GDUFA: FDA’s new Guidance on Self-Identification of Generic Drug Manufacturers

FDA’s new Guidance requesting generic drug manufacturers who want to export to the USA to self-identify has recently been published in a finalised form. Read more here about what types of generic drug manufacturers are affected and which company data are required by the FDA.

http://www.gmp-compliance.org/enews_05598_GDUFA-FDA-s-new-Guidance-on-Self-Identification-of-Generic-Drug-Manufacturers_15332,S-RGL_n.html

The GDUFA (Generic Drug User Fee Amendments) is a legislative package which came into force in 2012 and entitles the US-American FDA to collect fees from generic drug manufacturers, who strive for a marketing authorisation for the American market. An annual fee has to be paid after the successful registration.

The core of the document is the obligation to “Self-Identify” for those companies that have to submit essential site-related information to the FDA. The details of this self-identification are set in a Guidance for Industry entitled “Self-Identification of Generic Drug Facilities, Sites, and Organizations” published on 22 September 2016 by the FDA in the finalised form.

The Guidance describes the following elements:

1. Which types of generic facilities, sites, and organizations are required to self-identify?

2. What information is requested?

3. What technical standards are to be used for electronically submitting the requested information?

4. What is the penalty for failing to self-identify?

Hereinafter, you will find a short summary of these four topics:

1. Companies that manufacture finished generic medicinal products for human use or the APIs for them, or both are required to self-identify as well as companies that package the finished generic drug into the primary container and label it. Besides, sites that – pursuant to a contract with the applicant (generic drug manufacturer) – repack/redistribute the finished drug from a primary container  into a different primary container are also required to submit a self-identification as well as sites that perform bioequivalence/bioavailability studies. Last but not least, the obligation to self-identify also concerns sites that are listed in the application dossier as contract laboratories for the sampling and performing of analytical testing.

2. Essential data are: the D-U-N-S number (a unique nine-digit sequence specific for each site / each distinct physical location of an entity), the “Facility Establishment Identifier, FEI” (an identifier used by the FDA for the planning and tracking of inspections) and general information with regard to the facility (company owner, type of business operation, contact data, information about the manufacture of non generic drugs).

3. The HLS standard (Health Level Seven Structured Product Labeling) requested for generic applications (ANDAs)  has to be also used for the submission of self-identification information. A detailed description of this standard can be found in the Guidance “Providing Regulatory Submissions in Electronic Format – Drug Establishment Registration and Drug Listing“.

4. Companies that fail to self-identify do not have to expect an explicit penalty. However, such a failure leads to two drawbacks: first, the likelihood of a site inspection by the FDA prior to approval is higher. The second drawback which is much more serious is that all the APIs or finished drugs from a manufacturer who hasn’t self-identified are deemed misbranded. For the FDA, such products are not allowed for importation in the USA.

To the satisfaction of the FDA, the regulations set in the GDUFA and the provisions laid down in the new Guidance represent a major contribution to an enhanced transparency in particular of complex supply chains.

//////////GDUFA, FDA,  new Guidance,  Self-Identification, Generic Drug Manufacturers

BMT-145027

C23H14ClF3N4
Exact Mass: 438.0859

3-(4-chloro-3-(trifluoromethyl)phenyl)-4-cyclopropyl-6-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile

3-(4-chloro-3- (trifluoromethyl)phenyl)-4-cyclopropyl-6-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile

1H NMR (600 MHz, DMSO-d6) δ = 14.46 (br. s., 1H), 8.24 (s, 1H), 8.14 (d, J=8.1 Hz, 1H), 7.88 (d, J=8.3 Hz, 1H), 7.84 (dd, J=6.1, 2.7 Hz, 2H), 7.61 – 7.55 (m, 3H), 2.50 – 2.45 (m, 1H), 0.74 – 0.68 (m, 2H), 0.65 – 0.59 (m, 2H).

13C NMR (126 MHz, DMSO-d6) δ 160.5, 155.0, 153.0, 144.1, 138.3, 135.4, 133.9, 132.0, 131.2, 130.3, 129.7, 128.9, 128.9, 128.8, 127.0 (q, J=30.5 Hz), 118.1, 112.4, 103.9, 14.6, 9.4.

LCMS (method A) tR, 2.01 min, MS Anal. Calcd. for [M+H]+ C23H15ClF3N4: 439.09; found: 439.15.

LC/MS HPLC methods: method A: Column: Phenomenex Luna 30 x 2.0 mm 3um; Mobile Phase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10 acetonitrile:water with 0.1% TFA; Temperature: 40 °C; Gradient: 0-100% B over 2 min; Flow: 1 mL/min.

DETAILS WILL BE UPDATED…………

BMT-145027 is a potent mGluR5 PAM with no inherent mGluR5 agonist activity. BMT-145027 is a non-MPEP site PAM to demonstrate in vivo efficacy. BMT-145027 has mGluR5 PAM EC50 = 47 nM, with fold shit = 3.5, and is effective in mouse NOR. The metabotropic glutamate receptor 5 (mGluR5) is an attractive target for the treatment of schizophrenia due to its role in regulating glutamatergic signaling in association with the N-methyl-D-aspartate receptor (NMDAR).

The metabotropic glutamate receptor 5 (mGluR5) is an attractive target for the treatment of schizophrenia due to its role in regulating glutamatergic signaling in association with the N-methyl-d-aspartate receptor (NMDAR). We describe the synthesis of 1H-pyrazolo[3,4-b]pyridines and their utility as mGluR5 positive allosteric modulators (PAMs) without inherent agonist activity. A facile and convergent synthetic route provided access to a structurally diverse set of analogues that contain neither the aryl-acetylene-aryl nor aryl-methyleneoxy-aryl elements, the predominant structural motifs described in the literature. Binding studies suggest that members of our new chemotype do not engage the receptor at the MPEP and CPPHA mGluR5 allosteric sites. SAR studies culminated in the first non-MPEP site PAM, 1H-pyrazolo[3,4-b]pyridine 31 (BMT-145027), to improve cognition in a preclinical rodent model of learning and memory.

Development of 1H-Pyrazolo[3,4-b]pyridines as Metabotropic Glutamate Receptor 5 Positive Allosteric Modulators

Matthew D. Hill*, Haiquan Fang, Jeffrey M. Brown, Thaddeus Molski, Amy Easton, Xiaojun Han, Regina Miller, Melissa Hill-Drzewi, Lizbeth Gallagher, Michele Matchett, Michael Gulianello, Anand Balakrishnan, Robert L. Bertekap, Kenneth S. Santone, Valerie J. Whiterock, Xiaoliang Zhuo, Joanne J. Bronson, John E. Macor, and Andrew P. Degnan
Research and Development, Bristol-Myers Squibb, 5 Research Parkway, Wallingford, Connecticut 06492-7660, United States
ACS Med. Chem. Lett., Article ASAP
DOI: 10.1021/acsmedchemlett.6b00292, http://pubs.acs.org/doi/abs/10.1021/acsmedchemlett.6b00292

*Tel: 1-203-677-7102. Fax: 1-203-677-7884. E-mail: matthew.hill@bms.com.

///////////BMT-145027, glutamat,  mGluR5,  novel object recognition,  positive allosteric modulator,  schizophrenia

c1(c(c(c2c(n1)nnc2c3ccc(c(c3)C(F)(F)F)Cl)C4CC4)C#N)c5ccccc5

ClC(C=C1)=C(C(F)(F)F)C=C1C2=NNC3=C2C(C4CC4)=C(C#N)C(C5=CC=CC=C5)=N3

MCC 950

256373-96-3 (sodium salt); 210826-40-7 (free form).

MCC950; CP-456773; CAS 210826-40-7; DSSTox_CID_27301; DSSTox_RID_82252; DSSTox_GSID_47301;

CP-456,773; CRID3

1-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)-3-[4-(2-hydroxypropan-2-yl)furan-2-yl]sulfonylurea

CP-456773, also known as MCC950 and CRID3, is a potent and selective cytokine release inhibitor and NLRP3 inflammasome inhibitor for the treatment of inflammatory diseases. CP-456773 inhibits interleukin 1β (IL-1β) secretion and caspase 1 processing. MCC950 blocked canonical and noncanonical NLRP3 activation at nanomolar concentrations. MCC950 specifically inhibited activation of NLRP3 but not the AIM2, NLRC4 or NLRP1 inflammasomes. MCC950 reduced interleukin-1β (IL-1β) production in vivo and attenuated the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis. MCC950 is a potential therapeutic for NLRP3-associated syndromes, including autoinflammatory and autoimmune diseases, and a tool for further study of the NLRP3 inflammasome in human health and disease.

sodium ((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)((4-(2-hydroxypropan-2-yl)furan-2-yl)sulfonyl)amide

PAPER

Identification, Synthesis, and Biological Evaluation of the Major Human Metabolite of NLRP3 Inflammasome Inhibitor MCC950

Abstract

MCC950 is an orally bioavailable small molecule inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that exhibits remarkable activity in multiple models of inflammatory disease. Incubation of MCC950 with human liver microsomes, and subsequent analysis by HPLC–MS/MS, revealed a major metabolite, where hydroxylation of MCC950 had occurred on the 1,2,3,5,6,7-hexahydro-s-indacene moiety. Three possible regioisomers were synthesized, and coelution using HPLC–MS/MS confirmed the structure of the metabolite. Further synthesis of individual enantiomers and coelution studies using a chiral column in HPLC–MS/MS showed the metabolite was R-(+)- N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide (2a). Incubation of MCC950 with a panel of cytochrome P450 enzymes showed P450s 2A6, 2C9, 2C18, 2C19, 2J2, and 3A4 catalyze the formation of the major metabolite 2a, with a lower level of activity shown by P450s 1A2 and 2B6. All of the synthesized compounds were tested for inhibition of NLRP3-induced production of the pro-inflammatory cytokine IL-1β from human monocyte derived macrophages. The identified metabolite 2a was 170-fold less potent than MCC950, while one regioisomer had nanomolar inhibitory activity. These findings also give first insight into the SAR of the hexahydroindacene moiety.

PATENT

WO 2001019390

http://www.google.co.in/patents/WO2001019390A1?cl=en

Synthesis of precursors will be update soon……………

Novel synthesis of 1-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)-3-[4-(1-hydroxy-1-methylethyl)furan-2-sulfonyl]urea, an antiinflammatory agentPAPER

Synthetic Communications (2003), 33, (12), 2029-2043.

http://dx.doi.org/10.1081/SCC-120021029

Abstract

A novel synthesis of the anti-inflammatory agent 1-(1,2,3,5,6,7- hexahydro-s-indacen-4-yl)-3-[4-(1-hydroxy-1-methyl-ethyl)-furan-2-sulfonyl] urea 1 is described. Sulfonamide 5 was prepared starting from ethyl 3-furoate 2. Key steps were a one-pot sulfonylation with chlorosulfonic acid in methylene chloride followed by pyridinium salt formation and reaction with phosphorus pentachloride to provide ethyl 2-(chlorosulfonyl)-4-furoate 7. This sulfonyl chloride was treated with ammonium bicarbonate to form sulfonamide 8, followed by treatment with excess methyl magnesium chloride to provide 4-(1-hydroxy-1-methyl-ethyl)-furan-2-sulfonamide 5. 4-Isocyanato-1,2,3,5,6,7-hexahydro-s-indacene 16 was prepared from indan in five steps. The formation of the desired sulfonyl urea was carried out both with the isolated isocyanate 16 and via an in situ method.

1-(1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)-3-[4-(1-hydroxy-1-methylethyl)-furan-2-sulfonylurea 1 has been in development for treatment of inflammation. [1] The synthetic route to furan sulfonamide 5 used by its discoverer Mark Dombroski in Medicinal Chemistry is shown in Sch. 1. The starting material was ethyl 3-furoate 2. This was treated with excess methyl magnesium chloride to provide the 3-furanyl-tertiary alcohol 3. Furan alcohol 3 was deprotonated with methyl lithium followed by s-butyl lithium at low temperature and reacted with liquid sulfur dioxide to generate sulfinic acid 4. Without isolation, sulfinic acid 4 was oxidized to sulfonamide 5 with hydroxylamine O-sulfinic acid via a procedure described by workers at Merck.[2] We were interested in finding a synthesis of furan sulfonamide 5 and its conversion to sulfonylurea 1 that would be suitable for scale up. In this article, we describe the discovery of a better bulk process to sulfonamide 5 from the same starting material and a procedure to form the desired sulfonylurea without isolating the isocyanate of 4-amino-1,2,3,5,6,7-hexahydro-s-indacene.

1-(1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)-3-[4-(1-hydroxy- 1-methyl-ethyl)-furan-2-sulfonyl Urea (1) ………….. anhydrous sodium salt weighed 4.9 g. mp 239 C. 1 H NMR (D2O, 400 MHz) 7.35 (s, 1), 6.81 (s, 1), 6.65 (s, 1), 2.53 (m, 4), 2.41 (m, 4), 1.73 (m, 4), 1.31 (s, 6). 13C NMR (D2O, 100 MHz) 159.87, 151.82, 143.89, 140.49, 138.77, 134.87, 129.58, 118.38, 112.02, 68.24, 32.67, 30.10, 29.53, 25.34. Anal. calcd. for C20H23N2O5SNa: C, 56.33; H, 5.44; N, 6.57; S, 7.52. Found: C, 56.19; H, 5.40; N, 6.34; S, 7.42. N

CLIPS

Dr Rebecca Coll, PostDoctoral Researcher, Inflammasome Lab, UQ Fellow

Rebecca completed her PhD research under Prof. Luke O’Neill in Trinity College Dublin at one of the leading laboratories in the innate immunity field. For her work on the regulation of TLR signalling she received the International Endotoxin and Innate Immunity Society Young Investigator Award in 2012. However, her main research focus has been inflammasomes and their therapeutic targeting by small molecule drugs. Her recent first author publication on MCC950 in Nature Medicine has been widely acclaimed (the subject of seven commentaries in leading journals and attention from 24 international news outlets) and is already a highly cited paper. She joined the Schroder group in May 2014 with the goal of defining the molecular target of MCC950 as part of a broader collaboration between the Schroder, Cooper and O’Neill labs.

Email: r.coll@imb.uq.edu.au

Office Telephone: +61 7 3346 2351

Lab Telephone: +61 7 3346 2071

Institute for Molecular Bioscience

Google Scholar

Research Gate

Researcher ID

A collaboration between scientists from Dublin’s Trinity College (Ireland) and the University of Queensland (Australia) identified a compound able to inhibit an inflammatory process common to many diseases, including Alzheimer’s disease. The study entitled “A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases” was published on line in the journal Nature Medicine.

Pathogenesis of several diseases, including Alzheimer’s, have a strong inflammatory component. Inflammatory processes can be triggered by molecules of the NOD-like receptor (NLR) family such as NLRP3. Once activated, this molecule leads to a cascade of events known as the NLRP3 inflammasome that ultimately causes the production of inflammatory factors. Aberrant activation of NLRP3 is responsible for increased inflammatory responses in complex diseases such as multiple sclerosis, Muckle-Wells syndrome, type 2 diabetes, Alzheimer’s disease and atherosclerosis.

Targeting this molecule can overcome the side effects of other anti-inflammatory drugs commonly used: “Drugs like aspirin or steroids can work in several diseases, but can have side effects or be ineffective. What we have found is a potentially transformative medicine, which targets what appears to be the common disease-causing process in a myriad of inflammatory diseases,” said Luke A J O’Neill, one of the team leaders.

Previous studies identified NLRP3 inhibitors, though neither very potent nor specific. This research team now identified a specific inhibitor of NLRP3 inflammasome, the molecule MCC950. They observed that it inhibits NLRP3 in mouse models of multiple sclerosis with consequent attenuation of disease progression. MCC950 also blocks production of inflammatory factors in blood samples from patients with a severe inflammatory disorder, Muckle-Wells syndrome. These results demonstrated the pharmaceutical potential of this specific NLRP3 inhibitor.

“MCC950 is blocking what was suspected to be a key process in inflammation. There is huge interest in NLRP3 both among medical researchers and pharmaceutical companies and we feel our work makes a significant contribution to the efforts to find new medicines to limit it,” said Rebecca Coll, the paper’s first author.

The researchers were able to demonstrate the potential of MCC950 in multiple sclerosis, an inflammatory disease of the central nervous system (CNS). However, the target for MCC950 is strongly implicated in other diseases of the CNS such as Alzheimer’s and Parkinson’s diseases indicating that it has the potential to treat all of these conditions. The fact that MCC950 can be orally administered further enhances the potential of this molecule as a therapeutic drug.

“MCC950 is able to be given orally and will be cheaper to produce than current protein-based treatments, which are given daily, weekly, or monthly by injection. Importantly, it will also have a shorter duration in the body, allowing clinicians to stop the anti-inflammatory action of the drug if the patient ever needed to switch their immune response back to 100% in order to clear an infection.” said Matt Cooper, chemist and also co-senior author in this study.

REFERENCES

1: Shao BZ, Xu ZQ, Han BZ, Su DF, Liu C. NLRP3 inflammasome and its inhibitors: a review. Front Pharmacol. 2015 Nov 5;6:262. doi: 10.3389/fphar.2015.00262. eCollection 2015. Review. PubMed PMID: 26594174; PubMed Central PMCID: PMC4633676.

2: Baker PJ, Boucher D, Bierschenk D, Tebartz C, Whitney PG, D’Silva DB, Tanzer MC, Monteleone M, Robertson AA, Cooper MA, Alvarez-Diaz S, Herold MJ, Bedoui S, Schroder K, Masters SL. NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5. Eur J Immunol. 2015 Oct;45(10):2918-26. doi: 10.1002/eji.201545655. Epub 2015 Aug 24. PubMed PMID: 26173988.

3: Krishnan SM, Dowling JK, Ling YH, Diep H, Chan CT, Ferens D, Kett MM, Pinar A, Samuel CS, Vinh A, Arumugam TV, Hewitson TD, Kemp-Harper BK, Robertson AA, Cooper MA, Latz E, Mansell A, Sobey CG, Drummond GR. Inflammasome activity is essential for one kidney/deoxycorticosterone acetate/salt-induced hypertension in mice. Br J Pharmacol. 2016 Feb;173(4):752-65. doi: 10.1111/bph.13230. Epub 2015 Jul 31. PubMed PMID: 26103560; PubMed Central PMCID: PMC4742291.

4: Groß CJ, Groß O. The Nlrp3 inflammasome admits defeat. Trends Immunol. 2015 Jun;36(6):323-4. doi: 10.1016/j.it.2015.05.001. Epub 2015 May 16. PubMed PMID: 25991463.

5: Coll RC, Robertson AA, Chae JJ, Higgins SC, Muñoz-Planillo R, Inserra MC, Vetter I, Dungan LS, Monks BG, Stutz A, Croker DE, Butler MS, Haneklaus M, Sutton CE, Núñez G, Latz E, Kastner DL, Mills KH, Masters SL, Schroder K, Cooper MA, O’Neill LA. A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat Med. 2015 Mar;21(3):248-55. doi: 10.1038/nm.3806. Epub 2015 Feb 16. PubMed PMID: 25686105; PubMed Central PMCID: PMC4392179.

BMS 906024

cas 1401066-79-2

• MF C₂₆H₂₆F₆N₄O₃
• MW 556.500

(2R,3S)-N-[(3S)-1-Methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]-2,3-bis(3,3,3-trifluoropropyl)succinamide

Butanediamide, N1-((3S)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluorophenyl)-, (2R,3S)-

(2R,35)-N-((35)-l-Methyl-2-oxo-5-phenyl-2,3-dihydro-lH-l,4-benzodiazepin-3-yl)-3- (2,2,2-trifluoroethyl)-2-(3,3,3-trifluoropropyl)succinamide

Ashvinikumar Gavai

Claude Quesnelle
Senior Research Investigator/Chemist at Bristol-Myers Squibb

RICHARD LEE

BMS-906024 is a novel, potent Notch receptor inhibitor . Cancers have a tendency to relapse or to become resistant to treatments that once worked. A family of proteins called Notch is implicated in that resistance and in cancer progression more generally. BMS-906024 is in Phase I clinical trials, both alone and in combination with other agents. Patients with colon, lung, breast, and other cancers are receiving intravenous doses of the compound to determine its safety and optimum dose ranges.

New Phase I drug structure by Bristol-Myers Squibb disclosed at the spring 2013 American Chemical Society meeting in New Orleans to treat breast, lung, and colon cancers and leukemia.^[1] The drug works as an pan-Notch inhibitor. The structure is one of a set patented in 2012,^[2] and it currently being studied in clinical trials.^[3][4]

useful for the treatment of conditions related to the Notch pathway, such as cancer and other proliferative diseases.

Notch signaling has been implicated in a variety of cellular processes, such as cell fate specification, differentiation, proliferation, apoptosis, and angiogenesis. (Bray, Nature Reviews Molecular Cell Biology, 7:678-689 (2006); Fortini, Developmental Cell 16:633-647 (2009)). The Notch proteins are single-pass heterodimeric transmembrane molecules. The Notch family includes 4 receptors, NOTCH 1-4, which become activated upon binding to ligands from the DSL family (Delta-like 1, 3, 4 and Jagged 1 and 2).

The activation and maturation of NOTCH requires a series of processing steps, including a proteolytic cleavage step mediated by gamma secretase, a multiprotein complex containing Presenilin 1 or Presenilin 2, nicastrin, APH1, and PEN2. Once NOTCH is cleaved, NOTCH intracellular domain (NICD) is released from the membrane. The released NICD translocates to the nucleus, where it functions as a transcriptional activator in concert with CSL family members (RBPSUH, “suppressor of hairless”, and LAG1). NOTCH target genes include HES family members, such as HES- 1. HES- 1 functions as transcriptional repressors of genes such as HERP 1 (also known as HEY2), HERP2 (also known as HEY1), and HATH1 (also known as ATOH1).

The aberrant activation of the Notch pathway contributes to tumorigenesis. Activation of Notch signaling has been implicated in the pathogenesis of various solid tumors including ovarian, pancreatic, as well as breast cancer and hematologic tumors such as leukemias, lymphomas, and multiple myeloma. The role of Notch inhibition and its utility in the treatment of various solid and hematological tumors are described in Miele, L. et al, Current Cancer Drug Targets, 6:313-323 (2006); Bolos, V. et al, Endocrine Reviews, 28:339-363 (2007); Shih, I.-M. et al, Cancer Research, 67: 1879- 1882 (2007); Yamaguchi, N. et al., Cancer Research, 68: 1881-1888 (2008); Miele, L., Expert Review Anti-cancer Therapy, 8: 1 197-1201 (2008); Purow, B., Current Pharmaceutical Biotechnology, 10: 154-160 (2009); Nefedova, Y. et al, Drug Resistance Updates, 1 1 :210-218 (2008); Dufraine, J. et al, Oncogene, 27:5132-5137 (2008); and Jun, H.T. et al, Drug Development Research, 69:319-328 (2008).

There remains a need for compounds that are useful as Notch inhibitors and that have sufficient metabolic stability to provide efficacious levels of drug exposure. Further, there remains a need for compounds useful as Notch inhibitors that can be orally or intravenously administered to a patient.

U.S. Patent No. 7,053,084 Bl discloses succinoylamino benzodiazepine compounds useful for treating neurological disorders such as Alzheimer’s Disease. The reference discloses that these succinoylamino benzodiazepine compounds inhibit gamma secretase activity and the processing of amyloid precursor protein linked to the formation of neurological deposits of amyloid protein. The reference does not disclose the use of these compounds in the treatment of proliferative diseases such as cancer.

Applicants have found potent compounds that have activity as Notch inhibitors and have sufficient metabolic stability to provide efficacious levels of drug exposure upon intravenous or oral administration. These compounds are provided to be useful as pharmaceuticals with desirable stability, bioavailability, therapeutic index, and toxicity values that are important to their drugability.

PAPER

Structure–activity relationships in a series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides identified highly potent inhibitors of γ-secretase mediated signaling of Notch1/2/3/4 receptors. On the basis of its robust in vivo efficacy at tolerated doses in Notch driven leukemia and solid tumor xenograft models, 12 (BMS-906024) was selected as a candidate for clinical evaluation.

Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch Inhibitor for the Treatment of Leukemia and Solid Tumors

Patent

http://www.google.co.in/patents/WO2012129353A1?cl=en

Example 1

(2R,35)-N-((35′)-l-Methyl-2-oxo-5-phenyl-2,3-dihydro-lH-l,4-benzodiazepin-3-yl)-2,3- b -trifluoropropy l)succinamide

Preparation 1A: tert-Butyl 5, -trifluoropentanoate

[00219] To a stirred solution of 5,5,5-trifluoropentanoic acid (5 g, 32.0 mmol) in THF (30 mL) and hexane (30 mL) at 0 °C, was added tert-butyl 2,2,2-trichloroacetimidate (11.46 mL, 64.1 mmol). The mixture was stirred for 15 min at 0 °C. Boron trifluoride etherate (0.406 mL, 3.20 mmol) was added and the reaction mixture was allowed to warm to room temperature overnight. To the clear reaction mixture was added solid aHC0₃ (5 g) and stirred for 30 min. The mixture was filtered through MgS0₄ and washed with hexanes (200 mL). The solution was allowed to rest for 45 min, and the resulting solid material was removed by filtering on the same MgS0₄ filter again, washed with hexanes (100 mL) and concentrated under reduced pressure without heat. The volume was reduced to about 30 mL, filtered through a clean fritted funnel, washed with hexane (5 mL), and then concentrated under reduced pressure without heat. The resulting neat oil was filtered through a 0.45μηι nylon membrane filter disk to provide tert-butyl 5,5,5- trifluoropentanoate (6.6 g, 31.4 mmol 98% yield) as a colorless oil: ^XH NMR (400 MHz, CDC1₃) δ ppm 1.38 (s, 9 H) 1.74-1.83 (m, 2 H) 2.00-2.13 (m, 2 H) 2.24 (t, J=7.28 Hz, 2 H).

Preparation IB: (45)-4-(Propan-2- l)-3-(5,5,5-trifluoropentanoyl)-l,3-oxazolidin-2-one

[00220] To a stirred solution of 5,5,5-trifluoropentanoic acid (5.04 g, 32.3 mmol) in DCM (50 mL) and DMF (3 drops) was added oxalyl chloride (3.4 mL, 38.8 mmol) dropwise over 5 min and the solution was stirred until all bubbling subsided. The reaction mixture was concentrated under reduced pressure to give pale yellow oil. To a separate flask charged with a solution of (45)-4-(propan-2-yl)-l,3-oxazolidin-2-one (4.18 g, 32.4 mmol) in THF (100 mL) at -78 °C was added n-BuLi (2.5M in hexane) (13.0 mL, 32.5 mmol) dropwise via syringe over 5 min. After stirring for 10 min, the above acid chloride dissolved in THF (20 mL) was added via cannula over 15 min. The reaction mixture was warmed to 0 °C, and was allowed to warm to room temperature as the bath warmed and stirred overnight. To the reaction mixture was added saturated NH₄CI, and then extracted with EtOAc (2x). The combined organics were washed with brine, dried (Na₂S0₄), filtered and concentrated under reduced pressure. The crude material was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 5% to 60% solvent A/B=hexanes/EtOAc, REDISEP® S1O2 120g). Concentration of appropriate fractions provided Preparation IB (7.39 g, 86%) as a colorless oil: ^XH NMR (400 MHz, CDC1₃) δ ppm 4.44 (1 H, dt, J=8.31, 3.53 Hz), 4.30 (1 H, t, J=8.69 Hz), 4.23 (1 H, dd, J=9.06, 3.02 Hz), 2.98-3.08 (2 H, m), 2.32-2.44 (1 H, m, J=13.91, 7.02, 7.02, 4.03 Hz), 2.13-2.25 (2 H, m), 1.88-2.00 (2 H, m), 0.93 (3 H, d, J=7.05 Hz), 0.88 (3 H, d, J=6.80 Hz). Preparation 1C: (25′,3R)-tert-Butyl 6,6,6-trifluoro-3-((5)-4-isopropyl-2-oxooxazolidine- 3 -carbonyl)-2-(3 ,3,3 -trifluoropropyl)hexanoate, and

Preparation ID: (2R,3R)-tert-Butyl 6,6,6-trifluoro-3-((5)-4-isopropyl-2-oxooxazolidine- 3 -carbonyl)- -(3 ,3 ,3 -trifluoropropyl)hexanoate

(1 C) (1 D)

[00221] To a cold (-78 °C), stirred solution of diisopropylamine (5.3 mL, 37.2 mmol) in THF (59 mL) under nitrogen atmosphere was added n-BuLi (2.5M in hexane) (14.7 mL, 36.8 mmol), then warmed to 0 °C to give a 0.5M solution of LDA. A separate vessel was charged with Preparation IB (2.45 g, 9.17 mmol), the material was azeotroped twice with benzene (the RotoVap air inlet was fitted with nitrogen inlet to completely exclude humidity) then toluene (15.3 mL) was added. This solution was added to a flask containing dry lithium chloride (1.96 g, 46.2 mmol). To the resultant mixture, cooled to -78 °C, was added LDA solution (21.0 mL, 10.5 mmol) and stirred at -78 °C for 10 min, warmed to 0 °C for 10 min then recooled to -78 °C. To a separate reaction vessel containing Preparation 1A (3.41 g, 16.07 mmol), also azeotroped twice with benzene, was added toluene (15.3 mL), cooled to -78 °C and LDA (37.0 mL, 18.5 mmol) was added, the resulting solution was stirred at -78° for 25 min. At this time the enolate derived from the ester was transferred via cannula into the solution of the oxazolidinone enolate, stirred at -78 °C for an additional 5 min at which time the septum was removed and solid powdered bis(2-ethylhexanoyloxy)copper (9.02 g, 25.8 mmol) was rapidly added to the reaction vessel and the septum replaced. The vessel was immediately removed from the cold bath and immersed into a warm water bath (40 °C) with rapid swirling with a concomitant color change from the initial turquoise to brown. The reaction mixture was stirred for 20 min, was poured into 5% aqueous NH₄OH (360 mL) and extracted with EtOAc (2x). The combined organics were washed with brine, dried (Na₂S0₄), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 0% to 60% solvent A/B=hexanes/EtOAc, REDISEP® S1O₂ 120g). Concentration of appropriate fractions provided Preparation 1C (2.87 g, 66%) as pale yellow viscous oil. ^XH NMR showed the product was a 1.6: 1 mixture of diastereoisomers 1C: 1D as determined by the integration of the multiplets at 2.74 & 2.84 ppm: ^XH NMR (400 MHz, CDC1₃) δ ppm 4.43-4.54 (2 H, m), 4.23-4.35 (5 H, m), 4.01 (1 H, ddd, J=9.54, 6.27, 3.51 Hz), 2.84 (1 H, ddd, J=9.41, 7.28, 3.64 Hz), 2.74 (1 H, ddd, J=10.29, 6.27, 4.02 Hz), 2.37-2.48 (2 H, m, J=10.38, 6.98, 6.98, 3.51, 3.51 Hz), 2.20-2.37 (3 H, m), 1.92-2.20 (8 H, m), 1.64-1.91 (5 H, m), 1.47 (18 H, s), 0.88-0.98 (12 H, m). Preparation IE: (2R,35)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and

Preparation IF: (2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

(1 E) (1 F)

[00222] To a cool (0 °C), stirred solution of Preparation 1C and ID (4.54 g, 9.51 mmol) in THF (140 mL) and water (42 mL) was sequentially added hydrogen peroxide (30% in water) (10.3 g, 91 mmol) and LiOH (685.3 mg, 28.6 mmol) and the mixture was stirred for 1 hr. At this time the reaction vessel was removed from the cold bath and then stirred for 1.5 hr. The reaction was judged complete by HPLC. To the reaction mixture was added saturated NaHC0₃ (45 mL) and saturated a₂S0₃(15 mL), and then partially concentrated under reduced pressure. The resulting crude solution was extracted with DCM (3x). The aqueous phase was acidified to pH~l-2 with IN HC1, extracted with DCM (3x) and EtOAc (lx). The combined organics were washed with brine, dried (Na₂S0₄), filtered and concentrated under reduced pressure to provide a mixture of Preparation IE and IF (3.00 g, 86%) as colorless oil: ^XH NMR (400 MHz, CDC1₃) δ ppm 2.76-2.84 (1 H, m, diastereoisomer 2), 2.64-2.76 (3 H, m), 2.04-2.35 (8 H, m), 1.88-2.00 (4 H, m), 1.71-1.83 (4 H, m), 1.48 (9 H, s, diastereoisomer 1), 1.46 (9 H, s, diastereoisomer 2); ^XH NMR showed a 1.7: 1 mixture of 1E: 1F by integration of the peaks for the ?-butyl groups.

Preparation IE: (2R,35)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and

Preparation IF: (2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

(1 E) (1 F)

[00223] To a cold (-78 °C), stirred solution of diisopropylamine (1.7 mL, 11.93 mmol) in THF (19 mL) under nitrogen atmosphere was added n-BuLi (2.5M in hexanes) (4.8 mL, 12.00 mmol). The mixture was stirred for 5 min and then warmed to 0 °C. In a separate vessel, to a cold (-78 °C) stirred solution of the mixture of Preparation IE and IF (1.99 g, 5.43 mmol) in THF (18 mL) was added the LDA solution prepared above via cannula slowly over 25 min. The mixture was stirred for 15 min, then warmed to room temperature (placed in a 24 °C water bath) for 15 min, and then again cooled to -78 °C for 15 min. To the reaction mixture was added Et₂AlCl (1M in hexane) (11.4 mL, 1 1.40 mmol) via syringe, stirred for 10 min, warmed to room temperature for 15 min and then cooled back to -78 °C for 15 min. Methanol (25 mL) was rapidly added, swirled vigorously while warming to room temperature, then concentrated to ~l/4 original volume. The mixture was dissolved in EtOAc and washed with IN HCl (50 mL) and ice (75 g). The aqueous phase was separated, extracted with EtOAc (2x). The combined organics were washed with a mixture of KF (2.85g in 75 mL water) and IN HCl (13 mL) [resulting solution pH 3-4], then with brine, dried (Na₂S0₄), filtered and concentrated under reduced pressure to give a 9: 1 (IE: IF) enriched diastereoisomeric mixture (as determined by ^XH NMR) of Preparation IE and Preparation IF (2.13 g, >99%) as a pale yellow viscous oil: ^XH NMR (400 MHz, CDC1₃) δ ppm 2.64-2.76 (2 H, m), 2.04-2.35 (4 H, m), 1.88-2.00 (2 H, m), 1.71-1.83 (2 H, m), 1.48 (9 H, s). Preparation 1 G: (35)-3 -Amino- 1 -methyl-5-phenyl- 1 ,3 -dihydro-2H- 1 ,4-benzodiazepin-2- one, and

Preparation 1H: (3R)-3 -Amino- 1 -methyl-5-phenyl- 1 ,3-dihydro-2H- 1 ,4-benzodiazepin-2- one

(1G) (1 H)

[00224] Racemic 3-amino-l-methyl-5-phenyl-l,3-dihydro-2H-l,4-benzodiazepin-2- one (Rittle, K.E. et al, Tetrahedron Letters, 28(5):521-522 (1987)) was prepared according to the literature procedure. The enantiomers were separated under chiral-SFC conditions using the following method: CHIRALPAK® AS-H 5×25; Mobile phase: 30% MeOH+ 0.1% DEA in C0₂; Flow rate: 280 mL/min; Pressure: 100 bar; Temperature: 35 °C.

[00225] Obtained the S-enantiomer (Preparation 1G): HPLC: RT=1.75 min (30% MeOH + 0.1% DEA in C0₂ on CHIRALPAK® AS-H 4.6×250 mm, 3 mL/min, 35 °C, 100 bar, 230 nm, ΙΟμΙ injection); ¾ NMR (400 MHz, CDC1₃) δ ppm 7.58-7.63 (2 H, m), 7.55 (1 H, ddd, J=8.50, 7.1 1, 1.76 Hz), 7.40-7.47 (1 H, m), 7.34-7.40 (3 H, m), 7.31 (1 H, dd, J=7.81, 1.51 Hz), 7.14-7.22 (1 H, m), 4.46 (1 H, s), 3.44 (3 H, s), 3.42 (2 H, s); [a]_D= -155° (c=1.9, MeOH) (Lit. Rittle, K.E. et al, Tetrahedron Letters, 28(5):521-522 (1987): [a]_D=-236°).

