 70% MeCN / H₂O (0.1% TFA), gradient elution) gave the title compound as a white foam.

^gN NMR (400 MHz, CHLOROFORM-d) b ppm 7.25 (2 H, d, J = 8.2 Hz), 7.10-7.18 (2 H, m), 7.00-7.10 (2H, m), 6.97 (1H, s), 6.83 (1H, d, J = 7.2 Hz), 4.99 - 5.20 (1H, m), 4, 87- 4.97 (1H, m), 4.74 (1H, d, J = 10.4 Hz), 3.44 - 3.65 (1H, m), 3.10 - 3.33 (2H, m), 3.02-3.09 (1H, m), 2.99 (3H, s), 2.96 (1H, s), 2.77 (1H, s) , 2.36 (1H, s), 1.94 - 2.05 (1H, m), 1.77 -1.92 (1H, m), 1.52 -1.59 (1H, m), 1.50 (3H, s), 0.58 (3H, t, J = 7.3 Hz); MS (ESI) 541.0 [M + H]⁺, 539.0 [MH] &amp; lt; - &amp; gt ;.

REFERENCE EXAMPLE 6

2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((3S, 4R) -5-hydroxy-4,5-dimethylhexan-3-yl) -3-methyl-2-oxopiperidin-3-yl) acetic acid or 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (3S, 4S) - 5-hydroxy-

4,5-Dimethylhexan-3-yl) -3-methyl-2-oxopiperidin-3-yl) acetic acid [0134]

<img file="RS58366B1_D0040.tif" />

<img file="RS58366B1_D0041.tif" />

Step A. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1- ((S) -2-oxopentan-3-yl) piperidin-2-one [0135]

<img file="RS58366B1_D0042.tif" />

To a solution of oxalyl dichloride (78 μΙ, 0.87 mmol) in DCM (1.5 mL) at -60 ° C was added a solution of DMSO (93 μΙ, 1.30 mmol) in DCM (1 , 5 ml) in an atmosphere of N₂. After stirring for 2 minutes, a solution of (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (3S) -2-hydroxypentan-3-yl was added. ) -3-methylpiperidin-2-one prepared in reference example 7, step C (200 mg, 0.434 mmol) in DCM (1.5 ml) to give

58366 Β1 solution stirred for 15 minutes at -60 ° C. Triethylamine (305 μΙ, 2.17 mmol) was then added to the reaction mixture. After stirring for 20 minutes, the reaction was quenched (with water), the reaction mixture was extracted (2 h with EtOAc) and washed (2 h with aqueous NaCl solution). The combined organic layers were dried (Na₂SO₄) and concentrated under reduced pressure. Purification by gas chromatography (SiO2, 24 g, 20% and 30% EtOAc / hexanes) gave the title compound as a colorless foam.

Step B. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-methoxy-2-methylpent-1-ene -3-yl) -3methylpiperidin-2-one [0137]

<img file="RS58366B1_D0043.tif" />

(Methoxymethyl) triphenylphosphonium chloride was dried at 80 ° C in vacuo for 2 h. To a solution of dried (methoxymethyl) triphenylphosphonium chloride (673 mg, 1.96 mmol) in THF (3.5 mL) was added 0.5 M KHMDS in toluene (3.49 mL, 1.75 mmol) at -78 ° C. Dissolution led to the appearance of red blood.

After the addition, the reaction mixture was stirred at 0 ° C for 30 min and a solution (3S, 5R, 6S) was added dropwise.

3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1- ((S) -2-oxopentan-3-yl) piperidin-2-one obtained as described above in step B (200 mg, 0.436 mmol) in THF (3.5 mL), at 0 ° C. The reaction mixture was allowed to warm to rt and stirred for 1.5 h. The reaction was then quenched (sat. NH solution₄Cl), extracted (2 h EtOAc) and washed (aqueous brine). The combined organic layers were dried (Na₂SO₄) and concentrated under reduced pressure. Purification by combined pressure chromatography (SiO₂, 24 g, 15% and 20% EtOAc / hexanes) gave the title compound as a colorless film.

Step C. (2S, 3S) -3- (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1- yl) -2-methylpentanal and (2R, 3S) -3- (35,5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidine- 1-yl) -2-methylpentanal [0139]

58366 Yes

<img file="RS58366B1_D0044.tif" />

<img file="RS58366B1_D0045.tif" />

To a solution of (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (S) -1-methoxy-2-methylpent-1- en-3-yl) 3-methylpiperidin-2-one obtained above in step B (179 mg, 0.368 mmol) in acetonitrile (3.7 mL) was added 3 N hydrochloric acid (1.5 mL, 4.5 mmol) on st. After stirring at rt for 1.5 h, the reaction mixture was extracted (2 h EtOAc) and washed (2 h aqueous brine). The combined organic layers were dried (Na₂SO₄) and concentrated under reduced pressure to give the title compound as a mixture of isomers (dr = 7: 3) as a pale yellow film.

Step D. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1- (3S, 4R) -4-methyl-5- oxohexan-3-yl) piperidin-2-one (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1- (3S, 4S) -4-methyl-5-oxohexan3-yl) piperidin-2-one [0141]

<img file="RS58366B1_D0046.tif" />

<img file="RS58366B1_D0047.tif" />

To a solution of (2S, 3S) -3- (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidine- 1-yl) 2-methylpentanal and (2R, 3S) -3- (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2 -oxopiperidin-1-yl) 2-methylpentanal obtained as previously described in step C (177 mg, 0.375 mmol) in THF (3.076 ml) was added 1.4 M methylmagnesium bromide in toluene and THF (75 : 25) (0.803 mL, 1.12 mmol) at 0 ° C. The reaction mixture was allowed to warm to rt and stirred for 2 h. The reaction was then quenched (sat. NH solution₄Cl), extracted (2 h EtOAc) and washed (aqueous brine). The combined organic layers were dried (Na₂SO₄) and concentrated under reduced pressure to give the crude secondary alcohol product.

58366 Β1 To a solution of the crude secondary alcoholic product (183 mg, 0.375 mmol) in DCM (4.2 mL) was added water (14 μΙ, 0.75 mmol) and Des-Martin periodinanan (196 mg, 0.462 mmol). ), in that order. After stirring at rt overnight, the reaction was quenched (1 M aq₂S₂Oh₃), extracted (2 h DCM) and washed (2 h sat. NaHCO 3)₃ and 1 h water. salt solution). The combined organic layers were dried (Na₂SO₄) and concentrated under reduced pressure. Purification of the residue by chromatography on silica gel (24 g SiO₂, 13%, 27% and 37% EtOAc / Neh) gave a less polar and more polar isomer, respectively.

Less polar isomer: ^gN NMR (400 MHz, CHLOROFORM-d) b ppm 7.25 (2 H, d, J = 8.0 Hz), 7.03 7.17 (4 H, m), 6.94 - 6.99 ( 1 H, m), 6.81 - 6.87 (1 H, m), 5.12 - 5.23 (2 H, m), 4.58 (1 H, d, J = 10.8 Hz) , 3.14 (1H, s), 2.66 (1H, s), 2.58 (2H, d, J = 7.4 Hz), 2.22 (3H, s), 1, 81 (1H, d, J = 4.3 Hz), 1.75 (1H, d, J = 7.2 Hz), 1.51 -1.65 (2 H, m), 1.19 ( 3 H, s), 1.00 (3 H, d, J = 7.2 Hz), 0.30 (3 H, t, J = 1 J Hz); MS (ESI) 486.1 [M + H]⁺.

More polar isomer: ^:1 H NMR (400 MHz, CHLOROFORM-d) b ppm 7.22 - 7.27 (2 H, m), 7.01 - 7.17 (4 H, m), 6.89 - 6.95 (1 H , m), 6.71 (1H, dt, J = 7.5, 1.3 Hz), 5.81 - 5.93 (1H, m), 5.17 - 5.25 (2H, m), 4.32 (1H, d, J = 10.8 Hz), 3.50 (1H, no. s.), 3.23 - 3.32 (1H, m), 3.06 (1H, br. S.), 2.60 - 2.66 (2H, m), 2.13 - 2.20 (3H, m), 1.91 - 2.01 (2H, m) ), 1.64 -1.70 (2 H, m), 1.28 - 1.32 (3 H, m), 1.13 (3 H, d, J = 7.0 Hz), 0.34 (3 H, t, J = 7.5 Hz); MS (ESI) 486.1 [M + H]⁺.

Step E. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((3S, 4R) -5-hydroxy-4,5-dimethylhexane- 3-yl) -3-methylpiperidin-2-one or (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((3S, 4S) -5- hydroxy-4,5-dimethylhexan-3-yl) -3-methylpiperidin-2-one [0146]

<img file="RS58366B1_D0048.tif" />

To a solution of the less polar isomer obtained as previously described in Reference Example 6, step D (96 mg, 0.20 mmol) in THF (2.0 mL) was added 1.4 M methylmagnesium bromide in toluene and THF (75:25) (423 μΙ, 0.592 mmol) at 0 ° C. The reaction mixture was then allowed to warm to rt and stirred overnight. The reaction was stopped (sat. NH solution₄Cl), extracted (2 h EtOAc) and washed (aqueous brine). The combined organic layers were dried (Na₂SO₄) and concentrated under reduced pressure.

58366 Β1

Purification by combined gas pressure chromatography (combi-flash chromatography) (12 g SiO₂, 30% EtOAc / Neh) gave the title compound as a single isomer.

Step F. 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((3S, 4R) -5-hydroxy-4,5-dimethylhexane-3 -yl) -3-methyl2-oxopiperidin-3-yl) acetic acid or 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - (3S, 4S) -5-hydroxy-

4,5-Dimethylhexan-3-yl) -3-methyl-2-oxopiperidin-3-yl) acetic acid [0148]

<img file="RS58366B1_D0049.tif" />

<img file="RS58366B1_D0050.tif" />

The title compound was obtained from (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((3S, 4R) -5hydroxy-4,5 -dimethylhexan-3-yl) -3-methylpiperidin-2-one or (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -

1- (3S, 4S) -5-hydroxy-4,5-dimethylhexan-3-yl) -3-methylpiperidin-2-one (Reference Example 6, Step E) in a procedure similar to that described for Reference Example 5 , step D.

^gN NMR (400 MHz, CHLOROFORM-d) b ppm 7.01 - 7.27 (6 H, m), 6.96 (1 H, t, J = 1.7 Hz), 6.70 (3 H, d, J = 7.6 Hz), 4.59 (1H, d, J = 9.8 Hz), 3.63 - 3.91 (1H, m), 3.14 (1H, s) , 2.98 (1H, d, J = 14.5 Hz), 2.72 (1H, d, J = 14.5 Hz), 1.98 - 2.21 (2H, m), 1 , 84 -1.96 (1H, m), 1.56 -1.69 (2H, m), 1.48 (3H, s), 1.15 (3H, s), 1.06 (3H, s), 0.75 (3H, no. S.), 0.28 (3H, no. S.); MS (ESI) 520.2 [M + H]⁺, 518.2 [MH] &amp; lt; - &amp; gt ;.

REFERENCE EXAMPLE 7

2- (3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxo-1 - ((S) -2-oxopentan-3-yl) piperidine-3-acetic acid [0150]

58366 Yes

<img file="RS58366B1_D0051.tif" />

Step Α. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-hydroxybutan-2-yl) -3-methylpiperidin-2- on [0151]

<img file="RS58366B1_D0052.tif" />

Cl U 1004 g solution (1.47 mol) (3S, 5R, 6S) -3-allyl-1- ((S) -1 - ((tert-butyldiphenylsilyl) oxy) -butan-2-yl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methylpiperidin-2-one (reference example 9, step E) in THF (3.0 ml) was added 2.50 I (2.50) mol) 1 M solution of TBAF in THF, over a period of 10 minutes. The orange solution was stirred at room temperature for 4 h. The reaction was quenched with 1N HCl (3 L) and extracted with EtOAc (3 h). The combined organic layers were washed with a mixture of water and saturated aqueous sodium chloride (3: 1) (4 h) followed by saturated aqueous sodium chloride (1 h). The organic layer was dried over Na₂SO₄, filtered, and the filtrate was concentrated. Purification of the residue by chromatography (Biotage® Snap ™ column; Biotage, LLC, Charlotte, NC), 10 to 50% EtOAc / hexanes where EtOAc contains 2% MeCN, gradient elution) gave the title compound as a white foam.

Step B. (S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1-yl) butanal [0153]

<img file="RS58366B1_D0053.tif" />

58366 Β1 U solution 428 g (959 mmol) (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-hydroxybutane2 -yl) -3-methylpiperidin-2-one (Reference Example 7, Step A) in dichloromethane (4.55 L) was added 2.59 ml (1.44 mol) of water. A solution of 610 g (1.44 mol) of Des-Martin periodinane in dichloromethane (4.55 L) was added slowly over a period of 25 minutes so that the internal temperature of the reaction mixture did not exceed 25 ° C. The white slurry was stirred for 2.5 ha then the reaction was quenched by careful, slow addition of saturated aqueous sodium thiosulfate (5.2 L) so that the internal temperature of the reaction mixture did not exceed 30 ° C. Water was added and the mixture was extracted with dichloromethane (3 h). The combined organic layers were washed with saturated aqueous sodium bicarbonate solution (4 h) followed by saturated aqueous sodium chloride (1 h), dried over Na₂SO₄, filtered, and the filtrate was concentrated to give a yellow, oily solid. A mixture of ethyl ether and DCM was added, and the precipitated solid was separated by filtration. The precipitation / filtration process is repeated. The filtrate was concentrated to give the title compound as a white solid. The crude product was used directly in the next step.

Step C. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((3S) -2-hydroxypentan-3-yl) -3-methylpiperidine -2on [0155]

<img file="RS58366B1_D0054.tif" />

To a solution of 399 g (899 mmol) (S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl- 2-Oxopiperidin-1-yl) butanal (Reference Example 7, Step B) in THF (9 I) at 0 ° C was slowly added 1.93 I (2.70 mol) to a 1.4 M solution of methymagnesium bromide 75:25 in toluene / tetrahydrofuran for 30 minutes to keep the internal temperature of the reaction mixture below 6 ° C. The yellow solution was warmed to room temperature and stirred for 1.5 h. Thereafter, the reaction mixture was cooled to 0 ° C and quenched by careful, slow addition of saturated aqueous ammonium chloride (4.6 L) to keep the internal temperature of the reaction mixture below 15 ° C. The mixture was warmed to room temperature, ethyl acetate was added, and the layers were separated. The aqueous layer was extracted with EtOAc (2 h). The combined organic layers were washed with water (1 h) and then saturated aqueous sodium chloride (1 h), dried over Na₂SO₄, filtered, and the filtrate was concentrated to give a yellow oil. Purification of the residue by silica gel chromatography (Biotage® Snap® column; Biotage, LLC, Charlotte, NC), in a gradient

58366 Using a system of 5% acetone / 5% EtOAc / 90% hexanes to 5% acetone / 29% EtOAc / 66% hexanes) the title compound was obtained as a white solid.

