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Minisci reactions: Versatile CH-functionalizations for medicinal chemists

June 26, 2014 4:46 am / 1 Comment on Minisci reactions: Versatile CH-functionalizations for medicinal chemists

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DOI: 10.1039/C1MD00134E (Review Article) Med. Chem. Commun.,2011, 2, 1135-1161

Show Compounds Show Chemical Terms Show Biomedical Terms

Minisci reactions: Versatile CH-functionalizations for medicinal chemists

Matthew A. J. Duncton† *
Renovis, Inc. (a wholly-owned subsidiary of Evotec AG), Two Corporate Drive, South San Francisco, CA 94080, United States. E-mail: mattduncton@yahoo.com; Tel: +1 917-345-3183

Received 24th May 2011 , Accepted 3rd July 2011

First published on the web 22nd August 2011

http://pubs.rsc.org/en/content/articlehtml/2011/md/c1md00134e

The addition of a radical to a heteroaromatic base is commonly referred to as a Minsici reaction. Such reactions constitute a broad-set of selective CH-functionalization processes. This review describes some of the major applications of Minisci reactions and related processes to medicinal or biological chemistry, and highlights some potential developments within this area.

Introduction

The aim of this review is to summarize the use of Minisci reactions within medicinal chemistry, and to highlight some future opportunities to continue progression of this chemistry. As such, it is not an aim that detailed mechanistic information, or a comprehensive list of examples be described. For this, the reader is directed to excellent articles from Minisci, Harrowven and Bowman.^1–3 Rather, the review is written to show that Minisci reactions are extremely valuable CH-functionalization processes within medicinal chemistry. However, their use has been somewhat under-utilized when compared with other well-known selective transformations (e.g. palladium-catalysed cross-couplings). Therefore, it is hoped that in the future, Minisci chemistry will continue to develop, such that the reactions become a staple-set of methods for medicinal and biological chemists alike.

To aid discussion, the review is divided in to several sections. First, some historical perspective is given. This is followed by a discussion of scope and limitations. The main-body of the review describes some specific examples of Minisci reactions and related processes, with a focus on their use within medicinal, or biological chemistry. Finally, brief mention is given to potential future applications, some of which may be beneficial in providing ‘high-content’ diverse libraries for screening.

http://pubs.rsc.org/en/content/articlehtml/2011/md/c1md00134e

…………………….

WIKI

The Minisci reaction is a named reaction in organic chemistry. It is a radical substitution to an aromatic compound, in particular to a heteroaromatic base, that introduces an alkyl group. The reaction was published about in 1971 by F. Minisci.^[1] The aromatic compound is generally electron-deficient and with N-aromatic compounds the nitrogen atom is protonated.^[2] A typical reaction is that between pyridine and pivalic acid to 2-tert-butylpyridine with silver nitrate, sulfuric acid and ammonium persulfate. The reaction resembles Friedel-Crafts alkylation but with opposite reactivity and selectivity.^[3]

The Minisci reaction proceeds regioselectively and enables the introduction of a wide range of alkyl groups.^[4] A side-reaction is acylation.^[5] The ratio between alkylation and acylation depends on the substrate and the reaction conditions. Due to the simple raw materials and the simple reaction conditions the reaction has many applications in heterocyclic chemistry.^[6]^[7]

Mechanism

A free radical is formed from the carboxylic acid in an oxidative decarboxylation with silver salts and an oxidizing agent. The oxidizing agent reoxidizes the silver salt. The radical then reacts with the aromatic compound. The ultimate product is formed by rearomatisation. The acylated product is formed from the acyl radical.^[4]^[5]

References

F. Minisci, R. Bernardi, F. Bertini, R. Galli, M. Perchinummo: Nucleophilic character of alkyl radicals—VI : A new convenient selective alkylation of heteroaromatic bases, in: Tetrahedron 1971, 27, 3575–3579.
Minisci reaction Jie Jack Li in Name Reactions 2009, 361-362, doi:10.1007/978-3-642-01053-8_163
Strategic applications of named reactions in organic synthesis: background and detailed mechanisms László Kürti, Barbara Czakó 2005
F. Fontana, F. Minisci, M. C. N. Barbosa, E. Vismara: Homolytic acylation of protonated pyridines and pyrazines with α-keto acids: the problem of monoacylation, in: J. Org. Chem. 1991, 56, 2866–2869; doi:10.1021/jo00008a050.
M.-L. Bennasar, T. Roca, R. Griera, J. Bosch: Generation and Intermolecular Reactions of 2-Indolylacyl Radicals, in: Org. Lett. 2001, 3, 1697–1700; doi:10.1021/ol0100576.
P. B. Palde, B. R. McNaughton, N. T. Ross, P. C. Gareiss, C. R. Mace, R. C. Spitale, B. L. Miller: Single-Step Synthesis of Functional Organic Receptors via a Tridirectional Minisci Reaction, in: Synthesis 2007, 15, 2287–2290; doi:10.1055/s-2007-983792.
J. A. Joules, K. Mills: Heterocyclic Chemistry, 5. Auflage, S. 125–141, Blackwell Publishing, Chichester, 2010, ISBN 978-1-4051-9365-8.

NERATINIB, HKI 272, ..Puma presents positive results from phase II trial of its investigational drug PB272

April 11, 2014 4:25 am / Leave a comment

NERATINIB

(2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide

[(2E)-N-[4-[[3-chloro-4- [(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4- (dimethylamino)but-2-enamide].

(E)-N- {4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6- quinolinyl} -4-(dimethylamino)-2-butenamide

FOR METASTATIC BREAST CANCER.PHASE 3

CAS 698387-09-6,

PFIZER …….INNOVATOR

HKI-272, HKI 272, Neratinib(HKI-272), Neratinib, HKI-272, 698387-09-6, HKI272, HKI 272, HKI-272,

HKI-272
PB-272
PF-0528767
WAY-179272
WAY-179272-B (maleate)

http://www.pfizer.com/files/news/asco/neratinib_fact_sheet_2010.pdf

Molecular Formula: C₃₀H₂₉ClN₆O₃

Molecular Weight: 557.04266

Puma Biotechnology, a development stage biopharmaceutical company, announced the presentation of positive results from the phase II clinical trial of Puma’s investigational drug PB272 (neratinib) for the neoadjuvant treatment of breast cancer(I-SPY 2 TRIAL) in an oral presentation at the American Association for Cancer Research (AACR) Annual Meeting 2014 in San Diego, California.

READ AT

http://www.pharmabiz.com/NewsDetails.aspx?aid=81352&sid=2

Neratinib – малая молекула класса 6,7-дизамещенных-4-anilinoquinoline-3-карбонитрила –
ингибитор тирозинкиназы HER-2 с потенциальной противоопухолевой активностью.
Neratinib связывается с рецептором HER-2 необратимо, снижая аутофосфорилирование в клетках,
и направляя остаток цистеина в АТФ-связывающего кармана рецептора.
Обработка раковых клеток с этим агентом приводит к торможению передачи сигнала клеточного цикла и
в конечном счете уменьшает клеточную пролиферацию.
Neratinib ингибирует рецептор EGFR киназы и распространение EGFR-зависимых клеток.

Neratinib – small molecule 6,7-disubstituted class of 4-anilinoquinoline-3-carbonitrile –
inhibitor of the HER-2 tyrosine kinase with potential antitumor activity.
Neratinib binds to the receptor HER-2 irreversible, reducing autophosphorylation in cells
and directing the cysteine residue in the ATP-binding pocket of the receptor.
Treatment of cancer cells with this agent leads to inhibition of signal transduction and cell cycle ultimately reducescell proliferation.
Neratinib inhibit EGFR kinase receptor and distribution of EGFR-dependent cells.

EVER THE POST WAS WRITTEN IT GOT FDA APPROVAL

NERATINIB MALEATE

PUMA BIOTECH

Image result for NERATINIB

Nerlynx	FDA	7/17/2017	To reduce the risk of breast cancer returning Press Release Drug Trials Snapshot

LINK…https://newdrugapprovals.org/2014/04/11/neratinib-hki-272-puma-presents-positive-results-from-phase-ii-trial-of-its-investigational-drug-pb272/

Neratinib (HKI-272) is a tyrosine kinase inhibitor^[1]^[2] under investigation for the treatment breast cancer^[3] and other solid tumours.

It is in development for the treatment of early- and late-stage HER2-positive breast cancer.^[4]

Like lapatinib and afatinib, it is a dual inhibitor of the human epidermal growth factor receptor 2 (Her2) and epidermal growth factor receptor (EGFR) kinases.^[5]

Neratinib is a signal transduction pathway inhibitor and an irreversible inhibitor of HER-2 in early clinical trials for the treatment of advanced solid tumors in combination with paclitaxel. The company had also been developing the drug candidate for the treatment of non-small cell lung cancer (NSCLC); however, no recent development has been reported for the indication. In 2011, Pfizer discontinued development of the compound as monotherapy for the treatment of ErbB-2-positive breast cancer. A phase III clinical trial had been under way. Dana-Farber Cancer Institute is studying the compound for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer and brain metastases. Puma Biotechnology is conducting phase III trials for use as third-line treatment of HER2-positive metastatic breast cancer and phase II trials for the treatment of patients with HER2 activating mutations in Non-Small Cell Lung Cancer (as monotherapy or in combination with temsirolimus) as well as other solid tumors.

The drug candidate is a synthetic compound developed based on the chemical structure of EKB-569, an inhibitor of the epidermal growth factor receptor (EGFR) currently under clinical evaluation for the treatment of EGFR-positive tumors. In previous trials, neratinib inhibited kinase activity of HER-2 and EGFR by 50% while showing no effects on several serine-threonine kinases, and also inhibited the proliferation of two HER-2-positive breast cancer cell lines and a mouse fibroblast cell line transfected with the HER-2 oncogene.

In 2011, the compound was licensed to Puma by Pfizer for global development and commercialization.

HKI-272 (neratinib) has been described for the treatment of neoplasms [US Patent 6,288,082]. Neratinib is a potent irreversible pan erbB inhibitor. Neratinib is an orally available small molecule that inhibits erbB-1 , erbB-2 and erbB-4 at the intracellular tyrosine kinase domains, a mechanism of action that is different from trastuzumab. Neratinib reduces erbB-1 and erbB-2 autophosphorylation, downstream signaling, and the growth of erbB-1 and erbB-2 dependent cell lines.

Preclinical data suggest that neratinib will have antitumor activity in erbB-1 – and/or erbB 2-expressing carcinoma cell lines, with cellular IC50 <100 nM [Rabindran SK, et al. Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase. Cancer Research. 2004;64(1 1 ):3958-65].

Neratanib is being developed by Puma Biotechnology. It will be included in the forthcoming I-SPY2 breast cancer trial.^[6]

neratinib refers to HKI-272, which has the following core structure:

in its free base form. Optionally, a pharmaceutically acceptable salt or hydrate thereof may be used. The core structure represented above is a particular HKI-272 compound, called HKI-272 or neratinib, which has the chemical name [(2E)-N-[4-[[3-chloro-4- [(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4- (dimethylamino)but-2-enamide]. Although currently less preferred, another HKI-272 compound may be used in the place of neratinib. “A HKI-272 compound” refers, in one embodiment, to a compound derived from the core structure of neratinib shown above

The preparation of HKI-272 compounds, of which neratinib is a species, are described in detail in US Patent Application Publication No. 2005/0059678, which is hereby incorporated by reference. See, also, US Patent Nos. 6,288,082, US Patent No. 6,002,008, US Patent No. 6,297,258 and US Patent Application Publication No. 2007/0104721 , which are hereby incorporated by reference. The methods described in these documents can also be used to prepare neratinib and/or the other HKI-272 and substituted 3-quinoline compounds used herein and are hereby incorporated by reference. In addition to the methods described in these documents, International Patent Publication Nos. WO-96/33978 and WO-96/33980, which are hereby incorporated by reference, describe methods that are useful for the preparation of these HKI-272 compounds. Although these methods describe the preparation of certain quinazolines, they are also applicable to the preparation of correspondingly substituted 3- cyanoquinolines and are hereby incorporated by reference.

The term “treating” or “treatment” refers to the administration of the neratinib to a subject to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with neoplasms

(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4- (dimethylamino)-2-butenamide is an irreversible inhibitor to Her-2 (also known as ErbB-2 or neu) kinase, a member of the epidermal growth factor receptor (EGFR) family. EGFR family members have been implicated in tumorigenesis and associated with poor prognosis in tumor types in humans. The structure of the (E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano- 7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide in the form of a free base is shown below:

The compound (E)-N-{4-[3-chloro-4 J-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}- 4-(dimethylamino)-2-butenamide in the form of a free base is described in U.S. Patent No. 6,288,082. The compound is classified, based on the Biopharmaceutical Classification System, as a BCS Class IV compound (low water solubility and low permeability). The free base has low solubility in water, with a water solubility of about 1 μg/ml_ at about pH 7. The water solubility increases with decreasing pH as the compound becomes ionized. This compound is water soluble at gastrointestinal pH, and dissolution is not rate limiting.

Research on Chemical Intermediates, 2012, 09(22),6168
10.1007/s11164-012-0822-4
The Wittig–Horner reaction for the synthesis of neratinib

…………………

U.S. Patent No. 6,288,082

http://www.google.co.in/patents/US6288082

…………

WO2010048477A2

http://www.google.com/patents/WO2010048477A2?cl=en

U.S. Pat. No. 7,126,025 discloses certain novel 4-amino-2-butenoyl chlorides, processes for their preparation and their use as intermediates in the synthesis of pharmaceutically active protein kinase inhibitors, including but not limited to for example HKI-272 and EKB-569.

The sequence illustrated below and summarized in Scheme 1 describes one existing process for preparing HKI-272, (E)-Λ/-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-3- cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-enamide in the form of the maleate salt, also known as Neratinib™.

1 95 eq (COCI)₂, cat DMF

Step 5 OH 16 h HCI

Scheme 1

Scheme 2

Scheme 3. Formation of acid chloride with SOCI₂ in DMAc and coupling with a substituted aniline.

SOCl₂

_/^Nv^-^’C0₂H HCI DMAc HCI

Scheme 4. Formation of the MW 638 impurity.

Example 4: Process 3

4-Dimethylaminocrotonoyl chloride hydrochloride and its coupling with 6-amino- 4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-7-ethoxyquinoline-3-carbonitrile (procedure with thionyl chloride and DMAc).

A suspension of 4-dimethylaminocrotonic acid (17.0 g, 97.5 mmol) in DMAc (170 ml_) was cooled to -15 ⁰C under nitrogen atmosphere. Neat thionyl chloride (12.8 g, 7.83 mmol) was added to the slurry at a rate to maintain the temperature in the reactor in the range of -18 to -14 ⁰C (moderate exotherm). The reaction mixture was held at -17 to -15 ⁰C for 4 hrs. A solution of the aminoquinoline (36.2 g, 81.3 mmol) in DMAc (440 ml_) was added to the reactor maintaining the temperature in the -14 to -19 ⁰C range. The resulting mixture was held for 18 hr at approximately -15 ⁰C. At this point HPLC analysis showed residual aniline level at 2.5%. The thick suspension of the hydrochloride salt of the coupled product was quenched with water (200 ml_) maintaining the batch temperature between -5 and -16 ⁰C. The pH of the resulting clear solution was adjusted to 1 1 with a 13% aqueous solution of NaOH (approx. 210 ml_ of the solution was added). The suspension was further diluted with water (350 ml_) and the solids were filtered on a polypropylene cloth filter. The cake was washed with water until neutral pH of the washes and dried first in the nitrogen flow on the filter and then on a tray in vacuum at 45 to 50 ⁰C to afford crude (.=)-/\/-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-3-cyano-7- ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-enamide (42.0 g, 91 %) as a bright-yellow crystalline solid.

………………..

WO2004066919A2

http://www.google.com/patents/WO2004066919A2?cl=en

Reaction Scheme Example 1 :

SCHEME 1

(“)

6-(4-N,N-dimethylarninocrotonyt)amido- 4-(4-benzyioxy-3-chloro)arniπo-3-cyano- 7-ethoxyquiπoline, WAY-177820 C₃₁H_3[1CIN₅θ₃ MW 556.07

A suspension of 4-N,N-dimethylaminocrotonic acid hydrochloride in acetonitrile and a catalytic amount of DMF is cooled to 0-10° C. Oxalyl chloride (0.95 eq) is added dropwise and the mixture warmed to 25-30° C and stirred until the chlorinating agent is completely consumed. The light yellow solution is checked for complete consumption of oxalyl chloride by HPLC then cooled to 0-10° C. A cooled solution (0-10° C) of 4-[4-benzyloxy-3-chloro]amino-6-amino-3-cyano-7- ethoxyquinoline in NMP is added dropwise and the mixture is stirred until < 2% of the starting aniline remains. The mixture is added to saturated aqueous sodium bicarbonate, the yellow precipitates are filtered and washed with water. The wet solids are heated to reflux in acetonitrile and clarified hot to remove insolubles. The solution is cooled, the precipitated product filtered and washed with cold acetonitrile. The product is dried (40-50° C, 10 mm Hg, 24 hours) to obtain the final product. Reaction Scheme Example 2:

A solution of 4-N,N-dimethylaminocrotonic acid hydrochloride in tetrahydrofuran (THF) and a catalytic amount of dimethyiformamide (DMF) is cooled to 0-5^s C. Oxalyl chloride (0.95 eq) is added dropwise and the mixture warmed to 25-30²C and stirred until the chlorinating agent is completely consumed. The orange solution is checked for complete consumption of oxalyl chloride by high- pressure liquid chromatography (HPLC) then cooled to 0-5² C. A solution of 4-[4-(2- pyridylmethoxy)-3-chloro]amino-6-amino-3-cyano-7-ethoxyquinoline is added dropwise and the mixture is stirred until < 0.5% of the starting aniline remains. The reaction is quenched with water and the mixture warmed to 40^s C. Aqueous sodium hydroxide is added to bring the pH to 10-11. The resulting precipitates are filtered hot and washed with water. The wet solids are heated to reflux (70-75⁹ C) in acetonitrile:THF (1 :5:1) and the solution cooled slowly to room temperature. The product is filtered and washed with acetonitrile.THF. The product is dried (50^e C, 10 mm Hg, 24 hours) to 80-85% yield.

Reaction Scheme Example 3:

4-Dirnethy!amino-but-2-enoic acid |4-(3-chloro-4-fluoro-phenylamino)-3-cvano-7- ethoxy-quinolin-6-vHamide

A. 4-(dimethylamino)-2-butenoyl chloride hydrochloride

A 1 L multi-neck flask equipped with agitator, thermometer, addition funnel, and nitrogen protection is charged with acetonitrile (0.67 kg, 0.85 L) followed by adding dimethylformamide (0.00086 kg, 0.91 mL, d=0.944 g/mL). At ambient temperature, is added 4-dimethylaminocrotonic acid hydrochloride (0.0709 kg) and the mixture stirred until homogeneous. Cool the reaction mixture to (0-10° C) and add oxalyl chloride (0.0473 kg, 0.0325 L, d = 1.45 g/mL) dropwise over (20 minutes) at (0-10° C) followed by a rinse with acetonitrile (0.02 kg, 0.03 L). The temperature (0-10°C) is maintained for about (20 minutes). The temperature of the reaction mixture is adjusted to (22-26° C) over (20 minutes) and maintained over (2 hours). The temperature of reaction mixture is adjusted to (40-45° C) and held for about (5 minutes). Cool the light suspension to about (20-25° C) and check for reaction completion by high-pressure liquid chromatography (HPLC). The reaction is complete when there is < 15 % of the starting material (4-dimethylaminocrotonic acid hydrochloride) present and/or < 2 % of oxalyl chloride (detected as the dimethyl oxalate).

B. 4-Dimethy!amino-but-2-enoic acid |4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7- ethoxy-quinolin-6-yll-amide (crude)

A 3 L multi-neck flask equipped with agitator, thermometer, dip tube, and nitrogen protection is charged N-methyl pyrrolidinone (0.77 kg, 0.75 L, d=1.033 g/mL). At ambient temperature is added 4-[3-chloro-4-fluorophenyl]amino-6-amino-3-cyano-7- ethoxy quinoline (0.0748 kg). The reaction mixture is heated to 40-45° C and maintained for about (15 minutes). The reaction mixture is cooled to (0-10° C) and the light suspension of 4-(dimethylamino)-2-butenoyl chloride hydrochloride in CH₃CN added via dip tube and positive nitrogen pressure, over (30-45 minutes) while maintaining the temperature (0-10° C) for at least (2 hours). Reaction completion is monitored by HPLC. The reaction is complete when there is < 2 % of the starting material (4-[3-chloro-4-fluorophenyl]amino-6-amino-3-cyano-7-ethoxy quinoline) present. To a 12 L multi-neck flask equipped with agitator, thermometer, dip tube, and nitrogen protection is charged with water (2.61 kg, 2.61 L) and sodium bicarbonate (0.209 kg) with stirring until a solution is obtained followed by cooling to (20-24° C) to which is transferred the reaction mixture above which contains < 2 % of the starting material (4-[3-chloro-4-fluorophenyl]amino-6-amino-3-cyano-7-ethoxy quinoline), via dip tube and positive nitrogen pressure, to the 12 L flask over about (45-60 minutes) while maintaining the temperature at (20-24° C). The temperature is maintained at (20-24° C) for at least (1 hour). Filter the reaction mixture on a Buchner funnel, rinse with water (3 x 0.40 kg, 3 x 0.40 L), and maintain suction until dripping stops. Dry the product in a vacuum oven at about (50° C) and about (10 mm Hg) for about (28-30 hours). The yield is 78.5 g (86%) at 79.7% strength and 12.3% total impurities.

4-Dimethylamino-but-2-enoic acid r4-(3-chloro-4-fluoro-phenylamino -3-cyano-7- ethoxy-quinolin-6-vn-amide (purified small scale)

First crop: A 6 L multi-neck flask equipped with agitator, condenser, temperature probe, and nitrogen protection is charged with acetonitrile (3.14 kg, 4.00 L) followed by adding 4-dimethylamino-but-2-enoic acid [4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7- ethoxy-quinolin-6-yl]-amide (0.16 kg, 0.167 moles). Heat the mixture to (75-80° C) and hold it for (1 hour). Cool the mixture to (70-75° C) and filter on a pad of diatomaceous earth to remove inorganic salts. Wash the pad with acetonitrile (2 x 0.24 kg, 2x 0.30 L), preheated to (70-75° C). Concentrate the filtrate at (20-30 mm Hg) and a maximum temperature of (40-45° C) to a volume of ( 1.2 L). To the concentrate (slurry) add prefiltered tetrahydrofuran (0.53 kg, 0.60 L). Heat to (65-70° C) to obtain a complete solution. Cool the mixture to (40-45° C) over (0.3 hours). Add seeds and continue cooling to (20-25° C) over (1 hour). Hold at (20-25° C) for a minimum of (18 hours). Collect the solid on a Buchner funnel and wash the collected solid with a prefiltered and precooled at (0-5° C) mixture of acetonitrile/tetrahydrofuran (2/1 by volume) (2 x .06 kg, 2 x 0.08 L). Dry the product in a vacuum oven at (50° C) and (10 mm Hg) for (48 hours) to a loss on drying (LOD) of less than (0.5 %). All washes and concentrates (mother liquors) are saved for further purification.

Second crop:

A 3 L multi-neck flask equipped with agitator, temperature probe, nitrogen protection, and charge with the mother liquors and washes from above. Concentrate by distillation at (20-30 mm Hg) and a maximum temperature of (40-45° C) to a volume of (0.50 L). Collect the solid on a Buchner funnel and wash the solid with prefiltered acetonitrile (0.04 kg, 0.05 L). Dry the solid product in a vacuum oven at (50° C) and (10 mm Hg) for (18 hours). A 1 L multi-neck flask equipped with agitator, condenser, temperature probe, nitrogen protection and charge with prefiltered acetonitrile (0.47 kg, 0.60 L), and the collected solid is heated as a suspension to (70-75° C) over (0.5 hours). Add prefiltered tetrahydrofuran (0.03 kg, 0.03 L) to the suspension while maintaining the temperature at (70-75° C). Cool the solution to (40-45° C) and add seed crystals. Continue cooling to (20-25° C) over (1 hour) and hold for (2 hours). Collect the resulting solid on a Buchner funnel and wash the collected solid with a prefiltered and precooled to (5° C) mixture of acetonitrile/tetrahydrofuran (20/1 by volume) (2 x 0.02 kg, 2 x 0.03 L). Dry the collected solid in a vacuum oven at (50° C) and (10 mm Hg) for (24 hours) to an LOD of less than (0.5 %). The combined yield is 27.5 g + 30.5 g (73%) in 96.2-98.4% strength and 1.5-1.7% total impurities by high pressure liquid chromatography (HPLC).

4-Dimethylamino-but-2-enoic acid f4-(3-chloro-4-fluoro-phenylamino)-3-cvano-7- ethoxy-quinolin-6-vn-amide (purified larger scale)

Acetonitrile, practical (34.0 kg) and 4-dimethylamino-but-2-enoic acid [4-(3- chloro-4-fluoro-phenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide (2.69 kg crude, 1.53 kg at 100% strength) are charged to a purged (100 L) reactor. Acetonitrile, practical (2.0 kg) is used as rinse for funnel and vessel walls. The brown suspension is heated at 70 to 76° C using a jacket temperature not exceeding 85° C, then held at the latter temperature for a minimum of 45 minutes, not exceeding 60 minutes. The resulting suspension is then filtered on the warm-jacketed (70-76° C) 14″ Aurora filter, while maintaining the batch temperature at 70 to 76° C. The filtrates are collected by pump into a purged (100 L) receiver, while keeping their temperature below 50° C. The diatomaceous earth pad is then washed with warm (70 to 76° C) acetonitrile, practical (3 x 2.5 kg). The filtrates and washes in (100 L) receiver are cooled to 20 to 26° C, then transferred into a stainless steel drum. Acetonitrile, practical (2.0 kg) is used as rinse. After cleaning and purging both vessels, the contents of the stainless steel drum is transferred into the (100 L) receiver. Acetonitrile, practical (2.0 kg) is used as a rinse. The batch is heated at 70 to 76° C without exceeding jacket temperature of 85° C. The batch is filtered by pump through a .0 micron single cartridge filter, while maintaining the contents at 70 to 76° C. Warm (70-76° C) acetonitrile, practical (4.0 kg) is used as rinse for vessel, filters, pump and lines. The filtrate and rinse are collected and maintained below 50° C. The batch is adjusted to 10 to 16° C, then concentrated by vacuum distillation to 28 to 33 L volume: expected distillation temperature 20 to 30° C, distillate volume 32 to 37 L. The suspension is heated to 64 to 70° C without exceeding jacket temperature of 85° C. The resulting solution is cooled to 40 to 46° C, then seeded using 4-dimethylamino-but-2~enoic acid [4-(3-chloro-4-fluoro-phenylamino)-3-cyano- 7-ethoxy-quinolin-6-yl]-amide, purified (0.5 g). The mixture is cooled to 20 to 26° C over 1 hour, then held at the latter temperature for a minimum of 2 hours. The suspension is then cooled at -3 to 3° C over 1 hour, then held for a minimum of 1 hour. The solid product is collected on a 16″ Buchner, then washed with cold (0-5° C) acetonitrile-tetrahydrofuran (20-6 v/v) mixture (2 x 2.5 kg). The wet collected solid is recrystallized once more from acetonitrile-tetrahydrofuran (20-6 v/v) to desired purity. The material is dried in a vacuum oven first at 35 to 45° C (target 40° C) for 4 hours, liquid ring pump, then 45 to 55° C (target 50° C) for 4 hours. After high vacuum is applied at the latter temperature, until LOD <0.5% (90° C, 2 hours, full vacuum) and each of acetonitrile, tetrahydrofuran and 1-methyl-2-pyrrolidinone are below 0.2%. The purified drug substance is milled (Comil), then blended. The yield is 1.10 kg (70.1 %, corrected for starting material). The strength of the material is 98.3% and a total impurities of 1.27%.

………………….

N OXIDE

http://www.google.com/patents/US20130225594

EXAMPLE 19 Formula 57-Compound 19a

19a: (E)-4-((4-((3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)amino)-N,N-dimethyl-4-oxobut-2-en-1-amine oxide

To a solution of compound A (200 mg, 0.36 mmol, 1.0 eq) in CH₂Cl₂(20 mL) was added m-CPBA (74 mg, 0.43 mmol, 1.2 eq) and the resulting mixture was stirred at room temperature for 4 h. A saturated aqueous solution of NaHCO₃(20 mL) was then added and the organic layer was separated, dried over Na₂SO₄and concentrated under reduced pressure. The residue was purified by preparative TLC (CH₂Cl₂/MeOH, 10/1, v/v) to give (E)-4-((4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)amino)-N,N-dimethyl-4-oxobut-2-en-1-amine oxide (20 mg, 10%) as a yellow solid.

LC-MS (Agilent): R_t3.03 min; m/z calculated for C₃₀H₂₉ClN₆O₄[M+H]⁺573.19. found 573.2.

¹H NMR: (400 MHz, CD₃OD) δ (ppm): 8.98 (s, 1H), 8.57 (m, 1H), 8.39 (s, 1H), 7.92 (td, J=7.2, 1.6 Hz, 1H), 7.72 (d, J=8.0 Hz, 1H), 7.39 (m, 1H), 7.36 (d, J=2.4 Hz, 1H), 7.28 (s, 1H), 7.24-7.13 (m, 3H), 6.74 (d, J=15.6 Hz, 1H), 5.29 (s, 2H), 4.32 (q, J=6.8 Hz, 2H), 4.20 (d, J=7.2 Hz, 2H), 3.28 (s, 6H), 1.57 (t, J=6.8 Hz, 3H).

……………

http://www.google.fm/patents/EP1883631A1?cl=en

Scheme 2 and Scheme 3. Scheme 2

e-Acelamlno^chloro-S-cyano- 7-ethoxy quinoliπe C,₄Hi₂CIN₂O2 +

MW 289.72

25 °C, 5 h 3-Chloro-4-(3-fluorobenzyl)oxy- anillne

C₁₃Hi₁CIFNO

MW 251.69

2 h

free base

Scheme 3

6-Acetamlno-4-chloro-3-cyanc~ 7-elhoxy qulnollne C,₄H₁₂CIN₂O2 +

MW 28972

3-Chlorc-4-fluoronitrobenzene 2-Pyπdyl carblnol 3-Chloro-4-(3-pyndinylmethoxy) 3-Chloro-4-(2-pyrtdlnylmethewy)- C₆H₃CIFNO₂ C₆H₇NO nitrobenzene anlllne

MW 17555 MW 109 13 C₁₂H₉CIN₂O₃ C₁₂H₁₁CIN₂O d=1 1131 g/ml MW 26467 (EM 264) MW 23469

1 h

(HCI salt)

free base

maleate

Example 1

[0078] Synthesis of 3-chloro-4-(2-pyridylmethoxy)nitrobenzene

[0079] 2-pyridinyl carbinol (31.08 g, 1.05 eq) was dissolved in ACN (750 mL) and KOH flakes (85%) were added (20.6 g, 1.25 eq.). The resulting suspension was warmed to 35 °C. A solution of the 3-chloro-4-fluoronitrobenzene (50.0 g, 0.285 mol) in ACN (250 mL) was added at 35-40 °C. The mixture was held for 14 hours. The mixture was then cooled back to 20-25 °C, quenched with H₂O (IL) and the resulting slurry filtered and washed with H₂O (3 x 100 mL). The resulting product was isolated as a tan solid in 93% yield with a greater than 99.5% purity as determined by HPLC area. Example Ia

[0080] To accomplish the analogous synthesis of 3-chloro-4-(3-fluorobenzyloxy) nitrobenzene, 3-fluorobenzyl alcohol (0.30 kg, 2.39 mole, 1.05 eq) was dissolved in ACN (6.0 L) and to it was added potassium hydroxide flakes (85%) (0.16 kg, 1.25 eq). The resulting suspension was warmed to 35 ⁰C. A solution of the 3-chloro-4-fluoronitrobenzene (0.40 kg, 2.28 mol) in ACN (2.0 L) was added at 35-40 °C. The mixture was held for 18 hours. The mixture was then cooled back to 20-25 °C, quenched with water (8 L) and the resulting slurry filtered and washed with water (2 x 0.40 L). The resulting product was dried at 45 °C, under 10 mm Hg pressure, for 25 hours to give 0.59 kg (92% yield). Example Ib

[0081] To prepare 4-(benzyloxy)3-chloronitrobenzene, benzyl alcohol (0.34 kg, 3.14 mole, 1.10 eq) was dissolved in acetonitrile (1.70 L) and to it was added potassium hydroxide flakes (85%) (0.24 kg, 1.50 eq). The resulting suspension was warmed to 25 ⁰C. A solution of the 3- chloro-4-fluoronitrobenzene (0.50 kg, 2.85 mol, 1.0 eq) in acetonitrile (0.75 L) was added keeping the pot temperature < 45 ⁰C. The mixture was held for 14 h. The mixture was then cooled back to 0-15 ⁰C, quenched with water (2.5 L) and the resulting slurry was filtered and washed with water (2 x 0.50 L). The resulting product was dried at 50 ⁰C, under 10 mm Hg pressure, for 24 hours to give 0.73 kg (97% yield). [0082] Experimental results for the reaction of Example 1 with different bases and solvents are shown in Table 1. The last three entries on Table 1 are large scale runs in which a 5% excess of pyridyl carbinol was used. Table 1 – Preparation of Nitroaryl Intermediate

NA = not applicable

RT = room temperature (20-25 °C)

Example 2

[0083] Preparation of 3-chloro-4-(2-pyridyhnethoxy)aniline from the nitrobenzene product of

Example 1 was accomplished with catalytic hydrogenation using platinum on carbon.