[00226] Also obtained the R-enantiomer (Preparation 1H): HPLC: RT=1.71 min; [a]_D=+165° (c=2.1, MeOH) (Lit [a]_D= +227°).

Alternate procedure to make Preparation 1 G:

Preparation 1G^»CSA salt: (35)-3-Amino-l-methyl-5-phenyl-l,3-dihydro-2H-l,4- benzodiazepin-2-one, (15)-(+)-10-camphorsulfonic acid salt

[00227] Preparation lG’CSA was prepared from racemic 3-amino-l-methyl-5-phenyl- l,3-dihydro-2H-l,4-benzodiazepin-2-one (9.98g, 37.6 mmol) (prepared according to the literature as shown above) according to the literature procedure (Reider, P.J. et al, J. Org. Chem., 52:955-957 (1987)). Preparation lG’CSA (16.91g, 99%) was obtained as a colorless solid: Optical Rotation: [a]_D = -26.99° (c=l, H₂0) (Lit. [a]_D = -27.8° (c=l,

H₂0))

Preparation II: tert-Butyl (25^,,3R)-6,6,6-trifluoro-3-(((35)-l-methyl-2-oxo-5-phenyl-2,3- dihydro- 1 H- 1 ,4-benzodiazepin-3 -yl)carbamoyl)-2-(3 ,3 ,3 -trifluoropropyl)hexanoate, and Preparation 1J: tert-Butyl (2R,3R)-6,6,6-trifluoro-3-(((35)-l-methyl-2-oxo-5-phenyl-2,3- dihydro- 1 H- 1 ,4-benzodiazepin-3 -yl)carbamoyl)-2-(3 ,3 ,3-trifluoropropyl)hexanoate

(11) (U)

[00228] To a stirred solution of Preparation 1G (1.45 g, 5.47 mmol) and a 9: 1 mixture of Preparation IE and IF (1.989 g, 5.43 mmol) in DMF (19 mL) was added O- benzotriazol-l-yl-N,N,N’,N’-tetra-methyluronium tetrafluoroborate (1.79 g, 5.57 mmol) and triethylamine (3.0 mL, 21.52 mmol) and stirred overnight. The reaction was judged complete by LCMS. The reaction mixture was poured into water (125 mL) and the precipitated solid was collected by filtration, washed with water and air dried to provide an 8: 1 mixture of Preparation II and Preparation 1J (2.95 g, 89%) as a cream solid: MS (ES): m/z= 614 [M+H]⁺;^XH NMR (400 MHz, CDC1₃) δ ppm 7.55-7.65 (3 H, m), 7.44- 7.52 (2 H, m), 7.35-7.45 (4 H, m), 5.52 (1 H, d, J=8.03 Hz), 3.48 (3 H, s), 2.63 (2 H, ddd, J=9.35, 3.95, 3.76 Hz), 2.14-2.25 (4 H, m), 1.90-2.03 (3 H, m), 1.69-1.82 (1 H, m), 1.51 (9 H, s).

Preparation IK: (25^,,3R)-6,6,6-Trifluoro-3-(((35)-l-methyl-2-oxo-5-phenyl-2,3-dihydro- lH-l,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoic acid, and

Preparation 1L: (2R,3R)-6,6,6-Trifluoro-3-(((35)-l-methyl-2-oxo-5-phenyl-2,3-dihydro- 1 H- 1 ,4-

(1 K) (1 L)

[00229] To a cool (0 °C), stirred solution of the above mixture of Preparation II and Preparation 1 J (2.95 g, 4.81 mmol) in DCM (20 mL) was added TFA (20 mL, 260 mmol). The reaction mixture was stirred for lhr, then allowed to warm to room temperature and stirred for 2.5 hr. The reaction was judged complete by LCMS. The reaction mixture was diluted with toluene (50 mL) and concentrated under reduced pressure. The residue mixture was redissolved in toluene (50 mL) and concentrated under reduced pressure then dried under high vacuum. The crude product was dissolved in DCM, S1O2 (15g) was added, concentrated, then was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 0% to 45% solvent A/B=DCM/EtOAc, REDISEP® S1O2 80g). Concentration of appropriate fractions provided a mixture of Preparation IK and Preparation 1L (2.00 g, 75%) as a cream solid: HPLC: RT=2.770 min

(CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min grad) eluting with 10-90% aqueous MeOH over 4 minutes containing 0.1% TFA, 4 mL/min, monitoring at 254 nm); MS (ES): m/z= 558 [M+H]⁺; ^XH NMR (400 MHz, CDC1₃) δ ppm 8.32 (1 H, d, J=8.03 Hz), 7.65-7.71 (1 H, m), 7.50-7.60 (3 H, m), 7.41-7.49 (2 H, m), 7.39 (1 H, dd, J=7.91, 1.63 Hz), 7.23-7.35 (2 H, m), 5.59 (1 H, d, J=8.03 Hz), 3.51 (3 H, s), 2.81 (1 H, ddd, J=10.54, 6.90, 3.64 Hz), 2.67-2.76 (1 H, m), 2.22-2.33 (3 H, m), 1.99-2.12 (3 H, m), 1.85-1.94 (1 H, m), 1.79 (1 H, ddd, J=13.87, 7.84, 3.64 Hz). Example 1 :

[00230] To a stirred solution of an 8: 1 mixture of Preparation IK and Preparation 1L (3.46 g, 6.21 mmol) in DMF (25 mL) under nitrogen atmosphere was added ammonium chloride (3.32 g, 62.1 mmol), EDC (3.55 g, 18.52 mmol), HOBT (2.85 g, 18.61 mmol), and triethyl amine (16 mL, 1 15 mmol) and stirred overnight. The reaction was judged complete by LCMS. The reaction mixture was poured into water (200 mL) with vigorous swirling and then allowed to sit. The solid was collected by filtration, washed with water, allowed to dry to afford 3.6 g colorless solid. The solid was purified by preparative SFC chromatography (Lux-Cellulose-2 (3x25cm), 8% methanol in CO₂, 140ml/min @220nm and 35 °C; Sample: 3.6g in 50cc methanol, conc.=70mg/ml, Stack injection:

0.5cc/9.2min). Fractions containing product were concentrated, dried overnight under vacuum. Obtained Example 1 (2.74 g, 79%) as a colorless solid (Crystal Form -1): HPLC: RT=9.601 min (H₂0/CH₃CN with TFA, Sunfire CI 8 3.5um, 4.6x150mm, 4.6x150mm, gradient = 15 min, wavelength = 220 and 254 nm). MS (ES): m/z= 557 [M+H]⁺; ^XH NMR (400 MHz, DMSO-d₆) δ ppm 9.54 (1 H, d, J=7.28 Hz), 7.71-7.80 (1 H, m), 7.68 (2 H, d, J=8.78 Hz), 7.50-7.62 (3 H, m), 7.45 (2 H, t, J=7.28 Hz), 7.29-7.40 (2 H, m), 7.15 (1 H, br. s.), 5.30 (1 H, d, J=7.28 Hz), 3.39 (3 H, s), 2.74-2.86 (1 H, m), 2.02-2.32 (3 H, m), 1.45-1.79 (4 H, m); [a]_D = -107.0° (5.73 mg/mL, DMSO).

[00231] Crystal Form A-2 was prepared by adding approximately 1 mg of Example 1 to approximately 0.7 mL of acetone/acetonitrile/water solution (2:2: 1). A mixture of colorless needles and thin blades crystals were obtained after one day of slow evaporation of the solution at room temperature. The thin blade crystals were separated to provide crystal Form A-2.

[00232] Crystal Form EA-3 was prepared by adding approximately 1 mg of Example 1 to approximately 0.7 mL of ethyl acetate/heptane solution (1 : 1). Colorless blade crystals were obtained after three days of slow evaporation of the solution at room temperature.

[00233] Crystal Form THF-2 was obtained by adding approximately 5 mg of Example 1 to approximately 0.7 mL of THF/water solution (4: 1). Colorless blade-like crystals were obtained after one day of solvent evaporation at room temperature.

Alternate Procedure to Make Example 1 : Preparation 1M: 3,3,3-Trifluoropropyl trifluoromethanesulfonate

[00234] To a cold (-25 °C), stirred solution of 2,6-lutidine (18.38 mL, 158 mmol) in CH2CI2 (120 mL) was added Tf₂0 (24.88 mL, 147 mmol) over 3 min, and stirred for 5 min. To the reaction mixture was added 3,3,3-trifluoropropan-l-ol (12 g, 105 mmol) over an interval of 3 min. After 2 hr, the reaction mixture was warmed to room temperature and stirred for 1 hr. The reaction mixture was concentrated to half volume, then purified by loading directly on silica gel column (330g ISCO) and eluted with CH₂C1₂. Obtained Preparation 1M (13.74 g, 53%) as a colorless oil. ^XH NMR (400 MHz, CDCI₃) δ ppm 4.71 (2 H, t, J=6.15 Hz), 2.49-2.86 (2 H, m).

Preparation IN: (45)-4-Benzyl- -(5,5,5-trifluoropentanoyl)-l,3-oxazolidin-2-one

[00235] Preparation IN was prepared from 5,5,5-trifluoropentanoic acid (3.35 g, 21.46 mmol) and (45)-4-benzyl-l,3-oxazolidin-2-one (3.80 g, 21.46 mmol) by the general methods shown for Preparation IB. Preparation IN (5.67 g, 84%) was obtained as a colorless viscous oil: ^XH NMR (400 MHz, CDC1₃) δ ppm 7.32-7.39 (2 H, m), 7.30 (1 H, d, J=7.05 Hz), 7.18-7.25 (2 H, m), 4.64-4.74 (1 H, m), 4.17-4.27 (2 H, m), 3.31 (1 H, dd, J=13.35, 3.27 Hz), 3.00-3.1 1 (2 H, m), 2.79 (1 H, dd, J=13.35, 9.57 Hz), 2.16-2.28 (2 H, m), 1.93-2.04 (2 H, m).

Preparation 10: tert-Butyl (3R)-3-(((45)-4-benzyl-2-oxo-l,3-oxazolidin-3-yl)carbonyl)- 6,6,6-trifluorohexanoate

[00236] To a cold (-78 °C), stirred solution of Preparation IN (3.03 g, 9.61 mmol) in THF (20 mL) was added NaHMDS (1.0M in THF) (10.6 mL, 10.60 mmol) under nitrogen atmosphere. After 2 hours, tert-butyl 2-bromoacetate (5.62 g, 28.8 mmol) was added neat via syringe at -78 °C and stirring was maintained at the same temperature. After 6 hours, the reaction mixture was warmed to room temperature. The reaction mixture was partitioned between saturated NH₄C1 and EtOAc. The organic phase was separated, and the aqueous was extracted with EtOAc (3x). The combined organics were washed with brine, dried (Na₂S0₄), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 5% to 100% solvent A/B=hexanes/EtO Ac, REDISEP® Si0₂ 120g). Concentration of appropriate fractions provided Preparation 10 (2.79 g, 67.6%) as a colorless viscous oil: XH NMR (400 MHz, CDC1₃) δ ppm 7.34 (2 H, d, J=7.30 Hz), 7.24-7.32 (3 H, m), 4.62- 4.75 (1 H, m, J=10.17, 6.89, 3.43, 3.43 Hz), 4.15-4.25 (3 H, m), 3.35 (1 H, dd, J=13.60, 3.27 Hz), 2.84 (1 H, dd, J=16.62, 9.57 Hz), 2.75 (1 H, dd, J=13.35, 10.07 Hz), 2.47 (1 H, dd, J=16.62, 4.78 Hz), 2.1 1-2.23 (2 H, m), 1.90-2.02 (1 H, m), 1.72-1.84 (1 H, m), 1.44 (9 H, s). -2-(2-tert-Butoxy-2-oxoethyl)-5,5,5-trifluoropentanoic acid

[00237] Preparation IP was prepared from Preparation 10 (2.79 g, 6.50 mmol) by the general methods shown for Preparation IE. Preparation IP (1.45 g, 83%) was obtained as a colorless oil: ^XH NMR (400 MHz, CDC1₃) δ ppm 2.83-2.95 (1 H, m), 2.62-2.74 (1 H, m), 2.45 (1 H, dd, J=16.62, 5.79 Hz), 2.15-2.27 (2 H, m), 1.88-2.00 (1 H, m), 1.75-1.88 (1 H, m), 1.45 (9 H, s). Preparation IE: (2R,35′)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and

Preparation IF: (2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

(1 E) (1 F)

[00238] To a cold (-78 °C), stirred solution of Preparation IP (5.44 g, 20.13 mmol) in THF (60 mL) was slowly added LDA (24.60 mL, 44.3 mmol) over 7 min. After stirring for 2 hr, Preparation 1M (6.44 g, 26.2 mmol) was added to the reaction mixture over 3 min. After 45 min, the reaction mixture was warmed to -25 °C bath (ice/MeOH/dry ice) for 1 hr, and then warmed to 0 °C. After 45 min, Preparation 1M (lg) was added and the reaction mixture was stirred for 20 min. The reaction was quenched with water and IN NaOH and was extracted with (¾(¾. The organic layer was again extracted with IN NaOH (2x) and the aqueous layers were combined. The aqueous layer was cooled in ice/water bath and then acidified with concentrated HCl to pH 2. Next, the aqueous layer was extracted with EtOAc. The combined organics were washed with brine, dried over anhydrous sodium sulphate, and concentrated under reduced pressure. The residue was dried under high vacuum to provide a 1 :5 (IE: IF) mixture (as determined by ^XH NMR) of Preparation IE and Preparation IF (5.925 g, 80%) as a pale yellow solid. ^XH NMR (500 MHz, CDC1₃) 8 ppm 2.81 (1 H, ddd, J=10.17, 6.32, 3.85 Hz), 2.63-2.76 (1 H, m), 2.02- 2.33 (4 H, m), 1.86-1.99 (2 H, m), 1.68-1.85 (2 H, m), 1.47 (9 H, s).

Preparation IE: (2R,35)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and

Preparation IF: (2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

(1 E) (1 F)

[00239] A mixture of Preparation IE and Preparation IF (64 mg, 1.758 mmol) was taken in THF (6 mL) to give a colorless solution which was cooled to -78 °C. Then, LDA (2.149 mL, 3.87 mmol) (1.8M in heptane/THF/ethylbenzene) was slowly added to the reaction mixture over 10 min. After stirring for 15 min the reaction mixture was placed in a room temperature water bath. After 15 min the reaction mixture was placed back in -78 °C bath and then diethylaluminum chloride (3.87 mL, 3.87 mmol) (1M in hexane) was added slowly over 5 min. The reaction mixture was stirred at -78 °C. After 15 min the reaction mixture was placed in a room temperature water bath for 10 min and then cooled back to -78 °C bath. After 15 min the reaction was quenched with MeOH (8 mL, 198 mmol), removed from the -78 °C bath and concentrated. To the reaction mixture was added ice and HC1 (16 mL, 16.00 mmol), followed by extraction with EtOAc (2x). The organic layer was washed with potassium fluoride (920 mg, 15.84 mmol) (in 25 mL FLO) and HC1 (4.5 mL, 4.50 mmol). The organics were dried over anhydrous magnesium sulphate and concentrated under reduced pressure to provide a 9: 1 (IE: IF) enriched mixture of Preparation IE and Preparation IF (540 mg, 1.583 mmol, 90% yield) as light yellow/orange solid. ¾ NMR (400 MHz, CDC1₃) δ ppm 2.64-2.76 (2 H, m), 2.04-2.35 (4 H, m), 1.88-2.00 (2 H, m), 1.71-1.83 (2 H, m), 1.48 (9 H, s). It was converted to Example 1 by the sequence of reactions as outlined above.

Alternate procedure to make Preparation IE:

Preparation 1Q: (2R,35)- -Benzyl 4-tert-butyl 2,3-bis(3,3,3-trifluoropropyl)succinate

(1Q) [00240] A clean and dry 5 L four neck round bottom flask equipped with mechanical stirring, thermometer socket and nitrogen bubbler at room temperature was charged with Ν,Ν-dimethyl formamide (2.07 L), a 1.2: 1 mixture of Preparation IE and Preparation IF (207 g, 0.5651 moles), potassium carbonate (1 17.1 g, 0.8476 moles) followed by benzyl bromide (116 g, 0.6781 moles) over 15-20 min. The reaction mixture was stirred for 2-3 hr. After completion of the reaction, the reaction mixture was concentrated to dryness at 50-55 °C under vacuum. Ethyl acetate (3.1 L, 30 Vol.) was charged into the concentrated reaction mass and then washed with water (2.07 L), brine (0.6 L) then dried over anhydrous sodium sulfate (207 g), filtered and concentrated to dryness at 40-45 °C under vacuum. The residue was dissolved in dichloromethane (1.035 L, 5 vol.) and then absorbed onto silica gel (60-120) (607 g, 3.0 w/w), then was purified with column chromatography using petroleum ether and ethyl acetate as solvents. After pooling several batches, Preparation 1Q (235 g) was obtained. HPLC purity: 99.77%, Preparation IE: (2R,35)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

[00241] A clean and dry 2 L autoclave was charged with methanol (540 mL) and was purged with nitrogen for 5-10 minutes. To the autoclave was added 10% palladium on carbon (12 g, 20%), purged with nitrogen once again for 5-10 min then was charged with Preparation 1Q (60g, 0.1315 moles), the autoclave was flushed with methanol (60mL) and stirred for 4-6 hr at 20-25 °C under 5Kg hydrogen pressure. After completion of the reaction, the reaction mass was filtered through CELITE®, washed with methanol (180 mL), dried with anhydrous sodium sulfate (60 g), filtered and concentrated to dryness at 45-50 °C under vacuum. Obtained Preparation IE (45.8 g, 95%) as a colorless solid: HPLC purity: 98.9%.

Alternate procedure to make Preparation IE: Preparation IE: (2R,35)-3-(te^Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

[00242] Preparation IE was prepared in a procedure identical as above from a mixture of Preparations IE and IF (200g, 0.5460 moles) using LDA (1.8 M solution in THF, ethyl benzene and heptane) (698mL, 2.3equiv.) and diethyl aluminum chloride (1.0 M solution in hexane) (1256mL, 2.3equiv) in THF (2.0L). After workup as explained above, the resulting residue was treated as follows: The crude material was added to a 2L four neck round bottom flask, followed by the addition of MTBE (1.0L) charged below 30 °C. The resulting mixture was stirred for 5-10 minutes to obtain a clear solution.

Hexanes (600mL) was charged to the reaction mixture at a temperature below 30 °C. The reaction mixture was stirred for 10 min. Next, tert-butylamine (43.8g, l. leq) was charged slowly over a period of 15 minutes below 30 °C. This addition was observed to be exothermic. The reaction mixture was stirred for 2 hrs below 30 °C and filtered. The solid material was washed with 5:3 MTBE: hexane (200mL), the filtrate was

concentrated and transferred to an amber color bottle. The filtered solid was dissolved in dichloromethane (2.0L), washed with IN HC1 (2.0), the organic layer was washed with brine (1.0L x 2), then was concentrated under reduced pressure below 45 °C. This material was found to be 91.12% pure. The material was repurified by the above t- butylamine crystallization purification procedure. Obtained Preparation IE (78 g, 39%): HPLC purity: 99.54%.

Alternate procedure to make Example 1 :

Preparation II: tert-Butyl (25^,,3R)-6,6,6-trifluoro-3-(((35)-l-methyl-2-oxo-5-phenyl-2,3- dihydro- 1 H- 1 ,4-benzodiazepin-3 -yl)carbamoyl)-2-(3 ,3 ,3 -trifluoropropyl)hexanoate

[00243] A clean and dry 2 L four neck round bottom flask equipped with mechanical stirring, thermometer socket and nitrogen bubbler was charged with N,N- dimethylformamide (457 mL), Preparation IE (45.7g, 0.1248moles) and Preparation lG’CSA (62.08g, 0.1248moles) under nitrogen atmosphere at 20-25 °C. The reaction mixture was stirred for 15-20 minutes to make clear solution at 20-25 °C. To the reaction mixture was added TBTU (48.16g, 0.1498 moles) at 20-25 °C followed by triethylamine (50.51g, 0.4992 moles) over 15-20 minutes at 20-25 °C. The reaction mixture was stirred for 60-120 minutes at 20-25 °C under nitrogen atmosphere. After completion of the reaction, the reaction was quenched into water (1.37L, 30 Vol.) at 20-25 °C under stirring. The reaction mixture was stirred for 30 minutes at 20-25 °C. The reaction mixture was filtered and washed with water (228 mL). The resulting solid material was dissolved in ethyl acetate (457 mL), washed with water (2×137 mL), brine (137 mL), and then dried with anhydrous sodium sulfate (45.7g). Activated charcoal (9.14 g, 20%) was charged into the reaction mixture and stirred for 30 minutes. The mixture was filtered through CELITE® bed and 1 micron filter cloth, washed charcoal bed with ethyl acetate (137 mL), concentrated to 1.0 Vol. stage and then petroleum ether (457 mL, 10 Vol.) was charged and stirred for 30 minutes at 20-25 °C. The solid was collected by filtration, washed with petroleum ether (137 mL) and then dried under vacuum at 40-45 °C for 8 hr until loss on drying was less than 3.0%. Obtained Preparation II (65.2 g, 85%): HPLC purity: 98.26%.

Preparation IK: (25^,,3R)-6,6,6-Trifluoro-3-(((35)-l-methyl-2-oxo-5-phenyl-2,3-dihydro- 1 H- 1 ,4-benzodiazepin-3 -yl)carbamoyl)-2-(3 ,3 ,3 -trifluoropropyl)hexanoic acid

[00244] A clean and dry 3 L four neck round bottom flask equipped with mechanical stirring, thermometer socket and nitrogen bubbler was charged with dichloromethane (980 mL) under nitrogen atmosphere followed by Preparation II (140 g, 0.2282 moles) at 20-25 °C. The reaction mixture was cooled to 0-5 °C and trifluoroacetic acid (980 mL) was charged slowly for 30-40 minutes. The resulting mixture was stirred for 2 hr at 0-5 °C under nitrogen atmosphere. The reaction temperature was raised to 20 to 25 °C, and the reaction mixture was stirred for 1-2 hr at 20 to 25 °C. After completion of the reaction, the reaction mixture was concentrated to dryness at 50 to 55 °C under vacuum. Toluene (3×700 mL,) was charged into the concentrated reaction mass, and then distilled off at 50 to 55 °C under vacuum. After complete concentration from toluene, ethyl acetate (280 mL) was charged into the reaction mass at 20 to 25 °C, stirred for 60 minutes, then the solid was collected by filtration, washed with ethyl acetate (140 mL), dried under vacuum at 50 to 55 °C for 12 hr until loss on drying was less than 2.0%. Obtained Preparation IK (106 g, 84%): HPLC purity: 98.43%.

Example 1 :

[00245] A reaction vessel was charged with Preparation IK (30 g, 53.81 mmol), HOBt (8.7g, 64.38 mmol), and THF (150 mL) at room temperature. To the homogeneous solution was added EDCI (12.4g, 64.68 mmol), stirred for 15 min, then cooled to 8 °C. To the reaction mixture was added ammonia (2M in IP A) (81 mL, 162 mmol) over 5 min so as to maintain a temperature below 10 °C. The resulting heavy slurry was stirred for 10 min, warmed to room temperature over 30 min, then stirred for 4 hr. At the completion of the reaction, water (230 mL) was slowly added over 15 min to maintain a temperature below 20 °C, and then stirred for 2 hr. The solid was collected by filtration, washed with water (3X60 mL), then dried under vacuum 48 hr at 55 °C. The above crude product was charged into a 1 L 3 -necked round flask. IP A (200 mL) was added, then heated to 80 °C resulting in a homogeneous solution. Water (170 mL) was slowly added (15 min) to maintain an internal temperature >75 °C. The resulting slurry was stirred and cooled to room temperature for 2 hr. The solid was collected by filtration, washed with water (2 X 50 mL), then dried under vacuum (55 °C for 24 h, and 30 °C for 48 h).

Obtained Example 1 (23.4 g, 78% yield): HPLC purity: 99.43%.

Example 2 NOT SAME

WITHOUT METHYL GROUP

(2R,35)-N-((35)-2-Oxo-5-phenyl-2,3-dihydro-lH-l,4-benzodiazepin-3-yl)-2,3-bis(3,3,3- trifluoropropyl)succinamide

Preparation 2A: (35)-3-Amino-5-phenyl-l,3-dihydro-2H-l,4-benzodiazepin-2-one, and Preparation 2B: -3-Amino-5-phenyl-l,3-dihydro-2H-l,4-benzodiazepin-2-one

(2A) (2B)

[00246] Racemic 3-amino-5-phenyl-l,3-dihydro-2H-l,4-benzodiazepin-2-one (J. Med. Chem., 49:231 1-2319 (2006), compound# 5) was prepared according to the literature procedure. The enantiomers were separated on Berger SFC MGIII Column: Lux 25X3 cm, 5cm; Mobile phase: 30% MeOH+ 0.1% DEA in C0₂; Flow rate: 150 mL/min;

Temperature: 40 °C; Detector wavelength: 250 nM. Obtained the S-enantiomer

Preparation 2A as a white solid: ^XH NMR (400 MHz, DMSO-d₆) δ ppm 10.67 (1 H, br. s.), 7.58 (1 H, td, J=7.65, 1.76 Hz), 7.37-7.53 (5 H, m), 7.23-7.30 (2 H, m), 7.14-7.22 (1 H, m), 4.23 (1 H, s), 2.60 (2 H, br. s.); HPLC: RT=3.0625 min (30% MeOH + 0.1% DEA in C0₂ on OD-H Column, 3 mL/min, 35 °C, 96 bar, 230 nm, ΙΟμΙ inj); [a]_D = -208.3° (5.05 mg/niL, MeOH). Also obtained the R-enantiomer Preparation 2B as an off white solid: HPLC: RT=3.970 min; [a]_D = 182.1° (2.01 mg/mL, MeOH).

Preparation 2C: tert-Butyl (25^,,3R)-6,6,6-trifluoro-3-(((35)-2-oxo-5-phenyl-2,3-dihydro- 1 H- 1 ,4-benzodiazepin-3 -yl)carbamoyl)-2-(3 ,3 ,3 -trifluoropropyl)hexanoate, and

Preparation 2D: tert-Butyl (2R,3R)-6,6,6-trifluoro-3-(((35)-2-oxo-5-phenyl-2,3-dihydro- 1 H- -benzodiazepin-3 -yl)carbamoyl)-2-(3 ,3 ,3 -trifluoropropyl)hexanoate

(2C) (2D)

[00247] Preparation 2C was prepared from Preparation 2A (564 mg, 2.244 mmol) and a mixture of Preparation IE and Preparation IF (822 mg, 2.244 mmol) according to the general procedure shown for Preparation II. Obtained Preparation 2C and Preparation 2D (1.31 g, 97%): HPLC: RT=3.443 min (CHROMOLITH® ODS 4.6 x 50 mm (4 min grad) eluting with 10-90% aqueous MeOH over 4 minutes containing 0.% TFA, 4 mL/min, monitoring at 220 nm); MS (ES): m/z= 600.3 [M+H]⁺.

Preparation 2E: (25′,3R)-6,6,6-Trifluoro-3-(((35)-2-oxo-5-phenyl-2,3-dihydro-lH-l,4- benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoic acid, and

Preparation 2F: (2R,3R)-6,6,6-Trifluoro-3-(((35)-2-oxo-5-phenyl-2,3-dihydro-lH-l,4- benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoic acid

(2E) (2F) [00248] A mixture of Preparation 2E and Preparation 2F was prepared from a mixture of Preparation 2C and Preparation 2D (1.3 lg, 2.185 mmol) by the general methods shown for Preparation IK. Obtained a mixture of Preparation 2E and Preparation 2F (1.18 g, 99%): HPLC: RT=2.885 min (CHROMOLITH® ODS 4.6 x 50 mm (4 min grad) eluting with 10-90% aqueous MeOH over 4 minutes containing 0.% TFA, 4 mL/min, monitoring at 220 nm). MS (ES): m/z= 544.2 [M+H]⁺.

Example 2:

[00249] Example 2 was prepared from a mixture of Preparation 2E and Preparation 2F (354 mg, 0.651 mmol) by the general methods shown for Example 1. After separation of the diastereoisomers, Example 2 was obtained (188 mg, 52%) as a white solid: HPLC: RT=9.063 min (H₂0/CH₃CN with TFA, Sunfire C18 3.5um, 4.6x150mm, 4.6x150mm, gradient = 15 min, wavelength = 220 and 254 nm); MS (ES): m/z= 543 [M+H]⁺; ^XH NMR (400 MHz, DMSO-d₆) δ ppm 10.87 (1 H, br. s.), 9.50-9.55 (1 H, m), 7.62-7.69 (2 H, m), 7.40-7.57 (5 H, m), 7.29-7.36 (2 H, m), 7.22-7.28 (1 H, m), 7.16 (1 H, br. s.), 5.25 (1 H, d), 3.30-3.32 (1 H, m), 2.75-2.86 (1 H, m), 2.44-2.48 (1 H, m), 2.06-2.34 (3 H, m), 1.51- 1.77 (4 H, m); [a]_D = -114.4° (8.04 mg/mL, DMSO).

[00250] Crystal Form M2- 1 was prepared by adding approximately 1 mg of Example 2 to approximately 0.7 mL of MeOH/fluorobenzene solution (3 : 1). Colorless plate-like crystals were obtained after 2 days of solvent evaporation at room temperature.

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Company: Bristol-Myers Squibb

Target: pan-Notch

Disease: breast, lung, colon cancer; leukemia

http://cen.acs.org/articles/91/i16/BMS-906024-Notch-Signaling-Inhibitor.html

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BMS-906024
Company: Bristol-Myers Squibb
Meant to treat: cancers including breast, lung, colon, and leukemia
Mode of action: pan-Notch inhibitor
Medicinal chemistry tidbit: The BMS team used an oxidative enolate heterocoupling en route to the candidate– a procedure from Phil Baran’s lab at Scripps Research Institute. JACS 130, 11546
Status in the pipeline: Phase I
Relevant documents: WO 2012/129353

PAPER

An enantioselective synthesis of (S)-7-amino-5H,7H-dibenzo[b,d]azepin-6-one (S–1) is described. The key step in the sequence involved crystallization-induced dynamic resolution (CIDR) of compound 7 using Boc-d-phenylalanine as a chiral resolving agent and 3,5-dichlorosalicylaldehyde as a racemization catalyst to afford S–1 in 81% overall yield with 98.5% enantiomeric excess.

BMS-906024

Systematic (IUPAC) name
(2R,3S)-N-[(3S)-1-Methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]-2,3-bis(3,3,3-trifluoropropyl)succinamide
Identifiers
PubChem	CID 66550890
ChemSpider	28536138
Chemical data
Formula	C26H26F6N4O3
Molar mass	556.500 g/mol
SMILES CN1c2ccccc2C(=N[C@@H](C1=O)NC(=O)[C@H](CCC(F)(F)F)[C@H](CCC(F)(F)F)C(=O)N)c3ccccc3

Patent US8377886 – Use of gamma secretase inhibitors and notch …

RO4929097 | γ-secretase inhibitor – Cellagen Technology

BMS-986115
CAS 1584647-27-7

(2R,3S)-N-((3S)-5-(3-Fluorophenyl)-9-methyl-2-oxo-2,3-dihydro-lH-l,4-benzodiazepin- 3-yl)-2, -bis(3,3,3-trifluoropropyl)succinamide

MW: 574.4945,  C26-H25-F7-N4-O3, UNII: LSK1L593UU

10-Nitrooleate, CTK3B7458, CTK3C3167, 9-Octadecenoic acid, 10-nitro-, 875685-46-4, AG-L-63109, 9-Octadecenoic acid, 10-nitro-, (9E)-, 88127-53-1

FOR advanced solid tumors

• Originator Bristol-Myers Squibb
• Class Antineoplastics
• Mechanism of Action Amyloid precursor protein secretase inhibitors; Notch signalling pathway inhibitors

• Phase I Solid tumours

Most Recent Events

• 30 Aug 2016Bristol-Myers Squibb terminates a phase I trial for Solid tumours (late-stage disease, second-line therapy or greater) in USA, Australia and Canada (NCT01986218)
• 25 Jan 2016Bristol-Myers Squibb completes enrolment in its phase I trial for Solid tumours in USA, Australia and Canada (NCT01986218)
• 31 Dec 2013Phase-I clinical trials in Solid tumours (late-stage disease) in Canada & Australia (Oral)

DETAILS WILL BE UPDATED SOON………….

BMS-986115 is an orally bioavailable, gamma secretase (GS) and pan-Notch inhibitor, with potential antineoplastic activity. Upon administration, GS/pan-Notch inhibitor BMS 986115 binds to GS and blocks the proteolytic cleavage and release of the Notch intracellular domain (NICD), which would normally follow ligand binding to the extracellular domain of the Notch receptor. This prevents both the subsequent translocation of NICD to the nucleus to form a transcription factor complex and the expression of Notch-regulated genes. This results in the induction of apoptosis and the inhibition of growth of tumor cells that overexpress Notch. Overexpression of the Notch signaling pathway plays an important role in tumor cell proliferation and survival

Ashvinikumar Gavai

Claude Quesnelle
Senior Research Investigator/Chemist at Bristol-Myers Squibb

RICHARD LEE

Dan O’Malley (Rice University)
Currently: Bristol-Myers Squibb

PICTURES WILL BE UPDATED………….

useful for the treatment of conditions related to the Notch pathway, such as cancer and other proliferative diseases.

Notch signaling has been implicated in a variety of cellular processes, such as cell fate specification, differentiation, proliferation, apoptosis, and angiogenesis. (Bray, Nature Reviews Molecular Cell Biology, 7:678-689 (2006); Fortini, Developmental Cell 16:633-647 (2009)). The Notch proteins are single-pass heterodimeric transmembrane molecules. The Notch family includes 4 receptors, NOTCH 1-4, which become activated upon binding to ligands from the DSL family (Delta-like 1, 3, 4 and Jagged 1 and 2).

The activation and maturation of NOTCH requires a series of processing steps, including a proteolytic cleavage step mediated by gamma secretase, a multiprotein complex containing Presenilin 1 or Presenilin 2, nicastrin, APH1, and PEN2. Once NOTCH is cleaved, NOTCH intracellular domain (NICD) is released from the membrane. The released NICD translocates to the nucleus, where it functions as a transcriptional activator in concert with CSL family members (RBPSUH, “suppressor of hairless”, and LAG1). NOTCH target genes include HES family members, such as HES- 1. HES- 1 functions as transcriptional repressors of genes such as HERP 1 (also known as HEY2), HERP2 (also known as HEY1), and HATH1 (also known as ATOH1).

The aberrant activation of the Notch pathway contributes to tumorigenesis. Activation of Notch signaling has been implicated in the pathogenesis of various solid tumors including ovarian, pancreatic, as well as breast cancer and hematologic tumors such as leukemias, lymphomas, and multiple myeloma. The role of Notch inhibition and its utility in the treatment of various solid and hematological tumors are described in Miele, L. et al, Current Cancer Drug Targets, 6:313-323 (2006); Bolos, V. et al, Endocrine Reviews, 28:339-363 (2007); Shih, I.-M. et al, Cancer Research, 67: 1879- 1882 (2007); Yamaguchi, N. et al., Cancer Research, 68: 1881-1888 (2008); Miele, L., Expert Review Anti-cancer Therapy, 8: 1 197-1201 (2008); Purow, B., Current Pharmaceutical Biotechnology, 10: 154-160 (2009); Nefedova, Y. et al, Drug Resistance Updates, 1 1 :210-218 (2008); Dufraine, J. et al, Oncogene, 27:5132-5137 (2008); and Jun, H.T. et al, Drug Development Research, 69:319-328 (2008).