Step D. 2- (3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxo-1- (S) -2-oxopentan-3 -yl) piperidin-3-yl) acetic acid In solution (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((3S) -2 -hydroxypentan-3-yl) -3methylpiperidin-2-one (reference example 7, step C) (130.30 g, 283 mmol) and NalO₄ (61.5 g) in EtOAc (630 ml), CH₃CN (630 mL) and water (935 mL) at 18 ° C were added ruthenium (III) chloride hydrate (1,404 g, 6.23 mmol). The rest of NalO₄ (307.5 g) was added in five portions over 2.5 hours while maintaining the temperature below 26 ° C. 15 minutes after the final addition of NalO₄ the cooling bath was removed, and the reaction mixture was stirred at room temperature for 50 minutes. The sand-colored reaction mixture was filtered on a Bihner funnel and washed with EtOAc (500 mL) and CH₃CN (500 ml). The layers were separated, and the aqueous layer was extracted with EtOAc twice. The organic layers were combined, washed with 10% aq. NaHSO₃ (3x11), aqueous salt solution (1

l), dried₂SO₄), the liquid was drained and the mixture was concentrated in vacuo to give a green oil. The substance was dissolved in a minimum amount of DCM and purified using two 1.5 kg Biotage® Snap columns (Biotage, LLC, Charlotte, NC) eluting with 10-50% (15% MeOH / acetone) / hexanes to a pale pink foam (109.67 g) was obtained.

^g1 H NMR (400 MHz, CHLOROFORM-d) brrt 7.25 (2 H, d, J = 8.2 Hz), 6.93 - 7.18 (5 H, m), 6.73 - 6.80 1 H,

m), 4.47 (1H, d, J = 10.6 Hz), 3.28 (1H, ddd, J = 13.4, 10.5, 3.0 Hz), 3.16 (1 H, dd, J = 7.0, 5.5 Hz), 2.73 3.00 (2 H, m), 2.28 - 2.40 (1 H, m), 2.18 - 2.25 (1H, m), 2.16 (3H, s), 2.11 - 2.15 (1H, m), 1.83 (1H, ddd, J = 14.3, 7.8, 5.7 Hz), 1.47 (3 H, s), 0.64 (3 H, t, J = 7.5 Hz); MS (ESI) 476.2 [M + H]⁺.

REFERENCE EXAMPLE8

2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((2S, 3S) -2-hydroxypentan-3-yl) -3-methyl-2 -oxopiperidine-3-acetic acid [0158]

<img file="RS58366B1_D0055.tif" />

58366 Β1 U solution 3.86 g (8.13 mmol) 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxo- A 1 M solution of sodium tri-sec-butylborohydride (N-Selectride®) was added to 1 - ((S) -2-oxopentan-3-yl) piperidin-3-yl) acetic acid (reference example 7) in THF (102 ml). , Aldrich, St. Louis, MO) in THF (16.26 mL, 16.26 mmol) at -78 ° C dropwise over a period of 5 min. After stirring at -78 ° C for 30 minutes, the reaction mixture was allowed to warm to rt. The reaction mixture was stirred at rt for 2 h, then quenched (sat. NH solution₄CI), the mixture was extracted (3 h EtOAc) and washed (3 h ice-cold 1 N aqueous HCl solution and 3 h saturated aqueous sodium chloride). The combined organic layers were dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure. The crude material was purified by chromatography on a Biotage Isolera gas purification system (Biotage, Charlotte, NC) (columns 2 x 1500 g, using a gradient of 10-30% (15% MeOH / acetone) in hexanes. then recrystallized from 3: 1 hexanes / acetone (8 ml / g) to give the title compound.

³1 H NMR (500 MHz, DMSO-d 6) δ ppm 0.30 (t, J = 7.6 Hz, 3 H), 1.00 (d, J = 6.3 Hz, 3 H), 1.26 s, 3H (), 1,411.49 (m, 1H), 1.55-1.64 (m, 1H), 2.04-2.15 (m, 2H), 2.29-2, 33 (m, 1H), 2.48 (d, J = 13.7 Hz, 1H), 2.87 (d, J = 13.7 Hz, 1H), 3.35-3.40 m, 1H), 4.01-4.06 (m, 1H), 4.77 (d, J = 10.9 Hz, 1H), 4.80 (br. s. 1H), 6.936 , 95 (m, 1H), 7.08-7.10 (m, 1H), 7.17-7.27 (m, 4H), 7.33 (d, J = 8.4 Hz, 2H), 12.42 (br s, 1H); MS (ESI) 478.2 [M + H]⁺, 476.2 [MH] &amp; lt; - &amp; gt ;. [α] D = + 110 ° (Τ = 23 ° C, MeOH, c = 0.51).

Alternatively, the title compound can be obtained from (3S, 5R, 6R) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one which was prepared according to reference example 15, step F.

(3S, 5R, 6R) -3-Allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one were added to a 10 ml round flask. mg, 1,998 mmol), (2S, 3S) -3-aminopentan-2-ol hydrochloride (837 mg, 6.00 mmol, reference: J. Org Chem., 2003, 68 (26), 9948) and triethylamine (1966) μΙ, 13.99 mmol). The vessel was equipped with a reflux condenser and heated to 85 to 95 ° C for 2 days. The reaction mixture was cooled to RT and diluted with ethyl acetate, then washed with 1N HCl (2 x 20 mL) and aqueous brine. The organic layer was dried over MgSO 4₄, filtered, and the filtrate was concentrated. Purification by column chromatography using 40 to 50% ethyl acetate in hexane gave (S) -2 - ((2R, 3R) -2- (3-chlorophenyl) -3- (4-chlorophenyl) -3-hydroxypropyl) -N- (2S, 3S) -2-hydroxypentan-3-yl) -2-methylpent-4-enamide.

³1 H NMR (500 MHz, DMSO-d 6) δ 7.17 (m, 2H), 7.16 (m, 1H), 7.14-7.08 (series m, 2H), 6.97 (m, 2H) ), 6.88 (br d, J = 6.9 Hz, 1H), 5.96 (d, J = 8.3 Hz, 1H), 5.65 (ddt, J = 17,4,10,2 , 7.2 Hz, 1H), 5.07 (dd J = 10.3, 1.0 Hz, 1H), 5.02 (d, J = 17.6, 1H), 4.75 (t, J = 4.2 Hz, 1H), 3.79 (m, 1H), 3.66 (ddd, J = 8.8, 5.9, 4.2 Hz, 1H), 3.30 (d, J = 3.4 Hz, 1H), 3.03 (dt, J = 6.9, 5.4 Hz, 1H), 2.37 (dd, J = 13.9, 7.3 Hz, 1H), 2, 32 (dd, J = 14.7, 5.6 Hz, 1H), 2.12 (dd, J = 13.7, 7.1 Hz, 1H), 2.01 (d, J = 4.7 Hz) ,, 1H), 1.83 (dd, J = 14.7, 7.3 Hz, 1H), 1.58 (m,

58366 Β1

1Η), 1.42 (ddq, J = 14.9, 8.6, 7.3 Hz), 1.14 (s, ZN), 1.14 (d, J = 6.4 Hz, ZN), 0.90 (t, J = 7.3 Hz, ZN) ppm. LC / MS (M + H) = 478.2.

In solution (S) -2 - ((2R, 3R) -2- (3-chlorophenyl) -3- (4-chlorophenyl) -3-hydroxypropyl) -N - ((2S, 3S) -2-hydroxypentane- Ammonium molybdate (NH) was added to 3-yl) -2-methylpent-4-enamide (127 mg, 0.265 mmol) in toluene (5309 μΙ)₄)₂MoO₄) (5.20 mg, 0.027 mmol), and the mixture was heated at reflux under DinStark reaction conditions overnight. The reaction mixture was cooled to room temperature, diluted with ethyl acetate and washed with sat. NaHCO₃ and aqueous brine. The organic layer was dried over MgSO 4₄, filtered, and the filtrate was concentrated. Purification by column chromatography using 20 to 40% ethyl acetate in hexane gave (1R, 2R, 4S) -2- (3-chlorophenyl) -1- (4-chlorophenyl) -4- (4S, 5S) -4- ethyl-5-methyl-4,5-dihydrooxazol-2-yl) -4-methylhept-6-en-1-ol.

^g1 H NMR (500 MHz, DMSO-d 6) δ 7.25 (m, 2H), 7.12 (m, 2H), 7.07 (br s, 1H), 7.05 (m, 2H), 6, 96 (br d, J = 6.8 Hz, 1H), 5.53 (ddt, J = 17.4, 10.3, 7.4 Hz, 1H), 5.42 (d, J = 4.2) Hz, 1H), 4.95 (m, 2H), 4.66 (t, J = 4.9 Hz, 1H), 3.56 (dq, J = 7.6, 6.1 Hz, 1H), 3.12 (q, J = 7.1 Hz, 1 H), 2.90 (ddd, (9.5, 5.1, 2.3 Hz, 1 H), 2.20 (m, 2 H), 1, 93 (dd, J = 13.7, 7.8 Hz, 1H), 1.75 (dd, J = 14.3, 2.2 Hz, 1H), 1.11 (m, 1H), 1.10 (d, J = 6.4 Hz, ZN), 0.98 (m, 1H), 0.97 (s, ZN), 0.75 (t, J = 7.6 Hz, ZN) ppm. LC / MS (M + H) = 460.2.

To a solution of (1R, 2R, 4S) -2- (3-chlorophenyl) -1- (4-chlorophenyl) -4 - ((4S, 5S) -4-ethyl-5-methyl-4,5- dihydrooxazol-2yl) -4-methylhept-6-en-1-ol (80 mg, 0.174 mmol) in CH₂CI₂ (1737 μΙ) at -50 ° C was added 2,6-lutidine (46.4 μΙ, 0.400 mmol) followed by a solution of trifluoromethanesulfonic anhydride, 1 M in methylene chloride (191 μΙ, 0.191 mmol). The reaction mixture was stirred at -50 ° C for 30 minutes and then treated with an additional 25 [mu] l of a solution of trifluoromethanesulfonic anhydride, 1 M in methylene chloride, then with 2 ml of sat. CuSO₄. The reaction mixture was heated to rt and extracted with dichloromethane. The organic phase was dried over MgSO 4₄, filtered, and the filtrate was concentrated. Purification by column chromatography using 40 to 80% acetone in hexanes afforded (2S, 3S, 5S, 6R, 8S) -8-allyl-6- (3-chlorophenyl) -5- (4-chlorophenyl) -3-ethyl -2,8-dimethyl-2,3,5,6,7,8-hexahydrooxazolo [3,2-a] pyridin-4-yl triflate.

^g1 NMR (500 MHz, DMSO-d 6) δ 7.55-7.05 (series m, 8H), 5.88 (ddt, J = 17.3, 10.0, 7.8 Hz, 1H), δ , 36 (dd, J = 17.1, 2.0 Hz, 1H), 5.31 (d, J = 10.8 Hz, 1H), 5.28 (dd, J = 10.0, 2.0) Hz, 1H), 5.18 (quintet, J = 6.1 Hz, 1H), 4.10 (td, J = 6.6, 2.7 Hz, 1H), 3.98 (ddd, J = 13) , 7, 11.2, 3.4 Hz, 1H), 2.80 (AVH, J_AB = 13.7 Hz, J_AX = 7.3 Hz, 1H), 2.73 (AVH J)_AB = 13.7 Hz, J_ent = 7.8 Hz, 1H), 2.49 (m, 1H), 2.41 (t, J = 13.7 Hz, 1H), 2.00 (dd, J = 13.9, 3.7 Hz) , 1H), 1.55 (d, J = 6.1 Hz, 1H), 1.31 (s, ZN), 0.95 (dqd, J = 14.2, 7.8, 3.0 Hz, 1H), 0.58 (t, J = 7.3 Hz, 1H), 0.47 (ddq, J = 13.7, 6.3, 6.3 Hz, 1H) ppm. LC / MS (M + = 442.2).

In solution (2S, 3S, 5S, 6R, 8S) -8-allyl-6- (3-chlorophenyl) -5- (4-chlorophenyl) -3-ethyl-2,8-dimethyl-2,3 , 5,6,7,8hexahydrooxazolo [3,2-a] pyridin-4-yum triflate (60 mg, 0.101 mmol) in 1 ml dichloromethane at 0 ° C was added tetra-n-butylammonium chloride (2.81 mg, 10.13 μιτιοΙ) and acetic acid (116 μΙ, 2.025 mmol). That's it

58366 Β1 added ΚΜηΟ₄ (32.0 mg, 0.203 mmol) in 1 mL of water and then washed with 1 mL of water. Another 10 eq. acetic acid, followed by an additional 16 mg of KMnO₄ in 1 ml of water. This was repeated once more. A total of 4 eq. KMnO₄and 40 eq. acetic acid.

The reaction was quenched by the addition of 1 mL of sat. Na solution₂S₂Oh₃, and the reaction mixture was diluted with ethyl acetate. The layers were separated and the organic phase was washed with aqueous brine, dried over MgSO 4₄, filtered and concentrated to give crude (2S, 3S, 5S, 6R, 7aR) -6- (3-chlorophenyl) -5- (4-chlorophenyl) -3-ethyl-2,7a-dimethylhexahydrofuro [2,3-b ] oxazolo [3,2-a] pyridin-9 (5H) -one. This crude residue was redissolved in 2 ml of isopropyl acetate and treated with 2 ml of sat. NaHCO₃, and heated to 70 ° C. After 2 h, the reaction mixture was cooled to 0 ° C and treated with 10% acetic acid to a pH of about 3. The reaction mixture was diluted with ethyl acetate and washed with one 10% acetic acid solution, dried over MgSO 4.₄, filtered and concentrated. Purification by column chromatography using 10 to 50% of a mixture (15% MeOH / acetone) in hexanes gave 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 ((2S, 3S) -2-hydroxypentan-3-yl) -3-methyl-2-oxopiperidin-3-yl) acetic acid.