[0084] A typical hydrogenation was done using 6 volumes of THF, 2% by weight of 5%Pt/C (50% water wet), at 25 psi and at 25-30 ⁰C for approximately 4-6 hours. The reaction is slightly exothermic and the temperature will rise to about 30-35 °C. Cooling is necessary to maintain the temperature below 30 ⁰C.

[0085] As a specific example, a mixture of 3-chloro-4-(2-pyridylmethoxy)nitrobenzene (0.15 kg, 0.57 mole) and 2% (w/w) of 5% Pt/C (6.0 g) in tetrahydrofuran (0.90 L) was hydrogenated at 25 psi for at least 5 hours. The mixture was filtered through a celite pad and washed with tetrahydrofuran (0.60 L). The filtrate was distilled to a volume of about 0.75 L and ethanol (1.12 L) was added. Distillation was continued to a volume of about 0.75 L and ethanol (2.85 L) was added. The mixture may be used “as is” in the step of Example 3 below. Example 2 a

[0086] To accomplish an analogous synthesis of 3-chloro-4-(3-fluorobenzyloxy)aniline, zinc (0.464 kg) was added to a mixture of 3-chloro-4-(3-fluorobenzyloxy)nitrobenzene (0.40 kg, 1.42 mole) and ethanol (4.0 L). The mixture was heated to 40-50 ^°C. A solution of ammonium chloride (0.152 kg) in water (0.80 L) was added over 0.5 hour keeping the pot temperature at 40-50 ^°C. The mixture was stirred for 2 hours, filtered and washed with hot (40-50 ^°C) ethanol (2 x 0.40 L). The filtrate was distilled to a volume of about 0.80 L and 2- methyltetrahydrofuran (2.0 L) was added to dissolve the product. Water (0.80 L) and saturated brine (0.40 L) were added and the layers separated. The organic layer was washed with water (0.60 L), and distilled to a volume of about 0.40 L. Ethanol (2.0 L) was added and distillation continued to a volume of 1.2 L. Example 2b

[0087] To prepare 4-(benzyloxy)-3-chloroaniline, a mixture of 4-(benzyloxy)-3- chloronitrobenzene (0.325 kg, 1.23 mole, 1.0 eq) and 1% (w/w) of 5% Pt/C (3.25 g) in isopropanol (3.25 L) was hydrogenated at 25 psi for a minimum of 4.5 h. The mixture was filtered through a celite pad and washed with isopropanol (2.0 L). The filtrates were used as is in the next step.

[0088] Performing the hydrogenation in isopropyl alcohol (PA), methanol (MeOH), or ethanol

(EtOH) may result in the product being contaminated with late eluting impurity that partially precipitates out on standing in solution. It was found that performing the hydrogenation in a solvent where both the product and starting material are soluble, such as tetrahydrofuran

(THF), resulted in greater product purity and required much less solvent. Thus, THF is a preferred solvent for this step. Experimental results showing the effect of different reaction conditions are shown in Table 2. For the larger scale runs, the first aniline intermediate was not isolated (“NI”) before proceeding with the next step.

Table 2 – Hydrogenation to Form First Aniline Intermediate

* Solid impurities noted after reaction completion. ** percent by weight of starting material. Example 3

[0090] Following hydrogenation to form the first aniline intermediate, acid catalyzed coupling was performed to prepare 4~[3-chloro-4-(2-pyridylmethoxy)anilino]-3-cyano-7-ethoxy-6-N- acetylaminoquinoline, as shown below:

[0091] To perform the coupling reaction, the two reactants were heated together in alcohol at 65-78°C over 4-6 hours, yielding the product. The reaction begins as an amber slurry and thickens to a lighter beige slurry as it approaches completion. Upon scaling up from 75 g to 350 g, it proved necessary to add a catalytic amount (0.025 eq.) of methanesulfonic acid to initiate the reaction. As a specific example, 4-chloro-3-cyano-7-ethoxy-6-N- acetylaminoquinoline (0.141 kg, 0.49 mole) was added to the mixture of Example 2, followed by ethanol (0.037 L) to give a suspension. A catalytic amount of methanesulfonic acid (1.17 g) was added at 20-25 C. The resulting slurry was heated to 70-75 C and held for a minimum of 4 hours. Thickening of the slurry was evident after 1.5 hours. Following reaction completion, the mixture was cooled to room temperature and may be used “as is” in the telescoped reaction of Example 4 below. Example 3 a

[0092] To prepare 6-acetamido-4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-3-cyano-7- ethoxyquinoline, ethanol (4.80 L) was added to the aniline solution followed by 4-chloro-3- cyano-7-ethoxy-6-N-acetylaminoquinoline (0.350 kg, 1.11 mole). A catalytic amount of methanesulfonic acid (2.0 ml) was added at 20-25⁰C. The resulting suspension was heated to 70-75⁰C and held for a minimum of 2 h. Thickening of the slurry was evident during this holding period. Following reaction completion, the mixture was used as is in the following telescoped reaction. Example 3 b

[0093] To prepare 6-acetainido-4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-quinoline, isopropanol (6.75 L) was added to the aniline solution followed by 4-chloro-3-cyano-7-ethoxy- 6-N-acetylaminoquinoline (0.277 kg, 0.96 mole, 0.78 eq). A catalytic amount of methane sulfonic acid (3.50 ml) was added at 20-25⁰C. The resulting suspension was heated to 80-85⁰C and held for a minimum of 10 hr. Thickening of the slurry was evident during this holding period. Following reaction completion, the mixture was cooled to 25-35 ⁰C, filtered and the cake washed with isopropanol (3 x 0.25 L). The cake was used as is in the following telescoped reaction.

[0094] As solvents EtOH, DMF or other suitable solvent may be used. Experimental results obtained using different solvents and reaction conditions are shown in Table 3. Difficulty filtering the product of this step (noted in several entries on Table 3) was circumvented by not isolating the solid at this point, but telescoping the reaction with the next step. It has been found that on the order of 20 volumes of EtOH were necessary to achieve reasonable stirring, but that the reaction can proceed in only 10 volumes of DMF, without significant loss in purity. [0095] In Table 3, where the entry is labelled NI , the intermediate product was not isolated, but carried into the next reaction step. Table 3 – Coupling Reaction

NR = no reaction, NI = not isolated; ND = not determined; NA = not available

1. Carried through to the deprotection and generation of free base to give 69.5% overall yield.

2. The overall yield after the deprotection and generation of the free base is 76.1%

3. This reaction was not filtered at all but taken as slurry to the next step.

Example 4 – Deprotection

[0096] The deprotection of the quinoline intermediate formed by the coupling reaction using

2N HCl in water is preferred as noted in Table 4 below. As in the previous Examples, the intermediate product of this step is advantageously not isolated, but carried over as a wet cake into the next step.

[0097] Preparation of 4-[3-chloro-4-(2-pyridylmethoxy)anilino]-3-cyano-7-ethoxy-6- aminoquinoline hydrochloride.

[0098] The reaction mixture from the previous step (Example 3) was taken as is and to it was added 2.7N HCl (3.3L) in H₂O (16.0 L). The slurry was heated to 70⁰C and held for 19 hours. The resulting slurry was then filtered and rinsed with 1:1 EtOHTH₂O (4 x 1.0 L). The product was isolated as a wet cake and carried through to the next step. A small sample was dried at this stage and analyzed. The HCl salt had a strength of 98.9%. Example 4a

[0099] To prepare 6-amino-4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-3-cyano-7- ethoxyquinoline hydrochloride, the reaction mixture from the previous step was taken as is and to it was added ethanol (1.6 L) and concentrated hydrochloric acid (1.38 L) to bring the pH to 1-3. The suspension was held at 70-75 ⁰C for a minimum of 2 h. After 1 h, the mixture thickens and ethanol (0.80 L) was added. After 2 h, water (6.80 L) was added, the mixture stirred for 1 h and then cooled to 35-45 ⁰C and stirred overnight (12 h). The mixture was filtered and rinsed with 1 : 1 ethanol/water (2 x 0.84 L) at 35-45 ⁰C. The product was isolated as a wet cake and carried through to the next step. Example 4b

[00100] To prepare 6-amino-4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7- ethoxyquinoline hydrochloride, the wet cake from the previous step was taken as is and to it was added a 2 N solution of concentrated hydrochloric acid (1.16 L) in methanol (5.84 L). The suspension was heated to 63-68 ⁰C and held for a minimum of 30 h. The mixture was cooled to 20-30⁰C, filtered and rinsed with methanol (2 x 0.30 L). The product was isolated as a wet cake and carried through to the next step. Table 4 – Deprotection

ND = not determined (the product was used in the next step as a wet cake) NA = not available SM= starting material

Example 5 – Preparation of free base

[0100] The 4-[3-chloro-4-(2-pyridylmethoxy)anilino]-3-cyano-7-ethoxy-6-aminoquinoline HCl salt was converted to the corresponding free base by treatment with 10% potassium carbonate (1.8 L) in MeOH (2.82 L). The mixture was stirred for a minimum of 2.5 hours and the pH was 9-10. The product was filtered, washed with 1:1 methanol/water (3 x 0.19 L) and dried (at 45-50 C at a pressure of 10 mm Hg, for 24 hours) to give 0.186 kg of product with an overall yield of 86% over 4 steps.

Example 5 a

[0101] To prepare 6-amino-4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-3-cyano-7- ethoxyquinoline free base, the 6-amino-4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-3-cyano-7- ethoxyquinoline hydrochloride salt was converted to its corresponding free base by treatment with 10% potassium carbonate (0.22 kg in 2.27 L water) in methanol (7.21 L) until pH was 10. The mixture was stirred for a minimum of 2 h. The beige suspension was filtered, washed with 1:1 methanol/water (2 x 0.84 L) and dried (45-50 ⁰C, 10 mm Hg, 24 h) to give 0.51 kg of product with an overall yield of 99% over 4 steps. Example 5b

[0102] To prepare 6-amino-4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxyquinolme free base, the 6-amino-4-[4-(benzyloxy)-3-chloroamlino]-3-cyano-7-ethoxyqumoline hydrochloride salt was converted to its corresponding free base by treatment with 10% aqueous potassium carbonate (0.213 kg in 2.13 L) in methanol (6.40 L). The mixture was stirred for a minimum of 1.5 h keeping the pH at 9-10. The product was filtered, washed with water (2 x 0.50 L) and dried (50-60 ⁰C, 10 mm Hg, 20 h) to give 0.347 kg of product with an overall yield of 82% over 4 steps.

Example 6 – Side Chain Coupling

[0103] An acid chloride of formula RV(C=O)-Cl, a mixed anhydride or an activated carboxylase R’ 2-(C=O)-LG derived from the corresponding carboxylic acid, may be used to couple a side chain at the 6 position to form a 6-amido-4-amino-3 cyanoquinoline. R’₂ may be alkyl of 1-6 carbon atoms, which may be mono- or di-substituted with amino groups or cycloamino groups, or R’₂ may be alkenyl of 2-6 carbon atoms which may be mono- or di- substituted with amino groups or cycloamino groups. [0104] Using the 2-step sequence shown below, an activated carboxylate is prepared in situ and coupled with the aniline. Although the acid chloride can be prepared in acetonitile, a better yield was obtained when the acid chloride was prepared in THF. In both cases, the aniline should be dissolved in NMP before amidation. It is believed that formation of product is better due to better solubility of the aniline in a THF/NMP mixture rather than in an ACN/NMP combination.

[0105] The amount of 4-N,N-dimethylaminocrotonic acid needed was 2 equivalents with respect to aniline. A slight undercharge of 1.95 eq of oxalyl chloride was added along with a catalytic amount (3 mol %) of DMF. The acid chloride was formed via the Vilsmeier intermediate. The completion test for the acid chloride reaction consists of quenching an aliquot of the reaction into ethanol and detecting by HPLC the crotonic acid ethyl ester. This method serves as a check to ensure complete consumption of oxalyl chloride. Excess oxalyl chloride will form diethyl oxalate when quenched in ethanol. [0106] The acid chloride is stable after holding for up to 5 hours at 0-10 °C, when decomposition begins. After 20 hours, complete decomposition takes place. If the acid chloride is allowed to warm, decomposition occurs and its effectiveness is diminished. [0107] The quality of the starting crotonic acid also plays a role in this coupling reaction, as commercially available crotonic acid may contain acetic acid. Acetic acid is detrimental to this reaction. 6-N-acetyl quinoline can be formed which is difficult to remove from the final product. The acetic acid can be removed by re-slurrying the crotonic acid in 4 volumes of isopropanol at room tempature, filtering and drying preferably to a level of less than 0.01%. [0108] It was found that the addition of the aniline solution in NMP to the acid chloride gave a better yield as compared to adding the acid chloride to the aniline. The addition is done keeping the temperature at 0-5 °C. The coupling reaction is slow and requires holding overnight at this temperature. It is not desirable to raise the reaction temperature as the stability of the acid chloride diminishes.

[0109] The reaction is quenched using aqueous sodium hydroxide at 40 °C and then filtered at that temperature. Quenching the reaction at 40 ⁰C gives bigger crystals that are easily filterable. It was observed that filtration at 40 °C was faster than at room temperature. The product is recrystallized from a 1.5:1 mixture of acetonitrile:THF (15 volumes) at 70-75 ⁰C. This in-process purification beneficially removes unreacted aniline. The recovery yields are typically greater than 85%.

[0110] To demonstrate a specific synthesis of (E)-N- {4-[3-chloro-4-(2- pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide, a solution of 4-N,N-dimethylaminocrotonic acid hydrochloride (186 g, 1.12 mol) in THF (1.88 L) and a catalytic amount of DMF (2 mL) was cooled to 0-5 °C. Oxalyl chloride (97 mL, 1.09 mol, 0.95 eq) was added dropwise over 45 minutes. The mixture was then warmed to 25-30 °C and stirred for 2 hours. The yellow solution was checked for complete consumption of oxalyl chloride by HPLC, then cooled to 0-5 ⁰C.

[0111] When the reaction is deemed complete, a solution of 4-[4-(2-pyridylniethoxy)-3- chloro]amino-6-amino-3-cyano-7-ethoxyquinoline (250 g, 0.56 mol) in N-methyl-2- pyrolidinone (1.88 L) was added dropwise over 2 hours keeping the temperature at 0-5 °C. The mixture was stirred for at least 3 hours until less than about 2% of the starting aniline remains by HPLC, which takes about 3 hours.

[0112] Upon completion, the reaction was quenched with water (3.0 L), held for 30 minutes and then warmed to 40 °C. Aqueous sodium hydroxide (170 g in 1.25 L water) was added over 1.25 hours to bring the pH to 10-11. The mixture was stirred for an hour, then cooled to room temperature and held for 3 hours. The resulting precipitates were filtered and washed with water (100 mL) and heptane (100 mL). The wet solids were heated to reflux (70-75 °C) in acetonitrile:THF and the solution cooled over 3 hours to room temperature. The product was filtered and washed with cold acetonitrile:THF. The product was dried (40-50 ⁰C, 10 mm Hg, 24 hours) to give 83% uncorrected yield. Example 6a

[0113] In an analogous synthesis of (E)-N- {4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-3- cyano-7-ethoxy-6-qumolmyl}-4-(dimethylamino)-2-butenamide, a solution of 4-N₅N- dimethylaminocrotonic acid hydrochloride (108 g, 0.65 mole) in tetrahydrofuran (1.13 L) and a catalytic amount of dimethylformamide (1.2 mL) was cooled to 0-5 ^°c. Oxalyl chloride (55 mL, 0.62 mole, 0.95 eq) was added dropwise over 50 min. The mixture was then warmed to 25-30 ^°c and stirred for 2 h then cooled to 0-5 ^°c. N-methyl-2-pyrrolidinone (0.225 L) was added over 25 min followed by a solution of 6-amino-4-[3-chloro-4-(3- fluorobenzyloxy)]anilino-3-cyano-7-ethoxy-quinoline (150 g, 0.32 mol) in N-methyl-2- pyrrolidinone (1.20 L) added dropwise over 2 hours keeping the temperature 0-5 . The mixture was stirred for at least about 3 hours, warmed to 10-15 ^°c and stirred for a further 12 hours. The mixture is cooled to 0-10 ^c, quenched by adding water (1.8 L) over 2 hours, and stirred for 30 minutes. The mixture is warmed to 40 ^°c. Aqueous sodium hydroxide (101 g in 0.75 L water) was added over 1 hour to bring the pH to 10-11. The mixture was stirred for an hour, filtered warm (40 ^°c) and washed with water (2 x 0.30 L) until the pH of the last wash was about 7. The wet solids were recrystallized by heating to reflux (70-75 ^°c) in 60:40 acetonitrile:tetrahydrofuran (2.25 L) and the solution cooled over 3 hours to room temperature. The product was filtered and washed with cold 60:40 acetonitrile:tetrahydrofuran (2 x 0.30 L). The product was dried (40-50 ^°c, 10 mm Hg, 16 h) to give 0.154 kg (83% yield). Example 6b

[0114] To prepare (E)-N- {4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}- 4-(dimethylamino)-2-butenamide free base, a solution of 4-N,N-dimethylaminocrotonic acid hydrochloride (18.6 g, 112 mmole) in acetonitrile (295 ml) and a catalytic amount of dimethylformamide (0.25 mL) was cooled to 0-5 ^°c. Oxalyl chloride (9.3 mL, 106 mmole, 0.95

_Op eq) was added dropwise over 5 min. The mixture was then warmed to 25-30 and stirred for 1-1.5 h then cooled to 0-10 ^°c. A solution of 6-amino-4-[4-(benzyloxy)-3-cliloroanilino]-3- cyano-7-ethoxy-quinoline (25 g, 56 mmole) in N-methyl-2-pyrrolidinone (175 ml) was added dropwise over 30 min keeping the temperature 0-10 ^°c. The mixture was stirred for a minimum of 1 h at 0-10 ^°c. After reaction completion, the mixture was quenched by dropwise addition to a solution of sodium bicarbonate (69.7 g in 870 ml water) over 30 mins. and stirred overnight while warming to room temperature. The mixture was filtered and washed with water (3 x 25 ml). The crude product was recrystallized in refluxing (80-82 ^°c) acetonitrile (570 ml). The product was dried (45-50 ^°c, 10 mm Hg, 28 h) to give 12.81 g (41% yield). ¹H NMR : δ (DMSO-d6) 9.44 (s, IH, NH), 8.97 (s, IH, Ar), 8.44 (s, IH, Ar), 7.53-7.35 (m, 7H, Ar), 7.35- 7.10 (in, 2H, Ar), 6.78 (dt, IH, -CH₂CH=CH-), 6.59 (d, IH, -CH₂CH=CH-), 5.21 (s, 2H, OCH₂Ph), 4.30 (q, 2H, OCH₂CH₃), 3.07 (s, 2H, NCH₂), 2.18 (s, 6H, N(CHs)₂), 1-47 (t, 3H, OCH₂CH₃).

[0115] Results obtained with different reaction procedures at different degrees of scale-up for synthesis of the 2-pyridylmethoxy analog are shown in Table 5. Table 5 – Side Chain Coupling

* TI = total impurities

[0116] Purificatiuon of the product is conducted by recrystallization in a suitable solvent followed by reslurrying with water followed by additional recrystallization, as necessary. As noted in Table 6, in the synthesis of the 2-pyridylmethoxy analog, several trials in different solvents did not result in the isolation of a single polymorphic form of the product. Table 6

Example 7 – Formation of Salt

[0117] The free base is hygroscopic and undergoes hydrolysis readily. Forming a salt of the compound, such as a fumarate or mesylate salt, stabilizes the molecule and renders the compound more soluble. The most preferred salt is a maleate salt, which has been found to be highly crystalline and to exist substantially as a single polymorph as shown by DSC thermogram in Fig. 1.

[0118] Recrystallizing the product in the presence of an acid has been found to yield a stable salt form of the product. Experimental results achieved utilizing different solvents for the recrystallization are set forth in Table 7. As seen in Table 7, an improvement is observed when n-propanol/water is used as the solvent system. A maleate salt is the most preferred, as it exists in a single polymorphic form. Table 7 – Recrystallization

Preparation of (E)-N- {4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6- quinolinyl} -4-(dimethylamino)-2-butenamide maleate, WAY- 179272-B

[0120] (E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4- dimethylamino)-2-butenamide crude free base (0.1 kg, 0.159 mole) and maleic acid (0.019 kg, 0.164 mole) were dissolved at 40-50 in a 10% water/n-propanol mixture (1.20 L). The hot solution was clarified and cooled over 2 h to room temperature and held for 12-15 hr. The product was filtered and washed with 10% water/n-propanol (2 x 0.15 L). The product was dried (50 ^°c, 10 mm Hg, 24 h) to give 94.4 g (88% yield). DSC: 204 ^°c (single crystal form). ¹H NMR : δ (DMSO-d6) 9.73 (s, IH, NH), 9.62 (s, IH, NH), 8.93 (s, IH, Ar), 8.60 (dd, IH, Ar), 8.50 (s, IH, Ar), 7.88 (dd, IH, Ar), 7.58 (d, IH, Ar), 7.40 (m, 3H, Ar), 7.24 (m, 2H, Ar), 6.75 (d, 2H, -CH=CH-), 6.03 (s, 2H, HOOC-CH=CH-COOH), 5.29 (s, 2H, OCH₂PVr), 4.33 (q, 2H, OCH₂CH₃), 3.89 (s, 2H, NCH₂), 2.76 (s, 6H, N(CH₃)₂), 1.47 (t, 3H, OCH₂CH₃). ¹³C NMR : δ (DMSO-d6) 168.0, 163.2, 156.9, 154.2, 153.2, 151.9, 151.3, 149.8, 148.5, 137.8, 136.5, 134.7, 133.4, 132.2, 128.0, 126.6, 124.9, 123.8, 122.3, 122.2, 117.9, 116.4, 115.1, 113.9, 109.5, 88.1, 72.0, 65.3, 57.8, 43.1, 14.9.

Example 7a

To prepare (E)-N- {4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-3-cyano-7-ethoxy-6- quinolinyl}-4-(dimethylamino)-2-butenamide dimaleate,

(E)-N- {4-[3-chloro-4-(3- fluorobenzyloxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-dimethylamino)-2-butenamide crude free base (0.516 kg, 0.90 mole) and maleic acid (0.214 kg, 1.84 mole) were dissolved at 40-50 ^°c in a 6.5% water/n-propanol mixture (12.60 L). The hot solution was clarified, rinsed with 5% water/n-propanol (0.52 L) and n-propanol (2.0 L). The mixture was held at 45 for 3 hr, cooled over 2 h to room temperature and held overnight. The mixture was further cooled to 5-10 ^°c. The product was filtered and washed with cold 5% water/n-propanol (0.52 L). The product was dried (45 ^°c, 10 mm Hg, 16-24 h) to give 0.586 kg (81% yield). DSC: 184 ^°c (single crystal form). ¹HNMR : δ (DMSO-d6) 9.77 (s, IH, NH), 8.95 (s, IH, Ar), 8.53 (s, IH, Ar), 7.49-7.16 (m, 8H, Ar), 6.78 (m, 2H, -CH=CH-), 6.15 (s, 4H, 2 x HOOC-CH=CH-COOH), 5.26 (s, 2H, OCH₂PyT), 4.33 (q, 2H, OCH₂CH₃), 3.97 (dd, 2H, NCH₂), 2.82 (s, 6H, N(CEb)₂), 1.47 (t, 3H, OCH₂CH₃). ¹³C NMR : δ (DMS0-d6) 167.0, 163.8, 162.3, 160.6, 153.6, 152.2, 151.3, 150.8, 139.5, 139.4, 133.7, 133.2, 132.2, 131.8, 130.5, 130.4, 127.4, 126.1, 124.3, 123.3, 121.7, 116.9, 115.7, 114.8, 114.5, 114.4, 114.1, 113.8, 113.1, 108.1, 87.2, 69.5, 64.6, 56.9, 42.1, 14.2. Example 7b

[0122] To prepare (E)-N- {4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}- 4-(dimethylamino)-2-butenamide maleate, (E)-N- {4-[4-(benzyloxy)-3-chloroanilino]-3-cyano- 7-ethoxy-6-quinolinyl}-4-dimethylamino)-2-butenamide crude free base (2.0 g, 3.6 mmole) and maleic acid (0.43 g, 3.7 mmole) were mixed at 40-50 ^c in a 10% water/n-propanol mixture (24 ml) for 2 hr. The mixture was cooled to ambient temperature, filtered and washed with 10% water/n-propanol (2 x 3 ml). The product was dried (40 ^°c, 10 mm Hg, 24 h) to give 0.32 g (13% yield). ¹HNMR : δ (DMSO-d6) 9.75 (s, IH, NH), 8.95 (s, IH, Ar), 8.49 (s, IH, Ar), 7.49-7.37 (m, 7H, Ar), 7.23 (dd, 2H, Ar), 6.78 (s, 2H, -CH₂CH=CH-), 6.06 (s, 2H, HOOC- CH=CH-COOH), 5.22 (s, 2H, OCH₂Ph), 4.31 (q, 2H, OCH₂CH₃), 3.93 (s, 2H, NCH₂), 2.79 (s, 6H, N(CH₃)₂), 1.46 (t, 3H, OCH₂CH₃).¹³C NMR : δ (DMSO-d6) 167.9, 163.1, 154.2, 153.3, 152.1, 151.3, 148.5, 137.3, 136.3, 134.5, 133.2, 132.3, 129.3, 129.2, 128.7, 128.3, 128.2, 128.0, 126.7, 124.9, 122.4, 117.9, 116.4, 115.2, 113.9, 109.5, 88.0, 71.1, 65.3, 57.7, 43.0, 15.0. [0123] (E)-N-{4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}-4- dimethylamino)-2-butenamide crude free base (2.0 g, 3.6 mmole) and maleic acid (0.43 g, 3.7 mmole) were mixed at 40-50 ^°c in a 10% water/n-propanol mixture (24 ml) for 2 hr. The mixture was cooled to ambient temperature, filtered and washed with 10% water/n-propanol (2 x 3 ml). The product was dried (40 ^°c, 10 mm Hg, 24 h) to give 0.32 g (13% yield). ¹H NMR : δ (DMSO-d6) 9.75 (s, IH, NH), 8.95 (s, IH, Ar), 8.49 (s, IH, Ar), 7.49-7.37 (m, 7H, Ar), 7.23 (dd, 2H, Ar), 6.78 (s, 2H, -CH₂CH=CH-), 6.06 (s, 2H, HOOC-CH=CH-COOH), 5.22 (s, 2H, OCH₂Ph), 4.31 (q, 2H, OCH₂CH₃), 3.93 (s, 2H, NCH₂), 2.79 (s, 6H, N(CH₃)₂), 1.46 (t, 3H, OCH₂CH₃). ¹³C NMR : δ (DMSO-d6) 167.9, 163.1, 154.2, 153.3, 152.1, 151.3, 148.5, 137.3, 136.3, 134.5, 133.2, 132.3, 129.3, 129.2, 128.7, 128.3, 128.2, 128.0, 126.7, 124.9, 122.4, 117.9,

116.4, 115.2, 113.9, 109.5, 88.0, 71.1, 65.3, 57.7, 43.0, 15.0.

……………….

http://www.google.com/patents/WO2009052264A2?cl=en

TABLE 1 1. STRUCTURES OF DEGRADATION PRODUCT AND PROCESS IMPURITIES

N-{4-[3-chloro-4-(2- (E)-4-({4-[3-chloro-4-(2- N -{4-[3-chloro-4-(2- pyrιdιnylmethoxy)anιlιno]-3-cyano-7- pyrιdιnylmethoxy)anιlιno]-3-cyano-7- pyrιdιnylmethoxy)anιlιno]-3-cyano-7-ethoxy- ethoxy-6-quιnolιnyl}acetamιde ethoxy-6-quιnolιnyl}amιno)-N,N,N- 6-quιnolιnyl}-N²,N²-dιmethylethanedιamιde trιmethyl-4-oxo-2-buten-1-amιnιum

Exact Mass ₄87 14 Exact Mass 544 16

Exact Mass 571 22

Process Impurity I Process Impurity J

SCHEME 1

The reaction of the free base and maleic acid occurs at an elevated temperature of from about 40 ⁰C to about 60 ⁰C, preferably between about 4O⁰C to about 5O⁰C. The ratio of watenn- propanol may vary, for example between about 1 :10 to about 1 :5, and the optimal ratio of watenn-propanol is about 1 :9. The water-alcohol solution may comprise from about 5% to about 20% by volume water and from about 80% to about 95% by volume alcohol. The alcohol may be n-propanol. In one embodiment, the water-alcohol solution comprises about 10% by volume water and about 90% by volume n-propanol. The volume of the solvent solution may be between about 8 to about 25 volumes, including about 10 to about 12 volumes. About 1.0-1.2 equivalents of maleic acid is used per equivalent of the free base, preferably about 1.03 equivalents of maleic acid per equivalent of the free base.

The resulting solution of the maleate salt may be clarified by filtration prior to cooling. The cooling step may be continued until the solution reaches a temperature of about 45°C or less, including a temperature of about 39°C or less, and more preferably to about 30⁰C or less. In one embodiment, the solution is filtered after cooling to about room temperature, preferably from about 23⁰C to about 25 ⁰C. Typically, the maleate salt begins to crystallize out of solution once the temperature reaches 37⁰C or below. The solution may be allowed to sit for at least 12 hours, preferably about 12 to about 15 hours at room temperature, and is then filtered and washed to recover the crystalline maleate salt product. The resulting filter cake may be washed with the same or a different water-alcohol solution to obtain the product. The product may be dried to obtain crystalline (E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7- ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide maleate. At this point, the maleate salt product recovered and isolated is typically in the form of the monohydrate form of the maleate salt.