There remains a need for compounds that are useful as Notch inhibitors and that have sufficient metabolic stability to provide efficacious levels of drug exposure. Further, there remains a need for compounds useful as Notch inhibitors that can be orally or intravenously administered to a patient.

U.S. Patent No. 7,053,084 Bl discloses succinoylamino benzodiazepine compounds useful for treating neurological disorders such as Alzheimer’s Disease. The reference discloses that these succinoylamino benzodiazepine compounds inhibit gamma secretase activity and the processing of amyloid precursor protein linked to the formation of neurological deposits of amyloid protein. The reference does not disclose the use of these compounds in the treatment of proliferative diseases such as cancer.

Applicants have found potent compounds that have activity as Notch inhibitors and have sufficient metabolic stability to provide efficacious levels of drug exposure upon intravenous or oral administration. These compounds are provided to be useful as pharmaceuticals with desirable stability, bioavailability, therapeutic index, and toxicity values that are important to their drugability.

PATENTS

US-20150166489-A1

US-20140087992-A1

PATENT

WO-2014047372-A1

https://www.google.com/patents/WO2014047372A1?cl=en

Scheme 3

XII XI

Scheme 4

Intermediate S-l : (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

Intermediate S-IA: 3,3,3-Trifluoro ropyl trifluoromethanesulfonate

[00180] To a cold (-25 °C) stirred solution of 2,6-lutidine (18.38 mL, 158 mmol) in DCM (120 mL) was added Tf₂0 (24.88 mL, 147 mmol) over 3 min, and the mixture was stirred for 5 min. To the reaction mixture was added 3,3,3-trifluoropropan-l-ol (12 g, 105 mmol) over an interval of 3 min. After 2 hr, the reaction mixture was warmed to room temperature and stirred for 1 hr. The reaction mixture was concentrated to half its volume, then purified by loading directly on a silica gel column (330g ISCO) and the product was eluted with DCM to afford Intermediate S-IA (13.74 g, 53%) as a colorless oil. 1H NMR (400 MHz, CDC1₃) δ ppm 4.71 (2 H, t, J= 6.15 Hz), 2.49-2.86 (2 H, m).

Intermediate S-1B: (4S)-4-Benzyl-3-(5,5,5-trifluoropentanoyl)-l,3-oxazolidin-2-one

[00181] To a stirring solution of 5,5,5-trifluoropentanoic acid (14.76 g, 95 mmol) and DMF (0.146 rriL) in DCM (50 mL) was slowly added oxalyl chloride (8.27 mL, 95 mmol). After 2h, the mixture was concentrated to dryness. A separate flask was changed with (S)-4-benzyloxazolidin-2-one (16.75 g, 95 mmol) in THF (100 mL) and then cooled to -78 °C. To the solution was slowly added n-BuLi (2.5M, 37.8 mL, 95 mmol) over 10 min, stirred for 10 min, and then a solution of the above acid chloride in THF (50 mL) was slowly added over 5 min. The mixture was stirred for 30 min, and then warmed to room temperature. The reaction was quenched with sat aq NH₄C1. Next, 10% aq LiCl was then added to the mixture, and the mixture was extracted with Et₂0. The organic layer was washed with sat aq NaHC0₃ then with brine, dried (MgSC^), filtered and concentrated to dryness. The residue was purified by Si0₂ chromatography (ISCO, 330 g column, eluting with a gradient from 100% hexane to 100% EtOAc) to afford the product Intermediate S-IB; (25.25 g, 85%): 1H NMR (400 MHz, CDC1₃) δ ppm 7.32-7.39 (2 H, m), 7.30 (1 H, d, J= 7.05 Hz), 7.18-7.25 (2 H, m), 4.64-4.74 (1 H, m), 4.17-4.27 (2 H, m), 3.31 (1 H, dd, J= 13.35, 3.27 Hz), 3.00-3.11 (2 H, m), 2.79 (1 H, dd, J= 13.35, 9.57 Hz), 2.16-2.28 (2 H, m), 1.93-2.04 (2 H, m).

Intermediate S-IC: tert- utyl (3R)-3-(((4S)-4-benzyl-2-oxo-l,3-oxazolidin-3- yl)carbonyl)-6,6,6-trifluoroh xanoate

[00182] To a cold (-78 °C), stirred solution of Intermediate S-IB (3.03 g, 9.61 mmol) in THF (20 mL) was added NaHMDS (1.0M in THF) (10.6 mL, 10.60 mmol) under a nitrogen atmosphere. After 2 hours, tert-butyl 2-bromoacetate (5.62 g, 28.8 mmol) was added neat via syringe at -78 °C and stirring was maintained at the same temperature. After 6 hours, the reaction mixture was warmed to room temperature. The reaction mixture was partitioned between saturated NH₄C1 and EtOAc. The organic phase was separated, and the aqueous phase was extracted with EtOAc (3x). The combined organics were washed with brine, dried (Na₂s0₄), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (Teledyne ISCO

CombiFlash Rf, 5% to 100% solvent A/B = hexanes/EtOAc, REDISEP® Si0₂ 120g). Concentration of the appropriate fractions provided Intermediate S-1C (2.79 g, 67.6%) as a colorless viscous oil: 1H NMR (400 MHz, CDC1₃) δ ppm 7.34 (2 H, d, J= 7.30 Hz), 7.24-7.32 (3 H, m), 4.62-4.75 (1 H, m, J= 10.17, 6.89, 3.43, 3.43 Hz), 4.15-4.25 (3 H, m), 3.35 (1 H, dd, J= 13.60, 3.27 Hz), 2.84 (1 H, dd, J= 16.62, 9.57 Hz), 2.75 (1 H, dd, J = 13.35, 10.07 Hz), 2.47 (1 H, dd, J= 16.62, 4.78 Hz), 2.11-2.23 (2 H, m), 1.90-2.02 (1 H, m), 1.72-1.84 (1 H, m), 1.44 (9 H, s).

Intermediate S-ID: (2R)-2-( -tert-Butoxy-2-oxoethyl)-5,5,5-trifluoropentanoic acid

[00183] To a cool (0 °C), stirred solution of Intermediate S-1C (2.17 g, 5.05 mmol) in THF (50 mL) and water (15 mL) was added a solution of LiOH (0.242 g, 10.11 mmol) and H₂0₂ (2.065 mL, 20.21 mmol) in H₂0 (2 mL). After 10 min, the reaction mixture was removed from the ice bath, stirred for lh, and then cooled to 0 °C. Saturated aqueous NaHCC”3 (25 mL) and saturated aqueous Na₂s0₃ (25 mL) were added to the reaction mixture, and the mixture was stirred for 10 min, and then partially concentrated. The resulting mixture was extracted with DCM (2x), cooled with ice and made acidic with cone. HC1 to pH 3. The mixture was saturated with solid NaCl, extracted with EtOAc (3x), and then dried over MgS0₄, filtered and concentrated to a colorless oil to afford Intermediate S-ID, 1.2514g, 92%): 1H NMR (400 MHz, CDCI3) δ ppm 2.83-2.95 (1 H, m), 2.62-2.74 (1 H, m), 2.45 (1 H, dd, J= 16.62, 5.79 Hz), 2.15-2.27 (2 H, m), 1.88-2.00 (1 H, m), 1.75-1.88 (1 H, m), 1.45 (9 H, s). Intermediate S-l : (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and Intermediate S-1E: (2R,3R)-3-(tert-butoxycarbonyl)- 6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid

(S-1E)

[00184] To a cold (-78 °C) stirred solution of Intermediate S-1D (5 g, 18.50 mmol) in THF (60 mL) was slowly added LDA (22.2 mL, 44.4 mmol, 2.0M) over 7 min. After stirring for 2 hr, Intermediate S- 1 A (6.38 g, 25.9 mmol) was added to the reaction mixture over 3 min. After 60 min, the reaction mixture was warmed to -25 °C

(ice/MeOH/dry ice) and stirred for an additional 60 min at which time sat aq NH₄C1 was added. The separated aqueous phase was acidified with IN HC1 to pH 3, and then extracted with Et₂0. The combined organic layers were washed with brine (2x), dried over MgS04, filtered and concentrated to provide a 1 :4 (II :I1E) mixture (as determined by 1H NMR) of Intermediate S-l and Intermediate S-1E (6.00 g, 89%) as a pale yellow solid. 1H NMR (500 MHz, CDC1₃) δ ppm 2.81 (1 H, ddd, J = 10.17, 6.32, 3.85 Hz), 2.63- 2.76 (1 H, m), 2.02-2.33 (4 H, m), 1.86-1.99 (2 H, m), 1.68-1.85 (2 H, m), 1.47 (9 H, s).

[00185] To a cold (-78 °C), stirred solution of a mixture of Intermediate S-l and Intermediate S-1E (5.97 g, 16.30 mmol) in THF (91 mL) was added LDA (19 mL, 38.0 mmol, 2.0M in THF/hexane/ethyl benzene) dropwise via syringe over 10 min (internal temperature never exceeded -65 °C, J-KEM® probe in reaction solution). The mixture was stirred for 15 min, and then warmed to room temperature (24 °C water bath), stirred for 15 min, and then cooled to -78 °C for 15 min. To the reaction mixture was added Et₂AlCl (41 mL, 41.0 mmol, 1M in hexane) via syringe (internal temperature never exceeded -55 °C), and the mixture was stirred for 10 min, and then warmed to room temperature (24 °C bath) for 15 min and then back to -78 °C for 15 min. Meanwhile, a 1000 mL round bottom flask was charged with MeOH (145 mL) and precooled to -78 °C. With vigorous stirring the reaction mixture was transferred via cannula over 5 min to the MeOH. The flask was removed from the bath, ice was added followed by the slow addition of IN HC1 (147 mL, 147 mmol). Gas evolution was observed as the HC1 was added. The reaction mixture was allowed to warm to room temperature during which the gas evolution subsided. The reaction mixture was diluted with EtOAc (750 mL), saturated with NaCl, and the organic phase was separated, washed with a solution of potassium fluoride (8.52 g, 147 mmol) and IN HC1 (41 mL, 41.0 mmol) in water (291 mL), brine (100 mL), and then dried (Na₂s0₄), filtered and concentrated under vacuum. 1H NMR showed the product was a 9: 1 mixture of Intermediate S-l and Intermediate S- 1E. The enriched mixture of Intermediate S-l and Intermediate S-1E (6.12 g, >99% yield) was obtained as a dark amber solid: 1H NMR (400 MHz, CDC1₃) δ ppm 2.64-2.76 (2 H, m), 2.04-2.35 (4 H, m), 1.88-2.00 (2 H, m), 1.71-1.83 (2 H, m), 1.48 (9 H, s).

Alternate procedure to make Intermediate S-l :

Intermediate S-IF: (2R,3 -1 -Benzyl 4-tert-butyl 2,3-bis(3,3,3-trifluoropropyl)succinate

[00186] To a stirred solution of a 9: 1 enriched mixture of Intermediate S-l and Intermediate S-1E (5.98 g, 16.33 mmol) in DMF (63 mL) were added potassium carbonate (4.06 g, 29.4 mmol) and benzyl bromide (2.9 mL, 24.38 mmol), the mixture was then stirred overnight at room temperature. The reaction mixture was diluted with EtOAc (1000 mL), washed with 10% LiCl (3×200 mL), brine (200 mL), dried (Na_2S0₄), filtered, concentrated, and then dried under vacuum. The residue was purified by Si0₂ chromatography using a toluene:hexane gradient. Diastereomerically purified

Intermediate S-IF (4.81g, 65%) was obtained as a colorless solid: 1H NMR (400 MHz, chloroform-d) δ 7.32-7.43 (m, 5H), 5.19 (d, J= 12.10 Hz, 1H), 5.15 (d, J= 12.10 Hz, 1H), 2.71 (dt, J= 3.52, 9.20 Hz, 1H), 2.61 (dt, J= 3.63, 9.63 Hz, 1H), 1.96-2.21 (m, 4H), 1.69-1.96 (m, 3H), 1.56-1.67 (m, 1H), 1.45 (s, 9H).

Intermediate S-l : (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

[00187] To a solution of Intermediate S-1F (4.81 g, 10.54 mmol) in MeOH (100 mL) was added 10% palladium on carbon (wet, Degussa type, 568.0 mg, 0.534 mmol) in a H₂– pressure flask. The vessel was purged with N₂ (4x), then purged with H₂ (2x), and finally, pressurized to 50 psi and shaken overnight. The reaction vessel was

depressurized and purged with nitrogen. The mixture was filtered through CELITE®, washed with MeOH and then concentrated and dried under vacuum. Intermediate S-1 (3.81 g, 99% yield)) was obtained as a colorless solid: 1H NMR (400 MHz, chloroform-d) δ 2.62-2.79 (m, 2H), 2.02-2.40 (m, 4H), 1.87-2.00 (m, 2H), 1.67-1.84 (m, 2H), 1.48 (s, 9H).

Alternate procedure to make Intermediate S-1 :

Intermediate S-1 : (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

[00188] Intermediate S-1 as a mixture with Intermediate S-IE was prepared in a similar procedure as above from Intermediate S-1D to afford a 1 :2.2 mixture of

Intermediate S-1 and Intermediate S-IE (8.60 g, 23.48 mmol), which was enriched using LDA (2.0 M solution in THF, ethyl benzene and heptane, 28.2 mL, 56.4 mmol) and diethyl aluminum chloride (1.0 M solution in hexane, 59 mL, 59.0 mmol) in THF (91 mL). After workup as described above, the resulting residue was found to be a 13.2: 1 (by 1H NMR) mixture of Intermediate S-1 and Intermediate S-IE, which was treated as follows: The crude material was dissolved in MTBE (43 mL). Hexanes (26 mL) were slowly charged to the reaction mixture while maintaining a temperature below 30 °C. The reaction mixture was stirred for 10 min. Next, tert-butylamine (2.7 mL, 1.1 eq) was charged slowly over a period of 20 minutes while maintaining a temperature below 30 °C. This addition was observed to be exothermic. The reaction mixture was stirred for 2 hrs below 30 °C and then filtered. The solid material was washed with 5:3 MTBE: hexane (80 mL), and the filtrate was concentrated and set aside. The filtered solid was dissolved in dichloromethane (300 mL), washed with IN HC1 (lOOmL), and the organic layer was washed with brine (100 mL x 2), and then concentrated under reduced pressure below 45 °C to afford Intermediate S-l (5.46 g, 64%).

A second alternate procedure for preparing Intermediate S-l :

Intermediate S-1G: tert- utyl 5,5,5-trifluoropentanoate

[00189] To a stirred solution of 5,5,5-trifluoropentanoic acid (5 g, 32.0 mmol) in THF (30 mL) and hexane (30 mL) at 0 °C, was added tert-butyl 2,2,2-trichloroacetimidate (11.46 mL, 64.1 mmol). The mixture was stirred for 15 min at 0 °C. Boron trifluoride etherate (0.406 mL, 3.20 mmol) was added and the reaction mixture was allowed to warm to room temperature overnight. To the clear reaction mixture was added solid NaHC0₃ (5 g) and stirred for 30 min. The mixture was filtered through MgSC^ and washed with hexanes (200 mL). The solution was allowed to rest for 45 min, and the resulting solid material was removed by filtering on the same MgSC^ filter again, washed with hexanes (100 mL) and concentrated under reduced pressure without heat. The volume was reduced to about 30 mL, filtered through a clean fritted funnel, washed with hexane (5 mL), and then concentrated under reduced pressure without heat. The resulting neat oil was filtered through a 0.45μιη nylon membrane filter disk to provide Intermediate S-1G (6.6 g, 31.4 mmol 98% yield) as a colorless oil: 1H NMR (400 MHz, CDC1₃) δ ppm 1.38 (s, 9 H) 1.74-1.83 (m, 2 H) 2.00-2.13 (m, 2 H) 2.24 (t, J= 7.28 Hz, 2 H). Intermediate S-1H: (4S)-4-(Propan-2-yl)-3-(5,5,5-trifluoropentanoyl)-l,3-oxazolidin-2- one

[00190] To a stirred solution of 5,5,5-trifluoropentanoic acid (5.04 g, 32.3 mmol) in DCM (50 mL) and DMF (3 drops) was added oxalyl chloride (3.4 mL, 38.8 mmol) dropwise over 5 min. The solution was stirred until all bubbling subsided. The reaction mixture was concentrated under reduced pressure to give pale yellow oil. To a separate flask charged with a solution of (4S)-4-(propan-2-yl)-l,3-oxazolidin-2-one (4.18 g, 32.4 mmol) in THF (100 mL) at -78 °C was added n-BuLi (2.5M in hexane) (13.0 mL, 32.5 mmol) dropwise via syringe over 5 min. After stirring for 10 min, the above acid chloride, dissolved in THF (20 mL), was added via cannula over 15 min. The reaction mixture was warmed to 0 °C, and was allowed to warm to room temperature as the bath warmed and stirred overnight. To the reaction mixture was added saturated NH₄C1, and the mixture was extracted with EtOAc (2x). The combined organics were washed with brine, dried (Na₂s0₄), filtered and concentrated under reduced pressure. The crude material was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 5% to 60% solvent A/B = hexanes/EtOAc, REDISEP® Si0₂ 120g). Concentration of the appropriate fractions provided Intermediate S-1H (7.39 g, 86%) as a colorless oil: 1H NMR (400 MHz, CDC1₃) δ ppm 4.44 (1 H, dt, J= 8.31, 3.53 Hz), 4.30 (1 H, t, J= 8.69 Hz), 4.23 (1 H, dd, J= 9.06, 3.02 Hz), 2.98-3.08 (2 H, m), 2.32-2.44 (1 H, m, J= 13.91, 7.02, 7.02, 4.03 Hz), 2.13-2.25 (2 H, m), 1.88-2.00 (2 H, m), 0.93 (3 H, d, J= 7.05 Hz), 0.88 (3 H, d, J= 6.80 Hz).

Intermediate S-1I: (2S,3R)-tert-Butyl 6,6,6-trifluoro-3-((S)-4-isopropyl-2- oxooxazolidine-3-carbonyl)-2-(3,3,3-trifluoropropyl)hexanoate, and Intermediate S-U: (2R,3R)-tert-Butyl 6,6,6-trifluoro-3-((S)-4-isopropyl-2-oxooxazolidine-3-carbonyl)-2- (3 ,3 ,3 -trifluoropropyl)hexanoate

[00191] To a cold (-78 °C), stirred solution of diisopropylamine (5.3 mL, 37.2 mmol) in THF (59 mL) under a nitrogen atmosphere was added n-BuLi (2.5M in hexane) (14.7 mL, 36.8 mmol). The mixture was then warmed to 0 °C to give a 0.5M solution of LDA. A separate vessel was charged with Intermediate S-1H (2.45 g, 9.17 mmol). The material was azeotroped twice with benzene (the RotoVap air inlet was fitted with a nitrogen inlet to completely exclude humidity), and then toluene (15.3 mL) was added. This solution was added to a flask containing dry lithium chloride (1.96 g, 46.2 mmol). To the resultant mixture, cooled to -78 °C, was added the LDA solution (21.0 mL, 10.5 mmol) and the mixture was stirred at -78 °C for 10 min, then warmed to 0 °C for 10 min., and then cooled to -78 °C. To a separate reaction vessel containing Intermediate S-1G (3.41 g, 16.07 mmol), also azeotroped twice with benzene, was added toluene (15.3 mL), cooled to -78 °C and LDA (37.0 mL, 18.5 mmol) was added. The resulting solution was stirred at -78 °C for 25 min. At this time the enolate derived from the ester was transferred via cannula into the solution of the oxazolidinone enolate and stirred at -78 °C for an additional 5 min, at which time the septum was removed and solid powdered bis(2- ethylhexanoyloxy)copper (9.02 g, 25.8 mmol) was rapidly added to the reaction vessel and the septum was replaced. The vessel was immediately removed from the cold bath and immersed into a warm water bath (40 °C) with rapid swirling and with a concomitant color change from the initial turquoise to brown. The reaction mixture was stirred for 20 min, was then poured into 5% aqueous NH₄OH (360 mL) and extracted with EtOAc (2x). The combined organics were washed with brine, dried (Na₂s0₄), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 0% to 60% solvent A/B = hexanes/EtOAc, REDISEP® Si0₂ 120g). Concentration of the appropriate fractions provided a mixture of Intermediate S- II and Intermediate S-1J (2.87 g, 66%) as a pale yellow viscous oil. 1H NMR showed the product was a 1.6: 1 mixture of diastereomers S-1LS-1J as determined by the integration of the multiplets at 2.74 and 2.84 ppm: 1H NMR (400 MHz, CDC1₃) δ ppm 4.43-4.54 (2 H, m), 4.23-4.35 (5 H, m), 4.01 (1 H, ddd, J= 9.54, 6.27, 3.51 Hz), 2.84 (1 H, ddd, J = 9.41, 7.28, 3.64 Hz), 2.74 (1 H, ddd, J= 10.29, 6.27, 4.02 Hz), 2.37-2.48 (2 H, m, J = 10.38, 6.98, 6.98, 3.51, 3.51 Hz), 2.20-2.37 (3 H, m), 1.92-2.20 (8 H, m), 1.64-1.91 (5 H, m), 1.47 (18 H, s), 0.88-0.98 (12 H, m). Intermediate S-1 : (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and Intermediate S-IE: (2R,3R)-3-(tert-Butoxycarbonyl)- 6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid

(S-IE)

[00192] To a cool (0 °C), stirred solution of Intermediate S-1I and Intermediate S-1 J (4.54 g, 9.51 mmol) in THF (140 mL) and water (42 mL) were sequentially added hydrogen peroxide (30% in water) (10.3 g, 91 mmol) and LiOH (685.3 mg, 28.6 mmol). The mixture was stirred for 1 hr. At this time the reaction vessel was removed from the cold bath and then stirred for 1.5 hr. To the reaction mixture were added saturated NaHC0₃ (45 mL) and saturated Na₂s0₃ (15 mL), and then the mixture was partially concentrated under reduced pressure. The resulting crude solution was extracted with DCM (3x). The aqueous phase was acidified to pH~l-2 with IN HC1, extracted with DCM (3x) and then EtOAc (lx). The combined organics were washed with brine, dried (Na₂s0₄), filtered and concentrated under reduced pressure to provide a mixture of Intermediates S-1 and S-IE (3.00 g, 86%) as a colorless oil: 1H NMR (400 MHz, CDC1₃) δ ppm 2.76-2.84 (1 H, m, diastereomer 2), 2.64-2.76 (3 H, m), 2.04-2.35 (8 H, m), 1.88- 2.00 (4 H, m), 1.71-1.83 (4 H, m), 1.48 (9 H, s, diastereomer 1), 1.46 (9 H, s,

diastereomer 2); 1H NMR showed a 1.7: 1 mixture of S-1E:S-1F by integration of the peaks for the t-butyl groups. Intermediate S-1 : (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and Intermediate S-IF: (2R,3R)-3-(fert-Butoxycarbonyl)- 6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid

[00193] To a cold (-78 °C) stirred solution of diisopropylamine (1.7 mL, 11.93 mmol) in THF (19 mL) under a nitrogen atmosphere was added n-BuLi (2.5M in hexanes) (4.8 mL, 12.00 mmol). The mixture was stirred for 5 min and then warmed to 0 °C. In a separate vessel, to a cold (-78 °C) stirred solution of the mixture of Intermediates S-1 and S-1E (1.99 g, 5.43 mmol) in THF (18 mL) was added the LDA solution prepared above via cannula slowly over 25 min. The mixture was stirred for 15 min, then warmed to room temperature (placed in a 24 °C water bath) for 15 min, and then again cooled to -78 °C for 15 min. To the reaction mixture was added Et₂AlCl (1M in hexane) (11.4 mL, 11.40 mmol) via syringe. The mixture was stirred for 10 min, warmed to room

temperature for 15 min and then cooled back to -78 °C for 15 min. Methanol (25 mL) was rapidly added, swirled vigorously while warming to room temperature, and then concentrated to ~l/4 the original volume. The mixture was dissolved in EtOAc and washed with IN HC1 (50 mL) and ice (75 g). The aqueous phase was separated and extracted with EtOAc (2x). The combined organics were washed with a mixture of KF (2.85g in 75 mL water) and IN HC1 (13 mL) [resulting solution pH 3-4], then with brine, dried (Na₂s0₄), filtered and concentrated under reduced pressure to give a 9: 1 (S-LS-1E) enriched diastereomeric mixture (as determined by 1H NMR) of Intermediate S-1 and Intermediate S-1E (2.13 g, >99%) as a pale yellow viscous oil: 1H NMR (400 MHz, CDC1₃) δ ppm 2.64-2.76 (2 H, m), 2.04-2.35 (4 H, m), 1.88-2.00 (2 H, m), 1.71-1.83 (2 H, m), 1.48 (9 H, s).

Intermediate S-2: (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3- fluoropropyl)hexanoic acid

Intermediate S-2: (2R,3S)-3-(tert-Butoxycarbonyl)-7,7,7-trifluoro-2-(3,3,3- trifluoropropyl)heptanoic acid, and Intermediate S-2A: (2R,3R)-3-(tert-Butoxycarbonyl)- 7,7,7-trifluoro-2-(3,3,3-trifluoropropyl)heptanoic acid

(S-2A)

[00194] To a cold (-78 °C), stirred solution of Intermediate S-1D (1.72 g, 6.36 mmol) in THF (30 mL) was slowly added LDA (7.32 mL, 14.6 mmol) over 7 min. After stirring for 1 h, 4,4,4-trifluorobutyltrifluoromethanesulfonate (2.11 g, 8.11 mmol) was added to the reaction mixture over 2 min. After 15 min, the reaction mixture was warmed to -25 °C (ice/MeOH/dry ice) for lh, and then cooled to -78 °C. After 80 min, the reaction was quenched with a saturated aqueous NH₄C1 solution (10 mL). The reaction mixture was further diluted with brine and the solution was adjusted to pH 3 with IN HC1. The aqueous layer was extracted with ether. The combined organics were washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to provide a mixture of Intermediates S-2 and S-2A (2.29 g, 95%) as a colorless oil. 1H NMR (400MHz, chloroform-d) δ 2.83-2.75 (m, 1H), 2.64 (ddd, J = 9.9, 6.7, 3.6 Hz, 1H), 2.32-2.03 (m, 5H), 1.98-1.70 (m, 3H), 1.69-1.52 (m, 3H), 1.50-1.42 (m, 9H). 1H NMR showed a 1 :4.5 mixture (S-2:S-2A) of diastereomers by integration of the peaks for the t- Bu groups.

Intermediate S-2: (2R,3S)-3-(fert-Butoxycarbonyl)-7,7,7-trifluoro-2-(3,3,3- trifluoropropyl)heptanoic acid, and Intermediate S-2A: (2R,3R)-3-(tert-Butoxycarbonyl)- 7,7,7-trifluoro-2-(3,3,3-trifluoropropyl)heptanoic acid

[00195] A mixture of Intermediate S-2 and Intermediate S-2A (2.29 g, 6.02 mmol) was dissolved in THF (38 mL) to give a colorless solution which was cooled to -78 °C. Then, LDA (7.23 mL, 14.5 mmol) (2.0M in heptane/THF/ethylbenzene) was slowly added to the reaction mixture over 3 min. After stirring for 15 min, the reaction mixture was placed in a room temperature water bath. After 15 min the reaction mixture was placed back in a -78 °C bath and then diethylaluminum chloride (14.5 mL, 14.5 mmol) (1M in hexane) was added slowly over 5 min. The reaction mixture was stirred at -78 °C. After 15 min, the reaction mixture was placed in a room temperature water bath for 10 min, and then cooled back to -78 °C. After 15 min, the reaction was quenched with MeOH (30.0 mL, 741 mmol), removed from the -78 °C bath and concentrated. To the reaction mixture was added ice and HC1 (60.8 mL, 60.8 mmol) and the resulting mixture was extracted with EtOAc (2x 200 mL). The organic layer was washed with potassium fluoride (3.50g, 60.3 mmol) in 55 mL H₂0 and 17.0 mL of IN HC1. The organics were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to provide an enriched mixture of Intermediate S-2 and Intermediate S-2A (2.25g, 98% yield) as a light yellow oil. 1H NMR (400MHz, chloroform-d) δ 2.83-2.75 (m, 1H), 2.64 (ddd, J= 9.9, 6.7, 3.6 Hz, 1H), 2.32-2.03 (m, 5H), 1.98-1.70 (m, 3H), 1.69-1.52 (m, 3H), 1.50-1.42 (m, 9H). ¹H NMR showed a 9: 1 ratio in favor of the desired diastereomer Intermediate S-2.

Intermediate S-2B: (2R,3S)-1 -Benzyl 4-tert-butyl 2,3-bis(4,4,4-trifluorobutyl)succinate

[00196] To a stirred 9: 1 mixture of Intermediate S-2 and Intermediate S-2A (2.24 g, 5.89 mmoL) and potassium carbonate (1.60 g, 11.58 mmoL) in DMF (30 mL) was added benzyl bromide (1.20 mL, 10.1 mmoL)). The reaction mixture was stirred at room temperature for 19 h. The reaction mixture was diluted with ethyl acetate (400 mL) and washed with 10% LiCl solution (3 x 100 mL), brine (50 mL), and then dried over anhydrous magnesium sulfate, filtered and concentrated to dryness under vacuum. The residue was purified by flash chromatography (Teledyne ISCO CombiFlash 0%> to 100% solvent A/B = hexane/EtOAc, REDISEP® Si0₂ 220 g, detecting at 254 nm, and monitoring at 220 nm). Concentration of the appropriate fractions provided Intermediate S-2B (1.59 g, 57.5%). HPLC: RT = 3.863 min (CHROMOLITH® SpeedROD column 4.6 x 50 mm, 10-90% aqueous methanol over 4 minutes containing 0.1% TFA, 4 mL/min, monitoring at 220 nm), 1H NMR (400MHz, chloroform-d) δ 7.40-7.34 (m, 5H), 5.17 (d, J= 1.8 Hz, 2H), 2.73-2.64 (m, 1H), 2.55 (td, J= 10.0, 3.9 Hz, 1H), 2.16-1.82 (m, 5H), 1.79-1.57 (m, 3H), 1.53-1.49 (m, 1H), 1.45 (s, 9H), 1.37-1.24 (m, 1H).

Intermediate S-2: (2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(4,4,4- trifluorobutyl)hexanoic acid

[00197] To a stirred solution of Intermediate S-2B (1.59 g, 3.37 mmoL) in MeOH (10 mL) and EtOAc (10 mL) under nitrogen was added 10%> Pd/C (510 mg). The atmosphere was replaced with hydrogen and the reaction mixture was stirred at room temperature for 2.5 h. The palladium catalyst was filtered off through a 4 μΜ polycarbonate film and rinsed with MeOH. The filtrate was concentrated under reduced pressure to give intermediate S-2 (1.28 g, 99%). 1H NMR (400MHz, chloroform-d) δ 2.76-2.67 (m, 1H), 2.65-2.56 (m, 1H), 2.33-2.21 (m, 1H), 2.17-2.08 (m, 3H), 1.93 (dtd, J= 14.5, 9.9, 5.2 Hz, 1H), 1.84-1.74 (m, 2H), 1.70-1.52 (m, 3H), 1.48 (s, 9H).

Intermediate A- 1 : (2-Amino-3 -methylphenyl)(3 -fluorophenyl)methanone

Intermediate A-1 A: 2-Amino- -methoxy-N,3-dimethylbenzamide

[00198] In a 1 L round-bottomed flask was added 2-amino-3-methylbenzoic acid (11.2 g, 74.1 mmol) and Ν,Ο-dimethylhydroxylamine hydrochloride (14.45 g, 148 mmol) in DCM (500 mL) to give a pale brown suspension. The reaction mixture was treated with Et₃N (35 mL), HOBT (11.35 g, 74.1 mmol) and EDC (14.20 g, 74.1 mmol) and then stirred at room temperature for 24 hours. The mixture was then washed with 10% LiCl, and then acidified with IN HCl. The organic layer was washed successively with 10%> LiCl and aq NaHC0₃. The organic layer was decolorized with charcoal, filtered, and the filtrate was dried over MgSC^. The mixture was filtered and concentrated to give 13.22 g (92% yield) of Intermediate A-1A. MS(ES): m/z = 195.1 [M+H⁺]; HPLC: RT = 1.118 min. (H₂0/MeOH with TFA, CHROMOLITH® ODS S5 4.6 x 50 mm, gradient = 4 min, wavelength = 220 nm); 1H NMR (500MHz, chloroform-d) δ 7.22 (dd, J= 7.8, 0.8 Hz, 1H), 7.12-7.06 (m, 1H), 6.63 (t, J= 7.5 Hz, 1H), 4.63 (br. s., 2H), 3.61 (s, 3H), 3.34 (s, 3H), 2.17 (s, 3H).

Intermediate A- 1 : (2-Amino-3 -methylphenyl)(3 -fluorophenyl)methanone

[00199] In a 500 mL round-bottomed flask, a solution of l-fluoro-3-iodobenzene (13.61 mL, 116 mmol) in THF (120 mL) was cooled in a -78 °C bath. A solution of n- BuLi, (2.5M in hexane, 46.3 mL, 116 mmol) was added dropwise over 10 minutes. The solution was stirred at -78 °C for 30 minutes and then treated with a solution of

Intermediate A-1 A (6.43 g, 33.1 mmol) in THF (30 mL). After 1.5 hours, the reaction mixture was added to a mixture of ice and IN HCl (149 mL, 149 mmol) and the reaction flask was rinsed with THF (5 ml) and combined with the aqueous mixture. The resulting mixture was diluted with 10% aq LiCl and the pH was adjusted to 4 with IN NaOH. The mixture was then extracted with Et₂0, washed with brine, dried over MgS0₄, filtered and concentrated. The resulting residue was purified by silica gel chromatography (220g ISCO) eluting with a gradient from 10% EtOAc/hexane to 30% EtOAc/hexane to afford Intermediate A-l (7.11 g, 94% yield) as an oil. MS(ES): m/z = 230.1 [M+H⁺]; HPLC: RT = 2.820 min Purity = 99%. (H₂0/MeOH with TFA, CHROMOLITH® ODS S5 4.6 x 50 mm, gradient = 4 min, wavelength = 220 nm).

Intermediate B-1 : (S)-3-Amino-5-(3-fluorophenyl)-9-methyl-lH-benzo[e][l,4]diazepin- 2(3H)-one

Intermediate B-1 A: (S)-Benzyl (5-(3-fluorophenyl)-9-methyl-2-oxo-2,3-dihydro benzo[e] [ 1 ,4]diazepin-3-yl)carbamate

(B-1A)

[00225] In a 1 L round-bottomed flask, a solution of 2-(lH-benzo[d][l,2,3]triazol-l- yl)-2-((phenoxycarbonyl)amino)acetic acid (J. Org. Chem., 55:2206-2214 (1990)) (19.37 g, 62.0 mmol) in THF (135 mL) was cooled in an ice/water bath and treated with oxalyl chloride (5.43 mL, 62.0 mmol) and 4 drops of DMF. The reaction mixture was stirred for 4 hours. Next, a solution of Intermediate A- 1 (7.11 g, 31.0 mmol) in THF (35 mL) was added and the resulting solution was removed from the ice/water bath and stirred at room temperature for 1.5 hours. The mixture was then treated with a solution of ammonia, (7M in MeOH) (19.94 mL, 140 mmol). After 15 mins, another portion of ammonia, (7M in MeOH) (19.94 mL, 140 mmol) was added and the resulting mixture was sealed under N₂ and stirred overnight at room temperature. The reaction mixture was then concentrated to ~l/2 volume and then diluted with AcOH (63 mL) and stir at room temperature for 4 hours. The reaction mixture was then concentrated, and the residue was diluted with 500 mL water to give a precipitate. Hexane and Et₂0 were added and the mixture was stirred at room temperature for 1 hour to form an orange solid. Et₂0 was removed under a stream of nitrogen and the aqueous layer was decanted. The residue was triturated with 40 mL of iPrOH and stirred at room temperature to give a white precipitate. The solid was filtered and washed with iPrOH, then dried on a filter under a stream of nitrogen to give racemic Intermediate B-1A (5.4 g, 41.7%yield).