REFERENCE EXAMPLE9

2 - ((3R, 5R, 6S) -5- (3-Chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-hydroxybutan-2-yl) -3-methyl-2-oxopiperidine -3yl) acetic acid [0166]

<img file="RS58366B1_D0056.tif" />

Step A. (S) -Methyl 2 - ((2S, 3R) -3- (3-chlorophenyl) -2- (4-chlorophenyl) -6-oxopiperidin-1-yl) butanoate [0167]

58366 Β1

<img file="RS58366B1_D0057.tif" />

To a solution of 33.8 g (60% in mineral oil, 845 mmol) of sodium hydride in 2-methyltetrahydrofuran (550 ml) at 50 ° C was added a solution of 240 g (750 mmol) (5R, 6S) -5- 3-chlorophenyl) -6- (4-chlorophenyl) piperidin-2-one (reference example 1, step E) in 2-methyltetrahydrofuran (550 ml) over a period of 45 min. After an additional 1.25 h at 50 ° C, 105 ml (912 mmol) of methyl 2-bromobutyrate were added over a period of 20 minutes. The resulting slurry was stirred at 50 ° C for 3.5 h, then cooled to room temperature, and the reaction was quenched with sat. vod. NH solution₄CI. Water was added to dissolve the precipitate, and the resulting mixture was extracted with ethyl acetate (4x). The combined organic layers were washed with sat. vod. NaCl solution (1x), dried over Na₂SO₄, filtered, and the filtrate was concentrated. Purification of the residue by silica gel chromatography (Biotage® Snap® column (Biotage, LLC, Charlotte, NC), 0 to 35% EtOAc / DCM, gradient elution) gave the title compound as a white oily solid.

Step B. (5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-hydroxybutan-2-yl) piperidin-2-one [0169]

<img file="RS58366B1_D0058.tif" />

In an ice-cold solution of 48.5 g (115 mmol) (S) -methyl 2 - ((2S, 3R) -3- (3-chlorophenyl) -2- (4-chlorophenyl) -6-oxopiperidin-1-yl) ) of butanoate (reference example 9, step A) in ethyl ether (850 mL) was added 5.96 g (90%, 246 mmol) of lithium borohydride. The resulting pale yellow solution was stirred at 0 ° C for 3 h, then MeOH (2.5 mL) and more ethyl ether (100 mL) were added. After the addition of MeOH, gas evolution was observed. After 40 minutes, the reaction was quenched by the careful addition of 1 N HCl until gas evolution ceased. The mixture was extracted with EtOAc (2x), and the combined organic layers were washed with saturated aqueous sodium chloride (1x). The organic layer was dried over Na₂SO₄, filtered, and the filtrate was concentrated to

58366 Dobiί received the title compound in the form of a white foam. The crude product was used directly in the next step without further purification.

Step C. (5R, 6S) -1 - ((S) -1- (tert-butyldiphenylsilyloxy) butan-2-yl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) piperidin-2-one [0171]

<img file="RS58366B1_D0059.tif" />

To a solution of 44.7 g (114 mmol) (5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-hydroxybutan-2-yl) piperidin-2 -one (Reference Example 9, Step B) and 19.4 g (285 mmol) of imidazole in DMF (350 mL) were added 39.4 mL (154 mmol) of tert-butyldiphenylsilyl chloride. The colorless solution was stirred at room temperature for 17 h. The reaction mixture was partitioned between water and ethyl ether (3 h), and the combined organic layers were washed with saturated aqueous sodium chloride (1 h), dried over Na₂SO₄, filtered and the filtrate was concentrated. Purification of the residue by silica gel chromatography (Biotage® Snap® column (Biotage, LLC, Charlotte, NC), 0 to 60% EtOAc / hexanes, gradient elution) gave the title compound as a white foam.

Step D. (5R, 6S) -1 - ((S) -1- (tert-butyldiphenylsilyloxy) butan-2-yl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methylpiperidin-2 -on [0173]

<img file="RS58366B1_D0060.tif" />

To a solution of 98.2 g (156 mmol) (5R, 6S) -1 - ((S) -1- (tert-butyldiphenylsilyloxy) butan-2-yl) -5- (3-chlorophenyl) -6 (4 -chlorophenyl) piperidin-2-one (reference example 9, step C) and 10.0 ml (160 mmol) of methyl iodide in dry THF from which the gas (400 ml) was evacuated at -78 ° C was slowly added 200 ml (200 mmol) of a 1M solution of lithium bisphtrimethylsilyl) amide from which the gas in THF was evacuated, over 20 minutes. It is an orange solution

58366 Β1 stirred at -78 ° C for 1.5 ha then warmed to 0 ° C and stirred for another 1.5 h. The reaction was quenched with saturated aqueous ammonium chloride and extracted with EtOAc (Zh). The combined organic layers were dried over Na₂SO₄, filtered, and the filtrate was concentrated. Purification of the residue by silica gel chromatography (Biotage® Snap® column; Biotage, LLC, Charlotte, NC), 5-55% EtOAc / hexanes, gradient elution) gave the title compound as a light yellow foam.

Step E. (3S, 5R, 6S) -3-allyl-1- (S) -1- (tert-butyldiphenylsilyloxy) butan-2-yl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methylpiperidin-2-one

<img file="RS58366B1_D0061.tif" />

Cl To a solution of 37.3 (266 mmol) diisopropylamine in dry THF from which gas (150 ml) was evacuated at 78 ° C was slowly added via cannula 100 ml (250 mmol) of a 2.5 Μ solution of n-butyllithium in hexanes from which the gas was evacuated. The pale yellow solution was stirred at -78 ° C for 15 minutes, then warmed to 0 ° C and stirred for an additional 5 min. To the ice-cold LDA solution was added a solution of 85.7 g (133 mmol) (5R, 6S) -1 - ((S) -1- (tert-butyldiphenylsilyloxy) butan-2-yl) -5- (3-chlorophenyl) -6 - (4-chlorophenyl) -3-methylpiperidin-2-one (reference example 9, step D) in dry THF from which gas (210 ml) was evacuated, via cannula, for 15 minutes.

The dark orange solution was stirred at 0 ° C for 30 minutes, and 34.5 mL (399 mmol) of allyl bromide was added rapidly from a syringe. After 20 seconds, the ice bath was removed and the reaction mixture was left in a room temperature water bath and stirred for an additional 15 min. The reaction was quenched with saturated water. ammonium chloride and extracted with EtOAc (3 h). The combined organic layers were dried over Na₂SO₄, filtered, and the filtrate was concentrated. Purification of the residue by silica gel chromatography (Biotage® Snap® column; Biotage, LLC, Charlotte, NC), 6-14% EtOAc / hexanes, gradient elution) gave the title compound as a white foam.

Step F. 2- (3R, 5R, 6S) -1- (S) -1- (tert-butyldiphenylsilyloxy) butan-2-yl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl2-oxopiperidin-3-yl) acetic acid [0177]

58366 Yes

<img file="RS58366B1_D0062.tif" />

Cl The title compound was obtained from (3S, 5R, 6S) -3-allyl-1- ((S) -1- (tert-butyldiphenylsilyloxy) butan-2-yl) -5 (3-chlorophenyl) -6 - (4-chlorophenyl) -3-methylpiperidin-2-one (Reference Example 9, Step E) by a procedure similar to that described in Reference Example 3, Step F.

1 N NMR (400 MHz, CHLOROFORM-d) b ppm 0.30 (t, J = 7.53 Hz, 3 H) 1.17 (s, 9 H) 1.34 -1.48 (m, 1 H) ) 1.53 (s, 3H) 1.74 -1.88 (m, 1H) 1.93-1.03 (m, 1H) 2.29 (t, J = 13.69 Hz, 1H) ) 2.69 (d, J = 15.85 Hz, 1H) 2.81 2.93 (m, 1H) 2.98 - 3.08 (m, 1H) 3.12 (d, J = 15.65 Hz, 1H) 3.52 (dd, J = 10.66, 4.21 Hz, 1H) 4.32 (t, J = 10.27 Hz, 1H) 4.71 (d, J = 10.76 Hz, 1H) 6.56 - 6.66 (m, 1H) 6.91 - 6.97 (m, 1H) 7.02 - 7.09 (m, 1H) 7 , 12 - 7.18 (m, 1H) 7.20 - 7.30 (m, 4H) 7.33 - 7.51 (m, 6H) 7.64 (td, J = 7.83, 1.57 Hz, 4 H).

Step G. 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-hydroxybutan-2-yl) -3-methyl- 2-oxopiperidin3-yl) acetic acid In an ice-cold solution of 370 g (0.53 mmol) of 2 - ((3R, 5R, 6S) -1 - ((S) -1- (tert-butyldiphenylsilyloxybutan2-yl) -5- (3-Chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-3-yl) acetic acid (Reference Example 9, Step F) in THF (15 ml) was added 2 , 60 ml (2.60 mmol) of a 1 M solution of TBAF in THF The yellow solution was warmed to rt and stirred for 5 h. Then 2.60 ml (2.60 mmol) of a 1 M solution of TBAF in THF was added, and the reaction mixture was stirred for an additional 20 h. The reaction mixture was partitioned between 1 N HCl and EtOAc (4 h). The combined organic layers were dried over Na₂SO₄, filtered, and the filtrate was concentrated. The residue was purified by reverse phase preparative HPLC (Sunfire ™ Prep C18 OBD 10 pm column) (Waters, Milford, MA), gradient elution of 40% MeCN in water to 80% MeCN in water over a period of 30 min, where both solvents contained 0.1 l % TFA), and the title compound was obtained as a white solid.

^:H NIVIR (400 MHz, CDCl 3)₃b ppm 7.23-7.28 (2H, m), 7.15-7.20 (1H, m), 7.07-7.14 (1H, m), 6.98-7 , 06 (3 H, m), 6.74 (1 H, d, J = 7.1 Hz), 4.55 (1 H, dd J = 9.8 Hz, 2.9 Hz), 3.71 -3.79 (1H, m), 3.58-3.66 (1H, m), 3.19-3.28 (1H, m), 3.07-3.16 (1H, m), 2.96-3.03 (1H, m), 2.75 (1H, dd, J = 14.9 Hz, 2.9 Hz), 2.16-2.25 (1H, m), 2.03-2.10 (1H, m), 1.87-1.98 (1H, m), 1.46 (3H, s), 1.41-1.54 (m , 1H), 0.63 (3H, dd, J = 7.3 Hz, 3.3 Hz). Mass spectrum (ESI) m / z = 464.1 (M + 1).

REFERENCE EXAMPLE 10

58366 Yes

2 - ((3R, 5R, 6S) -1- (S) -2- (tert-Butoxy) -1-cyclopropyl-2-oxoethyl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -2-Oxopiperidin-3-yl) acetic acid [0180]

<img file="RS58366B1_D0063.tif" />

StepA. Ethyl 2-bromo-2-cyclopropyl acetate.

[0181]

<img file="RS58366B1_D0064.tif" />

To a solution of 2-cyclopropylsacetic acid (24.7 g, 247 mmol) in anhydrous DCE (250 mL) was added thionyl chloride (22 mL, 302 mmol) dropwise over 5 minutes at 25 ° C. . After refluxing for 2 h, the reaction mixture was cooled to room temperature, N-bromosuccinimide (53.6 g, 301 mmol) and hydrogen bromide (48% aqueous solution) (0.195 mL, 1.727 mmol) were added in this order at 25 ° C. . The resulting mixture was heated at reflux for 96 h. After the reaction mixture was cooled to room temperature, absolute EtOH (200 mL) was added, and the resulting dark brown solution was stirred at room temperature for one hour. The reaction mixture was concentrated under reduced pressure (35 ° C, 4.0 kilopascals) and the residue was suspended in carbon tetrachloride (300 ml) and passed through a glass filter. The filtrate was concentrated under reduced pressure (35 ° C, 4.0 kilopascals). Purification of the crude product by chromatography (silica gel, 330 g x 2.5% ethyl acetate / hexanes) and concentration of the desired fractions under reduced pressure (35 ° C, 4.0 kilopascals) gave the title compound as a pale yellow liquid.

1 NMR (400 MHz, CHLOROFORM-d) b ppm 4.20-4.32 (2 H, m), 3.59 (1 H, d, 7 = 10.4 Hz), 1.53 -1.66 (1H, m), 1.29 -1.36 (3H, m), 0.76-0.93 (2H, m), 0.51-0.61 (1H, m), 0 , 40 - 0.47 (1H, m).

Step B. (S) -ethyl 2 - ((2S, 3R) -3- (3-chlorophenyl) -2- (4-chlorophenyl) -6-oxopiperidin-1-yl) -2-cyclopropylacetate

58366 ]Ί [0183]

<img file="RS58366B1_D0065.tif" />

To a solution of (3S, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-piperidin-2-one (8.01 g, 25 mmol; Reference Example 1, step E) in DMF (60 ml) was added 60% sodium hydride in mineral oil (2.0 g, 50 mmol) at 0 ° C, and the resulting mixture was stirred at the same temperature for 30 min. Ethyl 2-bromo-2-cyclopropyl acetate (12.18 g, 50 mmol) in DMF (10 mL) was added dropwise to the mixture, and the mixture was stirred at room temperature for 2 h, after which the reaction was quenched with sat. ammonium chloride solution and the reaction mixture diluted with ethyl acetate. The organic layer was washed with 10% aq. citric acid, 5% aq. NaHCO solution₃, water, sat. vod. NaCl solution and then dried over MgSO 4₄. The solvent was evaporated under reduced pressure and the residue purified by chromatography on silica gel eluting with 20 to 50% ethyl acetate in hexane to give the title compound as the first eluted diastereomer as a white solid.

^g1 NMR (400 MHz, CHLOROFORM-d) δ ppm -0.34 (m, 1H), 0.23 (m, 1H), 0.38 (m, 1H), 0.62 (m, 1H), 1 , 26 (t, J = 8 Hz, ZN), 1.39 (m, 1H), 2.13 (m, 2H), 2.63 (m, 2H), 3.09 (m, 1H), 3 , 20 (d, J = 12 Hz, 1H), 4.07 (m, 2H), 4.81 (d, J = 8 Hz, 1H), 6.90 (dt, J = 7.1, 1, 7 Hz, 1H), 7.11-7.19 (m, 5H), 7.28 (m, 2H). Mass spectrum (ESI) m / z = 446.2 (M + 1).