……………

PAPTENT

http://www.google.com/patents/CN102731395A?cl=en

The present invention relates to a process for preparing that imatinib (neratinib, HKI-272) is a new method for its preparation and its intermediates in the preparation to the application that imatinib

[0155] Example 14 (E)-N-(4 – (3 – chloro-4 – (2 – pyridyl) phenyl) amino] _3_ ethoxy-quinolin-6-cyano-_7_ – yl) -4 – dimethylamino-2 – butene amide

[0156]

Compound of Example 13 (20mg, 0. 037mmol) was dissolved in DMF was added potassium carbonate (10mg, 0. 07mmol), dimethylamine hydrochloride (5mg, 0. 06mmol), at room temperature for I hour, after , the reaction mixture was dropped into water, stirred for 10 minutes, filtered, washed with water and dried to give the title compound 1511 ^ 75% yield.1HNMR (300MHz, DMS0_d6): δ I. 5 (t, 3H, J = 6 · 8,13. 8), 2. 2 (br s, 6H), 3. I (d, 2H, J = 3. 8 ), 4. 3 (q, 2H, J = 7. 0,14. 2), 5. 2 (s, 2H),

6. 6 (d, 1H, J = 15. 0), 6. 8 (m, 1H), 7. 1-7. 3 (m, 2H), 7. 3-7. 4 (m, 3H), 7. 6 (d, 1H, J = 3. 9),

7. 9 (d, 1H, J = 3. 9), 8. 5 (s, 1H), 8. 6 (d, 1H, J = 3. 9), 9. 0 (s, 1H), 9. 5 (s, 1H), 9. 6 (s, 1H). ESI-MS: [M + H] + = 557. 3.

GOING BACKWARDS…………………

Example 13 (E) -4 – bromo-N-(4 – (3 – chloro-4 – (2 – pyridyl) phenyl) amino] _3_ cyano _7_ ethoxyquin -6 – yl) -2 – butene amide

Example 12 Compound (100mg, 0. 2mmol) was suspended in carbon tetrachloride was added NBS (40mg,

O. 22mmol), benzoyl peroxide (2mg, 0. Olmmol), nitrogen, refluxed for 10 hours, the reaction solution directly mixed baby gel, silica gel column chromatography to obtain the title compound isolated 60mg, yield 51%. 1HnmrgoomHz, cdci3): δ i.6 (t, 3H, J = 6. 8,13. 7), 2. 0 (d, 2H, J = 6. 9), 4. 3 (q, 2H, J = 7. 2,13. 8), 5. 3 (s, 2H), 6. I (d, 1H, J =

15. 0), 7. 0 (m, 1H), 7. 2 (m, 1H), 7. 3 (s, 1H), 7. 4 (s, 1H), 7. 6 (d, 1H, J = 8. 2), 7. 8 (d, 1H, J =

7. 6), 8. 0 (s, 1H), 8. 5 (s, 1H), 8. 6 (d, 1H, J = 4. 7), 9. 2 (s, 1H). ESI-MS: [M + H] + = 594. I.

……………

PAPER

Optimization of 6,7-disubstituted-4-(arylamino)quinoline-3-carbonitriles as orally active, irreversible inhibitors of human epidermal growth factor receptor-2 kinase activity
J Med Chem 2005, 48(4): 1107

http://pubs.acs.org/doi/full/10.1021/jm040159c

Abstract Image

(E)-N-{4-[3-Chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide (25o).

This compound was prepared as a yellow solid (0.86 g, 85%) by the method described for 25g using 0.65 g (1.81 mmol) of 23 and 0.42 g (3.62 mmol) of 3-chloro-4-(2-pyridinylmethoxy)aniline:

HRMS (ES+) m/z 557.205 89 (M + H)⁺¹, Δ = −0.36 mmu;

¹H NMR (DMSO-d₆) δ 9.62 (s, 1H), 9.49 (s, 1H), 8.96 (s, 1H),

8.60 (d, 1H, J = 3.9 Hz), 8.47 (s, 1H),

7.88 (t, 1H, J = 3.9 Hz), 7.58 (d, 1H, J = 3.9 Hz),

7.39−7.35 (m, 3H), 7.26 (d, 1H, J = 7.8 Hz),

7.19 (d, 1H, J = 8.1 Hz), 6.81−6.73 (m, 1H),

6.59 (d, 1H, J = 7.8 Hz), 5.28 (s, 2H),

4.30 (q, 2H, J = 6.9 Hz),

3.07 (d, 2H, J = 3.9 Hz),

2.17 (s, 6H),

1.46 (t, 3H, J = 3.9 Hz).

Anal. (C₃₀H₂₉ClN₆O₃·1.1H₂O) C, H, N.

INTERPRETATION

¹H NMR : δ (DMSO-d6)

9.44 (s, IH, NH),

8.97 (s, IH, Ar),

8.44 (s, IH, Ar),

7.53-7.35 (m, 7H, Ar),

7.35- 7.10 (in, 2H, Ar),

6.78 (dt, IH, -CH₂CH=CH-),

6.59 (d, IH, -CH₂CH=CH-),

5.21 (s, 2H, OCH₂Ph),

4.30 (q, 2H, OCH₂CH₃),

3.07 (s, 2H, NCH₂),

2.18 (s, 6H, N(CHs)₂),

1-47 (t, 3H, OCH₂CH₃).

References

“Definition of neratinib – National Cancer Institute Drug Dictionary”. Retrieved 2008-12-01.
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ClinicalTrials.gov NCT00398567 A Phase 1/2 Study Of HKI-272 In Combination With Herceptin In Subjects With Advanced Breast Cancer
“Puma Acquires Global Rights to Pfizer’s Phase III Breast Cancer Drug Neratinib”.
Minami Y, Shimamura T, Shah K, et al. (July 2007). “The major lung cancer-derived mutants of ERBB2 are oncogenic and are associated with sensitivity to the irreversible EGFR/ERBB2 inhibitor HKI-272”. Oncogene 26 (34): 5023–7. doi:10.1038/sj.onc.1210292.PMID 17311002.
http://www.reuters.com/article/idUSN1612347120100317 “Breast cancer study aims to speed drugs, cooperation” March 2010
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TSOU H-R ET AL: “Optimization of 6,7-Disubstituted-4-(arylamino)quinoline-3 -carbonitr iles as Orally Active, Irreverible Inhibitors of HEGFR-2 Kinase Activity” JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, WASHINGTON, US, vol. 48, 27 January 2005 (2005-01-27), pages 1107-1131, XP002414228 ISSN: 0022-2623 cited in the application
Optimization of 6,7-disubstituted-4-(arylamino)quinoline-3-carbonitriles as orally active, irreversible inhibitors of human epidermal growth factor receptor-2 kinase activity
J Med Chem 2005, 48(4): 1107

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US7126025	10-25-2006	Synthesis of 4-(amino)-2-butenoyl chlorides and their use in the preparation of 3-cyano quinolines

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NMR

1H NMRMASS

1H NMRLCMS

Nemonoxacin….TaiGen’s pneumonia antibiotic Taigexyn 奈诺沙星 gets marketing approval in Taiwan

March 18, 2014 4:07 am / Leave a comment

Nemonoxacin 奈诺沙星

378746-64-6 CAS

TG-873870

C20-H25-N3-O4

371.4345

WARNER CHILCOTT ORIGINATOR

CLINICAL TRIALS http://clinicaltrials.gov/search/intervention=Nemonoxacin

(3S,5S)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4- dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid

7-[3(S)-Amino-5(S)-methylpiperidin-1-yl]-1-cyclopropyl-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
Taigexyn has been approved in Taiwan IN 2014

太景生物

“TAIPEI, MARCH 13, 2014 /PRNEWSWIRE/ — TAIGEN BIOTECHNOLOGY …”
13.03.14 |

TaiGen Biotechnology Receives Marketing Approval from the Taiwan Food and Drug Administration for Taigexyn in Taiwan

TAIPEI, March 13, 2014 /PRNewswire/ — TaiGen Biotechnology Company, Limited (“TaiGen”) today announced that the Taiwan Food and Drug Administration (TFDA) has approved the new drug application (NDA) of Taigexyn® (nemonoxacin) oral formulation (500 mg) for the treatment of community-acquired bacterial pneumonia (CAP). With this NDA approval, Taiwan is the first region to grant marketing approval to Taigexyn®. An NDA for Taigexyn® was also submitted to China FDA (CFDA) in April 2013 and is currently under review.

http://www.ad-hoc-news.de/taigen-biotechnology-receives-marketing-approval-from-the-taiwan-food-and-drug-administration-for-taigexyn-in-taiwan–/de/News/35824729

Nemonoxacin is a novel non-fluorinated quinolone antibiotic undergoing clinical trials.

Taigexyn Granted QIDP and Fast Track Designations

TaiGen Biotechnology announced that the FDA has granted nemonoxacin (Taigexyn) Qualified Infectious Disease Product (QIDP) and Fast Track designations for community-acquired bacterial pneumonia (CAP) and acute bacterial skin and skin structure infections (ABSSSI).

Safety and clinical pharmacokinetics of nemonoxacin, a novel non-fluorinated quinolone, in healthy Chinese volunteers following single and multiple oral doses

Nemonoxacin is a novel non-fluorinated quinolone broad spectrum antibiotic available in both oral and intravenous formulations. Nemonoxacin demonstrates activity against gram-positive and gram-negative bacteria and atypical pathogens. Nemonoxacin also possesses activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant pathogens.

Nemonoxacin is a novel non-flourinated quinolone antibiotic registered in Taiwan for the oral treatment of community-acquired pneumonia. Clinical trials are in development at TaiGen Biotechnology for the treatment of diabetic foot infections and for the treatment of moderate to severe community-acquired pneumonia with an intravenous formulation. The drug is thought to accomplish its antibacterial action through topoisomerase inhibition.

Originally developed at Procter & Gamble, nemonoxacin was the subject of a strategic alliance formed in January 2005 between P&G and TaiGen to further the development and commercialization of nemonoxacin. In 2012, the product was licensed by TaiGen Biotechnology to Zhejiang Medicine in China for manufacturing, sales and marketing. In 2014, TaiGen out-licensed the exclusive rights of the product in Russian Federation, Commonwealth Independent States and Turkey to R-Pharm.

TaiGen has completed two Phase 2 clinical studies, one in CAP and the other in diabetic foot infections with demonstrated efficacy and safety. In the clinical trials conducted to date, nemonoxacin has shown activity against drug-resistant bacteria such as MRSA, quinolone-resistant MRSA, as well as quinolone-resistant Streptococcus pneumoniae.

Malate salt

Nemonoxacin malate anhydrous
951163-60-3 CAS NO, MW: 505.5209

Nemonoxacin malate hemihydrate
951313-26-1, MW: 1029.0566

Chemical structure of nemonoxacin as a malate salt (C20H25N3O4·C4H6O5·H2O). Nemonoxacin is the free base, and its molecular mass is 371.44 g/mol. The molecular mass of the salt, nemonoxacin malate, is 514.53 g/mol.

……………………..

isomeric compounds are:

(3S,5S)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4-dihydro-8- methoxy-4-oxo-3 -quinolinecarboxylic acid

COMPD1…….DESIRED

(3S,5R)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4-dihydro-8- methoxy-4-oxo-3 -quinolinecarboxylic acid

COMPD 1’….NOT DESIRED

EP2303271A1

Example 1

Malate salts of (3S,5S)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4- dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (Compound 1) and (3S,5R)-7- [3-ammo-5-methyl-piperidinyl]- 1 -cyclopropyl- 1 ,4-dihydro-8-methoxy-4-oxo-3- quinolinecarboxylic acid (Compound 1′) were synthesized as follows:

(A) Synthesis of (3S,5S)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (Compound 9) and (3S,5R)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (Compound 9′): Compound 9′ was synthesized as shown in Scheme 1 below:

Scheme 1

3 4 Boc

A 50-L reactor was charged with Compound 2 (5.50 kg, 42.60 mol), methanol (27 L) and cooled to 10-15⁰C. Thionyl chloride (10.11 kg, 2.0 equiv.) was added via an addition funnel over a period of 65 min, with external cooling to keep temperature below 30°. The resulting solution was stirred at 25⁰C for 1.0 hour, after which methanol was removed under reduced pressure. The oily residue was azeotroped with ethyl acetate (3 x 2.5 L) to remove residual methanol, dissolved in ethyl acetate (27.4 L), charged into a 50 L reactor, and neutralized by slow addition of triethylamine (3.6 kg) below 3O⁰C. The resulting suspension was filtered to remove triethylamine hydrochloride.

The filtrate was charged to a 50 L reactor, along with DMAP (0.53 kg). Di- fert-butyl dicarbonate (8.43 kg) was added via hot water heated addition funnel, over a period of 30 min at a temperature of 20-30⁰C. The reaction was complete after 1 hour as determined by TLC analysis. The organic phase was washed with ice cold IN HCl (2 x 7.5 L), saturated sodium bicarbonate solution (1 x 7.5 L), dried over magnesium sulfate, and filtered. After ethyl acetate was removed under reduced pressure, crystalline slurry was obtained, triturated with MTBE (10.0 L), and filtered to afford Compound 3 as a white solid (5.45 kg, 52.4%).

Anal. Calcd for C_HH_I7NO₅ : C, 54.3; H, 7.04; N, 5.76. Found: C, 54.5; H, 6.96; N, 5.80. HRMS (ESI⁺) Expected for C_HH_I8NO₅, [M+H] 244.1185. Found

244.1174; ¹H NMR (CDCl₃, 500 MHz):δ=4.54 (dd, J= 3.1, 9.5 Hz, IH), 3.7 (s, 3H), 2.58-2.50 (m, IH), 2.41 (ddd, IH, J= 17.6, 9.5, 3.7), 2.30-2.23 (m, IH), 1.98-1.93 (m, IH), 1.40 (s, 9H); ¹³C NMR (CDCl₃, 125.70 MHz) δ 173.3, 171.9, 149.2, 83.5, 58.8, 52.5, 31.1, 27.9, 21.5. Mp 70.2⁰C.

A 50-L reactor was charged with Compound 3 (7.25 kg, 28.8 mol), DME (6.31 kg), and Bredereck’s Reagent (7.7 kg, 44.2 mole). The solution was agitated and heated to 75⁰C + 5⁰C for three hours. The reaction was cooled to O⁰C over an hour, during which time a precipitate formed. The mixture was kept at O⁰C for an hour, filtered, and dried in a vacuum oven for at least 30 hours at 3O⁰C + 5⁰C to give compound 4 as a white crystalline solid (6.93 kg, 77.9%).

Anal. Calcd for Ci₄H₂₂N₂O₅: C, 56.4; H, 7.43; N, 9.39. Found C, 56.4; H, 7.32; N, 9.48; HRMS (ESI⁺) Expected for Ci₄H₂₂N₂O₅, [M+H] 299.1607. Found 299.1613; ¹H NMR (CDCl₃, 499.8 MHz) δ = 7.11 (s, IH), 4.54 (dd, IH, J= 10.8, 3.6), 3.74 (s, 3H), 3.28-3.19 (m, IH), 3.00 (s, 6H), 2.97-2.85 (m,lH), 1.48 (s, 9H); ¹³C NMR (CDCl₃, 125.7 MHz) δ = 172.6, 169.5, 150.5, 146.5, 90.8, 82.2, 56.0, 52.3, 42.0, 28.1, 26.3. MP 127.9⁰C. A 10-gallon Pfaudler reactor was charged with ESCAT 142 (Engelhard Corp.

N.J, US) 5% palladium powder on carbon (50% wet, 0.58 kg wet wt), Compound 4 (1.89 kg, 6.33 mol), and isopropanol (22.4 Kg). After agitated under a 45-psi hydrogen atmosphere at 45⁰C for 18 hrs, the reaction mixture was cooled to room temperature and filtered though a bed of Celite (0.51 kg). The filtrate was evaporated under reduced pressure to give a thick oil, which was solidified on standing to afford Compound 5 (1.69 kg, 100%) as a 93:7 diastereomeric mixture.

A sample of product mixture was purified by preparative HPLC to give material for analytical data. Anal. Calcd for Ci₂Hi₉NO₅: C, 56.0; H, 7.44; N, 5.44. Found C, 55.8; H, 7.31; N, 5.44; MS (ESI⁺) Expected for Ci₂Hi₉NO₅, [M+H] 258.1342. Found 258.1321; ¹H NMR (CDCl₃, 499.8 MHz) δ = 4.44 (m, IH), 3.72 (s, 3H), 2.60-2.48 (m, 2H), 1.59-1.54 (m, IH), 1.43 (s, 9H), 1.20 (d, j = 6.8 Hz,3H); ¹³C NMR (CDCl₃, 125.7 MHz) δ = 175.7, 172.1, 149.5, 83.6, 57.4, 52.5, 37.5, 29.8, 27.9, 16.2. Mp 89.9⁰C.

A 50-L reactor was charged with Compound 5 (3.02 kg, 11.7 mol), absolute ethanol (8.22 kg), and MTBE (14.81 kg). Sodium borohydride (1.36 kg, 35.9 mol) was added in small portions at 0⁰C + 5⁰C. A small amount of effervescence was observed. The reaction mixture was warmed to 1O⁰C + 5⁰C and calcium chloride dihydrate (2.65 kg) was added in portions at 1O⁰C + 5⁰C over an hour. The reaction was allowed to warm to 2O⁰C + 5⁰C over one hour and agitated for an additional 12 hours at 20⁰C + 5⁰C. After the reaction was cooled to -5⁰C + 5⁰C, ice-cold 2N HCl (26.9 kg) was added slowly at of O⁰C + 5⁰C. Agitation was stopped. The lower aqueous phase was removed. The reactor was charged with aqueous saturated sodium bicarbonate (15.6 kg) over five minutes under agitation. Agitation was stopped again and the lower aqueous phase was removed. The reactor was charged with magnesium sulfate (2.5 kg) and agitated for at leastlO minutes. The mixture was filtered though a nutsche filter, and concentrated under reduced pressure to afford Compound 6 (1.80 kg, 66%). Anal. Calcd for CnH₂₃NO₄: C, 56.6 H, 9.94; N, 6.00. Found C, 56.0; H, 9.68;

N, 5.96; HRMS (ESI⁺) Expected for CnH₂₄NO₄, [M+H] 234.1705. Found 234.1703; ¹H NMR (CDCl₃, 500 MHz) δ = 6.34 (d, J= 8.9 Hz, IH, NH), 4.51 (t, J= 5.8, 5.3 Hz, IH, NHCHCH₂OH), 4.34 (t, J= 5.3, 5.3 Hz, IH, OBCHCH₂OH), 3.46-3.45, (m, IH, NHCH), 3.28 (dd, J= 10.6, 5.3 Hz, NHCHCHHOH), 3.21 (dd, J= 10.2, 5.8 Hz , IH, CH₃CHCHHOH), 3.16 (dd, J = 10.2, 6.2 Hz, IH, NHCHCHHOH), 3.12 (dd, J= 10.6, 7.1 Hz , IH, CH₃CHCHHOH), 1.53-1.50 (m, IH, CH₃CHCHHOH), 1.35 (s, 9H, 0(CH_B)₃, 1.30 (ddd, J = 13.9, 10.2, 3.7 Hz, IH, NHCHCHHCH), 1.14 (ddd, J= 13.6, 10.2, 3.4 Hz, IH, NHCHCHHCH), 0.80 (d, J= 6.6 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 125.7 MHz) δ 156.1, 77.9, 50.8, 65.1, 67.6, 65.1, 35.6, 32.8, 29.0, 17.1. Mp 92.1⁰C. A 50 L reactor was charged with a solution of Compound 6 (5.1 kg) in isopropyl acetate (19.7 kg). The reaction was cooled to 15⁰C + 5°C and triethylamine (7.8 kg) was added at that temperature. The reactor was further cooled to O⁰C + 5⁰C and methanesulfonyl chloride (MsCl) (6.6 kg) was added. The reaction was stirred for a few hours and monitored for completion by HPLC or TLC. The reaction was quenched by saturated aqueous bicarbonate solution. The organic phase was isolated and washed successively with cold 10% aqueous triethylamine solution, cold aqueous HCl solution, cold saturated aqueous bicarbonate solution, and finally saturated aqueous brine solution. The organic phase was dried, filtered, and concentrated in vacuo below 55⁰C + 5⁰C to afford compound 7 as a solid/liquid slurry, which was used in the subsequent reaction without further purification.

After charged with 9.1 kg of neat benzylamine, a 50 L reactor was warmed to 55⁰C, at which temperature, a solution of compound 7 (8.2 kg) in 1,2- dimethoxyethane (14.1 kg) was added. After the addition, the reaction was stirred at 6O⁰C + 5⁰C for several hours and monitored for completion by TLC or HPLC. The reaction was cooled to ambient temperature and the solvent was removed under vacuum. The residue was diluted with 11.7 kg of 15% (v/v) ethyl acetate/hexanes solution and treated, while agitating, with 18.7 kg of 20% (wt) aqueous potassium carbonate solution. A triphasic mixture was obtained upon standing. The upper organic layer was collected. The isolated middle layer was extracted twice again with 11.7 kg portions of 15% (v/v) ethyl acetate/hexanes solution. The combined organic layers were concentrated under vacuum to give an oily residue. The residue was then purified by chromatography to afford Compound 8 as an oil. A 40 L pressure vessel was charged with 0.6 kg 50% wet, solid palladium on carbon (ElOl, 10 wt. %) under flow of nitrogen. A solution of Compound 8 (3.2 kg) in 13.7 kg of absolute ethanol was then added to the reactor under nitrogen. The reactor was purged with nitrogen and then pressurized with hydrogen at 45 psi. The reaction was then heated to 45°C. It was monitored by TLC or LC. Upon completion, the reaction was cooled to ambient temperature, vented, and purged with nitrogen. The mixture was filtered through a bed of Celite and the solid was washed with 2.8 kg of absolute ethanol. The filtrate was concentrated under vacuum to afford Compound 9 as a waxy solid.

TLC R/(Silica F₂₅₄, 70:30 v/v ethyl acetate-hexanes, KMnO₄ stain) = 0.12; ¹H NMR (300 MHz, CDCl₃) δ 5.31 (br s, IH), 3.80-3.68 (m, IH), 2.92 (d, J=I 1.4 Hz,

IH), 2.77 (AB quart, J_AB=12.0 Hz, v=50.2 Hz, 2H), 2.19 (t, J=10.7 Hz, IH), 1.82-1.68 (m, 2H), 1.54 (br s, IH), 1.43 (s, 9H), 1.25-1.15 (m, IH), 0.83 (d, J=6.6 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ: 155.3, 78.9, 54.3, 50.8, 45.3, 37.9, 28.4, 27.1, 19.2; MS (ESI+) m/z 215 (M+H), 429 (2M+H). Similarly, (3S,5R)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester

(Compound 9′) was synthesized as shown in Scheme 2.

Scheme 2

_HN Boc HN Boc

NaBH₄,EtOH _w – ^“ MsCI₁TEA . „ _. – – _. „ Benzyl Amine

THF EA₁CoId

(B) Synthesis of l-Cyclopropyl-7-fluoro-8-methoxy-4-oxo-l,4-dihydro-quinoline-3- carboxylic acid (Compound 10): Compound 10 was prepared according to the method described in U.S. Patent

6,329,391.

(C) Synthesis of borone ester chelate of l-Cyclopropyl-7-fluoro-8-methoxy-4-oxo- l,4-dihydro-quinoline-3-carboxylic acid (Compound 11):

Scheme 3

Toluene, tert-Butylmethyl ether 20-50⁰C, filter

A reactor was charged with boron oxide (2.0 kg, 29 mol), glacial acetic acid (8.1 L, 142 mol), and acetic anhydride (16.2 L, 171 mol). The resulting mixture was refluxed at least 2 hours, and then cooled to 40⁰C, at which temperature, 7- fluoroquinolone acid compound 10 (14.2 kg, 51 mol) was added. The mixture was refluxed for at least 6 hours, and then cooled to about 90⁰C. Toluene (45 L) was added to the reaction. At 5O⁰C, terϊ-butylmethyl ether (19 L) was added to introduce precipitation. The mixture was then cooled to 20⁰C and filtered to isolate the precipitation. The isolated solid was then washed with teτt-butylmethyl ether (26 L) prior to drying in a vacuum oven at 4O⁰C (50 torr) to afford Compound 11 in a yield of 86.4%. Raman (cm ¹): 3084.7, 3022.3, 2930.8, 1709.2, 1620.8, 1548.5, 1468.0, 1397.7, 1368.3, 1338.5, 1201.5, 955.3, 653.9, 580.7, 552.8, 384.0, 305.8. NMR (CDCl₃, 300 MHz) δ (ppm): 9.22 (s, IH), 8.38-8.33 (m, IH), 7.54 (t, J=9.8 Hz, IH), 4.38-4.35 (m, IH), 4.13 (s, 3H), 2.04 (s, 6H), 1.42-1.38 (m, 2H), 1.34-1.29 (m, 2H). TLC (Whatman MKC18F Silica, 6θA, 200 μm), Mobile Phase: 1 :1 (v/v) CH₃CN : 0.5N NaCl (aq), UV (254/366 nm) visualization; R^O.4-0.5. (D) Synthesis of malate salt of (3S,5S)-7-[3-amino-5-methyl-piperidmyl]-l- cyclopropyl-l,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (Compound 1) and malate salt of (3S,5R)-7-[3-amino-5-methyl-piperidmyl]-l-cyclopropyl-l,4- dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (Compound 1′)

Compound 1 was synthesized from compound 9 as shown in Scheme 4 below:

Scheme 4

5O⁰C 3 d

a 6 0 N HCI (aq) CH₂CI₂ 35°40°C 12 h t> Extract pH ad]ust to ~7-8 50″-65″C filter

A reactor was charged with Compound 11 (4.4 kg, 10.9 mol), Compound 9 (2.1 kg, 9.8 mol), triethylamine (TEA) (2.1 L, 14.8 mol), and acetonitrile (33.5 L, 15.7 L/kg). The resulting mixture was stirred at approximately 50⁰C till completion of the reaction, as monitored by HPLC or reverse phase TLC. It was cooled to approximately 35°C and the reaction volume was reduced to approximately half by distillation of acetonitrile under vacuum between 0-400 torr. After 28.2 kg of 3.0 N NaOH (aq) solution was added, the reaction mixture was warmed to approximately 4O⁰C, distilled under vacuum until no further distillates were observed, and hydro lyzed at room temperature. Upon completion of hydrolysis, which was monitored by HPLC or reverse phase TLC, 4-5 kg of glacial acetic acid was added to neutralize the reaction mixture.

The resulting solution was extracted 3 times with 12.7 kg (9.6 L) of dichloromethane. The organic layers were combined and transferred to another reactor. The reaction volume was reduced to approximately a half by evaporation at 40⁰C. After 20.2 Kg 6.0N HCl (aq) solution was added, the reaction mixture was stirred for at least 12 hours at 35°C. After the reaction was completed as monitored by HPLC or reverse phase TLC, agitation was discontinued to allow phase separation. The organic phase was removed and the aqueous layer was extracted with 12.7 kg (9.6 L) of dichloromethane. The aqueous layer was diluted with 18.3 kg distilled water and warmed to approximately 50⁰C. Dichloromethane was further removed by distillation under vacuum (100-400 torr).

The pH of the aqueous solution was then adjusted to 7.8-8.1 by adding about 9.42 kg of 3.0 N NaOH (aq) below 65°C. The reaction mixture was stirred at 50⁰C for at least an hour and then cooled to room temperature. The precipitate was isolated by suction filtration, washed twice with 5.2 kg of distilled water, and dried with suction for at least 12 hours and then in a convection oven at 55°C for additional 12 hours. Compound 12 (3.2 kg, 79%) was obtained as a solid.

A reactor was charged with 3.2 kg of Compound 12 and 25.6 kg of 95% ethanol. To the reactor was added 1.1 kg of solid D,L-malic acid. The mixture was refluxed temperature (~80°C). Distilled water (-5.7 L) was added to dissolve the precipice and 0.2 kg of activated charcoal was added. The reaction mixture was passed through a filter. The clear filtrate was cooled to 45°C and allowed to sit for at least 2 hours to allow crystallization. After the reaction mixture was further cooled to 5°C, the precipitate was isolated by suction filtration, washed with 6.6 kg of 95% ethanol, and dried with suction for at least 4 hours. The solid was further dried in a convection oven at 45⁰C for at least 12 hours to afford 3.1 kg of Compound 1 (yield: 70%). NEMONOXACIN

NMR (D₂O, 300 MHz) δ (ppm): 8.54 (s, IH), 7.37 (d, J=9.0 Hz, IH), 7.05 (d, J=9.0 Hz, IH), 4.23-4.18 (m, IH), 4.10-3.89 (m, IH), 3.66 (br s, IH), 3.58 (s, 3H), 3.45 (d, J=9.0 Hz, IH), 3.34 (d, J=9.3 Hz, IH), 3.16 (d, J=12.9 Hz, IH), 2.65 (dd, J=16.1, 4.1 Hz, IH), 2.64-2.53 (m, IH), 2.46 (dd, J=16.1, 8.0 Hz, IH), 2.06 (br s, IH), 1.87 (d, J=14.4 Hz, IH), 1.58-1.45 (m, IH), 1.15-0.95 (m, 2H), 0.91 (d, J=6.3 Hz, 3H), 0.85-0.78 (m, 2H).

Similarly, Compound 1′ was synthesized from Compound 9′ as shown in Scheme 5 below:

Scheme 5

(3S,5R)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4-dihydro-8- methoxy-4-oxo-3 -quinolinecarboxylic acid

COMPD 1’….NOT DESIRED

…………………

US20070232650

US2007/232650 A1,

malate salts of

(3S,5S)-7-[3-amino-5-methyl-piperidinyl]-1-cyclopropyl-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (hereinafter Compound I, see also intermediate (23) in Section D, of Detailed Description of the Invention).

EXAMPLES Example 1 Synthesis of (3S,5S)-7-[3-amino-5-methyl-piperidinyl]-1-cyclopropyl-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid and malate salt thereof A. Synthesis of (3S,5S)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (8)

(2S)-1-(1,1-Dimethylethyl)-5-oxo-1,2-pyrrolidinedicarboxylic acid-2-methyl ester, (2). A 50-L reactor is charged with compound (1) (5.50 Kg, 42.60 mol), methanol (27 L) and cooled to 10-15° C. Thionyl chloride (10.11 Kg, 2.0 equiv.) is added via addition funnel over a period of 65 min, with external cooling to maintain temperature at <30°. The resulting solution is stirred at 25° C.+5° C. for 1.0 hour, after which the methanol is distilled off under reduced pressure. The resulting thick oil is azeotroped with ethyl acetate (3×2.5 L) to remove residual methanol. The residue is dissolved in ethyl acetate (27.4 L), charged into a 50 L reactor, and neutralized by the addition of triethylamine (3.6 Kg) from an addition funnel over 30 minutes. The temperature of the neutralization is maintained below 30° C. via external cooling. The resulting suspension of triethylamine hydrochloride is removed by filtration, and the clarified mother liquor solution is charged to a 50 L reactor, along with DMAP (0.53 Kg). Di-tert-butyl dicarbonate (8.43 Kg) is added via hot water heated addition funnel, over a period of 30 min with external cooling to maintain temperature at about 20-30° C. The reaction is complete after 1 hour as determined by TLC analysis. The organic phase is washed with ice cold 1N HCl (2×7.5 L), saturated sodium bicarbonate solution (1×7.5 L), and dried over magnesium sulfate. The mixture is filtered through a nutsche filter and ethyl acetate is removed under reduced pressure to yield a crystalline slurry that is triturated with MTBE (10.0 L) and filtered to afford intermediate (2) as a white solid (5.45 Kg, 52.4%). Anal. Calcd for C₁₁H₁₇NO₅: C, 54.3; H, 7.04; N, 5.76. Found: C, 54.5; H, 6.96; N, 5.80. HRMS (ESI⁺) Expected for C₁₁H₁₈NO₅, [M+H] 244.1185. Found 244.1174; ¹H NMR (CDCl₃, 500 MHz): δ=4.54 (dd, J=3.1, 9.5 Hz, 1H), 3.7 (s, 3H), 2.58-2.50 (m, 1H), 2.41 (ddd, 1H, J=17.6, 9.5, 3.7), 2.30-2.23 (m, 1H), 1.98-1.93 (m, 1H), 1.40 (s, 9H); ¹³C NMR (CDCl₃, 125.70 MHz) δ 173.3, 171.9, 149.2, 83.5, 58.8, 52.5, 31.1, 27.9, 21.5; Mp 70.2° C.