[00226] Racemic Intermediate B-1A (5.9 g, 14.3 mmol) was resolved using the Chiral SFC conditions described below. The desired stereoisomer was collected as the second peak in the elution order: Instrument: Berger SFC MGIII, Column: CHIRALPAK® IC 25 x 3 cm, 5 cm; column temp: 45 °C; Mobile Phase: C0₂/MeOH (45/55); Flow rate: 160 mL/min; Detection at 220 nm.

[00227] After evaporation of the solvent, Intermediate B-1A (2.73 g, 46% yield) was obtained as a white solid. HPLC: RT = 3.075 min. (H₂0/MeOH with TFA,

CHROMOLITH® ODS S5 4.6 x 50 mm, gradient = 4 min, wavelength = 220 nm).

Chiral HPLC RT: 8.661 min (AD, 60% (EtOH/MeOH)/heptane) > 99%ee. MS(ES): m/z = 418.3 [M+H⁺];1H NMR (500MHz, DMSO-d₆) δ 10.21 (s, 1H), 8.38 (d, J= 8.3 Hz, 1H), 7.57-7.47 (m, 2H), 7.41-7.29 (m, 8H), 7.25-7.17 (m, 2H), 5.10-5.04 (m, 3H), 2.42 (s, 3H).

Intermediate B-l : (S)-3-Amino-5-(3-fluorophenyl)-9-methyl-lH-benzo[e][l,4]diazepin- 2(3H)-one.

[00228] In a 100 mL round-bottomed flask, a solution of Intermediate B-1A (2.73 g, 6.54 mmol) in acetic acid (12 mL) was treated with HBr, 33% in HOAc (10.76 mL, 65.4 mmol) and the mixture was stirred at room temperature for 1 hour. The solution was diluted with Et₂0 to give a yellow precipitate. The yellow solid was filtered and rinsed with Et₂0 under nitrogen. The solid was transferred to 100 mL round bottom flask and water was added (white precipitate formed). The slurry was slowly made basic with saturated NaHC0₃. The resulting tacky precipitate was extracted with EtOAc. The organic layer was washed with water, dried over MgS0₄, and then filtered and

concentrated to dryness to give Intermediate B-l (1.68 g, 91% yield) as a white foam solid. MS(ES): m/z = 284.2 [M+H⁺]; HPLC: RT = 1.72 min (H₂0/MeOH with TFA, CHROMOLITH® ODS S5 4.6 x 50 mm, gradient = 4 min, wavelength = 220 nm). 1H NMR (400MHz, DMSO-d₆) δ 10.01 (br. s., 1H), 7.56-7.44 (m, 2H), 7.41-7.26 (m, 3H), 7.22-7.11 (m, 2H), 4.24 (s, 1H), 2.55 (br. s., 2H), 2.41 (s, 3H). [00229] The compounds listed below in Table 6 (Intermediates B-2 to B-3) were prepared according to the general synthetic procedure described for Intermediate B-l , using the starting materials Intermediate A- 10 and Intermediate A-4, respectively.

Example 1

(2R,3S)-N-((3S)-5-(3-Fluorophenyl)-9-methyl-2-oxo-2,3-dihydro-lH-l,4-benzodiazepin- 3-yl)-2, -bis(3,3,3-trifluoropropyl)succinamide

Intermediate 1A: (2S,3R)-tert-Butyl 6,6,6-trifluoro-3-(((S)-5-(3-fluorophenyl)-9-methyl- 2-0X0-2, 3-dihydro-lH-benzo[e][l,4]diazepin-3-yl)carbamoyl)-2-(3,3 ,3- trifluoropropyl)hexanoat

[00240] In a 100 mL round-bottomed flask, a solution of Intermediate B-l (1683 mg, 5.94 mmol), Et₃N (1.656 mL, 11.88 mmol), and Intermediate S-l in DMF (20 mL) was treated with o-benzotriazol-l-yl-A .A .N’.N’-tetramethyluronium tetrafluoroborate (3815 mg, 11.88 mmol) and stirred at room temperature for 1 hour. The reaction mixture was diluted with water and saturated aqueous NaHC0₃. An off white precipitate formed and was filtered and washed with water. The resulting solid was dried on the filter under a stream of nitrogen to give Intermediate 1A (3.7 g, 99% yield). MS(ES): m/z =

632.4[M+H⁺]; HPLC: RT = 3.635 min Purity = 98%. (H₂0/MeOH with TFA,

CHROMOLITH® ODS S5 4.6 x 50 mm, gradient = 4 min, wavelength = 220 nm). 1H NMR (400MHz, methanol-d₄) δ 7.53 (t, J = 4.5 Hz, 1H), 7.46-7.30 (m, 3H), 7.28-7.23 (m, 1H), 7.23-7.18 (m, 2H), 5.37 (s, 1H), 2.88 (td, J = 10.4, 3.4 Hz, 1H), 2.60 (td, J =

10.2, 4.1 Hz, 1H), 2.54-2.40 (m, 1H), 2.47 (s, 3 H), 2.33-2.12 (m, 3H), 1.98-1.69 (m, 4H), 1.51 (s, 9H). Intermediate IB: (2S,3R)-6,6,6-Trifluoro-3-(((S)-5-(3-fluorophenyl)-9-methyl-2-oxo-

2,3-dihydro-lH-benzo[e][l,4]diazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoic acid

[00241] In a 250 mL round-bottomed flask, a solution of Intermediate 1A (3.7 g, 5.86 mmol) in DCM (25 mL) was treated with TFA (25 mL) and the resulting pale orange solution was stirred at room temperature for 1.5 hours. The reaction mixture was then concentrated to give Intermediate IB. HPLC: RT = 3.12 min (H₂0/MeOH with TFA, CHROMOLITH® ODS S5 4.6 x 50 mm, gradient = 4 min, wavelength = 220 nm).

MS(ES): m/z = 576.3 (M+H)⁺. 1H NMR (400MHz, methanol-d₄) δ 7.54 (t, J= 4.5 Hz, 1H), 7.49-7.29 (m, 3H), 7.28-7.15 (m, 3H), 5.38 (br. s., 1H), 2.89 (td, J= 10.3, 3.7 Hz, 1H), 2.67 (td, J= 9.9, 4.2 Hz, 1H), 2.56-2.38 (m, 1H), 2.48 (s, 3 H), 2.34-2.13 (m, 3H), 2.00-1.71 (m, 4H).

Example 1 :

[00242] In a 250 mL round-bottomed flask, a solution of Intermediate IB (4.04 g, 5.86 mmol) in THF (50 mL) was treated with ammonia (2M in iPrOH) (26.4 mL, 52.7 mmol), followed by HOBT (1.795 g, 11.72 mmol) and EDC (2.246 g, 11.72 mmol). The resulting white suspension was stirred at room temperature overnight. The reaction mixture was diluted with water and saturated aqueous NaHC0₃. The resulting solid was filtered, rinsed with water and then dried on the filter under a stream of nitrogen. The crude product was suspended in 20 mL of iPrOH and stirred at room temperature for 20 min and then filtered and washed with iPrOH and dried under vacuum to give 2.83 g of solid. The solid was dissolved in re fluxing EtOH(100 mL) and slowly treated with 200 mg activated charcoal added in small portions. The hot mixture was filtered through CELITE® and rinsed with hot EtOH. The filtrate was reduced to half volume, allowed to cool and the white precipitate formed was filtered and rinsed with EtOH to give 2.57 g of white solid. A second recrystallization from EtOH (70 mL) afforded Example 1 (2.39 g, 70% yield) as a white solid. HPLC: RT = 10.859 min (H₂0/CH₃CN with TFA, Sunfire C18 3.5μπι, 3.0x150mm, gradient = 15 min, wavelength = 220 and 254 nm); MS(ES): m/z = 575.3 [M+H⁺]; 1H NMR (400MHz, methanol-d₄) δ 7.57-7.50 (m, 1H), 7.47-7.30 (m, 3H), 7.29-7.15 (m, 3H), 5.38 (s, 1H), 2.85-2.75 (m, 1H), 2.59 (td, J= 10.5, 4.0 Hz, 1H), 2.53-2.41 (m, 4H), 2.31-2.10 (m, 3H), 1.96-1.70 (m, 4H).

PAPER RELATED

Structure–activity relationships in a series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides identified highly potent inhibitors of γ-secretase mediated signaling of Notch1/2/3/4 receptors. On the basis of its robust in vivo efficacy at tolerated doses in Notch driven leukemia and solid tumor xenograft models, 12 (BMS-906024) was selected as a candidate for clinical evaluation.

Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch Inhibitor for the Treatment of Leukemia and Solid Tumors

Patent

http://www.google.co.in/patents/WO2012129353A1?cl=en

PATENT RELATED

US-20160060232-A1

https://patentscope.wipo.int/search/en/detail.jsf?docId=US159930181&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

PATENTS RELATED

US-20150284342-A1

US-20140357605-A1

US-20140100365-A1

Clip RELATED

Company: Bristol-Myers Squibb

Target: pan-Notch

Disease: breast, lung, colon cancer; leukemia

(From left, front row) Gavai, Weifeng Shan, (second row) Aaron Balog, Patrice Gill, Gregory Vite, (third row) Francis Lee, Claude Quesnelle, (rear row) Wen-Ching Han, Richard Westhouse.

Credit: Catherine Stroud Photography

http://cen.acs.org/articles/91/i16/BMS-906024-Notch-Signaling-Inhibitor.html

PAPER RELATED

An enantioselective synthesis of (S)-7-amino-5H,7H-dibenzo[b,d]azepin-6-one (S–1) is described. The key step in the sequence involved crystallization-induced dynamic resolution (CIDR) of compound 7 using Boc-d-phenylalanine as a chiral resolving agent and 3,5-dichlorosalicylaldehyde as a racemization catalyst to afford S–1 in 81% overall yield with 98.5% enantiomeric excess.

//////////BMS-986115, BMS 986115, 3,5-dichlorosalicylaldehyde, Alzheimer’s disease, Boc-D-phenylalanine, CIDR;dibenzoazepenone,  DKR; Notch inhibitors, Notch inhibitor, SAR,  T-acute lymphoblastic leukemia, triple-negative breast cancer, γ-secretase inhibitor, PHASE 1, BMS, Bristol-Myers Squibb,  Ashvinikumar Gavai, 1584647-27-7, UNII: LSK1L593UU

Cc1cccc2c1NC(=O)[C@H](N=C2c3cccc(c3)F)NC(=O)[C@H](CCC(F)(F)F)[C@H](CCC(F)(F)F)C(=O)N

Organizational Initiatives Towards Developing Greener Processes for Generic Active Pharmaceutical Ingredients
– Dr. Vilas H. Dahanukar, Chief Scientist-Process R&D, Integerated Product Development Organization, Dr. Reddy’s Laboratories Ltd., India

presented at

4th Industrial Green Chemistry World Convention & Ecosystem (IGCW-2015) on 4^th – 5^th December 2015

A PRESENTATION

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A PRESENTATION

Innovative Techniques, To Synthesize Breakthrough Molecules, See DOE On pae 4 onwards

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WHAT YOU NEED TO KNOW…..

Quality by Design in Drug Product Development

Introduction to drug product development – setting the scene

• Drug product development at a glance – from first in man to marketing authorization
• Pharmaceutical QbD: Quo vadis?
• Application of QbD principles to drug product development

Expectations from regulatory agencies

• Regulatory initiatives and approaches for supporting emerging technologies
• Concepts of Real Time Release Testing (Draft Annex 17 EU GMP Guideline)
• Harmonization of regulatory requirements (QbD parallel-assessment FDA-EMA, ICH Q8 -> Draft Q12?)
• Regulatory expectations: Lessons learned from applications so far

Knowledge Management

• Knowledge Management (KM) System – Definition and Reason
• Knowledge Management Cycle
• Explicit and Tacit Knowledge – The Knowledge Spiral
• Correlation between KM and other Processes
• Enabling Knowledge Management
• Knowledge Review – integral part of the Management Review (ICH Q10)

Quality Risk Assessment and Control Strategy

• Objectives of Quality Risk Assessment (QRA) as part of development
• Overview to risk assessment tools
• Introduction of Process Risk Map
• Introduction of risk based control strategy development

QbD Toolbox: Case studies DoE, PAT, and Basic Statistics

• Value-added use of QbD tools – generic approaches and tailored solutions
• Case studies and examples for different unit operations and variable problems

Reports and Documentation

• Development Reports
• Transfer protocols and reports
• Control Strategy and link to the submission dossier

Wrap-up & Final Discussion
The concepts and tools used over the two days will be summarized and future implications and opportunities of applying QbD principles to process development will be discussed. Delegates will be given time to ask questions on how they can apply what they have learned to their own drug product development and manufacturing.

Workshop Process Risk Map & link to Control Strategy
Based on a risk assessment tool tailored to cover development needs, delegates will work on case studies of process development for a solid oral dosage form.
From QTPP and CQA to relationship analysis of process parameters and material attributes
Process mapping for integrated documentation of the development work
Process Risk Map as a tool for development-focussed risk assessment

Quality by Design in API Manufacturing

General framework and key elements of QbD for APIs – background and potential strategies

• What is it all about?
• What are the benefits?
• When and how should you use it?
• Practical examples with typical points of discussion

How to identify and control Critical Quality Attributes (CQAs) in API synthesis – a risk-based approach to developing a control strategy

• Severity assessment of quality attributes
• Impact levels for critical process parameters (CPPs) and critical material attributes (CMAs)
• Considerations for the API Starting material
• Design of an effective risk-based control strategy
• Examples

How to provide information on the development of the API manufacturing process – dossier requirements

• What should be done at which stage?
• Which information is relevant for the dossier?
• What are the key-points to be considered for APIs (NCE/Biotech) and their formulations
• Typical questions from Authorities

Process Evaluation and Design Space

• Changing Validation Approach
• Validation Life Cycle
• Design Space Concept

Application of PAT in the API industry

• PAT at development stages of a QbD-based development
• PAT as part of the Control Strategy in a GMP environment
• Practical examples of PAT implementations at a commercial scale in a GMP environmen

t
Control strategies – Case studies and examples

• HA definitions
• Why and When is a control strategy needed
• Different types/elements of a control strategy
• Practical examples

///////////

BMS 986001

Censavudine, Festinavir

Has anti-HIV activity. IN PHASE 2

CAS: 634907-30-5, UNII: 6IE83O6NGA, OBP 601, 4′-Ethynyl D4T, 4′-Ed4T, TDK-4-114

Molecular Formula, C12-H12-N2-O4, Molecular Weight, 248.2368

2′,3′-Didehydro-3′-deoxy-4′-ethynylthymidine, 

1-((2R,5R)-5-Ethynyl-5-(hydroxymethyl)-2H-furan-2-yl)-5-methyl-pyrimidine-2,4-dione, 

2′,3′-Didehydro-3′-deoxy-4′-ethynylthymidine

INNOVATOR= YALE UNIVERSITY

Festinavir is a nucleoside reverse transcriptase inhibitor

(NRTI) which is being developed for the treatment of HIV infection. The drug has shown considerable efficacy in early development, and with perhaps less toxicity than some other NRTIs, such as the drug stavudine (marketed under the trade name ZERIT^®).

Festinavir has the chemical form and the structural formula:

Festinavir was developed by Yale University in conjunction with two Japanese research scientists, and is protected by U.S. Patent No. 7,589,078, the contents of which are incorporated herein by reference. The ‘078 patent sets forth the synthesis of the primary compound, and other structural analogs. In addition, Oncolys BioPharma, Inc. of Japan has now published US 2010/0280235 for the production of 4′ ethynyl D4T. As starting raw material, the Oncolys method utilizes a substituted furan compound, furfuryl alcohol. In another publication by Nissan Chemical Industries of Japan, and set forth in WO 201 1/099443, there is disclosed a method for producing a beta-dihydrofuran deriving compound or a beta-tetrahydrofuran deriving compound. In this process, a diol compound is used as the starting material. Nissan has also published WO 2011/09442

directed to a process for the preparation of a β-glycoside compound. Two further publications, each to Hamari Chemicals of Japan, WO 2009/1 19785 and

WO 2009/125841, set forth methods for producing and purifying ethynyl thymide compounds. Pharmaset, Inc. of the U.S. has also published US 2009/0318380,

WO 2009/005674 and WO 2007/038507 for the production of 4’ -nucleoside analogs for treating HIV infection. Reference is also made to the BMS application entitled

“Sulfilimine and Sulphoxide Methods for Producing Festinavir” filed as a PCT application, PCT/US2013/042150 on May 22, 2013 (now WO2013/177243).

PAPER

Haraguchi, Kazuhiro; Bioorganic & Medicinal Chemistry Letters 2003, V 13(21), PG 3775-3777 

http://dx.doi.org/10.1016/j.bmcl.2003.07.009

http://www.sciencedirect.com/science/article/pii/S0960894X0300831X

white crystalline solid. 1: Rf = 0.8 (silica, MeOH:CH2Cl2,1:4);

M.P. = 196-207°C;

1 H NMR (d6-DMSO, 500 MHz): δ = 11.34 (s, 1 H), 6.88 (s, 1 H), 6.35 (d, J = 6.0 Hz, 6.05 (d, J = 6.0 Hz, 1 H), 5.45 (t, J = 5.5 Hz, 1 H), 3.69 (dd, J = 12.0, 1.5 Hz, 1 H), 3.64 (s, 1 H), 3.59 (dd, J = 12.0, 1.5 Hz, 1 H) 1.70 (s, 3 H) ppm;

13C NMR (d6-DMSO, 125 MHz): δ = 163.85, 150.82, 136.81, 135.54, 127.13, 109.04, 88.94, 86.60, 81.45, 77.39, 65.76, 12.23 ppm;

HRMS calcd for C12H12N2O4H+ [M + H+] 249.09 found 249.08.

PATENT

WO 2014172264

https://www.google.ch/patents/WO2014172264A1?cl=en

invention:

Step#l: Acetal Formation

Compound 1

85% yield

The starting material is 5-methylurdine, which is commercially available. The first step of the process is an acetal formation. 5-methyluridine is utilized and is treated with H2SO4 and acetaldehyde. Other acids available to the scientist, such as perchloric acid, will also work for this transformation. The solvent utilized for this step is acetonitrile (ACN), and other solvents may also be utilized as well. Once the starting material is consumed, a slurry is obtained and the product can be simply filtered off and dried to provide Compound 1 as a solid.

Acetal formation

Preparation of l-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2-methyltetrahydrofuro [3,4-d] [1,3] dioxol-4-yl)-5-methylpyrimidine-2,4(lH,3H)-dione

The following were added to a flask: 5-methyluridine (10 g, 38.70 mmol), acetonitrile (20 mL) and 70% perchloric acid (4.01 mL, 47.63 mmol). A solution of acetaldehyde (3.26 mL, 58.10 mmol) in acetonitrile (20 mL) was added dropwise over 1 h. The resulting solution was allowed to stir at 20 °C for 18 h. The resulting slurry was filtered and dried (50 °C, 25 mmHg) to afford Acetal (9.30 g, 84% yield) as white solid

^XH NMR (400MHz, DMSO-d₆) δ = 11.39 (s, 1H), 7.72 – 7.63 (m, 1H), 5.82 (d, J=3.0 Hz, 1H), 5.21 – 5.07 (m, 2H), 4.84 (dd, J=6.6, 2.5 Hz, 1H), 4.68 (dd, J=6.6, 3.0 Hz, 1H), 4.12 – 4.05 (m, 1H), 3.65 – 3.51 (m, 2H), 3.36 (s, 2H), 1.77 (s, 3H), 1.37 (d, J=5.1 Hz, 3H) ¹³C NMR (101MHz, DMSO-d₆) δ = 163.77, 150.32, 137.64, 109.39, 104.50, 90.79, 86.16, 83.83, 81.37, 61.25, 19.76, 12.06

Step #2: Acetate protection

Compound 2

85% yield

The next step of the sequence is installation of a 4-biphenylacetate. Without being bound by any particular theory, this protecting step may be chosen for two reasons:

1) To provide a solid intermediate that can be easily isolated, and

2) Act as a directing group in the next step (set forth later on).

This reaction consists of reacting Compound 1 with 4-biphenyl acid chloride and pyridine in acetonitrile. In this reaction, pyridine is preferred as it allows the reaction to occur only at the -OH moiety of the molecule. It should also be noted that other polar solvents could be used, but acetonitrile allowed the desired product Compound 2 to be isolated as s solid.

Ac lation

Preparation of ((3aR,4R,6R,6aR)-2-methyl-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)tetrahydrofuro[3,4-d] [l,3]dioxol-4-yl)methyl [1,1′-biphenyl]-4-carboxylate.

Acetal (9.30 g, 32 mmol) was dissolved into acetonitrile (100 mL). Pyridine (1.3 eq) was added followed by the addition of 4-biphenylcarbonyl chloride (1.05 eq). The solution was heated to 50 °C and held for 2 h. The slurry was cooled to 20 °C and held for 2 h. The slurry was filtered and washed with acetonitrile (100 mL). The solids were dried (50 °C, 25 mmHg) to Compound 2 (85% yield).

^XH NMR (400MHz, CHLOROFORM-d) δ = 8.10 (d, J=8.1 Hz, 2H), 7.62 (d, J=7.6 Hz, 2H), 7.67 (d, J=8.1 Hz, 2H), 7.55 – 7.36 (m, 3H), 7.09 (s, 1H), 5.71 (s, 1H), 5.26 (q, J=4.7 Hz, 1H), 5.03 (dd, J=6.6, 2.0 Hz, 1H), 4.91 (dd, J=6.7, 3.2 Hz, 1H), 4.73 – 4.63 (m, 1H), 4.61 – 4.50 (m, 2H), 2.02 (s, 3H), 1.85 – 1.76 (m, 3H), 1.52 (d, J=4.8 Hz, 3H)

^1JC MR (101MHz, CHLOROFORM-d) δ = 164.02, 161.94, 148.20, 144.18, 137.85, 135.89, 128.20, 127.05, 126.36, 126.30, 125.35, 125.26, 1 14.49, 109.20, 103.88, 92.51, 83.36, 83.29, 79.87, 75.45, 75.13, 74.81, 62.54, 17.92, 10.32, -0.01

With the acetal and 4-biphenylacetate groups in place, the next reaction is a regioselective acetal opening utilizing TMSOTf (Trimethylsilyl trifluoromethane sulfonate, or other available Lewis acids)/Et₃N to afford the corresponding silyl ether, which is cleaved in situ, to afford the 2-vinyloxy compound as Compound 3. Compound 3 may be prepared in a step-wise fashion (shown below), but in order to reduce the number of steps, it is possible to take Compound 3 and selectively form the desired 2-vinyl oxy regioisomer Compound 3. Those skilled in the art may recognize that the 4-biphenylacetate can be important to obtain high selectivity for this transformation.

Although a variety of Lewis acids may be utilized, TMSOTf is generally found to be more effective. Et₃ is also a preferred reactant, as other amine bases are generally less effective. The ratio of TMSOTf to Ets is preferably within the range of about 1 : 1.3; if the reaction medium became acidic, Compound 3 would revert back to Compound 2. In terms of solvents, DCM (Dichloromethane) may be particularly effective, but toluene, CF₃-PI1, sulfolane, and DCE (Dichloroethene) are also effective. The reaction can be worked up using aqueous acid, preferably K₂HP0₄, or methanolic NH₄F to quench the reaction, as well as remove the TMS-ether in situ.

TMSOTf-opening

Preparation of ((2R,3R,4R,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-4-(vinyloxy)tetrahydrofuran-2-yl)methyl [1,1′-biphenyl]-4-carboxylate

Compound 2 (20 g, 43.06 mmol) was dissolved into DCM (160 mL). Triethylamine (78 mL, 560 mmol) was added followed by the addition of TMSOTf (80.30 mL, 431 mmol). This solution was heated to 45 °C and held there until complete by HPLC analysis (6 h). Once complete, this solution was added to ammonium acetate (66.40 g, 861 mmol) in water (200 mL). After stirring for 20 min, the layers were separated. The organics were concentrated and the resulting residue was dissolved into EtOAc (200 mL). The organics were washed with the following solution (potassium phosphate monobasic (118 g, 861 mmol) in water (400 mL). The organics were then dried ( a₂S0₄), filtered and concentrated. The resulting residue was purified by column chromatography [Silica gel; 20% to 90% EtOAc in Hexanes] to afford Compound 3 (15.8 g, 79% yield) as a solid.

^XH NMR (400MHz, CHLOROFORM-d) 6 = 9.18 (br. s., IH), 8.18 – 8.06 (m, 2H), 7.73 -7.56 (m, 4H), 7.55 – 7.38 (m, 3H), 7.24 (d, J=1.3 Hz, IH), 6.59 (dd, J=14.0, 6.4 Hz, IH), 5.81 (d, J=2.0 Hz, IH), 4.84 (dd, J=12.6, 2.5 Hz, IH), 4.63 (dd, J=12.5, 4.2 Hz, IH), 4.59 – 4.44 (m, 3H), 4.40 – 4.26 (m, 2H), 1.70 (d, J=1.0 Hz, 3H)

¹³C MR (101MHz, CHLOROFORM-d) δ = 166.13, 163.65, 150.00, 149.67, 146.39, 139.66, 135.67, 130.16, 129.01, 128.40, 128.06, 127.32, 127.28, 111.43, 91.93, 89.44, 81.60, 80.19, 69.32, 63.06, 12.32

Step #4: Iodiiiation

Compound 4

Compound 3 _{75% yie|d}

Next, Compound 3 is transformed into the iodide compound which is Compound 4. This can be accomplished by treating Compound 3 with (2.0 eq), PPI13 (2.0 eq.) and imidazole (4.0 eq). Other methods to install the iodide may also be utilized, such as mesylation/Nal, etc., but these may be less preferred. In addition, other halogen-bearing compounds such as Br₂ and CI2 may be considered by the skilled scientist. Premixing imidazole, , and PPh₃, followed by addition of Compound 3 in THF and heating at 60 °C allows smooth conversion to Compound 4. It is highly preferred to add all reagents prior to the addition of Compound 3; if not, the vinyloxy group will be cleaved. Other solvents, such as 2-MeTHF and PhMe may be utilized, but THF often provides the best yield.

Iodiiiation

Preparation of ((2R,3S,4S,5R)-3-iodo-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-4-(vinyloxy)tetrahydrofuran-2-yl)methyl [l,l’-biphenyl]-4-carboxylate

The following were added to a flask: imidazole (8.79 g, 129 mmol),

triphenylphosphine (16.94 g, 65 mmol), iodine 16.39 g, 65 mmol) and THF (525 mL). A solution of Compound 3 (15 g, 32 mmol) in THF (375 mL) was added. The solution was heated to 60 °C and was held at 60 °C for 4 h. Once complete by HPLC analysis (4 h), the solution was concentrated and the residue was purified by column chromatography [Silica gel; 10% to 60% EtOAc in Hexanes] to afford Compound 4 (17.0 g, 92% yield) as a solid.

^XH NMR (400MHz, CHLOROFORM-d) δ = 9.25 (br. s., IH), 8.16 (d, J=8.3 Hz, 2H), 7.75 – 7.61 (m, 5H), 7.54 – 7.40 (m, 3H), 7.32 – 7.24 (m, 2H), 7.23 – 7.16 (m, 2H), 6.56 -6.45 (m, IH), 6.06 (d, J=1.5 Hz, IH), 4.89 (s, IH), 4.66 (dd, J=12.0, 6.9 Hz, IH), 4.56 (dd, J=12.0, 3.9 Hz, IH), 4.46 (d, J=4.0 Hz, IH), 4.39 – 4.26 (m, 2H), 4.13 (dt, J=7.1, 3.8 Hz, 1H), 2.06 – 1.97 (m, 3H)

^1JC MR (101MHz, CHLOROFORM-d) δ = 165.96, 163.94, 150.27, 149.29, 146.28, 139.81, 137.88, 135.84, 130.37, 129.06, 129.01, 128.34, 128.25, 127.94, 127.31, 127.22, 125.32, 1 11.07, 91.37, 90.32, 89.18, 78.43, 69.15, 25.81, 21.49, 12.71

Step #5: Iodide Elimination

Compound 4

The next step of the sequence is to install the allyic moiety. Heating a solution of Compound 4 in toluene in the presence of DABCO (l,4-Diazabicyclo[2.2.2]octane) allows for elimination of the iodide. Other solvents, such as THF and DCE may be utilized, but toluene often provides the best conversion and yield. Other amine bases may be used in this transformation, but generally DABCO is preferred.

Elimination

Preparation of ((4R,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l (2H)-yl)-4-(vinyloxy)-4,5-dihydrofuran-2-yl)methyl [l,l’-biphenyl]-4-carboxylate

Compound 4 (17 g, 30 mmol) was dissolved into toluene (255 niL), and DABCO (10 g, 89 mmol) was added. The solution was heated to 90 °C and held there for 2 h. Once complete, the organics were washed with sat. aq. a₂S₂03 (200 mL). The organics were then dried ( a₂S0₄), filtered, and concentrated. The resulting residue was purified by column chromatography [Silica gel; 5% to 60% EtOAc in Hexanes] to yield

Compound 5 (10.9, 85% yield) as a foam.

^XH NMR (400MHz, CHLOROFORM-d) δ = 8.93 (br. s., IH), 8.18 – 8.11 (m, 2H), 7.75 -7.61 (m, 5H), 7.55 – 7.39 (m, 4H), 6.95 (d, J=1.0 Hz, IH), 6.54 (d, J=2.0 Hz, IH), 6.46 (dd, J=14.3, 6.7 Hz, IH), 5.53 (d, J=2.5 Hz, IH), 5.09 (d, J=2.8 Hz, IH), 5.04 (d, J=6.6 Hz, 2H), 4.29 (dd, J=14.3, 2.4 Hz, IH), 4.23 (dd, J=6.7, 2.4 Hz, IH), 1.88 (d, J=1.0 Hz, 3H)

^1JC MR (101MHz, CHLOROFORM-d) δ = 165.73, 159.58, 149.10, 146.49, 139.70, 134.51, 132.17, 132.07, 131.94, 131.92, 130.30, 129.01, 128.56, 128.44, 128.40, 127.73, 127.30, 127.28, 112.50, 99.16, 90.57, 90.23, 84.81, 58.68, 12.44

Step #6: Claisen Rearrangement

An important reaction in the sequence is the Claisen rearrangement. This reaction is utilized to install the quaternary stereocenter and the olefin geometry in the ring. Heating Compound 5 in benzonitrile at 190 °C for 2-3 hours allows for smooth conversion to Compound 6, and after chromatography, a 90% yield can be achieved.

Toluene (110 °C, 8 h) also works to provide the desired Compound 6 as a solid by simply cooling the reaction to 20 °C (no chromatography). Other solvents with boiling points over about 100°C may also be utilized.

Claisen Rearrangement

Preparation of ((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-2-(2-oxoethyl)-2,5-dihydrofuran-2-yl)methyl [l,l’-biphenyl]-4-carboxylate

Compound 5 (1 mmol) was dissolved into benzonitrile (10 mL). The solution was heated to 190 °C for 3 h. After cooling to 20 °C, the solution was purified by column chromatography [silica gel, 50:50 Hexanes:EtOAc] to afford Compound 6 (1 mmol).

Alternatively, Compound 5 (1 mmol) was dissolved into toluene (10 mL). The solution was heated to 110 °C and held for 12 h. Upon cooling to 20 °C, a slurry formed. The solids were filtered, washed (PhMe) and dried (50 °C, 25 mmHg) to afford

Compound 6 (1 mmol) as a white solid.

^XH NMR (400MHz, CHLOROFORM-d) δ = 9.84 (t, J=1.8 Hz, 1H), 8.53 (br. s., 1H), 8.13 – 8.03 (m, J=8.3 Hz, 2H), 7.73 – 7.67 (m, 2H), 7.67 – 7.60 (m, 2H), 7.56 – 7.38 (m, 3H), 7.14 (d, J=1.3 Hz, 1H), 7.04 (t, J=1.5 Hz, 1H), 6.57 (dd, J=6.1, 2.0 Hz, 1H), 6.02 (dd, J=5.9, 1.1 Hz, 1H), 4.68 – 4.52 (m, 2H), 3.06 – 2.89 (m, 2H), 1.59 (d, J=1.0 Hz, 3H)

¹³C MR (101MHz, CHLOROFORM-d) δ = 198.33, 165.83, 163.35, 150.65, 146.56, 139.63, 136.24, 135.02, 130.21, 129.04, 128.44, 127.86, 127.49, 127.41, 127.28, 111.59, 90.03, 89.61, 67.33, 50.06, 12.06

ne Formation via elimination of Enol Nonaflate

The alkyne formation is performed by first treating Compound 6 with TMSCl (Trimethylsilyl chloride)/Et₃N. NfF (Nonafluoro- 1 -butanesulfonyl fluoride) and P-base () are then added at -20 °C. After warming to 20 °C, the desired alkyne Compound 7 can be isolated in about 80 % yield. Initially, TMSCl is presumed to react at the NH moiety. NfF/P-base then reacts with the aldehyde to form the enol Nonaflate. Upon warming to 20 °C in the presence of P-base, the enol Nonaflate eliminates smoothly to the alkyne Compound 7. Without the TMSCl/Et₃N, the yields are only -25%.

Alkyne formation

Preparation of ((2R,5R)-2-ethynyl-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-2,5-dihydrofuran-2-yl)methyl [l,l’-biphenyl]-4-carboxylate

Compound 6 (1 g, 2.24 mmol) was dissolved into DMF (Dimethylformamide) (5 mL). (Other polar solvents could also have been used.) Triethylamine (406 uL, 2.91 mmol) was added and the solution was cooled to 0 °C. TMSCl (314 uL, 2.46 mmol) was added and the solution was allowed to stir at 0 °C for 30 min. The solution was then cooled to -20 °C, and NfF (484 uL, 2.69 mmol) was added and the solution was allowed to stir at -20 °C for 5 min. Phosphazane P l-base (1.54 mL, 4.93 mmol) was added

dropwise over 20 min. The solution was then allowed to warm to 20 °C and held for 20 h. The solution was then poured into water (50 mL) and extracted with DCM (100 mL). The organics were concentrated and the resulting residue was purified by column chromatography [Silica gel; 10% to 60% EtOAc in Hexanes] to afford Compound 7 (816 mg, 85% yield) as a solid.

^XH NMR (400MHz, DMSO-d₆) δ = 11.46 (s, 1H), 8.08 – 7.97 (m, J=8.6 Hz, 2H), 7.92 -7.80 (m, 2H), 7.73 (d, J=7.1 Hz, 2H), 7.59 – 7.39 (m, 3H), 7.06 (d, J=1.0 Hz, 1H), 6.89 (d, J=1.5 Hz, 1H), 6.61 (dd, J=5.6, 2.0 Hz, 1H), 6.23 (dd, J=5.6, 1.0 Hz, 1H), 4.66 (d, J=12.1 Hz, lH), 4.57 (d, J=11.6 Hz, 1H), 3.87 (s, 1H), 1.37 (s, 3H)

¹³C MR (101MHz, DMSO-d₆) δ = 164.89, 163.57, 150.61, 145.13, 138.73, 135.30, 134.40, 129.94, 129.12, 128.49, 127.84, 127.78, 127.18, 126.98, 110.01, 89.37, 83.69, 80.01, 78.23, 66.89, 11.46

90% yield

The final step of the sequence is to remove the aromatic ester protecting group. This consists of hydrolysis by NaOH in aq. THF solution. The API is extracted into THF and then crystallized from THF/PhMe.

Deprotection

Preparation of l-((2R,5R)-5-ethynyl-5-(hydroxymethyl)-2,5-dihydrofuran-2-yl)-5-methylpyrimidine-2,4(lH,3H)-dione (Ed4T)

Compound 7 (10 g, 23.40 mmol) was dissolved into THF (100 mL). 3N NaOH (10 mL) was added. The solution was allowed to stir at 20 °C for 12 h. The layers were split and the organics were kept. The organics were concentrated to reach a KF <1 wt%. Toluene (100 mL) was added, and solids crashed out of solution. The solids were filtered and washed with Toluene (100 mL). The solids were then dried (50 °C, 25 mmHg) to afford Festinavir (5.21 g, 90% yield) as a white solid.

