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DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with GLENMARK PHARMACEUTICALS LTD, Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 29 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international, etc in organic chemistry are some most read blogs He has hands on experience in initiation and developing novel routes for drug molecules and implementation them on commercial scale over a 29 year tenure till date Aug 2016, Around 30 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 9 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 25 Lakh plus views on dozen plus blogs, He makes himself available to all, contact him on +91 9323115463, email amcrasto@gmail.com, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 13 lakh plus views on New Drug Approvals Blog in 212 countries......https://newdrugapprovals.wordpress.com/ , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc

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Commercial Production of Semi-Synthetic Artemisinin


STR1

Figure 1. Production of artemisinic acid or β-farnesene by engineered yeast. The sesquiterpene alkenes β-farnesene and amorphadiene are both derived from FPP (farnesyl diphosphate) by the action of specific enzymes introduced from plants: amorphadiene synthase (ADS) generates amorphadiene and β-farnesene synthase (FS) generates β-farnesene. Production strains express either ADS or FS, not both. Oxidation of amorphadiene to artemisinic acid is accomplished by the action of five plant enzymes expressed in the engineered yeast.17 Conversion of purified artemisinic acid to artemisinin is accomplished by in vitro organic chemistry. Isoprenoid production strains make little ethanol.

 

The antimalarial drug artemisinin and the specialty chemical β-farnesene are examples of natural product isoprenoids that can help solve global challenges, but whose usage has previously been limited by supply and cost impediments. This review describes the path to commercial production of these compounds utilizing fermentation of engineered yeast. Development of commercially viable yeast strains was a substantial challenge that was addressed by creation and implementation of an industrial synthetic biology pipeline. Using the engineered strains, production of β-farnesene from Brazilian sugarcane offers several environmental advantages. Among the many commercial applications of β-farnesene, its use as a feedstock for making biodegradable lubricants is highlighted. This example, along with others, highlight a powerful new suite of technologies that will become increasingly important for production of chemicals, spanning from pharmaceuticals through commodity chemicals.

 

STR1

Figure 2. Sanofi industrial semi-synthesis of artemisinin. The process starts with a moderate pressure catalytic diastereoselective hydrogenation of artemisinic acid to produce a high (95:5) ratio of the desired (R)-isomer. To avoid formation of a lactone byproduct, dihydro-epi-deoxyarteannuin B, during the photooxidation, the carboxylic acid is protected as a mixed anhydride. The final step combines formation of the intermediate hydroperoxide via photoxidation using a Hg vapor lamp and commercially available tetraphenylporphyin (TPP) as sensitizer with a Hock cleavage and rearrangement catalyzed by trifluoroacetic acid to give, after workup, the best yield reported to date of pure isolated artemisinin (55%).

Synthetic Biology and the Development of Commercial β-Farnesene Production Strains Semi-synthetic artemisinin is a pharmaceutical with a price point comparable to plant-derived artemisinin,20 namely above $150 per kg. β-Farnesene, however, is a specialty chemical with multiple uses (more details below); most specialty and commodity chemicals have significantly lower price points, often below $10 per kg. For these product categories, it is of paramount importance that fermentative production be as efficient as possible, with high yields (namely, grams of product made per gram of feed substrate), productivities (grams of product/liter of culture/hour) and concentration (also known as titer; grams of product per liter of culture). Developing yeast strains capable of the yield, productivity and titer required for chemical production requires extensive development, and has been enabled over the last decade by the new discipline of synthetic biology. Synthetic biology seeks to extend approaches and concepts from engineering and computation to redesign biology for a chosen function;21recent advances in the application of design automation, i.e., the use of software, hardware and robotics22 have enabled the creation and screening of hundreds of thousands of strain variants (created by both design and random mutagenesis) for the properties required for commercial production of β-farnesene. Notable enabling technologies developed for routine usage include rapid and reliable assembly of large (i.e., multiple kilobase) deoxyribonucleic acid (DNA) constructs;23-25 high throughput, cost effective, verification of structural DNA assemblies by both initial restriction digest26 and by low-cost DNA sequencing;27 and whole genome sequencing of yeast strains.28 In addition, there is a need to effectively identify the best new strains (akin to panning for gold!) through high throughput, rapid, and accurate methods to screen thousands of strains. Further, the results of small-scale (< 1 milliliter) tests must correspond to the results of large-scale (> 50,000 liter) production. Development and implementation of these technologies required considerable investment by Amyris. The outcome is a robust pipeline for efficient, cost-effective strain generation allied with screening for the properties required for commercial production of β-farnesene by fermentation (i.e., at a price point required for its use as a specialty chemical).

 

As the world’s population and economies grow, the demand for a wide variety of specialty, commodity, and pharmaceutical chemicals will outpace the supply available from current sources. There is an urgent need to develop alternative, sustainable sources of many existing chemicals and to develop abundant sources of currently scarce chemicals with novel beneficial properties. Synthetic biology and industrial fermentation, combined with synthetic chemistry, will be an increasingly important source of chemicals in the decades ahead; artemisinin and β-farnesene provide good examples of this relatively new approach to chemical production. Brazil’s plentiful sugar cane feedstock and fermentation expertise make it an excellent location for this type of manufacturing, which can expand and diversify the nation’s industrial base and international importance.

J. Braz. Chem. Soc. 2016, 27(8), 1339-1345

Developing Commercial Production of Semi-Synthetic Artemisinin, and of β-Farnesene, an Isoprenoid Produced by Fermentation of Brazilian Sugar

Kirsten R. Benjamin; Iris R. Silva; João P. Cherubim; Derek McPhee; Chris J. Paddon

How to cite this article

Genes encoding the biosynthetic pathway for production of a valuable product (e.g., farnesene) in a native organism are expressed in a heterologous microbial host (e.g., yeast). The engineered yeast produces farnesene by commercial fermentation. Copyright © 2016 Amyris, inc. All rights reserved.

http://dx.doi.org/10.5935/0103-5053.20160119

http://jbcs.sbq.org.br/imagebank/pdf/v27n8a04.pdf

Benjamin KR, Silva IR, Cherubim JP, Mcphee D, Paddon CJ. Developing Commercial Production of Semi-Synthetic Artemisinin, and of β-Farnesene, an Isoprenoid Produced by Fermentation of Brazilian Sugar. J. Braz. Chem. Soc. 2016;27(8):1339-1345

Kirsten R. Benjamin,a Iris R. Silva,b João P. Cherubim,c Derek McPheea and Chris J. Paddon*,a a Amyris, Inc., 5885 Hollis Street, Suite 100, CA 94608 Emeryville, USA b Amyris Brasil Ltda, Rua John Dalton 301-Bloco B-Edificio 3, Condominio Techno Plaza, 13069-330 Campinas-SP, Brazil c Amyris Brasil Ltda, Rodovia Brotas/Torrinha-km 7.5, 17380-000 Brotas-SP, Brazil

*e-mail: paddon@amyris.com
Chris Paddon

Chris Paddon, PhD

Dr. Paddon has a PhD in Biochemistry from Imperial College, London, but now considers himself a synthetic biologist. After postdoctoral work at the National Institutes of Health in Bethesda, MD, he worked in the pharmaceutical industry (GlaxoSmithKline), and then for two Bay Area biopharmaceutical companies (Affymax and Xenoport) before joining Amyris, Inc. in 2005 as its sixth employee and first scientist. He was project leader for the semi-synthetic artemisinin project at Amyris, Inc. and has subsequently led a number of other projects and programs there.

Chris Paddon is a Principal Scientist at Amyris, Inc. in Emeryville, CA. He was project leader for the Semi-Synthetic Artemisinin project, and subsequently led a number of projects at Amyris using synthetic biology for the production of natural products. He received his Bachelor’s degree in Microbiology from The University of Surrey (UK), and doctorate in Biochemistry from Imperial College (London, UK). Following postdoctoral work at The National Institutes for Health (Bethesda, MD) he joined the pharmaceutical industry, working for GSK (London, UK). He subsequently worked for Affymax (Palo Alto, CA) and Xenoport (Santa Clara, CA) before joining Amyris.

//////////// Commercial Production, Semi-Synthetic , Artemisinin,  farnesene, fermentation, natural product, lubricant

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UCT Drug Discovery and Development Centre, H3D, pioneers world-class drug discovery in Africa.


H3D

UCT’s H3D is a center of excellence for research and innovation with an already strong track record in malaria drug  discovery. The vision of H3D is to be the leading organization for integrated drug discovery and development on the African continent.

ABOUT H3D

H3D is Africa’s first integrated drug discovery and development centre. The Centre was founded at the University of Cape Town in April 2011 and pioneers world-class drug discovery in Africa.

Our Vision

To be the leading organisation for integrated drug discovery and development from Africa, addressing global unmet medical needs.

Our Mission

To discover and develop innovative medicines for unmet medical needs on the African continent and beyond, by performing state-of-the-art research and development and bridging the gap between basic science and clinical studies.

We embrace partnerships with local and international governments, pharmaceutical companies, academia, and the private sector, as well as not-for-profit and philanthropic organisations, while  training scientists to be world experts in the field.

The H3D collaboration with the Medicines for Malaria Venture (MMV) focuses on delivering potential agents against malaria that will be affordable and safe to use. In line with the global aim to eradicate malaria, projects are pursued that not only eliminates blood-stage Plasmodium falciparum and Plasmodium vivax infection, but also acts against liver stages and blocks transmission of the infection. The projects embrace multidisciplinary activities to optimise hit compounds from screening libraries through the drug discovery pipeline and deliver clinical candidates.

Merck Serono Announces Recipients of the Second Annual €1 Million Grant for Multiple Sclerosis Innovation

Darmstadt, Germany, September 12, 2014 – Merck Serono, the biopharmaceutical division of Merck, today announced the recipients of the second annual Grant for Multiple Sclerosis Innovation (GMSI) at MS Boston 2014, the joint meeting of the Americas Committee for Treatment and Research in MS (ACTRIMS) and European Committee for Treatment and Research in MS (ECTRIMS), taking place September 10-13 in Boston, U.S.A.

Merck signed a research agreement with the University of Cape Town (UCT), South Africa, to co-develop a new R&D platform. It aims at identifying new lead programs for potential treatments against malaria, with the potential to expand it to other tropical diseases. It combines Merck’s R&D expertise and the drug discovery capabilities of the UCT Drug Discovery and Development Centre, H3D.
UCT’s H3D is a center of excellence for research and innovation with an already strong track record in malaria drug  discovery. The vision of H3D is to be the leading organization for integrated drug discovery and development on the African continent. They say that working with partners like Merck is critical to build up a comprehensive pipeline to tackle malaria and related infectious diseases.