Step C. (S) -tert-Butyl 2 - ((2S, 3R) -3- (3-chlorophenyl) -2- (4-chlorophenyl) -6-oxopiperidin-1-yl) -2-cyclopropylacetate [0185]

<img file="RS58366B1_D0066.tif" />

58366 U1 (S) -ethyl 2- (2S, 3R) -3- (3-chlorophenyl) -2- (4-chlorophenyl) -6-oxopiperidin-1-yl) -2-cyclopropylacetate solution (500) mg, 1.12 mmol) (Reference Example 10, Step B) in a THF / MeOH / H mixture₂O (5/5 / 5.15 mL) was added lithium hydroxide (1.68 mL, 3.36 mmol) per mL, and then the mixture was heated to 60 ° C. After stirring at 60 ° C for 1.5 h, the reaction was quenched with saturated aqueous NH₄CI, and the mixture was extracted (2 h DCM). The combined organic layers were washed (1 h brine with NaCl solution), dried over Na₂SO4, filtered, and the filtrate was concentrated under reduced pressure.

The crude acid (450 mg, 1.076 mmol) which was synthesized as previously described was dissolved in DCM (10 mL), and sulfuric acid (115 [mu] l, 2.151 mmol) was added followed by 2-methylprop. 1-ene (1.207 g, 21.51 mmol) at -78 ° C. The reaction vessel was sealed and the mixture was slowly warmed to room temperature. After vigorous stirring for 4 days, the reaction was quenched with saturated water. NH solution₄CI and extracted with ethyl acetate. The combined organic layers were washed with sat. vod. NaCl solution, dried over sodium sulfate, filtered, the filtrate concentrated under reduced pressure. Purification of the residue by chromatography on silica gel under gas pressure (eluent: 40% EtOAc / hexanes) gave the title compound.

Step D. (S) -tert-Butyl 2- (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -2-oxopiperidin-1-yl) - 2cyclopropyl acetate [0188]

<img file="RS58366B1_D0067.tif" />

A solution of lithium bis (trimethylsilyl) amide (1 M in THF, 0.165 mL, 0.165 mmol) was added dropwise to a solution of (S) -tert-butyl 2- (2S, 3R) -3- (3 -chlorophenyl) -2- (4-chlorophenyl) -6-oxopiperidin-1-yl) -2-cyclopropylacetate (Reference Example 10, Step C, 71 mg, 0.15 mmol) and allyl bromide (15.54 uL, 0.165 mmol) in 0.5 ml of THF at -78 ° C. The reaction mixture was allowed to cool to room temperature. After stirring for 2 h, the reaction was quenched with saturated aq. with ammonium chloride solution and extracted with ethyl acetate.

The combined organic layers were washed with sat. vod. NaCl solution, dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purification of the residue by chromatography on silica gel eluting with ethyl acetate / hexane gave the title compound.

58366 Β1

Step E. 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1- (N-cyclopropylacetamido) butan-2-yl) -3- methyl 2-oxopiperidin-3-yl) acetic acid The title compound was obtained from (S) -tert-butyl 2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) 2-oxopiperidin-1-yl) -2-cyclopropylacetate (Reference Example 10, Step D) by the procedure of Reference Example 3, Step F.

^gN NMR (400 MHz, CHLOROFORM-d) b ppm 0.08 (m, 1 H), 0.49 (m, 1 H), 0.58 (m, 1 H), 0.66 (m, 1 H) ), 1.02 (m, 1H), 1.44 (s, 9H), 1.99 (m, 1H), 2.19 (m, 1H), 2.58 (dd, J = 16.0, 4.0 Ηζ, Ι H), 2.66 (m, 1 H), 2.93 (dd, J = 12.0, 12.0 HZ, 1 H), 3.20 (s, 1 H), 3.34 (d, J = 12 Hz, 1 H), 5.37 (s, 1 H), 7.14 (m, 1 H), 7.25-7.33 (m, 3 H), 7.37-7.45 (m, 4H). Mass spectrum (ESI) m / z = 532.2 (M + 1).

REFERENCE EXAMPLE 11

2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- (S) -1-cyclopropyl-2-hydroxyethyl) -3-methyl-2-oxopiperidine -3-acetic acid [0191]

<img file="RS58366B1_D0068.tif" />

Step A. (5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-cyclopropyl-2-hydroxyethyl) piperidin-2-one [0192]

<img file="RS58366B1_D0069.tif" />

58366 Β1 A solution of lithium borohydride (2 M in THF, 15.46 mL, 30.9 mmol) was added to a solution of (S) -ethyl 2 (2S, 3R) -3- (3-chlorophenyl) - 2- (4-chlorophenyl) -6-oxopiperidin-1-yl) -2-cyclopropyl acetate (Reference Example 10, Step B, 2.3 g, 5.15 mmol) in ether (40 mL) at 0 ° C. After stirring at room temperature for 20 hours, the reaction was quenched with sat. vod. NH solution₄CI, and the reaction mixture was extracted with EtOAc. The organic layer was washed with sat. vod. NaCl solution, dried over sodium sulfate and concentrated to give the title compound as a solid, which was used without further purification.

^T1 NMR (400 MHz, CHLOROFORM-d) b ppm 0.00 (m, 1H), 0.23 (m, 1H), 0.48-0.57 (m, 2H), 0.85 (m, 1H) ), 1.99 (m, 1H), 2.07 (m, 1H), 2.61 (m, 2H), 2.64 (m, 1H), 3.22 (dd, 7 = 11.2, 9.8 Hz, 1H), 3.42 (td, 7 = 10.1, 4.3 Hz, 1H), 3.60 (m, 1H), 4.93 (d, 7 = 6.5 Hz, 1 H), 6.89 (m, 1 H), 7.09 (m, 4 H), 7.18 (m, 2 H), 7.27 (m, 1 H). Mass spectrum (ESI) m / z = 404.0 (M + 1).

Step B. (SR, 6S) -1 - ((S) -2 - ((tert-butyldiphenylsilyl) oxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) piperidin-2-one [0194]

<img file="RS58366B1_D0070.tif" />

The product of Reference Example 11 step A was converted to the title compound by a procedure similar to that described in Reference Example 9 step C.

^g1 NMR (400 MHz, CHLOROFORM-d) δ ppm -0.70 (m, 1H), -0.40 (m, 1H), 0.02 (m, 1H), 0.13 (m, 1H), 0.91 (s, 9H), 1.09 (m, 1H), 1.92-1.91 (m, 2H), 2.47-2.50 (m, 2H), 2.78 (s, br, 1H), 2.80 (m, 1H), 3.31 (m, 1H), 3.98 (m, 1H), 4.62 (d, 7 = 7.8 Hz, 1H), 6, 60 (m, 1 H), 6.82-6.91 (m, 4 H), 6.91-7.03 (m, ZN), 7.18-7.26 (6 H), 7.37-7, 45 (m, 4 H). Mass spectrum (ESI) m / z = 642.3 (M + 1).

Step C. (SR, 6S) -1 - ((S) -2 - ((tert-butyldiphenylsilyl) oxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methylpiperidine- 2-on [0196]

58366 Β1

<img file="RS58366B1_D0071.tif" />

(5R, 6S) -1 - ((S) -2 - ((tert-butyldiphenylsilyl) oxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) piperidin-2-one (Reference Example 11, Step B, 7.9 g, 12.29 mmol) was converted to the title compound, a mixture of diastereomers, according to the procedure of Reference Example 9, Step D.

³1 H NMR (400 MHz, CHLOROFORM-d) δ ppm -0.15 (m, 1H), 0.00 (m, 1H), 0.41-0.52 (m, 2H), 1.26 (s, 9H), 1.41 (m, 1H), 1.62 (d, J = 7.2 Hz, ZN), 2.09 (m, 1H), 2.30 (m, 1H), 2.87 m, 1H), 3.28 (m, 2H), 3.68 (m, 1H), 4.23 (m, 1H), 5.11 (d, J = 6.1 Hz, 1H), 7, 16-7.34 (m, 4H), 7.37-7.46 (m, 4H), 7.51-7.65 (m, 6H), 7.74-7.78 (m, 4H).

Step D. (SR, 6S) -3-allyl-1- (S) -2 - ((tert-butyldiphenylsilyl) oxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) )

3-methylpiperidin-2-one

Ph-Si-Ph

<img file="RS58366B1_D0072.tif" />

(5R, 6S) -1 - ((S) -2- (tert-butyldiphenylsilyloxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methylpiperidin-2-one (5.02 g, 7.64 mmol, Reference Example 11, Step C) was converted to the title compound by the procedure described for Reference Example 9, Step E. After work-up, the crude product was further used as obtained.

58366 Β1

Step E. 2 - ((3R, 5R, 6S) -1 - ((S) -2 - ((tert-butyldiphenylsilyl) oxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) 3 -methyl-2-oxopiperidin-3-yl) acetic acid [0200]

<img file="RS58366B1_D0073.tif" />

[0201] (5R, 6S) -3-allyl-1- (S) -2 - ((tert-butyldiphenylsilyl) oxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) ) -3-methylpiperidin-2-one (Reference Example 11, Step D, 106 mg, 0.15 mmol) was treated according to the procedure of Reference Example 9, Step F to give 2 - ((3R, SR, 6S) -1 - ((S) -2 - ((tert-butyldiphenylsilyl) oxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-3-yl) acetic acid. Mass spectrum (ESI) m / z = 714.3 (M + 1).

Step F. 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-cyclopropyl-2-hydroxyethyl) -3-methyl-2-oxopiperidin-3 -yl) acetic acid A solution of tetrabutylammonium fluoride (1.0 M in THF, 0.453 mL, 0.453 mmol) was added to solution 2 (3 R, 5R, 6S) -1 - ((S) -2 - (tert-butyldiphenylsilyloxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-3-yl) acetic acid (Reference Example 11, Step E, 108 mg , 0.151 mmol) in THF (4ml), and the reaction mixture was stirred at room temperature for 20 hours. Analysis by LC-MS showed that the reaction was not complete, and an additional 0.225 ml of tetrabutylammonium fluoride solution was added, and the reaction mixture was stirred for another 26 hours. The mixture was diluted with ethyl acetate and then washed with water and sat. vod. NaCl solution. The organic layer was dried over sodium sulfate and concentrated. Purification by RP-HPLC (Sunfire Prep C_lgOBD 10 pm column, gradient elution of 10% MeCN in water to 80% MeCN in water over 30 min, where both solvents contain 0.1% TFA) the title compound was obtained as a solid.

³1 H NMR (400 MHz, CHLOROFORM-d) brrt -0.30 (s, m, 1H), 0.00 (s, m, 1H), 0.37 (m, 2H), 0.79 (m, 1H) ), 1.22 (s, ZN), 2.00 (m, 2H), 2.52 (d, J = 14.1 Hz, 1H), 2.70 (d, J = 13.9 Hz, 1H) ), 3.00 (m, 2H), 3.20 (s, br, ZN), 3.29 (m, 1H), 4.65 (d, J = 10, Hz, 1H), 6.61 (m, 1H), 6.84 (s, br, 2H), 6.97 (m, ZN), 7.04 (m, 2H). Mass spectrum (ESI) m / z = 476.2 (M + 1).

58366 Β1

REFERENCE EXAMPLE 12

2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((1S, 2S) -1-cyclopropyl-2-hydroxybutyl) -3-methyl- 2-Oxopiperidin-3-yl) acetic acid or 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((1S, 2R) -1-cyclopropyl2-hydroxybutyl -3-methyl-2-oxopiperidin-3-yl) acetic acid [0203]

<img file="RS58366B1_D0074.tif" />

<img file="RS58366B1_D0075.tif" />

Step A. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-cyclopropyl-2-hydroxyethyl) -3-methylpiperidin-2 -on [0204]

<img file="RS58366B1_D0076.tif" />

A solution of tetrabutylammonium fluoride in THF (1M, 2.10 mL, 2.10 mmol) was added to a solution of the diastereomer (5R, 6S) -3-allyl-1- (S) -2- (tert- butyldiphenylsilyloxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methylpiperidin-2-one (reference example 11, step D, 488 mg, 0.700 mmol) in THF (10 ml) . The reaction mixture was stirred at room temperature for 2 hours. The mixture was diluted with ethyl acetate and then washed with water and sat. vod. NaCl solution. The organic layer was dried over sodium sulfate.

Chromatography on silica gel eluting with ethyl acetate / hexane gave the title compound as a single diastereomer.

Mass spectrum (ESI) m / z = 458.0 (M + 1)

58366 Yes

Step Β. (S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1-yl) -2cyclopropylacetaldehyde [ 0206]

<img file="RS58366B1_D0077.tif" />

[(3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- ((S) -1-cyclopropyl-2-hydroxyethyl) -3-methylpiperidine2 -one (Reference Example 12, Step A, 80 mg, 0.17 mmol) was converted to the title compound as a white foam by the procedure described in Reference Example 4, Step C. Mass Spectrum (ESI) m / z = 456.1 (M + l)

StepC. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((1S, 2S) -1-cyclopropyl-2-hydroxybutyl) -3-methylpiperidine-2- he or (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((1S, 2R) -1-cyclopropyl-2-hydroxybutyl) -3-methylpiperidine- 2-on [0208]

<img file="RS58366B1_D0078.tif" />

<img file="RS58366B1_D0079.tif" />

According to the procedure of reference example 6, step A, with methylmagnesium bromide substitution by ethylmagnesium bromide, (S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- 4-Chlorophenyl) -3-methyl-2-oxopiperidinyl-yl) -2-cyclopropylacetaldehyde (Reference Example 12, Step B, 90 mg, 0.20 mmol) was converted to the title compound which was obtained as a diastereomer which eluted second after chromatography.

³1 H NMR (400 MHz, CHLOROFORM-d) δ ppm -0.43 (m, 1H), -0.16 (m, 1H), 0.32 (m, 1H), 0.51 (m, 1H), 0.78 (t, J = .3 Hz, ZN), 1.18 (s, ZN), 1.21-1.35 (m, 1H), 2.54 (m, 2H), 1.57 s, br, 1H), 1.87-2.0 (m, 2H), 2.21 (s, br, 1H), 2.50-2.62 (m, 2H), 3.22 (ddd, J = 12.8, 10.2, 4.0 Hz, 1H), 3.68 (s, br, 1H), 4.34 (d, J = 10.0 Hz, 1H), 5.12

58366 Βί (s, 1Η), 5.14 (d, J = 8Ηζ, 1H), 5.79 (m, 1H), 6.65 (dt, J = 7.6, 1.6Ηζ, 1H), 6.87-6.91 (m, ZN), 7.01-7.07 (m,

2H), 7.16-7.18 (m, 2H).