^XH NMR (400MHz, DMSO-d₆) δ = 1 1.36 (s, 1H), 7.58 (s, 1H), 6.89 (s, 1H), 6.36 (d, J=6.1 Hz, 1H), 6.05 (d, J=6.1 Hz, 1H), 5.48 (t, J=5.6 Hz, 1H), 3.78 – 3.49 (m, 3H), 3.46 3.31 (m, 1H), 1.71 (s, 3H)

^1JC MR (101MHz, DMSO-d₆) δ = 163.80, 150.76, 136.75, 135.47, 127.06, 108.98, 88.87, 86.52, 81.37, 77.33, 65.68, 12.17.

PAPER

Tetrahedron (2009), 65(36), 7630-7636.

Tetrahedron

Volume 65, Issue 36, 5 September 2009, Pages 7630–7636

PAPER

Nucleophilic Substitution at the 4‘-Position of Nucleosides: New Access to a Promising Anti-HIV Agent 2‘,3‘-Didehydro-3‘-deoxy-4‘-ethynylthymidine

Journal of Organic Chemistry (2006), 71(12), 4433-4438.

http://pubs.acs.org/doi/abs/10.1021/jo060194m

For the synthesis of 2‘,3‘-didehydro-3‘-deoxy-4‘-ethynylthymidine (8:  4‘-Ed4T), a recently reported promising anti-HIV agent, a new approach was developed. Since treatment of 1-(2,5-dideoxy-β-l–glycero-pent-4-enofuranosyl)thymine with Pb(OBz)₄ allowed the introduction of the 4‘-benzoyloxy leaving group, nucleophilic substitution at the 4‘-position became feasible for the first time. Thus, reaction between the 4‘-benzoyloxy derivative (14) and Me₃SiC⋮CAl(Et)Cl as a nucleophile led to the isolation of the desired 4‘-“down”-ethynyl derivative (18) stereoselectively in 62% yield. As an application of this approach, other 4‘-substituted nucleosides, such as the 4‘-allyl (24a) and 4‘-cyano (26a) derivatives, were synthesized using organosilicon reagents. In these instances, pretreatment of 14 with MeAlCl₂ was necessary.

PATENTS

US75890782009-09-15Anti-viral nucleoside analogs and methods for treating viral infections, especially HIV infections

/////////BMS 986001, 634907-30-5, UNII: 6IE83O6NGA, OBP 601, 4′-Ethynyl D4T, 4′-Ed4T, TDK-4-114, PHASE 2

Cc1cn(c(=O)[nH]c1=O)[C@H]2C=C[C@](O2)(CO)C#C

Inaugural ACS Industry Symposium, 11-12 November 2016 in Hyderabad, India

Recent Advances in Drug Development

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CB-618, CB-238618

sodium salt of (2S,5R)-sulfuric acid mono-(2-[1,3,4]oxadiazol-2-yl-7-oxo-1,6-diaza-bicyclo[3.2.1]oct-6-yl)ester

WO2013149121

US 20140275001

US 20150094472

WO 2016081452

Infection, multidrug resistant bacteria (MDR) in  phase 1 at  Merck
CB-618 is in phase I clinical trails by Cubist for the treatment of resistant bacterial infections, including carbapenem-resistant Enterobacteriaceae and Klebsiella pneumonia carbapenemases infection.

CB-618 is a beta-Lactamase inhibitor in phase I clinical trials at Merck & Co. for the treatment of multidrug resistant bacterial infections, including those caused by carbapenem-resistant Enterobacteriaceae and Klebsiella pneumoniae carbapenemases.

The product was originally developed at Cubist. In 2015, Merck & Co. acquired the company

Bacterial resistance to β-lactam antibiotics, especially in Gram-negative bacteria, is most commonly mediated by β-lactamases. β-lactamases are enzymes that catalyze the hydrolysis of the β-lactam ring, which inactivates the antibacterial activity of the β-lactam antibiotic and allows the bacteria to become resistant. Inhibition of the β-lactamase with a BLI slows or prevents degradation of the β-lactam antibiotic and restores β-lactam antibiotic susceptibility to β-lactamase producing bacteria. Many of these β-lactamases are not effectively inhibited by BLIs currently on the market rendering the β-lactam antibiotics ineffective in treating bacteria that produce these β-lactamases. There is an urgent need for novel BLIs that inhibit β-lactamases that are not effectively inhibited by the current clinical BLIs (e.g. KPC, class C and class D β-lactamases) and that could be used in combination with β-lactam antibiotics to treat infections caused by β-lactam resistant bacteria.

PATENT

WO2013149121

Yu Gui Gu, Yong He, Ning Yin, Dylan C. ALEXANDER, Jason B. CROSS, Chester A. Metcalf, Robert Busch
Applicant	Cubist Pharmaceuticals, Inc.

Example 3: Synthesis of (2S,5R)-2-(l ,3,4-oxadiazol-2-yl)-7-oxo-l ,6- diazabicyclo[3.2.1 loctan-6-yl hydrogen sulfate (Compound 701 )

Step 1: Ι,Γ-Carbonyldiimidazole (5.8 g, 36.2 mmol) was added to a 0 °C solution of (2S,5R)- 6-(benzyloxy)-7-oxo-l,6-diazabicyclo[3.2.1]octane-2-carboxylic acid (5.0 g, 18.1 mmol) in dry THF (200 mL). The reaction mixture was allowed to warm to rt then was stirred at rt for 3 hrs. Formohydrazide (5.4 g, 90.5 mmol) was added in one portion, and the reaction mixture was stirred for additional 3 hrs. The mixture was then diluted with saturated sodium chloride and exatracted with EtOAc (3x). The combined organic layer was washed with saturated sodium chloride (2x), dried over Na₂S0₄, and concentrated to afford crude (25,5 ?)- 6-(benzyloxy)-N-formyl-7-oxo-l,6-diazabicyclo[3.2.1]octane-2-carbohydrazide (-11 g), which was directly used in the next step. ESI-MS (Ef , m/z): 319.1 [M+H]⁺.

Step 2: To a -10 °C solution of (25′,5«)-6-(benzyloxy)-N-formyl-7-oxo-l,6- diazabicyclo[3.2.1]octane-2-carbohydrazide (11 g) in dry DCM (200 mL) was added pyridine (28 mL), followed by dropwise addition of (CF₃S0₂)₂0 (28 mL). The reaction mixture was allowed to warm to rt and was stirred for 3 hrs. The reaction mixture was then cooled to -10 °C and quenched with sat. NaHCC>3. The organic layer was separated and the aqueous layer was extracted with EtOAc (3x). The combined organic layer was dried over Na₂S0₄, concentrated and purified by silica gel column chromatography (gradient elution 1 :3 to 2: 1 EtOAc/hexanes) to give (25,5/?)-6-(benzyloxy)-2-(l,3,4-oxadiazol-2-yl)-l ,6- diazabicyclo[3.2.1]octan-7 -one (4.6 g, 86% for two steps) as a slightly yellow solid. ESI-MS (EI⁺, m/z): 301.0 [M+H]⁺.

Step 3: To a solution of (25,5/?)-6-(benzyloxy)-2-(l,3,4-oxadiazol-2-yl)-l ,6- diazabicyclo[3.2.1]octan-7-one (4.6 g, 15.3 mmol) in THF (150 mL) was added 10% Pd/C (1 g). The mixture was stirred under H₂ atmosphere at rt for 3 hrs. The reaction mixture was then filtered and concentrated to afford (25,5/?)-6-hydroxy-2-(l,3,4-oxadiazol-2-yl)-l,6- diazabicyclo[3.2.1]octan-7-one (2.9 g, 91 %), which was used directly in the next step. ESI- MS (EI⁺, m/z): 211.1 [M+H]⁺. Step 4: To a solution of (25,5fl)-6-hydroxy-2-(l,3,4-oxadiazol-2-yl)-l,6- diazabicyclo[3.2.1]octan-7-one (2.9 g, 13.8 mmol) in dry pyridine (60 mL) was added SC>3- Py (11.0 g, 69.0 mmol). The reaction mixture was stirred at rt for 8 hrs and then concentrated under vacuum. The residue was re-dissolved in aqueous NaH₂PC>4 (1.5 M, 100 mL) then tetrabutylammonium hydrogensulphate (5.88 g, 17.3 mmol) was added. The mixture was stirred at rt for 20 minutes, then was extracted with EtOAc (4x). The combined organic layer was dried and concentrated and the residue was purified by silica gel column chromatography (gradient elution 10:1 to 2:1 DCM/acetone) to afford tetrabutylammonium (25,5/?)-2-(l ,3,4-oxadiazol-2-yl)-7-oxo-l,6-diazabicyclo[3.2.1]octan-6-yl sulfate (4.1 g, 97%) as a white solid. ESI-MS (EL, m/z): 289.0 [M-H]\ ^lH NMR (400 MHz, CDC1₃): δ 8.48 (s, 1H), 4.75 (d, / = 6.5 Hz, 1H), 4.40 (br s, 1H), 3.34-3.26 (m, 9H), 2.82 (d, / = 12.0 Hz, 1H), 2.37-2.25 (m, 3H), 2.06-1.98 (m, 1H), 1.71-1.65 (m, 8H), 1.49-1.42 (m, 8H), 1.01 (t, / = 7.5 Hz, 12H).

Step 5: Resin Exchange: Tetrabutylammonium (25, 5R)-2-(l, 3, 4-oxadiazol-2-yl)-7-oxo-l, 6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (4.1 g, 7.72 mmol) was dissolved in a minimum amount of HPLC grade water (~ 40 mL) and passed through a column of 80 g of DOWEX 50WX 8 Na⁺ resin (the resin was prewased with >4 L of HPLC grade water) and eluted with HPLC grade water to afford sodium (25,5fl)-2-(l,3,4-oxadiazol-2-yl)-7-oxo-l,6- diazabicyclo[3.2.1]octan-6-yl sulfate (2.2 g, 91 %) as a white solid after lyophilization. ESI- MS (EI⁺, m/z): 291.2 [M+H]⁺. ^lH NMR (300 MHz, D₂0) δ 8.92 (s, 1H), 4.84 (d, J = 6.1 Hz, 1H), 4.20 (br s, 1H), 3.25-3.16 (m, 1H), 2.92 (d, / = 12.3 Hz, 1H), 2.41-2.26 (m, 1H), 2.26- 2.11 (m, 2H), 2.04-1.89 (m, 1H).

PATENT

WO-2016157057

Wockhardt Ltd

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016157057&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

A compound of Formula (I), chemically known as sodium salt of 2S, 5R) mono-(2-[l,3,4]oxadiazol-2-yl-7-oxo-l,6-diazabicyclo[3.2.1 ]oct-6-yl)ester has antibacterial properties and is disclosed in PCT International Patent Application No. PCT/US2013/034562. The compound of Formula (I) is also generically disclosed in PCT International Patent Application No. PCT/IB2012/054296. The present invention discloses a process for preparation of a compound of Formula (I).

Formula (I)

Scheme 1.

(VI) Compound of Formula (I)

Example 1

Sodium salt of (25, 5R) sulfuric acid mono-(2-[l,3,4]oxadiazol-2-yl-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl) ester

Step I: Synthesis of (25,5R)-2-(iV’-formyl-hydrazinocarbonyl)-6-benzyloxy-7-oxo-l,6-diaza-bicyclo[3.2.1] octane (III):

To a turbid solution of sodium salt of (2<S’,5i?)-6-benzyloxy-7-oxo-l,6-diaza-bicyclo[3.2.1 ] octane-2-carboxylic acid (II, 20 g, 0.067 mol) (prepared according to process disclosed in PCT/IB2013/059264) in dimethylformamide (200 ml) was added EDC hydrochloride (19.44 g, 0.10 mol) followed by formyl hydrazide (4.02 g, 0.067 mol) and N-hydroxybenzotriazole (9 g, 0.67 mol) at about 25°C under stirring. Diisopropylethylamine (35.62 ml, 0.20 mol) was added to the reaction mixture and stirred at 25°C temperature for 18 hours. The reaction mixture was evaporated under vacuum to provide a residue. The residue was dissolved in ethyl acetate (500 ml) and washed with water (500 ml ^χ 2), followed by saturated aqueous sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulphate and evaporated under vacuum to provide a crude intermediate, which was purified by silica gel column chromatography to provide 11 g of the titled compound as solid in 52% yield.

Analysis:

Mass: 319.1 (M+l); for Molecular Formula of C15H18N4O4 and Molecular Weight of 318.34;

H¹ NMR (DMSO-d6): δ 9.93 (s, 1H), 9.87 (s, 1H), 8.01 (s, 1H), 7.36-7.46 (m, 5H), 4.91-4.97 (dd, 2H), 3.83-3.84 (br s, 1H), 3.70 (s, 1H), 3.15-3.18 (br s, 1H), 2.90-2.95 (m, 1H), 1.99-2.03(m, 1H), 1.86(br s, 1H), 1.73-1.75 (m, 1H), 1.66 (m, 1H).

Step II: Synthesis of (25,5R)-2-([l,3,4]-oxadiazol-2-yl)-6-benzyloxy-7-oxo-l,6-diaza-bicyclo[3.2.1] octane (IV):

To a clear solution of (2<S’,5i?)-2-(N’-formyl-hydrazinocarbonyl)-6-benzyloxy-7-oxo-l ,6-diaza-bicyclo[3.2.1 ] octane (III, 11 g, 0.0345 mol) in chloroform (120 ml) was added diisopropylethylamine (18.31 ml, 0.1035 mol) and p-tolylsulfonylchloride (9.83 g, 0.0517 mol). The solution was stirred at 60°C for 15 hours. Reaction mixture was cooled to room temperature and water (100 ml) was added. Organic layer was dried over anhydrous sodium sulphate and evaporated under vacuum to provide a crude residue, which was purified by silica gel column chromatography to provide 7 g of the titled compound as a solid in 68% yield.

Analysis:

Mass: 301.3 (M+l); for Molecular Formula of Ci₅Hi₆N₄0₃ and Molecular Weight of 300.32;

H¹ NMR (CDC1₃): δ 8.45 (s, 1H), 7.25-7.44 (m, 5H), 5.07-5.10 (dd, 1H), 4.92-4.95 (dd, 1H), 4.76-4.78 (br s, 1H), 3.37 (br s, 1H), 2.93-.95 (br s, 1H), 2.75-2.77 (m, 1H), 2.32-2.33 (m, 2H), 2.13-2.16 (m, 1H), 1.93-2.01 (m, 1H).

Step III: Synthesis of (25,5R)-2-([l,3,4]-oxadiazol-2-yl)-6-hydroxy-7-oxo-l,6-diaza-bicyclo[3.2.1] octane (V):

To a clear solution of (2<S’,5i?)-2-([l,3,4]-oxadiazol-2-yl)-6-benzyloxy-7-oxo-l,6-diaza-bicyclo[3.2.1 ] octane (IV, 7.0 g, 0.0233 mmol) in methanol (70 ml) was added 10% palladium on carbon (2.5 g). The suspension was stirred under atmospheric hydrogen pressure at a temperature 25° C for 2 hrs. The catalyst was filtered over a celite bed and the bed was washed with methanol (30 ml). The filtrate was concentrated under vacuum to provide an oily residue. The residue was triturated with cyclohexane (100 ml) to effect solid formation. The suspension was filtered under suction and the wet cake was washed with additional cyclohexane (50 ml). The soild was dried under vacuum to provide 4.5 g of the titled compound as a whitish solid in 92% yield, which was used for the next reaction immediately.

Analysis:

Mass: 211.2 (M+l); for Molecular Formula of C₈Hi₀N₄O₃ and Molecular Weight of 210.19; 1H NMR (DMSO-d6): δ 9.88 (br s, 1H), 9.29 (s, 1H), 4.65 (d, 1H ), 4.64 (br s, 1H), 2.94-2.97 (br d, 1H), 2.63-2.66 (d, 1H), 1.89-2.09 (m,3H), 1.82-1.86 (m, 1H).

Step IV: Synthesis of tetrabutylammonium salt of (25, 5R)-sulfuric acid mono-(2-[l,3,4]oxadiazol-2-yl-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl) ester (VI):

To a clear solution of (2<S’,5i?)-2-([l,3,4]-oxadiazol-2-yl)-6-hydroxy-7-oxo-l ,6-diaza-bicyclo[3.2.1 ] octane (V, 4.5 g, 0.0214 mol) in dichloromethane (50 ml) was added triethylamine (9 ml, 0.642 mol), followed by the addition of sulfur trioxide pyridine complex (6.83 g, 0.428 mol). The resulting reaction mixture was stirred for 2 hours. Tetrabutylammonium hydrogen sulfate (7.26 g,

0.0214 mol) was added to the reaction mixture and it was stirred for 1.5 hours. A solution of aqueous 0.5 N KH₂PO₄ (100 ml) was added to the reaction mixture. Layers were separated and the aqueous layer was washed with dichloromethane (125 ml). Combined organic layer was dried over Na2S04, and was evaporated under vacuum to yield crude foam, which was purified on silica gel column chromatography to give 7 g of the titled compound as white foam in 98% yield.

Analysis:

Mass: 289.1 (M-l); for Molecular Formula
and Molecular Weight of 517.26;

¹H NMR (DMSO-d6): δ 9.30 (s, 1H), 4.69 (d, 1H), 4.06 (br s, 1H ), 3.14-3.18 (m, 8H), 2.94-2.97 (br d, 1H), 2.67-2.70 (d, 1H), 1.98-2.05 (m,lH), 2.85-2.92 (m, 1H), 1.53-1.60 (m, 8H), 1.27-1.36 (m, 8H), 0.91-0.95 (m, 12H).

Step V: Sodium salt of (25, 5R)-sulfuric acid mono-(2-[l,3,4]oxadiazol-2-yl-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl) ester (I):

The compound sodium salt of (2S, 5i?)-sulfuric acid mono-(2-[l,3,4]oxadiazol-2-yl-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl) ester of Formula (I) was prepared by loading tetrabutylammonium salt of sulfuric acid mono-(2-[l,3,4]oxadiazol-2-yl-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl) ester (VI, 7 g) on a column packed with Amberlite IR 120 Na form of resin, and by eluting the column with methanol water mixture (9: 1). Fractions containing compound were collected and solvent was evaporated under vacuum below 40°C, to provide formula- 1 compound in 4 gm (62%) quantity as a white solid.

Analysis:

Mass: 289.3 (M-l) as free acid; for Molecular Formula
and Molecular Weight 290.26;

H¹ NMR (DMSO-d6): δ 9.29 (s, 1H), 4.70(d, 1H), 4.061 (d, 1H), 2.95 (d, 1H), 2.69 (d, 1H), 2.19 (m, 1H), 2.07 (m, 2H), 1.90 (m, 1H);

Purity as determined by HPLC: 89 86%;

//////////CB-618, phase 1

O=S(=O)(O)ON3[C@H]1C[N@]([C@@H](CC1)c2nnco2)C3=O

Ranitidine

Ranitidine, sold under the trade name Zantac among others, is a medication that decreases stomach acid production.^[1] It is commonly used in treatment of peptic ulcer disease, gastroesophageal reflux disease, and Zollinger–Ellison syndrome.^[1] There is also tentative evidence of benefit for hives.^[2] It can be taken by mouth, by injection into a muscle, or into a vein.^[1]

Common side effects include headaches and pain or burning if given by injection. Serious side effects may include liver problems, a slow heart rate, pneumonia, and the potential of masking stomach cancer.^[1] It is also linked to an increased risk ofClostridium difficile colitis.^[3] It is generally safe in pregnancy. Ranitidine is an H₂ histamine receptor antagonist that works by blocking histamine and thus decreasing the amount of acid released by cells of the stomach.^[1]

Ranitidine was discovered in 1976 at Glaxo Pharmaceuticals, now a part of GlaxoSmithKline.^[4][5] It is on the World Health Organization’s List of Essential Medicines, the most important medications needed in a basic health system.^[6] It is available as a generic medication.^[1] The wholesale price in the developing world is about 0.01 to 0.05 USD per pill.^[7] In the United States it is about 0.05 USD per dose.^[1]

Laboratory Synthesis Of Ranitidine

Synthesis Of Ranitidine

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Ranitidine Synthetic procedure/method of synthesis

The reaction of 5-dimethylaminomethyl-2-furanylmethanol (I) with 2-mercaptoethylamine (II) by means of aqueous HCl gives 2-[[(5-dimethylamino-methyl-2-furanyl)methylthio]ethaneamine (III), which is then condensed with N-methyl-1-methylthio-2-nitrotheneamine (IV) by heating at 120 C. Compound (IV) is obtained by reaction of 1,1-bis(methylthio)-2-nitroethene (V) with methylamine in refluxing ethanol

Ranitidine reference

1. Serradell, M.N.; Blancafort, P.; Casta馿r, J.; Hillier, K.; Ranitidine. Drugs Fut 1979, 4, 9, 663
2.  Price, B.J. et al. (Allen and Hanburys, Ltd.); US 4128658.
3. Price, B.J.; Bradshaw, J.; Clitherow, J.W. (Allen & Hansburys Ltd.); Aminoalkyl furan derivatives.. DE 2734070; FR 2360587; US 4128658 ,DE 2734070; FR 2360587; US 4128658.

PAPER

The biomass-derived platform chemical 5-(chloromethyl)furfural is converted into the blockbuster antiulcer drug ranitidine (Zantac) in four steps with an overall 68% isolated yield.

PROCESS

2. Experimental Procedures

5-[[(2-Acetamidoethyl)thio]methyl]furfural 14

Sodium hydride (95%) (103 mg, 4.08 mmol) was added to a solution of Nacetylcysteamine (0.4051 g, 3.40 mmol) in dry THF (20 mL) under argon. The resulting suspension was stirred at RT for 30 min and a solution of CMF 12 (0.4912 g, 3.40 mmol) in dry THF (10 mL) was added dropwise over a 10 min period. The resulting light yellow solution was allowed to stir overnight at RT. The solvent was evaporated and saturated brine (50 mL) was added. The mixture was extracted with CH2Cl2 (2 × 50 mL) and the organic layers were combined and washed with saturated brine (100 mL). The organic layer was dried over Na2SO4. Charcoal (100 mg) was added and the mixture was stirred for 20 min and filtered. The solvent was evaporated to give 14 as a yellow liquid (0.7042 g, 91 %). 1H NMR (CDCl3, 300 MHz) 9.58 (1H, s), 7.21 (1H, d, J = 3.6 Hz), 6.48 (1H, s, br), 5.95 (1H, d, J = 3.6 Hz), 3.79 (2H, s), 3.45 (2H, q, J = 6.3 Hz), 2.72 (2H, t, J = 6.6 Hz), 2.00 (3H, s); 13C NMR (CDCl3, 75 MHz) 23.1, 27.8, 31.7, 38.4, 110.7, 121.9, 152.2, 158.9, 170.7, 177.4; IR (neat) 3298, 3101, 1663, 1548, 1512, 1287, 1022, 772 cm-1; HRMS (ESI): calculated for C10H14O3NS: [M+H]+ 228.0694: found 228.0690.

5-[[(2-Acetamidoethyl)thio]methyl]-N,N-dimethyl-2-furanmethanamine 15

Me2NH (1.0 mL) was added to a solution of 14 (0.2105 g, 0.926 mmol) in dry methanol (20 mL) and the mixture was stirred at RT for 1 h. The resulting red solution was cooled to 0 °C and NaBH4 (98 %) (55 mg, 1.42 mmol) was added over a 5 min period. The mixture was allowed to come to RT and stirred for 30 min. The solvent was evaporated while keeping the bath temperature below 45 °C. The residue was dissolved in CH2Cl(50 mL) and filtered to remove inorganic impurities. The solvent was evaporated to give 15 (0.2145 g, 90 %) as a pale yellow oil. 1H NMR (CDCl3, 300 MHz) 6.42 (1H, s, br), 6.09 (1H, s), 3.67 (2H, s), 3.37 (2H, s), 3.26 (2H, q, J = 6.0 Hz), 2.62 (2H, t, J = 6.4 Hz) 2.21 (6H, s), 1.93 (3H, s); 13C NMR (CDCl3, 75 MHz) 23.5, 28.4, 31.9, 38.7, 45.4, 56.2, 108.4, 109.9, 151.4, 152.1, 170.5; IR (neat) 3273, 2944, 1656, 1545, 1291, 1019, 729 cm- 1 ; HRMS (ESI): calculated for C12H21O2N2S: [M+H]+ 257.1322: found 257.1323.

5-[[(2-aminoethyl)thio]methyl]-N,N-dimethyl-2-furanmethanamine 5

A solution of 15 (0.2473 g, 0.965 mmol) in freshly prepared 2N aq NaOH (10 mL) was heated at reflux for 2 h. The mixture was cooled to RT and extracted with CH2Cl2 (3×30 mL). The organic layers were combined and washed with saturated brine, dried over Na2SO4, and evaporated to give 5 (0.1934 g, 94 %) as a pale yellow oil. 1H NMR (CDCl3, 300 MHz) 6.02 (2H, s), 3.61 (2H, s), 3.33 (2H, s), 2.74 (2H, t, J = 6.3 Hz), 2.52 (2H, t, J = 6.6 Hz), 2.16 (6H, s); 13C NMR (CDCl3, 75 MHz) 28.2, 35.9, 40.9, 45.1, 55.9, 108.1, 109.5, 151.4, 152.1; IR (neat) 3359 cm-1, 2947, 2769, 1559, 1459, 1015, 797 cm-1; HRMS (ESI): calculated for C10H19ON2S: [M+H]+ 215.1212: found 215.1218.

N-[2-[[[5-[(dimethylamino)methyl]-2-furanyl]methyl]thio]ethyl]-N’-methyl-2-nitro- 1-Ethenediamine (Ranitidine) 1 The experimental procedure is modified from existing literature:2 A solution of 5 (0.1501 g, 0.700 mmol ) in distilled water (10 mL) was added dropwise over a period of 10 min to a suspension of 1-methylthio-1-methylamino-2-nitroethylene 7 (0.1041 g, 0.703 mmol) in distilled water (5 mL) with stirring. The resulting light yellow solution was placed in an oil bath at 55 °C and the mixture was stirred at that temperature overnight. Saturated brine (30 mL) was added and the mixture was extracted with CHCl3 (3×20 mL). The combined organic layer was dried over Na2SO4. Evaporation of the solvent gave 1 as a pale yellow oil (0.1935 g, 88 %). 1H NMR (CDCl3, 300 MHz, 56 oC) 10.23-10.15 (1H, br, NH), 6.57 (1H, s), 6.13 (2H, d, 6.0 Hz), 5.04 (1H, br, NH), 3.73 (2H, s), 3.41 (4H, s), 2.92 (2H, s), 2.76 (2H, t, 6.0 Hz), 2.24 (6H, s); 13C NMR (CDCl3, 75 MHz, 56 °C) 28.2, 30.6, 40.7, 44.6, 55.6, 97.9, 108.1, 109.1, 150.4, 152.1, 156.6; IR (neat) 3209, 2944, 2815, 2776, 1620, 1574, 1384, 1230, 1019, 761 cm-1; HRMS (ESI): calculated for C13H23O3N4S: [M+H]+ 315.1491: found 315.1497.

SEE NMR AT http://www.rsc.org/suppdata/gc/c1/c1gc15537g/c1gc15537g.pdf

PATENT

Patent EP0796256B1 – Process for preparing ranitidine – Google Patents

HPLC

An Improved HPLC Method for the Determination of Ranitidine …

CLIP

The paper was found in Green Chemistry,“Synthesis of ranitidine (Zantac) from cellulose-derived 5-(chloromethyl)furfural” by Mark Mescal et al, Green Chemistry,  2011,13, 3101-3102, DOI: 10.1039/c1gc15537g.  Once again, I am beating the press before they print so I supplied the Digital Object Identifier.  I am sure the sales for Ranitidine are quite large; who doesn’t get heartburn at one time or another.  I think it is very fortunate the author shows you can use a starting material that can be derived from just about any source of cellulose.  I find it interesting how renewable feedstocks can be utilized in industry and become part of important commodities, such as plastics, pharmaceuticals, etc.  This paper refers to another discussing where the starting material was derived from.  Starting material can be sugars, cellulose or raw cellulosic biomass and the reaction can produce yields of 80-90 %. M.Mascal and E. B. Nikitin, Angew. Chem., Int. Ed., 2008, 47, 7924;On with the show, though.  The original synthetic route was provided in the paper and I will provide it to you.

Furfural 1 was reduced to give the furfuryl alcohol 2.  The furfuryl alcohol is methylaminated to give 3, which is reacted with cysteamine in concentrated HCl to give 4.  This is condensed with 1-methylthio-1-methylamino-2-nitroethylene to give the final product.  The patent literature has the yield < 50 % for the aminomethylation and subsequent reaction with cysteamine, but recently, these steps have been reported to have higher conversions.

This new synthesis, apart from using a renewable feedstock as a starting material, has synthetic steps with an average yield of 91 %, and requires no chromatography.  Note that N-acetylcysteamine was used as opposed to cysteamine in the first step, in high yield.  A reductive amination with methylamine gives 8 again in high yield.  Treatment with KOH provides the free amine 9 and  the final step is the condensation with the nitroethylene used in the previous synthesis

https://developingtheprocess.wordpress.com/2014/06/22/got-heartburn-here-is-a-synthesis-to-satisfy-that-appetite-for-good-chemistry/

References

Ranitidine

Systematic (IUPAC) name
N-(2-[(5-[(dimethylamino)methyl]furan-2-yl)methylthio]ethyl)-N’-methyl-2-nitroethene-1,1-diamine
Clinical data
Pronunciation	/rəˈnɪtᵻdiːn/
Trade names	Zantac, others
AHFS/Drugs.com	Monograph
MedlinePlus	a601106
License data	US FDA: Ranitidine
Pregnancy category	AU: B1 US: B (No risk in non-human studies)
Routes of administration	Oral, IV
Legal status
Legal status	AU: S2 (Pharmacy only) UK: General sales list (GSL, OTC) US: OTC/RX
Pharmacokinetic data
Bioavailability	39 to 88%
Protein binding	15%
Metabolism	Hepatic: FMOs, including FMO3; other enzymes
Biological half-life	2–3 hours
Excretion	30–70% Renal
Identifiers
CAS Number	66357-35-5
ATC code	A02BA02 (WHO) A02BA07 (WHO) (ranitidine bismuth citrate)
PubChem	CID 3001055
IUPHAR/BPS	1234
DrugBank	DB00863
ChemSpider	4863
UNII	884KT10YB7
KEGG	D00422
ChEBI	CHEBI:8776
ChEMBL	CHEMBL1790041
Synonyms	Dimethyl [(5-{[(2-{[1-(methylamino)- 2-nitroethenyl]amino}ethyl)sulfanyl] methyl}furan-2-yl)methyl]amine
Chemical data
Formula	C13H22N4O3S
Molar mass	314.4 g/mol

//////////

WAY-315193

7-Fluoro-1-[(1S,2R)-1-(3-fluorophenyl)-2-hydroxy-3-(methylamino)propyl]-3,3-dimethylindolin-2-one Hydrochloride

Drugs that possess norepinephrine reuptake inhibition, either selectively or in combination with serotonin reuptake inhibition, have been used for multiple indications including major depressive disorder, attention deficit hyperactivity disorder, stress urinary incontinence, vasomotor symptoms, and pain disorders such as diabetic neuropathy and fibromyalgia.1 In the search for new candidates with improvements in both potency and selectivity, one of the lead compounds in the 1-(3-amino- 2-hydroxy-1-phenylpropyl)indolin-2-one series, WAY-315193 (1), was identified.2

Paper

Organic Process Research & Development 2009, 13, 880–887

Large-Scale Synthesis of a Selective Inhibitor of the Norepinephrine Transporter:
Mechanistic Aspects of Conversion of Indolinone Diol to Indolinone Aminoalcohol
and Process Implications
Asaf Alimardanov,* Alexander Gontcharov, Antonia Nikitenko, Anita W. Chan, Zhixian Ding, Mousumi Ghosh,
Mahmut Levent, Panolil Raveendranath,† Jianxin Ren, Maotang Zhou, Paige E. Mahaney,‡ Casey C. McComas,‡
Joseph Ashcroft, and John R. Potoski
Wyeth Research, 401 North Middletown Road, Pearl RiVer, New York 10965, U.S.A., and Wyeth Research, 500 Arcola Road,
CollegeVille, PennsylVania 19426, U.S.A.

TREATMENT OF GYNECOLOGICAL DISORDERS
WAY-315193 (Wyeth Pharmaceuticals)

Development of a scalable synthesis of WAY-315193 is described.
Use of LiHMDS as a base and Ti(O-i-Pr)4 as a Lewis acid was optimal for efficient and reproducible addition of indolinone anion to epoxyalcohol. Conversion of indolinone diol to indolinone aminoalcohol was achieved via monotosylationmethylamination.
The possibility of selective formation of the amidine side product, as well as its utilization for alternative selective preparation of the target aminoalcohol, was demonstrated.

The synthetic route used initially for preparation of 1 is shown in Scheme 1. The key step of the synthesis was the
Sharpless epoxidation of fluorocinnamic alcohol 3 which selectively introduced both relative and absolute configurations at the C-2 and C-3 positions. At the early stages of the project, allylic alcohol 3 was prepared in two steps from commercially available fluorocinnamic acid 2 by treatment with MeI in the presence of Cs2CO3 in acetone, followed by DIBAL reduction at -78 °C. The epoxide 4 was opened with the sodium salt of dimethylfluoroindolinone in DMF to afford the diol. The diol 6 was further elaborated into the final aminoalcohol hydrochloride 1 in 30-34% yield via tosylation with p-toluenesulfonyl chloride (TsCl) in pyridine, isolation of the intermediate monotosylate, treatment with MeNH2, and conversion to HCl salt. Dimethylfluoroindolinone was prepared by reduction and bis-methylation of 7-fluoroisatin by a process developed earlier as described in a prior publication.3

white solid (58% yield). Mp 209-212 °C.
[R]D25°)+10.7°.

1H NMR (D2O, 400 MHz) δ: 7.40-7.25 (m,3H), 7.16-6.97 (m, 4H), 5.47-5.25 (2H, broad m), 3.27-3.20
(2H, broad m), 2.76 (s, 3H), 1.37 (s, 3H), 1.24 (broad s, 3H).
ES+ MS, m/z 361 (MH+). Anal. Calc’d for C20H23ClF2N2O2:C, 60.53; H, 5.84; N, 7.06. Found: C, 60.43; H, 5.69; N, 6.84.
Sn content: <1 ppm. Enantiomeric purity: 99.1% ee. Chiral SFCanalysis conditions: column: Chiralcel OF 250 mm × 4.6 mm;mobile phase: 30% ethanol, 0.4% diethylamine in CO2; detection wavelength: 254 nm; 2 mL/min, 40 °C.

* Corresponding author. E-mail: alimara@wyeth.com.
† Deceased.
‡ Wyeth Research, Collegeville, PA.
(1) (a) For a review on norepinephrine reuptake inhibitors, see: Babu,R. P. K.; Maiti, S. N. Heterocycles 2006, 69, 539. (b) Krell, H. V.;Leuchter, A. F.; Cook, I. A.; Abrams, M. Psychosomatics 2005, 46,379. (c) Hajos, M.; Fleishaker, J. C.; Filipiak-Reisner, J. K.; Brown,M. T.; Wong, E. H. W. CNS Drug ReV. 2004, 10, 23. (d) McCormack,
P. L.; Keating, G. M. Drugs 2004, 64, 2567.
(2) Kim, C. Y.; Mahaney, P. E.; Trybulski, E. J.; Zhang, P.; Terefenko,E. A.; McComas, C. C.; Marella, M. A.; Coghlan, R. D.; Heffernan,G. D.; Cohn, S. T.; Vu, A. T.; Sabatucci, J. P.; Ye, F. Phenylaminopropanol
Derivatives and Methods of Their Use. U.S. Patent 7,517,899,2009.