(2S,4E)-1-(1,1-Dimethylethyl)-4-[(dimethylamino)methylene]-5-oxo-1,2-pyrrolidinedicarboxylic acid-2-methyl ester (3). A 50-L reactor is charged with intermediate (2) (7.25 Kg, 28.8 mol), DME (6.31 Kg), and Bredereck’s Reagent (7.7 Kg, 44.2 mole). The solution is agitated and heated to 75° C.±5° C. for at least three hours. The progress of the reaction is monitored by HPLC. The reaction is cooled to 0° C.±5° C. over on hour during which time a precipitate forms. The mixture is held at 0° C.±5° C. for one hour and filtered though a nutsche filter and the product dried in a vacuum oven for at least 30 hours at 30° C.±5° C. to give intermediate (3) as a white crystalline solid (6.93 Kg, 77.9%). Anal. Calcd for C₁₄H₂₂N₂O₅: C, 56.4; H, 7.43; N, 9.39. Found C, 56.4; H, 7.32; N, 9.48; HRMS (ESI⁺) Expected for C₁₄H₂₂N₂O₅, [M+H] 299.1607. Found 299.1613; ¹H NMR(CDCl₃, 499.8 MHz)δ=7.11 (s, 1H), 4.54 (dd, 1H, J=10.8, 3.6), 3.74 (s, 3H), 3.28-3.19 (m, 1H), 3.00 (s, 6H), 2.97-2.85 (m, 1H), 1.48 (s, 9H); ¹³C NMR (CDCl₃, 125.7 MHz) δ=172.6, 169.5, 150.5, 146.5, 90.8, 82.2, 56.0, 52.3, 42.0, 28.1, 26.3. Mp 127.9° C.

(2S,4S)-1-(1,1-Dimethylethyl)-4-methyl-5-oxo-1,2-pyrrolidinedicarboxylic acid-2-methyl ester (4). A 10-gallon Pfaudler reactor is inerted with nitrogen and charged with ESCAT 142 5% palladium powder on carbon (50% wet, 0.58 Kg wet wt.), intermediate (3) (1.89 Kg, 6.33 mol) and isopropanol (22.4 Kg). The reaction mixture is agitated under a 45-psi hydrogen atmosphere at 45° C. for 18 hrs. The reaction mixture is then cooled to room temperature and filtered though a bed of Celite (0.51 Kg) in a nutsche filter to remove catalyst. The mother liquor is evaporated under reduced pressure to give a thick oil that crystallizes on standing to afford 4 (1.69 Kg, 100%) as a 93:7 diastereomeric mixture. A sample of product mixture is purified by preparative HPLC to give material for analytical data. Anal. Calcd for C₁₂H₁₉NO₅: C, 56.0; H, 7.44; N, 5.44. Found C, 55.8; H, 7.31; N, 5.44; MS (ESI⁺) Expected for C₁₂H₁₉NO₅, [M+H] 258.1342. Found 258.1321; ¹H NMR (CDCl₃, 499.8 MHz) δ=4.44 (m, 1H), 3.72 (s, 3H), 2.60-2.48 (m, 2H), 1.59-1.54 (m, 1H), 1.43 (s, 9H), 1.20 (d, j=6.8 Hz,3H); ¹³C NMR (CDCl₃, 125.7 MHz) δ=175.7, 172.1, 149.5, 83.6, 57.4, 52.5, 37.5, 29.8, 27.9, 16.2. Mp 89.9° C.

(1S,3S)-(4-Hydroxyl-1-hydroxymethyl-3-methyl-butyl)-carbamic acid tert-butyl ester (5). A 50-L reactor is charged with intermediate (4) (3.02 Kg, 11.7 mol), absolute ethanol (8.22 Kg), and MTBE (14.81 Kg). The solution is agitated and cooled to 0° C.±5° C. and sodium borohydride (1.36 Kg, 35.9 mol) is added in small portions so as to maintain reaction temperature at 0° C.±5° C. A small amount of effervescence is observed. The reaction mixture is warmed to 10° C.±5° C. and calcium chloride dihydrate (2.65 Kg) is added portion wise at a slow rate over an hour so as to maintain a reaction temperature of 10° C.±5° C. The reaction is allowed to warm to 20° C.±5° C. over one hour and agitated for an additional 12 hours at 20° C.±5° C. The reaction is cooled to −5° C.±5° C., ice-cold 2N HCl (26.9 Kg) is added at a rate to maintain a reaction temperature of 0° C.±5° C. Agitation is stopped to allow phases to separate. The lower aqueous phase (pH=1) is removed. The reactor is charged with aqueous saturated sodium bicarbonate (15.6 Kg) over five minutes. Agitation is stopped to allow phases to separate. The lower aqueous phase (pH=8) is removed. The reactor is charged with magnesium sulfate (2.5 Kg) and agitated for at least 10 minutes. The mixture is filtered though a nutsche filter, and condensed under reduced pressure to afford intermediate (5) (1.80 Kg, 66%). Anal. Calcd for C₁₁H₂₃NO₄: C, 56.6; H, 9.94; N, 6.00. Found C, 56.0; H, 9.68; N, 5.96; HRMS (ESI⁺) Expected for C₁₁H₂₄NO₄, [M+H] 234.1705. Found 234.1703; ¹H NMR (CDCl₃, 500 MHz)δ=6.34(d, J=8.9 Hz, 1H, NH), 4.51 (t, J=5.8, 5.3 Hz, 1H, NHCHCH₂OH), 4.34 (t, J=5.3, 5.3 Hz, 1H, CH3CHCH₂OH), 3.46-3.45, (m, 1H, NHCH), 3.28 (dd, J=10.6, 5.3 Hz, NHCHCHHOH), 3.21 (dd, J=10.2, 5.8 Hz, 1H, CH₃CHCHHOH), 3.16 (dd, J=10.2, 6.2 Hz, 1H, NHCHCHHOH), 3.12 (dd, J=10.6, 7.1 Hz, 1H, CH₃CHCHHOH), 1.53-1.50 (m, 1H, CH₃CHCHHOH), 1.35 (s, 9H, O(CH ₃)₃, 1.30 (ddd, J=13.9, 10.2, 3.7 Hz, 1H, NHCHCHHCH), 1.14 (ddd, J=13.6, 10.2, 3.4 Hz, 1H, NHCHCHHCH), 0.80 (d, J=6.6 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 125.7 MHz) δ 156.1, 77.9, 50.8, 65.1, 67.6, 65.1, 35.6, 32.8, 29.0, 17.1. Mp 92.1° C.

(2S,4S)-Methanesulfonic acid 2-tert-butoxycarbonylamino-5-methanesulfonyloxy-4-methyl-pentyl ester (6). A 50 L reactor is charged with a solution of intermediate (5) (5.1 Kg) in isopropyl acetate (i-PrOAc) 11.8 Kg followed by a rinse with an additional 7.9 Kg i-PrOAc. The reaction is cooled to 15° C.±5° C. and triethylamine (TEA) (7.8 Kg) is added while maintaining the set temperature. The reactor is further cooled to 0° C.±5° C. and methanesulfonyl chloride (MsCl) (6.6 Kg) is added to the reaction solution while maintaining the set temperature. The reaction is stirred for a few hours and monitored for completion by HPLC or TLC. The reaction is quenched by the addition of a saturated aqueous bicarbonate solution and the resulting isolated organic phase is washed successively with cold 10% aqueous triethylamine solution, cold aqueous HCl solution, cold saturated aqueous bicarbonate solution, and finally saturated aqueous brine solution. The organic phase is dried, filtered, and concentrated in vacuo below 55° C.±5° C. until a solid/liquid slurry containing intermediate (6) is obtained. The slurry is used crude in subsequent reaction without further characterization.

(3S,5S)-(1-Benzyl-5-methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (7). A 50 L reactor is charged with 9.1 Kg of neat benzylamine. The reactor is brought to 55° C. and a solution of intermediate (6) (8.2 Kg) in 1,2-dimethoxyethane (DME) (14.1 Kg) is added to the reactor while maintaining a temperature of 60° C.±5° C. After complete addition of this solution, the reaction is stirred at 60° C.±5° C. for several hours and monitored for completion by TLC or HPLC. The reaction is cooled to ambient temperature and volatiles (DME) are removed by rotary evaporation under vacuum. The residue is diluted with 11.7 Kg of 15% (v/v) ethyl acetate/hexanes solution and treated, while agitating, with 18.7 Kg of 20% (wt) aqueous potassium carbonate solution. A triphasic mixture is obtained upon settling. The bottom aqueous phase is removed and the middle phase is set aside. The upper organic phase is collected and held for combination with extracts from additional extractions. The isolated middle phase is extracted twice again with 11.7 Kg portions of 15% (v/v) ethyl acetate/hexanes solution, each time combining the extracts with original organic phase. The combined organic extracts are transferred into a rotary evaporator and solvent is removed under vacuum until an oily residue remains. The residue is then purified via large-scale preparative chromatography to afford purified intermediate (7) as an oil.

(3S,5S)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (8). A 40 L pressure vessel is charged with 0.6 Kg 50% wet, solid palladium on carbon (E101, 10 wt. %) under flow of nitrogen. A solution of 3.2 Kg intermediate (7) in 13.7 Kg of absolute ethanol is then charged to the reactor under nitrogen. The reactor is purged with nitrogen and is then pressurized with hydrogen at 45 psi. The reaction is then heated to 45° C. while maintaining a hydrogen pressure of 45 psi. The reaction is monitored by TLC or LC until complete. The reaction is cooled to ambient temperature, vented, and purged with nitrogen. The reactor contents are filtered through a bed of Celite and the solids are washed with 2.8 Kg of absolute ethanol. The filtrate is concentrated by rotary evaporation under vacuum until a waxy solid is obtained to afford intermediate (8): TLC R_f(Silica F₂₅₄, 70:30 v/v ethyl acetate-hexanes, KMnO₄stain)=0.12; ¹H NMR (300 MHz, CDCl₃) δ 5.31 (br s, 1H), 3.80-3.68 (m, 1H), 2.92 (d, J=11.4 Hz, 1H), 2.77 (AB quart, J_AB=12.0 Hz, Δν=50.2 Hz, 2H), 2.19 (t, J=10.7 Hz, 1H), 1.82-1.68 (m, 2H), 1.54 (br s, 1H), 1.43 (s, 9H), 1.25-1.15 (m, 1H), 0.83 (d, J=6.6 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 155.3, 78.9, 54.3, 50.8, 45.3, 37.9, 28.4, 27.1, 19.2; MS (ESI⁺) m/z 215 (M+H), 429 (2M+H).

B. Synthesis of 1-Cyclopropyl-7-fluoro-8-methoxy-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (19)

Intermediate (12): A reactor is charged with a solution of intermediate (11) (1.2 Kg, 7.7 mol, 1.0 eq) in anhydrous toluene (12 L) followed by ethylene glycol (1.8 L, 15.7 mol, 4.2 eq) and solid p-toluenesulfonic acid (120 g, 10 wt. %). The reaction mixture is stirred at ambient temperature for at least 30 minutes and then heated to reflux, collecting the water/toluene azeotrope in a Dean Stark type trap apparatus until the reaction is complete as determined by TLC analysis (15% EtOAc/Hexanes v/v). Upon completion, the reaction is cooled to ambient temperature and poured into an aqueous solution of sodium bicarbonate (6 L). The organic toluene phase was removed and washed with saturated sodium bicarbonate solution (6 L), distilled water (2×6 L), and saturated aqueous brine (6 L). The organic phase was removed and dried over MgSO₄, filtered, and evaporated under reduced pressure to afford intermediate (12) as an oil (1.3 Kg, 86%). The material is used without further purification in subsequent reaction steps.

Intermediate (13): A reactor is charged with a solution of intermediate (12) (1.2 Kg, 6.0 mol, 1.0 eq) in anhydrous tetrahydrofuran (12 L) and n-butyllithium (2.5M in hexanes, 2.6 L, 6.6 mol, 1.1 eq) is added at −40° C., while maintaining this temperature throughout the addition. The reaction is stirred for at least one hour at −40° C. and trimethylborate (0.9 L, 7.8 mol, 1.3 eq) is added to the mixture while maintaining the temperature at or below −40° C. The reaction mixture is stirred for at least one hour at −40° C. until complete as determined by TLC analysis (30% EtOAc/Hexanes v/v). The reaction is warmed slightly to −30° C. and acetic acid (3 L) is added slowly. Upon complete addition, water is added (0.5 L) to the reaction and the mixture is allowed to quickly warm to ambient temperature while stirring overnight. Organic solvent is removed from the reaction by distillation under reduced pressure at 45° C. To the reaction residue is added 3-4 volumes of water (6 L) and 30% hydrogen peroxide (0.7 L, 1.0 eq) slowly at ambient temperature with cooling provided to control the exotherm. The reaction is stirred for at least an hour at ambient temperature until complete as determined by TLC (15% EtOAc/Hexanes v/v). The reaction mixture is cooled to 0-5° C. and excess peroxide is quenched with the addition of 10% aqueous sodium bisulfite solution (2 L). The mixture is tested to ensure a negative peroxide result and the reaction is acidified by the addition of 6N HCl (aq) (1.2 L). The reaction is stirred until the hydrolysis reaction is complete as determined by TLC or NMR analysis. The resulting solids are collected by suction filtration to afford intermediate (13) as a yellow solid (1.0 Kg, 79%).

Intermediate (14): A reactor is charged with intermediate (13) (0.53 Kg, 3.0 mol, 1.0 eq) and dissolved in dry toluene (2.7 Kg, 3.1 L). To this solution is added dimethylsulfate (0.49 Kg, 3.9 mol, 1.30 eq) followed by solid potassium carbonate (0.58 Kg, 4.2 mol, 1.4 eq). The reaction mixture is heated to reflux and held for at least 1 hour until complete as determined by HPLC. During this time, vigorous gas evolution is observed. The reaction is then cooled to ambient temperature and diluted with distilled water (3.2 L) along with 30% NaOH (aq) (0.13 Kg, 0.33 eq). The aqueous phase is separated and the remaining toluene phase is extracted twice more with distilled water (3.2 L) combined with 30% NaOH (aq) (0.13 Kg, 0.33 eq), removing the aqueous phase each time. The organic upper phase is concentrated by distillation in vacuo (<100 mbar) at approximately 40° C. until a concentrated toluene solution is achieved. The resulting solution is cooled to ambient temperature, checked for quality and yield by HPLC, and carried forward to the next step in the synthesis without further purification (theoretical yield for intermediate (14) assumed, 0.56 Kg).

Intermediate (15a,b): A reactor is charged with 1.8 Kg (2.1 L) anhydrous toluene along with sodium hydride (0.26 Kg, 6.6 mol, 2.20 eq) as a 60 wt. % dispersion in mineral oil. To this mixture is added (0.85 Kg, 7.2 mol, 2.4 eq) diethylcarbonate as the reaction mixture is heated to 90° C. over 1 hour. A solution of intermediate (14) (˜1.0 eq) in toluene from the previous step is added to the reaction while maintaining a temperature of 90° C.±5° C. Gas evolution can be observed during this addition. After complete addition, the reaction is stirred for at least 30 minutes or until complete as determined by HPLC analysis. Upon completion, the mixture is cooled to ambient temperature and diluted with 10 wt. % aqueous sulfuric acid (3.8 Kg, 3.9 mol, 1.3 eq) with agitation. The phases are allowed to separate and the lower aqueous phase is removed. The remaining organic phase is concentrated in vacuo (<100 mbar) at approximately 40° C. until a concentrated toluene solution is achieved. The resulting solution is cooled to ambient temperature and carried forward to the next step in the synthesis without further purification (theoretical yield for intermediate (15a,b) assumed, 0.85 Kg).

Intermediate (16a,b; 17a,b): A reactor is charged with a solution of intermediate (15a,b) (0.85 Kg, ˜3.0 mol, ˜1.0 eq) in toluene from the previous step. To the reactor is then added dimethylformamide-dimethylacetal (0.54 Kg, 4.5 mol, 1.5 eq) and the resulting solution is heated to reflux temperature (˜95-105° C.). The lower boiling solvent (methanol from reaction) is allowed to distill off while the temperature is maintained at ≧90° C. Heating is continued for at least 1 hour or until complete as determined by HPLC analysis. Upon completion, the reaction containing the mixture of intermediate (16a,b), is cooled to ambient temperature and toluene (1.8 Kg, 2.1 L) along with cyclopropylamine (0.21 Kg, 3.6 mol, 1.2 eq) are added to the reaction. The reaction is stirred at ambient temperature for at least 30 minutes until complete as determined by HPLC. Upon completion, the reaction is diluted with 10 wt. % aqueous sulfuric acid (2.9 Kg, 3.0 mol, 1.0 eq) with agitation, and the phases are then allowed to separate. The aqueous phase is removed and the organic phase is concentrated under reduced pressure (<100 mbar) at approximately 40° C. by distillation. When the desired concentration is achieved, the solution is cooled to ambient temperature and the toluene solution containing the mixture of intermediate (17a,b) is carried forward to the next step in the synthesis without further purification (theoretical yield for intermediate (17a,b) assumed, ˜1.1 Kg).

Intermediate (18): A reactor is charged with a solution of the mixture of intermediate (17a,b) (˜4.7 Kg, ˜3.0 mol) at ambient temperature. To the reactor is added N,O-bis(trimethylsilyl)acetamide (0.61 Kg, 3.0 mol, 1.0 eq) and the reaction is heated to reflux temperature (˜105-115° C.) for at least 30 minutes or until complete as determined by HPLC analysis. If not complete, an additional amount of N,O-bis(trimethylsilyl)acetamide (0.18 Kg, 0.9 mol, 0.3 eq) is added to the reaction to achieve completion. Upon completion, the reaction is cooled to below 40° C. and organic solvent is removed under reduced pressure (<100 mbar) at approximately 40° C. by distillation until a precipitate is formed. The reaction is cooled to ambient temperature and the precipitated solids are isolated by suction filtration and washed with distilled water twice (1×1.8 L, 1×0.9 L). The solid is dried to afford intermediate (18) as a white solid (0.76 Kg, 82%). The material is used without further purification in the next reaction step.

Intermediate (19): A reactor is charged with solid intermediate (18) (0.76 Kg, ˜2.5 mol, ˜1.0 eq) at ambient temperature followed by ethanol (5.3 Kg, 6.8 L) and 32 wt. % aqueous hydrochloric acid (1.1 Kg, 10 mol). The reaction mixture is brought to reflux temperature (76-80° C.) during which time the mixture first becomes homogeneous and later becomes heterogeneous. The mixture is heated at reflux for at least 5 hours or until complete as determined by TLC analysis (15% EtOAc/Hexanes v/v). Upon completion, the reaction is cooled to 0° C.±5° C. and the precipitated solid is isolated by filtration and washed with distilled water (1.7 Kg) followed by ethanol (1.7 Kg). The isolated solid is dried to afford intermediate (19) as a white solid (0.65 Kg, ˜95%). ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 14.58 (s, 1H), 8.9 (s, 1H), 8.25 (m, 1H), 7.35 (m, 1H), 4.35 (m, 1H), 4.08 (s, 3H), 1.3 (m, 2H), 1.1 (m, 2H) ¹⁹F NMR (CDCl₃+CFCl₃, 292 MHz) δ (ppm): −119. HPLC: 99.5% by area.

C. Synthesis of borone ester chelate of 1-Cyclopropyl-7-fluoro-8-methoxy-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (20)

A reactor is charged with boron oxide (2.0 Kg, 29 mol) followed by dilution with glacial acetic acid (8.1 L, 142 mol) and acetic anhydride (16.2 L, 171 mol). The resulting mixture is heated to reflux temperature for at least 2 hours. The reaction contents are cooled to 40° C. and the solid 7-fluoroquinolone acid intermediate (19) (14.2 Kg, 51 mol) is added to the reaction mixture. The mixture is again heated to reflux temperature for at least 6 hours. Reaction progress is monitored by HPLC and NMR. The mixture is cooled to approximately 90° C. and toluene (45 L) is added to the reaction. The reaction is further cooled to 50° C. and tert-butylmethyl ether (19 L) is added to the reaction mixture to bring about precipitation of the product. The mixture is then cooled to 20° C. and the solid product 19 is isolated by filtration. The isolated solids are then washed with tert-butylmethyl ether (26 L) prior to drying in a vacuum oven at 40° C. (50 torr). The product yield obtained for intermediate (20) in this reaction is 86.4%. Raman (cm⁻¹): 3084.7, 3022.3, 2930.8, 1709.2, 1620.8, 1548.5, 1468.0, 1397.7, 1368.3, 1338.5, 1201.5, 955.3, 653.9, 580.7, 552.8, 384.0, 305.8. NMR (CDCl₃, 300 MHz) δ (ppm): 9.22 (s, 1H), 8.38-8.33 (m, 1H), 7.54 (t, J=9.8 Hz, 1H), 4.38-4.35 (m, 1H), 4.13 (s, 3H), 2.04 (s, 6H), 1.42-1.38 (m, 2H), 1.34-1.29 (m, 2H). TLC (Whatman MKC18F Silica, 60 Å, 200 μm), Mobile Phase: 1:1 (v/v) CH₃CN:0.5N NaCl (aq), UV (254/366 nm) visualization; R_f=0.4-0.5.

D. Coupling of 1-Cyclopropyl-7-fluoro-8-methoxy-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (20) to (3S,5S)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (8), and synthesis of malate salt of (3S,5S)-7-[3-amino-5-methyl-piperidinyl]-1-cyclopropyl-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (25)

A reactor is charged with solid intermediate (20) (4.4 Kg, 10.9 mol) followed by dilution with a solution of triethylamine (TEA) (2.1 L, 14.8 mol) and piperidine side chain intermediate (8) (2.1 Kg, 9.8 mol) in acetonitrile (33.5 L, 15.7 L/Kg) at room temperature. The resulting mixture is warmed to approximately 50° C. until reaction is judged complete. Reaction progress is monitored by HPLC or reverse phase TLC. When complete, the reaction is cooled to approximately 35° C. and reaction volume is reduced to approximately half by distillation of acetonitrile under vacuum between 0-400 torr. The reactor is then charged with 28.2 Kg of 3.0N NaOH (aq) solution and the temperature is raised to approximately 40° C. Distillation under vacuum is continued between 1-4 hours or until no further distillates are observed. The reaction is then cooled to room temperature and the hydrolysis reaction is monitored by HPLC or reverse phase TLC. Upon completion, the reaction mixture is neutralized to a pH of between 6-8 by adding ˜4-5 Kg of glacial acetic acid. The reactor is then charged with 12.7 Kg (9.6 L) of dichloromethane as an extraction solvent, the mixture is agitated, phases are allowed to separate, and the organic dichloromethane phase is removed. The extraction process is repeated two additional times using 12.7 Kg (9.6 L) of dichloromethane, collecting the lower, organic phase each time. The aqueous phase is discarded and the organic extracts are combined in a single reactor. The reactor contents are heated to 40° C. and the reaction volume is reduced to approximately one half by distillation. The reactor is then charged with 20.2 Kg 6.0N HCl (aq) solution, the temperature is adjusted to 35° C., and agitation is allowed for at least 12 hours to permit the Boc deprotection reaction to occur. The reaction is monitored by HPLC or reverse phase TLC. When complete, agitation is discontinued and the phases are allowed to separate. The lower, organic phase is removed and set aside. The reactor is then charged with 12.7 Kg (9.6 L) of dichloromethane as an extraction solvent, the mixture is agitated, phases are allowed to separate, and the organic dichloromethane phase is removed. The organic extracts are combined and discarded. The remaining aqueous phase is diluted with 18.3 Kg distilled water and the temperature is raised to approximately 50° C. Distillation under vacuum (100-400 torr) is performed to remove residual dichloromethane from the reaction. The pH of the reaction is then adjusted to between 7.8-8.1 using about 9.42 Kg of 3.0N NaOH (aq) solution while keeping the temperature of the reaction below 65° C. The reaction is cooled to 50° C. and the precipitated solids are aged for at least an hour prior to cooling the mixture to room temperature. The solids are isolated by suction filtration and washed twice with 5.2 Kg portions of distilled water. The solids are dried for at least 12 hours with suction and then for an additional 12 hours in a convection oven at 55° C. The yield achieved for intermediate (23) in this example is 3.2 Kg (79%). A reactor is charged with 3.2 Kg solid intermediate (23) and the solids are suspended in 25.6 Kg of 95% ethanol as solvent. To the reactor is then added 1.1 Kg of solid D,L-malic acid (24), and the mixture is heated to reflux temperature (˜80° C.). Distilled water (˜5.7 L) is added to the reaction until a complete solution is achieved and 0.2 Kg of activated charcoal is added. The reaction mixture is passed through a filter to achieve clarification, cooled to 45° C. and held for a period of at least 2 hours to allow crystallization to occur. The reaction mixture is further cooled to 5° C. and the suspended solids are isolated by suction filtration. The solids are then washed with 6.6 KG of 95% ethanol and dried for at least 4 hours with suction under vacuum. The solids are then further dried in a convection oven for at least 12 hours at 45° C. to afford 3.1 Kg of intermediate (24) (70%). NMR (D₂O, 300 MHz) δ (ppm): 8.54 (s, 1H), 7.37 (d, J=9.0 Hz, 1H), 7.05 (d, J=9.0 Hz, 1H), 4.23-4.18 (m, 1H), 4.10-3.89 (m, 1H), 3.66 (br s, 1H), 3.58 (s, 3H), 3.45 (d, J=9.0 Hz, 1H), 3.34 (d, J=9.3 Hz, 1H), 3.16 (d, J=12.9 Hz, 1H), 2.65 (dd, J=16.1, 4.1 Hz, 1H), 2.64-2.53 (m, 1H), 2.46 (dd, J=16.1, 8.0 Hz, 1H), 2.06 (br s, 1H), 1.87 (d, J=14.4 Hz, 1H), 1.58-1.45 (m, 1H), 1.15-0.95 (m, 2H), 0.91 (d, J=6.3 Hz, 3H); 0.85-0.78 (m, 2H). TLC (Whatman MKC18F Silica, 60 Å, 200 μm), Mobile Phase: 1:1 (v/v) CH₃CN:0.5N NaCl (aq), UV (254/366 nm) visualization. HPLC: Mobile Phase H₂O with 0.1% formic acid/Acetonitrile with 0.1% formic acid, gradient elution with 88% H₂O/formic acid to 20% H₂O/formic acid, Zorbax SB-C8 4.6 mm×150 mm column, Part No. 883975.906, 1.5 ml/min rate, 20 min run time, 292 nm, Detector Model G1314A, S/N JP72003849, Quat Pump Model G1311A, S/N US72102299, Auto Sampler Model G1313A, S/N DE14918139, Degasser Model G1322A, S/N JP73007229; approximate retention time for intermediate (19): 13.0 min; approximate retention time for intermediate (20): 11.6 min; approximate retention time for intermediate (21): 16.3 min; approximate retention time for intermediate (22): 18.2 min; approximate retention time for intermediate (23): 8.6 min; approximate retention time for compound (25): 8.6 min.

………………..

REF

A. ARJONA ET AL: “Nemonoxacin“, DRUGS OF THE FUTURE, vol. 34, no. 3, 1 January 2009 (2009-01-01), page 196, XP55014485, ISSN: 0377-8282, DOI: 10.1358/dof.2009.034.03.1350294


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3	*	ANONYMOUS: “TaiGen Biotechnology Initiates Phase II Trial Of Nemonoxacin For Treatment Of Adult Community Acquired Pneumonia (CAP)“, 20070108, [Online] 8 January 2007 (2007-01-08), page 1, XP007919910, Retrieved from the Internet: URL:http://www.taigenbiotech.com/news.html#11> [retrieved on 2011-12-12]
4	*	ANONYMOUS: “TaiGen Initiates Phase 1B Trial of a Novel Quinolone Antibiotic“, 20050618, 18 June 2005 (2005-06-18), pages 1-2, XP007919904,
5	*	See also references of WO2010002415A1

WO2007110834A2 *	Mar 26, 2007	Oct 4, 2007	Procter & Gamble	Malate salts, and polymorphs of (3s,5s)-7-[3-amino-5-methyl-piperidinyl]-1-cyclopropyl-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid
WO2009023473A2 *	Aug 5, 2008	Feb 19, 2009	Chi-Hsin Richard King	Antimicrobial parenteral formulation
WO2010009014A2 *	Jul 10, 2009	Jan 21, 2010	Taigen Biotechnology Co., Ltd.

US8211909	7-4-2012	TREATMENT OF ANTIBIOTIC-RESISTANT BACTERIA INFECTION
US8158798	4-18-2012	Coupling Process For Preparing Quinolone Intermediates
US8039485	10-19-2011	Malate salts, and polymorphs of (3S,5S)-7-[3-amino-5-methyl-piperidinyl]-1-cyclopropyl-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid
US2010152452	6-18-2010	STEREOSELECTIVE SYNTHESIS OF PIPERIDINE DERIVATIVES
US2010041697	2-19-2010	PNEUMONIA TREATMENT
US7528264	5-6-2009	Hydride reduction process for preparing quinolone intermediates
US2009042932	2-13-2009	ANTIMICROBIAL PARENTERAL FORMULATION
US7456279	11-26-2008	Coupling process for preparing quinolone intermediates

US8158798	Oct 27, 2008	Apr 17, 2012	Taigen Biotechnology Co., Ltd.	Coupling process for preparing quinolone intermediates
US8211909	Sep 8, 2008	Jul 3, 2012	Taigen Biotechnology Co., Ltd.	Treatment of antibiotic-resistant bacteria infection
WO2010002965A2 *	Jul 1, 2009	Jan 7, 2010	Taigen Biotechnology Co., Ltd.	Pneumonia treatmen

WO 2007110834

WO 2007110835

WO 2007110836

WO 1999014214

WO 2010077798

1, nemonoxacin; 2, delafloxacin; 3, finafloxacin; 4, zabofloxacin; 5, JNJ-Q2; 6, DS-8587; 7, KPI-10; 8, ozenoxacin; 9, chinfloxacin; 10, ACH-702.

Vorapaxar …FDA advisory panel votes to approve Merck & Co’s vorapaxar

January 17, 2014 8:31 am / 2 Comments on Vorapaxar …FDA advisory panel votes to approve Merck & Co’s vorapaxar

VORAPAXAR

Thrombosis, Antiplatelet Therapy, PAR1 Antagonists , MERCK ..ORIGINATOR

Ethyl N-[(3R,3aS,4S,4aR,7R,8aR,9aR)-4-[(E)-2-[5-(3-fluorophenyl)-2-pyridyl]vinyl]-3-methyl-1-oxo-3a,4,4a,5,6,7,8,8a,9,9a-decahydro-3H-benzo[f]isobenzofuran-7-yl]carbamate

618385-01-6 CAS NO

Also known as: SCH-530348, MK-5348

Molecular Formula: C₂₉H₃₃FN₂O₄

Molecular Weight: 492.581723

Vorapaxar (formerly SCH 530348) is a thrombin receptor (protease-activated receptor, PAR-1) antagonist based on the natural product himbacine. Discovered by Schering-Plough and currently being developed by Merck & Co., it is an experimental pharmaceutical treatment for acute coronary syndrome chest pain caused by coronary artery disease.^[1]

In January 2011, clinical trials being conducted by Merck were halted for patients with stroke and mild heart conditions.^[2] In a randomized double-blinded trial comparing vorapaxar with placebo in addition to standard therapy in 12,944 patients who had acute coronary syndromes, there was no significant reduction in a composite end point of death from cardiovascular causes, myocardial infarction, stroke, recurrent ischemia with rehospitalization, or urgent coronary revascularization. However, there was increased risk of major bleeding.^[3]

A trial published in February 2012, found no change in all cause mortality while decreasing the risk of cardiac death and increasing the risk of major bleeding.^[4]

SCH-530348 is a protease-activated thrombin receptor (PAR-1) antagonist developed by Schering-Plough and waiting for approval in U.S. for the oral secondary prevention of cardiovascular events in patients with a history of heart attack and no history of stroke or transient ischemic attack. The drug candidate is being investigated to determine its potential to provide clinical benefit without the liability of increased bleeding; a tendency associated with drugs that block thromboxane or ADP pathways. In April 2006, SCH-530348 was granted fast track designation in the U.S. for the secondary prevention of cardiovascular morbidity and mortality outcomes in at-risk patients.