Journal Publications:

  1. Aminopyrazolo[1,5-a]pyrimidines as potential inhibitors of Mycobacterium tuberculosis: Structure activity relationships and ADME characterization C. Soares de Melo, T-S. Feng, R. van der Westhuyzen, R.K. Gessner, L. Street, G. Morgans, D. Warner, A. Moosa, K. Naran, N. Lawrence, H. Boshoff, C. Barry, C. Harris, R. Gordon, K. Chibale. Biorg. Med. Chem. 2015, 23, 7240-7250.
  2. A Novel Pyrazolopyridine with in Vivo Activity in Plasmodium berghei- and Plasmodium falciparum- Infected Mouse Models from Structure−Activity Relationship Studies around the Core of Recently Identified Antimalarial Imidazopyridazines. C. Le Manach, T. Paquet, C. Brunschwig, M. Njoroge, Z. Han, D. Gonzàlez Cabrera, S. Bashyam, R. Dhinakaran, D. Taylor, J. Reader, M. Botha, A. Churchyard, S. Lauterbach, T. Coetzer, L-M. Birkholtz, S. Meister, E. Winzeler, D. Waterson, M. Witty, S. Wittlin, M-B. Jiménez-Díaz, M. Santos Martínez, S. Ferrer, I. Angulo-Barturen, L. Street, and K. Chibale, J. Med. Chem. 2015, XX, XXXX
  3. Structure−Activity Relationship Studies of Orally Active Antimalarial 2,4-Diamino-thienopyrimidines. D. Gonzàlez Cabrera, F. Douelle, C. Le Manach, Z. Han, T. Paquet, D. Taylor, M. Njoroge, N. Lawrence, L. Wiesner, D. Waterson, M. Witty, S. Wittlin, L. Street and K. Chibale. J Med Chem. 2015, 58, 7572-7579.
  4. Medicinal Chemistry Optimization of Antiplasmodial Imidazopyridazine Hits from High Throughput Screening of a SoftFocus Kinase Library: Part 2. Le Manach, T. Paquet, D. Gonzalez Cabrera, Y. Younis, D. Taylor, L. Wiesner, N. Lawrence, S. Schwager, D. Waterson, M.J. Witty, S. Wittlin, L. Street, and K. Chibale. J. Med. Chem. 2014, 57, 8839−8848.
  5. Medicinal Chemistry Optimization of Antiplasmodial Imidazopyridazine Hits from High Throughput Screening of a SoftFocus Kinase Library: Part 1. Le Manach, D. González Cabrera, F. Douelle, A.T. Nchinda, Y. Younis, D. Taylor, L. Wiesner, K. White, E. Ryan, C. March, S. Duffy, V. Avery, D. Waterson, M. J. Witty, S. Wittlin; S. Charman, L. Street, and K. Chibale. J. Med. Chem. 2014, 57, 2789-2798.
  6. 2,4-Diamino-thienopyrimidines as Orally Active Antimalarial Agents. D. González Cabrera, C. Le Manach, F. Douelle, Y. Younis, T.-S. Feng, T. Paquet, A.T. Nchinda, L.J. Street, D. Taylor, C. de Kock, L. Wiesner, S. Duffy, K.L. White, K.M. Zabiulla, Y. Sambandan, S. Bashyam, D. Waterson, M.J. Witty, A. Charman, V.M. Avery, S. Wittlin, and K. Chibale. J. Med. Chem. 2014,57, 1014-1022.
  7. Effects of a domain-selective ACE inhibitor in a mouse model of chronic angiotensin II-dependent hypertension. Burger, T.L. Reudelhuber, A. Mahajan, K. Chibale,E.D. Sturrock, R.M. Touyz. Clin. Sci. (Lond). 2014, 127(1), 57-63.
  8. Pharmacokinetic evaluation of lisinopril-tryptophan, a novel C-domain ACE inhibitor. Denti, S.K. Sharp, W.L. Kröger, S.L. Schwager, A. Mahajan, M. Njoroge, L. Gibhard, I. Smit, K. Chibale, L. Wiesner, E.D. Sturrock, N.H. Davies. Eur. J. Pharm. Sci.2014, 56, 113-119.
  9. Fragment-based design for the development of N-domain-selective angiotensin-1-converting enzyme inhibitors. R.G. Douglas, R.K. Sharma, G. Masuyer, L. Lubbe, I. Zamora, K.R. Acharya, K. Chibale, E.D. Sturrock. Sci. (Lond). 2014, 126(4),305-313.
  10. Fast in vitro methods to determine the speed of action and the stage-specificity of anti-malarials in Plasmodium falciparum. Le Manach, C. Scheurer, S. Sax, S. Schleiferböck, D. González Cabrera, Y. Younis, T. Paquet, L. Street, P.J. Smith, X. Ding, D. Waterson, M.J. Witty, D. Leroy, K. Chibale and S. Wittlin*. Malaria Journal, 2013, 12, 424.
  11. Structure-Activity-Relationship Studies Around the 2-Amino Group and Pyridine Core of Antimalarial 3,5-Diarylaminopyridines Lead to a Novel Series of Pyrazine Analogues with Oral in vivo Activity. Y. Younis, F. Douelle, González Cabrera, C. Le Manach, A.T. Nchinda, T. Paquet, L.J. Street, K.L. White, K. M. Zabiulla, J.T. Joseph,  S. Bashyam, D. Waterson, M.J. Witty, S. Wittlin, S.A. Charman, and K. Chibale*   J. Med. Chem. 2013, 56, 8860−8871.
  12. Cell-based Medicinal Chemistry Optimization of High Throughput Screening (HTS) Hits for Orally Active Antimalarials-Part 2: Hits from SoftFocus Kinase and other Libraries. Y. Younis, L. J. Street, D. Waterson, M.J. Witty, and K. Chibale. J. Med. Chem. 2013, 56, 7750−7754.
  13. Structure-Activity Relationship Studies of Orally active Antimalarial 3,5-Substituted 2-Aminopyridines. D. González Cabrera, F. Douelle, Y. Younis, T.-S. Feng, C. Le Manach, A.T. Nchinda, L.J. Street, C. Scheurer, J. Kamber, K. White, O. Montagnat, E. Ryan, K. Katneni, K.M. Zabiulla, J. Joseph, S. Bashyam, D. Waterson, M.J. Witty, S. Charman, S. Wittlin, and K. Chibale* J. Med. Chem. 2012, 55, 11022– 11030.
  14. 3,5-Diaryl-2-aminopyridines as a Novel Class of Orally Active Antimalarials Demonstrating Single Dose Cure in Mice and Clinical Candidate Potential. Y. Younis, F. Douelle, T.-S. Feng, D. González Cabrera, C. Le Manach, A.T. Nchinda, S. Duffy, K.L. White, M. Shackleford,  J. Morizzi, J. Mannila, K. Katneni, R. Bhamidipati, K. M. Zabiulla, J.T. Joseph,  S. Bashyam, D. Waterson, M.J. Witty, D. Hardick, S. Wittlin, V. Avery, S.A. Charman, and K. Chibale*.  J. Med. Chem.  2012, 55, 3479−3487.
  15. Novel Orally Active Antimalarial Thiazoles. D. González Cabrera, F. Douelle, T.-S Feng, A.T. Nchinda, Y. Younis, K.L. White, Wu,E. Ryan, J.N. Burrows,D. Waterson, M.J. Witty,S. Wittlin,S.A. Charman and K. Chibale.  J. Med. Chem. 2011, 54, 7713–7719.
  16. Synthesis and molecular modeling of a lisinopril-tryptophan analogue inhibitor of angiotensin I-converting enzyme. A.T. Nchinda, K. Chibale, P. Redelinghuys and E.D. Sturrock. Med. Chem. Lett. 2006, 16(17), 4616-4619.

Patents

  1. Anti-Malarial Agents. Y. Younis, K. Chibale, M.J. Witty, D. Waterson. (2016) US9266842 B2.
  2. New Anti-Malarial Agents. D. Waterson, M.J. Witty, K. Chibale, L. Street, D. González Cabrera, T. Paquet. EP patent application (2015), No. 15 176 514.6.
  3. Preparation of aminopyrazine compounds as antimalarial agents for treatment of malaria. Y. Younis, K. Chibale, M.J. Witty, D. Waterson. PCT Int Appl. (2013), WO 2013121387 A1 20130822.
  4. Preparation of peptides as angiotensin I-converting enzyme (ACE) inhibitors. E.D. Sturrock, A.T. Nchinda, K. Chibale. PCT Int. ppl. (2006), WO 2006126087 A2 20061130.
  5. Preparation of peptides as angiotensin I-converting enzyme (ACE) inhibitors, E.D. Sturrock, A.T. Nchinda, K. Chibale. PCT Int. ppl. (2006), WO 2006126086 A2 20061130.

Head Office, Medicinal Chemistry Unit

Physical Address:
Department of Chemistry
7.32 H3D Lab Suite, PD Hahn Building, Level 7
North Lane off Ring Road
Upper Campus, University of Cape Town
Rondebosch, 7700, South Africa

T | 021 650 5495
F | 021 650 5195

Postal Address:
University of Cape Town
Private Bag X3
Rondebosch 7701
South Africa

P. D. Hahn Bldg, Rondebosch, Cape Town,
Map of P. D. Hahn Bldg, Rondebosch, Cape Town, 7700, South Africa
P. D. Hahn Bldg, Rondebosch, Cape Town, 7700, South Africa

//////H3D, Africa,  integrated drug discovery and development centre,  University of Cape Town 

Recovery of Artemisinin from a Complex Reaction Mixture Using Continuous Chromatography and Crystallization


Figure

Figure

Recovery of Artemisinin from a Complex Reaction Mixture Using Continuous Chromatography and Crystallization

Articles ASAP (As Soon As Publishable)
Publication Date (Web): May 8, 2015 (Article)
DOI: 10.1021/acs.oprd.5b00048
*E-mail: seidel-morgenstern@mpi-magdeburg.mpg.de. Tel.: +49-(0)391-6110 401. Fax: +49-(0)391-6110 521.
Artemisinin, a secondary metabolite of sweet wormwood, is the basis for the production of the most effective antimalarial drugs. Since the amount of artemisinin currently produced from plants is not sufficient to treat the worldwide malaria cases, an effective semisynthetic method was developed recently that is capable of producing artemisinin from dihydroartemisinic acid (DHAA). DHAA is a byproduct obtained during the extraction of artemisinin from plant leaves. The photocatalytic reaction to convert DHAA to artemisinin can be performed continuously in a tubular reactor using toluene as a solvent. The reactor effluent contains besides artemisinin the photocatalyst (dicyanoanthracene) and several compounds that are structurally similar to artemisinin, including unreacted DHAA starting material. To isolate artemisinin from the reaction mixture, two separation techniques were applied, crystallization and chromatography. The solid obtained by seeded cooling crystallization was highly enriched in artemisinin but contained also traces of the photocatalyst. In contrast, using a variant of continuously operated multicolumn simulated moving bed (SMB) chromatography, which splits the feed into three fractions, we were able to recover efficiently the photocatalyst in the raffinate stream. The extract stream provided already almost pure artemisinin, which could be finally further purified in a simple crystallization step.
Magdeburg, Germany
Map of magdeburg germany
 
 
 
 