(3) Wu, Y.; Wilk, B. K.; Ding, Z.; Shi, X.; Wu, C. C.; RaveendranathP.; Durutlic, H. Process for the Synthesis of Progesterone ReceptorModulators. U.S. Patent Publ. Appl. US 2007/027327, 2007.
(4) (a) Gao, Y.; Hanson, R. M.; Klunder, J. M.; Ko, S. Y.; Masamune,H.; Sharpless, K. B. J. Am. Chem. Soc. 1987, 109, 5765. (b) For a recent example of large-scale asymmetric epoxidation, see: Henegar,
K. E.; Cebula, M. Org. Process Res. DeV. 2007, 11, 354.

(5) (a) For indolinone deprotonation for epoxide opening, see: Proudfoot,J. R.; Regan, J. R.; Thomson, D. S.; Kuzmich, D.; Lee, T. W.;Hammach, A.; Ralph, M. S.; Zindell, R.; Bekkali, Y. Preparation ofPropanol and Propylamine Derivatives and Their Use as Glucocorticoid Ligands. WO 2004/063163, 2004. (b) Gillman, K.; Bocchino, D. M.
Preparation of Monosaccharides Prodrugs of Fluorooxindoles Useful in Treatment of Disorders Which are Responsive to the Opening of Potassium Channels. U.S. Patent Publ. Appl. US 2004/0152646, 2004.
(c) For amide deprotonation for epoxide opening, see: Albanese, D.; Landini, D.; Penso, M. Tetrahedron 1997, 53, 4787. (d) Chan, W. N.; Evans, J. M.; Hadley, M. S.; Herdon, H. J.; Jerman, J. C.; Morgan,H. K. A.; Stean, T. O.; Thompson, M.; Upton, N.; Vong, A. K. J. Med.Chem. 1996, 39, 4537.
(6) Bordwell, F. G.; Fried, H. E. J. Org. Chem. 1991, 56, 4218.
(7) (a) Smith, J. G. Synthesis 1984, 629. (b) Parker, R. E.; Isaacs, N. S.Chem. ReV. 1959, 59, 737.

//////////WAY-315193

Onions contain a powerful cancer fighting compound Onions contain a powerful cancer fighting compound We review the study” Anti-cancer effects found in natural compound derived from onions ” This study was done in regard to Ovarian Cancer, but should have potential in a variety of cancers. * Tsuboki, J. et al. Onionin A inhibits ovarian […]

via Onions contain a powerful cancer fighting compound — ClinicalNews.Org

BMS-741672

N-((1R,2S,5R)-5-(Isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide BMS-741672

N-((lR,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6- (trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-l-yl)cyclohexyl)acetamide

N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide;

CAS 1004757-96-3

PHASE 2, , Treatment of Type 2 Diabetes, Agents for Neuropathic Pain

Chemokine CCR2 (MCP-1 Receptor) Antagonists

• Originator Bristol-Myers Squibb
• Class Analgesics; Antihyperglycaemics
• Mechanism of Action CCR2 receptor antagonists

• Discontinued Diabetic neuropathies; Type 2 diabetes mellitus

Most Recent Events

• 10 Apr 2007 Preclinical trials in Inflammation in USA (unspecified route)

BMS-741672, 1 , is a highly selective CCR2 antagonist (IC₅₀ = 1.4 nM) featuring a complex array of four stereocenters. The key synthetic challenge was efficient assembly of the densely functionalized 1,2,4-triaminocyclohexane (TACH) core in a minimum number of linear steps.

N-((1R,2S,5R)-5-(Isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide BMS-741672

Mp 161.3 °C.

¹H NMR (400 MHz, CDCl₃) δ 9.50–9.20 (1H), 9.04 (s, 1H), 8.68 (s, 1H), 8.41 (d, J = 7.1 Hz, 1H), 7.87 (s, 1H), 5.04 (dt, J = 1.3, 7.3 Hz, 1H), 4.9 (m, 1H), 4.07 (dt, J = 3.7, 12.9 Hz, 1H), 3.53 (dt, J = 1.4, 9.9 Hz, 1H), 3.44–3.30 (m, 2H), 2.39 (dq, J = 13.6, 8.4 Hz, 1H), 2.26 (m, 1H), 2.21 (s, 3H), 2.17 (q, J = 2.9 Hz, 1H), 2.03–1.91 (m, 5H), 1.71–1.54 (m, 5H), 1.04 (s, br., 6H).

¹³C NMR (100 MHz, d₆-DMSO) δ 171.46, 169.49, 159.62, 156.92, 151.22, 129.28, 128.27 (q, ⁴J_CF = 3 Hz), 125.78 (q, ²J_CF = 32 Hz), 124.11 (q, ¹J_CF = 272 Hz), 121.57 (q, ³J_CF = 4 Hz), 114.33, 54.83, 53.54, 52.36, 47.34, 46.94, 43.13, 30.76, 30.24, 26.94, 26.38, 23.28, 20.87, 17.65 (br.), 16.73 (br.).

¹³C NMR (100 MHz, CDCl₃) δ 172.17. 170.73, 159.89, 156.91, 151.16, 128.68, 128.06 (q,⁴J_CF = 3.0 Hz), 127.25 (q, ²J_CF = 32 Hz), 123.98 (q, ¹J_CF = 272 Hz), 121.78 (q, ³J_CF = 4 Hz), 115.11, 54.89, 53.21, 52.40, 47.40, 46.98, 43.72, 30.84, 30.70, 29.96, 27.80, 23.55, 19.96, 17.70 (2C).

LCMS (ESI, pos.): 508 (16.8), 507 (66.2), 254 (5.0). HR-ESI(pos)-MS: calcd for C₂₅H₃₄F₃N₆O₂ 507.2690 [M + H]⁺, found 507.2694.

IR (KBr): ν = 3428 (m, br.), 2966, 1686 (s), 1635 (m), 1584 (s), 1540 (m), 1334 (m), 1307 (s), 1164 (m), 1121 (m), 870, 845.

[α]²⁰_D−187.9 (c 1.0, CHCl₃).

Anal. Calcd for C₂₅H₃₃F₃N₆O₂: C, 59.28; H, 6.57; F, 11.25; N, 16.59. Found: C, 59.21; H, 6.43; F, 11.07; N, 16.53.

PATENT

WO 2008014381

http://www.google.ch/patents/WO2008014381A2?cl=en&hl=de

EXAMPLE 1

N-((lR,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6- (trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-l-yl)cyclohexyl)acetamide

[00212] Example 1, Step 1: (IR, 2S, 5R)-tert-Butyl 2-benzyloxycarbonylamino- 7-oxo-6-aza-bicyclo[3.2.1]octane-6-carboxylate (89.6 g, 0.24 mol, see: P. H. Carter, et al. PCT application WO 2005/021500) was dissolved in ethyl acetate (1.5 L) and the resulting solution was washed with sat. NaHCCh (2 x 0.45 L) and sat. NaCl (I x 0.45 L). The solution was dried (Na₂SO₄) and then filtered directly into a 3 -necked 3 L round-bottom flask. The solution was purged with direct nitrogen injection before being charged with 10% Pd/C (13.65 g) under nitrogen atmosphere. The flask was evacuated and back-filled with hydrogen; this was repeated twice more. Hydrogen was bubbled through the solution for 30 min and then the reaction was stirred under 1 atm H₂ for 18 h. The flask was evacuated, back-filled with nitrogen, and charged with fresh catalyst (6 g of 10% Pd/C). Hydrogen was bubbled through the solution for 30 min and then the reaction was stirred under 1 atm H₂ for 18 h. The flask was evacuated and back-filled with nitrogen. The mixture was filtered through Celite; the filter pad was then washed with ethyl acetate. The filtrate (-1.6 L EtOAc volume) was diluted with acetonitrile (0.3 L) and charged sequentially with Z-N-Cbz- methionine (68 g, 0.24 mol), TBTU (77 g, 0.24 mol), and Ν,Ν-diisopropylethylamine (42 mL, 0.24 mol). The reaction was stirred at room temperature for 4 h, during which time it changed from a suspension to a clear solution. The reaction was quenched with the addition of sat. NH₄Cl (0.75 L) and water (0.15 L); the mixture was diluted further with EtOAc (0.75 L). The phases were mixed and separated and the organic phase was washed with sat. Na₂Cθ3 (2 x 0.9 L) and sat. NaCl (1 x 0.75 L). The solution was dried (Na₂SO₄), filtered, and concentrated in vacuo to give (IR,2S,5R)- tert-butyl 2-((5)-2-(benzyloxycarbonylamino)-4-

(methylthio)butanamido)-7-oxo-6-aza-bicyclo[3.2.1]octane-6-carboxylate as an oil, which was taken into the next step without further purification. LC/MS for primary peak: [M-Boc+H]⁺ = 406.3; [M+Naf = 528.3. ¹H-NMR (400 MHz, d₄-Me0H): δ 7.36 (m, 5H), 5.11 (s, 2H), 4.32 (m, IH), 4.2 (m, IH), 4.0 (m, IH), 2.5 – 2.7 (m, 3H), 2.25 (m, IH), 2.11 (s, 3H), 2.05 (m, 4H), 1.9 (m, IH), 1.7 (m, 2H), 1.54 (s, 9H). Also present are EtOAc [1.26 (t), 2.03 (s), 4.12 (q)] and N,N,N,N-tetramethylurea [2.83

(^S)].

[00213] Example 1, Step 2: A sample of (1^,25,5^)- tert-butyl 2-((5)-2- (benzyloxycarbonylamino)-4-(methylthio)butanamido)-7-oxo-6-aza- bicyclo[3.2. l]octane-6-carboxylate (0.24 mol assumed; see previous procedure) was dissolved in iodomethane (1,250 g) and stirred for 48 h at room temperature. The reaction was concentrated in vacuo. The residue was dissolved in dichloromethane and concentrated in vacuo. This was repeated twice more. The resultant sludge was dissolved in dichloromethane (0.4 L) and poured into a rapidly stirring solution of MTBE (4.0 L). The resultant yellow solids were collected via suction filtration and dried under high vacuum to afford the sulfonium salt (179 g). This material was taken into the next step without further purification. LC/MS for primary peak: [M- Me₂S+H]⁺ = 458.4; [M]⁺ = 520.4. ¹H-NMR (400 MHz, d₄-Me0H): δ 7.35 (m, 5H), 5.09 (s, 2H), 4.33 (m, IH), 4.28 (m, IH), 3.98 (m, IH), 3.3 – 3.45 (m, 2H), 2.97 (s, 3H), 2.94 (s, 3H), 2.78 (m, IH), 2.0 – 2.3 (m, 4H), 1.7 (m, 2H), 1.52 (s, 9H). Also present are MTBE [1.18 (s), 3.2 (s)] and traces of N,N,N,N-tetramethylurea [2.81 (s)]. [00214] Example 1, Step 3: All of the sulfonium salt from the previous step (0.24 mol assumed) was dissolved in DMSO (2.0 L). The resultant solution was stirred under nitrogen at room temperature and charged with cesium carbonate (216 g) portionwise. The suspension was stirred at room temperature for 3 h and then filtered to remove the solids. The solution was divided into -0.22 L portions and worked up as follows: the reaction mixture (-0.22 L) was diluted with ethyl acetate (1.5 L) and washed successively with water (3 x 0.5 L) and brine (1 x 0.3 L). The organic phase was dried (Na₂SO₄), filtered, and concentrated in vacuo. The desired (\R,2S,5R)- tert-bvXyl 2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-l-yl)-7-oxo-6- azabicyclo[3.2.1]octane-6-carboxylate (90.8 g, 83%) was obtained as a microcrystalline foam, free from tetramethyl urea impurity. LC/MS for primary peak: [M-Boc+H]⁺ = 358.4; [M+Na]⁺ = 480.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.35 (m, 5H), 5.12 (s, 2H), 4.35 (m, 2H), 4.2 (m, IH), 3.6 (m, IH), 3.3 (m, IH), 2.64 (m, IH), 2.28 – 2.42 (m, 2H), 2.15 (m, IH), 1.7 – 2.0 (m, 5H), 1.55 (s, 9H). If desired, this material can be isolated as a solid by dissolving in MTBE (1 volume), adding to heptane (3.3 volumes), and collecting the resultant precipitate.

[00215] Example 1, Step 4: A stirring solution of (\R,2S,5R)- tert-butyl 2-((S>3- (benzyloxycarbonylamino)-2-oxopyrrolidin-l-yl)-7-oxo-6-azabicyclo[3.2.1]octane-6- carboxylate (108 g, 0.236 mol) in THF (1 L) was charged with lithium hydroxide monohydrate (21.74 g, 0.519 mol). Water (0.3 L) was added slowly, such that the temperature did not exceed 20 ⁰C. The reaction was stirred at room temperature overnight and the volatiles were removed in vacuo. The pH was adjusted to -4 through the addition of IN HCl (450 mL) and NaH₂PO₄. The resultant white precipitates were collected by filtration and washed with water (2 x 1 L). The solid was dissolved in dichloromethane (1.5 L) and water (~ 1 L). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was dissolved in EtOAc (0.7 L) and the resultant solution was heated at reflux for 1 h. Solids separated after cooling to RT, and were collected via filtration. These solids were purified by recrystallization in isopropanol to afford the desired (\R,2S,5R)-2-((S)-3- (benzyloxycarbonylamino)-2-oxopyrrolidin-l-yl)-5-(tert- butoxycarbonylamino)cyclohexanecarboxylic acid as a white solid (104.5 g, 93% yield). LC/MS for primary peak: [M-tBu+H]⁺ = 420.2; [M-Boc+H]⁺ = 376.2; [M+H]⁺ = 476.2. ¹H-NMR (400 MHz, d₄-Me0H): δ 7.35 (m, 5H), 5.11 (s, 2H), 4.35 (m, 2H), 3.71 (m, IH), 3.45 – 3.6 (m, 2H), 2.99 (m, IH), 2.41 (m, IH), 2.15 (m, IH), 2.0 (m, 2H), 1.6 – 1.9 (m, 4H), 1.46 (s, 9H).

[00216] Example 1, Step 5: A 3 L round bottom flask was charged with (lR,25′,5R)-2-((5)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-l-yl)-5-(tert- butoxycarbonylamino)cyclohexanecarboxylic acid (75.5 g, 0.158 mol), EDOHCl (33.5 g, 0.175 mol), 1 -hydroxybenzotriazole (23.6 g, 0.175 mol), and dichloromethane (1 L). The reaction was stirred at room temperature for 2 h, during which time it changed from a white suspension to a clear solution. Ammonia (gas) was bubbled into the solution until the pH was strongly basic (paper) and the reaction was stirred for 10 min; this ammonia addition was repeated and the reaction was stirred for an additional 10 min. Water was added. The organic phase was washed with sat. NaHCθ3, NaH₂PO₄, and brine before being concentrated in vacuo. The residue was slurried with acetonitrile (0.5 L) and then concentrated in to give (lR,2S,5R)-2-((5)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-l-yl)-5-(tert- butoxycarbonylamino)cyclohexanecarboxamide as a white solid (75.9 g, -100%), which was used in the next step without further purification. LC/MS for primary peak: [M-Boc+H]⁺ = 375.3; [M+H]⁺ = 475.4; [M-tBu+H]⁺ = 419.3. ¹H-NMR (400 MHz, Cl₄-MeOH): δ 7.35 (m, 5H), 5.11 (s, 2H), 4.25 (m, 2H), 3.70 (m, IH), 3.6 (m, IH), 3.45 (m, IH), 2.91 (m, IH), 2.38 (m, IH), 2.12 (m, IH), 1.9 – 2.05 (m, 2H), 1.65 – 1.9 (m, 4H), 1.46 (s, 9H).

[00217] Example 1, Step 6: The reaction was run in three equal portions and combined for aqueous workup. A 5 L, 3-necked round bottom flask was charged with (lR,2S,5R)-2-((5)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-l-yl)-5-(tert- butoxycarbonylamino)cyclohexanecarboxamide (25.3 g, 53 mmol), acetonitrile (1.9 L), and 2.6 L of water/ice. The mixture was stirred and cooled to 0 ⁰C. Iodobenzene diacetate (25.77 g, 80 mmol) was added and the reaction was stirred for 2 h; another 0.5 eq of iodobenzene diacetate was added. The reaction was stirred for 9 h (reaction temp < 10 ⁰C). The mixture was charged with 8 eq N,N-diisopropylethylamine and 2 eq acetic anhydride. Over the next thirty minutes, 4 eq N,N-diisopropylethylamine and 2 eq acetic anhydride were added every ten minutes, until the reaction had proceeded to completion (HPLC). The acetonitrile was removed in vacuo; some solid separated from the residue, and this was collected by filtration. The remaining residue was extracted with dichloromethane (3 L, then 1 L). The organic phase was washed sequentially with water, sat. NaHCθ3, and brine. The collected solids were added to the organic phase, along with activated carbon (15 g). The mixture was stirred for 30 minutes at 40 ⁰C before being filtered and concentrated in vacuo. The residue was dissolved in EtOAc (1 L), and the resultant solution was stirred at 75 ⁰C for 1 h before being allowed to cool to room temperature. A solid separated and was collected by filtration. This solid was purified further by recrystallization: it was first dissolved in 0.5 L CH₂CI₂, then concentrated in vacuo, then re-crystallized from 1 L EtOAc; this was repeated three times. The solids obtained from the mother liquors of the above were recrystallized three times using the same method. The combined solids were recrystallized twice more from acetonitrile (0.7 L) to provide 66 g (84%) of tert-bυXyl (lR,3R,45)-3-acetamido-4-((5)-3-(benzyloxycarbonylamino)-2- oxopyrrolidin-l-yl)cyclohexylcarbamate (purity >99.5% by HPLC). LC/MS for primary peak: [M+H]⁺ = 489.4; [M-tBu+H]⁺ = 433.3. ¹H-NMR (400 MHz, d₄– MeOH): δ 7.3 – 7.4 (m, 5H), 5.11 (s, 2H), 4.35 (m, IH), 4.15 (m, IH), 4.04 (m, IH), 3.8 (m, IH), 3.6 (m, 2H), 2.44 (m, IH), 2.12 (m, IH), 1.87 – 2.05 (m, 4H), 1.87 (s, 3H), 1.55 – 1.7 (m, 2H), 1.46 (s, 9H). The stereochemical fidelity of the Hofmann rearrangement was confirmed through X-ray crystal structure analysis of this compound, as shown in Figure 1. [00218] Example 1, Step 7: A stirring solution of tert-butyl (\R,3R,4S)-3- acetamido-4-((5′)-3 -(benzyloxycarbonylamino)-2-oxopyrrolidin- 1 – yl)cyclohexylcarbamate (66 g, 0.135 mol) in dichloromethane (216 mL) was charged with trifluoroacetic acid (216 mL). The reaction was stirred for 2 h at room temperature and concentrated in vacuo. The residue was dissolved in methanol and the resultant solution was concentrated in vacuo; this was repeated once. Benzyl («S)-l-((l«S,2R,4R)-2-acetamido-4-aminocyclohexyl)-2-oxopyrrolidin-3-ylcarbamate was obtained as an oil and used directly in Step 8 below. LC/MS found [M + H]⁺ = 389.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.3 – 7.4 (m, 5H), 5.12 (s, 2H), 4.41 (br. s, IH), 4.15 (m, IH), 4.00 (t, J= 9.3 Hz, IH), 3.81 (t, J= 9.1 Hz, IH), 3.65 (q, J= 8.4 Hz, IH), 3.3 – 3.4 (m, IH), 2.45 (m, IH), 1.95 – 2.24 (m, 5H), 2.00 (s, 3H), 1.6 – 1.8 (m, 2H). [00219] Example 1, Step 8: A stirring solution of benzyl (S)- 1-(( \S,2R,4R)-2- acetamido-4-aminocyclohexyl)-2-oxopyrrolidin-3-ylcarbamate (-0.135 mol) in methanol (675 mL) was charged sequentially with acetone (37.8 g, 4 eq), sodium acetate (33.2 g, 3 eq), and sodium cyanoborohydride (16.9 g, 2 eq). The mixture was stirred at room temperature for 6 h and filtered. The filtrate was dissolved in dichloromethane (1 L); this solution was washed with IN NaOH (1 L). The solids collected in the filtration were dissolved in IN NaOH (IL) at 0 ⁰C and then extracted with dichloromethane (1 L). The organic extracts were combined and extracted with aqueous HCl (200 mL IN HCl + 800 mL water). The aqueous phase was basified with sat. NaHCO₃ (500 mL) and then IN NaOH (100 mL) until pH 11. The aqueous phase was extracted with dichloromethane (2 L). The organic extracts were combined, dried (Na₂SO₄), filtered, and concentrated in vacuo to give benzyl (S)-I- ((lS,2R,4R)-2-acetamido-4-(isopropylamino)cyclohexyl)-2-oxopyrrolidin-3- ylcarbamate as an oil. LC/MS found [M + H]⁺ = 431.45. ¹H-NMR (400 MHz, d₄– MeOH): δ 7.3 – 7.4 (m, 5H), 5.12 (s, 2H), 4.31 (m, IH), 4.24 (t, J= 9.4 Hz, IH), 4.11 (m, IH), 3.61 (t, J= 9.1 Hz, IH), 3.52 (q, J= 8.6 Hz, IH), 3.04 (br. s, IH), 2.96 (sep, J= 6.3 Hz, IH), 2.40 (m, IH), 2.15 (m, IH), 1.92 (s, 3H), 1.7 – 1.9 (m, 5H), 1.65 (m, IH), 1.12 (app. dd, J= 6.3, 1.1 Hz, 6H).

[00220] Example 1, Step 9 (See Alternative Step 9, below): A stirring solution of benzyl (S)-I -((lS’,2R,4R)-2-acetamido-4-(isopropylamino)cyclohexyl)-2- oxopyrrolidin-3-ylcarbamate (-115 mmol) in dichloromethane (600 mL) was cooled to 0 ⁰C and charged sequentially with formaldehyde (18.6 g, 37 wt% solution), triethylamine (23 mL), and sodium triacetoxyborohydride (28.7 g). The mixture was stirred at room temperature for 30 minutes and diluted with dichloromethane (up to 1.2 L). This solution was washed thrice with 500 mL sat. NaHCθ3 + NaOH (sat. NaHCO₃, pH to 11 w/ IN NaOH). The organic layer was extracted with aq. HCl (200 mL IN HCl + 600 mL water). The aqueous phase was basified with sat. NaHCO₃ (500 mL) and then IN NaOH (100 mL) until pH 11. The aqueous phase was extracted with dichloromethane (1.2 L). The organic extracts were combined, dried (Na2SO4), filtered, and concentrated in vacuo to give benzyl {S)-\-{{\S,2R,AR)-2- acetamido-4-(isopropyl(methyl)amino)cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate as an oil, which was used directly in Step 10 below. LC/MS found [M + H]⁺ = 445.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.3 – 7.4 (m, 5H), 5.12 (s, 2H), 4.33 (br s, IH), 4.25 (t, J= 9.2 Hz, IH), 4.11 (br s, IH), 3.5 – 3.6 (m, 2H), 2.77 (v br s, 2 H), 2.41 (m, IH), 2.26 (s, 3H), 2.0 – 2.1 (m, 2H), 1.92 (s, 3H), 1.7 – 1.9 (m, 5H), 1.10 (app. dd, J = 17, 6.4 Hz, 6H). [00221] Example 1, Step 10: To a solution of benzyl (S)- 1-(( 15″,2R,4R)-2- acetamido-4-(isopropyl(methyl)amino)-cyclohexyl)-2-oxopyrrolidin-3 -ylcarbamate (-0.115 mol) in methanol (600 mL) was added 10% Pd/C (6 g of 50% wet catalyst). The flask was evacuated and back-filled with hydrogen. The mixture was stirred under 1 atm H₂ for 2 h and the catalyst was removed by filtration through Celite. The filtrate was concentrated in vacuo to provide N-((li?,25,5i?)-2-((S)-3-amino-2- oxopyrrolidin-l-yl)-5-(isopropyl(methyl)amino)cyclohexyl)acetamide as an oil, which was taken on to the next step without further purification. LC/MS found [M + H]⁺ = 311.47. ¹H-NMR (400 MHz, (I₄-MeOH): δ 4.39 (br s, IH), 4.00 (m, IH), 3.3 –

3.5 (m, 4H), 2.73 (m, IH), 2.38 (m, IH), 2.25 (s, 3H), 2.0 – 2.2 (m, 3H), 1.94 (s, 3H),

1.6 – 1.75 (m, 4H), 1.07 (app. dd, J= 21, 6.4 Hz, 6H). [00222] Example 1, Step 11: To a solution of N-((lR,25′,5R)-2-((S)-3-amino-2- oxopyrrolidin-l-yl)-5-(isopropyl(methyl)amino)cyclohexyl)acetamide (~35 g, 0.115 mol) in isopropanol (600 mL) was added 4-chloro-6-(trifluoromethyl)quinazoline (32 g, 0.138 mol, 1.2 eq, see: P.H. Carter et al, PCT application WO 2005/021500). The mixture was stirred at room temperature overnight before being charged with triethylamine (46 g, 0.46 mol, 4 eq). The mixture was stirred at 60 ⁰C for 10 h. The solvent was removed under reduced pressure to give an oil. Azeotropic distillation with isopropanol was performed twice. The residue was dissolved in dichloromethane (600 mL) and extracted with water (250 mL, containing 4 eq acetic acid). Dichloromethane (600 mL) was added to the combined aqueous washes, and the mixture was cooled to 0 ⁰C. Aqueous NaOH (50% by weight) was added with stirring until the pH reached 11. The water layer was extracted with dichloromethane twice (2 x 600 mL). The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated in vacuo to give the amorphous free base of the title compound (99% purity by HPLC). LC/MS found [M+H]⁺ = 507.3. ¹H-NMR (400 MHz, U₄– MeOH): δ 8.82 (s, IH), 8.59 (s, IH), 8.05 (dd, J= 8.8, 1.8 Hz, IH), 7.9 (d, J= 8.7 Hz, IH), 5.28 (t, J= 8.6 Hz, IH), 4.58 (br s, IH), 4.06 (m, IH), 3.52 – 3.68 (m, 2H), 3.43 (m, IH), 2.76 (br s, IH), 2.55 (m, IH), 2.28 (s, 3H), 2.1 – 2.3 (m, 3H), 2.0 (s, 3H), 2.0 (m, IH), 1.65 – 1.8 (m, 3H), 1.09 (app. dd, J= 24, 6.4 Hz, 6 H).

Example 1, Alternative Step 9

[00223] Example 1, Alternative step 9a¹: To a hydrogenator were charged ethyl (7R,SS)-S-((S)- l-phenyl-ethylamino)-l,4-dioxa-spiro[4.5]decane-7-carboxylate A- toluenesulfonate salt I A (1417 g, 2.8 moles, c.f : WO2004098516, prepared analogous to US Pat.6,835,841), ethanol (200 proof, 11.4 L), and 10% Pd/C catalyst (50% wet, 284 g). The mixture was inerted with nitrogen, then pressurized with hydrogen gas (45 psig) and agitated vigorously at approx. 40 ⁰C until starting material was consumed (HPLC). The suspension was cooled, purged with nitrogen gas and the catalyst was removed by filtration while inerted. The spent catalyst was washed with ethanol (4.3 L). The filtrate and washings were combined and concentrated under vacuum to a volume of 2-3 L while maintaining the batch between 40°-60 ⁰C. Isopropyl acetate (5 L) was charged and the mixture was concentrated to a volume of ~2 L until most ethanol was removed (<0.5%) and residual moisture content was <l,000 ppm. Batch volume was adjusted to -7.5 L by the addition of isopropyl acetate. The mixture was heated to 80 ⁰C until clear, then cooled 65°-70 ⁰C. Seed crystals of 1 (5 g) were added and the batch was cooled to 50⁰C over 2 hours, then further cooled to 20 ⁰C over 4 hours and held for ~10 hours. The resulting slurry was filtered and the cake was washed with isopropyl acetate (2 L). The product was dried under vaccum at -35 ⁰C until volatiles were recduced below -1% (LOD). Ethyl (7R,85′)-8-amino-l,4-dioxa-spiro[4.5]decane-7-carboxylate 4-toluenesulfonate salt 1 was obtained as a white, crystalline solid (936 g, 83% yield; HPLC purity: 99.8%). ¹H-NMR: (300MHz, CDCl₃) 8.14-7.89 (brs, 3H), 7.75 (d, J 9.0Hz, 2H), 7.15 (d, J 8.0Hz, 2H), 4.22-4.04 (m, 2H), 4.01-3.77 (m, 4H), 3.55-3.43 (m, IH,), 3.20-3.13 (m, IH), 2.40-2.27 (m, 4H), 2.21-1.94 (m, 2H), 1.81-1.51 (m, 3H), 1.23 (t, J 7.0Hz, 3H); HPLC: Waters Xterra MS C18 4.6 mm x 150 mm Ld., 3.5μm particle size, 0.05% NH40H (5% ACN, 95% H₂O, solvent A), to 0.05% NH₄OH (95% ACN, 5% H₂O, solvent B), 5% B to 20% B in 10 minutes, changed to 95% B in 25 minutes, and then changed to 5% B in 1 minute; 11.1 minutes (aminoester 1).

Example 1, Alternative Step 9a”: Aminoester 1 (63g, 0.16M, leq.; the product of reductive deprotection of a known compound – (See e.g. R. J. Cherney, WO 2004/098516 and G. V. Delucca & S. S. Ko, WO 2004/110993) was placed in a round bottom flask and MeCN (50OmL) was added. EDAC (33.1g, 0.17M, l. leq), HOBt-H₂O (21.2g, 0.16M, l.Oeq) and N-Cbz-Z-methionine (46.7g, 0.17M, 1.05eq) were then added followed by TEA (48.OmL, 0.35M, 2.2eq). An exotherm to 38 ⁰C was observed. The reaction mass was left to stir at RT. After 30mins, HPLC indicated complete conversion. The reaction mass was diluted with EtOAc (2.5L) and washed with KHCO₃ (4x500mL, 20wt% aq. solution) and brine (50OmL). The organic phase was separated, dried over MgSO₄ and concentrated. The residue was dissolved in TBME and reconcentrated to give ethyl (7R,85)-8- {(2S)-2-benzyloxycarbonylamino- 4-methylsulfanyl-butyr-yl-amino}-l,4-dioxa-spiro[4.5]decane-7-carboxylate 2 as a sticky semi-solid (76.2g, 98% yield, 93AP purity). ¹H-NMR: (300MHz, CDCl₃) δ 7.36-7.30 (m, 5H), 7.03 (d, J9.0Hz, IH), 5.66 (d, J 8.0Hz, IH), 5.10 (s, 2H), 4.35- 4.25 (m, 2H), 4.19-4.04 (m, 2H,), 3.98-3.86 (m, 4H), 2.87-2.80 (m, IH), 2.55-2.45 (m, 2H), 2.18 (dd, J 14.0Hz, 7.0Hz, IH), 2.08 (s, 3H), 2.05-1.67 (m, 6H), 1.26 (t, J 7.0Hz, 3H). HPLC: YMC-Pack Pro C18 5μm 4.6 x 150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% lOmin gradient. lO.Olmin (Compound 2, 93.1 AP). HRMS: m/z 495.2166 [CaIc: C₂₄H₃₅N₂O₇S 495.2165].

2 3 [00224] Example 1, Alternative Step 9b: Methionine amide 2 (75.Og, 0.15M) was dissolved in MeI (225mL, 3mL/g) – some off gassing was noted but no exotherm. The reaction mass was left to stir in the dark for 16.5h. After this time a thick light yellow precipitate had formed. The flask was then evacuated to 200mmHg and some of the MeI removed. The remaining material was slurried in TBME (50OmL), after a 30min stir-out the slurry was filtered, the cake washed with TBME (50OmL). NMR analysis of this material indicated a small amount of MeI remaining. The cake was re-slurried in TBME (50OmL), filtered, washed with TBME (50OmL) and dried under vacuum to give [(35)-3-benzyloxycarbonylamino-3-{(7R,85′)-7- ethoxycarbonyl-l,4-di-oxa-spiro[4.5]dec-8-ylcarbamoyl}-propyl]-dimethylsulfonium iodide 3 as a free flowing off-white solid (93.5g, 97%, 99 area% purity). ¹H-NMR: (300MHz, CDCl₃) δ 7.75 (d, J 9.0Hz, IH), 7.38-7.27 (m, 5H), 6.40 (d, J 7.0Hz, IH), 5.10 (s, 2H), 4.76-4.65 (m, IH), 4.48-4.39 (m, IH), 4.14-3.85 (m, 6H), 3.84-7.73 (m, IH), 3.68-3.55 (m, IH), 3.21 (s, 3H), 3.12 (s, 3H), 2.90-2.83 (s, IH), 2.52-1.55 (m, 8H), 1.24 (t, J7.0Hz, 3H). HPLC: YMC-Pack Pro C18 5μm 4.6 x 150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% lOmin gradient. 2.45min (I-), 8.14min (Compound 3, 43.6AP, I^“ 54.6AP). HRMS: m/z 509.2341 [CaIc: C₂₅H₃₇N₂O₇S 509.2321].

[00225] Example 1, Alternative Step 9c: Cs₂CO₃ (61.5g, 0.19M, 1.5eq) was placed in an round bottom flask and anhydrous DMSO (2.4L) was added. Sulfonium salt 3 (80.Og, 0.13M, 1.Oeq) was then added portionwise. Once the addition was complete the reaction mass was left to stir in the dark for 2Oh. The reaction mass was then split in half and each half worked up separately: the reaction mass was diluted with EtOAc (2.0L) and washed with brine (2L), the organic phase was washed with brine (50OmL). The combined aq. layers were then washed EtOAc (50OmL). The combined organic phases were then washed with brine (3x750mL). The second half of the reaction mass was treated in an identical manner and the combined organics dried over MgSO₄ and concentrated to give ethyl (7R,8S)-8-{(3S>3- Benzyloxycarbonylamino-2-oxo-pyrrolidin-l-yl}-l,4-dioxa-spiro[4.5]decane-7- carboxylate 4 as a light colored oil (56.5g, 0.13M, -100 area-% purity) pure by NMR analysis. ¹H-NMR: (300MHz, CDCl₃) δ 7.38-7.30 (m, 5H), 5.37 (br d, J4.0Hz, IH), 5.11 (s, 2H), 4.27-4.18 (m, IH), 4.17-3.82 (m, 8H), 3.32 (td, J 10.0Hz, 60.0Hz, IH), 3.23 (q, J5.0Hz, IH), 2.63-2.57 (m, IH), 2.42-2.25 (m, 2H), 1.94-1.68 (m, 5H), 1.25 (t, J 7.0Hz, 3H). HPLC: YMC-Pack Pro Cl 8 5μm 4.6 x 150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% lOmin gradient. 8.99min (Compound 5, produced on column, 4.2AP), 9.48 (Compound 4, 74.3AP). HRMS: m/z 447.2127 [CaIc: C₂₃H₃₁N₂O₇ 447.2131].

4 5

[00226] Example 1, Alternative Step 9d: Pyrrolidinone 4 (50.Og, 0.1 IM) was dissolved in acetone (50OmL) and IN HCl (50OmL) was added. The reaction mass was then heated to 65°C. After 20mins HPLC indicated complete reaction. The reaction mass was allowed to cool to RT and the acetone was removed on a rotary evaporator. During this distillation the product precipitated from solution as a white solid. This was isolated by filtration and the cake washed with water. The cake was then dried azeotropically with toluene (3x3OOmL) to give ethyl (\R,2S)-2-((3S)-3- Benzyloxycarbonylamino-2-oxo-pyrrolidin-l-yl)-5-oxo-cyclohexanecarboxylate 5 as a white solid (39.8g, 88%, 97 area-% purity). ¹H-NMR: (300MHz, CDCl₃) δ 7.37- 7.32 (m, 5H), 6.65 (br d, J4.0Hz, IH), 5.12 (s, 2H), 4.54-4.47 (m, IH), 4.34-4.26 (m, IH), 4.18 (dq, J 11.0Hz, 7.0Hz, IH), 4.09 (dq, J 11.0Hz, , 7.0Hz, IH), 3.36-3.20 (m, 3H), 2.70-2.35 (m, 6H), 2.05-1.96 (m, IH), 1.81 (quin., J l l.OHz, IH), 1.24 (t, J 7.0Hz, 3H). HPLC: YMC-Pack Pro C18 5μm 4.6 x 150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% lOmin gradient. 8.95min (Compound 5). HRMS: m/z 403.1864 [CaIc: C₂iH₂₇N₂O₆403.1869].