Vorapaxar was recommended for FDA approval on January 15, 2014.^[5]

VORAPAXAR

17 JAN 2014
FDA advisory panel votes to approve Merck & Co’s vorapaxar REF 6

VORAPAXAR SULPHATE

CAS Number: 705260-08-8

Molecular Formula: C29H33FN2O4.H2O4S

Molecular Weight: 590.7

Chemical Name: Ethyl [(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-2-[5-(3-fluorophenyl)pyridin-2- yl]ethenyl]-1-methyl-3-oxododecahydronaphtho[2,3-c]furan-6-yl]carbamate sulfate

Synonyms: Carbamic acid, [(1R,3aR,4aR,6R,8aR,9S,9aS)-9-[(1E)-2-[5-(3-fluorophenyl)-2- pyridinyl]ethenyl]dodecahydro-1-methyl-3-oxonaphtho[2,3-c]furan-6-yl]-,ethyl ester,sulfate; SCH-530348

Vorapaxar Sulfate (SCH 530348) a thrombin receptor (PAR-1) antagonist for the prevention and treatment of atherothrombosis.

……………………

GENERAL INTRO

SIMILAR NATURAL PRODUCT

+ HIMBACINE

Himbacine is an alkaloid muscarinic receptor antagonist displaying more potent activity associated with M2 and M2 subtypes over M1 or M3. Observations show himbacine bound tightly to various chimeric receptors in COS-7 cells as well as possessed the ability to bind to cardiac muscarinic receptors allosterically. Recent studies have produced series of thrombin receptor (PAR1) antagonists derived from himbacine Himbacine is an inhibitor of mAChR M2 and mAChR M4.

Technical Information

Physical State:	Solid
Derived from:	Australian pine Galbulimima baccata
Solubility:	Soluble in ethanol (50 mg/ml), methanol, and dichloromethane. Insoluble in water.
Storage:	Store at -20° C
Melting Point:	132-134 °C
Boiling Point:	469.65 °C at 760 mmHg
Density:	1.08 g/cm³
Refractive Index:	n²⁰_D 1.57
Optical Activity:	α20/D +51.4º, c = 1.01 in chloroform

Application:	An alkaloid muscarinic receptor antagonist
CAS Number:	6879-74-9

Molecular Weight:	345.5
Molecular Formula:	C₂₂H₃₅NO₂

general scheme:

……………………………

SYNTHESIS

WO2003089428A1

THE EXACT BELOW COMPD IS 14

Example 2

Step 1 :

Phosphonate 7, described in US 6,063,847, (3.27 g, 8.1 mmol) was dissolved in THF (12 ml) and C(O)Oled to 0 °C, followed by addition of 2.5 M n- BuLi (3.2 ml, 8.1 mmol). The reaction mixture was stirred at 0 °C for 10 min and warmed up to rt. A solution of aldehyde 6, described in US 6,063,847, in THF (12 ml) was added to the reaction mixture. The reaction mixture was stirred for 30 min. Standard aqueous work-up, followed by column chromatography (30-50% EtOAc in hexane) afforded product 8. ¹HNMR (CDCI₃): δ 0.92-1.38 (m, 31 H), 1.41 (d, J= 6 Hz, 3H), 1.40-1.55 (m, 2H), 1.70-1.80 (m, 2H), 1.81-1.90 (m, 2H), 2.36 (m, 2H), 2.69 (m, 1 H), 3.89 (m, 4H), 4.75 (m, 1 H), 6.28-6.41 (m, 2H), 7.05-7.15 (m, 2H), 8.19 (br s, 1 H). Step 2:

Compound 8 (2.64 g, 4.8 mmol) was dissolved in THF (48 ml). The reaction mixture was C(O)Oled to 0 °C followed by addition of 1 M TBAF (4.8 ml). The reaction mixture was stirred for 5 min followed by standard aqueous work-up. Column chromatography (50% EtOAc/hexane) afforded product 9 (1.9 g, 100%). ¹HNMR (CDCI₃): δ 1.15-1.55 (m, 6H), 1.41 (d, J= 6 Hz, 3H), 1.70-1.82 (m, 3H), 1.85-1.90 (m, 1 H), 2.36 (m, 2H), 2.69 (m, 1 H), 3.91 (m, 4H), 4.75 (m, 1 H), 6.18- 6.45 (m, 2H), 7.19 (br s, 2H), 8.19 (br s, 1 H). Step 3:

To a solution of compound 9 (250 mg, 0.65 mmol) in pyridine (5 ml) C(O)Oled to 0 °C was added Tf₂O (295 μL, 2.1 mmol). The reaction mixture was stirred overnight at rt. Standard aqueous work-up followed by column chromatography afforded product 10 (270 mg, 80%). ¹HNMR (CDCI₃): δ 1.15-1.55 (m, 6H), 1.41 (d, J= 6 Hz, 3H), 1.70-1.82 (m, 3H), 1.85-1.90 (m, 1 H), 2.36 (m, 2H), 2.69 (m, 1 H), 3.91 (m, 4H), 4.75 (m, 1 H), 6.42-6.68 (m, 2H), 7.25 (m, 1 H), 7.55 (m, 1 H), 8.49 (d, J= 2.8 Hz, 1 H).

Compound 10 (560 mg, 1.1 mmol), 3-fluorophenyl boronic acid (180 mg, 1.3 mmol) and K₂CO₃ (500 mg, 3.6 mmol) were mixed with toluene (4.4 ml), H₂O (1.5 ml) and EtOH (0.7 ml) in a sealed tube. Under an atmosphere of N₂, Pd(Ph₃P)₄ (110 mg, 0.13 mmol) was added. The reaction mixture was heated at 100 °C for 2 h under N₂. The reaction mixture was C(O)Oled down to rt, poured to EtOAc (30 ml) and washed with water (2X20 ml). The EtOAc solution was dried with NaHCO₃ and concentrated at reduced pressure to give a residue. Preparative TLC separation of the residue (50% EtOAc in hexane) afforded product 11 (445 mg, 89%). ¹HNMR (CDCI₃): δ 1.15-1.59 (m, 6H), 1.43 (d, J= 6 Hz, 3H), 1.70-1.79 (m, 2H), 1.82 (m, 1H), 1.91 (m, 2H), 2.41 (m, 2H), 2.69 (m, 1 H), 3.91 (m, 4H), 4.75 (m, 1 H), 6.52-6.68 (m, 2H), 7.15 (m, 1 H), 7.22 (m, 2H), 7.35 (m, 1 H), 7.44 (m, 1 H), 7.81 (m, 1 H), 8.77 (d, J= 1.2 Hz, 1 H). Step 5:

Compound 11 (445 mg, 0.96 mmol) was dissolved in a mixture of acetone (10 ml) and 1 N HCI (10 ml). The reaction mixture was heated at 50 °C for 1 h.

Standard aqueous work-up followed by preparative TLC separation (50% EtOAc in hexane) afforded product 12 (356 mg, 89%). ¹HNMR (CDCI₃): δ 1.21-1.45 (m, 2H), 1.47 (d, J= 5.6 Hz, 3H), 1.58-1.65 (m, 2H), 2.15 (m, 1 H), 2.18-2.28 (m, 2H), 2.35- 2.51 (m, 5H), 2.71 (m, 1 H), 4.79 (m, 1 H), 6.52-6.68 (m, 2H), 7.15 (m, 1 H), 7.22 (m, 2H), 7.35 (m, 1 H), 7.44 (m, 1 H), 7.81 (m, 1 H), 8.77 (d, J= 1.2 Hz, 1 H). Step 6:

Compound 12 (500 mg, 4.2 mmol) was dissolved in EtOH (40 ml) and CH₂CI₂ (15 ml) NH₃ (g) was bubbled into the solution for 5 min. The reaction mixture was C(O)Oled to 0 °C followed by addition of Ti(O/^‘Pr)₄ (1.89 ml, 6.3 mmol). After stirring at 0 °C for 1 h, 1 M TiCI (6.3 ml, 6.3 mmol) was added. The reaction mixture was stirred at rt for 45 min and concentrated to dryness under reduced pressure. The residue was dissolved in CH₃OH (10 ml) and NaBH₃CN (510 mg, 8 mmol) was added. The reaction mixture was stirred overnight at rt. The reaction mixture was poured to 1 N NaOH (100 ml) and extracted with EtOAc (3x 100 ml). The organic layer was combined and dried with NaHC0₃. Removal of solvent and separation by PTLC (5% 2 M NH₃ in CH₃OH/ CH₂CI₂) afforded β-13 (spot 1 , 30 mg, 6%) and α-13 (spot 2, 98 mg, 20%). β-13: ¹HNMR (CDCI₃): δ 1.50-1.38 (m, 5H), 1.42 (d, J= 6 Hz, 3H), 1.51-1.75 (m, 5H), 1.84 (m, 2H), 2.38 (m, 1 H), 2.45 (m, 1 H), 3.38 (br s, 1 H), 4.78 (m, 1 H), 6.59 (m, 2H), 7.15 (m, 1 H), 7.26 (m, 2H), 7.36 (m, 1 H), 7.42 (m, 1 H), 7.82 (m, 1 H), 8.77 (d, J= 2 Hz, 1 H). α-13:¹HNMR (CDCI₃): δ 0.95 (m, 2H), 1.02-1.35 (m, 6H), 1.41 (d, J= 6 Hz, 3H), 1.82-1.95 (m, 4H), 2.37 (m; 2H), 2.69 (m, 2H), 4.71 (m, 1 H), 6.71 (m, 2H), 7.11 (m, 1 H), 7.25 (m, 2H), 7.38 (m, 1 H), 7.42 (m, 1 H), 7.80 (m, 1 H), 8.76 (d, J= 1.6 Hz, 1 H). Step 7:

Compound α-13 (300 mg, 0.71 mmol) was dissolved in CH₂CI₂ (10 ml) followed by addition of Et₃N (0.9 ml). The reaction mixture was C(O)Oled to 0 °C and ethyl chloroformate (0.5 ml) was added. The reaction mixture was stirred at rt for 1 h. The reaction mixture was directly separated by preparative TLC (EtOAc/ hexane, 1 :1) to give the title compound (14) VORAPAXAR (300 mg, 86%). MS m/z 493 (M+1).

HRMS Calcd for C₂₉H₃₄N₂O₄F (M+1 ): 493.2503, found 493.2509.

…………………

SYNTHESIS 1

http://www.google.com/patents/WO2006076564A1

VORAPAXAR= COMPD A

Example 6 – Preparation of Compound A

To a three-neck flask equipped with an agitator, thermometer and nitrogen inertion was added 7A (13.0 g), THF (30 mL). The mixture was cooled to below -20⁰C after which lithium diisopropylamide (2M, 20 mL) was slowly added. The reaction mixture was agitated for an additional hour (Solution A). To another flask was added 6 (10.0 g) and THF (75 mL) . The mixture was stirred for about 30 minutes and then slowly transferred into the solution A while maintaining the temperature below 20⁰C. The mixture was stirred at below -20⁰C for an additional hour before quenching the reaction by adding 20 mL of water. The reaction mixture was warmed to 0⁰C and the pH was adjusted to about 7 by addition of 25% HaSO₄ (11 mL). The mixture was further warmed to 20⁰C and then diluted with 100 mL of ethyl acetate and 70 mL of water. The two phases that had formed were separated and the aqueous layer was extracted with 50 mL of ethyl acetate. The solvents THF and ethyl acetate were then replaced with ethanol, and the Compound A was precipitated out as a crystalline solid from ethanol with seeding at 35 to 4O⁰C. After cooling to O⁰C, the suspension was stirred for an additional hour and then the product was filtered and washed with cold ethanol. The product was dried at 50 – 60⁰C under vacuum to provide an off-white solid. VORAPAXAR

Yield: 12.7 g, (90%). m.p. 104.9⁰C (DSC onset point).

¹H NMR (CDCl₃) δ 8.88 (d, J = 2.4 Hz, IH), 8.10 (dd, J = 8.2, 2.4 Hz, IH), 7.64 (IH), 7.61 (d, J = 8.8 Hz, IH), 7.55 (m, J = 8.2, 6.2 Hz, IH), 7.51 (d, J = 8.0 Hz, IH), 7.25 (dt, J = 9.0, 2.3 Hz, IH), 7.08 (d, J = 8.0 Hz, IH), 6.68 (dd, J = 15.4, 9.4 Hz, IH), 6.58 (d, J = 9.6 Hz, IH), 4.85 (dd, J = 14.2, 7.2 Hz, IH), 3.95 (dd, J = 14.2, 7.1 Hz, 2H), 3.29 (m, IH), 2.66 (m, J = 12.0, 6.4 Hz, IH), 2.33 (m, 2H), 1.76 (m, 4H), 1.30 (d, J = 5.6 Hz, 3H), 1.19 (m, 4H), 1.14 (t, J = 7.2 Hz, 3H), 0.98 (m, IH), 0.84 (m, IH). MS (EI) m/z: calcd. 492, found 492.

BISULPHATE SALT

Example 7 – Preparation of an Acid Salt (bisulfate) of Compound A:

Compound IA (5 g) was dissolved in about 25 mL of acetonitrile.

The solution was agitated for about 10 minutes and then heated to about 50 ⁰C. About 6 mL of 2M sulfuric acid in acetonitrile was added into the heated reaction mixture. The solid salt of Compound A precipitated out during the addition of sulfuric acid in acetonitrile. After addition of sulfuric acid solution, the reaction mixture was agitated for 1 hour before cooling to room temperature. The precipitated solid was filtered and washed with about 30 mL of acetonitrile. The wet solid was dried under vacuum at room temperature for 1 hour and at 80 ⁰C for about 12 hours to provide about 5 g white solid (yield 85%). m.p. 217.0 ⁰C. ¹H NMR (DMSO) 9.04 (s, IH), 8.60 (d, J = 8.1 Hz, IH), 8.10 (d, J = 8.2 Hz, IH), 7.76 (d, J = 10.4, IH), 7.71 (d, J = 7.8 Hz, IH), 7.60 (dd, J = 8.4, 1.8 Hz, IH), 7.34 (dd, 8.4, 1.8 Hz, IH), 7.08 (d, J = 8.0 Hz, IH), 7.02 (m, IH), 6.69 (d, J = 15.8 Hz, IH), 4.82 (m, IH), 3.94 (dd, J = 14.0, 7.0 Hz, 2H), 3.35 (brs, IH), 2.68 (m, IH), 2.38 (m, 2H), 1.80-1.70 (m, 4H), 1.27 (d, J = 5.8 Hz, 3H), 1.21 (m, 2H), 1.13 (t, J = 7.0 Hz, 3H), 0.95 (m, IH, 0.85 (m, IH). MS (EI) m/z calcd. 590, found 492.

INTERMEDIATE 6

Example 5- Preparation of Compound 6

To a three-neck flask equipped with an agitator, thermometer and nitrogen inert were added the crude product solution of Compound 5 (containing about 31 g. of Compound 5 in 300 mL solution) and anhydrous DMF (0.05 mL). After the mixture was agitated for 5 minutes, oxalyl chloride (12.2 mL) was added slowly while maintaining the batch temperature between 15 and 25°C. The reaction mixture was agitated for about an hour after the addition and checked by NMR for completion of reaction. After the reaction was judged complete, the mixture was concentrated under vacuum to 135 mL while maintaining the temperature of the reaction mixture below 30⁰C. The excess oxalyl chloride was removed completely by two cycles of vacuum concentration at below 50⁰C with replenishment of toluene (315 mL) each time, resulting in a final volume of 68 mL. The reaction mixture was then cooled to 15 to 25°C, after which THF (160 mL) and 2,6-lutidine (22 mL) were added. The mixture was agitated for 16 hours at 20 to 25°C under 100 psi hydrogen in the presence of dry 5% Pd/C (9.0 g). After the reaction was judged complete, the reaction mixture was filtered through celite to remove catalyst. More THF was added to rinse the hydrogenator and catalyst, and the reaction mixture was again filtered through celite. Combined filtrates were concentrated under vacuum at below 25°C to 315 mL. MTBE (158 mL) and 10% aqueous solution of phosphoric acid (158 mL) were added for a thorough extraction at 10⁰C to remove 2,6- lutidine. Then phosphoric acid was removed by extracting the organic layer with very dilute aqueous sodium bicarbonate solution (about 2%), which was followed by a washing with dilute brine. The organic solution was concentrated atmospherically to a volume of 90 mL for solvent replacement. IPA (315 mL) was added to the concentrated crude product solution. The remaining residual solvent was purged to <_ 0.5% of THF (by GC) by repeated concentration under vacuum to 68 mL, with replenishment of IPA (315 mL) before each concentration. The concentrated (68 mL) IPA solution was heated to 50°C, to initiate crystallization. To this mixture n-heptane (68 mL) was added very slowly while maintaining the batch temperature at 50°C. The crystallizing mixture was cooled very slowly over 2.5 hours to 25°C. Additional n- heptane (34 mL) was added very slowly into the suspension mixture at 25⁰C. The mixture was further cooled to 20⁰C, and aged at that temperature for about 20 hours. The solid was filtered and washed with a solvent mixture of 25% IPA in n-heptane, and then dried to provide

19.5 g of a beige colored solid of Compound 6. (Yield: 66%) m.p. 169.3⁰C. IH NMR (CD₃CN) δ 9.74 (d, J = 3.03 Hz, IH), 5.42 (br, IH), 4.69 (m, IH), 4.03 (q, J = 7.02 Hz, 2H), 3.43 (qt, J = 3.80, 7.84 Hz, IH), 2.67 (m, 2H), 2.50 (dt, J = 3.00, 8.52 Hz, IH), 1.93 (d, J = 12.0 Hz, 2H), 1.82 (dt, J = 3.28, 9.75 Hz, 2H), 1.54 (qd, J = 3.00, 10.5 Hz, IH), 1.27 (d, J = 5.97 Hz, 3H), 1.20 (m, 6H), 1.03 – 0.92 (m, 2H). MS (ESI) m/z (M⁺+1): calcd. 324, found 324.

INTERMEDIATE 7A

Example 4 – Preparation of Compound 7A

+ 1-Pr₂NLi + (EtO)₂POCI – + LiCI

To a 10 L three-necked round bottomed flask equipped with an agitator, thermometer and a nitrogen inlet tube, was added 20Og of

Compound 8 (1.07 mol, from Synergetica, Philadelphia, Pennsylvania). THF (1000 mL) was added to dissolve Compound 8. After the solution was cooled to -80 ⁰C to -50 ⁰C, 2.0 M LDA in hexane/THF(1175 mL, 2.2 eq) was added while maintaining the batch temperature below -50 ⁰C. After about 15 minutes of agitation at -80⁰C to -50 ⁰C, diethyl chlorophosphate (185 mL, 1.2 eq) was added while maintaining the batch temperature below -50 ⁰C. The mixture was agitated at a temperature from -80⁰C to – 50 ⁰C for about 15 minutes and diluted with n-heptane (1000 mL). This mixture was warmed up to about -35 ⁰C and quenched with aqueous ammonium chloride (400 g in 1400 mL water) at a temperature below -10 ⁰C. This mixture was agitated at -15⁰C to -10 ⁰C for about 15 minutes followed by agitation at 15⁰C to 25 ⁰C for about 15 minutes. The aqueous layer was split and extracted with toluene (400 mL). The combined organic layers were extracted with 2N hydrochloric acid (700 mL) twice. The product-containing hydrochloric acid layers were combined and added slowly to a mixture of toluene (1200 mL) and aqueous potassium carbonate (300 g in 800 mL water) at a temperature below 30 ⁰C. The aqueous layer was extracted with toluene (1200 mL). The organic layers were combined and concentrated under vacuum to about 600 ml and filtered to remove inorganic salts. To the filtrate was added n-heptane (1000 ml) at about 55 ⁰C. The mixture was cooled slowly to 40 ⁰C, seeded, and cooled further slowly to -10 ⁰C. The resulting slurry was aged at about -10 ⁰C for 1 h, filtered, washed with n- heptane, and dried under vacuum to give a light brown solid (294 g, 85% yield), m.p. 52 ⁰C (DSC onset point).¹H NMR (CDCl₃) δ 8.73 (d, J = 1.5 Hz, IH), 7.85 (dd, Ji = 8.0 Hz, J₂ = 1.5 Hz, IH), 7.49 (dd, Ji = 8.0 Hz, J₂ = 1.3 Hz, IH), 7.42 (m, IH), 7.32 (d, J = 7.8 Hz, IH), 7.24 (m, IH), 7.08 (dt, Ji = 8.3 Hz, J₂ = 2.3 Hz, IH), 4.09 (m, 4H), 3.48 (d, J = 22.0 Hz, 2H), 1.27 (t, J = 7.0 Hz, 6H). MS (ESI) for M+H calcd. 324, found 324.

Example 3 – Preparation of Compound 5:

4 5

To a three-necked round bottomed flask equipped with an agitator, thermometer and a nitrogen inlet tube was added a solution of Compound 4 in aqueous ethanol (100 g active in 2870 ml). The solution was concentrated to about 700 ml under reduced pressure at 35⁰C to 40°C to remove ethyl alcohol. The resultant homogeneous mixture was cooled to 20⁰C to 30⁰C and its pH was adjusted to range from 12 to 13 with 250 ml of 25% sodium hydroxide solution while maintaining the temperature at 20-30⁰C. Then 82 ml of ethyl chloroformate was slowly added to the batch over a period of 1 hour while maintaining the batch temperature from 20⁰C to 30⁰C and aged for an additional 30 minutes. After the reaction was judged complete, the batch was acidified to pH 7 to 8 with 10 ml of concentrated hydrochloric acid (37%) and 750 ml of ethyl acetate. The pH of the reaction mixture was further adjusted to pH 2 to 3 with 35% aqueous hydrochloric acid solution. The organic layer was separated and the aqueous layer was extracted again with 750 ml of ethyl acetate. The combined organic layers were washed twice with water (200 ml) . Compound 5 was isolated from the organic layer by crystallization from ethyl acetate and heptane mixture (1: 1 mixture, 1500 ml) at about 70⁰C to 80 ⁰C. The solid was filtered at 50⁰C to 60 °C, washed with heptane and then dried to provide an off-white solid (yield 50%). m.p. 197.7°C. ¹HNMR (CD₃CN) δ 5.31 (brs, IH), 4.67 (dt, J = 16.1, 5.9 Hz, IH), 4.03 (q, J = 7.1 Hz, 2H), 3.41 (m, IH), 2.55 – 2.70 (m, 2H), 1.87 – 1.92 (m, IH), 1.32 – 1.42 (m, IH), 1.30 (d, J = 5.92 Hz, 3H), 1.30 – 1.25 (m, 6H), 0.98 (qt, J = 15.7, 3.18 Hz, 2H). MS (ESI) M+l m/z calculated 340, found 340.

Example 2 – Preparation of Compound 4;

3 4

7.4 kg of ammonium formate was dissolved in 9L of water at 15- 25⁰C, and then cooled to 0-10⁰C. 8.9 kg of Compound 3 was charged at 0-15⁰C followed by an addition of 89L of 2B ethyl alcohol. The batch was cooled to 0-5⁰C 0.9 kg of 10% Palladium on carbon (50% wet) and 9 L of water were charged. The batch was then warmed to 18-28⁰C and agitated for 5 hours, while maintaining the temperature between 18-28 ⁰C. After the reaction was judged complete, 7 IL of water was charged. The batch was filtered and the wet catalyst cake was then washed with 8OL of water. The pH of the filtrate was adjusted to 1-2 with 4N aqueous hydrochloric acid solution. The solution was used in the next process step without further isolation. The yield is typically quantiative. m.p. 216.4⁰C. IH NMR (D₂O+1 drop HCl) δ 3.15 (m, IH), 2.76 (m, IH), 2.62 (m, IH), 2.48 (dd,J-5.75Hz, IH), 1.94 (m, 2H), 1.78 (m, 2H), 1.38 (m, 2H), 1.20 (m, 6H), 1.18 (m, IH), 0.98 (q,J=2.99Hz, IH).

Example 1 – Preparation of Compound 3

2B 3

To a reactor equipped with an agitator, thermometer and nitrogen, were added about 10.5 kg of 2B, 68 L of acetone and 68 L of IN aqueous hydrochloric acid solution. The mixture was heated to a temperature between 50 and 60⁰C and agitated for about 1 hour before cooling to room temperature. After the reaction was judged complete, the solution was concentrated under reduced pressure to about 42 L and then cooled to a temperature between 0 and 5⁰C. The cooled mixture was agitated for an additional hour. The product 3 was filtered, washed with cooled water and dried to provide an off-white solid (6.9 kg, yield 76%). m.p. 251⁰C. Η NMR (DMSO) δ 12.8 (s, IH), 4.72 (m, J = 5.90 Hz, IH), 2.58 (m, 2H), 2.40 (m, J = 6.03 Hz, 2H), 2.21 (dd, J = 19.0, 12.8 Hz, 3H), 2.05 (m, IH), 1.87 (q, J = 8.92 Hz, IH), 1.75 (m, IH), 1.55 (m, IH), 1.35 (q, J = 12.6 Hz, IH), 1.27 (d, J = 5.88 Hz, 3H). MS (ESI) M+l m/z calcd. 267, found 267.

NOTE

Compound 7A may be prepared from Compound 8 by treating Compound 8 with diethylchlorophosphate:

Compound 8 may be obtained by the process described by Kyoku, Kagehira et al in “Preparation of (haloaryl)pyridines,” (API Corporation, Japan). Jpn. Kokai Tokkyo Koho (2004). 13pp. CODEN: JKXXAF JP

2004182713 A2 20040702. Compound 8 is subsequently reacted with a phosphate ester, such as a dialkyl halophosphate, to yield Compound 7A. Diethylchlorophosphate is preferred. The reaction is preferably conducted in the presence of a base, such as a dialkylithium amide, for example diisopropyl lithium amide.

…………………………………..

J Med Chem 2008, 51(11): 3061

http://pubs.acs.org/doi/abs/10.1021/jm800180e

The discovery of an exceptionally potent series of thrombin receptor (PAR-1) antagonists based on the natural product himbacine is described. Optimization of this series has led to the discovery of 4 (SCH 530348), a potent, oral antiplatelet agent that is currently undergoing Phase-III clinical trials for acute coronary syndrome (unstable angina/non-ST segment elevation myocardial infarction) and secondary prevention of cardiovascular events in high-risk patients.

Ethyl [(3aR,4aR,8aR,9aS)-9(S)-[(E)-2-[5-(3-fluorophenyl)-2-
pyridinyl]ethenyl]dodecahydro-1(R)-methyl-3-oxonaphtho[2,3-c]furan-6(R)-yl]carbamate (4).

4 (300 mg, 86%). MS m/z 493 (M+1).

HRMS Calcd for C29H34N2O4F
(M+1): 493.2503, found 493.2509; mp125 °C;

[]D20 6.6 (c 0.5, MeOH).

1HNMR (CDCl3):

http://pubs.acs.org/doi/suppl/10.1021/jm800180e/suppl_file/jm800180e-file002.pdf

0.88-1.18 (m, 5 H), 1.22-1.30 (m, 3 H), 1.43 (d, J = 5.85 Hz, 3 H), 1.88-2.10 (m, 4 H), 2.33-2.42 (m, 2 H),
2.75-2.67 (m, 1 H), 3.52-3.60 (m, 1 H), 4.06-4.14 (m, 2 H), 4.54-4.80 (m, 1 H), 4.71-4.77 (m, 1 H),
6.55-6.63 (m, 2 H), 7.07-7.12 (m, 1 H), 7.26-7.29 (m, 2 H), 7.34 (d, J = 8.05 Hz, 1 H), 7.41-7.46 (m, 1 H), 7.80-7.82 (m, 1 H), 8.76-8.71 (m, 1 H).

……………………..

References

Samuel Chackalamannil; Wang, Yuguang; Greenlee, William J.; Hu, Zhiyong; Xia, Yan; Ahn, Ho-Sam; Boykow, George; Hsieh, Yunsheng et al. (2008). “Discovery of a Novel, Orally Active Himbacine-Based Thrombin Receptor Antagonist (SCH 530348) with Potent Antiplatelet Activity”. Journal of Medicinal Chemistry 51 (11): 3061–4.doi:10.1021/jm800180e. PMID 18447380.
Merck Blood Thinner Studies Halted in Select Patients, Bloomberg News, January 13, 2011
Tricoci et al. (2012). “Thrombin-Receptor Antagonist Vorapaxar in Acute Coronary Syndromes”. New England Journal of Medicine 366 (1): 20–33.doi:10.1056/NEJMoa1109719. PMID 22077816.
Morrow, DA; Braunwald, E; Bonaca, MP; Ameriso, SF; Dalby, AJ; Fish, MP; Fox, KA; Lipka, LJ; Liu, X; Nicolau, JC; Ophuis, AJ; Paolasso, E; Scirica, BM; Spinar, J; Theroux, P; Wiviott, SD; Strony, J; Murphy, SA; TRA 2P–TIMI 50 Steering Committee and, Investigators (Apr 12, 2012). “Vorapaxar in the secondary prevention of atherothrombotic events.”. The New England Journal of Medicine 366 (15): 1404–13. doi:10.1056/NEJMoa1200933.PMID 22443427.
“Merck Statement on FDA Advisory Committee for Vorapaxar, Merck’s Investigational Antiplatelet Medicine”. Merck. Retrieved 16 January 2014.
http://www.forbes.com/sites/larryhusten/2014/01/15/fda-advisory-panel-votes-in-favor-of-approval-for-mercks-vorapaxar/
SCH-530348 (Vorapaxar) is an investigational candidate for the prevention of arterial thrombosis in patients with acute coronary syndrome and peripheral arterial disease. “Convergent Synthesis of Both Enantiomers of 4-Hydroxypent-2-ynoic Acid Diphenylamide for a Thrombin Receptor Antagonist Sch530348 and Himbacine Analogues.” Alex Zaks et al.: Adv. Synth. Catal. 2009, 351: 2351-2357 Full text;
Discovery of a novel, orally active himbacine-based thrombin receptor antagonist (SCH 530348) with potent antiplatelet activity
J Med Chem 2008, 51(11): 3061

Stu Borman (2005). “Hopes Ride on Drug Candidates: Researchers reveal potential new medicines for thrombosis, anxiety, diabetes, and cancer”. Chemical & Engineering News 83 (16): 40–44.