Mass spectrum (ESI) m / z = 486.3 (M + 1)

Step D. 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((1S, 2S) -1-cyclopropyl-2-hydroxybutyl) -3- methyl-2-oxopiperidin-3-yl) acetic acid or 2 - ((3R, 5R, 6S) -5- (3-Chlorophenyl) -6- (4-chlorophenyl) -1- (1S, 2R) -1-cyclopropyl2 -hydroxybutyl) -3-methyl-2-oxopiperidin-3-yl) acetic acid [0210]

<img file="RS58366B1_D0080.tif" />

<img file="RS58366B1_D0081.tif" />

The title compound was obtained by treating the compound of Reference Example 12, Step C by the method described in Reference Example 3, Step F.

^g1 NMR (400 MHz, methanol-d 4) δ ppm -0.16 (s, br, 1H), 0.26 (s, br, 1H), 0.55 (s, br, 1H), 0.67 s, br, 1H),

0.86 (m, ZN), 1.32 (m, 4H), 1.41 (s, ZN), 1.68 (m, 1H), 1.92 (m, 2H), 2.64 (d) , J = 12 Ηζ, 1H), 2.99 (d, J = 12 Hz, 1H), 3.51 (m, 1H), 4.97 (m, 1H), 6.97 (m, 1H), 7.06 (m, 1 H), 7.17 (m, 4 H), 7.28 (m, 2 H). Mass spectrum (ESI) m / z = 504.1 (M + 1).

REFERENCE EXAMPLE 13

2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((1S, 2R) -1-cyclopropyl-2-hydroxypropyl) -3-methyl-2-oxopiperidine -3-yl) acetic acid [0212]

<img file="RS58366B1_D0082.tif" />

58366 Yes

Step Α. (3S, 5R, 6S) -3-allyl-1- (S) -2- (tert-butyldiphenylsilyloxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methylpiperidine- 2-on [0213]

<img file="RS58366B1_D0083.tif" />

A mixture of diastereomers obtained from (5R, 6S) -1 - ((S) -2- (tert-butyldiphenylsilyloxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methylpiperidine The 2-one (4.34 g, 6.61 mmol) was purified by chromatography on silica gel eluting with ethyl acetate / hexanes by the procedure of Reference Example 11, step D. Fractions containing the desired epimer were combined and concentrated to give (3S, 5R, 6S) -3-allyl-1- ((S) -2- (tert-butyldiphenylsilyloxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) - 6- (4-chlorophenyl) -3-methylpiperidin-2-one in the form of a white foam, weighing 3.01 g (yield 65%). MS (ESI) m / z = 696 [M + H]⁺.

Step B. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-cyclopropyl-2-hydroxyethyl) -3-methylpiperidin-2 -He. (see also reference example 12, step A) [0215]

<img file="RS58366B1_D0084.tif" />

By treatment with (3S, 5R, 6S) -3-allyl-1- (S) -2- (tert-butyldiphenylsilyloxy) -1-cyclopropylethyl) -5- (3-chlorophenyl) -6 (4-chlorophenyl) -3-methylpiperidin-2-one (Reference Example 13, Step A, 3.00 g, 4.31 mmol) according to the procedure of Reference Example 12, Step A, gave (3S, 5R, 6S) -3- allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-cyclopropyl-2-hydroxyethyl) -3-methylpiperidin-2-one as a white foam (1.905 g, 97%) . ^gN NMR (500 MHz, CHLOROFORM-d) b 0.00 - 0.15 (m, 1H), 0.18 - 0.35 (m, 1H), 0.44 - 0.69 (m, 2 H), 0.75 0.87 (m, 1H), 1.28 (s, 3H), 1.87 - 2.03 (m, 2H), 2.48 - 2.72 (m, 2 H), 3.01 - 3.22 (m, 2 H), 3.41 (td, J = 10.33, 4.52 Hz, 1H), 3.60 (dd, J = 11.00, 4.40 Hz, 1H), 4.86 (d, J = 10.03 Hz, 1H), 5.06 - 5.24 (m, 2H), 5.74

58366 Β1

5.97 (m, 1H), 6.74 (d, J = 7.58 Hz, 1H), 6.86 - 7.10 (m, 3H), 7.10 - 7.26 (m, 4 H). MS (ESI) m / z = 458 [M + H]⁺

Step C. (S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1-yl) -2cyclopropylacetaldehyde. (see also reference example 12, step B) [0217]

<img file="RS58366B1_D0085.tif" />

(3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-cyclopropyl-2-hydroxyethyl) -3-methylpiperidine2 -one (Reference Example 13, Step B, 1.01 g, 2.2 mmol) was converted to the title compound as a white foam (866 mg, 86%) by the procedure described in Reference Example 4, Step C. MS (ESI) m / z = 456 [M + 1]⁺.

Step D. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((1S, 2R) -1-cyclopropyl-2-hydroxypropyl) -3-methylpiperidine -2-one and (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((1S, 2S) -1-cyclopropyl-2-hydroxypropyl) -3 -methylpiperidin-2-one [0219]

<img file="RS58366B1_D0086.tif" />

<img file="RS58366B1_D0087.tif" />

According to the procedure of reference example 6, step A, (S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl- 2-Oxopiperidin-1-yl) -2-cyclopropylacetaldehyde (Reference Example 13, Step C, 866 mg, 1,897 mmol) was treated with methylmagnesium bromide to give diastereomeric alcohols (3S, 5R, 6S) -3-allyl-5 (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((1S, 2R) -1-cyclopropyl-2-hydroxypropyl) -3-methylpiperidin-2-one and (3S, 5R, 6S) -3

58366 Allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((1S, 2S) -1-cyclopropyl-2-hydroxypropyl) -3-methylpiperidin-2-one in the form of a white foam. MS (ESI) m / z = 472 [M + H] &amp; lt; + &amp; gt ;.

Step E. 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-cyclopropyl-2-oxopropyl) -3-methyl- 2-oxopiperidin-3-yl) acetic acid.

[0221]

<img file="RS58366B1_D0088.tif" />

(3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-cyclopropyl-2-hydroxypropyl) -3-methylpiperidin-2 -one (reference example 13, step D, 809 mg, 1.71 mmol) was treated according to the procedure described in reference example 3, step F, to obtain, after purification by SFC, 2 - ((3R, 5R , 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-cyclopropyl-2-oxopropyl) -3-methyl-2-oxopiperidin-3-yl) acetic acid in the form of white solids.

^gN NMR (500 MHz, methanol-D4) δ -0.70 - -0.47 (m, 1H), 0.10 (dq, J = 9.78, 5.05 Hz, 1H), 0.31 - 0.48 (m, 1H), 0.63 (tt, J = 8.59, 5.35 Hz, 1H), 1.34 (s, 3H), 1.47 -1.58 m, 1H), 2.15 - 2.35 (m, 6H), 2.65 (d, J = 13.69 Hz, 1H), 2.79 (d, J = 10.03 Hz, 1 H), 2.98 (d, J = 13.69 Hz, 1H), 3.48 - 3.57 (m, 1H), 4.65 (d, J = 10.51 Hz, 1H) , 6.94 - 7.01 (m, 1H), 7.08 (s, 1H), 7.11 - 7.54 (m, 6H); MS (ESI) m / z = 488 [M + H]⁺.

Step F. 2- (3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((1S, 2R) -1-cyclopropyl-2-hydroxypropyl) -3- methyl-2-oxopiperidin-3-yl) acetic acid [0223]

<img file="RS58366B1_D0089.tif" />

58366 Redί Reduction of 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-cyclopropyl-2-oxopropyl) -3- methyl 2-oxopiperidin-3-yl) acetic acid in methanol at 0 ° C sodium borohydride gave a mixture of diastereomeric alcohols in a ratio of 22: 1. The residue was purified by chromatography on silica gel, eluting with a gradient of isopropanol in hexanes. The fractions containing the most abundant isomer were concentrated and then lyophilized from an acetonitrile / water mixture to give the title compound as a fluffy white solid.

1 N NMR (500 MHz, methanol-d 4) δ ppm -0.29 (br s, 1 H), 0.20 (br s, 1 H), 0.46 (br s, 1 H), 0.60 (br s, 1 H), 1.19 (br s, 3 H), 1.24 -1.35 (m, 1 H), 1.39 (s, 3 H), 2.10 - 2.29 (m, 2H), 2.63 (d, J = 13.69 Hz, 1H), 2.82 (br s, 1H), 2.98 (d, J = 13.94 Hz, 1H) ), 3.40 - 3.50 (m, 1H), 3.57 (br s, 1H), 4.82 (d, J = 11.00 Hz, 1H), 6.61 7.64 (m, 8H). MS (ESI) m / z = 490 [M + H]⁺.

REFERENCE EXAMPLE 14

2 - ((3R, 5R, 6S) -5- (3-Chlorophenyl) -6- (4-chlorophenyl) -1 - ((1S, 2S) -1-cyclopropyl-2-hydroxypropyl) -3-methyl-2-oxopiperidine -3-yl) acetic acid [0225]

<img file="RS58366B1_D0090.tif" />

L-Selectride® (Aldrich, St. Louis, MO), (1M in THF, 5.0 mL, 5.00 mmol) was added dropwise over minutes to 2 - (3R, 5R) solution. , 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- ((S) -1-cyclopropyl-2-oxopropyl) -3-methyl2-oxopiperidin-3-yl) acetic acid (reference Example 13, step E, 1, 035g, 2.12 mmol) in THF (35 mL) at -78 ° C. After 90 minutes, the mixture was allowed to warm to 0 ° C, and the reaction was carefully quenched by the addition of saturated ammonium chloride. The aqueous phase was extracted three times with ethyl acetate. The combined organic layers were washed with 1M HCl, water, sat. vod. NaCl solution and dried over sodium sulfate. After concentration in vacuo, the residue was purified by chromatography on silica gel, eluting with a gradient of isopropanol in hexanes. The fractions containing the most abundant isomer were concentrated and then lyophilized from an acetonitrile / water mixture to give the title compound as a white powder. Stereochemistry was determined by analogy with reference example 8.

58366 Β1 ³Η NMR (500 MHz, DMSO-d 6)_e) b -0.69 (br s, 1 H), -0.35 (br s, 1 H), 0.17 (br s, 1 H), 0.36 (br s, 1 H), 1, 07 (br s, 1H), 1.27 (s, 4H), 1.98 - 2.23 (m, 2H), 2.53 - 2.58 (m, 1H), 2.93 (d, J = 13.94 Hz, 1H), 3.36 - 3.49 (m, 1H), 3.74 - 4.44 (m, 1H), 4.46 - 5.11 (m , 2H), 6.60 - 7.59 (m, 8H). MS (ESI) m / z = 490 [M + H]⁺.

REFERENCE EXAMPLE 15

2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((2S, 3S) -2-hydroxy-4-methylpentan-3-yl) -3 -methyl-2-oxopiperidin-3-yl) acetic acid [0227]

<img file="RS58366B1_D0091.tif" />

Step A: Methyl 4- (3-chlorophenyl) -5- (4-chlorophenyl) -2-methyl-5-oxopentanoate [0228]

<img file="RS58366B1_D0092.tif" />

Methyl methacrylate (82 mL, 773 mmol) was added to a solution of 2- (3-chlorophenyl) -1- (4-chlorophenyl) ethanone (195.2 g, 736 mmol; reference example 1, step A) in anhydrous To THF (1.5 L) under a nitrogen atmosphere. A suspension of potassium t-butoxide (8.26 g, 73.6 mmol) in anhydrous THF (340 mL) was then prepared (passed through an ultrasonic bath to break up the solid), and this suspension was added from the cannula to the solution. containing 2- (3-chlorophenyl) -1- (4-chlorophenyl) ethanone. The solution was cooled to 1616 ° C, and the orange solution was allowed to stir at room temperature for 2.5 d. (After 2 d, TLC showed no starting materials). The mixture was concentrated in vacuo. The remaining red-brown oil was diluted with ethyl acetate (900 mL) and washed with water (4 x 190 mL) and then sat. vod. NaCl solution. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to give the title compound as a racemic mixture of diastereomers.

58366 Yes ¹1 H-NMR (500 ΜΗζ, CDCl 3)₃) δ 7.88 (m, 4Η), 7.39 (m, 2N), 7.27-7.12 (series m, 4H), 4.62 (dd, J = 9.0, 5.6 Hz ,,

0.5H), 4.59 (dd, J = 9.3, 5.4 Hz, 0.5H), 3.69 (s, 1.5H), 3.60 (s, 1.5H), 2.46 (m, 1 H), 2.33 (m, 1 H), 2.08 (ddd, J = 13.9, 9.3, 5.4 Hz, 0.5 H), 1.97 (ddd , J = 13.7, 9.0, 4.4 Hz, 0.514), 1.23 (d, J = 6.9 Hz, 1.5 H), 1.16 (d, J = 7.1 Hz) , 1.5H) ppm.

Step B: Racemic mixture of (4R, 5R) -methyl 4- (3-chlorophenyl) -5- (4-chlorophenyl) -5-hydroxy-2-methylpentanoate and (4S, 5S) -methyl 4- (3-chlorophenyl) - 5- (4-chlorophenyl) -5-hydroxy-2-methylpentanoate [0230]

<img file="RS58366B1_D0093.tif" />

<img file="RS58366B1_D0094.tif" />

Anhydrous methanol (600 ml) was poured into a three-necked round balloon of 31 equipped with a mechanical stirrer and a thermometer, in an atmosphere of N₂, then cooled to about -20 ° C. Sodium borohydride (26.2 g, 693 mmol) was added in 5 g portions. A solution of methyl 4- (3-chlorophenyl) -5- (4-chlorophenyl) -2-methyl-5-oxopentanoate (253 g, 693 mmol; reference example 15, step A) in methanol was added via addition funnel to the reaction mixture. (600 ml) in drops, maintaining the temperature between -27 ° C and -30 ° C. The reddish solution was stirred at -30 ° C for 30 minutes and then allowed to warm to -15 ° C. The reaction was monitored by TLC until complete. The reaction was quenched by light addition of water (68.6 mL, 3.8 mol) through a feed funnel. The reaction mixture was allowed to warm to room temperature. The solvents were removed in vacuo. The remaining yellowish oil was diluted with ethyl acetate (1.2 L) and washed with water (400 mL). The organic layer was washed with sat. vod. NaCl solution (2 x 300 mL), building an emulsion. After the emulsion was allowed to substantially separate into layers, the organic layer was dried over magnesium sulfate. The solution was filtered through filter paper and concentrated in vacuo to give a racemic mixture of diastereomers.