[00227] Example 1, Alternative Step 9e: Cyclohexanone 5 (22.5g, 0.06M, leq), DMSO (3OmL) and Ti(O-ZPr)₄ (33.7mL, 0.1 IM, 2.04eq) were placed in a round bottom flask. N-isopropyl-N-methylamine (11.6mL, 0.1 IM, 2.0eq) was then added in one portion. The mixture was left to stir for 30mins at room temperature before being cooled to <3°C in ice/water. MeOH (3OmL) was then added followed by the portionwise addition OfNaBH₄ (4.33g, 0.1 IM, 2.04eq) – temperature kept <8°C. 30mins after the addition was completed the reaction mass was diluted with methylene chloride (30OmL) and then NaOH (IN, 4OmL). The resulting slurry was filtered through Celite, and the cake washed with methylene chloride (10OmL). The resulting liquor was concentrated under reduced pressure and the residue dissolved in EtOAc (50OmL). This solution was extracted with IN HCl (2x400mL), the combined aqueous layers were then basified with Na₂CO3. Extraction with EtOAc (4x250mL) provided a clear and colorless organic phase which was dried over Na₂SO₄ and concentrated to give a white powder (24.6g, 96%, 7: 1 d.r.). This material was then slurried overnight in hexane (67OmL). The solid was isolated by filtration and dried under reduced pressure to give ethyl (lR,25′,5R)-2-((3S)-3-benzyloxycarbonylamino- 2-oxo-pyrrolidin-l-yl)-5-(isopropyl-methyl-amino)-cyclohexanecarboxylate 6 as a while solid (20.9g, 81%, 24: 1 d.r.). ¹H-NMR: (300MHz, CDCl₃) δ 7.37-7.28 (m, 5H), 5.55 (d, J4.5, IH), 5.10 (s, 2H), 4.42 (q, J4.5, IH), 4.23-4.12 (m, IH), 4.08 (dq, J 10.5, 7.0, IH), 4.02 (dq, J 10.5, 7.0, IH), 3.84 (t, J9.0, IH), 3.46-3.36 (m, IH), 3.04 (septet, J6.5, IH), 2.86-2.80 (m, IH), 2.63-2.48 (m, 2H), 2.17 (s, 3H, Me), 2.10-1.63 (m, 7H), 1.22 (t, J 7.0, 3H), 1.00 (d, J 6.5, 3H), 0.97 (d, J 6.5, 3H). HPLC: YMC- Pack Pro C18 5μm 4.6 x 150 mm, 0.01M NH₄OAc (MeOH:water 20:80) to 0.01M NH₄OAc (MeOH:water:MeCN 20:5:75) 10 to 100% 15min gradient. 8.23 (Compound 6), 8.88 (5-e/«-Compound 6). HRMS: 460.2798 [CaIc: C₂₅H₃₈N₃O₅ 460.2811].

[00228] Example 1, Alternative Step 9f: The aminoester 6 (9.76 g, 2.12 mmol) was dissolved in 2N HCl (80 mL), then heated to -55 ⁰C under inert atmosphere. The reaction was stirred for 20 h, then cooled to room temperature. The reaction solution was washed twice with toluene (25 mL portions), neutralized to pH 6 – 7 by the addition of KOH pellets, then extracted eight times with methylene chloride (100 mL portions). The combined extracts were dried (Na₂SO₄), filtered, and concentrated under reduced pressure to 50 mL total volume. The concentrated solution was then slowly added to methyl tert-butyl ether (300 mL) over 15 min in an addition funnel with vigorous stirring. The resulting white slurry was stirred at ambient temperature for Ih, then cooled to 0 ⁰C and stirred for Ih. The product was filtered, and washed twice with methyl tert-butyl ether (25 mL portions). Water from the wet cake was removed by azeotropic distillation with acetonitrile (300 mL). The product was dried under reduced pressure to provide (li?,25^r,5R)-2-((35′)-3-Benzyloxycarbonylamino-2- oxo-pyrrolidin-l-yl)-5-(isopropyl-methyl-amino)-cyclohexanecarboxylic acid 7, (7.69 g, 84% yield) as a white foam. ¹H-NMR: (400 MHz, 50⁰C, CDCl₃) δ 7.44-7.32 (m, 5H), 6.10 (broad s, IH), 5.19 (app s, 2H), 4.42 (dd, J= 15.6, 7.8 Hz, IH), 4.29-4.23 (m, IH), 3.68-3.60 (m, 2H), 3.33-3.27 (m, 2H), 3.20 (broad s, IH), 2.99 (broad s, IH), 2.51 (s, 3H), 2.49-2.45 (m, 3H), 2.33-2.31 (m, IH), 2.00 (ddd, J= 9.0, 8.6, 3.9 IH), 1.95-1.78 (m, 2H), 1.36-1.21 (m, 6H). LCMS: m/z 432.20 [CaIc: C₂₃H₃₄N₃O₅ 432.25].

NHCbz

[00229] Example 1, Alternative Step 9g: Amino acid 7 (6.3g, 14.7mmol, l.Oeq) was dissolved in THF (8OmL) under N₂ and NaH (584mg, 14.7mmol, l.Oeq, 60wt% dispersion in mineral oil) was added portionwise. When the addition was complete, and the evolution of gas had ceased, the reaction mass was concentrated under reduced pressure and the resulting solid azeotroped with toluene (50 mL) to give a white solid (KF 0.59wt%). This solid was slurried in toluene (100 mL) under N₂and heated to 90⁰C. DPPA (3.32 mL, 15.3 mmol, 1.05 eq) was added dropwise over ~2min. After ~5min all the solids had dissolved, after lOmins precipitation of a white solid was observed. After 30mins HPLC analysis indicated complete reaction. The reaction mass was allowed to cool to RT before being filtered, the cake was washed with toluene. The liquors where then slowly added into ACOH/AC₂O (80/20, 168mL) solution at 90⁰C. After 45mins HPLC still indicated some isocyanate. At 1.15h , the reaction mass was cooled to RT and diluted with toluene (10OmL) and water (10OmL). The organic layer was removed and the toluene washed with IN HCl

(10OmL). The combined aq. phases were then basified with K₂Cθ3(s) and brought to pH 12 with NaOH (10N), keeping the temperature below 20⁰C. The aq layer was then extracted with methylene chloride (4xl50mL), the combined organic layers dried over K₂CO₃ and concentrated to give benzyl (S)-l-((lS,2R,4R)-2-acetamido-4- (isopropyl(methyl)amino)cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate 8 as a white foam (4.5g, 70%, 94AP purity). The ¹H-NMR was identical to material obtained from the route described above (Example 1, Step 9). HPLC: YMC-Pack Pro Cl 8 5μm 4.6 x 150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% lOmin gradient. 7.20min (Compound 8), 7.85min (urea dimer). HRMS: 445.2809 [CaIc: C₂₄H₃₇N₄O₄ 445.2815].

Alternative Preparation of Example 1

2 3

[00230] Example 1, Alternative Preparation, Step 1: Ethyl (7R,85)-8-amino- l,4-dioxa-spiro[4.5]decane-7-carboxylate 4-toluenesulfonate salt 1 (450. Ig), was combined with l-ethyl-3-(3-dimethyl-amino-propyl)carbo-diimide hydrochloride (236.3g), 1-hydroxy benzotriazole hydrate (171.9g), N-carbobenzyloxy-Z -methionine (333.4g) and acetonitrile (3.1 L). To the stirred mixture was added triethylamine (249.5g) below 30 ⁰C. Upon reaction completion (HPLC), the mixture was diluted with ethyl acetate (8.2 L) and washed with aqueous 25% potassium bicarbonate solution (2×4.5 L) followed by water (4.5 L). The organic phase was separated and concentrated under reduced pressure to obtain a solution of ethyl (7R,85)-8-((5)-2- benzyloxycarbonylamino-4-methylsulfanyl-butyrylamino)-l,4-dioxa- spiro[4.5]decane-7-carboxylate 2 (1.4 L). Methyl iodide (2.39 kg) was added, the vessel was shielded from light and the mixture was held under slow agitation for approx. 24 h. To the thick yellow precipitate was added methyl tert-butyl ether (2.7 L) and the mixture was held for approx. 1 h. The product was isolated by filtration and the cake was washed with methyl tert-butyl ether (2×1.4 L), then dried under vacuum, yielding [(5)-3-benzyloxy-carbonylamino-3-((7R,8«S’)-7-ethoxycarbonyl-l,4- dioxa-spiro[4.5]dec-8-ylcarbamoyl)-propyl]-dimethylsulfonium iodide 3 (671.4 g, -94% yield) as an off-white solid (HPLC purity 99.9%).

[00231] Example 1, Alternative Preparation, Step 2: Sulfonium salt 3 (619.4 g), and cesium carbonate (416.8 g) and anhydrous dimethyl sulfoxide (6.2 L) were combined in a reactor equipped with a scrubber to neutralize volatile sulfides.

Vigorous agitation was maintained until complete conversion was obtained (HPLC). Ethyl acetate (12.4 L) was added, followed by 20 % brine (3 L). The organic phase was separated, washed twice with brine (2×3 L) and evaporated to obtain a solution of ethyl (7R,8«S)-8-((«S)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-l-yl)-l,4-dioxa- spiro[4.5]decane-7-carboxylate 4 in ethyl acetate (~0.8 L). Acetone (2.55 L) was added, followed by aqueous 0.5 M hydrochloric acid solution (2.3 L). With good mixing, the solution was heated to 50 to 60 ⁰C until conversion of 4 to ethyl (IR,2S)- 2-((5)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-l-yl)-5-oxo- cyclohexanecarboxylate 5 was complete (HPLC). The mixture was concentrated under reduced pressure while below 40 ⁰C, cooled to -30 ⁰C, and water (4.1 L) was added. The resulting slurry was cooled to 5 to 10 ⁰C and agitated for ~1 hour. The product was filtered and the cake was washed with water (2×2.5 L). Upon deliquoring, the cake was dried to a constant weight below 40 ⁰C in a vacuum oven. Cyclohexanone 5 (272g, 70% yield) was obtained (HPLC purity 98.7%).

[00232] Example 1, Alternative Preparation, Step 3: Cyclohexanone 5 (206 g) was dissolved in dichloromethane (1.1 L) and charged to a hydrogenator. Titanium tetraisopropoxide (218.2 g) and N-isopropyl N-methylamine (63.64 g) were added and the mixture was stirred at ambient temperature (23 to 25 ⁰C) for at least 5 h. Platinum catalyst (5% Pt/S/C, 15 g, approx. 7.5 % relative to 5) was added and hydrogenation was performed at -30 psig for at least 6 h, yielding a mixture of ethyl (lR,25′,5R)-2-((5)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-l-yl)-5-(isopropyl- methyl-amino)-cyclohexanecarboxylate 6 and its 5-epz-isomer (-7%). The catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to approx. -600 mL. Wet ethyl acetate (-3% water, 2.0 L) was added with vigorous agitation over a period of at least 1.5 h. Stirring was continued for at least an additional 6 h. The slurry was filtered. Filter cake was washed with ethyl acetate (1.0 L) and discarded. The combined filtrate and washings were concentrated to -400 mL. Toluene (2.0 L) was added and the solution was washed with 2M aqueous hydrochloric acid (2 x 400 mL). The aqueous layer was warmed to 50° to 60 ⁰C for approx. 20 h or hydrolysis of 6 was deemed complete (HPLC). Aqueous sodium hydroxide solution was added to adjust to pH -10, and mixture was extracted with toluene (3×600 mL). The organic phase was discarded and pH was readjusted to ~6 by addition of aqueous hydrochloric acid. The aqueous phase was concentrated to -600 mL under reduced pressure and extracted with methylene chloride (at least 3×2.0 L). The combined methylene chloride layers were evaporated under reduced pressure and continuously replaced with THF to obtain a solution of (\R,2S,5R)-2- ((5^*)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-l-yl)-5-(isopropyl-methyl-amino)- cyclohexane carboxylic acid 7 (-148 g) in THF (-4 L). Seed crystals of 8 were added, followed by 25 % solution of sodium methoxide in methanol (81.24 g) below 25 ⁰C. The slurry was held for at least additional 16h with agitation. The product was isolated by filtration and the cake was washed with THF (4×200 mL) and dried to a constant weight in vacuo below 30 ⁰C. Dry (lR,25′,5R)-2-((5)-3-benzyloxycarbonyl- amino-2-oxo-pyrrolidin-l-yl)-5-(isopropyl-methyl-amino)-cyclohexane-carboxylate sodium salt 8 was obtained (139g, -60% yield from 5).

[00233] Example 1, Alternative Preparation, Step 4: Aminoester sodium salt 8 (10Og), diphenyl phosphate (3.86g), tert-BuOH (1275 mL) and toluene (225 mL) were combined and heated to reflux under reduced pressure. Approx. 500 mL of distillate were collected and discarded while being continuously replaced with a solution of toluene in tert-BuOH. Vacuum was removed and distillate was switched to percolate through a column filled with molecular sieves and allowed to return to the vessel. After drying was complete, DPPA (52.4mL; dissolved in 60 mL toluene) was added slowly to the slurry at 80 ⁰C. Upon complete conversion (HPLC), tert- BuOH was removed by vacuum distillation and continuously replaced with toluene. The mixture was cooled to room temperature and washed twice with 10% aqueous K2HPO4 (lx800mL, 1×400 mL) and water (40OmL). The organic phase was heated and concentrated in vacuo to approx. 27OmL. Vacuum was removed and heptane (1.1 L) was added slowly at approx. 80 ⁰C, followed by seeds of 9 (~lg). The slurry was slowly cooled to room temperature and benzyl {(S)-l-[(lS,2R,4R)-2- tert- butoxycarbonylamino-4-(isopropyl-methyl-amino)-cyclo-hexyl]-2-oxo-pyrrolidin-3- yl} -carbamate 9 was isolated by filtration as a white solid (86.76g, 78% yield).

[00234] Example 1, Alternative Preparation, Step 5: The tert-Butyl carbamate 9 (5Og) was dissolved in Toluene (50OmL) and /-PrOH (15OmL). The resulting solution was then heated to 6O⁰C. Methanesulfonic acid (19.6mL) was added below 65°C. Upon reaction completion (HPLC), the mixture was cooled to RT and triethylamine (69.4mL) added slowly below 25°C. Acetic anhydride was then added below 25°C. After Ih acetic acid (25OmL) was added below 25°C. The toluene phase was discarded and 2-methyl-THF (50OmL) was added to the aqueous phase. The mixture was stirred vigorously and basified with NaOH (25% aqueous solution) to pH 12. The aqueous phase was discarded and the organic layer was washed with brine (25OmL). The organic layer was concentrated under reduced pressure and continuously replaced with /-PrOH. The solution was cooled and filtered to provide benzyl {(5′)-l-[(15^r,2R,4R)-2-acetylamino-4-(isopropyl-methyl-amino)-cyclohexyl]-2- oxo-pyrrolidin-3-yl} -carbamate 10 in /-PrOH solution which was used directly in the hydrogenation.

[00235] Example 1, Alternative Preparation, Step 6: To a solution containing acetamide 10 (~61g) in /-PrOH (-625 mL) was added 10% Pd/C wet catalyst (2.5 g) and the suspension was hydrogenated at 30 psig and approx. 25 ⁰C for at least 2 h. Upon completion (HPLC), the catalyst was removed by filtration and the filtrate was concentrated to approx. 550 mL. Water (8.8 mL) was added, followed by 5.6 N hydrochloric acid in /-PrOH solution (69.5 mL). The resulting slurry was held at room temperature overnight. The product was isolated by filtration and the cake was rinsed with /-PrOH (2×100 mL) and dried in vacuo to constant weight at -50 ⁰C to give N-[(li?,25^r,5R)-2-((5′)-3-amino-2-oxo-pyrrolidin-l-yl)-5-(isopropyl-methyl- amino)-cyclohexyl]-acetamide 11 (55.6 g, 97% yield) as its hydrochloric acid salt (73.6% free base assay, HPLC).

NH,

CL,

Example 1

[00236] Example 1, Alternative Preparation, Step 7: To 6-trifluoromethyl- quinazolin-4-ol 12 (20.1 g) in MeCN (400 mL) was added 5.5 M solution of sodium methoxide in methanol (17.0 mL). The resulting suspension was distilled under reduced pressure and continuously replaced by MeCN to remove methanol. To the slurry was added DMF (1.4 g), followed by oxalyl chloride (13.0 mL) below 50 ⁰C. Upon reaction completion (HPLC), excess reagent was removed under reduced pressure to give -400 mL of slurry. The mixture was cooled to room temperature and washed with 10 % aqueous K₂HPO₄ (lxl.O L, 1×0.5 L) to afford 4-chloro-6- trifluoromethyl-quinazoline 13 (-21.2 g) in approx. 450 mL of wet MeCN solution, which was used directly in the subsequent coupling reaction (HPLC purity 99.8 %). [00237] Example 1, Alternative Preparation, Step 8: To a mixture of acetamide 11 (28.5 g, HCl salt, 73.6% free base assay), acetonitrile (100 mL), N,N,-di-isopropyl- N-ethylamine (61 mL) at room temperature was added a solution of 13 (-21.2 g) in MeCN (-450 mL). The homogeneous mixture was held overnight. Upon reaction completion (HPLC), the mixture was concentrated in vacuo to approx. 125 mL. A 9.5% aqueous solution of acetic acid (240 mL) was added and the aqueous phase was extracted with methylene chloride. The aqueous phase was separated and methyl tert- butyl ether (450 mL) was added, followed by 2N aqueous lithium hydroxide solution to adjust to pH >11.5. The organic layer was separated, washed with water and filtered. Approx. half of the ether phase was diluted with methyl tert-bvAyl ether (-250 mL) and concentrated in vacuo. Heptane (45 mL) was added slowly below 60 ⁰C, followed by seed crystals of Example 1 (0.4 g). Additional heptane (125 mL) was added and the mixture was slowly cooled to room temperature and the resulting slurry was held overnight. The product was isolated by filtration, the cake was washed with heptane and dried in vacuo to constant weight to give N-((lR,25′,5R)-5- (isopropylamino)-2-((5′)-2-oxo-3-(6-(trifluoromethyl)-quin-azolin-4- ylamino)pyrrolidin-l-yl)cyclohexyl)acetamide 14 (15.0 g, 85% yield).

Crystallization Procedures for Example 1

[00238] Example 1, Production of bis-BSA salt and purification: The entirety of the amorphous free base from Example 1, Step 11 was dissolved in methanol (600 mL). The resultant solution was heated at 60 ⁰C and charged with benzenesulfonic acid (2.5 eq). The mixture was cooled to room temperature and the resultant white solid was collected by filtration to yield the bis-benzene sulfonic acid salt of the title compound (95 g, 86%). This material was >99% pure by HPLC. This material was further purified by re-crystallization from 80/20 EtOH/H₂θ, which provided the salt free from any residual methanol. HPLC purity = 99.8%. ¹H ΝMR (500 MHz, D₂O) δ ppm 8.75 (1 H, s), 8.66 (1 H, s), 8.25 (1 H, d, J=8.80 Hz), 7.90 (1 H, d, J=8.80 Hz), 7.75 (4 H, d, J=8.25 Hz), 7.43 – 7.57 (6 H, m), 5.42 (1 H, t), 4.33 – 4.44 (1 H, m), 4.09 – 4.19 (1 H, m), 3.83 – 3.91 (1 H, m), 3.74 – 3.83 (2 H, m), 3.61 (1 H, t, J=I 1.55 Hz), 2.75 (3 H, d, J=6.60 Hz), 2.61 – 2.70 (1 H, m), 2.31 – 2.44 (1 H, m), 2.20 – 2.27 (1 H, m), 2.17 (2 H, d, J=12.10 Hz), 1.94 – 2.04 (1 H, m, J=12.65 Hz), 1.90 – 1.95 (3 H, m), 1.72 – 1.91 (2 H, m), 1.37 (3 H, d, J=6.05 Hz), 1.29 (3 H, d, J=6.60 Hz). Differential scanning calorimetry utilized a heating rate of 10 °C/min and revealed a melting / decomposition endotherm with an onset temperature of 297.6 ⁰C and a peak temperature at 299.1 ⁰C. [00239] Example 1, Crystallization of the Free Base: A sample of the amorphous free base of N-((lR,25^r,5R)-5-(isopropyl(methyl)amino)-2-((5′)-2-oxo-3- (6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin- 1 -yl)cyclohexyl)acetamide ( 1 g) was dissolved in dichloromethane (5 mL). The solution was charged with heptane (30 mL) and then warmed to distill the dichloromethane. The solution was cooled to 40 ⁰C; a white solid precipitated. The suspension was heated to 90 ⁰C and stirred for 2 h. The suspension was cooled to room temperature and filtered to provide the pure free base of the title compound. No residual solvent was apparent by ¹H-NMR.

PATENT

US 7671062

http://google.com/patents/US7671062

or a pharmaceutically acceptable salt, solvate or prodrug, thereof, having an unexpected combination of desirable pharmacological characteristics. Crystalline forms of the present invention are also provided. Pharmaceutical compositions containing the same and methods of using the same as agents for the treatment of inflammatory diseases, allergic, autoimmune, metabolic, cancer and/or cardiovascular diseases is also an objective of this invention. The present disclosure also provides a process for preparing compounds of Formula (I), including N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide:

wherein R¹, R⁸, R⁹, R¹⁰, and

are as described herein. Compounds that are useful intermediates of the process are also provided herein.

1^st embodiment, the disclosure provides a process for preparing a compound of formula IV, or a salt thereof:

Example 1 N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide

Example 1, Step 1: (1R,2S,5R)-tert-Butyl 2-benzyloxycarbonylamino-7-oxo-6-aza-bicyclo[3.2.1]octane-6-carboxylate (89.6 g, 0.24 mol, see: P. H. Carter, et al. PCT application WO 2005/021500) was dissolved in ethyl acetate (1.5 L) and the resulting solution was washed with sat. NaHCO₃ (2×0.45 L) and sat. NaCl (1×0.45 L). The solution was dried (Na₂SO₄) and then filtered directly into a 3-necked 3 L round-bottom flask. The solution was purged with direct nitrogen injection before being charged with 10% Pd/C (13.65 g) under nitrogen atmosphere. The flask was evacuated and back-filled with hydrogen; this was repeated twice more. Hydrogen was bubbled through the solution for 30 min and then the reaction was stirred under 1 atm H₂ for 18 h. The flask was evacuated, back-filled with nitrogen, and charged with fresh catalyst (6 g of 10% Pd/C). Hydrogen was bubbled through the solution for 30 min and then the reaction was stirred under 1 atm H₂ for 18 h. The flask was evacuated and back-filled with nitrogen. The mixture was filtered through Celite; the filter pad was then washed with ethyl acetate. The filtrate (˜1.6 L EtOAc volume) was diluted with acetonitrile (0.3 L) and charged sequentially with L-N-Cbz-methionine (68 g, 0.24 mol), TBTU (77 g, 0.24 mol), and N,N-diisopropylethylamine (42 mL, 0.24 mol). The reaction was stirred at room temperature for 4 h, during which time it changed from a suspension to a clear solution. The reaction was quenched with the addition of sat. NH₄Cl (0.75 L) and water (0.15 L); the mixture was diluted further with EtOAc (0.75 L). The phases were mixed and separated and the organic phase was washed with sat. Na₂CO₃ (2×0.9 L) and sat. NaCl (1×0.75 L). The solution was dried (Na₂SO₄), filtered, and concentrated in vacuo to give (1R,2S,5R)-tert-butyl 2-((S)-2-(benzyloxycarbonylamino)-4-(methylthio)butanamido)-7-oxo-6-aza-bicyclo[3.2.1]octane-6-carboxylate as an oil, which was taken into the next step without further purification. LC/MS for primary peak: [M-Boc+H]⁺=406.3; [M+Na]⁺=528.3. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.36 (m, 5H), 5.11 (s, 2H), 4.32 (m, 1H), 4.2 (m, 1H), 4.0 (m, 1H), 2.5-2.7 (m, 3H), 2.25 (m, 1H), 2.11 (s, 3H), 2.05 (m, 4H), 1.9 (m, 1H), 1.7 (m, 2H), 1.54 (s, 9H). Also present are EtOAc [1.26 (t), 2.03 (s), 4.12 (q)] and N,N,N,N-tetramethylurea [2.83 (s)].

Example 1, Step 2: A sample of (1R,2S,5R)-tert-butyl 2-((S)-2-(benzyloxycarbonylamino)-4-(methylthio)butanamido)-7-oxo-6-aza-bicyclo[3.2.1]octane-6-carboxylate (0.24 mol assumed; see previous procedure) was dissolved in iodomethane (1,250 g) and stirred for 48 h at room temperature. The reaction was concentrated in vacuo. The residue was dissolved in dichloromethane and concentrated in vacuo. This was repeated twice more. The resultant sludge was dissolved in dichloromethane (0.4 L) and poured into a rapidly stirring solution of MTBE (4.0 L). The resultant yellow solids were collected via suction filtration and dried under high vacuum to afford the sulfonium salt (179 g). This material was taken into the next step without further purification. LC/MS for primary peak: [M-Me₂S+H]⁺=458.4; [M]⁺=520.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.35 (m, 5H), 5.09 (s, 2H), 4.33 (m, 1H), 4.28 (m, 1H), 3.98 (m, 1H), 3.3-3.45 (m, 2H), 2.97 (s, 3H), 2.94 (s, 3H), 2.78 (m, 1H), 2.0-2.3 (m, 4H), 1.7 (m, 2H), 1.52 (s, 9H). Also present are MTBE [1.18 (s), 3.2 (s)] and traces of N,N,N,N-tetramethylurea [2.81 (s)].

Example 1, Step 3: All of the sulfonium salt from the previous step (0.24 mol assumed) was dissolved in DMSO (2.0 L). The resultant solution was stirred under nitrogen at room temperature and charged with cesium carbonate (216 g) portionwise. The suspension was stirred at room temperature for 3 h and then filtered to remove the solids. The solution was divided into ˜0.22 L portions and worked up as follows: the reaction mixture (˜0.22 L) was diluted with ethyl acetate (1.5 L) and washed successively with water (3×0.5 L) and brine (1×0.3 L). The organic phase was dried (Na₂SO₄), filtered, and concentrated in vacuo. The desired (1R,2S,5R)-tert-butyl 2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)-7-oxo-6-azabicyclo[3.2.1]octane-6-carboxylate (90.8 g, 83%) was obtained as a microcrystalline foam, free from tetramethyl urea impurity. LC/MS for primary peak: [M-Boc+H]⁺=358.4; [M+Na]⁺=480.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.35 (m, 5H), 5.12 (s, 2H), 4.35 (m, 2H), 4.2 (m, 1H), 3.6 (m, 1H), 3.3 (m, 1H), 2.64 (m, 1H), 2.28-2.42 (m, 2H), 2.15 (m, 1H), 1.7-2.0 (m, 5H), 1.55 (s, 9H). If desired, this material can be isolated as a solid by dissolving in MTBE (1 volume), adding to heptane (3.3 volumes), and collecting the resultant precipitate.

Example 1, Step 4: A stirring solution of (1R,2S,5R)-tert-butyl 2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)-7-oxo-6-azabicyclo[3.2.1]octane-6-carboxylate (108 g, 0.236 mol) in THF (1 L) was charged with lithium hydroxide monohydrate (21.74 g, 0.519 mol). Water (0.3 L) was added slowly, such that the temperature did not exceed 20° C. The reaction was stirred at room temperature overnight and the volatiles were removed in vacuo. The pH was adjusted to ˜4 through the addition of 1N HCl (450 mL) and NaH₂PO₄. The resultant white precipitates were collected by filtration and washed with water (2×1 L). The solid was dissolved in dichloromethane (1.5 L) and water (˜1 L). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was dissolved in EtOAc (0.7 L) and the resultant solution was heated at reflux for 1 h. Solids separated after cooling to RT, and were collected via filtration. These solids were purified by recrystallization in isopropanol to afford the desired (1R,2S,5R)-2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)-5-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid as a white solid (104.5 g, 93% yield). LC/MS for primary peak: [M-tBu+H]⁺=420.2; [M-Boc+H]⁺=376.2; [M+H]⁺=476.2. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.35 (m, 5H), 5.11 (s, 2H), 4.35 (m, 2H), 3.71 (m, 1H), 3.45-3.6 (m, 2H), 2.99 (m, 1H), 2.41 (m, 1H), 2.15 (m, 1H), 2.0 (m, 2H), 1.6-1.9 (m, 4H), 1.46 (s, 9H).

Example 1, Step 5: A 3 L round bottom flask was charged with (1R,2S,5R)-2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)-5-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid (75.5 g, 0.158 mol), EDC.HCl (33.5 g, 0.175 mol), 1-hydroxybenzotriazole (23.6 g, 0.175 mol), and dichloromethane (1 L). The reaction was stirred at room temperature for 2 h, during which time it changed from a white suspension to a clear solution. Ammonia (gas) was bubbled into the solution until the pH was strongly basic (paper) and the reaction was stirred for 10 min; this ammonia addition was repeated and the reaction was stirred for an additional 10 min. Water was added. The organic phase was washed with sat. NaHCO₃, NaH₂PO₄, and brine before being concentrated in vacuo. The residue was slurried with acetonitrile (0.5 L) and then concentrated in to give (1R,2S,5R)-2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)-5-(tert-butoxycarbonylamino)cyclohexanecarboxamide as a white solid (75.9 g, ˜100%), which was used in the next step without further purification. LC/MS for primary peak: [M-Boc+H]⁺=375.3; [M+H]⁺=475.4; [M-tBu+H]⁺=419.3. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.35 (m, 5H), 5.11 (s, 2H), 4.25 (m, 2H), 3.70 (m, 1H), 3.6 (m, 1H), 3.45 (m, 1H), 2.91 (m, 1H), 2.38 (m, 1H), 2.12 (m, 1H), 1.9-2.05 (m, 2H), 1.65-1.9 (m, 4H), 1.46 (s, 9H).

Example 1, Step 6: The reaction was run in three equal portions and combined for aqueous workup. A 5 L, 3-necked round bottom flask was charged with (1R,2S,5R)-2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)-5-(tert-butoxycarbonylamino)cyclohexanecarboxamide (25.3 g, 53 mmol), acetonitrile (1.9 L), and 2.6 L of water/ice. The mixture was stirred and cooled to 0° C. Iodobenzene diacetate (25.77 g, 80 mmol) was added and the reaction was stirred for 2 h; another 0.5 eq of iodobenzene diacetate was added. The reaction was stirred for 9 h (reaction temp<10° C.). The mixture was charged with 8 eq N,N-diisopropylethylamine and 2 eq acetic anhydride. Over the next thirty minutes, 4 eq N,N-diisopropylethylamine and 2 eq acetic anhydride were added every ten minutes, until the reaction had proceeded to completion (HPLC). The acetonitrile was removed in vacuo; some solid separated from the residue, and this was collected by filtration. The remaining residue was extracted with dichloromethane (3 L, then 1 L). The organic phase was washed sequentially with water, sat. NaHCO₃, and brine. The collected solids were added to the organic phase, along with activated carbon (15 g). The mixture was stirred for 30 minutes at 40° C. before being filtered and concentrated in vacuo. The residue was dissolved in EtOAc (1 L), and the resultant solution was stirred at 75° C. for 1 h before being allowed to cool to room temperature. A solid separated and was collected by filtration. This solid was purified further by recrystallization: it was first dissolved in 0.5 L CH₂Cl₂, then concentrated in vacuo, then re-crystallized from 1 L EtOAc; this was repeated three times. The solids obtained from the mother liquors of the above were recrystallized three times using the same method. The combined solids were recrystallized twice more from acetonitrile (0.7 L) to provide 66 g (84%) of tert-butyl (1R,3R,4S)-3-acetamido-4-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)cyclohexylcarbamate (purity>99.5% by HPLC). LC/MS for primary peak: [M+H]⁺=489.4; [M-tBu+H]⁺=433.3. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.3-7.4 (m, 5H), 5.11 (s, 2H), 4.35 (m, 1H), 4.15 (m, 1H), 4.04 (m, 1H), 3.8 (m, 1H), 3.6 (m, 2H), 2.44 (m, 1H), 2.12 (m, 1H), 1.87-2.05 (m, 4H), 1.87 (s, 3H), 1.55-1.7 (m, 2H), 1.46 (s, 9H). The stereochemical fidelity of the Hofmann rearrangement was confirmed through X-ray crystal structure analysis of this compound, as shown in FIG. 1.

Example 1, Step 7: A stirring solution of tert-butyl (1R,3R,4S)-3-acetamido-4-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)cyclohexylcarbamate (66 g, 0.135 mol) in dichloromethane (216 mL) was charged with trifluoroacetic acid (216 mL). The reaction was stirred for 2 h at room temperature and concentrated in vacuo. The residue was dissolved in methanol and the resultant solution was concentrated in vacuo; this was repeated once. Benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-aminocyclohexyl)-2-oxopyrrolidin-3-ylcarbamate was obtained as an oil and used directly in Step 8 below. LC/MS found [M+H]⁺=389.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.3-7.4 (m, 5H), 5.12 (s, 2H), 4.41 (br. s, 1H), 4.15 (m, 1H), 4.00 (t, J=9.3 Hz, 1H), 3.81 (t, J=9.1 Hz, 1H), 3.65 (q, J=8.4 Hz, 1H), 3.3-3.4 (m, 1H), 2.45 (m, 1H), 1.95-2.24 (m, 5H), 2.00 (s, 3H), 1.6-1.8 (m, 2H).

Example 1, Step 8: A stirring solution of benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-aminocyclohexyl)-2-oxopyrrolidin-3-ylcarbamate (˜0.135 mol) in methanol (675 mL) was charged sequentially with acetone (37.8 g, 4 eq), sodium acetate (33.2 g, 3 eq), and sodium cyanoborohydride (16.9 g, 2 eq). The mixture was stirred at room temperature for 6 h and filtered. The filtrate was dissolved in dichloromethane (1 L); this solution was washed with 1N NaOH (1 L). The solids collected in the filtration were dissolved in 1N NaOH (1 L) at 0° C. and then extracted with dichloromethane (1 L). The organic extracts were combined and extracted with aqueous HCl (200 mL 1N HCl+800 mL water). The aqueous phase was basified with sat. NaHCO₃ (500 mL) and then 1N NaOH (100 mL) until pH 11. The aqueous phase was extracted with dichloromethane (2 L). The organic extracts were combined, dried (Na₂SO₄), filtered, and concentrated in vacuo to give benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-(isopropylamino)cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate as an oil. LC/MS found [M+H]⁺=431.45. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.3-7.4 (m, 5H), 5.12 (s, 2H), 4.31 (m, 1H), 4.24 (t, J=9.4 Hz, 1H), 4.11 (m, 1H), 3.61 (t, J=9.1 Hz, 1H), 3.52 (q, J=8.6 Hz, 1H), 3.04 (br. s, 1H), 2.96 (sep, J=6.3 Hz, 1H), 2.40 (m, 1H), 2.15 (m, 1H), 1.92 (s, 3H), 1.7-1.9 (m, 5H), 1.65 (m, 1H), 1.12 (app. dd, J=6.3, 1.1 Hz, 6H).