PATENTS

WO 2003089428
WO 2006076452
US 6063847
WO 2006076565
WO 2008005344
WO2010/141525
WO2008/5353
US2008/26050
WO2006/76564 mp, nmr

US8138180	3-21-2012	EXO-SELECTIVE SYNTHESIS OF HIMBACINE ANALOGS
US2011251392	10-14-2011	EXO- AND DIASTEREO- SELECTIVE SYNTHESIS OF HIMBACINE ANALOGS
US7989653	8-3-2011	Exo- and diastereo-selective syntheses of himbacine analogs
US2011065676	3-18-2011	COMBINATION THERAPIES COMPRISING PAR1 ANTAGONISTS WITH NAR AGONISTS
US7772276	8-11-2010	Exo-selective synthesis of himbacine analogs
US2010137597	6-4-2010	SYNTHESIS Of DIETHYLPHOSPHONATE
US7713999	5-12-2010	THROMBIN RECEPTOR ANTAGONISTS
US7687631	3-31-2010	Synthesis of diethyl{[5-(3-fluorophenyl)-pyridine-2yl]methyl}phosphonate
US2009297576	12-4-2009	Local Delivery of PAR-1 Antagonists to Treat Vascular Complications
US7626045	12-2-2009	SYNTHESIS OF HIMBACINE ANALOGS

US7605275	10-21-2009	Exo- and diastereo- selective syntheses of himbacine analogs
US7553987	6-31-2009	Synthesis of 3-(5-nitrocyclohex-1-enyl) acrylic acid and esters thereof
US7541471	6-3-2009	Synthesis of himbacine analogs
US2009022729	1-23-2009	METHODS AND COMPOSITIONS FOR TREATING CARDIAC DYSFUNCTIONS
US2008234236	9-26-2008	REDUCTION OF ADVERSE EVENTS AFTER PERCUTANEOUS INTERVENTION BY USE OF A THROMBIN RECEPTOR ANTAGONIST
US2008031943	2-8-2008	IMMEDIATE-RELEASE TABLET FORMULATIONS OF A THROMBIN RECEPTOR ANTAGONIST
US2008026050	1-32-2008	SOLID DOSE FORMULATIONS OF A THROMBIN RECEPTOR ANTAGONIST
US7304078	12-5-2007	Thrombin receptor antagonists
US2007270439	11-23-2007	THROMBIN RECEPTOR ANTAGONISTS
US2007202140	8-31-2007	THROMBIN RECEPTOR ANTAGONISTS AS PROPHYLAXIS TO COMPLICATIONS FROM CARDIOPULMONARY SURGERY

US2007203193	8-31-2007	CRYSTALLINE POLYMORPH OF A BISULFATE SALT OF A THROMBIN RECEPTOR ANTAGONIST
US7235567	6-27-2007	Crystalline polymorph of a bisulfate salt of a thrombin receptor antagonist
US2006172397	8-4-2006	Preparation of chiral propargylic alcohol and ester intermediates of himbacine analogs
US2004192753	9-31-2004	Methods of use of thrombin receptor antagonists

US6063847 *	Nov 23, 1998	May 16, 2000	Schering Corporation	Thrombin receptor antagonists
US6326380 *	Apr 7, 2000	Dec 4, 2001	Schering Corporation	Thrombin receptor antagonists
US20030216437 *	Apr 14, 2003	Nov 20, 2003	Schering Corporation	Thrombin receptor antagonists
US20040176418 *	Jan 9, 2004	Sep 9, 2004	Schering Corporation	Crystalline polymorph of a bisulfate salt of a thrombin receptor antagonist

WO2011128420A1

Apr 14, 2011

Oct 20, 2011

Sanofi

Pyridyl-vinyl pyrazoloquinolines as par1 inhibitors

Rapamycin (Sirolimus) For the prophylaxis of organ rejection in patients receiving renal transplants.

January 15, 2014 12:43 pm / Leave a comment

Rapamycin (Sirolimus)

(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25, 26,27,32,33,34,34a-Hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone

Wyeth Pharmaceuticals (Originator)

M.Wt:914.18

Formula:C₅₁H₇₉NO₁₃

53123-88-9 cas no

Antifungal and immunosuppressant. Specific inhibitor of mTOR (mammalian target of Rapamycin). Complexes with FKBP-12 and binds mTOR inhibiting its activity. Inhibits interleukin-2-induced phosphorylation and activation of p70 S6 kinase. Induces autophagy in yeast and mammalian cell lines.

Rapamycin is a triene macrolide antibiotic, which demonstrates anti-fungal, anti-inflammatory, anti-tumor and immunosuppressive properties. Rapamycin has been shown to block T-cell activation and proliferation, as well as, the activation of p70 S6 kinase and exhibits strong binding to FK-506 binding proteins. Rapamycin also inhibits the activity of the protein, mTOR, (mammalian target of rapamycin) which functions in a signaling pathway to promote tumor growth. Rapamycin binds to a receptor protein (FKBP12) and the rapamycin/FKB12 complex then binds to mTOR and prevents interaction of mTOR with target proteins in this signaling pathway. Rapamycin name is derived from the native word for Easter Island, Rapi Nui.

(-)-Rapamycin
Antibiotic AY 22989
AY 22989
AY-22989
CCRIS 9024
HSDB 7284
NSC 226080
Rapammune
Rapamune
Rapamycin
SILA 9268A
Sirolimus
UNII-W36ZG6FT64
WY-090217
A 8167

A macrolide compound obtained from Streptomyces hygroscopicus that acts by selectively blocking the transcriptional activation of cytokines thereby inhibiting cytokine production. It is bioactive only when bound to IMMUNOPHILINS. Sirolimus is a potent immunosuppressant and possesses both antifungal and antineoplastic properties.

Sirolimus (INN/USAN), also known as rapamycin, is an immunosuppressant drug used to prevent rejection in organ transplantation; it is especially useful in kidney transplants. It prevents activation of T cells and B cells by inhibiting their response to interleukin-2 (IL-2). Sirolimus is also used as a coronary stent coating. Sirolimus works, in part, by eliminating old and abnormal white blood cells.^{[citation needed]} Sirolimus is effective in mice with autoimmunity and in children with a rare condition called autoimmune lymphoproliferative syndrome (ALPS).

sirolimus

A macrolide, sirolimus was discovered by Brazilian researchers as a product of the bacterium Streptomyces hygroscopicus in a soil sample fromEaster Island^[1] — an island also known as Rapa Nui.^[2] It was approved by the FDA in September 1999 and is marketed under the trade nameRapamune by Pfizer (formerly by Wyeth).

Sirolimus was originally developed as an antifungal agent. However, this use was abandoned when it was discovered to have potent immunosuppressive and antiproliferative properties. It has since been shown to prolong the life of mice and might also be useful in the treatment of certain cancers.

Unlike the similarly named tacrolimus, sirolimus is not a calcineurin inhibitor, but it has a similar suppressive effect on the immune system. Sirolimus inhibits the response tointerleukin-2 (IL-2), and thereby blocks activation of T and B cells. In contrast, tacrolimus inhibits the secretion of IL-2.

The mode of action of sirolimus is to bind the cytosolic protein FK-binding protein 12(FKBP12) in a manner similar to tacrolimus. Unlike the tacrolimus-FKBP12 complex which inhibits calcineurin (PP2B), the sirolimus-FKBP12 complex inhibits themammalian target of rapamycin (mTOR, rapamycin being an older name for sirolimus) pathway by directly binding the mTOR Complex1 (mTORC1).

mTOR has also been called FRAP (FKBP-rapamycin associated protein), RAFT (rapamycin and FKBP target), RAPT1, or SEP. The earlier names FRAP and RAFT were coined to reflect the fact that sirolimus must bind FKBP12 first, and only the FKBP12-sirolimus complex can bind mTOR. However, mTOR is now the widely accepted name, since Tor was first discovered via genetic and molecular studies of sirolimus-resistant mutants of Saccharomyces cerevisiae that identified FKBP12, Tor1, and Tor2 as the targets of sirolimus and provided robust support that the FKBP12-sirolimus complex binds to and inhibits Tor1 and Tor2.

rapamycin

Unlike the similarly named tacrolimus, sirolimus is not a calcineurin inhibitor, but it has a similar suppressive effect on the immune system. Sirolimus inhibits the response to interleukin-2 (IL-2), and thereby blocks activation of T and B cells. In contrast, tacrolimus inhibits the secretion of IL-2.

The mode of action of sirolimus is to bind the cytosolic protein FK-binding protein 12 (FKBP12) in a manner similar to tacrolimus. Unlike the tacrolimus-FKBP12 complex which inhibits calcineurin (PP2B), the sirolimus-FKBP12 complex inhibits the mammalian target of rapamycin(mTOR, rapamycin being an older name for sirolimus) pathway by directly binding the mTOR Complex1 (mTORC1).

SIROLIMUS

Rapamycin and its preparation are described in US Patent No. 3,929,992, issued December 30, 1975. Alternatively, rapamycin may be purchased commercially [Rapamune®, Wyeth].

Rapamycin (Sirolimus) is a 31-member natural macrocyclic lactone [C51H79N1O13; MWt=914.2] produced by Streptomyces hygroscopicus and found in the 1970s (U.S. Pat. No. 3,929,992; 3,993,749). Rapamycin (structure shown below) was approved by the Food and Drug Administration (FDA) for the prophylaxis of renal transplant rejection in 1999.

Rapamycin resembles tacrolimus (binds to the same intracellular binding protein or immunophilin known as FKBP-12) but differs in its mechanism of action. Whereas tacrolimus and cyclosporine inhibit T-cell activation by blocking lymphokine (e.g., IL2) gene transcription, sirolimus inhibits T-cell activation and T lymphocyte proliferation by binding to mammalian target of rapamycin (mTOR). Rapamycin can act in synergy with cyclosporine or tacrolimus in suppressing the immune system.

Rapamycin is also useful in preventing or treating systemic lupus erythematosus [U.S. Pat. No. 5,078,999], pulmonary inflammation [U.S. Pat. No. 5,080,899], insulin dependent diabetes mellitus [U.S. Pat. No. 5,321,009], skin disorders, such as psoriasis [U.S. Pat. No. 5,286,730], bowel disorders [U.S. Pat. No. 5,286,731], smooth muscle cell proliferation and intimal thickening following vascular injury [U.S. Pat. Nos. 5,288,711 and 5,516,781], adult T-cell leukemia/lymphoma [European Patent Application 525,960 A1], ocular inflammation [U.S. Pat. No. 5,387,589], malignant carcinomas [U.S. Pat. No. 5,206,018], cardiac inflammatory disease [U.S. Pat. No. 5,496,832], anemia [U.S. Pat. No. 5,561,138] and increase neurite outgrowth [Parker, E. M. et al, Neuropharmacology 39, 1913-1919, 2000].

Although rapamycin can be used to treat various disease conditions, the utility of the compound as a pharmaceutical drug has been limited by its very low and variable bioavailability and its high immunosuppressive potency and potential high toxicity. Also, rapamycin is only very slightly soluble in water. To overcome these problems, prodrugs and analogues of the compound have been synthesized. Water soluble prodrugs prepared by derivatizing rapamycin positions 31 and 42 (formerly positions 28 and 40) of the rapamycin structure to form glycinate, propionate, and pyrrolidino butyrate prodrugs have been described (U.S. Pat. No. 4,650,803). Some of the analogues of rapamycin described in the art include monoacyl and diacyl analogues (U.S. Pat. No. 4,316,885), acetal analogues (U.S. Pat. No. 5,151,413), silyl ethers (U.S. Pat. No. 5,120,842), hydroxyesters (U.S. Pat. No. 5,362,718), as well as alkyl, aryl, alkenyl, and alkynyl analogues (U.S. Pat. Nos. 5,665,772; 5,258,389; 6,384,046; WO 97/35575).

………………………………………..

Synthesis

ref are independent of body…see below for this clip

Several total synthese of rapamycin have been reported^3,4as well as many fragments and part-syntheses. Rapamycin is a complicated molecule comprising a 31-membered ring including a pipecolinyl group and pyranose ring, a conjugated triene system and a tri-carbonyl region. It also has 15 chiral centres, meaning the number of possible stereoisomers is enormous. The synthesis of rapamycin therefore presents a huge challenge to synthetic chemists.

In the following synthesis, published in three separate papers^5,6,7two fragments of C10-C21 and C22-C42 are prepared separately, before being combined to give the total synthesis of rapamycin. Only the main outline of the synthesis will be shown as it is too long and complicated to show in great detail. For the full experimental details of the synthesis see the literature (ref. nos. given above).

In the retro-synthesis shown the molecule is disconnected at the ester group next to carbon 1 and the C₂₁-C₂₂ double bond of the triene to give the synthetic precursors 2 and 3. Further disconnections of 3 will be shown later. First the C₁₀-C₂₁ fragment is synthesised.

Synthesis of C10-C21 fragment

The synthesis uses (R)-methyl 3-hydroxy-2-methylpropionate (8) as a starting material.

The starting material 8 is converted to an alcohol by a four-step process; protection of the alcohol as aTHP ether followed by reduction, ether formation and deprotection steps. Substitution of the hydroxyl group in the product for a bromine leads to the formation of the bromide 9. Reaction of 9 with methyl acetoacetate gave ester 10.

Catalytic reduction of 10 using the conditions of Noyori produced ester 11, which was then converted to its Weinreb amide 12. Overall, compound 12 was produced in 54% yield from an inexpensive starting material. Vinyl bromide 13 was metalated with t-BuLi and the resulting vinyllithium was combined with 12 and the PMB-protecting group was removed to give 14. The remaining carbonyl group in 14 was selectively reduced to a hyrdoxy group. In order to differentiate the 1,3-diol a lactol was formed, where one hydroxy group ended up in the ring. To acheive this an oxidation was performed using RuCl₂(PPh₃)₃ resulting in formation of a lactol. The two remaining alcohol groups could then be methylated using MeI forming 15.

The lactol ring opening was achieved using TiCl₄ and thiol HS(CH₂)₂SH to form a dithiolane. The freed alcohol was then protected as its TBS ether and the same protecting group selectively removed from the primary alcohol to form 16. To avoid removing the dithiolane group at a later stage in the synthesis the thio-acetal was converted to the dimethyl acetal 17 using PhI(OCOCF₃)₂ and methanol.

The next stage in the synthesis was to extend 17 for the building of the triene region. The terminal alcohol was oxidised to its aldehyde using BaMnO₄ , then a Wittig reaction was carried out using Ph₃P=CHCO₂Et and CH₂Cl₂ to form the second double bond. Reduction of the ester group to an alcohol was carried out using DIBAL-H, then treatment with PPh₃ and exposure to the air gave rapamycin fragment 2.

Synthesis of C22-C42 fragment

Here the retro-synthesis of 3 is shown, giving the three synthetic precursors 5, 6 and 7

It was thought 4 could be obtained by alkylative coupling of a vinyllithium species generated from 7 to the Weinreb amide 6. The nucleophilic opening of epoxide 5 by the lithiated sulfone from phenyl sulfone 4 would then produce the desired fragment.

The ester 18 was used as a starting material to make fragment 6.

A Wittig reaction followed by reduction and protection steps produced 19. This was hydrogenated using a rhodium catalyst to give syn-dimethyl product 20. The minor anti diastereomer was successfully separated off. 20 was oxidised then underwent an aldol condensation to give adduct 21.

Transamination of 21 and protection of the alcohol with PMB resulted in amide 6, corresponding to the C₂₂-C₂₈ segment of rapamycin.

The vinyl bromide 7 was prepared using ester 22 as a starting material.

Reduction of 22 followed by dibromoolefination resulted in product 23. Acetylene 24 was prepared using n-BuLi, THF and MeI, then sulfenylation with Ph₂S₂ and bromination gave fragment 7.

Iodination and alkylation of starting material 25 with the lithiated allylic sulfide shown followed by a number of further steps resulted in its conversion to fragment 5.

Fragments 7 was first converted to its vinyllithium using t-BuLi then combined with 6 forming an enone in 78% yield. Stereoselective reduction of the carbonyl group using Zn(BH₄)₂ gave an alcohol which was protected with DEIPS giving 28. The phenyl sulfide was oxidised to a sulfone using m-CPBA in excess pyridine.

Lithiation and addition of the epoxide 5 resulted in the hydroxy sulfone in a 4:1 ratio of two diastereomers which were separated by HPLC. Metalation using n-BuLi followed by oxidation formed the total C₂₂-C₄₂ fragment.

Total synthesis of rapamycin through the combination of C₁₀-C₂₁ and C₂₂-C₄₂ fragments.

Fragment 3 (C₂₂-C₄₂) was treated with (S)-Boc-pipecolinal, followed by a Swern oxidation resulted in the aldehyde 29.

Condensation with the lithium salt of phosphine oxide 2 (C₁₀-C₂₁) produced the triene shown below.

The triene was hydrolysed with pyridinium p-toluenesulfonic acid and an aldol reaction was performed. Treatment with triethylsilyl triflate produced an amino acid which was subjected to Mukaiyama macrocyclization conditions to form the 31-membered ring. Finally, deprotection steps were performed to give synthetic rapamyin (1). This was judged to be identical to natural rapamycin by comparison of physical properties, ¹H-NMR, ¹³C-NMR, IR and UV spectral data.

3. K. C. Nicolaou, T. K. Chakraborty, A. D. Piscopio, N. Minowa, P. Bertinato; J. Am. Chem. Soc.; 115; 1993; 4419

4. C. M. Hayward, D. Yohannes, S. J. Danishefsky; J. Am. Chem. Soc.; 115; 1993; 9345

5. S. D. Meyer, T. Miwa, M. Nakatsuka, S. L. Schreiber; J. Org. Chem.; 57; 1992; 5058-5060

6. D. Romo, D. D. Johnson, L. Plamondon, T. Miwa, S. L. Schreiber; J. Org. Chem.; 57; 1992; 5060-5063

7. S. D. Meyer, D. Romo, D. D. Johnson, S. L. Schreiber; J. Am. Chem. Soc.; 115; 1993; 7906-7907

………………………………………….

Synthesis

http://www.google.co.in/patents/US3929992

PREPARATION

CUT PASTE FROM TEXT

In one embodiment of this invention rapamycin is prepared in the followingmanner: 4

A suitable fermenter is charged with production meis reached in the fermentation mixture after 2-8 days,

usually after about 5 days, as determined by the cup plate method and Candida albicans as the test organism. The mycelium is harvested by filtration with diatomaceous earth. Rapamycin is then extracted from the mycelium with a water-miscible solvent, for example a lower alkanol, preferably methanol or ethanol. The latter extract is then concentrated, preferably under reduced pressure, and the resulting aqueous phase is extracted with a water-immiscible solvent. A preferred water-immiscible solvent for this purpose is methylene dichloride although chloroform, carbon tetrachloride, benzene, n-butanol and the like may also be used. The latter extract is concentrated, preferably under reduced pressure, to afford the crude product as an oil.

The product may be purified further by a variety of methods. Among the preferred methods of purification is to dissolve the crude product in a substantially nonpolar, first solvent, for example petroleum ether or hexane, and to treat the resulting solution with a suit able absorbent, for example charcoal or silica gel, so that the antibiotic becomes absorbed on the absorbant. The absorbant is then separated and washed or eluted with a second solvent more polar than the first solvent, for example ethyl acetate, methylene dichloride, or a mixture of methylene dichloride and ether (preferred). Thereafter, concentration of the wash solution or eluate affords substantially pure rapamycin. Further purification is obtained by partial precipitation with a nonpolar solvent, for example, petroleum ether, hexane, pentane and the like, from a solution of the rapamycin in a more polar solvent, for example, ether, ethyl acetate, benzene and the like. Still-further purification is obtained by column chromatography, preferably employing silica gel, and by crystallization of the rapamycin from ether.

In another preferred embodiment of this invention a first stage inoculum of S treptomyces hygroscopicus NRRL 5491 is prepared in small batches in a medium containing soybean flour, glucose, ammonium sulfate, and calcium carbonate incubated at about 25C at pH 7.l-7.3 for 24 hrs. with agitation, preferably on a gyrotary shaker. The growth thus obtained is used to inoculate a number of somewhat larger batches of the same medium as described above which are incubated at about 25C and pH 7.1-7.3 for 18 hrs. with agitation, preferably on a reciprocating’shaker, to obtain a sec- “ond stagc inoculum which is used to inoculate the production stage fermenters.

6 5.86′.2.-The fermenters are inoculated with the second stage inoculum described above and incubated at about 25C with’ agitationand aeration while controlling and ‘mai’ntaining the mixture at approximately pH 6.0 by

addition offa base, for example, sodium hydroxide, potassium hydroxide or preferably ammonium hydroxide, as required from time to time. Addition of a source -of assimilable carbon, preferably glucose, is started when theconcentrationof the latter in the broth has dropped to about 0.5% wt/vol, normally about 48 hrs after. the start of fermentation, and is maintained until the end ofthe particular run. In this manner a fermentation broth containing about 60 ug/ml of rapamycin as determined by the assay method described above is obtained in 45 days, when fermentation is stopped.

‘ Filtration of the’mycelium, mixing the latter with a watef-miscible ‘lower’ alkanol, preferably methanol, followed by extraction with a halogenated aliphatic hydrocarbon, preferably trichloroethane, and evaporation of the solvents yields a first oily residue. This first oily residue is dissolved in a lower aliphatic ketone, preferably acetone, filtered from insoluble impurities, the filtrate evaporated to yield a second oily residue which is extractedjwith a water-miscible lower alkanol,

preferably methanol, and the latter extract is evaporated to yield crude rapamycin as a third oily residue. This third oily residue is dissolved in a mixture of a lower aliphatic ketone and a lower aliphatic hydrocarbon, preferably acetone-hexane, an absorbent such as charcoal or preferably silica gel is added to adsorb the rapamycin, the latter is eluted from the adsorbate with a similar but more polar solvent mixture, for example a mixture as above but containing a higher proportion of the aliphatic ketone, the eluates are evaporated and the residue is crystallized from diethyl ether, to yield pure crystalline rapamycin. In this manner a total of 45-5 8% of the rapamycin initially present in the fermentation mixture is recovered as pure crystalline rapamycin.

CHARACTERIZATION solvent systems; for example, ether-hexane 40:60 (Rf 0.42), ‘isopropyl alcoholvbenzene 15:85 (Rf= 0.5) and ethanol-benzene 20:80 (Rf f 0.43);

d. rapamycin obtained from four successive fermentation batchesgave the following values on repeated The production stage fermenters are equipped with 7 devices for controlling and maintaining pH at a predetermined level and for continuous metered addition of elemental analyses:

AVER- e. rapamycin exhibits the following characteristic absorption maxima in its ultraviolet absorption spectrum ethanol):

f. the infrared absorption spectrum of rapamycin in chloroform is reproduced in FIG. 1 and shows characteristic absorption bands at 3560, 3430, 1730, 1705 and 1630-1610 cm;

Further infrared absorption bands are characterized by the following data given in reciprocal centimeters with (s) denoting a strong, (m) denoting a medium, and denoting a weak intensity band. This classification is arbitrarily selected in such a manner that a band is denoted as strong (s) if its peak absorption is more than two-thirds of the background in the same region; medium (m) if its peak is between one-third and twothirds of the background in the same region; and weak if its peak is less than one-third of the background in the same region.

2990 cm (m) 1158 cm” (m) 2955 cm (s) 1129 cm (s) 2919 cm (s) 1080 cm (s) 2858 cm (s) 1060 cm (s) 2815 cm (m) 1040 cm (m) 1440 cm (s) 1020 crn’ (m) 1365 cm (m) 978 cm” (s) 1316 cm (in) 905 cm (m) 1272 cm (m) 888 cm” 1178 cm (s) 866 cm- g. the nuclear magnetic resonance spectrum of rapamycinin deuterochloroform is reproduced in FIG. 2; SEE PATENT

CLAIMS

l. Rapamycin, an antibiotic which a. is a colourless, crystalline compound with a melting point of 183 to l8SC, after recrystallization from ether;

b. is soluble in ether, chloroform, acetone, methanol and dimethylformamide, very sparingly soluble in hexane and petroleum ether and substantially insoluble in water;

c. shows a uniform spot on thin layer plates of silica gel”,

d. has a characteristic elemental analysis of about C,

e. exhibits the following characteristic absorption maxima in its ultraviolet absorption spectrum (95% ff has ‘a characteristic infrared absorption spectrum shown in accompanying FIG. 1; SEE PATENT

……………………………………………..

Rapamycin synthetic studies. 1. Construction of the C(27)-C(42) subunit. Tetrahedron Lett 1994, 35, 28, 4907

A partial synthesis of rapamycin has been reported: The condensation of sulfone (I) with epoxide (II) by means of butyllithium followed by desulfonation with Na/Hg gives the partially protected diol (III), which is treated with methanesulfonyl chloride and NaH to afford the epoxide (IV). Ring opening of epoxide (IV) with LiI and BF3.Et2O followed by protection of the resulting alcohol with PMBOC(NH)CCl3 yields the primary iodo compound (V). The condensation of (V) with the fully protected dihydroxyaldehyde (VI) (see later) by means of butyllithium in THF/HMPT gives the fully protected trihydroxyketone (VII), which is hydrolyzed with camphorsulfonic acid (CSA) to the corresponding gemdiol and reprotected with pivaloyl chloride (the primary alcohol) and tert-butyldimethylsilyl trifluoromethanesulfonate (the secondary alcohol), yielding a new fully protected trihydroxyketone (VIII). Elimination of the pivaloyl group with DIBAL and the dithiane group with MeI/CaCO3 affords the hydroxyketone (IX), which is finally oxidized with oxalyl chloride to the ketoaldehyde (X), the C(27)-C(42) fragment [the C(12)-C(15) fragment with the C(12)-substituent based on the IUPAC nomenclature recommendations]. The fully protected dihydroxyaldehyde (VI) is obtained as follows: The reaction of methyl 3-hydroxy-2(R)-methylpropionate (XI) with BPSCl followed by reduction with LiBH4 to the corresponding alcohol and oxidation with oxalyl chloride gives the aldehyde (XII), which is protected with propane-1,3-dithiol and BF3.Et2O to afford the dithiane compound (XIII). Elimination of the silyl group with TBAF followed by esterification with tosyl chloride, reaction with NaI and, finally, with sodium phenylsulfinate gives the sulfone (XIV), which is condensed with the partially protected dihydroxyaldehyde (XV), oxidized with oxalyl chloride and desulfonated with Al/Hg to afford the dithianyl ketone (XVI). The reaction of (XVI) with lithium hexamethyldisilylazane gives the corresponding enolate, which is treated with dimethyllithium cuprate to yield the fully protected unsaturated dihydroxyaldehyde (VI).

……………………………………………

http://www.google.com/patents/US8088789

……………………………

The Ley Synthesis of Rapamycin

Rapamycin (3) is used clinically as an immunosuppressive agent. The synthesis of 3 (Angew. Chem. Int. Ed. 2007, 46, 591. DOI: 10.1002/anie.200604053) by Steven V. Ley of the University of Cambridge was based on the assembly and subsequent coupling of the iododiene 1 and the stannyl alkene 2.

The lactone of 1 was prepared by Fe-mediated cyclocarbonylation of the alkenyl epoxide 5, following the protocol developed in the Ley group.

The cyclohexane of 2 was constructed by SnCl₄-mediated cyclization of the allyl stannane 9, again employing a procedure developed in the Ley group. Hydroboration delivered the aldehyde 11, which was crotylated with 12, following the H. C. Brown method. The alcohol so produced (not illustrated) was used to direct the diastereoselectivity of epoxidation, then removed, to give 13. Coupling with 14 then led to 2.

Combination of 1 with 2 led to 15, which was condensed with catechol to give the macrocycle 16. Exposure of 16 to base effected Dieckmann cyclization, to deliver the ring-contracted macrolactone 17, which was carried on to (-)-rapamycin (3).

……………………………….

Total Synthesis of Rapamycin

Angewandte Chemie International Edition

Volume 46, Issue 4, pages 591–597, January 15, 2007

PREVIEW THIS ARTICLE WITH READCUBE

http://www.readcube.com/articles/10.1002%2Fanie.200604053?r3_referer=wol

……………………..

Ley, Maddess, Tackett, Watanabe, Brennan, Spilling, Scott and Osborn. ACIEE, 2006, EarlyView. DOI:10.1002/anie.200604053.

It’s been in the works for quite a while, but Steve Ley’s synthesis of Rapamycin has just been published. This complex beast has a multitude of biological activities, including an interesting immunosuppressive profile, resulting in clinical usage following organ transplantation. So, unsurprisingly, it’s been the target of many projects, with complete total syntheses published by Smith, Danishefsky, Schreiber and KCN.

So what makes this one different? Well, it does have one of the most interesting macrocyclisations I’ve seen since Jamison’s paper, and a very nice demonstration of the BDA-aldol methodology. The overall strategy is also impressive, so on with the retro:

First stop is the BDA-aldol; this type of chemistry is interesting, because the protecting group for the diol is also the stereo-directing group. The stereochemistry for this comes from a glycolic acid, and has been usedin this manner by the group before. The result is as impressive as ever, with a high yield, and presumably a very high d.r. (no mention of actual numbers).

The rest of the fragment synthesis was completed in a succinct and competent manner, but using relatively well known chemistry. However, I was especially impressed with the macrocyclisation I mentioned:

Tethering the free ends of the linear precursor with a simple etherification/esterification onto catechol gave then a macrocycle holding the desired reaction centres together. Treatment of this with base then induces a Dieckmann-condensation type cyclisation to deliver the desired macrocycle. Of course, at this stage, only a few more steps were required to complete the molecule, and end an era of the Wiffen Lab.

………………………………

Drugs Fut 1999, 24(1): 22

DOI: 10.1358/dof.1999.024.01.474036

¹H and ¹³C NMR assignments

ref2. J. B. McAlpine, S. J. Swanson, M. Jackson, D. N. Whittern; J.Antibiot.; 44; 1991; 688-690;

In CDCl₃ rapamycin exists as a mixture of conformers in a 3:1 ratio, which complicates the NMR spectrum. In the table below the chemical shifts of the carbons and hydrogens of the major isomer only are given.

Carbon No.	Carbon Type	Major carbon	Major proton	Carbon No.	Carbon Type	Major carbon	Major proton
1	C=O	169.2	–	28	CH-OH	77.3	4.17
2	CH	51.3	5.29	29	C=C	136.1	–
3	CH₂	27.0	2.34, 1.76	30	CH=C	126.8	5.42
4	CH₂	20.6	1.78, 1.47	31	CH	46.6	3.33
5	CH₂	25.3	1.75, 1.48	32	C=O	208.2	–
6	CH₂	44.2	3.59, 3.44	33	CH₂	40.7	2.74, 2.60
8	C=O	166.8	–	34	CH-OCO	75.7	5.17
9	C=O	192.5	–	35	CH	33.1	1.98
10	O-C-OH	98.5	–	36	CH₂	38.4	1.22, 1.12
11	CH	33.7	1.98	37	CH	33.2	1.39
12	CH₂	27.3	1.60, 1.60	38	CH₂	34.2	2.10, 0.68
13	CH₂	31.3	1.62, 1.33	39	CH-OCH₃	84.4	2.93
14		67.2	3.86	40	CH-OH	73.9	3.37
15	CH₂	38.8	1.85, 1.52	41	CH₂	31.3	1.99, 1.33
16	CH-OCH₃	84.4	3.67	42	CH₂	31.7	1.70, 1.00
17	C=C	135.5	–	43	11-CH₃	16.2	0.95
18	CH=C	129.6	5.97	44	17-CH₃	10.2	1.65
19	CH=C	126.4	6.39	45	23-CH₃	21.5	1.05
20	CH=C	133.6	6.32	46	25-CH₃	13.8	1.00
21	CH=C	130.1	6.15	47	29-CH₃	13.0	1.74
22	CH=C	140.2	5.54	48	31-CH₃	16.0	1.11
23	CH	35.2	2.32	49	35-CH₃	15.9	0.92
24	CH₂	40.2	1.50, 1.20	50	16-OCH₃	55.8	3.13
25	CH	41.4	2.74	51	27-OCH₃	59.5	3.34
26	C=O	215.6	–	52	39-OCH₃	56.5	3.41
27	CH-OCH₃	84.9	3.71

REFERENCES

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3. Creating diverse target-binding surfaces on FKBP12: synthesis and evaluation of a rapamycin analogue library.

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Graziani EI.

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8 Total synthesis of rapamycin.

Ley SV, Tackett MN, Maddess ML, Anderson JC, Brennan PE, Cappi MW, Heer JP, Helgen C, Kori M, Kouklovsky C, Marsden SP, Norman J, Osborn DP, Palomero MA, Pavey JB, Pinel C, Robinson LA, Schnaubelt J, Scott JS, Spilling CD, Watanabe H, Wesson KE, Willis MC.

Chemistry. 2009;15(12):2874-914. doi: 10.1002/chem.200801656.

9 Highly diastereoselective desymmetrisation of cyclic meso-anhydrides and derivatisation for use in natural product synthesis.