Arteflene


Arteflene
Arteflene
CAS : 123407-36-3 (Z-form)
 [1S-[1a,4b(Z),5a,8b]]-4-[2-[2,4-Bis(trifluoromethyl)phenyl]ethenyl]-4,8-dimethyl-2,3-dioxabicyclo[3.3.1]nonan-7-one
(1S,4R,5R,8S)-4-[(Z)-2,4-bis(trifluoromethyl)styryl]-4,8-dimethyl-2,3-dioxabicyclo[3.3.1]nonan-7-one
(1S,4R,5R,8S)-4-[(Z)-2,4-Bis(trifluoromethyl)styryl]-4,8-dimethyl-2,3-dioxabicyclo[3.3.1]nonan-7-one
Manufacturers’ Codes: Ro-42-1611
Properties: Crystalline stable material, mp 124°. Highly lipophilic, not sol in water. Stable in soln except in the presence of strong bases or strong reducing agents.
Melting point: mp 124°
Therap-Cat: Antimalarial
 
The oxidation of (5R)-(-)-carvone (I) with 3-chloroperbenzoic acid (3-CPB) in dichloromethane gives 5(R)-acetyl-2-methyl-2-cyclohexen-1-one (II), which is condensed with ethyltriphenylphosphonium bromide (III) by means of butyllithium in THF yielding 2-methyl-5(Z)-(1-methyl-1-propenyl)-2-cyclohexen-1-one (IV). The photochemical oxidation of (IV) in acetonitrile catalyzed by methylene blue affords (1R,4RS,5R,8S)-4,8-dimethyl-4-vinyl-2,3-dioxabicyclo[3.3.1]nonan-7-one (V), which is ozonolyzed with O3 in methanol to the corresponding aldehyde as a mixture of enantiomers, which is submitted to crystallization giving the (1S,4R,5R,8S)-isomer (VI). Finally, this compound is submitted to a Wittig condensation with 2,4-bis(trifluoromethyl)benzyltriphenylphosphonium bromide (VII) by means of sodium bis(trimethylsilyl)amide (NaBTSA) in dichloromethane.
……………………….
Literature References:
Synthetic sesquiterpene peroxide; structurally derived from the natural peroxides artemisinin, q.v. and yingzhaosu. Prepn: W. Hofheinz et al., EP 311955; eidem, US 4977184 (1989, 1990 both to Hoffmann-La Roche).
Series of articles on prepn, biological activities, pharmacokinetics and clinical evaluations: Trop. Med. Parasitol. 45, 261-291 (1994).

KAE 609, NITD 609, Cipargamin for Malaria


 

NITD609.svg
Cipargamin, NITD 609
IUPAC Name: (3R,3’S)-5,7′-dichloro-6′-fluoro-3′-methylspiro[1H-indole-3,1′-2,3,4,9-tetrahydropyrido[3,4-b]indole]-2-one |
CAS Registry Number: 1193314-23-6
Synonyms: NITD609, NITD 609, NITD-609, GNF-609
KAE-609
NITD-609  
 390.238, C19 H14 Cl2 F N3 O
(1’R,3’S)-5,7′-Dichloro-6′-fluoro-3′-methyl-1,2,2′,3′,4′,9′-hexahydrospiro[indole-3,1′-pyrido[3,4-b]indole]-2-one
(1R,3S)-5′,7-Dichloro-6-fluoro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-1,3′-indol]-2′(1′H)-one
CURRENTLY IN -PHASE2
NITD609 is an experimental synthetic antimalarial molecule belonging to the spiroindolone class.[1][2] The compound was developed at the Novartis Institute for Tropical Diseases in Singapore, through a collaboration with the Genomics Institute of the Novartis Research Foundation (GNF), the Biomedical Primate Research Centre and the Swiss Tropical Institute. NITD609 is a novel, synthetic antimalarial molecule belonging to the spiroindolone class, awarded MMV Project of the Year 2009.
It is structurally related to GNF 493, a compound first identified as a potent inhibitor of Plasmodium falciparum growth in a high throughput phenotypic screen of natural products conducted at the Genomics Institute of the Novartis Research Foundation in San Diego, California in 2006. NITD609 was discovered by screening the Novartis library of 12,000 natural products and synthetic compounds to find compounds active against Plasmodium falciparum. The first screen turned up 275 compounds and the list was narrowed to 17 potential candidates.
KAE609 (cipargamin; formerly NITD609, Novartis Institute for Tropical Diseases) is a new synthetic antimalarial spiroindolone analogue with potent, dose-dependent antimalarial activity against asexual and sexual stages of Plasmodium falciparum.http://www.nejm.org/doi/full/10.1056/NEJMoa1315860
ChemSpider 2D Image | cipargamin | C19H14Cl2FN3O

KAE609 shows promise as next generation treatment for malaria

http://www.novartis.com/newsroom/media-releases/en/2014/1843976.shtml

  • KAE609 is the first antimalarial drug candidate with a novel mechanism of action to achieve positive clinical proof-of-concept in over 20 years
  • KAE609 was tested in adult patients with uncomplicated malaria and showed a median parasite clearance time of 12 hours, including in patients with resistant infections[1]
  • For more than a decade, Novartis has been a leader in the fight against malaria, setting the current gold standard for treatment and building one of the strongest malaria pipelines in the industry

KAE609 shows promise as next generation treatment for malaria

  • KAE609 is the first antimalarial drug candidate with a novel mechanism of action to achieve positive clinical proof-of-concept in over 20 years
  • KAE609 was tested in adult patients with uncomplicated malaria and showed a median parasite clearance time of 12 hours, including in patients with resistant infections[1]
  • For more than a decade, Novartis has been a leader in the fight against malaria, setting the current gold standard for treatment and building one of the strongest malaria pipelines in the industry

The digital press release with multimedia content can be accessed here:

Basel, Switzerland, July 30, 2014 Today, Novartis published clinical trial results in the New England Journal of Medicine showing that KAE609 (cipargamin), a novel and potent antimalarial drug candidate, cleared the parasite rapidly in Plasmodium falciparum (P. falciparum) and Plasmodium vivax (P. vivax) uncomplicated malaria patients[1]. Novartis currently has two drug candidates in development. Both KAE609 and KAF156 are new classes of anti-malarial compounds that treat malaria in different ways from current therapies, important to combat emerging drug resistance. Novartis has also identified PI4K as a new drug target with potential to prevent, block and treat malaria.

“Novartis is in the fight against malaria for the long term and we are committed to the continued research and development of new therapies to eventually eliminate the disease,” said Joseph Jimenez, CEO of Novartis. “With two compounds and a new drug target currently under investigation, Novartis has one of the strongest malaria pipelines in the industry.”

Malaria is a life-threatening disease primarily caused by parasites (P. falciparum and P. vivax) transmitted to people through the bites of infected Anopheles mosquitoes. Each year it kills more than 600,000 people, most of them African children[2].

“KAE609 is a potential game-changing therapy in the fight against malaria,” said Thierry Diagana, Head of the Novartis Institute for Tropical Diseases (NITD), which aims to discover novel treatments and prevention methods for major tropical diseases. “Novartis has given KAE609 priority project status because of its unique potential of administering it as a single-dose combination therapy.”

In June 2012, 21 patients infected by one of the two main malaria-causing parasite types took part in a proof-of-concept clinical study conducted in Bangkok and Mae Sot near the Thailand/Burma border where resistance to current therapies had been reported. Researchers saw rapid parasite clearance in adult patients (median of 12 hours)[2] with uncomplicated P. vivax or P. falciparum malaria infection including those with resistant parasites. No safety concerns were identified, however the study was too small for any safety conclusions.

“The growing menace of artemisinin resistance threatens our current antimalarial treatments, and therefore our attempts to control and eliminate falciparum malaria,” said Nick White, Professor of Tropical Medicine at Mahidol University in Thailand and lead author of the NEJM article. “This is why we are so enthusiastic about KAE609; it is the first new antimalarial drug candidate with a completely novel mechanism of action to reach Phase 2 clinical development in over 20 years.”

KAE609, the first compound in the spiroindolone class of treatment, works through a novel mechanism of action that involves inhibition of a P-type cation-transporter ATPase4 (PfATP4), which regulates sodium concentration in the parasite. Because KAE609 also appears to be effective against the sexual forms of the parasite, it could potentially help prevent disease transmission. The clinical trial was done in collaboration with the Wellcome Trust-Mahidol University – Oxford Tropical Medicine Research Programme. Research was supported by the Wellcome Trust, Singapore Economic Development Board, and Medicines for Malaria Venture.

KAE609 represents one of two new classes of antimalarial compounds that Novartis has discovered and published in the last four years.[3],[4] This drug candidate has shown potent in vitro activity against a broad range of parasites that have developed drug resistance against current therapies. KAE609 is currently being planned for Phase 2b trials.

References
[1] http://www.nejm.org/doi/full/10.1056/NEJMoa1315860
[2] World Health Organization, http://www.who.int/mediacentre/factsheets/fs094/en/
[3] Spiroindolones, a Potent Compound Class for the Treatment of Malaria, KAE609, Science, Sept. 2010
[4] Imaging of Plasmodium liver stages to drive next generation antimalarial drug discovery. Science Express, Nov. 17, 2011

http://www.ukmi.nhs.uk/applications/ndo/record_view_open.asp?newDrugID=6368

The current spiroindolone was optimized to address its metabolic liabilities leading to improved stability and exposure levels in animals. As a result, NITD609 is one of only a handful of molecules capable of completely curing mice infected withPlasmodium berghei (a model of blood-stage malaria).
Given its good physicochemical properties, promising pharmacokinetic and efficacy profile, the molecule was recently approved as a preclinical candidate and is now entering GLP toxicology studies with the aim of entering Phase I studies in humans in late 2010. If its safety and tolerability are acceptable, NITD609 would be the first antimalarial not belonging to either the artemisinin or peroxide class to go into a proof-of-concept study in malaria.
If NITD609 behaves similarly in people to the way it works in mice, it may be possible to develop it into a drug that could be taken just once – far easier than current standard treatments in which malaria drugs are taken between one and four times a day for up to seven days. NITD609 also has properties which could enable it to be manufactured in pill form and in large quantities. Further animal studies have been performed and researchers have begun human-stage trials.
NITD609
NITD609.svg
Identifiers
ChemSpider 24662493
Jmol-3D images Image 1
Properties
Molecular formula C19H14Cl2FN3O
Molar mass 390.24 g mol−1

Malaria is an old infectious disease caused by four protozoan parasites, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. These four parasites are typically transmitted by the bite of an infected female Anopheles mosquito. Malaria is a problem in many parts of the world, and over the last few decades the malaria burden has steadily increased. An estimated 1 to 3 million people die every year from malaria – mostly children under the age of 5. This increase in malaria mortality is due in part to the fact that Plasmodium falciparum, the deadliest malaria parasite, has acquired resistance against nearly all available antimalarial drugs, with the exception of the artemisinin derivatives.

Leishmaniasis is caused by one of more than twenty (20) varieties of parasitic protozoa that belong to the genus Leishmania, and is transmitted by the bite of female sandflies. Leishmaniasis is endemic in some 90 countries, including many tropical and sub-tropical areas.