Example 1, Step 9 (See Alternative Step 9, below): A stirring solution of benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-(isopropylamino)cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate (˜115 mmol) in dichloromethane (600 mL) was cooled to 0° C. and charged sequentially with formaldehyde (18.6 g, 37 wt % solution), triethylamine (23 mL), and sodium triacetoxyborohydride (28.7 g). The mixture was stirred at room temperature for 30 minutes and diluted with dichloromethane (up to 1.2 L). This solution was washed thrice with 500 mL sat. NaHCO₃+NaOH (sat. NaHCO₃, pH to 11 w/1N NaOH). The organic layer was extracted with aq. HCl (200 mL 1N HCl+600 mL water). The aqueous phase was basified with sat. NaHCO₃ (500 mL) and then 1N NaOH (100 mL) until pH 11. The aqueous phase was extracted with dichloromethane (1.2 L). The organic extracts were combined, dried (Na₂SO₄), filtered, and concentrated in vacuo to give benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-(isopropyl(methyl)amino)cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate as an oil, which was used directly in Step 10 below. LC/MS found [M+H]⁺=445.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.3-7.4 (m, 5H), 5.12 (s, 2H), 4.33 (br s, 1H), 4.25 (t, J=9.2 Hz, 1H), 4.11 (br s, 1H), 3.5-3.6 (m, 2H), 2.77 (v br s, 2H), 2.41 (m, 1H), 2.26 (s, 3H), 2.0-2.1 (m, 2H), 1.92 (s, 3H), 1.7-1.9 (m, 5H), 1.10 (app. dd, J=17, 6.4 Hz, 6H).

Example 1, Step 10: To a solution of benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-(isopropyl(methyl)amino)-cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate (0.115 mol) in methanol (600 mL) was added 10% Pd/C (6 g of 50% wet catalyst). The flask was evacuated and back-filled with hydrogen. The mixture was stirred under 1 atm H₂ for 2 h and the catalyst was removed by filtration through Celite. The filtrate was concentrated in vacuo to provide N-((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl)acetamide as an oil, which was taken on to the next step without further purification. LC/MS found [M+H]⁺=311.47. ¹H-NMR (400 MHz, d₄-MeOH): δ 4.39 (br s, 1H), 4.00 (m, 1H), 3.3-3.5 (m, 4H), 2.73 (m, 1H), 2.38 (m, 1H), 2.25 (s, 3H), 2.0-2.2 (m, 3H), 1.94 (s, 3H), 1.6-1.75 (m, 4H), 1.07 (app. dd, J=21, 6.4 Hz, 6H).

Example 1, Step 11: To a solution of N-((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl)acetamide (˜35 g, 0.115 mol) in isopropanol (600 mL) was added 4-chloro-6-(trifluoromethyl)quinazoline (32 g, 0.138 mol, 1.2 eq, see: P. H. Carter et al., PCT application WO 2005/021500). The mixture was stirred at room temperature overnight before being charged with triethylamine (46 g, 0.46 mol, 4 eq). The mixture was stirred at 60° C. for 10 h. The solvent was removed under reduced pressure to give an oil. Azeotropic distillation with isopropanol was performed twice. The residue was dissolved in dichloromethane (600 mL) and extracted with water (250 mL, containing 4 eq acetic acid). Dichloromethane (600 mL) was added to the combined aqueous washes, and the mixture was cooled to 0° C. Aqueous NaOH (50% by weight) was added with stirring until the pH reached 11. The water layer was extracted with dichloromethane twice (2×600 mL). The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated in vacuo to give the amorphous free base of the title compound (99% purity by HPLC). LC/MS found [M+H]⁺=507.3. ¹H-NMR (400 MHz, d₄-MeOH): δ 8.82 (s, 1H), 8.59 (s, 1H), 8.05 (dd, J=8.8, 1.8 Hz, 1H), 7.9 (d, J=8.7 Hz, 1H), 5.28 (t, J=8.6 Hz, 1H), 4.58 (br s, 1H), 4.06 (m, 1H), 3.52-3.68 (m, 2H), 3.43 (m, 1H), 2.76 (br s, 1H), 2.55 (m, 1H), 2.28 (s, 3H), 2.1-2.3 (m, 3H), 2.0 (s, 3H), 2.0 (m, 1H), 1.65-1.8 (m, 3H), 1.09 (app. dd, J=24, 6.4 Hz, 6 H).

Example 1 Alternative Step 9

Example 1, Alternative step 9aⁱ: To a hydrogenator were charged ethyl (7R,8S)-8-((S)-1-phenyl-ethylamino)-1,4-dioxa-spiro[4.5]decane-7-carboxylate 4-toluenesulfonate salt 1A (1417 g, 2.8 moles, c.f.: WO2004098516, prepared analogous to U.S. Pat. No. 6,835,841), ethanol (200 proof, 11.4 L), and 10% Pd/C catalyst (50% wet, 284 g). The mixture was inerted with nitrogen, then pressurized with hydrogen gas (45 psig) and agitated vigorously at approx. 40° C. until starting material was consumed (HPLC). The suspension was cooled, purged with nitrogen gas and the catalyst was removed by filtration while inerted. The spent catalyst was washed with ethanol (4.3 L). The filtrate and washings were combined and concentrated under vacuum to a volume of 2-3 L while maintaining the batch between 40°-60° C. Isopropyl acetate (5 L) was charged and the mixture was concentrated to a volume of ˜2 L until most ethanol was removed (<0.5%) and residual moisture content was <1,000 ppm. Batch volume was adjusted to ˜7.5 L by the addition of isopropyl acetate. The mixture was heated to 80° C. until clear, then cooled 65°-70° C. Seed crystals of 1 (5 g) were added and the batch was cooled to 50° C. over 2 hours, then further cooled to 20° C. over 4 hours and held for ˜10 hours. The resulting slurry was filtered and the cake was washed with isopropyl acetate (2 L). The product was dried under vaccum at ˜35° C. until volatiles were reduced below ˜1% (LOD). Ethyl (7R,8S)-8-amino-1,4-dioxa-spiro[4.5]decane-7-carboxylate 4-toluenesulfonate salt 1 was obtained as a white, crystalline solid (936 g, 83% yield; HPLC purity: 99.8%). ¹H-NMR: (300 MHz, CDCl₃) 8.14-7.89 (brs, 3H), 7.75 (d, J 9.0 Hz, 2H), 7.15 (d, J 8.0 Hz, 2H), 4.22-4.04 (m, 2H), 4.01-3.77 (m, 4H), 3.55-3.43 (m, 1H,), 3.20-3.13 (m, 1H), 2.40-2.27 (m, 4H), 2.21-1.94 (m, 2H), 1.81-1.51 (m, 3H), 1.23 (t, J 7.0 Hz, 3H); HPLC: Waters Xterra MS C18 4.6 mm×150 mm i.d., 3.5 μm particle size, 0.05% NH4OH (5% ACN, 95% H₂O, solvent A), to 0.05% NH₄OH (95% ACN, 5% H₂O, solvent B), 5% B to 20% B in 10 minutes, changed to 95% B in 25 minutes, and then changed to 5% B in 1 minute; 11.1 minutes (aminoester 1).

Example 1, Alternative Step 9aⁱⁱ: Aminoester 1 (63 g, 0.16M, 1 eq.; the product of reductive deprotection of a known compound—(See e.g. R. J. Cherney, WO 2004/098516 and G. V. Delucca & S. S. Ko, WO 2004/110993) was placed in a round bottom flask and MeCN (500 mL) was added. EDAC (33.1 g, 0.17M, 1.1 eq), HOBt.H₂O (21.2 g, 0.16M, 1.0 eq) and N-Cbz-L-methionine (46.7 g, 0.17M, 1.05 eq) were then added followed by TEA (48.0 mL, 0.35M, 2.2 eq). An exotherm to 38° C. was observed. The reaction mass was left to stir at RT. After 30 mins, HPLC indicated complete conversion. The reaction mass was diluted with EtOAc (2.5 L) and washed with KHCO₃ (4×500 mL, 20 wt % aq. solution) and brine (500 mL). The organic phase was separated, dried over MgSO₄ and concentrated. The residue was dissolved in TBME and reconcentrated to give ethyl (7R,8S)-8-{(2S)-2-benzyloxycarbonylamino-4-methylsulfanyl-butyr-yl-amino}-1,4-dioxa-spiro[4.5]decane-7-carboxylate 2 as a sticky semi-solid (76.2 g, 98% yield, 93 AP purity). ¹H-NMR: (300 MHz, CDCl₃) δ 7.36-7.30 (m, 5H), 7.03 (d, J 9.0 Hz, 1H), 5.66 (d, J 8.0 Hz, 1H), 5.10 (s, 2H), 4.35-4.25 (m, 2H), 4.19-4.04 (m, 2H,), 3.98-3.86 (m, 4H), 2.87-2.80 (m, 1H), 2.55-2.45 (m, 2H), 2.18 (dd, J 14.0 Hz, 7.0 Hz, 1H), 2.08 (s, 3H), 2.05-1.67 (m, 6H), 1.26 (t, J 7.0 Hz, 3H). HPLC: YMC-Pack Pro C18 5 μm 4.6×150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% 10 min gradient. 10.01 min (Compound 2, 93.1 AP). HRMS: m/z 495.2166 [Calc: C₂₄H₃₅N₂O₇S 495.2165].

Example 1, Alternative Step 9b: Methionine amide 2 (75.0 g, 0.15M) was dissolved in MeI (225 mL, 3 mL/g)—some off gassing was noted but no exotherm. The reaction mass was left to stir in the dark for 16.5 h. After this time a thick light yellow precipitate had formed. The flask was then evacuated to 200 mmHg and some of the MeI removed. The remaining material was slurried in TBMF (500 mL), after a 30 min stir-out the slurry was filtered, the cake washed with TBMF (500 mL). NMR analysis of this material indicated a small amount of MeI remaining. The cake was re-slurried in TBMF (500 mL), filtered, washed with TBMF (500 mL) and dried under vacuum to give [(3S)-3-benzyloxycarbonylamino-3-{(7R,8S)-7-ethoxycarbonyl-1,4-di-oxa-spiro[4.5]dec-8-ylcarbamoyl}-propyl]-dimethylsulfonium iodide 3 as a free flowing off-white solid (93.5 g, 97%, 99 area % purity). ¹H-NMR: (300 MHz, CDCl₃) δ 7.75 (d, J 9.0 Hz, 1H), 7.38-7.27 (m, 5H), 6.40 (d, J 7.0 Hz, 1H), 5.10 (s, 2H), 4.76-4.65 (m, 1H), 4.48-4.39 (m, 1H), 4.14-3.85 (m, 6H), 3.84-7.73 (m, 1H), 3.68-3.55 (m, 1H), 3.21 (s, 3H), 3.12 (s, 3H), 2.90-2.83 (s, 1H), 2.52-1.55 (m, 8H), 1.24 (t, J 7.0 Hz, 3H). HPLC: YMC-Pack Pro C18 5 μm 4.6×150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% 10 min gradient. 2.45 min (I−), 8.14 min (Compound 3, 43.6 AP, I⁻54.6 AP). HRMS: m/z 509.2341 [Calc: C₂₅H₃₇N₂O₇S 509.2321].

Example 1, Alternative Step 9c: Cs₂CO₃ (61.5 g, 0.19M, 1.5 eq) was placed in an round bottom flask and anhydrous DMSO (2.4 L) was added. Sulfonium salt 3 (80.0 g, 0.13M, 1.0 eq) was then added portionwise. Once the addition was complete the reaction mass was left to stir in the dark for 20 h. The reaction mass was then split in half and each half worked up separately: the reaction mass was diluted with EtOAc (2.0 L) and washed with brine (2 L), the organic phase was washed with brine (500 mL). The combined aq. layers were then washed EtOAc (500 mL). The combined organic phases were then washed with brine (3×750 mL). The second half of the reaction mass was treated in an identical manner and the combined organics dried over MgSO₄ and concentrated to give ethyl (7R,8S)-8-{(3S)-3-Benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl}-1,4-dioxa-spiro[4.5]decane-7-carboxylate 4 as a light colored oil (56.5 g, 0.13M, ˜100 area-% purity) pure by NMR analysis. ¹H-NMR: (300 MHz, CDCl₃) δ 7.38-7.30 (m, 5H), 5.37 (br d, J 4.0 Hz, 1H), 5.11 (s, 2H), 4.27-4.18 (m, 1H), 4.17-3.82 (m, 8H), 3.32 (td, J 10.0Hz, 60.0 Hz, 1H), 3.23 (q, J 5.0 Hz, 1H), 2.63-2.57 (m, 1H), 2.42-2.25 (m, 2H), 1.94-1.68 (m, 5H), 1.25 (t, J 7.0 Hz, 3H). HPLC: YMC-Pack Pro C18 5 μm 4.6×150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% 10 min gradient. 8.99 min (Compound 5, produced on column, 4.2 AP), 9.48 (Compound 4, 74.3 AP). HRMS: m/z 447.2127 [Calc: C₂₃H₃₁N₂O₇ 447.2131].

Example 1, Alternative Step 9d: Pyrrolidinone 4 (50.0 g, 0.11M) was dissolved in acetone (500 mL) and 1N HCl (500 mL) was added. The reaction mass was then heated to 65° C. After 20 mins HPLC indicated complete reaction. The reaction mass was allowed to cool to RT and the acetone was removed on a rotary evaporator. During this distillation the product precipitated from solution as a white solid. This was isolated by filtration and the cake washed with water. The cake was then dried azeotropically with toluene (3×300 mL) to give ethyl (1R,2S)-2-((3S)-3-Benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-5-oxo-cyclohexanecarboxylate 5 as a white solid (39.8 g, 88%, 97 area-% purity). ¹H-NMR: (300 MHz, CDCl₃) δ 7.37-7.32 (m, 5H), 6.65 (br d, J 4.0 Hz, 1H), 5.12 (s, 2H), 4.54-4.47 (m, 1H), 4.34-4.26 (m, 1H), 4.18 (dq, J 11.0 Hz, 7.0 Hz, 1H), 4.09 (dq, J 11.0 Hz, 7.0 Hz, 1H), 3.36-3.20 (m, 3H), 2.70-2.35 (m, 6H), 2.05-1.96 (m, 1H), 1.81 (quin., J 11.0 Hz, 1H), 1.24 (t, J 7.0 Hz, 3H). HPLC: YMC-Pack Pro C18 5 μm 4.6×150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% 10 min gradient. 8.95 min (Compound 5). HRMS: m/z 403.1864 [Calc: C₂₁H₂₇N₂O₆403.1869].

Example 1, Alternative Step 9e: Cyclohexanone 5 (22.5 g, 0.06M, 1 eq), DMSO (30 mL) and Ti(O-iPr)₄ (33.7 mL, 0.11M, 2.04 eq) were placed in a round bottom flask. N-isopropyl-N-methylamine (11.6 mL, 0.11M, 2.0 eq) was then added in one portion. The mixture was left to stir for 30 mins at room temperature before being cooled to <3° C. in ice/water. MeOH (30 mL) was then added followed by the portionwise addition of NaBH₄ (4.33 g, 0.11M, 2.04 eq)—temperature kept <8° C. 30 mins after the addition was completed the reaction mass was diluted with methylene chloride (300 mL) and then NaOH (1N, 40 mL). The resulting slurry was filtered through Celite, and the cake washed with methylene chloride (100 mL). The resulting liquor was concentrated under reduced pressure and the residue dissolved in EtOAc (500 mL). This solution was extracted with 1N HCl (2×400 mL), the combined aqueous layers were then basified with Na₂CO₃. Extraction with EtOAc (4×250 mL) provided a clear and colorless organic phase which was dried over Na₂SO₄ and concentrated to give a white powder (24.6 g, 96%, 7:1 d.r.). This material was then slurried overnight in hexane (670 mL). The solid was isolated by filtration and dried under reduced pressure to give ethyl (1R,2S,5R)-2-((3S)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-5-(isopropyl-methyl-amino)-cyclohexanecarboxylate 6 as a while solid (20.9 g, 81%, 24:1 d.r.). ¹H-NMR: (300 MHz, CDCl₃) δ 7.37-7.28 (m, 5H), 5.55 (d, J 4.5, 1H), 5.10 (s, 2H), 4.42 (q, J 4.5, 1H), 4.23-4.12 (m, 1H), 4.08 (dq, J 10.5, 7.0, 1H), 4.02 (dq, J 10.5, 7.0, 1H), 3.84 (t, J 9.0, 1H), 3.46-3.36 (m, 1H), 3.04 (septet, J 6.5, 1H), 2.86-2.80 (m, 1H), 2.63-2.48 (m, 2H), 2.17 (s, 3H, Me), 2.10-1.63 (m, 7H), 1.22 (t, J 7.0, 3H), 1.00 (d, J 6.5, 3H), 0.97 (d, J 6.5, 3H). HPLC: YMC-Pack Pro C18 5 μm 4.6×150 mm, 0.01M NH₄OAc (MeOH:water 20:80) to 0.01M NH₄OAc (MeOH:water:MeCN 20:5:75) 10 to 100% 15 min gradient. 8.23 (Compound 6), 8.88 (5-epi-Compound 6). HRMS: 460.2798 [Calc: C₂₅H₃₈N₃O₅ 460.2811].

Example 1, Alternative Step 9f: The aminoester 6 (9.76 g, 2.12 mmol) was dissolved in 2N HCl (80 mL), then heated to ˜55° C. under inert atmosphere. The reaction was stirred for 20 h, then cooled to room temperature. The reaction solution was washed twice with toluene (25 mL portions), neutralized to pH 6-7 by the addition of KOH pellets, then extracted eight times with methylene chloride (100 mL portions). The combined extracts were dried (Na₂SO₄), filtered, and concentrated under reduced pressure to 50 mL total volume. The concentrated solution was then slowly added to methyl tert-butyl ether (300 mL) over 15 min in an addition funnel with vigorous stirring. The resulting white slurry was stirred at ambient temperature for Ih, then cooled to 0° C. and stirred for 1 h. The product was filtered, and washed twice with methyl tert-butyl ether (25 mL portions). Water from the wet cake was removed by azeotropic distillation with acetonitrile (300 mL). The product was dried under reduced pressure to provide (1R,2S,5R)-2-((3S)-3-Benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-5-(isopropyl-methyl-amino)-cyclohexanecarboxylic acid 7, (7.69 g, 84% yield) as a white foam. ¹H-NMR: (400 MHz, 50° C., CDCl₃) δ 7.44-7.32 (m, 5H), 6.10 (broad s, 1H), 5.19 (app s, 2H), 4.42 (dd, J=15.6, 7.8 Hz, 1H), 4.29-4.23 (m, 1H), 3.68-3.60 (m, 2H), 3.33-3.27 (m, 2H), 3.20 (broad s, 1H), 2.99 (broad s, 1H), 2.51 (s, 3H), 2.49-2.45 (m, 3H), 2.33-2.31 (m, 1H), 2.00 (ddd, J=9.0, 8.6, 3.9 1H), 1.95-1.78 (m, 2H), 1.36-1.21 (m, 6H). LCMS: m/z 432.20 [Calc: C₂₃H₃₄N₃O₅ 432.25].

Example 1, Alternative Step 9g: Amino acid 7 (6.3 g, 14.7 mmol, 1.0 eq) was dissolved in THF (80 mL) under N₂ and NaH (584 mg, 14.7 mmol, 1.0 eq, 60 wt % dispersion in mineral oil) was added portionwise. When the addition was complete, and the evolution of gas had ceased, the reaction mass was concentrated under reduced pressure and the resulting solid azeotroped with toluene (50 mL) to give a white solid (KF 0.59 wt %). This solid was slurried in toluene (100 mL) under N₂and heated to 90° C. DPPA (3.32 mL, 15.3 mmol, 1.05 eq) was added dropwise over ˜2 min. After ˜5 min all the solids had dissolved, after 10 mins precipitation of a white solid was observed. After 30 mins HPLC analysis indicated complete reaction. The reaction mass was allowed to cool to RT before being filtered, the cake was washed with toluene. The liquors where then slowly added into AcOH/Ac₂O (80/20, 168 mL) solution at 90° C. After 45 mins HPLC still indicated some isocyanate. At 1.15 h, the reaction mass was cooled to RT and diluted with toluene (100 mL) and water (100 mL). The organic layer was removed and the toluene washed with 1N HCl (100 mL). The combined aq. phases were then basified with K₂CO₃(s) and brought to pH 12 with NaOH (10N), keeping the temperature below 20° C. The aq layer was then extracted with methylene chloride (4×150 mL), the combined organic layers dried over K₂CO₃ and concentrated to give benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-(isopropyl(methyl)amino)cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate 8 as a white foam (4.5 g, 70%, 94AP purity). The ¹H-NMR was identical to material obtained from the route described above (Example 1, Step 9). HPLC: YMC-Pack Pro C18 5 μm 4.6×150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% 10 min gradient. 7.20 min (Compound 8), 7.85 min (urea dimer). HRMS: 445.2809 [Calc: C₂₄H₃₇N₄O₄445.2815].

Alternative Preparation of Example 1

Example 1, Alternative Preparation, Step 1: Ethyl (7R,8S)-8-amino-1,4-dioxa-spiro[4.5]decane-7-carboxylate 4-toluenesulfonate salt 1 (450.1 g), was combined with 1-ethyl-3-(3-dimethyl-amino-propyl)carbo-diimide hydrochloride (236.3 g), 1-hydroxy benzotriazole hydrate (171.9 g), N-carbobenzyloxy-L-methionine (333.4 g) and acetonitrile (3.1 L). To the stirred mixture was added triethylamine (249.5 g) below 30° C. Upon reaction completion (HPLC), the mixture was diluted with ethyl acetate (8.2 L) and washed with aqueous 25% potassium bicarbonate solution (2×4.5 L) followed by water (4.5 L). The organic phase was separated and concentrated under reduced pressure to obtain a solution of ethyl (7R,8S)-8-((S)-2-benzyloxycarbonylamino-4-methylsulfanyl-butyrylamino)-1,4-dioxa-spiro[4.5]decane-7-carboxylate 2 (1.4 L). Methyl iodide (2.39 kg) was added, the vessel was shielded from light and the mixture was held under slow agitation for approx. 24 h. To the thick yellow precipitate was added methyl tert-butyl ether (2.7 L) and the mixture was held for approx. 1 h. The product was isolated by filtration and the cake was washed with methyl tert-butyl ether (2×1.4 L), then dried under vacuum, yielding [(S)-3-benzyloxy-carbonylamino-3-((7R,8S)-7-ethoxycarbonyl-1,4-dioxa-spiro[4.5]dec-8-ylcarbamoyl)-propyl]-dimethylsulfonium iodide 3 (671.4 g, ˜94% yield) as an off-white solid (HPLC purity 99.9%).

Example 1, Alternative Preparation, Step 2: Sulfonium salt 3 (619.4 g), and cesium carbonate (416.8 g) and anhydrous dimethyl sulfoxide (6.2 L) were combined in a reactor equipped with a scrubber to neutralize volatile sulfides. Vigorous agitation was maintained until complete conversion was obtained (HPLC). Ethyl acetate (12.4 L) was added, followed by 20% brine (3 L). The organic phase was separated, washed twice with brine (2×3 L) and evaporated to obtain a solution of ethyl (7R,8S)-8-((S)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-1,4-dioxa-spiro[4.5]decane-7-carboxylate 4 in ethyl acetate (˜0.8 L). Acetone (2.55 L) was added, followed by aqueous 0.5 M hydrochloric acid solution (2.3 L). With good mixing, the solution was heated to 50 to 60° C. until conversion of 4 to ethyl (1R,2S)-2-((S)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-5-oxo-cyclohexanecarboxylate 5 was complete (HPLC). The mixture was concentrated under reduced pressure while below 40° C., cooled to ˜30° C., and water (4.1 L) was added. The resulting slurry was cooled to 5 to 10° C. and agitated for ˜1 hour. The product was filtered and the cake was washed with water (2×2.5 L). Upon deliquoring, the cake was dried to a constant weight below 40° C. in a vacuum oven. Cyclohexanone 5 (272 g, 70% yield) was obtained (HPLC purity 98.7%).

Example 1, Alternative Preparation, Step 3: Cyclohexanone 5 (206 g) was dissolved in dichloromethane (1.1 L) and charged to a hydrogenator. Titanium tetraisopropoxide (218.2 g) and N-isopropyl N-methylamine (63.64 g) were added and the mixture was stirred at ambient temperature (23 to 25° C.) for at least 5 h. Platinum catalyst (5% Pt/S/C, 15 g, approx. 7.5% relative to 5) was added and hydrogenation was performed at ˜30 psig for at least 6 h, yielding a mixture of ethyl (1R,2S,5R)-2-((S)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-5-(isopropyl-methyl-amino)-cyclohexanecarboxylate 6 and its 5-epi-isomer (˜7%). The catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to approx. ˜600 mL. Wet ethyl acetate (˜3% water, 2.0 L) was added with vigorous agitation over a period of at least 1.5 h. Stirring was continued for at least an additional 6 h. The slurry was filtered. Filter cake was washed with ethyl acetate (1.0 L) and discarded. The combined filtrate and washings were concentrated to ˜400 mL. Toluene (2.0 L) was added and the solution was washed with 2M aqueous hydrochloric acid (2×400 mL). The aqueous layer was warmed to 50° to 60° C. for approx. 20 h or hydrolysis of 6 was deemed complete (HPLC). Aqueous sodium hydroxide solution was added to adjust to pH ˜10, and mixture was extracted with toluene (3×600 mL). The organic phase was discarded and pH was readjusted to ˜6 by addition of aqueous hydrochloric acid. The aqueous phase was concentrated to ˜600 mL under reduced pressure and extracted with methylene chloride (at least 3×2.0 L). The combined methylene chloride layers were evaporated under reduced pressure and continuously replaced with THF to obtain a solution of (1R,2S,5R)-2-((S)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-5-(isopropyl-methyl-amino)-cyclohexane carboxylic acid 7 (˜148 g) in THF (˜4 L). Seed crystals of 8 were added, followed by 25% solution of sodium methoxide in methanol (81.24 g) below 25° C. The slurry was held for at least additional 16 h with agitation. The product was isolated by filtration and the cake was washed with THF (4×200 mL) and dried to a constant weight in vacuo below 30° C. Dry (1R,2S,5R)-2-((S)-3-benzyloxycarbonyl-amino-2-oxo-pyrrolidin-1-yl)-5-(isopropyl-methyl-amino)-cyclohexane-carboxylate sodium salt 8 was obtained (139 g, ˜60% yield from 5).

Example 1, Alternative Preparation, Step 4: Aminoester sodium salt 8 (100 g), diphenyl phosphate (3.86 g), tert-BuOH (1275 mL) and toluene (225 mL) were combined and heated to reflux under reduced pressure. Approx. 500 mL of distillate were collected and discarded while being continuously replaced with a solution of toluene in tert-BuOH. Vacuum was removed and distillate was switched to percolate through a column filled with molecular sieves and allowed to return to the vessel. After drying was complete, DPPA (52.4 mL; dissolved in 60 mL toluene) was added slowly to the slurry at 80° C. Upon complete conversion (HPLC), tert-BuOH was removed by vacuum distillation and continuously replaced with toluene. The mixture was cooled to room temperature and washed twice with 10% aqueous K₂HPO₄ (1×800 mL, 1×400 mL) and water (400 mL). The organic phase was heated and concentrated in vacuo to approx. 270 mL. Vacuum was removed and heptane (1.1 L) was added slowly at approx. 80° C., followed by seeds of 9 (˜1 g). The slurry was slowly cooled to room temperature and benzyl {(S)-1-[(1S,2R,4R)-2-tert-butoxycarbonylamino-4-(isopropyl-methyl-amino)-cyclo-hexyl]-2-oxo-pyrrolidin-3-yl}-carbamate 9 was isolated by filtration as a white solid (86.76 g, 78% yield).

Example 1, Alternative Preparation, Step 5: The tert-Butyl carbamate 9 (50 g) was dissolved in Toluene (500 mL) and i-PrOH (150 mL). The resulting solution was then heated to 60° C. Methanesulfonic acid (19.6 mL) was added below 65° C. Upon reaction completion (HPLC), the mixture was cooled to RT and triethylamine (69.4 mL) added slowly below 25° C. Acetic anhydride was then added below 25° C. After 1 h acetic acid (250 mL) was added below 25° C. The toluene phase was discarded and 2-methyl-THF (500 mL) was added to the aqueous phase. The mixture was stirred vigorously and basified with NaOH (25% aqueous solution) to pH 12. The aqueous phase was discarded and the organic layer was washed with brine (250 mL). The organic layer was concentrated under reduced pressure and continuously replaced with i-PrOH. The solution was cooled and filtered to provide benzyl {(S)-1-[(1S,2R,4R)-2-acetylamino-4-(isopropyl-methyl-amino)-cyclohexyl]-2-oxo-pyrrolidin-3-yl}-carbamate 10 in i-PrOH solution which was used directly in the hydrogenation.

Example 1, Alternative Preparation, Step 6: To a solution containing acetamide 10 (˜61 g) in i-PrOH (˜625 mL) was added 10% Pd/C wet catalyst (2.5 g) and the suspension was hydrogenated at 30 psig and approx. 25° C. for at least 2 h. Upon completion (HPLC), the catalyst was removed by filtration and the filtrate was concentrated to approx. 550 mL. Water (8.8 mL) was added, followed by 5.6 N hydrochloric acid in i-PrOH solution (69.5 mL). The resulting slurry was held at room temperature overnight. The product was isolated by filtration and the cake was rinsed with i-PrOH (2×100 mL) and dried in vacuo to constant weight at ˜50° C. to give N-[(1R,2S,5R)-2-((S)-3-amino-2-oxo-pyrrolidin-1-yl)-5-(isopropyl-methyl-amino)-cyclohexyl]-acetamide 11 (55.6 g, 97% yield) as its hydrochloric acid salt (73.6% free base assay, HPLC).

Example 1, Alternative Preparation, Step 7: To 6-trifluoromethyl-quinazolin-4-ol 12 (20.1 g) in MeCN (400 mL) was added 5.5 M solution of sodium methoxide in methanol (17.0 mL). The resulting suspension was distilled under reduced pressure and continuously replaced by MeCN to remove methanol. To the slurry was added DMF (1.4 g), followed by oxalyl chloride (13.0 mL) below 50° C. Upon reaction completion (HPLC), excess reagent was removed under reduced pressure to give ˜400 mL of slurry. The mixture was cooled to room temperature and washed with 10% aqueous K₂HPO₄ (1×1.0 L, 1×0.5 L) to afford 4-chloro-6-trifluoromethyl-quinazoline 13 (˜21.2 g) in approx. 450 mL of wet MeCN solution, which was used directly in the subsequent coupling reaction (HPLC purity 99.8%).

Example 1, Alternative Preparation, Step 8: To a mixture of acetamide 11 (28.5 g, HCl salt, 73.6% free base assay), acetonitrile (100 mL), N,N,-di-isopropyl-N-ethylamine (61 mL) at room temperature was added a solution of 13 (˜21.2 g) in MeCN (˜450 mL). The homogeneous mixture was held overnight. Upon reaction completion (HPLC), the mixture was concentrated in vacuo to approx. 125 mL. A 9.5% aqueous solution of acetic acid (240 mL) was added and the aqueous phase was extracted with methylene chloride. The aqueous phase was separated and methyl tert-butyl ether (450 mL) was added, followed by 2N aqueous lithium hydroxide solution to adjust to pH>11.5. The organic layer was separated, washed with water and filtered. Approx. half of the ether phase was diluted with methyl tert-butyl ether (˜250 mL) and concentrated in vacuo. Heptane (45 mL) was added slowly below 60° C., followed by seed crystals of Example 1 (0.4 g). Additional heptane (125 mL) was added and the mixture was slowly cooled to room temperature and the resulting slurry was held overnight. The product was isolated by filtration, the cake was washed with heptane and dried in vacuo to constant weight to give N-((1R,2S,5R)-5-(isopropylamino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)-quin-azolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide 14 (15.0 g, 85% yield).

Crystallization Procedures for Example 1Example 1, Production of bis-BSA salt and purification: The entirety of the amorphous free base from Example 1, Step 11 was dissolved in methanol (600 mL). The resultant solution was heated at 60° C. and charged with benzenesulfonic acid (2.5 eq). The mixture was cooled to room temperature and the resultant white solid was collected by filtration to yield the bis-benzene sulfonic acid salt of the title compound (95 g, 86%). This material was >99% pure by HPLC. This material was further purified by re-crystallization from 80/20 EtOH/H₂O, which provided the salt free from any residual methanol. HPLC purity=99.8%. ¹H NMR (500 MHz, D₂O) δ ppm 8.75 (1H, s), 8.66 (1H, s), 8.25 (1H, d, J=8.80 Hz), 7.90 (1H, d, J=8.80 Hz), 7.75 (4H, d, J=8.25 Hz), 7.43-7.57 (6H, m), 5.42 (1H, t), 4.33-4.44 (1H, m), 4.09-4.19 (1H, m), 3.83-3.91 (1H, m), 3.74-3.83 (2H, m), 3.61 (1H, t, J=11.55 Hz), 2.75 (3H, d, J=6.60 Hz), 2.61-2.70 (1H, m), 2.31-2.44 (1H, m), 2.20-2.27 (1H, m), 2.17 (2H, d, J=12.10 Hz), 1.94-2.04 (1H, m, J=12.65 Hz), 1.90-1.95 (3H, m), 1.72-1.91 (2H, m), 1.37 (3H, d, J=6.05 Hz), 1.29 (3H, d, J=6.60 Hz). Differential scanning calorimetry utilized a heating rate of 10° C./min and revealed a melting/decomposition endotherm with an onset temperature of 297.6° C. and a peak temperature at 299.1° C.

Example 1, Crystallization of the Free Base: A sample of the amorphous free base of N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide (1 g) was dissolved in dichloromethane (5 mL). The solution was charged with heptane (30 mL) and then warmed to distill the dichloromethane. The solution was cooled to 40° C.; a white solid precipitated. The suspension was heated to 90° C. and stirred for 2 h. The suspension was cooled to room temperature and filtered to provide the pure free base of the title compound. No residual solvent was apparent by ¹H-NMR.

PAPER

A concise bulk synthesis of stereochemically complex CCR2 antagonist BMS-741672 is reported. A distinct structural feature is the chiral all-cis 1,2,4-triaminocyclohexane (TACH) core, which was assembled through consecutive stereocontrolled heterogeneous hydrogenations: efficient Pt-catalyzed reduction of a β-enaminoester, directed by (S)-α-methylbenzylamine as a low-cost chiral template, and reductive amination of a 3,4-cis-disubstituted cyclohexanone over sulfided Pt/C introduced a tert-amine, setting the third stereocenter in the all-cis cyclohexane core. The heterogeneous catalysts were recycled. Ester hydrolysis produced a γ-amino acid, isolated as its Na salt. A challenging Curtius reaction to introduce the remaining C–N bond at C-2 was strongly influenced by the presence of the basic tert-amine, providing a stereoelectronically highly activated isocyanate. Detailed mechanistic and process knowledge was required to enable clean trapping with an alcohol (t-BuOH) while avoiding formation of side products, particularly an unusual carbamoyl phosphate. Deprotection, N-acetylation, and uncatalyzed S_NAr coupling with known 4-chloroquinazoline provided the final product. The resulting 12-step synthesis was used to prepare 50 kg of the target compound in an average yield of 82% per step.

Stereoselective Bulk Synthesis of CCR2 Antagonist BMS-741672: Assembly of an All-cis (S,R,R)-1,2,4-Triaminocyclohexane (TACH) Core via Sequential Heterogeneous Asymmetric Hydrogenations

Patents

Bristol-Myers Squibb, Paul Biondi Senior Vice President, Head of Business Development

About Bristol-Myers Squibb

Bristol-Myers Squibb is a global biopharmaceutical company whose mission is to discover, develop and deliver innovative medicines that help patients prevail over serious diseases. For more information, please visit www.bms.com or follow us on Twitter at http://twitter.com/bmsnews.

//////////   ////////////////BMS-741672, BMS 741672, BRISTOL MEYER SQUIB, PHASE 2,  type 2 Diabetes, Neuropathic Pain, Bristol-Myers Squibb
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