Evans AC, Longbottom DA, Matsuoka M, Davies JE, Turner R, Franckevicius V, Ley SV.

Org Biomol Chem. 2009 Feb 21;7(4):747-60. doi: 10.1039/b813494d. Epub 2009 Jan 6.

10 Total synthesis studies on macrocyclic pipecolic acid natural products: FK506, the antascomicins and rapamycin.

Maddess ML, Tackett MN, Ley SV.

Prog Drug Res. 2008;66:13, 15-186. Review.

11 Determination of sirolimus in rabbit arteries using liquid chromatography separation and tandem mass spectrometric detection.

Zhang J, Rodila R, Watson P, Ji Q, El-Shourbagy TA.

Biomed Chromatogr. 2007 Oct;21(10):1036-44.

12 Saccharomyces cerevisiae FKBP12 binds Arabidopsis thaliana TOR and its expression in plants leads to rapamycin susceptibility.

Sormani R, Yao L, Menand B, Ennar N, Lecampion C, Meyer C, Robaglia C.

BMC Plant Biol. 2007 Jun 1;7:26.

13 Total synthesis of rapamycin.

Maddess ML, Tackett MN, Watanabe H, Brennan PE, Spilling CD, Scott JS, Osborn DP, Ley SV.

Angew Chem Int Ed Engl. 2007;46(4):591-7. No abstract available.

14 Drug evaluation: AP-23573–an mTOR inhibitor for the treatment of cancer.

Elit L.

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15 lipase-catalyzed regioselective esterification of rapamycin: synthesis of temsirolimus (CCI-779).

Gu J, Ruppen ME, Cai P.

Org Lett. 2005 Sep 1;7(18):3945-8.

16 CCI-779 Wyeth.

Elit L.

Curr Opin Investig Drugs. 2002 Aug;3(8):1249-53. Review.

17 Everolimus. Novartis.

Dumont FJ.

Curr Opin Investig Drugs. 2001 Sep;2(9):1220-34. Review.

18 Kuo et al (1992) Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature 358 70. PMID:1614535.

19 Huang et al (2003) Rapamycins: mechanism of action and cellular resistance. Cancer Biol.Ther. 2 221. PMID:12878853.

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21 Fleming et al (2011) Chemical modulators of autophagy as biological probes and potential therapeutics. 7 9. PMID:21164513.

22 J Am Chem Soc1993,115,(10):4419

23 Tetrahedron Lett1994,35,(28):4911

24 Chemistry (Weinheim)1995,1,(5):318

Figure imgf000004_0001 SIROLIMUS

FEMALE FERTILITY

http://amcrasto.theeurekamoments.com/2013/02/11/immunosuppressant-drug-rapamycin-helps-preserving-female-fertility/

PATENTS

Canada	2293793	APPROVED2006-07-11	EXP 2018-06-11
Canada	2103571	2003-04-29	2012-02-21
United States	5989591	1998-09-11	2018-09-11
United States	5212155	1993-05-18	2010-05-18

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A plaque, written in Brazilian Portuguese, commemorating the discovery of sirolimus on Easter Island, near Rano Kau

mTOR inhibitor

temsirolimus (CCI-779), everolimus (RAD001), deforolimus (AP23573), AP21967, biolimus, AP23102, zotarolimus (ABT 578), sirolimus (Rapamune), and tacrolimus (Prograf).\

SIROLIMUS

1H NMR

13 C NMR

HPLC

TIDEGLUSIB ..An NSAID and neuroprotective agent.

January 10, 2014 5:11 am / 2 Comments on TIDEGLUSIB ..An NSAID and neuroprotective agent.

Tideglusib

M.Wt: 334.39
Formula: C19H14N2O2S
CAS No.: 865854-05-3
4-Benzyl-2-(naphthalen-1-yl)-1,2,4-thiadiazolidine-3,5-dione

Glycogen Synthase Kinase 3 beta (GSK-3beta; tau Protein Kinase I) Inhibitors

Treatment of Neurologic Drugs (Miscellaneous)
Alzheimer’s Dementia, Treatment ofCerebrovascular Diseases, NP031112; NP-031112, Nypta Zentylor

NP 031112
NP-12
NP031112
Tideglusib
UNII-Q747Y6TT42

Noscira (Originator)
Tideglusib (NP-12, NP031112) is a potent, selective and irreversible^[1] small molecule non-ATP-competitive GSK3 inhibitor that has been investigated as a potential treatment for Alzheimer’s disease and paralysis supranuclear palsy in Phase IIa^[2] and IIb clinical trials.^[3]^[4]^[5]^[6] The first clinical trial conducted with tideglusib to be published (in English, at least) was phase II and demonstrated that overall tideglusib was well tolerated, except for some moderate, asymptomatic, fully reversible increases in liver enzymes (≥2.5xULN; where ULN=Upper Limit of Normal).^[4]

tideglusib

NP-031112 is an inhibitor of glycogen synthase kinase-3 beta (GSK-3beta) in early clinical development for the oral treatment of Alzheimer’s disease. The compound had been in phase II clinical trials for the treatment of progressive supranuclear palsy and for the treatment of Alzheimer’s disease; however the development was discontinued in 2011 and 2012 respectively, due to lack of efficacy.

The neuroprotective effects demonstrated in animal studies have also suggested its potential use in stroke and other brain disorders. It is being developed by Noscira (formerly known as NeuroPharma). In 2009, orphan drug designation was received in the E.U. and the U.S. for the treatment of progressive supranuclear palsy. In 2010, fast track designation was assigned in the U.S. by Noscira for this indication.

Fast Track status is granted to facilitate development and expedite the review of a drug for a serious or potentially fatal illness and to meet an unmet medical need

The Phase II trial for Progressive Supranuclear Palsy (PSP) commenced in December 2009 and is currently in progress

Belen Sopesen, CEO of Noscira: ‘Fast Track status is very positive for the company and is an incentive to continue advancing in the clinical development of Tideglusib (ZentylorTM) in Progressive Supranuclear Palsy’

Overexpression of GSK-3 leads to hyperphosphorylation of the tau protein, an anomaly which occurs in a number of neurodegenerative diseases known collectively as tauopathies, which include Alzheimer’s disease (AD), Progressive Supranuclear Palsy (PSP) and Pick disease. NP-12 is a GSK-3 inhibitor with oral bioavailability and great therapeutic potential as a disease-modifying treatment for Alzheimer’s.

NP-12 is currently undergoing clinical trials for Alzheimer’s disease in the EU. NP-12, the only GSK-3 inhibitor under clinical development for AD, has proven to be capable of acting on all of the histopathological lesions associated with the disease in experimental models: it reduces phosphorylation of the tau protein and hippocampal and entorhinal cortex neuron loss, improves spatial memory deficits and significantly reduces the accumulation of amyloid plaques in the brain. NP-12 also provides neuroprotection in vivo and has a potent anti-inflammatory effect in a range of animal models.

About Progressive Supranuclear Palsy

PSP is a neurodegenerative disease characterized by oculomotor disturbances, specifically difficulties in moving the eye vertically, falling down and Parkinsonian symptoms.

The disease affects an estimated 5-6.4 out of every 100,000 people.

There is currently no treatment capable of delaying or altering the progression of the illness.

TIDEGLUSIB

Domínguez, JM; Fuertes, A; Orozco, L; del Monte-Millán, M; Delgado, E; Medina, M (January 2012). “Evidence for Irreversible Inhibition of Glycogen Synthase Kinase-3 by Tideglusib”. The Journal of Biological Chemistry 287 (2): 893–904.doi:10.1074/jbc.M111.306472. PMC 3256883. PMID 22102280.
Teodoro Del Ser (2010). “Phase IIa clinical trial on Alzheimer’s disease with NP12, a GSK3 inhibitor”. Alzheimer’s & Dementia 6 (4): S147. doi:10.1016/j.jalz.2010.05.455.
Eldar-Finkelman, H; Martinez, A (2011). “GSK-3 Inhibitors: Preclinical and Clinical Focus on CNS”. Frontiers in Molecular Neuroscience 4: 32.doi:10.3389/fnmol.2011.00032. PMC 3204427. PMID 22065134.
Del Ser, T; Steinwachs, KC; Gertz, HJ; Andrés, MV; Gómez-Carrillo, B; Medina, M; Vericat, JA; Redondo, P et al. (2013). “Treatment of Alzheimer’s disease with the GSK-3 inhibitor tideglusib: A pilot study”. Journal of Alzheimer’s disease 33 (1): 205–15.doi:10.3233/JAD-2012-120805. PMID 22936007.
“FDA Grants Fast Track Status to Tideglusib (ZentylorTM) for Progressive Supranuclear Palsy”. PR Newswire Europe Including UK Disclose. 10 September 2010. Retrieved 11 August 2013.
Dominguez, JM; Fuertes, A; Orozco, L; Del Monte-Millan, M; Delgado, E; Medina, M (2011). “Evidence for Irreversible Inhibition of Glycogen Synthase Kinase-3 by Tideglusib”. Journal of Biological Chemistry 287 (2): 893–904.doi:10.1074/jbc.M111.306472. PMC 3256883. PMID 22102280.
WO 2005097117
WO 2006045581
WO 2006084934
WO 2008057933
WO 2011151359
Evidence for irreversible inhibition of glycogen synthase kinase-3β by tideglusib.

Domínguez JM, Fuertes A, Orozco L, del Monte-Millán M, Delgado E, Medina M.

J Biol Chem. 2012 Jan 6;287(2):893-904. doi: 10.1074/jbc.M111.306472. Epub 2011 Nov 18

13. MARTINEZ A ET AL.: “First Non-ATP Competitive Glycogen Synthase Kinase 3.beta. (GSK-3.beta.) Inhibitors: Thiadiazolidinones (TDZD) as Potential Drugs for the Treatment of Alzheimer’s Disease” JOURNAL OF MEDICINAL CHEMISTRY, vol. 45, no. 6, 2002, pages 1292-1299

US8158661	4-18-2012	GSK-3 Inhibitors
US7531561	5-13-2009	GSK-3 inhibitors
US2008153886	6-27-2008	Use Of Heterocyclic Compounds As Neurogenic Agents

CLINICAL TRIALS

http://clinicaltrials.gov/search/intervention=NP+031112

http://clinicaltrials.gov/show/NCT01350362

………….

http://www.google.com/patents/WO2005097117

For example, the following procedure can be used to produce 4-N-benzyl substituted thiadiazolidinones :

The general experimental procedure of Scheme 1 is described for example in Slomczynska,

U.; Barany, G., “Efficient Synthesis of l,2,4-Dithiazolidine-3,5-diones (Dithiasuccinoyl- amines) and observations on formation of l,2,4-Thiadiazolidine-3,5-dione by related

Chemistry”, J. Heterocyclic Chem., 1984, 21, 241-246.

For example, sulfuryl chloride is added dropwise with stirring, under nitrogen atmosphere, preferably at low temperature, preferably at about 5 °C, to a solution of benzyl isothiocyanate and the isocyanate indicated in each case, in a suitable solvent such as hexane, ether or THF. When the addition is finished, the mixture is left to react, for example by stirring for 20 hours at room temperature. After this time, the resulting product is isolated by conventional methods such as suction filtration or solvent evaporation and then, the purification is performed (e.g. by recristallization or silica gel column chromatography using the appropriate eluent). Other alternative procedures will be apparent to the person skilled in the art, such as the use of any other chlorinating agent instead of sulfuryl chloride, variations in the order of addition of the reactants and reaction conditions (solvents, temperature, etc).

Example 2

4-Benzyl-2-naphthalen-l-yl-[l,2,4]thiadiazolidine-3,5-dione (2)

Reagents: Benzyl-isothiocianate (13 mmol, 1.72 mL), 1-naphthyl-isocyanate (13 mmol, 1.9 mL) and SO₂CI₂ (13 mmol, 1.04 mL) in hexane (50 mL). Isolation: filtration of reaction mixture. Purification: recrystallization from EtOH. Yield: 3.8 g (87%), white needles. mp= 150 °C

1H-RMN (CDC1₃): 4.9 (s, 2H, CH2PI1); 7.3-7.9 (m, 12Η, arom.) ¹³C-RMN (CDCI3): 46.5 (CH2Ph); 128.3; 128.6; 129.0; 135.0 (C arom, Ph); 122.0; 125.3; 126.8; 127.2; 127.5; 128.5; 130.8; 134.4 (C arom, naphthyl); 152.2 (3-00); 165.9 (5- C=O).

Anal (C₁₉H₁₄N₂O₂S), C, H, N, S

Sulfuryl chloride is added dropwise with stirring, under nitrogen atmosphere, at 5 °C to a solution of benzyl isothiocyanate and the isocyanate indicated in each case, in hexane, ether or THF. When the addition is finished, the mixture is stirred for 20 hours at room temperature. After this time, the resulting product is isolated by suction filtration or by solvent evaporation and then, the purification is performed by recristallization or silica gel column chromatography using the appropriate eluent. More details can be found in Slomczynska, U.; Barany, G., “Efficient Synthesis of l,2,4-Dithiazolidine-3,5-diones (Dithiasuccinoyl-amines) and observations on formation of l,2,4-Thiadiazolidine-3,5-dione by related Chemistry”, J Heterocyclic Client., 1984, 21, 241-246.

…………

WO2006045581A1 *	Oct 21, 2005	May 4, 2006	Neuropharma Sa	The use of 1, 2, 4-thiadiazolidine-3, 5-diones as ppar activators
WO2011151359A1	Jun 1, 2011	Dec 8, 2011	Noscira, S.A.	Combined treatment with a cholinesterase inhibitor and a thiadiazolidinedione derivative
WO2013124413A1	Feb 22, 2013	Aug 29, 2013	Noscira, S.A.	Thiadiazolidinediones as gsk-3 inhibitors
EP2177510A1	Oct 17, 2008	Apr 21, 2010	Universität des Saarlandes	Allosteric protein kinase modulators
EP2527323A1	May 24, 2011	Nov 28, 2012	Noscira, S.A.	Urea carbonyl disulfide derivatives and their therapeutic uses

………..

THANKS AND REGARD’S
DR ANTHONY MELVIN CRASTO Ph.D

GLENMARK SCIENTIST , NAVIMUMBAI, INDIA

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MIDAZOLAM …A short-acting hypnotic-sedative drug with anxiolytic and amnestic properties

January 10, 2014 3:49 am / Leave a comment

MIDAZOLAM

8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine

59467-70-8^{CAS NO OF FREE BASE}

59467-94-6 MALEATE, Launched – 1982, Roche (Originator)

59467-96-8 (HCl)

A short-acting hypnotic-sedative drug with anxiolytic and amnestic properties. It is used in dentistry, cardiac surgery, endoscopic procedures, as preanesthetic medication, and as an adjunct to local anesthesia. The short duration and cardiorespiratory stability makes it useful in poor-risk, elderly, and cardiac patients. It is water-soluble at pH less than 4 and lipid-soluble at physiological pH.

Midazolam (/m ɪ ˈ d æ z ə l æ m /, marketed in English-speaking countries and Mexico under the trade names Dormicum, Hypnovel, andVersed,) is a short-acting drug in the benzodiazepine class developed by Hoffmann-La Roche in the 1970s. The drug is used for treatment of acute seizures, moderate to severe insomnia, and for inducing sedation and amnesia before medical procedures. It possesses profoundly potentanxiolytic, amnestic, hypnotic, anticonvulsant, skeletal muscle relaxant, and sedative properties.^[6]^[7]^[8] Midazolam has a fast recovery time and is the most commonly used benzodiazepine as a premedication for sedation; less commonly it is used for induction and maintenance of anesthesia.Flumazenil, a benzodiazepine antagonist drug, can be used to treat an overdose of midazolam, as well as to reverse sedation.^[7] However, flumazenil can trigger seizures in mixed overdoses and in benzodiazepine-dependent individuals, so is not used in most cases.^[9]^[10]

midazolam

Administration of midazolam by the intranasal or the buccal route (absorption via the gums and cheek) as an alternative to rectally administereddiazepam is becoming increasingly popular for the emergency treatment of seizures in children. Midazolam is also used for endoscopyprocedural sedation and sedation in intensive care. The anterograde amnesia property of midazolam is useful for premedication before surgery to inhibit unpleasant memories. Midazolam, like many other benzodiazepines, has a rapid onset of action, high effectiveness and low toxicity level. Drawbacks of midazolam include drug interactions, tolerance, and withdrawal syndrome, as well as adverse events including cognitive impairment and sedation. Paradoxical effects occasionally occur, most commonly in children and the elderly, particularly after intravenous administration. The drug has also recently been hastily introduced for use in executions in the USA in combination with other drugs.

Midazolam is a short-acting benzodiazepine in adults with an elimination half-life of one to four hours; however, in the elderly, as well as young children and adolescents, the elimination half-life is longer. Midazolam is metabolised into an active metabolite alpha1-hydroxymidazolam. Age related deficits, renal and liver status affect the pharmacokinetic factors of midazolam as well as its active metabolite. However, the active metabolite of midazolam is minor and contributes to only 10 percent of biological activity of midazolam. Midazolam is poorly absorbed orally with only 50 percent of the drug reaching the bloodstream. Midazolam is metabolised by cytochrome P450 (CYP) enzymes and by glucuronide conjugation. The therapeutic as well as adverse effects of midazolam are due to its effects on the GABA_A receptors; midazolam does not activate GABA_A receptors directly but, as with other benzodiazepines, it enhances the effect of the neurotransmitter GABA on the GABA_A receptors (↑ frequency of Cl− channel opening) resulting in neural inhibition. Almost all of the properties can be explained by the actions of benzodiazepines on GABA_A receptors. This results in the following pharmacological properties being produced: sedation, hypnotic, anxiolytic, anterograde amnesia, muscle relaxation and anti-convulsant.Midazolam maleate is a benzodiazepine that is commercialized by Astellas Pharma and Roche as an intravenous or intramuscular injection for the long-term sedation of mechanically ventilated patients under intensive care. The drug is also available in a tablet formulation, and is currently distributed in various markets, including Germany, Japan, Switzerland and the U.K. In March 2002, two lots of a syrup formulation were recalled in the U.S. due to the potential presence of a crystalline precipitate of an insoluble complex of midazolam and saccharin. Subsequently, the injection and syrup formulations of the product were both withdrawn from the U.S. market. In 2010, a Pediatric Use Marketing Authorization (PUMA) was filed for approval in the E.U. by ViroPharma for the treatment of prolonged, acute, convulsive seizures in infants, toddlers, children and adolescents, from 3 months to less than 18 years. In 2011, a positive opinion was assigned to the PUMA and final approval was assigned in June 2011. The product was launched in the U.S. in November 2011. This product was filed for approval in Japan in 2013 by Astellas Pharma for the conscious sedation in dentistry and dental surgery. In the same year the product was approved for this indication.

In terms of clinical development, a nasal formulation of the drug is in phase III clinical trials at Upsher-Smith for rescue treatment of seizures in patients on stable anti-epileptic drug regimens who require control of intermittent bouts of increased seizure activity (seizure clusters). The Hopitaux de Paris had been developing a sublingual tablet formulation of midazolam to be used in combination with morphine for the treatment of pain in children following bone fractures; however, no recent development has been reported for this indication. NovaDel Pharma had been developing the compound preclinically for the treatment of generalized anxiety, however no recent developments have been reported.

Midazolam achieves its therapeutic effect through interaction with the gamma-aminobutyric acid benzodiazepine (GABA-BZ) receptor complex. Subunit modulation of the GABA-BZ receptor chloride channel macromolecular complex is hypothesized to be responsible for some of the pharmacological properties of benzodiazepines, which include sedative, anxiolytic, muscle relaxant, and anticonvulsive effects in animal models. GABA acts at inhibitory synapses in the brain by binding to specific transmembrane receptors in the plasma membrane of both pre- and post-synaptic neurons, opening ion channels and bringing about a hyperpolarization via either chloride or potassium ion flow.

In 2008, fast track designation was assigned to midazolam maleate in the U.S. for the treatment of seizure disorders.

In 2009, Orphan Drug Designation was received in the U.S. by for the treatment of seizure disorders in patients who require control of intermittent bouts of increased seizure activity (e.g. acute repetitive seizures, seizure clusters). This designation was assigned in the U.S. for the treatment of nerve agent-induced seizures.

In 2010, midazolam maleate was licensed to Upsher-Smith by Ikano Therapeutics for the treatment of acute repetitive seizure in patients with epilepsy. However, in 2010, Ikano closed and dissolved its business. Previously, Ikano had transferred to Upsher-Smith ownership of it nasal midazolam maleate program.

Midazolam is among about 35 benzodiazepines which are currently used medically, and was synthesised in 1975 by Walser and Fryer at Hoffmann-LaRoche, Inc in the United States.Owing to its water solubility, it was found to be less likely to cause thrombophlebitis than similar drugs.The anticonvulsant properties of midazolam were studied in the late 1970s, but not until the 1990s did it emerge as an effective treatment for convulsive status epilepticus. As of 2010, it is the most commonly used benzodiazepine in anesthetic medicine. In acute medicine, midazolam has become more popular than other benzodiazepines, such as lorazepam and diazepam, because it is shorter lasting, is more potent, and causes less pain at the injection site.Midazolam is also becoming increasingly popular in veterinary medicine due to its water solubility.

Midazolam is a water-soluble benzodiazepine available as a sterile, nonpyrogenic parenteral dosage form for intravenous or intramuscular injection. Each mL contains midazolam hydrochloride equivalent to 1 mg or 5 mg midazolam compounded with 0.8% sodium chloride and 0.01% edetate disodium with 1% benzyl alcohol as preservative, and sodium hydroxide and/or hydrochloric acid for pH adjustment. pH 2.9-3.7.

Midazolam is a white to light yellow crystalline compound, insoluble in water. The hydrochloride salt of midazolam, which is formed in situ, is soluble in aqueous solutions. Chemically, midazolam HCl is 8-chloro-6-(2-fluorophenyl)-1-methyl-4H– imidazo[1,5-a] [1,4] benzodiazepine hydrochloride. Midazolam hydrochloride has the molecular formula C₁₈H₁₃ClFN₃•HCl, a calculated molecular weight of 362.25 and the following structural formula:

Midazolam HCl structural formula illustration

In the Netherlands, midazolam is a List II drug of the Opium Law. Midazolam is a Schedule IV drug under the Convention on Psychotropic Substances. In the United Kingdom, midazolam is a Class C controlled drug. In the United States, midazolam (DEA number 2884) is on the Schedule IV list of the Controlled Substances Act as a non-narcotic agent with low potential for abuse.

midaolam hydrochloride NDA 018654, 075154

REF

U.S. Pat. No. 4,280,957

U.S. Pat. No. 5,693,795

U.S. Pat. No. 6,512,114

Midazolam Maleate
Drugs Fut 1978, 3(11): 822

Bioorganic and Medicinal Chemistry, 2012 , vol. 20, 18 pg. 5658 – 5667

Journal of Heterocyclic Chemistry, 1983 , vol. 20, 3 pg. 551 – 558.. 32 maleate

WO 2001070744

WO 2001002402

WO 2012075286

US2011/275799 A1… no 5

Journal of Organic Chemistry, 1978 , vol. 43, p. 936,942, mp free base, nmr

US4280957	May 15, 1978	Jul 28, 1981	Hoffmann-La Roche Inc.	Imidazodiazepines and processes therefor
US6262260 *	Mar 23, 2000	Jul 17, 2001	Abbott Laboratories	Process for the preparation of midazolam
US6512114	Jun 30, 1999	Jan 28, 2003	Abbott Laboratories	Process for the preparation of Midazolam

……………………….

introduction

4H-imidazo[1,5-a][1,4]benzodiazepines or, more simply, imidazobenzodiazepines, are a class of benzodiazepines having the general formula (I),

wherein the 1,4-diazepine ring is fused with a 1,3-imidazole ring. The main compounds part of the 4H-imidazo[1,5-a][1,4]benzodiazepines are Midazolam of formula (IV):

an active ingredient currently commercially available as a hydrochloride salt under the name of Versed or Hypnovel for anaesthetic and sedative use and the maleate salt currently commercially available under the name Dormicum or Flormidal.
Other important compounds are Climazolam of formula (VII):

Imidazenil of formula (VIII):

1-Hydroxymidazolam of formula (IX):

and Desmethyl midazolam of formula (X):

all these being biologically active substances and having psychotropic and sedative action.
The synthesis of the Midazolam as described in U.S. Pat. No. 4,280,957 of Hoffmann-La Roche provides for the decarboxylation reaction of the 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid of formula (VI) according to the following scheme:

The process for preparing the intermediate (VI) via basic hydrolysis of the corresponding ester is described in such patent publication and it is well known in the art.
The thermal decarboxylation reaction in high boiling solvent such as mineral oil at 230° C. for 5 min results in a mixture of products of Midazolam of formula (IV) and of Isomidazolam of formula (IV-bis), a non-pharmacologically active isomer, at a 80:20 ratio. The two products are separated by chromatography.
At industrial level, the formation of the Isomidazolam isomer impurity requires a further isomerisation reaction performed on the mixture of the two compounds to convert the isomer into the active product. The reaction mixture obtained from the thermal decarboxylation is thus subjected to basic treatment under the action of KOH in EtOH followed by an acid treatment which thus provides a mixture of Midazolam-Isomidazolam at a 95:5 ratio. The final removal of the Isomidazolam impurity from the product occurs through crystallisation of the product from AcOEt and EtOH. In order to limit this isomerisation treatment, in the subsequent U.S. Pat. No. 5,693,795 of Hoffmann-La Roche dated 1999, there is described a process for performing the decarboxylation of the compound of formula (VI) in n-butanol in a continuous tubular reactor with a 4 minutes permanence period with a yield between 47-77%. However, the reaction, performed at high temperature and pressure (280° C., 100 bars) results in the formation of a considerable percentage of Isomidazolam (85:15 Midazolam/Isomidazolam ratio) which still requires the basic isomerisation step.
Lastly, in U.S. Pat. No. 6,512,114 of Abbott Laboratories there is described the decarboxylation of the compound of formula (VI) in mineral oil or in N,N-Dimethylacetamide (DMA) at 160-230° C. for at least 3 hours obtaining a 3/1 to 6/1 Midazolam/Isomidazolam ratio with a yield of isolated product equal to just 54%.
Though performed using dedicated apparatus and in extreme conditions, the prior art processes do not allow selectively performing the decarboxylation reaction of the intermediate (VI) to Midazolam thus requiring a further synthetic passage followed by crystallisation with ensuing reduction of the overall yield.

Midazolam (8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine) is represented by the following structural formula (I):

Midazolam is a central nervous system (CNS) depressant, used for short term treatment of insomnia. Like other benzodiazepines, midazolam binds to benzodiazepine receptors in the brain and spinal cord and is thus used as a short-acting hypnotic-sedative drug with anxiolytic and amnestic properties. It is currently used in dentistry, cardiac surgery, endoscopic procedures, as a preanesthetic medication, as an adjunct to local anesthesia and as a skeletal muscle relaxant. Depending on the pH value, midazolam can exist in solution as a closed ring form (I) as well as an open ring form (IA), which are in equilibrium, as shown in Scheme 1:

The amount of the open ring form (IA) is dependent upon the pH value of the solution. At a pH value of about 3, the content of the open ring form (IA) can be 40%, while at pH value of 7.5, the closed ring form (I) can be more than 90%.

Clinical studies have demonstrated that there are no significant differences in the clinical activity between midazolam hydrochloride and midazolam maleate, however the use of intravenous midazolam hydrochloride has been associated, in some cases, with respiratory depression and arrest.

U.S Pat. No. 4,280,957 (hereinafter the ‘957 patent) describes a synthetic process for preparing midazolam, which is depicted in Scheme 2 below. This process includes reacting 2-aminomethyl-7-chloro-2,3-dihydro-5-(2-fluorophenyl)-1H-1,4-bezodiazepine (II) with acetic anhydride in dichloromethane to produce 2-acetamido-methyl-7-chloro-2,3-dihydro-5-(2-fluorophenyl)-1H-1,4-bezodiazepine (III). The latter is heated with polyphosphoric acid at 150° C. to produce 8-chloro-6-(2-fluorophenyl)-3a,4-dihydro-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine of formula (IV), which is purified by column chromatography. Compound IV is then mixed with toluene and manganese dioxide and heated to reflux to afford midazolam base, which is crystallized from ether to yield a product with mp of 152-154° C.

The ‘957 patent further describes an alternative process which includes reacting 2-aminomethyl-7-chloro-2,3-dihydro-5-(2-fluorophenyl)-1H-1,4-bezodiazepine (II) (optionally as a dimaleate salt) with triethylorthoacetate in ethanol and in the presence of p-toluenesulfonic acid to afford 8-chloro-6-(2-fluorophenyl)-3a,4-dihydro-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine (IV). This product is dissolved in xylene and treated with activated manganese dioxide to afford the crude base, which is reacted in situ with maleic acid in ethanol and crystallized by addition of ether to produce the midazolam maleate having melting point of 148-151° C. The process is depicted in Scheme 3 below.

The preparation of midazolam maleate from the isolated midazolam base is also described in a further example of the ‘957 Patent, wherein a warm solution of midazolam base in ethanol is combined with a warm solution of maleic acid in ethanol. The mixture is diluted with ether and at least part of the solvents is evaporated using a steam bath to obtain crystalline midazolam maleate having melting point of 148-151° C. The yield and the purity of the obtained midazolam maleate are not disclosed.

U.S. Pat. No. 6,512,114 (hereinafter the ‘114 patent) describes another synthetic process for preparing midazolam, which is depicted in Scheme 4 below. According to this Process, the starting material 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid (V) is heated in mineral oil for 3 hours at 230° C. until it is decarboxylated, followed by treatment with potassium tert-butoxide, to afford midazolm (I), isomidazolam (VI) and a midazolam dimmer (VII). Midazolam base is obtained in 54.5% yield after two re-crystallizations from ethyl acetate and heptane; however, the purity of the product is not disclosed.

The preparation of midazolam by conventional routes is liable to produce impurities such as isomidazolam (VI) and a midazolam dimmer (VII), and possibly other impurities. There is, therefore, a need in the art for a midazolam purification process that will provide highly pure midazolam containing minimal amounts of impurities produced. The present invention provides such a process.

This example describes the preparation of midazolam base as taught in the ‘957 patent.

16 g (0.03 mol) of 2-aminomethyl-7-chloro-5-(2-fluorophenyl)-2,3-dihydro-1H-1,4-bezodiazepine dimaleate was dissolved in 200 ml of toluene and 10 ml of 25% ammonium hydroxide solution was added and mixing was maintained for an hour. Then, the phases were separated and the toluene phase was dried by azeotropic distillation using a Dean Stark apparatus. 7 ml (0.038 mol) of triethylorthoacetate was added and the solution was heated to reflux for 4 hours, after which time the solution was left to cool to ambient temperature. 25 ml of methyl tert-butyl ether was added and the mixture was cooled overnight to produce 8-chloro-6-(2-fluorophenyl)-3a,4-dihydro-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine, which was isolated by filtration. The product was mixed with 200 ml of toluene and dried by azeotropic distillation using a Dean Stark apparatus. Then, 30 g of manganese dioxide was added and the mixture was heated to reflux for two hours. The excess manganese dioxide was filtered off to afford a solution of midazolam base in toluene, which was evaporated to obtain a product having 97.9% purity and containing 0.44% of impurity VIII and 1.14% of impurity IX (according to HPLC).