There are four main forms of leishmaniasis. Visceral leishmaniasis, also called kala-azar, is the most serious form and is caused by the parasite Leishmania donovani. Patients who develop visceral leishmaniasis can die within months unless they receive treatment. The two main therapies for visceral leishmaniasis are the antimony derivatives sodium stibogluconate (Pentostam®) and meglumine antimoniate (Glucantim®). Sodium stibogluconate has been used for about 70 years and resistance to this drug is a growing problem. In addition, the treatment is relatively long and painful, and can cause undesirable side effects. Human African Trypanosomiasis, also known as sleeping sickness, is a vector-bome parasitic disease. The parasites concerned are protozoa belonging to the Trypanosoma Genus. They are transmitted to humans by tsetse fly {Glossina Genus) bites which have acquired their infection from human beings or from animals harbouring the human pathogenic parasites.

Chagas disease (also called American trypanosomiasis) is another human parasitic disease that is endemic amongst poor populations on the American continent. The disease is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to humans by blood-sucking insects. The human disease occurs in two stages: the acute stage, which occurs shortly after the infection, and the chronic stage, which can develop over many years. Chronic infections result in various neurological disorders, including dementia, damage to the heart muscle and sometimes dilation of the digestive tract, as well as weight loss. Untreated, the chronic disease is often fatal.

The drugs currently available for treating Chagas disease are nifurtimox and benznidazole. However, problems with these current therapies include their adverse side effects, the length of treatment, and the requirement for medical supervision during treatment. Furthermore, treatment is really only effective when given during the acute stage of the disease. Resistance to the two frontline drugs has already arisen. The antifungal agent amphotericin b has been proposed as a second-line drug, but this drug is costly and relatively toxic.

PAPER

Stereoselective Total Synthesis of KAE609 via Direct Catalytic Asymmetric Alkynylation to Ketimine

Institute of Microbial Chemistry (BIKAKEN), Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
JST, ACT-C, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
Org. Lett., 2015, 17 (19), pp 4762–4765
DOI: 10.1021/acs.orglett.5b02300
Publication Date (Web): September 14, 2015
Copyright © 2015 American Chemical Society

Abstract

Abstract Image

A direct catalytic asymmetric alkynylation protocol is applied to provide the requisite enantioenriched propargylic α-tertiary amine, allowing for the stereoselective total synthesis of KAE609 (formerly NITD609 or cipargamin).

STR1

STR1

CLICK ON IMAGE TO VIEW

http://pubs.acs.org/doi/abs/10.1021/acs.orglett.5b02300?journalCode=orlef7

http://pubs.acs.org/doi/suppl/10.1021/acs.orglett.5b02300/suppl_file/ol5b02300_si_001.pdf

 

 STR1.jpg
STR1.jpg

PATENT

WO 2009/132921

Figure

In this process, the chiral amine is installed via an enzymatic resolution via deacylation of the acetamide 2. In addition to the wasteful resolution, other inefficiencies of this route include protection/deprotection (Ac/Boc, 2 to 4, and 5 to 6) and a three-step sequence to reduce the carboxylic acid to a methyl group (3 to 6).

Patent

US 2015/0045562

Figure

Improved Route to Cipargamin Employing Transaminase Reaction

For the transamination step, the enzyme ATA-256 was engineered by Codexis to accommodate the non-natural indole substrate 12. Since the substrate is not water-soluble, PEG 200 (approximately 20 vol %) is used as a cosolvent, an interesting selection given that DMSO or methanol are the most common cosolvents for enzymatic reactions. Isopropylamine is employed as the amine donor, a strategy that was adopted from the work of Merck and Codexis for the transamination of sitagliptin ketone.(2) During the transamination, which is a reversible reaction,i-PrNH2 is converted to acetone, which can be readily removed by evaporation to drive the reaction to completion. The workup involves filtration to remove enzyme residues followed by pH swings in which the product is extracted into the aqueous layer under acidic conditions, then basified for extraction into the organic layer. Addition of (+)-camphorsulfonic acid (CSA) provides the amine 14 as the crystalline CSA salt. No details are provided on enantioselectivity for the transamination, and it is not clear if the (+)-CSA is required to upgrade the ee or whether this salt was selected based on physical properties and the ability to develop a scalable crystallization process.
The final step to generate the spiroindole involves a diastereoselective condensation of the chiral amine with 5-chloroisatin (7) under acidic conditions. The diastereoselectivity of this reaction is not provided, nor any ee or de data for the final product. The spiroindole is also isolated as a (+)-CSA salt, which is then converted to the crystalline free base hemihydrate as the final form of cipargamin.

Example 12: Process for Preparing a Compound of formula (IVA) 1/z Hydrate

622.54 399.25

In a 750ml reactor with impeller stirrer 50g of compound (IVB) salt were dissolved in 300ml Ethanol (ALABD) and 100 ml deionised Water (WEM). The clear, yellowish sollution was heated to 58°C internal temperature. To the solution 85 g of a 10% aqueous sodium carbonate solution was added within 10 minutes. The clear solution was particle filtered into a second reaction vessel. Vessel and particle filter were each rinsed with 25 ml of a mixture of ethanohwater (3:1 v/v) in the second reaction vessel. The combined particle filtered solution is heated to 58°C internal temperature and 200ml water (WEM) were added dropwise within 15 minutes. Towards the end of the addition the solution gets turbid. The mixture is stirred for 10 minutes at 58°C internal temperature and is then cooled slowely to room temperature within 4hours 30 minutes forming a thick, well stirable white suspension. To the suspension 200 ml water are added and the mixture is stirred for additional 15hours 20 minutes at room temperature. The suspension is filtered and the filter cake is washed twice with 25 ml portions of a mixture of ethanohwater 9: 1 (v/v). The colourless crystals are dried at 60°C in vacuum yielding 26.23g (=91.2% yield). H NMR (400 MHz, DMSO-d6)

0.70 (s, 1H), 10.52 (s, 1H), 7.44 (d, J = 10.0 Hz, 1H), 7.33 (dd, J = 8.4, 2.1 Hz, 1H),.26 (d, J = 6.5 Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 3.83 – 4.00 (m,H), 3.13 (d, J = 6.0 Hz, 1H), 2.77 (dd, J = 15.1, 3.8 Hz, 1H), 2.38 (dd, J = 15.1, 10.5 Hz,H), 1.17 (d, J = 6.3 Hz, 3H).

 

 

Patent

http://www.google.com/patents/WO2009132921A1?cl=en

 

SCHEME G: Preparation of (lR,3S)-5′,7-dichloro-6-fluoro-3-methyl-2,3,4,9- tetrahydrospiro[β-carboline-l,3′-indol-2′(l’iϊ)-one (35) and (lR,3S)-5′-chloro-6-fluoro-3- methyl-2,3,4,9-tetrahydrospiro[β-carboline-l,3′-indoI-2′(l’H0-one (36)

Step 1 : POCl3 (2.43 mL, 26.53 mmol) was added dropwise to N, N-dimethylformamide (15.0 mL) at -20 °C and stirred below -5 0C for one hour. A solution of 6-chloro-5-fluoroindole (3.0 g, 17.69 mmol) in dimethylformamide (5.0 mL) was added dropwise to the above reaction mixture at -20 °C. The salt-ice bath was removed and the reaction mixture was warmed to 35 0C, After one hour, the reaction was poured onto ice and basified by solid sodium bicarbonate and extracted with ethyl acetate. The combined organic layer was washed with water and then concentrated to give 6-chloro-5-fluoro-1H-indole-3-carbaldehyde (3.4 g, 97 %) as a light brown solid. 1H ΝMR (500 MHz, CDCl3): δ 10.02 (s, 1 H), 8.10 (d, IH, J = 9.5 Hz), 7.87 (s, 1 H), 7.49 (d, IH, J= 5.5 Hz).

Step 2: The solution (0.2 M) of 6-chloro-5-fluoro-1H-indole-3-carbaldehyde (4.0 g, 20.24 mmol) in nitroethane (100 mL) was refluxed with ammonium acetate (1.32 g, 0.85 mmol) for 4 hours. The reaction mixture was concentrated under vacuum to remove nitroethane, diluted with ethylacetate and washed with brine. The organic layer was concentrated to give 6-chloro-5- fluoro-3-(2-nitro-propenyl)-1H-indole (5.0 g, 97 %) as a reddish orange solid. 1H ΝMR (500 MHz, CDCl3): δ 8.77 (s, IH), 8.32 (s, IH), 7.58 (d, IH, J= 2.5 Hz), 7.54 (d, IH, J = 9 Hz), 7.50 (d, IH, J= 5.9 Hz), 2.52 (s, 3H). Step 3: A solution of 6-chloro-5-fluoro-3-(2-nitro-propenyl)-1H-indole (5.0 g, 19.63 mmol) in tetrahydrofuran (10 mL) was added to the suspension of lithium aluminium hydride (2.92 g, 78.54 mmol) in tetrahydrofuran (20 mL) at 0 0C and then refluxed for 3 hours. The reaction mixture was cooled to 0 °C, and quenched according to the Fischer method. The reaction mixture was filtered through celite and the filtrate concentrated to give 2-(6-chloro-5-fluoro-1H-indol-3- yl-1-methyl-ethylamine (4.7 g crude) as a viscous brown liquid. The residue was used without further purification. 1H NMR (500 MHz, CDCl3): δ 8.13 (s, IH), 7.37 (d, IH, 6.Hz), 7.32 (d, IH, J = 10 Hz), 7.08 (s, IH), 3.23-3.26 (m, IH), 2.77-2.81 (m, IH), 2.58-2.63 (m, IH), 1.15 (d, 3H, J= 6.5 Hz).

Step 4: A mixture of 2-(6-chloro-5-fluoro-1H-indol-3-yl-l-methyl-ethylamine (4.7 g, 20.73 mmol), 5-chloroisatin (3.76 g, 20.73 mmol) and p-toluenesulphonic acid (394 mg, 2.07 mmol) in ethanol (75 mL) was refluxed overnight. The reaction mixture was concentrated to remove ethanol, diluted with ethyl acetate and washed with saturated aqueous NaHCO3. The organic layer was concentrated to give a brown residue, which was purified by silica gel chromatography (20 % ethyl acetate in hexane) to provide the corresponding racemate (4.5 g, 56 %) as a light yellow solid. The racemate was separated into its enantiomers by chiral chromatography to provide 35.

Compound 36 can be obtained in a similar fashion from 5-fluoroindole.

Alternatively 35 and 36 were be prepared in enantiomerically pure form by the following scheme.

SCHEME H: Alternative preparation of (lR,3S)-5′,7-dichloro-6-fluoro-3-methyl-2,3,4,9- tetrahydrospiro[β-carboline-l,3′-indol-2′(1’H)-one (35)

Step 1 : To a solution of 6-chloro-5-fluoroindole (1.8 g, 10.8 mmol) and Ac2O (10 niL) in AcOH (3OmL) was added L-serine (2.2 g, 20.9 mmol), the mixture was heated to 80 °C. After TLC indicated the reaction was complete, the mixture was cooled to 0 °C, neutralized to pH 11 , and washed with MTBE. The aqueous phase was acidified to pH 2 and extracted with EtOAc. The combined organic layers were washed with water and bπne, dπed with Na2SO4, filtered, and concentrated. The residue was purified with chromatography (Petroleum ether /EtOAc 1:1) to give 2-acetylamino-3-(6-chloro-5-fluoro-1H-mdol-3-yl)-propπonic acid as a light yellow solid (1.2 g, 37% yield).