¹1 H-NMR (500 MHz, DMSO-d 6) δ 7.33 (m, 2H), 7.27-7.17 (series m, 5H), 7.04 (m, 1H), 5.43 (d, J = 4.4 Hz, 0.5H), 5.37 (d, J = 4.6 Hz, 0.5H), 4.77 (t, J = 5.4 Hz, 0.5H), 4, 71 (dd, J = 6.6, 4.9 Hz, 0.5H), 5.33 (s, 1.5H), 3.46 (s, 1.5H), 2.87 (dt, J = 10.2, 4.7 Hz, 0.5H), 2.75 (ddd, J = 11.2, 6.6, 4.9 Hz, 0.5H), 2.04 (m, 1.5 H), 1.71 (m, 1H), 1.46 (m, 0.5H), 0.97 (d, J = 6.6 Hz, 1.5H), 0.94 (d, J = 7) , 1 Hz, 1.5H) ppm; TLC (20% EtOAc / hexane) R_f = 0,34.

StepC. (4R, 5R) -4- (3-chlorophenyl) -5- (4-chlorophenyl) -5-hydroxy-2-methylpentanoic acid and (4S, 5S) -4- (3-chlorophenyl) -5- (4-chlorophenyl) -5-hydroxy-2-methylpentanoic acid [0232]

58366 Yes

<img file="RS58366B1_D0095.tif" />

<img file="RS58366B1_D0096.tif" />

Solution of racemic mixture (4R, 5R) -methyl 4- (3-chlorophenyl) -5- (4-chlorophenyl) -5-hydroxy-2-methylpentanoate and (4S, 5S) -methyl 4- (3-chlorophenyl) - 5- (4-Chlorophenyl) -5-hydroxy-2-methylpentanoate (245.7 g, 669 mmol; reference Example 15, step B) in THF (1.17 L) was prepared by heating to 40 ° C. The vessel was cooled until the internal temperature reached ~ 14 ° C. To a solution in THF was carefully added lithium hydroxide hydrate (42.1 g, 1.0 mol) in water (585 mL). The mixture was allowed to stir at room temperature and monitored by LC / MS to detect the absence of starting materials (~ 2.5 h). After completion of the reaction, the solution was cooled again to a temperature of ~ 14 ° C. 2 N HCl (526 mL) was added slowly. The layers were separated, and the aqueous layer (pH ~ 2) was washed with ethyl acetate (1 x 500 mL then 1 x 250 mL). The combined organic layers were dried over magnesium sulfate and concentrated to give 264 g of a racemic mixture of (4R, 5R) -4- (3-chlorophenyl) -5- (4-chlorophenyl) -5-hydroxy-2-methylpentanoic acid and ( 4S, 5S) -4- (3-chlorophenyl) -5- (4-chlorophenyl) -5-hydroxy-2-methylpentanoic acid. The crude material containing some residual solvent was used in this form in the next reaction. The product (estimated after solvent correction at 227g) is, roughly speaking, a 1: 1 mixture of diastereomers in position 2.

¹1 H-NMR (500 MHz, CDCl 3)₃) 5 7.31 (m, 2H), 7.25 (m, ZN), 7.17 (m, 2H), 7.05 (m, 1H) 4.74 (m, 1H), 2.99 ( ddd, J = 11.2, 1.7, 3.7 Hz, 0.5H), 2.90 (ddd, J = 11.5, 7.3, 4.6 Hz, 1H), 2.15 ( m, 1.5 H), 1.85 (m, 0.5 H), 1.67 (ddd, J = 14.3, 11.5, 3.4 Hz, 0.5 H), 1.52 m, 0.5 H), 1.08 (d, J = 7.1 Hz, 1.5 H), 1.05 (d, J = 6.9 Hz, 1.5 H) ppm.

Alternatively, (4R, 5R) -4- (3-chlorophenyl) -5- (4-chlorophenyl) -5-hydroxy-2-methylpentanoic acid, in the form of a mixture of methyl diastereomers, can be obtained from a racemic mixture of methyl 4- (3-chlorophenyl) -5- (4-chlorophenyl) -2-methyl-5-oxopentanoate.

A solution of the racemic mixture of methyl 4- (3-chlorophenyl) -5- (4-chlorophenyl) -2-methyl-5-oxopentanoate (500 g, 1.37 mol, 1 eq) in anhydrous 2-propanol was poured into a three-necked flask. (2.5 I), to which KO was added^lBoo (46.1 g, 0.41 mol, 0.3 eq) and the mixture was stirred for 30 minutes until a clear yellow solution formed. The solution was then treated with a solution of dichloro {(S) - (-) - 2,2'-bis [di (3,5-xylyl) phosphino] -1,1'-binaphthyl} [(2S) - (+) - 1 , 1-bis (4-methoxyphenyl) -3-methyl-1,2-butanediamine] ruthenium (11) (5 g, 4.1 mmol, 0.003 eq, Strem Chemicals inc., Newburyport, MA) in anhydrous toluene (250 ml) , and the mixture was stirred at RT for 2 hours (note: most of the methyl ester was converted to isopropyl). The solution was transferred to two pairs of shakers, which were hermetically sealed and bubbled with hydrogen 3 times. The reaction mixture was shaken on RT under a hydrogen pressure of 414 kilopascals. After 18h, the reaction was quenched by the addition of sat. NH₄CI, it's a mixture

58366 Β1 concentrated and extracted with EtOAc (2 I x 2). The combined organic layers were washed with brine and concentrated to a brown oil, which was used as such in the next step.

The crude intermediate (542 g, 1.37 mol) was dissolved in THF (3 L) and MeOH (11) and 2 M LiOH (11) was added. The solution was rotated at RT overnight, concentrated to remove most of the THF and MeOH, and the reaction was quenched by the addition of 11 2 IVI HCl. After phase separation, the aqueous layer was extracted with EtOAc (11 x 2). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. This product, (4R, 5R) -4- (3-chlorophenyl) -5- (4-chlorophenyl) -5-hydroxy-2-methylpentanoic acid, in the form of a mixture of methyl diastereomers, was used crude in the next step.

Step D. (5R, 6R) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one and (5S, 6S) -5- (3-chlorophenyl) -

6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one [0237]

<img file="RS58366B1_D0097.tif" />

<img file="RS58366B1_D0098.tif" />

A mixture of hydroxy acid diastereomers (227 g, 643 mmol; reference example 15, step C) was lactonized under Din-Stark reaction conditions in toluene (1.07 I) with pyridine 4-methylbenzenesulfonate (PPTS, 4.84 g, 19, 28 mmol) under a nitrogen atmosphere. After vigorous reflux for 2 h, the solution was cooled to room temperature and transferred to a separatory funnel. The residue in the flask was washed with ethyl acetate. The combined organic phases were washed, respectively, with water (1 x 250ml), sat. sodium bicarbonate solution (1 x 250ml) and sat. vod. NaCl solution (1 x 250 mL). After drying over magnesium sulfate, a mixture of diastereomeric lactones was obtained in the form of a light brown solid by concentration under reduced pressure.

¹1 H-NMR (500 MHz, CDCl 3)₃) 5 7.24-6.95 (series m, 6H), 6.91 (d, J = 7.6 Hz, 0.5H), 6.82 (m, 1.5H), 6.73 (d , J = 7.6 Hz, 0.5H), 5.77 (d, J = 3.9 Hz, 0.5H), 5.69 (d, J = 4.6 Hz, 0.5H), 3 , 67 (dt, J = 7.6, 4.2 Hz, 0.5H), 3.55 (td, J = 7.8, 4.6 Hz, 0.5H), 2.97 (m, 0 , 5 H), 2.81 (quintet doublet, J = 14.4, 7.1 Hz, 0.5 H), 2.56 (dt, 16.1, 8.0 Hz, 0.5H), 2 , 32 (dt, J = 13.7, 6.9 Hz, 0.5 H), 2.07 (ddd, J = 13.2, 8.6, 4.4 Hz, 0.5 H), 1.85 (ddd, J = 14.2, 12.7, 7.6 hz, 0.5H), 1.41 (d, J = 7.1 Hz, 1.5H), 1.39 (d, J = 6) .9 Hz, 1.5H) ppm.

58366 Yes

Step E: (3S, 5R, 6R) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one and (3R, 5S, 6S) - 3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one [0239]

<img file="RS58366B1_D0099.tif" />

<img file="RS58366B1_D0100.tif" />

A solution of the racemic lactone from the previous step (reference example 15, step D, 190.28 g, 568 mmol) in THF (946 ml) was prepared in a round 1-neck balloon equipped with a Kleisen adapter, a 500-funnel ml and an internal thermometer, under a nitrogen atmosphere. The solution was cooled to -35 ° C. Allyl bromide (120 mL, 1.42 mol) was added from the addition funnel, maintaining the temperature below -30 ° C during the addition of the reagent. A solution of LHMDS (1 M in THF, 738 mL, 738 mmol) was added dropwise to the reaction mixture while maintaining the temperature below -30 ° C. The reaction was allowed to slowly warm to -5 ° C over a period of 1 h. The solution was re-cooled to about -20 ° C and the cannula was added to a solution of ammonium chloride in water at about 5C. After separating the layers, the aqueous layers were extracted twice with ethyl acetate. The combined organic layers were washed with sat. vod. NaCl solution and dried over sodium sulfate. Concentration in vacuo gave 219 g of a light yellow solid. The solids were mixed with hexane (2 L) at room temperature for 2 h. The solid was then filtered off, washed with hexane (2 x 100 mL) and dried to give the title compounds as a racemic mixture.

¹1 H-NMR (500 MHz, CDCl 3)₃) 7.24 (m, 1H), 7.20-7.15 (m, ZN), 6.91 (t, J = 1.7 Ηζ, 1H), 6.77 (d, J = 7, 6 Hz, 1H), 6.59 (m, 2H), 5.84 (ddt, J = 17.6, 10.3, 7.6 Ηζ, 1H), 5.71 (d, J = 5.4) Ηζ, 1H), 5.21-5.13 (m, 2H), 3.81 (dt, J = 12.0, 4.2 Ηζ, 1H), 2.62 (AVH J_AB = 14.0, J_AX = 7.8 Ηζ, 1H), 2.52 (AVH, J_AB = 13.9, J_AX = 7.3 Ηζ, 1H), 1.98 (dd, J = 14.0, 12.0 Ηζ, 1H), 1.91 (ddd, J = 14.0, 3.7, 1.2 Ηζ, 1H), 1.42 (s, ZN) ppm.

Step F: Separation of (3S, 5R, 6R) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one and (3R, 5S, 6S) ) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one

58366 Β1 [0241]

<img file="RS58366B1_D0101.tif" />

Hiraina SFC

--------- * Chromatography

<img file="RS58366B1_D0102.tif" />

<img file="RS58366B1_D0103.tif" />

Racemic mixture (3S, 5R, 6R) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one and (3R, 5S, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one can be separated by supercritical-liquid chromatography (SFC) as follows : using a 250 h 30 mm Lux2® column (Phenomenex, Torrance, CA 90501, USA) with 20 g / min methanol (20 mM NH3) + 60 g / min CO₂ and Thar 80 SFC. Outlet pressure = 100 bar; temperature = 23C; wavelength = 220 nm. Injections of 0.3 ml 5.0 g / 80 ml (62.5 mg / ml sample solution in methanol / dichloromethane (75: 5), ie 18.75 mg / injection were used. Duration = 8 min, cycle time = 3 min.

The first maximum collected was assigned to (3R, 5S, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one. The second maximum collected was found to be in fact (3S, 5R, 6R) 3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one by further chemical by derivatization with (S) -2-amino-1-butanol and conversion to the same compound obtained in reference example 4, step B, in the process described for reference example 15, steps G and X. The NMR of the separated enantiomers coincided with the spectra of the racemates described above.

Alternatively, (3S, 5R, 6R) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one can be obtained from racemic a mixture of methyl 4- (3-chlorophenyl) -5- (4-chlorophenyl) -2-methyl-5-oxopentanoate.

A solution of racemic mixture of methyl 4- (3-chlorophenyl) -5- (4-chlorophenyl) -2-methyl-5-oxopentanoate (500 g, 1.37 mol, 1 eq) in anhydrous 2-propanol was poured into a three-necked flask. (2.5 I), to which KO was added^lBoo (46.1 g, 0.41 mol, 0.3 eq) and the mixture was stirred for 30 minutes until a clear yellow solution formed. The solution was then treated with a solution of dichloro {(S) - (-) - 2,2'-bis [di (3,5-xylyl) phosphino] -1,1'-binaphthyl} [(2S) - (+) - 1 , 1-bis (4-methoxyphenyl) -3-methyl-1,2-butanediamine] ruthenium (11) (5 g, 4.1 mmol, 0.003 eq, / Strem Chemicals inc., Newburyport, MA) in anhydrous toluene (250 ml ), and the mixture was stirred at RT for 2 hours (note: most of the methyl ester was converted to isopropyl). The solution was transferred to two pairs of shakers, which were hermetically sealed and bubbled with hydrogen 3 times. The reaction mixture was shaken on RT under a hydrogen pressure of 414 kilopascals. After 18 h, the reaction was quenched by the addition of sat. NH₄CI, it's a mixture

58366 Βί concentrated and extracted with EtOAc (2 I x 2). The combined organic layers were washed with brine and concentrated to a brown oil, which was used as such in the next step.

The crude intermediate (542 g, 1.37 mol) was dissolved in THF (3 L) and MeOH (11) and 2M LiOH (11) was added. The solution was rotated at RT overnight, concentrated to remove most of the THF and MeOH, and the reaction was quenched by the addition of 11 2M HCl. After phase separation, the aqueous layer was extracted with EtOAc (11 x 2). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo.