…………………………

US4280957

EXAMPLE 28

2-Aminomethyl-7-chloro-2,3-dihydro-5-(2-fluorophenyl)-1H-1,4-benzodiazepine dimaleate

A suspension of 17 g (0.05 m) of 7-chloro-1,3-dihydro-5-(2-fluorophenyl)-2-nitromethylene-2H-1,4-benzodiazepine-4-oxide in 200 ml of tetrahydrofuran and 100 ml of methanol was hydrogenated in presence of 17 g of Raney nickel at an initial pressure of 155 psi for 24 hrs. The catalyst was removed by filtration and the filtrate was evaporated. The residue was dissolved in 50 ml of 2-propanol and warmed on the steambath. A warm solution of 17 g of maleic acid in 60 ml of ethanol was added and the salt was allowed to crystallize by cooling in the ice bath. The final product consisted of yellow crystals with mp 196

EXAMPLE 14

8-Chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine

Acetic anhydride, 7 ml., was added to a solution of 6.16 g. of crude 2-aminomethyl-7-chloro-2,3-dihydro-5-(2-fluorophenyl)-1H-1,4-benzodiazepine in 200 ml. of methylene chloride. The solution was layered with 200 ml. of saturated aqueous sodium bicarbonate and the mixture was stirred for 20 minutes. The organic layer was separated, washed with sodium bicarbonate, dried over sodium sulfate and evaporated to leave 6.2 g. resinous 2-acetaminomethyl-7-chloro-2,3-dihydro-5-(2-fluorophenyl)-1H-1,4-benzodiazepine. This material was heated with 40 g. of polyphosphoric acid at 150 water, made alkaline with ammonia and ice and extracted with methylene chloride. The extracts were dried and evaporated and the residue (5.7 g.) was chromatographed over 120 g. of silica gel using 20% methanol in methylene chloride. The clean fractions were combined and evaporated to yield resinous 8-chloro-3a,4-dihydro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[ 1,5-a][1,4]benzodiazepine. A mixture of this material with 500 ml. of toluene and 30 g. of manganese dioxide was heated to reflux for 11/2 hours. The manganese dioxide was separated by filtration over celite. The filtrate was evaporated and the residue was crystallized from ether to yield a product with m.p. 152 was recrystallized from methylene chloride/hexane

EXAMPLE 49

8-Chloro-6-(2-fluorophenyl)-1-methyl-6H-imidazo[1,5-a][1,4]benzodiazepine

Potassium t-butoxide, 0.625 g. (5.5 mmol), was added to a solution of 1.625 g. (5 mmol) of 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine in 20 ml. of dimethylformamide cooled to -30 nitrogen for 10 min. at -30 ml. of glacial acetic acid and was then partitioned between aqueous bicarbonate and toluene/methylene chloride (3:1 v/v). The organic layer was separated, dried and evaporated. The residue was chromatographed over 60 g. of silica gel using 25% (v/v) methylene chloride in ethyl acetate. The less polar product was eluted first and was crystallized from ethylacetate/hexane to yield product with m.p. 180

EXAMPLE 50

8-Chloro-6-(2-fluorphenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine

Potassium t-butoxide, 0.125 g. (1.1 mmol) was added to a solution of 0.325 g. (1 mmol) of 8-chloro-6-(2-fluorophenyl)-1-methyl-6H-imidazo[1,5-a][1,4]benzodiazepine in 20 ml. of dimethylformamide cooled to -30 -30 by addition of 0.2 ml. of glacial acetic acid and was partitioned between aqueous sodium bicarbonate and methylene chloridetoluene (1:3). The organic phase was washed with water, dried and evaporated. The residue was chromatographed over 20 g. of silica gel using ethyl acetate for elution. After elution of starting material, product was collected and crystallized from ether/hexane, m.p. 156

hyd and dihydrochloride

EXAMPLE 24

8-Chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine dihydrochloride

A solution of 0.32 g (1 mmol) of 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine in 5 ml of ethanol was treated with excess ethanolic hydrogen chloride. The salt was crystallized by addition of 2-propanol and ether. The colorless crystals were collected, washed with ether and dried to leave a final product with mp 290

EXAMPLE 258-Chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine hydrochloride

A solution of 0.325 g (1 mmol) of 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine in 3 ml of ethanol was combined with a suspension of 0.4 g (1 mmol) of the dihydrochloride of this compound in 5 ml of ethanol. After filtration, the solution was treated with ether and heated on the steambath for 5 min to crystallize. The crystals were collected, washed with ether and dried to leave the monohydrochloride with mp 295

maleate

EXAMPLE 22

8-Chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine maleate

A warm solution of 6.5 g (0.02 m) of 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine in 30 ml of ethanol was combined with a warm solution of 2.6 g (0.022 m) of maleic acid in 20 ml of ethanol. The mixture was diluted with 150 ml of ether and heated on the steam bath for 3 min. After cooling, the crystals were collected, washed with ether and dried in vacuo to yield a final product with mp 148

…

Synthesis

US20110275799

Midazolam, can be described according to scheme 4 indicated below:

EXPERIMENTAL PART

Materials and Methods

8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepin-3-carboxylic acid of formula (VI)

was prepared according to processes known in the art (e.g. U.S. Pat. No. 4,280,957) which comprise the basic hydrolysis of the corresponding ester.
For the reactions performed in the microreactor, the solutions containing the substrates to be decarboxylated were loaded into 5 and 10 mL gastight glass syringes (Hamilton, item n. 81527, 81627) mounted on syringe pumps (KD Scientifics, model KDS100). A pipe made of PTFE® (OD=1.58 mm, ID=0.8 mm, Supelco, item n. 58696-U) was used for making the reaction channel.A counterpressure valve sold by Swagelok (item n. SS-SS1-VH) was used for regulating the flow within the channel.Example 1Synthesis of the Compound of Formula (V)—Example of the Invention

50 g (0.135 mol) of 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepin-3-carboxylic acid of formula (VI) and 250 mL of ethanol were loaded into a two-neck 500 mL flask, equipped with a magnetic stirrer. 40 mL of an aqueous solution of 1 M HCl are dripped in about 10 minutes. The open di-hydrochloride intermediate of formula (V) starts precipitating into the reaction environment already after 3 minutes from the beginning of the addition of the acid solution. The mixture is maintained stirred at RT for 3 hrs and then it is filtered on buckner washing the solid with ethanol. The moist product is dried in an oven under vacuum at 60° C. up to reaching a constant weight. A light yellow crystalline product is obtained (51.5 g, 83% yield). The crude product was used for the decarboxylation without further purifications.

ESI-MS [MeCN+0.1% HCOOH]: m/z 388 (V); 370 (VI).

¹H-NMR (250 MHz, CD3OD): 2.52 (s, 3H); 4.27-4.41 (m, 2H); 7.22-8.1 (m, 7H). M.p.: 217° C.

Example 2

Synthesis of Midazolam of Formula (IV)—Performed in Batch—Example of the Invention

30 g (0.065 mol) of 5-(aminomethyl)-1-{(4-chloro-2-[(2-fluorophenyl)carbonyl]phenyl}-2-methyl-1H-imidazole-4-carboxylic acid dihydrochloride of formula (V) and 90 mL of NMP are loaded into a three-neck 250 mL flask, equipped with a magnetic stirrer and coolant. The mass is heated using an oil bath at T=195-203° C. for one hour. Thus, 1 mL of solution is collected for performing HPLC analysis. The reaction product is Midazolam having 82% titre (w/w) (determined via HPLC titre correcting it using the solvent) and it contains 1% of Isomidazolam. The product is extracted using Isopropyl acetate after raising the pH to 10 by adding aqueous Na2CO3.

Example 3

Synthesis of Midazolam of Formula (IV)—Performed in a Micro-Reactor—Example of the Invention

3.22 g (7 mmol) of 5-(aminomethyl)-1-{4-chloro-2-[(2-fluorophenyl)carbonyl]phenyl}-2-methyl-1H-imidazole-4-carboxylic acid dihydrochloride of formula (V) and 10 mL of NMP are loaded into a 10 mL flask equipped with a magnetic stirrer. In order to facilitate the complete solubilisation of the substrate, it is necessary to slightly heat the reaction mixture (about 40° C.) for a few minutes. The solution thus obtained is transferred into a 10 mL gastight glass syringe mounted on a KDS100 syringe pump (FIG. 1) and the flow is regulated at 1.0 mL/h so as to set a residence period of 30 minutes at 200° C. The reaction product is Midazolam having an 89% titre (w/w) (determined via HPLC titre correcting it using the solvent) and containing 3% (w/w) of Isomidazolam.

Example 4Synthesis of Midazolam of formula (IV)—Comparison of the InventionA table is reported which summarises the results of the decarboxylation of the compound of formula (V) and (V-bis) (for the latter see Examples 6 and 7) obtained according to some embodiments of the invention and those obtained by way of experiment through the decarboxylation of the intermediate of formula (VI) (process of the prior art) both performed in 3 volumes of NMP at 200° C., both in batch method (Example 4) and in continuous method with the microreactor (MR) made of PTFE of FIG. 1. (Examples 4-1, 4-2, 4-3).


Example	substrate	Mode	Solv.	T° C.	t min.	Midazolam (p/p)	Isomidaz. (P/P)


2	(V)	Batch	NMP	200	60	82	1
3	(V)	MR	NMP	200	30	89	3
7	(V-bis)	Batch	NMP	200	60	68	3
4	(VI)	Batch	NMP	200	60	78	18
4-1	(VI)	MR	NMP	200	38	81	17
4-2	(VI)	MR	NMP	200	20	77	18
4-3	(VI)	MR	NMP	200	15	58	22
U.S. Pat. No.	(VI)	Tubular	n-BuOH	290	4	85 *	15 *
5,693,795		reactor
U.S. Pat. No.	(VI)	Batch	Olio	230	180	75 *	25 *
6,512,114			min.			87.5 *	12.5 *
			or DMA

* = Midazolam/Isomidazolam ratio only (other impurities not considered).

The product of the comparative experiments 4, 4-1, 4-2, 4-3 and of the two USA patents should be subjected to a further isomerisation process to reduce the high amount of Isomidazolam so as to be able to obtain Midazolam free of Isomidazolam after further crystallization, which would not be required for the product obtained according to the invention (examples 2 and 3).

Midazolam maleate, dihydrochloride and monohydrochloride

MIDAZOLAM MALEATE

Example 8

Preparation of 8-Chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine maleate (Midazolam Maleate)

A 4-neck RBF was charged under nitrogen flow with: 10 g of Midazolam (IV) (prepared according to example 2) and 40 mL of Ethanol. The slurry was stirred until complete dissolution at 25/30° C. In an other flask was prepared the following solution: 3.72 g of maleic acid are dissolved in 15 mL of Ethanol. The slurry was stirred until complete dissolution at 25/30° C. The maleic acid solution is dropped in 30/40 minutes and keeping T=25/30° C. into the solution containing Midazolam. The slurry was cooled down at −15° C. in one hour and kept at that temperature for at least 2 hours. The slurry was then filtered and the cake was washed with 40 mL of cool Ethanol. The filter was discharged and the product was dried at 40° C. under vacuum for 2 hours and then at 60° C. for 8 hours. 12.8 g of Midazolam Maleate as white solid were collected (Molar yield=94.5%). m.p.=149-152° C. (by DSC).

MIDAZOLAM DIHYDROCHLORIDE

Example 9

Preparation of 8-Chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine dihydrochloride (Midazolam dihydrochloride)

A 4-neck RBF was charged under nitrogen flow with: 1 g of Midazolam (IV) (prepared according to example 2) and 15 mL of Ethanol. The slurry was stirred until complete dissolution at 25/30° C. 5 mL of a ethanolic solution of Hydrochloric acid 2N were slowly added. 20 mL of Isopropanol were added over 30 minutes at RT. The slurry was cooled down at −15° C. in one hour and kept at that temperature for at least 2 hours. The slurry was then filtered and the cake was washed with 10 mL of cool isopropanol. The filter was discharged and the product was dried at 40° C. under vacuum for 2 hours and then at 60° C. for 8 hours. Midazolam dihydrochloride as white solid was collected.

MIDAZOLAM HYDROCHLORIDE

Example 10

Preparation of 8-Chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine hydrochloride (Midazolam hydrochloride)

A 4-neck RBF was charged under nitrogen flow with: 1 g of Midazolam (IV) (prepared according to example 2) and 10 mL of Ethanol. The slurry was stirred until complete dissolution at 25/30° C. In an other flask was prepared the following suspension: 1.22 g of Midazolam dihydrochloride (prepared according to example 9) and 15 mL of Ethanol. The Midazolam ethanolic solution was added to the Midazolam dihydrochloride suspension. After filtration, the solution was treated with MTBE and heated at 60° C. until crystallization. After cooling to RT, the slurry was filtered, the cake washed with MTBE and the product was dried to provide Midazolam (mono)hydrochloride as a white solid.

…..

DAPAGLIFLOZIN…FDA approves AZ diabetes drug Farxiga

January 10, 2014 3:05 am / Leave a comment

DAPAGLIFLOZIN, BMS-512148

The US Food and Drug Administration has finally approved AstraZeneca’s diabetes drug Farxiga but is insisting on six post-marketing studies, including a cardiovascular outcomes trial.

The approval was expected given that the agency’s Endocrinologic and Metabolic Drugs Advisory Committee voted 13-1 last month that the benefits of Farxiga (dapagliflozin), already marketed in Europe as Forxiga, outweigh identified risks. The FDA rejected the drug in January 2012 due to concerns about possible liver damage and the potential link with breast and bladder cancer.

READ ABOUT SYNTHESIS AT

https://newdrugapprovals.wordpress.com/2013/12/18/dapagliflozin-sees-light/

AVOSENTAN

January 9, 2014 8:08 am / Leave a comment

AVOSENTAN

N-[6-Methoxy-5-(2-methoxyphenoxy)-2-(4-pyridyl)pyrimidin-4-yl]-5-methylpyridine-2-sulfonamide

5-methyl-pyridine-2-sulfonic acid [6-methoxy-5-(2-methoxy-phenoxy)-2-(pyridin-4-yl)-pyrimidin-4-yl]-amide,

5-methyl-pyridine-2-sulfonic acid [6-methoxy-5-(2-methoxy-phenoxy)-2-pyridin-4-yl-pyrimidin-4-yl]-amide,

Endothelin ETA Receptor Antagonists

M.Wt: 479.51
Formula: C23H21N5O5S

Roche (Originator)

CAS No.: 290815-26-8

RO 67-0565
SPP 301
UNII-L94KSX715K

PHASE 3

CLINICAL TRIALS

http://clinicaltrials.gov/search/intervention=spp301+OR+Avosentan

SPP-301 is an oral, once-daily, second-generation endothelin ETA receptor antagonist which had been in phase III clinical development at Speedel for the treatment of diabetic nephropathy. In December 2006, the company reported that the phase III trial had been stopped based on the recommendation from the trial’s Data Safety Monitoring Board (DSMB) to stop the trial following incidence of a significant imbalance in fluid retention in patients in the study arms. Speedel reported that the compound will be evaluated for potential new clinical development for the treatment of diabetic kidney disease and other indications.

Originally developed by Roche and specifically optimized for improved liver safety, SPP-301 was licensed to Speedel in October 2000. In 2003, Speedel exercised its option to license from Roche all rights to SPP-301, including exclusive worldwide rights for the full development and commercialization of the ETA antagonist. SPP-301 has fast track designation and has undergone a special protocol assessment (SPA) by the FDA. Speedel had been studying the drug for the treatment of hypertension.

AVOSENTAN

290815-26-8 CAS

PATENTS

1. WO2000052007A1

2. WO 2004078104

3. WO 2005113543

4. WO 2007031501

5. WO 2008077916

6. Channels and transporters. Mini-symposium of the Division of Medicinal Chemistry (DMC) of the Swiss Chemical Society (SCS) at the Department of Chemistry, University of Basel, May 27, 2010.

Dutzler R, Ernstb B, Hediger MA, Keppler D, Mohr P, Neidhart W, Märki HP.Chimia (Aarau). 2010;64(9):662-6.

………………………

INTRODUCTION

5-methyl-pyridine-2-sulfonic acid [6-methoxy-5-(2-methoxy-phenoxy)-2-pyridin-4-yl-pyrimidin-4-yl]-amide corresponding to the formula

is an inhibitor of endothelin receptors. WO00/52007 describes the preparation of said compound which is crystallized from Me2Cl2.
Own investigations have shown that there exist two distinct crystalline forms, hereinafter referred to as form A and form B, as well as a number of further solvates, in particular the methanol, ethanol, isopropanol, dichloromethane, acetone, methyl ethyl ketone and tetrahydrofuran solvates.
It was further surprisingly found that the thermodynamically stable crystalline form – form B – can be prepared under controlled conditions and that said form B can be prepared with a reliable method in an industrial scale, which is easy to handle and to process in the manufacture and preparation of formulations.

………………..

US20020137933

4,6-Dichloro-5-(2-methoxy-phenoxy)-2-(pyridin-4-yl)-pyrimidine (described in EP 0 799 209) can be transformed to the intermediate of formula (III)—according to scheme 1—on reaction with an appropriate sulfonamide of formula (II), wherein R¹is as defined in claim 1, in a suited solvent such as DMSO or DMF at room temperature or at elevated temperature and in the presence of a suited base such as potassium carbonate.

EXAMPLE 1

[0064] a) To a solution of 6.9 g sodium in MeOH (300 ml) were added 14.52 g of 5-methyl-pyridine-2-sulfonic acid [6-chloro-5-(2-methoxy-phenoxy)-2-pyridin-4-yl-pyrimidin-4-yl]-amide at RT and the mixture was refluxed for 5 days until completion of the reaction according to TLC analysis. The reaction mixture was concentrated in vacuo to half its volume upon which the crude reaction product precipitated as a sodium salt. It was filtered off by suction and dried in a high vacuum. The solid was dissolved in water, which was then made acidic by addition of acetic acid. The precipitating free sulfonamide was extracted into Me₂Cl₂. The organic layer was dried over Mg₂SO₄, concentrated on a rotary evaporator, and the crystalline solid that had formed was filtered off. It was then dried in a high vacuum for 12 h at 120° C. to give the desired 5-methyl-pyridine-2-sulfonic acid [6-methoxy-5-(2-methoxy-phenoxy)-2-pyridin-4-yl-pyrimidin-4-yl]-amide as white crystals. Melting point 225-226° C. ISN mass spectrum, m/e 478.2 (M-1 calculated for C₂₃H₂₁N₅O₅S₁: 478).

[0065] C₂₃H₂₁N₅O₅S₁: Calc: C 57.61; H 4.41; N 14.61; S 6.69. Found: C 57.56; H 4.38; N 14.61; S 6.83

[0066] Preparation of the starting material:

[0067] b) 11.3 g of 4,6-dichloro-5-(2-methoxy-phenoxy)-2-pyridin-4-yl)-pyrimidine and 19.66 g of 5-methylpyridyl-2-sulfonamide potassium salt (preparations described in EP 0 799 209) were dissolved in DMF (255 ml) under argon. The solution was stirred for 2 h at 40° C. until completion of the reaction according to TLC analysis. The reaction mixture was cooled to RT and the solvent removed in a high vacuum. The residue was suspended in water (850 ml), acetic acid (85 ml) was added and the mixture was stirred for 30 minutes at RT. The solid that precipitated was collected by filtration and dried in a high vacuum at 60° C. for 16 h to give 5-methyl-pyridine-2-sulfonic acid [6-chloro-5-(2-methoxy-phenoxy)-2-pyridin-4-yl-pyrimidin-4-yl]-amide ( CHLORO STARTING MATERIAL) as yellow crystals. Melting point 177-179° C. ISN mass spectrum, m/e 482.2 (M-1 calculated for C₂₂H₁₈ClN₅O₅S₁: 482).

……………………………….

http://www.google.com/patents/US6417360

EXAMPLE 1

a) To a solution of 6.9 g sodium in MeOH (300 ml) were added 14.52 g of 5-methyl-pyridine-2-sulfonic acid [6-chloro-5-(2-methoxy-phenoxy)-2-pyridin-4-yl-pyrimidin-4-yl]-amide at RT and the mixture was refluxed for 5 days until completion of the reaction according to TLC analysis. The reaction mixture was concentrated in vacuo to half its volume upon which the crude reaction product precipitated as a sodium salt. It was filtered off by suction and dried in a high vacuum. The solid was dissolved in water, which was then made acidic by addition of acetic acid. The precipitating free sulfonamide was extracted into Me₂Cl₂. The organic layer was dried over Mg₂SO₄, concentrated on a rotary evaporator, and the crystalline solid that had formed was filtered off. It was then dried in a high vacuum for 12 h at 120° C. to give the desired 5-methyl-pyridine-2-sulfonic acid [6-methoxy-5-(2-methoxy-phenoxy)-2-pyridin-4-yl-pyrimidin-4-yl]-amide as white crystals. Melting point 225-226° C. ISN mass spectrum, m/e 478.2 (M-1 calculated for C₂₃H₂₁N₅O₅S₁: 478).

C₂₃H₂₁N₅O₅S₁: Calc: C 57.61; H 4.41; N 14.61; S 6.69. Found: C 57.56; H 4.38; N 14.61; S 6.83

Preparation of the starting material:

b) 11.3 g of 4,6-dichloro-5-(2-methoxy-phenoxy)-2-pyridin-4-yl)-pyrimidine and 19.66 g of 5-methylpyridyl-2-sulfonamide potassium salt (preparations described in EP 0 799 209) were dissolved in DMF (255 ml) under argon. The solution was stirred for 2 h at 40° C. until completion of the reaction according to TLC analysis. The reaction mixture was cooled to RT and the solvent removed in a high vacuum. The residue was suspended in water (850 ml), acetic acid (85 ml) was added and the mixture was stirred for 30 minutes at RT. The solid that precipitated was collected by filtration and dried in a high vacuum at 60° C. for 16 h to give 5-methyl-pyridine-2-sulfonic acid [6-chloro-5-(2-methoxy-phenoxy)-2-pyridin-4-yl-pyrimidin-4-yl]-amide as yellow crystals. Melting point 177-179° C. ISN mass spectrum, m/e 482.2 (M-1 calculated for C₂₂H₁₈ClN₅O₅S₁: 482).

…………………….

http://www.google.com/patents/EP0799209B1

SYNTHESIS OF

4,6-dichloro-5-(2-methoxy-phenoxy)-2-pyridin-4-yl)-pyrimidine

A BASIC STARTING MATERIAL FOR AVOSENTAN

- b) 53.1 g of 4-cyano-pyridine (98%) are added all at once to a solution of 1.15 g of sodium in 200 ml of abs. MeOH. After 6 hours 29.5 g of NH₄Cl are added while stirring vigorously. The mixture is stirred at room temperature overnight. 600 ml of ether are added thereto, whereupon the precipitate is filtered off under suction and thereafter dried at 50°C under reduced pressure. There is thus obtained 4-amidino-pyridine hydrochloride (decomposition point 245-247°C).
- c) 112.9 g of diethyl (2-methoxyphenoxy)malonate are added dropwise within 30 minutes to a solution of 27.60 g of sodium in 400 ml of MeOH. Thereafter, 74.86 g of the amidine hydrochloride obtained in b) are added all at once. The mixture is stirred at room temperature overnight and evaporated at 50°C under reduced pressure. The residue is treated with 500 ml of ether and filtered off under suction. The filter cake is dissolved in 1000 ml of H₂O and treated little by little with 50 ml of CH₃COOH. The precipitate is filtered off under suction, washed with 400 ml of H₂O and dried at 80°C under reduced pressure. There is thus obtained 5-(2-methoxy-phenoxy)-2-(pyridin-4-yl)-pyrimidine-4,6-diol (or tautomer), melting point above 250°C.
- d) A suspension of 154.6 g of 5-(2-methoxy-phenoxy)-2-(pyridin-4-yl)-pyrimidine-4,6-diol (or tautomer) in 280 ml of POCl₃ is heated at 120°C in an oil bath for 24 hours while stirring vigorously. The reaction mixture changes gradually into a dark brown liquid which is evaporated under reduced pressure and thereafter taken up three times with 500 ml of toluene and evaporated. The residue is dissolved in 1000 ml of CH₂Cl₂, treated with ice and H₂O and thereafter adjusted with 3N NaOH until the aqueous phase has pH 8. The organic phase is separated and the aqueous phase is extracted twice with CH₂Cl₂. The combined CH₂Cl₂ extracts are dried with MgSO₄, evaporated to half of the volume, treated with 1000 ml of acetone and the CH₂Cl₂remaining is distilled off at normal pressure. After standing in a refrigerator for 2 hours the crystals are filtered off under suction and dried at 50°C overnight. There is thus obtained 4,6-dichloro-5-(2-methoxy-phenoxy)-2-pyridin-4-yl)-pyrimidine, melting point 178-180°C.

…………………………

http://www.google.com/patents/WO2000052007A1

Preparation of the starting material:

5-methyl-pyridine-2-sulfonic acid [6-chloro-5-(2- methoxy-phenoxy)-2-pyridin-4-yl-pyrimidin-4-yl] -amide IE THE 6 CHLORO COMPD

b) 11.3 g of 4,6-dichloro-5-(2-methoxy-phenoxy)-2-pyridin-4-yl)-pyrimidine and 1 .66 g of 5-methylpyridyl-2-sulfonamide potassium salt (preparations described in EP 0 799 209) were dissolved in DMF (255 ml) under argon. The solution was stirred for 2 h at 40°C until completion of the reaction according to TLC analysis. The reaction mixture was cooled to RT and the solvent removed in a high vacuum. The residue was suspended in water (850 ml), acetic acid (85 ml) was added and the mixture was stirred for 30 minutes at RT. The solid that precipitated was collected by filtration and dried in a high vacuum at 60 °C for 16 h to give 5-methyl-pyridine-2-sulfonic acid [6-chloro-5-(2- methoxy-phenoxy)-2-pyridin-4-yl-pyrimidin-4-yl] -amide as yellow crystals. Melting point 177-179 °C. ISN mass spectrum, m/e 482.2 (M-l calculated for C₂₂Hi₈ClN₅O₅Sι: 482).

………………………………………………………………………………………….

Example 1AVOSENTAN

a) To a solution of 6.9 g sodium in MeOH (300 ml) were added 14.52 g of

5-methyl-pyridine-2-sulfonic acid [6-chloro-5-(2-methoxy-phenoxy)-2-pyridin-4-yl- pyrimidin-4-yl] -amide at RT and the mixture was refluxed for 5 days until completion of the reaction according to TLC analysis. The reaction mixture was concentrated in vacuo to half its volume upon which the crude reaction product precipitated as a sodium salt. It was filtered off by suction and dried in a high vacuum. The solid was dissolved in water, which was then made acidic by addition of acetic acid. The precipitating free sulfonamide was extracted into Me₂Cl₂. The organic layer was dried over Mg SO , concentrated on a rotary evaporator, and the crystalline solid that had formed was filtered off. It was then dried in a high vacuum for 12 h at 120 °C to give the desired 5-methyl-pyridine-2-sulfonic acid [6- methoxy-5-(2-methoxy-phenoxy)-2-pyridin-4-yl-pyrimidin-4-yl] -amide as white crystals. Melting point 225-226 °C. ISN mass spectrum, m/e 478.2 (M-l calculated for

C₂₃H₂₁N₅O₅S₁: Calc: C 57.61; H 4.41; N 14.61; S 6.69. Found: C 57.56; H 4.38; N 14.61; S 6.83

…………………………………………….

IS DESCRIBED IN

http://www.google.com/patents/EP2331513A1?cl=en

ALSO

Diabetic nephropathy is the principle cause of end stage renal disease in the western world. It is a major cause of morbidity and mortality in Type-I Diabetes, but is an increasing problem in Type-II Diabetes and because the incidence of this is five times that of Type-I Diabetes, it contributes at least 50% of diabetics with end stage renal disease.
The initial stage of subtle morphologic changes in the renal glomeruli is followed by microalbuminuria. This is associated with a modestly rising blood pressure and an increased incidence of cardiovascular disease. There follows a continued increase in urinary protein excretion and declining glomerular filtration rate. Diabetic nephropathy has many possible underlying pathophysiological causes including metabolic, glycosylation of proteins, haemodynamics, altered flow/pressure in glomeruli, the development of hypertension and cytokine production; all of these are associated with the development of extracellular matrix and increased vascular permeability leading to glomerular damage and proteinuria.

WO2005113543A1 *	May 12, 2005	Dec 1, 2005	Alexander Bilz	Crystalline forms of a pyridinyl-sulfonamide and their use as endothelin receptor antagonists
WO2007031501A2 *	Sep 11, 2006	Mar 22, 2007	Speedel Pharma Ag	Pyridylsulfonamidyl-pyrimidines for the prevention of blood vessel graft failure
WO2008077916A1 *	Dec 21, 2007	Jul 3, 2008	Ovidiu Baltatu	Pharmaceutical composition using aliskiren and avosentan
EP1454625A1 *	Mar 6, 2003	Sep 8, 2004	Speedel Development AG	Pyridylsulfonamidyl-pyrimidines for the treatment of diabetic nephropathies
EP1595880A1 *	May 13, 2004	Nov 16, 2005	Speedel Pharma AG	Crystalline forms of a pyridinyl-sulfonamide and their use as endothelin receptor antagonists
EP1938812A1 *	Dec 22, 2006	Jul 2, 2008	Speedel Pharma AG	Pharmaceutical composition using aliskiren and avosentan
US6951856	Jul 10, 2001	Oct 4, 2005	Actelion Pharmaceuticals Ltd.	Arylethene-sulfonamides
US7402587	May 12, 2005	Jul 22, 2008	Speedel Pharma Ag	Crystalline forms of a pyridinyl-sulfonamide and their use as endothelin receptor antagonists

WO1996019459A1 *	Dec 8, 1995	Jun 27, 1996	Volker Breu	Novel sulfonamides
EP0713875A1 *	Nov 13, 1995	May 29, 1996	F. Hoffmann-La Roche AG	Sulfonamides
EP0897914A1 *	Aug 10, 1998	Feb 24, 1999	F. Hoffmann-La Roche Ag	Process for the preparation of 2,5-disubstitued pyridines

	DR ANTHONY MELVIN CR… on Tozinameran, Pfizer–BioNTech C…
	DR ANTHONY MELVIN CR… on Evinacumab
	DR ANTHONY MELVIN CR… on Tozinameran, Pfizer–BioNTech C…
	DR ANTHONY MELVIN CR… on Lurbinectedin
	DR ANTHONY MELVIN CR… on AZITHROMYCIN, アジスロマイシン;

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Minisci reactions: Versatile CH-functionalizations for medicinal chemists

Introduction

Mechanism

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NERATINIB MALEATE

(E)-N-{4-[3-Chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide (25o).

This compound was prepared as a yellow solid (0.86 g, 85%) by the method described for 25g using 0.65 g (1.81 mmol) of 23 and 0.42 g (3.62 mmol) of 3-chloro-4-(2-pyridinylmethoxy)aniline:

HRMS (ES+) m/z 557.205 89 (M + H)+1, Δ = −0.36 mmu;

1H NMR (DMSO-d6) δ 9.62 (s, 1H), 9.49 (s, 1H), 8.96 (s, 1H),

8.60 (d, 1H, J = 3.9 Hz), 8.47 (s, 1H),

7.88 (t, 1H, J = 3.9 Hz), 7.58 (d, 1H, J = 3.9 Hz),

7.39−7.35 (m, 3H), 7.26 (d, 1H, J = 7.8 Hz),

7.19 (d, 1H, J = 8.1 Hz), 6.81−6.73 (m, 1H),

6.59 (d, 1H, J = 7.8 Hz), 5.28 (s, 2H),

4.30 (q, 2H, J = 6.9 Hz),

3.07 (d, 2H, J = 3.9 Hz),

2.17 (s, 6H),

1.46 (t, 3H, J = 3.9 Hz).

Anal. (C30H29ClN6O3·1.1H2O) C, H, N.

INTERPRETATION

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Taigexyn Granted QIDP and Fast Track Designations

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The Ley Synthesis of Rapamycin

Angewandte Chemie International Edition

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HRMS (ES+) m/z 557.205 89 (M + H)⁺¹, Δ = −0.36 mmu;

¹H NMR (DMSO-d₆) δ 9.62 (s, 1H), 9.49 (s, 1H), 8.96 (s, 1H),

Anal. (C₃₀H₂₉ClN₆O₃·1.1H₂O) C, H, N.