Step 2: 2-Acetylamino-3-(6-chloro-5-fluoro-1H-indol-3-yl)-proprionic acid (2.5g, 8.4mmol) was dissolved in aqueous NaOH (IN, 10 niL) and water added (70 mL). The mixture was heated to 37-380C and neutralized with HCl (IN) to pΗ 7.3-7.8. L-Aminoacylase (0.5 g) was added to the mixture and allowed to stir for 2 days, maintaining 37-380C and pΗ 7.3-7.8. The mixture was heated to 60 °C for another hour, concentrated to remove part of water, cooled and filtered. The filtrate was adjusted to pΗ 5.89 and filtered again. The filtrate was adjusted to pΗ 2.0 and extracted with EtOAc. The combined organic layer was dried over Na2SO4, filtered, concentrated and the residue was purified with chromatography (petroleum ether /EtOAc 1 : IEtOAc) to give R- 2-acetylamino-3-(6-chloro-5-fluoro-1H-mdol-3-yl)-propπonic acid as a light yellow solid (1.2 g, 48% yield). Step 3: R-2-acetylamino-3-(6-chloro-5-fluoro-1H-indol-3-yl)-proprionic acid (1.2 g, 4.0 mmol) was dissolved in HCl (6N, 10 mL) and the mixture heated to reflux for 4 hours, and then concentrated to dryness. Toluene (50 mL) was added to the residue and concentrated to dryness to remove water and HCl. The residue was dried under vacuum and then dissolved in MeOH (20 mL). To the solution was added dropwise SOCl2 (0.5 mL, 6.8 mmol) at 0 °C, and the mixture was stirred overnight. After removal of solvent, the residue was dissolved in THF/water (40/10 mL) and NaHCO3 (1.0 g, 11.9 mmol) was added portionwise. Upon basifϊcation, BoC2O (1.2 g, 5.5 mmol) added at 0 °C and allowed to stir at room temperature. After TLC indicated the reaction was finished, EtOAc was added and separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with water and brine, dried with Na2SO4, filtered, concentrated and the residue was purified with chromatography (petroleum ether /EtOAc: 5/1) to give R-2-tert-butoxycarbonylamino-3-(6-chloro-5-fluoro-l/-/-indol-3-yl)-proprionic acid methyl ester 460 g, 31% yield for 3 steps).

Step 4: To a solution of R-2-tert-butoxycarbonylamino-3-(6-chloro-5-fluoro-l//-indol-3-yl)- proprionic acid methyl ester (460mg, 1.2mmol) in dry ether (20 mL) was added portionwise LiAlH4 (92 mg, 2.4 mmol) at 0 °C. The mixture was heated to reflux for 2 hours. After TLC indicated the reaction was finished, the mixture was cooled and carefully quenched with Na2SO4. The mixture was filtered and the filtrate was washed with saturated aqueous NH4Cl and water, dried with Na2SO4, filtered, concentrated to give a crude product (400 mg), which was used without further purification.

Step 5: To a solution of the crude product (400 mg, 1.2mmol) and Et3N (0.3 mL, 2.2 mmol) in CH2Cl2 (5 mL) was added MsCl (160 mg, 1.4 mmol) dropwise at 0 °C. The mixture was stirred for 2 hours at room temperature. After TLC indicated the reaction was completed, the mixture was washed with water and brine, dried with Na2SO4, filtered, concentrated and the residue was purified with chromatography (petroleum ether/EtOAc 5:1) to give methansulfonic acid (R)-2- ?ert-butoxycarbonylamino-3-(6-chloro-5-fluoro-1H-indol-3-yl)-propyl ester as a light yellow solid (300 mg, 57% yield, 2 steps)

Step 6: To a solution of mesylate (300 mg, 0.7mmol) in dry ether (20 mL) was added portionwise LiAlH4 (55 mg, 1.4 mmol) at 0 °C. The mixture was stirred at room temperature overnight. After TLC indicated the reaction was finished, the mixture was cooled and carefully quenched with Na2SO4. The mixture was filtered and the filtrate was washed with saturated aqueous NH4Cl and water, dried with Na2SO4, filtered, concentrated and the residue was purified with chromatography (petroleum ether/EtOAc 10: 1) to give [(5)-2-(6-chloro-5-fluoro-1H-indol-3-yl)- 1 -methyl-ethyl] -carbamic acid tert-butyl ester as a light yellow solid (200 mg, 87% yield).

Step 7: A solution of [(S)-2-(6-chloro-5-fluoro-1H-indol-3-yl)-l-methyl-ethyl]-carbamic acid tert-butyl ester (200 mg, 0.6 mmol) in HCl/MeOH (10 mL) was stirred at room temperature. After TLC indicated the reaction was finished, the mixture was concentrated to remove the solvent. To the residue was added EtOAc (5OmL), and the mixture was neutralized with saturated NaHCO3 to pH 8~9, and then extracted with EtOAc. The combined organic phases were dried with Na2SO4, filtered, concentrated to give a crude (S)-2-(6-chloro-5-fluoro-1H-indol-3-yl)-l- methyl-ethylamine which was used without further purification.

Step 8: To a solution of (5)-2-(6-chloro-5-fluoro-1H-indol-3-yl)-l-methyl-ethylamine (120 mg, 0.5 mmol) in EtOH (1OmL) was added 5-chloroisatin (90 mg, 0.5 mmol) and p-TsOΗ (8 mg, 0.04 mmol). The mixture was heated in a sealed tube at 1100C for 16 hours. After TLC indicated the reaction was finished, the mixture was cooled and concentrated. The residue was dissolved in EtOAc (2OmL) and washed with NaOH (IN) and brine, dried with Na2SO4, filtered, concentrated and the residue was purified with chromatography (petroleum ether/EtOAc 5:1) to give 36 (150mg, 64% yield over two steps).

 

Example 48 (15,3R)-5′-Chloro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-l,3′-indol]-2′(l’JH)-one

(35)

35

Compound 35 may be prepared according to Scheme F using the same or analogous synthetic techniques and/or substituting with alternative reagents.

(lS^RVS’-Chloro-S-methyl-l^^^-tetrahydrospirotβ-carboline-l.S’-indoll-l^l’ZO-one: 1H NMR (300 MHz, DMSO-^6): δ 10.45 (s, IH), 10.42 (s, IH), 7.43 (d, J= 7.5 Hz, IH), 7.31 (dd, J = 2.1, 8.4 Hz, IH), 7.16 (d, J = 7.2 Hz, IH), 7.05-7.02 (m, 2H), 7.00-6.96 (m, IH), 6.92 (d, J = 8.1 Hz, IH), 3.98-3.86 (m, IH), 2.78 (dd, J= 3.6, 14.9 Hz, IH), 2.41 (dd, J= 4.5, 25.5 Hz, IH), 1.18 (d, J= 6.3 Hz, 3H); MS (ESI) m/z 338.0 (M+H)+.

Chiral compounds such as 36 and 37 can be prepared according to Scheme G or H using the same or analogous synthetic techniques and/or substituting with alternative reagents. Example 49

(IR^^-S’.T-Dichloro-ό-fluoro-S-methyl-l^^^-tetrahydrospiroIβ-carboline-l^’-indol]- 2\VH)-one (36)

36

35: 1H NMR (500 MHz, DMSO-Jd) δ 10.69 (s, IH), 10.51 (s, IH), 7.43 (d, J = 10.0 Hz, IH), 7.33 (dd, J= 8.4, 2.2 Hz, IH), 7.27 (d, J= 6.5 Hz, IH), 7.05 (d, J= 2.3, IH), 6.93 (d, J= 8.5 Hz, IH), 3.91 (m, IH), 3.13 (bd, J= 6.2 Hz, IH), 2.74 (dd, J= 15.0 , 3.0 Hz, IH), 2.35 (dd, J= 15.0, 10.3, IH), 1.15 (d, J= 6.0, 3H);

MS (ESI) m/z 392.0 (M+2H)+;

[α]25 D = + 255.4°

Example 50

(lS,3R)-5′,7-Dichloro-6-fluoro-3-methyI-2,3,4,9-tetrahydrospiro[β-carboline-l,3′-indol]- 2′(l’H)-one (37)

37

(lS^^-S’^-Dichloro-o-fluoro-S-methyl^jS^^-tetrahydrospirojP-carboline-l-S’-indol]- 2′(l’H)-one: 1H NMR (500 MHz, CDCl3) δ 8.49 (s, IH), 7.54 (s, IH), 7.24 (d, J= 9.7 Hz, IH), 7.21 (dd, J = 8.6, 2.0 Hz, IH), 7.14 (d, J= 6.0 Hz, IH), 7.11 (d, J= 1.8, IH), 6.77 (d, J= 8.3 Hz, IH), 4.14 (m, IH), 2.89 (dd, J = 15.4, 3.7 Hz, IH), 2.49 (dd, J = 15.3, 10.5, IH), 1.68 (bs, IH), 1.29 (d, J= 6.4 Hz, 3H); MS (ESI) m/z 392.0 (M+2H)+; [α]25D -223.3°

PATENT

US 2011275613

http://www.google.com/patents/WO2013139987A1?cl=en

 

Prior art:

(1 ‘R, 3’S)-5, 7′-dichloro-6′-fIuoro-3′-methyl-2′, 3′,4′, 9’-tetrahydrospiro[indoline-3, 1 – pyrido[3,4-b]indol]-2-one (eg. a compound of formula (IV), which comprises a spiroindolone moiety) and a 6-steps synthetic method for preparing, including known chiral amine intermediate compound (MA) are known (WO 2009/132921 ):

he present invention relates to processes for the preparation of spiroindolone compounds, such as (1’R,3’S)-5, 7′-dichloro-6′-fIuoro-3′-methyl-2′,3′,4′,9′- tetrahydrospiro[indoline-3, 1 ‘-pyhdo[3.4-b]indol]-2-one.

(1 ‘R, 3’S)-5, 7′-dichloro-6′-fluoro-3′-methyl-2′, 3′,4 9’-tetrahydrospiro[indoline-3, 1 ‘- pyrido[3, 4-b]indol]-2-one is useful in the treatment and/or prevention of infections such as those caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, Trypanosoma cruzi and parasites of the Leishmania genus such as, for example, Leishmania donovani., and it has the following structure:

(IVA)

(1 ‘R, 3’S)-5, 7′-dichloro-6′-fluoro-3′-methyl-2 3′, 4′, 9’-tetrahydrospiro[indoline-3, 1 – pyhdo[3, 4-b]indol]-2-one and a synthesis thereof are described in WO 2009/132921 Al in particular in Example 49 therein.