Step 1: (S) -2 - ((2R, 3R) -2- (3-chlorophenyl) -3- (4-chlorophenyl) -3-hydroxypropyl) -N - (S) -1-hydroxy-3-methylbutane -2yl) -2-methylpent-4-enamide [0247]

<img file="RS58366B1_D0104.tif" />

Mixture of (S) -2-amino-3-methylbutan-1-ol (550 mg, 5.33 mmol) and (3S, 5R, 6R) -3-allyl-5- (3-chlorophenyl) -6 (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one (Reference Example 15, Step H, Compound 2, 500 mg, 1,332 mmol) was heated at 100 ° C for 24 h. After cooling to room temperature, the residue was dissolved in ethyl acetate and washed with 3 h 1 N HCl (5 mL) and then sat. vod. NaCl solution (5 ml). The organic phase was dried over MgSO 4₄, filtered, and the filtrate was concentrated to give the title compound. ¹1 H-NMR (500 MHz, DMSO-d 6)₆) b 7.21 (m, 2H), 7.10 (m, 2H), 7.06 (br s, 1H), 6.99 (m, 2H), 6.86 (br d, J = 8, 8 Hz, 1H), 6.84 (br d, J = 7.1 Hz, 1H), 5.53 (dddd, J = 16.9, 10.3, 8.1, 6.6 Hz, 1H) , 5.46 (d, J = 4.4 Hz, 1H), 4.90 (m, 2H), 4.78 (t, J = 4.2 Hz, 1H), 4.56 (t, J = 5.1 Hz, 1H), 3.56 (m, 1H), 3.37 (m, 2H), 2.87 (dt, J = 7.8, 4.2 Hz, 1H), 2.29 ( dd, J = 13.7, 6.4 Hz, 1H), 2.14 (dd, J = 14.4, 7.8 Hz, 1H), 1.97 (dd, J = 14.4, 3, 9 Hz, 1H), 1.88 (dd, J = 13.9, 8.1 Hz, 1H), 1.76 (octet, J = 6.4 Hz, 1H), 0.97 (s, ZN) ,, 0.81 (d, J = 6.8 Hz, ZN), 0.75 (d, J = 6.6 Hz, ZN) ppm.

Step H. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-hydroxy-3-methylbutan-2-yl) -3-methylpiperidin-2-one

58366 ]Ί [0249]

<img file="RS58366B1_D0105.tif" />

(S) -2 - ((2R, 3R) -2- (3-chlorophenyl) -3- (4-chlorophenyl) -3-hydroxypropyl) -N - ((S) -1-hydroxy-3-) methylbutan-2-yl) 2-methylpent-4-enamide (Reference Example 15, Step G) was transferred, as a solution in anhydrous benzene, to a pre-weighed, dried 50 ml round flask and evaporated to dryness. Azeotropic distillation of benzene and water was performed two more times, and the residue was dried under high vacuum for 2 h, after which its mass was 550 mg. A dried mechanical stirrer was placed in the balloon. The vessel was sealed and air was purged with nitrogen, followed by anhydrous dichloromethane (23 mL) and triethylamine (1.3 mL, 9.33 mmol). The resulting solution was cooled to 0 ° C with stirring. Using a microsyringe, methanesulfonyl chloride (0.270 mL, 3.49 mmol) was added dropwise to the mixture. After 1 h the reaction was quenched by the addition of HCl (1.2M, 12 mL) and the reaction mixture was diluted with ethyl acetate. The organic layer was washed with 1.2M HCl (30 mL), saturated sodium bicarbonate (2 x 25 mL) and sat. vod. NaCl solution. After drying over magnesium sulfate and concentration in vacuo, an intermediate product was obtained in the form of an off-white foam (0.64 g). To this intermediate were added 1.8bis (dimethylamino) naphthalene (314 mg, 1.465 mmol) and water (0.104 mL, 5.75 mmol) followed by dioxane (23 mL). The mixture was heated under nitrogen at 110 ° C overnight. After cooling, the mixture was dissolved in ethyl acetate and washed with saturated ammonium chloride solution. The aqueous phase was re-extracted with ethyl acetate. The combined organic layers were washed with sat. vod. NaCl solution, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by chromatography on silica gel eluting with ethyl acetate in hexanes. The chromatographic fractions containing the largest proportion of the desired product were combined. The product was again purified by column chromatography on 40 g of silica gel, eluting with a gradient of 0 to 50% ethyl acetate in hexanes, to give the title compound.

^gN NMR (400 MHz, CHLOROFORM-d) b 0.73 (d, J = 6.46 Hz, 3 H), 0.83 (d, J = 6.65 Hz, 3 H), 1.28 (s , 3H), 1.91 - 2.00 (m, 1H), 2.00 - 2.10 (m, 1H), 2.26 - 2.45 (m, 1H), 2.56 - 2 , 74 (m, 2H), 3.16 (br.s, 1H), 3.26 (ddd, J = 13.50,10.47, 3.42 Hz, 1H), 3.43 (br. s, 1H), 3.76 (dd, J = 11.25, 3.42 Hz, 1H), 4.49 (d, J = 10.56 Hz, 1H), 5.18 (s, 1H) , 5.21 (d, J = 6.46 Hz, 1H), 5.87 (ddt, J = 16.95, 9.85, 7.53 Hz, 1H), 6.72 (seemingly d, J = 7.63 Hz, 1H), 6.95 (t, J = 1.66 Hz, 1H), 6.97 - 7.17 (m, 4H), 7.23 (d, J = 8.41 Hz, 2H); MS (ESI) m / z = 460 [M + H]⁺.

Step I. (S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1-yl) -3-methylbutanal

58366 ]Ί [0251]

<img file="RS58366B1_D0106.tif" />

To a solution of (3S, 5R, 6S) -3-al yl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-hydroxy-3-methylbutan-2 -yl) -3-methylpiperidin-2-one (reference example 15, step H, 365.4 mg, 0.794 mmol) in dichloromethane (8 ml) and water (0.04 ml, 2.220 mmol) was added Des-Martin periodin mg, 2.212 mmol) in the solid state. The resulting suspension was stirred vigorously at room temperature for 1.5 h. The reaction was quenched with sodium thiosulfate solution (1 M aq., 6 mL). An additional amount of sodium sulfate solution (1 M aq., 6 ml) was added and the mixture was stirred until the lime suspension became a slightly turbid two-phase mixture. The aqueous phase was separated and further extracted with dichloromethane. The organic layer was washed with sodium thiosulfate solution, saturated aqueous sodium bicarbonate and sat. vod. NaCl solution. After drying over sodium sulfate and concentration, the residue was purified on silica gel, eluting with a gradient of 0 to 30% ethyl acetate in hexanes. Fractions containing the desired product were combined to give the title compound as a white foam. MS (ESI) m / z = 458 [M + H]⁺.

Step J. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -2-hydroxy-4-methylpentan-3-yl) -3-methylpiperidin-2-one.

[0253]

ΗΩ. Me

<img file="RS58366B1_D0107.tif" />

In a pre-dried solution of (S) -2 - ((3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-2-oxopiperidin-1- il) -3-methylbutanal (Reference Example 15, Step I, 286 mg, 0.624 mmol) in THF (6.5 mL) at 0 ° C and methylmagnesium bromide (1.4 mL, 1,960 mmol, 1.4 M in a mixture of THF: toluene 1: 3) from a syringe. The ice bath has been removed. After 2 h, the solution was re-cooled to 0 ° C, and

58366 Β1 the reaction was stopped by careful addition of saturated ammonium chloride solution. The resulting mixture was extracted with ethyl acetate. The organic layer was washed with sat. vod. NaCl solution, dried over sodium sulfate, filtered and concentrated. The residue was purified by chromatography on silica gel eluting with a gradient of ethyl acetate in hexanes from 0 to 40%. The fractions containing the desired product were combined and purified again to give the title compound as a mixture of diastereomeric alcohols, in the form of a white foam. MS (ESI) m / z = 474 [M + H]⁺.

Step K. 2- (3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3-methyl-1- (S) -2-methyl-4-oxopentan-3 -yl) -2-oxopiperidin-3-yl) acetic acid [0255]

<img file="RS58366B1_D0108.tif" />

Using a procedure similar to that described in Reference Example 13, step E, (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -2 -hydroxy-4-methylpentan-3-yl) -3-methylpiperidin-2-one (reference example 15, step J, 244 mg, 0.51 mmol) was converted to the title compound obtained after purification by chromatography on silica gel eluting with ethyl acetate. in hexanes, in the form of a white solid. MS (ESI) 490 [M + H]⁺.

Step L. 2- (3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((2S, 3S) -2-hydroxy-4-methylpentan-3-yl ) -3-methyl-2-oxopiperidin-3-yl) acetic acid The title compound was obtained from 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -3 -methyl-1- ((S) -2methyl-4-oxopentan-3-yl) -2-oxopiperidin-3-yl) acetic acid (Reference Example 15, Step K, 79.9 mg, 0.16 mmol) similar to that described in reference example 13. After work-up, the substance was purified by chromatography on a 24 g silica gel column, eluting with a gradient of 10 to 20% isopropanol in hexanes. The purest fractions obtained were combined, concentrated, redissolved in MeCN / water 1: 1, passed through a textile microfilter, frozen and lyophilized to give the title compound as a white solid. Stereochemistry was determined by analogy with reference example 8.

58366 Β1 ^gN NMR (400 MHz, methanol-d4) b 0.62 (d, J = 7.04 Hz, 3 H), 0.67 (d, J = 6.65 Hz, 3 H), 1.26 (d , J = 6.46 Hz, 3H), 1.42 (s, 3H), 2.13 - 2.29 (m, 3H), 2.49 (t, J = 7.14 Hz, 1H) ), 2.62 (d, J = 13.69 Hz, 1H), 3.01 (d, J = 13.69 Hz, 1H), 3.57 (td, J = 10.81, 6.16) Hz, 1H), 4.23 (t, J = 6.65 Hz, 1 H), 4.70 (d, J = 10.95 Hz, 1 H), 6.65 - 7.51 (m, 8 H). MS (ESI) m / z = 492 [M + H]⁺.

REFERENCE EXAMPLE 16

2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1- (ethylsulfonyl) butan-2-yl) -3-methyl- 2-oxopiperidin-3-yl) acetic acid [0258]

<img file="RS58366B1_D0109.tif" />

StepA. (3S, 5S, 6R, 8S) -8-allyl-6- (3-chlorophenyl) -5- (4-chlorophenyl) -3-ethyl-8-methyl-2,3,5,6,7,8hexahydrooxazolo [ 3,2-a] pyridin-4-yl trifluoromethanesulfonate [0259]

<img file="RS58366B1_D0110.tif" />

The title compound was prepared by the method of Reference Example 17, step A, using (S) -2-aminobutanol instead of L-valinol, as the first eluted diastereomer.

^gN NMR (500 MHz, DMSO-d 6)_eb ppm 7.95 (1H, br, s), 7.34 - 7.60 (2H, m), 7.18-7.34 (4H, m), 7.13 (1H, dt, J = 7.5, 1.3 Hz), 5.88 (1 H, m), 5.37 (1 H, dd, J = 16.8, 1.6 Hz), 5.28 (1 H, dd, J = 10.0, 2.0 Hz), 5.16 (1 H, d, J = 10.8 Hz), 5.06 (1 H, t, J = 9.8 Hz), 4.78 (1H, dd, J = 9.5, 7.1 Hz), 4.45 (1H, m, J = 2.7 Hz), 3.88 - 3.98 (1H,

58366 Β1

m), 2.66 - 2.85 (2 Η, m), 2.33 (1 Η, t, 1 = 13.4 Ηζ), 1.99 (1 Η, dd, 1 = 13.7, 3) , 4 Ηζ), 1.32 (3 Η, s), 0.94 (1 Η, m),

0.59 (3 Η, t, 1 = 7.2 Ηζ), 0.41 - 0.53 (1 Η, m); Mass spectrum (ESI) m / z = 428.2 (M⁺).

Step B. (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1- (ethylthio) butan-2-yl) - 3-methylpiperidin-2-one [0261]

<img file="RS58366B1_D0111.tif" />

In solution (3S, 5S, 6R, 8S) -8-allyl-6- (3-chlorophenyl) -5- (4-chlorophenyl) -3-ethyl-8-methyl-2,3,5,6 , 7,8hexahydrooxazolo [3,2-a] pyridin-4-yl trifluoromethanesulfonate (86 mg, 0.15 mmol; reference example 16, step A) in DMF (0.74 ml) was added sodium ethantiolate (38 mg , 0.45 mmol). After stirring at 25 ° C for 1.5 h, the reaction was stopped (sat. Aq. NH₄CI), the reaction mixture was extracted (2 h EtOAc) and washed (2 h aqueous brine). The combined organic layers were dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure. Purification of the residue by chromatography on silica gel (12 g SiO₂, 10% and 20% EtOAc / hexanes) gave the title compound as a colorless liquid.

StepC. 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1- (ethylsulfonyl) butan-2-yl) -3-methyl- 2-Oxopiperidin-3-yl) acetic acid In solution (3S, 5R, 6S) -3-allyl-5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1-) (ethylthio) butan-2-yl) -3methylpiperidin-2-one (60 mg, 0.12 mmol; reference example 16, step B) in a mixture of water (0.66 ml), acetonitrile (0.44 ml) and CCl₄ Sodium periodate (157 mg, 0.734 mmol) was added (0.44 mL) which was stirred rapidly, followed by ruthenium (III) chloride hydrate (2.8 mg, 0.012 mmol). After stirring vigorously for 5 h, the reaction mixture was acidified (10% citric acid) and diluted (EtOAc). The mixture was filtered through a pad of Celite® (JT Baker, Phillipsberg, NJ, diatomaceous earth), and the filtrate was extracted (2 h EtOAc). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure. Purification of the residue by reverse-phase preparative HPLC (Gemini ™ Prep C₁₈ 5 pm column, Phenomenex, Torrance, CA; gradient elution with 40% to 60% MeCN in water, where both solvents contain 0.1% TFA) the title compound is obtained in the form of a white foam.

³1 H NMR (400 MHz, CHLOROFORM-d) b ppm 7.24 - 7.26 (2 H, m), 7.01 - 7.20 (4 H, m), 6.93 - 6.98 (1 H , m), 6.85 (1H, d, 1 = 7.0 Hz), 4.94 (1H, d, 1 = 10.6 Hz), 4.15 (1H, t, 1 = 12 , 1 Hz), 3.24 - 3.37 (1 H, m), 2.92 - 3.18 (4

58366 Yes

<img file="RS58366B1_D0112.tif" />

1.48 (3 Η, s), 1.42 -1.46 (1 Η, m) 1.44 (3 Η, t, J = 7.5 Ηζ), 0.41 (3 Η, t, 7 = 7.5 Hz); Mass spectrum (ESI) m / z =

540.1 [M + H].