 

Example 10: Process for Conversion of Compound (IA) to Compound (IIA) in 30g Scale

458.97

152.48g /so-propylamine hydrochloride and 0.204g pyridoxalphosphate monohydrate were dissolved in 495ml water while stirring. To this yellow clear solution a solution of 30. Og ketone in 85ml poly ethylene glycol (average mol weight 200) within 15 minutes. Upon addition the ketone precipitates as fine particles which are evenly distributed in the reaction media. To the suspension 180ml triethanolamine buffer (0.1 mol/l, pH 7) were added and the pH was adjusted to 7 by additon of aqueous sodium hydroxide solution (1 mol/l). The reaction mixture is heated to 50°C and a solution of 1.62g transaminase SEQ ID NO: 134 dissolved in 162ml triethanolamine buffer (0 1 mol/l, pH 7) is added. The reaction mixture is continiously kept at pH 7 by addition of 1 mol/l aqueous sodium hydroxide solution. The reaction mixture is stirred 24h at 50°C and a stream of Nitrogen is blown over the surface of the reaction mixture to strip off formed acetone. The reaction mixture is then cooled to 25°C and filtered over a bed of cellulose flock. The pH of the filtrate is adjusted to «1 by addition of concentrated sulfuric acid. The acidified filtrated is extracted with 250 ml /so-Propyl acetate. The layers are separated and the pH of the aqueous phase is adjusted to ¾10 by additon of concentrated aqueous sodium hydroxide solution. The basified aqueous phase is extracted with /so-propyl acetate. The layers are seperated and the organic phase is washed with 100 ml water. The organic phase is concentrated by distillation to 2/3 of its origin volume. In a second reactor 33.98g (+)- camphor sulfonic acid is dissolved in 225 ml /so-propyl acetate upon refluxing and the concentrated organic phase is added within 10 minutes. After complete addition the formed thin suspension is cooled to 0°C within 2 hours and kept at 0°C for 15 hours. The precipitated amine-(+)-camphor sulfonate salt is filtered, washed with 70 ml /so-propyl acetate and dried at 40°C in vaccuum yielding 51.57g of colourless crystals (84.5% yield t.q.)

Analytical Data

IR:

v (crn 1)=3296, 3061 , 2962, 2635, 2531 , 2078, 1741 , 1625, 1577, 1518, 1461 , 1415, 1392, 1375, 1324, 1302, 1280, 1256, 1226, 1 170, 1 126, 1096, 1041 , 988, 966, 937, 868, 834, 814, 790, 766, 746, 719, 669, 615.

LC-MS (ESI +):

Ammonium ion: m/z =227 ([M+H]), 268 ([M+H+CH3CN]), 453 ([2M+H]).

Camphorsulfonate ion: m/z =250 ([M+NH4]), 482 ([2M+NH4]).

LC-MS (ESI -):

Camphorsulfonate ion: m/z=231 ([M-H]), 463 ([2M-H]).

1H-NMR (DMSO-d6, 400 MHz):

1 1.22 (br. s., 1 H), 7.75 (br. s., 3H), 7.59 (d, J = 10.3 Hz, 1 H), 7.54 (d, J = 6.5 Hz, 1 H), 7.36 (d, J = 2.3 Hz, 1 H), 3.37 – 3.50 (m, 1 H), 2.98 (dd, J = 14.3, 5.8 Hz, 1 H), 2.91 (d, J = 14.8 Hz, 1 H), 2,81 (dd, J = 14.3, 8.0 Hz, 1 H), 2.63 – 2.74 (m, 1 H), 2.41 (d, J = 14.6 Hz, 1 H), 2.24 (dt, J = 18.3, 3.8 Hz, 1 H), 1 .94 (t, J = 4.4 Hz, 1 H), 1.86 (dt, J = 7.4, 3 6 Hz, 1 H), 1.80 (d, J = 18.1 Hz, H), 1.23 – 1 .35 (m, 2H), 1.15 (d, J = 6.3 Hz, 3H), 1.05 (s, 3H), 0.74 (s, 3H)

Free Amine (obtained by evaporatig the iso-Propylacetate layer after extraction of the basified aqueous layer):

1H NMR (400MHz, DMSO-d6): 11 .04 (br. s., 1 H), 7.50 (d, J = 10.5 Hz, 1 H), 7.48 (d, J = 6.5 Hz, 1 H), 7.25 (s, 1 H), 3.03 (sxt, J = 6.3 Hz, 1 H), 2.61 (dd, J – 14.3, 6.5 Hz, 1 H), 2.57 (dd, J = 14.1 , 6.5 Hz, 1 H), 1.36 (br. s., 2H), 0.96 (d, J = 6.3 Hz, 3H)

Example 11: Process for Conversion of Compound (HA) to Compound (IVB)

3. solvent exchange to TP

13.62 g 5-chloroisatin is suspended in 35 ml /so-propanol and 2.3 g triethyl amine is added. The suspension is heated to reflux and a solution of 34.42g amine-(+)-camphor sulfonate salt dissolved in 300 ml /so-propanol is added within 50 minutes. The reaction mixture is stirred at reflux for 17 hours. The reaction mixture is cooled to 75°C and 17.4g (+)-camphorsulfonic acid are added to the reaction mixture. Approximately 300 ml /so- propanol are removed by vacuum distillation. Distilled off /so-propanol is replaced by iso- propyl acetate and vacuum distillation is continued. This is distillation is repeated a second time. To the distillation residue 19 ml ethanol and 265 ml ethyl acetate is added and the mixture is heated to reflux. The mixture is cooled in ramps to 0°C and kept at 0°C for 24 hours. The beige to off white crystals are filtered off, washed with 3 portions (each 25 ml) precooled (0°C) ethylacetate and dried in vacuum yielding 40.3 g beige to off white crystals. (86.3% yield t.q.)

IR:

v (crrr)= 3229, 3115, 3078, 3052, 2971 , 2890, 2841. 2772. 2722, 2675, 2605, 2434. 1741 , 1718, 1621 , 1606, 1483, 1460, 1408, 1391 , 1372, 1336, 1307, 1277, 1267, 1238, 1202, 1 184, 1 162, 1 149, 1 128, 1067, 1036, 987, 973, 939, 919, 896, 871 , 857, 843, 785, 771 , 756, 717, 690, 678, 613.

LC-MS (ESI +):

Ammonium ion: m/z =390 ([M+H]), 431 ([M+H+CH3CN]) Camphorsulfonate ion: m/z =250 ([M+NH4]), 482 ([2M+NH4])

LC-MS (ESI -):

Camphorsulfonate ion: m/z=231 ([M-H]), 463 ([2M-H])

1H NMR (DMSO-d6, 600 MHz):

11.49 (s, 1 H), 1 1.23 (s, 1 H), 10.29 – 10.83 (m, 1 H), 9.78 – 10.31 (m, 1 H), 7.55 – 7.60 (m, 2H), 7.52 (s, 1 H), 7.40 (d, J = 6.2 Hz, H), 7.16 (d, J = 8.8 Hz, 1 H), 4.52 – 4.63 (m, 1 H). 3.20 (dd, J = 16.3, 4.2 Hz, 1 H), 2.96 (dd, J = 16.1 , 11.3 Hz, 1 H), 2.90 (d, J = 15.0 Hz, 1 H), 2.56 – 2.63 (m, 1 H), 2.39 (d, J = 14.6 Hz, 1 H), 2.21 (dt, J = 18.0, 3.8 Hz, 1 H), 1.89 – 1.93 (m, 1 H), 1.81 (ddd, J = 15.3, 7.8, 3.7 Hz, 1 H), 1.76 (d, J = 18.3 Hz, 1 H), 1 .53 (d, J = 6.6 Hz, 3H), 1.20 – 1.33 (m, 2H), 0.98 (s, 3H), 0.70 (s, 3H)

Example 12: Process for Preparing a Compound of formula (IVA) 1/z Hydrate

mw622.54 …………………………………………………………………..mw399.25

In a 750ml reactor with impeller stirrer 50g of compound (IVB) salt were dissolved in 300ml Ethanol (ALABD) and 100 ml deionised Water (WEM). The clear, yellowish sollution was heated to 58°C internal temperature. To the solution 85 g of a 10% aqueous sodium carbonate solution was added within 10 minutes. The clear solution was particle filtered into a second reaction vessel. Vessel and particle filter were each rinsed with 25 ml of a mixture of ethanohwater (3:1 v/v) in the second reaction vessel. The combined particle filtered solution is heated to 58°C internal temperature and 200ml water (WEM) were added dropwise within 15 minutes. Towards the end of the addition the solution gets turbid.

The mixture is stirred for 10 minutes at 58°C internal temperature and is then cooled slowely to room temperature within 4hours 30 minutes forming a thick, well stirable white suspension. To the suspension 200 ml water are added and the mixture is stirred for additional 15hours 20 minutes at room temperature. The suspension is filtered and the filter cake is washed twice with 25 ml portions of a mixture of ethanohwater 9: 1 (v/v). The colourless crystals are dried at 60°C in vacuum yielding 26.23g (=91.2% yield). H NMR (400 MHz, DMSO-d6)

0.70 (s, 1H), 10.52 (s, 1H), 7.44 (d, J = 10.0 Hz, 1H), 7.33 (dd, J = 8.4, 2.1 Hz, 1H),.26 (d, J = 6.5 Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 3.83 – 4.00 (m,H), 3.13 (d, J = 6.0 Hz, 1H), 2.77 (dd, J = 15.1, 3.8 Hz, 1H), 2.38 (dd, J = 15.1, 10.5 Hz,H), 1.17 (d, J = 6.3 Hz, 3H).

 

PAPER
 Journal of Medicinal Chemistry, 2010 ,  vol. 53,   14  p. 5155 – 5164

(1R,3S)-5′,7-Dichloro-6-fluoro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-1,3′-indol]-2′(1′H)-one (19a)

1H NMR (500 MHz, DMSO-d6): δ 10.69 (s, 1H), 10.51 (s, 1H), 7.43 (d, J = 10.0 Hz, 1H), 7.33 (dd, J = 8.0, 2.2 Hz, 1H), 7.27 (d, J = 6.5 Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H), 6.93 (d, J = 8.5 Hz, 1H), 3.91 (m, 1H), 3.13 (bd, J = 6.2 Hz, 1H), 2.74 (dd, J = 15.0, 3.0 Hz, 1H), 2.35 (dd, J = 15.0, 10.3 Hz, 1H), 1.15 (d, J = 6.0 Hz, 3H). MS (ESI) m/z 392.0 (M + 2H)+; [α]D25 = +255.4° (c = 0.102 g/L, methanol).
CLIPS

Z.Zhang, WO 2007 / 104714,2007).

 

Figure CN102432526AD00051

[0008] (2) year 2008 Roche pharmaceutical company disclosed a spiro [oxindole – cyclohexenone] skeleton biomedicine, PCT International Application No. W02008 / 055812. It also announced the preparation of anti-cancer agents and antagonists of the application of the compound is used as the interaction with MDM2 (reference:. Liu, J.-J; Zhang, Z; (Hoffmann-LaRoche AG), PCT Int App 1. . W02008 / 055812, 2008), its structural formula is as follows:

[0009]

Figure CN102432526AD00052

(3) Melchiorre research group abroad chiral amines and o-fluoro-3-benzyl benzoate as catalyst methylene-indole-2-one (3-benzylideneindolin-2-one, CAS Number: 3359-49- 7) with α, β – unsaturated ketone synthesis of chiral spiro [cyclohexane _1,3′- indole] _2,4 ‘- dione [s pir0 [cycl0hexane-l, 3’ -indoline] – 2 ‘, 4-diones] compounds (see:.. Bencivenni, G; ffu, LY; Mazzanti, A .; Giannichi, B.; Pesciaioli, F; Song, Μ P.; Bartoli, G.; Melchiorre, P …. .Angew Chem Int Ed 2009,48,7200), the structure of the total formula is as follows:

 

Figure CN102432526AD00061

(4) Gong Flow column team found to cyclohexanediamine derived Bronsted acid – a bifunctional catalyst Lewis base catalysis of 3-benzyl-methylene-indole-2-one and α, β- unsaturated 1,3 tandem reaction dicarbonyl compound (Nazarov reagent) can be obtained with high stereoselectivity chiral spiro [cyclohexane _1,3′- indol] -2 ‘, 4-dione [spiro [cyclohexane-l, 3 ‘-indoline] -2’, 4-diones] compounds; and by this method successfully synthesized 7 Roche pharmaceutical companies to develop chiral anti-tumor agents (see: Q Wei, L -Z Gong, Org Lett 2010….. , 12, 1008.).

(5) Wang Lixin research group recently reported that primary amines derived from cinchona alkaloids and Bronsted acid as catalyst N- protected indolone compounds and double Michael addition reaction of diketene generate hand spiro [cyclohexane-1, 3′-indol] -2 ‘, 4-dione [spiro [cyclohexane-l, 3’ -indoline] -2 ‘, 4-diones] type of tx ^ (: L. -L. Wang, L. Peng, J. -F. Bai, L. -N. Jia, X. -Y. Luo, QC Huang, L. -X. Wang, Chem. Commum. 2011,47, 5593.).

WO2009132921A1 * Apr 1, 2009 Nov 5, 2009 Novartis Ag Spiro-indole derivatives for the treatment of parasitic diseases
WO2010081053A2 * Jan 8, 2010 Jul 15, 2010 Codexis, Inc. Transaminase polypeptides
WO2012007548A1 * Jul 14, 2011 Jan 19, 2012 Dsm Ip Assets B.V. (r)-selective amination
AT507050A1 * Title not available
EP0036741A2 * Mar 17, 1981 Sep 30, 1981 THE PROCTER &amp; GAMBLE COMPANY Phosphine compounds, transition metal complexes thereof and use thereof as chiral hydrogenation catalysts
EP0120208A2 * Jan 24, 1984 Oct 3, 1984 Degussa Aktiengesellschaft Microbiologically produced L-phenylalanin-dehydrogenase, process for obtaining it and its use
EP0135846A2 * Aug 31, 1984 Apr 3, 1985 Genetics Institute, Inc. Production of L-amino acids by transamination
GB974895A * Title not available
US3282959 * Mar 21, 1962 Nov 1, 1966 Parke Davis & Co 7-chloro-alpha-methyltryptamine derivatives
US4073795 * Jun 22, 1976 Feb 14, 1978 Hoffmann-La Roche Inc. Synthesis of tryptophans
WO2005009370A2 * Jul 22, 2004 Feb 3, 2005 Pharmacia Corp Beta-carboline compounds and analogues thereof and their use as mitogen-activated protein kinase-activated protein kinase-2 inhibitors
EP0466548A1 * Jun 27, 1991 Jan 15, 1992 Adir Et Compagnie 1,2,3,4,5,6-Hexahydroazepino[4,5-b]indole and 1,2,3,4-tetrahydro-beta-carbolines, processes for their preparation, and pharmaceutical compositions containing them

Рисунок из Science 2010, 329, 1175

Исследовательская группа Элизабет Винцелер (Elizabeth A. Winzeler) разработала новый препарат, первоначально проведя скрининг библиотеки, состоящей из 12000 соединений, а затем получив производные наиболее перспективных кандидатов. В результате долгой работы исследователи отобрали единственное соединение спироиндолоновой структуры, получившее регистрационный номер NITD609. В случае успешного прохождения экспертизы фармакологических и токсикологических свойств нового соединения исследователи надеются приступить к первой фазе его клинических испытаний уже в конце этого года.

Было обнаружено, что NITD609 быстро останавливает белковый синтез в организме возбудителя малярии, ингибируя ген аденозинтрифосфатазы, ответственной за транспорт катионов через мембрану клетки возбудителя. То, что механизм действия нового соединения отличается от механизма, характерного для других средств лечения малярии, объясняет причины успешного действия нового препарата в том числе и против штаммов малярии, выработавших резистентность.

 HPLC
Analyte quantization was performed byLC/MS/MS. Liquid chromatography was performed using an Agilent
1100 HPLC system(Santa Clara, CA), with the Agilent Zorbax XDB Phenyl (3.5μ, 4.6 x75 mm) column at
an oven temperature of 35 °C, coupled with a QTRAP4000 triple quadruple mass
spectrometer (Applied Biosystems, Foster City, CA). Instrumentcontrol and dataacquisition were performed using Applied Biosystems software Analyst 1.4.2. Themobile phases used were A: water:acetic acid (99.8:0.2, v/v) and B: acetonitrile:aceticacid (99.8:0.2, v/v), using a gradient, with flow rate of 1.0 mL/min, and run time of 5minutes. Under these conditions the retention time of9a
was 3.2 minutes. Compounddetection on the mass spectrometer was performed in electrospraypositive ionizationmode and utilized multiple reaction monitoring (MRM) for specificity (9atransitions338.3/295.1, 338.3/259.2) together with their optimized MS parameters. The lower limitof quantification for9awas 70 ng/mL.
Extraction and LCMS analysis of 20a.Plasma samples were extracted withacetonitrile:methanol-acetic acid (90:9.8:0.2 v/v) for the analyte and internal standard(17a) using a 3.6 to 1 extractant to plasma ratio. Analyte quantitation was performed by
LC/MS/MS. Liquid chromatography was performed using an Agilent1100 HPLC systemS7(Santa Clara, CA), with the Agilent Zorbax XDB-Phenyl (3.5μ, 4.6x75mm) column atan oven temperature of 45 °C coupled with a QTRAP 4000 triple quadruple massSpectrometer (Applied Biosystems, Foster City, CA). Instrumentcontrol and dataacquisition were performed using Applied Biosystems software Analyst 1.4.2. Themobile phases used were A: water:acetic acid (99.8:0.2, v/v) and B: methanol:acetic acid
(99.8:0.2, v/v), using gradient elution conditions with a flow rate of 1.0 mL/min and a runtime of 6 minutes
++++++++++++++++++++++==
+++++++++++++++++++++++++++=

References

  1.  “NITD 609”. Medicines for Malaria Venture.
  2.  Rottmann M, McNamara C, Yeung BK, Lee MC, Zou B, Russell B, Seitz P, Plouffe DM, Dharia NV, Tan J, Cohen SB, Spencer KR, González-Páez GE, Lakshminarayana SB, Goh A, Suwanarusk R, Jegla T, Schmitt EK, Beck HP, Brun R, Nosten F, Renia L, Dartois V, Keller TH, Fidock DA, Winzeler EA, Diagana TT (2010). “Spiroindolones, a potent compound class for the treatment of malaria”. Science329 (5996): 1175–80. doi:10.1126/science.1193225. PMC 3050001. PMID 20813948.

Ang, S. H., Krastel, P., Leong, S. Y., Tan, L. J., Wong, W. L. J., Yeung, B. K., and Zou, B. Spiro-indole derivatives for the treatment of parasitic diseases. WO2009132921 A1, November 5, 2009.

Cipargamin
NITD609.svg
Names
IUPAC name

(1R,3S)-5’,7-Dichloro-6-fluoro-3-methyl-spiro[2,3,4,9-tetrahydropyrido[3,4-b]indole-1,3’-indoline]-2’-one
Identifiers
1193314-23-6
ChemSpider 24662493
Jmol interactive 3D Image
PubChem 44469321
Properties
C19H14Cl2FN3O
Molar mass 390.24 g·mol−1

SEE……….http://apisynthesisint.blogspot.in/2016/02/kae-609-nitd-609-cipargamin-for-malaria.html

////

C[C@H]1Cc2c3cc(c(cc3[nH]c2[C@]4(N1)c5cc(ccc5NC4=O)Cl)Cl)F

Artesunate, the antimalarial


Artesunate.svg

ARTESUNATE
Butanedioic acid mono[(3R,5aS,6R,8aS,9R,10R,12R,12aR)-decahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10-yl] ester
(3R,5aS,6R,8aS,9R,10S,12R,12aR)-Decahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano(4,3-j)-1,2-benzodioxepin-10-ol, hydrogen succinate
Butanedioic acid, mono((3R,5aS,6R,8aS,9R,10S,12R,12aR)-decahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano(4,3-j)-1,2-benzodioxepin-10-yl) ester
Additional Names: artesunic acid; dihydroqinghaosu hemisuccinate, Succinic ester of artemether.
Molecular Formula: C19H28O8
Molecular Weight: 384.42
Percent Composition: C 59.36%, H 7.34%, O 33.30%
Artesunate (superseded RN); Dihydroartemisinine-12-alpha-succinate; Succinyl dihydroartemisinin; Quinghaosu reduced succinate ester
Therap-Cat: Antimalarial.

Artesunate (INN) is part of the artemisinin group of drugs that treat malaria. It is a semi-synthetic derivative of artemisinin that is water-soluble and may therefore be given by injection. It is sometimes abbreviated AS.