REFERENCE EXAMPLE 17

2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1- (ethylsulfonyl) -3-methyl butan-2-yl) - 3-methyl-2-oxopiperidin-3-yl) acetic acid [0264]

Oh_no , O .0

Step A. (3S, 5S, 6R, 8S) -8-Allyl-6- (3-chlorophenyl) -5- (4-chlorophenyl) -3-isopropyl-8-methyl-2,3,5,6,7 , 8 hexahydrooxazolo [3,2-a] pyridin-4-yl trifluoromethanesulfonate [0265]

TfO L-valinol (Sigma Aldrich, St. Louis, MO) (3.64 g), racemic mixture (3S / R, 5R / S, 6R / S) -3-allyl-5- (3-chlorophenyl) - 6- (4-chlorophenyl) -3-methyltetrahydro-2H-pyran-2-one (3.17 g, reference example 15, step E) and lithium t-butoxide (0.025 g) were heated as a solid mixture for 17 h , on an oil bath set at 135 ° C. After cooling, the glassy solid was dissolved in dichloromethane. The organic layer was washed with sat. with ammonium chloride solution, then 1N sodium hydroxide solution, and then aqueous brine. The organic layer was dried over magnesium sulfate and concentrated to give 3.88 g of a mixture of diastereomers. A portion of 2.61 g (67% of the total amount) of the approximately 1: 1 mixture, obtained as previously described, was dissolved in toluene and evaporated to dryness three times to

58366 It removes residual moisture. Dichloromethane (55ml) and 2,6-dimethylpyridine (3.3ml, 28.5mmol) were added and the resulting solution was cooled to -50 ° C in a dry ice / acetonitrile bath with stirring. Trifluoromethanesulfonic anhydride (2.4 mL, 14.27 mmol) was added over 10 minutes so that the internal temperature of the reaction mixture did not exceed -45 ° C. After 40 minutes, the reaction was quenched by the addition of 2 M HCl.

The mixture was warmed to room temperature, diluted with dichloromethane, washed with 2 N HCl, water and finally aqueous brine. The organic layer was dried over magnesium sulfate, filtered and concentrated to a yellow foam weighing approximately 2.6 g. A portion of this substance (1.92 g, 74%) was purified by liquid chromatography under moderate pressure with a 120 g column eluting with a gradient of 20 to 100% acetone in hexanes. Fractions containing the faster eluting diastereomer (less polar) were concentrated to give the title compound as a white foam.

D NMR (400 MHz, CDCl 3)₃) b ppm 0.62 (d, J = 2.4 Hz, 3 H), 0.64 - 0.74 (m, 4 H), 1.51 (s, 3 H), 2.04 (dd, J = 14.1, 3.5 Hz, 1 H), 2.54 - 2.79 (m, 3 H), 3.58 (ddd, J = 13.7, 10.8, 3.5 Hz, 1H), 4.59 (dd, J = 10.2, 4.7 Hz, 1H), 4.67 (dd, J = 9.2, 4.9 Hz, 1H), 5.23 - 5.45 (m, 3H), 5.71 (d, J = 11 Hz, 1H), 5.83 (ddt, J = 17, 9.9, 7.4 Hz, 1H), 7.06 (t , J = 1.8 Hz, 1H), 7.11 - 7.16 (m, 1H), 7.19 (t, J = 7.7 Hz, 1H), 7.23 - 7.32 (m, 3H), 7.39 (br s, 2H); Mass spectrum (ESI) m / z = 442.2 (M⁺).

Step B. 2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1- (ethylsulfonyl) -3-methylbutan-2-yl ) -3-Methyl-2-oxopiperidin-3-yl) acetic acid The title compound was obtained from (3S, 5S, 6R, 8S) -8-allyl-6- (3-chlorophenyl) -5- (4-chlorophenyl) ) -3-Isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo [3,2-a] pyridin-4-yl trifluoromethanesulfonate (reference example 17, step A), by procedures similar to those described in reference Example 16, using an equivalent amount of entanediol in step B.

D NMR (500 MHz, methanol-d₄) b ppm 0.51 (d, J = 7.1 Hz, 3 H), 0.66 (d, J = 6.6 Hz, 3 H), 1.37 (s, 3 H), 1.41 (t, J = 7.5 Hz, 3 H), 2.05 (dd, J = 13.7, 2.9 Hz, 1 H), 2.18 (dq, J = 14.2, 6.9 Hz) , 1H), 2.31 (t, J = 13.7 Hz, 1H), 2.62 (d, J = 13.7 Hz, 1H), 3.00 (d, J = 13.7 Hz, 1H), 3.14 - 3.24 (m, 3H), 3.25 - 3.30 (m, 1H), 3.57 (ddd, J = 13.8, 10.9, 2 , 9 Hz, 1H), 4.02 (dd, J = 13.9, 10.5 Hz, 1H), 5.12 (d, J = 11 Hz, 1H), 7.00 (dt, J = 7.3, 1.5 Hz, 1H), 7.04 - 8.17 (m, 7H); Mass spectrum (ESI) m / z = 554.2 (M + 1).

EXAMPLE 1

2 - ((3R, 5R, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1 - ((S) -1- (isopropylsulfonyl) -3-methylbutan-2-yl) -3 -methyl-2-oxopiperidin-3-yl) acetic acid [0268]

58366 Yes

<img file="RS58366B1_D0113.tif" />

The title compound was obtained from (3S, 5S, 6R, 8S) -8-allyl-6- (3-chlorophenyl) -5- (4-chlorophenyl) -3-isopropyl-8methyl-2,3,5, 6,7,8-Hexahydrooxazolo [3,2-a] pyridin-4-yl trifluoromethanesulfonate (reference example 17, step A), by procedures similar to those described in reference example 16, using an equivalent amount of propane-2-thiol in step B.

³1 H NMR (500 MHz, methanol-d₄) b ppm 0.50 (d, J = 6.9 Hz, 3 H), 0.65 (d, J = 6.6 Hz, 3 H), 1.37 (s, 3 H), 1.41 (d, J = 6.9 Hz, 6 H), 2.05 (dd, J = 13.6, 2.8 Hz, 1 H), 2.18 (dq, J = 14, 6.9 Hz, 1 H), 2.31 (t, J = 13.7 Hz, 1 H), 2.61 (d, J = 13.5 Hz, 1 H), 2.99 (d, J = 13.7 Hz) , 1H), 3.11 (d, J = 13.7 Hz, 1H), 3.25 - 3.29 (m, 1H), 3.32 3.37 (m, 1H), 3 , 49 - 3.65 (m, 1H), 4.01 (dd, J = 13.7, 10.5 Hz, 1H), 5.13 (d, J = 11 Hz, 1H), 6.93 - 7.03 (m, 1H), 7.03 - 8.23 ​​(m, 7H); Mass spectrum (ESI) m / z = 568.0 (M + 1).

The compounds of the present invention show inhibition of the interaction between HDM2 and p53 in the following assays.

Homogeneous fluorescence test as a function of time (HTRF1 test) Standard test conditions for the in vitro HTRF test involved a total reaction volume of 50 [mu] l in a black Costar 384 field polypropylene plate in 1 h PBS buffer pH 7.4.1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53, 1.8 nM SA-XLent (Cisbio; Bedford, MA), 0.6 nM for anti- GST cryptate monoclonal antibody (Cisbio; Bedford, MA) and 200 mM CF.

Amino acid residues 1-188 of human MDM2 were expressed as aminiterminal fusion protein glutathione-S-transferase (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as the aminoterminal fusion protein AviTag ™ -TrxA-6xHis (biotinylated p53) in E. coli. Each protein was purified from the cell homogenate by affinity chromatography.

Specifically, 10 μl of GST-hMDM2 was incubated with 10 μl of diluted compound (various concentrations, serial dilutions) in 10% DMSO for 20 min at room temperature. 20 [mu] L of biotinylated p53 was added to the GST-hMDM2 + compound and then incubated at room temperature for 60 minutes. To the reaction mixture of GST-hMDM2, biotinylated p53 and compound was added 10 μl

58366 Β1 detection buffer, consisting of SA-XLent, anti-GST cryptate antibody and KF, and the mixture was left at room temperature for> 4 h to reach equilibrium. The final concentration of DMSO in the reaction mixture was 2%. Fluorescence readings as a function of time were performed on a microtiter plate reader. The percentage of inhibition was calculated relative to nutlin-3.

As the potency of HDM2 inhibitors increased, an improved HTRF assay (HTRF2 assay) was developed. All test conditions remained the same as previously described, except for the following changes in reagent concentrations: 0.2 nM GST-hMDM2 (1-188), 0.5 nM biotinylated p53 (1-83), 0.18 nM SA -XLent, and 100 mM KF.

The results are listed in the table below.

Table 1

<td>Example</td><td>HTRFl IC₅₀(pM)</td><td>HTRF2 IC₅₀(pM)</td>

<td>Reference example 1</td><td> 0,04</td><td> 0,004</td>

<td>Reference example 2</td><td> 0,06</td><td> 0,01</td>

<td>Reference example 3</td><td> 0,02</td><td> 0,003</td>

<td>Reference example 4</td><td> 0,01</td><td> 0,004</td>

<td></td><td></td><td></td>

<td>Example</td><td>HTRFl IC₅o (pM)</td><td>HTRF2 IC₅o (pM)</td>

<td>Reference example 5</td><td> 0,01</td><td> 0,001</td>

<td>Reference example 6</td><td> 0,04</td><td> 0,006</td>

<td>Reference example 7</td><td> 0,02</td><td> 0,006</td>

<td>Reference example 8</td><td> 0,01</td><td> 0,001</td>

<td>Reference example 9</td><td> 0,02</td><td> 0,002</td>

<td>Reference example 10</td><td> 0,03</td><td> 0,006</td>

<td>Reference example 11</td><td> 0,01</td><td> 0,001</td>

<td>Reference example 12</td><td> 0,06</td><td></td>

<td>Reference example 13</td><td></td><td> 0,001</td>

<td>Reference example 14</td><td> 0,08</td><td> 0,002</td>

<td>Reference example 15</td><td></td><td> 0,001</td>

<td></td><td></td><td></td>

<td>Reference example 16</td><td></td><td> 0,0003</td>

<td>Reference example 17</td><td></td><td> 0,0005</td>

<td>Example 1</td><td></td><td> 0,0010</td>

58366 Jedί The compounds of the present invention show activation of cyclin-dependent kinase p22 inhibitors^WAF1/ ciPi r21 TaqMan® test Inhibition of the interaction between hMDM2 and p53 leads to activation of p53 pathways through stabilization and accumulation of p53. p53 activates the transcription of many genes, one of which is p2i^{WAF1 / clpl}. to assess the potency of hMDM2 inhibitors, quantitative reverse transcription by polymerase chain reaction (qRT-PCR or TaqMan®) was applied to measure concentrations of r21 transcript in compound-treated cells relative to dimethylsulfoxide-treated control cells.

On day 1, SJSA-1 cells were seeded in plates at a density of 3 χ 10⁴ cell / field in 96-well culture plates, in 100 [mu] l of culture medium (RPMI 1640; 10 mM HEPES; 1 tM sodium pyruvate; 1 h penicillin-streptomycin-glutamine (PSQ); and 10% fetal bovine serum (all reagents obtained from Invitrogen; Carlsbad, CA)). Cells were grown overnight at 37 ° C and 5% CO₂.

On day 2, hMDM2 inhibitors were serially diluted in DMSO (Sigma-Aldrich; St. Louis, MO). 5 [mu] l of each dilution of compound was added to 245 [mu] l of filtered test medium (RPMI1640.10 mM HEPES, 1 mM sodium pyruvate, and IX PSQ), which contained 10% FBS. Alternatively, the test was also performed in the presence of 10% human serum or 10% mouse serum, or in the absence of any serum. The culture medium was removed from the SJSA-1 cells grown in the fields and replaced with 100 [mu] l / field of test medium. Then, 100 [mu] l of medium containing diluted inhibitor was added to each field, to a final volume of 200 [mu] l. Dose titration of the compound showed final concentrations ranging from 0.049 μM to 50 μM, plus DMSO control. Cells were incubated in the presence of inhibitor at 37 ° C and with 5% CO₂ within 7 hours. At the end of the incubation period, the medium was separated from the cells, and the plates were stored at -80 ° C.

On day 3, total RNA was purified from SJSA-1 cells treated with inhibitor and DMSO using a Qiagen BioRobot Universal workstation, according to the manufacturer's RNeasy 96 BioRobot 8000 protocol (Qiagen; Valencia, CA), with the following exceptions: by the addition of RLT lysis buffer, DNase treatment was skipped, the top of the Elute fluid was skipped and the final elution volume was changed to 120 [mu] l. After BioRobot Universal completed the RNA extraction procedure, the collection plate containing the total RNA from each field was briefly centrifuged to collect the eluate at the bottom of the cuvette.

58366 Β1 qRT-PCR was used to measure the concentrations of the present r21 transcript. Concentrations of both r21 and the host gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured from the total RNA for each inhibitor- and DMSO-treated field in technical duplicates. Each qRT-PCR field contained the following ingredients from the TaqMan® One-Step RT-PCR Master Mix Reagents Kit (Invitrogen): 10 ul 2X TaqMan® Universal PCR Master Mix, 0.5 ul 40H Multiscribe ™ Reverse Transcriptase / RNase Inhibitor Mix, 1 ul or r21 20XTaqMan® Gene Expression Assay (Invitrogen) or 1 ul GAPDH 20XTaqMan® Gene Expression Assay (Invitrogen), plus 5 ul total RNA and 3.5 ul DEPC-H₂O (EMD Chemicals; Gibbstown, NJ). qRT-PCR reaction mixtures were tested on an Applied Biosystems Prism 7900HT instrument, using a relative quantification procedure (delta delta Ct) with the following cycle conditions: 30 minutes at 48 ° C, then 10 minutes at 95 ° C, then 40 cycles of 15 seconds each 95 ° C and 1 minute at 60 ° C. Data were analyzed using the Applied Biosystems SDS2.2 software package, using GAPDH as an endogenous control and DMSO-treated samples as a calibrator. The SDS2.2 software calculated the relative quantification (RQ) or number of products of concentration r21 relative to the DMSO control for each treated sample. The maximum (100%) product of induction r21 was defined as the maximum of the fitted curve of the reference compound. The product of r21 induction for each dose of test inhibitor was converted to a value representing the percentage of the maximum. Dose response curves were generated by the XLFit software package (ID Business Solutions, Alameda, CA) to calculate IC₅₀ transit values ​​for each inhibitor tested.