It is on the World Health Organization’s List of Essential Medicines, a list of the most important medication needed in a basic health system.[1]

Artesunate

  • Arinate
  • Armax 200
  • Arsuamoon
  • Arsumax
  • Artesunata
  • Artesunate
  • Artesunato
  • Artesunato [INN-Spanish]
  • Artesunatum
  • Artesunatum [INN-Latin]
  • Artesunic acid
  • Asumax
  • Cosinate
  • Dihydroqinghasu hemsuccinate
  • Gsunate Forte
  • HSDB 7458
  • Plasmotrin
  • Qinghaozhi
  • Quinghaosu reduced succinate ester
  • Saphnate
  • SM 804
  • Succinyl dihydroartemisinin
  • UNII-60W3249T9M
  • WR 256283
  • Zysunate
SODIUM ARTESUNATE Structure
SODIUM ARTESUNATE;
Dihydroartemisinin alpha-hemisuccinate sodium salt;
Sodium dihydroarteannuin hydrogen succinate monoester;
Butanedioic acid 1-[(3R,5aα,8aα,12aR)-decahydro-3,6α,9β-trimethyl-3β,12α-epoxypyrano[4,3-j]-1,2-benzodioxepin-10α-yl]4-sodium salt;
Butanedioic acid, mono(decahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano(4,3-J)-1,2-benzodioxepin-10-yl)ester, sodium salt, (3R-(3-alpha,5A-beta,6-beta,8A-beta,9-alpha,10-alpha,12-beta,12ar*))-
CAS 82864-68-4
Derivative Type: Sodium salt
Manufacturers’ Codes: SM-804
Molecular Formula: C19H27NaO8
Molecular Weight: 406.40
Percent Composition: C 56.15%, H 6.70%, Na 5.66%, O 31.49%
Properties: Poor stability in aqueous solutions. LD50 in mice (mg/kg): 520 i.v.; 475 i.m. (China Cooperative Research Group); also reported as 699 ± 58.5 i.v. (Zhao, 1985).
Toxicity data: LD50 in mice (mg/kg): 520 i.v.; 475 i.m. (China Cooperative Research Group); also reported as 699 ± 58.5 i.v. (Zhao, 1985)

Medical uses

The World Health Organization recommends intramuscular or intravenous artesunate as the first line treatment for severe malaria.[2]Artesunate was shown to prevent more deaths from severe malaria than quinine in two large multicentre randomized controlled trials from Africa[3] and Asia.[4] A subsequent systematic review of seven randomized controlled trials found this beneficial effect to be consistent across all trials.[5]

For severe malaria during pregnancy, there is less certainty about the safety of artesunate during the first trimester but artesunate is recommended as first-line therapy during the second and third trimesters.[6]

Artesunate is also used to treat less severe forms of malaria when it can be given orally, but should always be taken with a second antimalarial such as mefloquine or amodiaquine to avoid the development of resistance.[2]

While artesunate is used primarily as treatment for malaria, there is some evidence that it may also have some beneficial effects inSchistosoma haematobium infection,[7] but this needs confirming in large randomized trials.

Adverse effects

Artesunate is generally safe and well-tolerated. The best recognised side effect of the artemesinins that they lower reticulocyte counts.[8] This is not usually of clinical relevance.

Delayed haemolysis (occurring around two weeks after treatment) has been observed in patients treated with artesunate for severe malaria.[9] Whether or not this haemolysis is due to artesunate, or to the malaria itself is unclear.[10]

The safety of artesunate in pregnancy is unclear. There is evidence of embryotoxicity in animal models (defects in long bones and ventricular septal defects in the heart in rates and monkeys). However, observational evidence from 123 human first-trimester pregnancies showed no evidence of damage to the fetus.[11]

Synthesis

Artesunate is prepared from dihydroartemisinin (DHA) by reacting it with succinic acid anhydride in basic medium. Pyridine as base/solvent, sodium bicarbonate in chloroform and catalyst DMAP (N,N-dimethylaminopyridine) and triethylamine in 1,2-dichloroethane have been used, with yields of up to 100%. A large scale process involves treatment of DHA indichloromethane with a mixture of pyridine, a catalytic amount of DMAP and succinic anhydride. The dichloromethane mixture is stirred for 6–9 h to get artesunate in quantitative yield. The product is further re-crystallized from dichloromethane. alpha-Artesunate is exclusively formed (m.p 135–137˚C).

Artemisinin and its ether and ester derivatives show antimalarial activity against multidrug resistant strains. Ether derivatives like arteether and artemether shows better activity but they suffer from some limitation like solubility, short half life. Unlike ether derivatives, ester derivatives like artesunate has increased solubility and improved pharmacokinetic properties. The water insoluble dihydroartimisinin hemisuccinate is given orally in tablet form and water soluble artesunate sodium is given as LV.

Artesunate was first prepared by Chinese scientists, using different methods. One of them describes acylation of dihydroartemisinin by succinic anhydride in pyridine at 300C for 24 hr with yield of 60%. In another method, described in Acta. Chim. Sinica 40(6), 557-561., ester derivatives of dihydroartemisinin was prepared in presence of 4- (N, N-dimethylamino) pyridine and triethylamine as basic catalyst and 1 ,2 dichloroethane as solvent. The reaction is continued until complete conversion of dihydroartemisinin and product is isolated and purified by silica gel column giving overall yield 60-90%.

Another improved method disclosed in US patent 5654446, describes preparation of artesunate from dihydroartemisinin and succinic anhydride in presence of triethylamine as basic catalyst and in low boiling water miscible dry solvent like acetone. After completion of reaction, mixture is acidified and diluted with water to get artesunate. The yield of esterification is 96%.

U.S. patent 6677463 discloses one pot method for preparation of artesunate from artemisinin. Method describes reduction of artermisinin to dihydroartemisinin in presence of polyhydroxy compound and sodium borohydride. After completion of reaction succinic anhydride and anion exchange resin was added to reaction mass and stirred for 2 hrs. Then cold water was added and product was extracted with ethylacetate hexane mixture in pH range of 6-7. Distilling off the solvent yields the crude artesunate which on silica gel column purification gives 96 % of pure artesunate. The process is complex and time consuming as it involves chromatographic purification step.

……………………………..

Chemical structure for artesunate

http://www.google.com/patents/WO2008087667A1?cl=en

Example 1 discloses the process for obtaining artesunate. The process involves reducing artemisinin to dihydroartemisinin in presence of 1, 2-propanediol and sodium borohydride in a solvent mixture of hexane and isopropanol to give dihydroartemisinin in a yield of 92%. The ratio of artemisinin to 1 , 2-propanediol is 1 :0.66 w/w and the ratio of artemisinin to sodium borohydride is 1 :0.33 w/w. The high yield is attributed to the combination of 1 , 2-propanediol and sodium borohydride in a solvent mixture of hexane and isopropanol that could not be derived from prior art. The dihydroartemisinin is esterified using succinic anhydride and imidazole to give the artesunate in a yield of 100% in 40 min. The ratio of artemisinin to succinic anhydride is 1 :0.52 w/w and that of artemisinin to imidazole is 1 :0.2 w/w. Further, high yield of artesunate obtained in less time was due to imidazole catalyst that accelerates the rate of reaction. Moreover, the process of the present disclosure does not employ purification over silica gel as is in the prior art, but the pure compound is obtained by simple crystallization using suitable solvent.

Example 2 describes the process for obtaining artesunate. The process involves reducing artemisinin to dihydroartemisinin as in example 1. The dihydroartemisinin is esterified using succinic anhydride and imidazole to give the artesunate in a yield of

100% in 25 min. The ratio of artemisinin to succinic anhydride is 1 :0.52 w/w and that of artemisinin to imidazole is 1 :0.3 w/w.

Example 3 describes the process for obtaining artesunate involving reducing artemisinin to dihydroartemisinin in presence of 1, 2-propanediol and sodium borohydride in a solvent mixture of hexane and isopropanol to give dihydroartemisinin in a yield of 88% in 40 min. The ratio of artemisinin to 1, 2-propanediol is 1 :0.8 w/w and the ratio of artemisinin to sodium borohydride is 1 :0.4 w/w. The dihydroartemisinin is esterified using succinic anhydride and imidazole to give the artesunate in a yield of 86%. The ratio of artemisinin to succinic anhydride is 1 :0.52 w/w and that of artemisinin to imidazole is 1 :0.2 w/w.

Example 4 describes the process for obtaining artesunate involving reducing artemisinin to dihydroartemisinin as in example 1. The dihydroartemisinin is esterified using succinic anhydride and imidazole to give the artesunate in a yield of 90% in 210 min. The ratio of artemisinin to succinic anhydride is 1 :0.52 w/w and that of artemisinin to imidazole is 1 :0.1 w/w.

Example 5 describes the process for obtaining artesunate involving reducing artemisinin to dihydroartemisinin as in example 1. The dihydroartemisinin is esterified using succinic anhydride and imidazole in dichloromethane to give the artesunate in a yield of 92% in 60 min. The ratio of artemisinin to succinic anhydride is 1:0.44 w/w and the ratio of artemisinin to imidazole is 1 :0.2 w/w.

Example 6 describes the process for obtaining artesunate involving reducing artemisinin to dihydroartemisinin as in example 1. The dihydroartemisinin is esterified using succinic anhydride and imidazole in acetonitrile to give the artesunate in a yield of 92% in 180 min. The ratio of artemisinin to succinic anhydride is 1:0.52 w/w and that of artemisinin to imidazole is 1 :0.2 w/w.

Example 1 Artemisinin (1.0 g) and 1, 2-propanediol (0.66 g) was added to a mixture of isopropanol (3.5 ml) and hexane (10 ml) and the suspension was stirred for 2 minutes at 2O0C followed by the addition of Sodium borohydride (0.33 gm). After 2 minutes of stirring, dihydroartemisinin started precipitating and the reaction mixture was further stirred for about 8 minutes at 2O0C. Water (10 ml) was added to the reaction mixture and stirred for 10 minutes at 100C. Solid was filtered, washed with hexane (2 * 20 ml) and dried to yield 0.92 g (92% w/w) dihydroartemisinin.

Dihydroartemisinin (0.92 g) was stirred in dichloromethane (10 ml) for 2 minutes at room temperature. Succinic anhydride (0.52 g) and imidazole (0.2 g) were added to this solution and stirred for 40 minutes. The pH of reaction mixture was adjusted to 5-6 and organic layer was washed with water, dried and concentrated to oily mass. The oily mass was dissolved in methanol (1.5 ml) and stirred for 2 min to obtain a clear solution. Water (ImI) was added dropwise to this solution to start the precipitation of artesunate and the suspension was stirred for 5 minutes. The solid was filtered, washed with cold water (2 x 2ml) and dried to yield 1.0 g of artesunate. The overall yield of artesunate was 100 % w/w.

Example 2

Reduction of artemisinin to dihydroartemisinin was carried out as described in Example 1. Dihydroartemisinin (0.92 g) was stirred in dichloromethane (10 ml) for 2 minutes at room temperature. Succinic anhydride (0.52 g) and imidazole (0.3 g) were added to this solution and stirred for 25 minutes. The pH of reaction mixture was adjusted to 5-6 and organic layer was washed with water, dried and concentrated to oily mass. The oily mass was dissolved in methanol (1.5 ml) and stirred for 2 min to obtain a clear solution. Water (ImI) was added dropwise to this solution to start the precipitation of artesunate and the suspension was stirred for 5 minutes. The solid was filtered, washed with cold water (2 χ 2 ml) and dried to yield 1.0 g of artesunate. The overall yield of artesunate was 100 % w/w.

Example 3

Artemisinin (1.0 g) and 1, 2-propanediol (0.8 g) was added to a mixture of isopropanol (3.5 ml) and hexane (10 ml) and the suspension was stirred for 2 minutes at 2O0C followed by the addition of Sodium borohydride (0.4 g). After 2 minutes of stirring, dihydroartemisinin started precipitating and the reaction mixture was further stirred for about 8 minutes at 200C. Water (7.5 ml) was added to the reaction mixture and stirred for 10 minutes at 100C. Solid was filtered, washed with hexane (2 ^ 2 ml) and dried to yield 0.88 g (88% w/w) dihydroartemisinin.

Dihydroartemisinin (0.88 g) was stirred in dichloromethane (10 ml) for 2 minutes at room temperature. Succinic anhydride (0.52 g) and imidazole (0.2 g) were added to this solution and stirred for 40 minutes. The pH of reaction mixture was adjusted to 5-6 and organic layer was washed with water, dried and concentrated to oily mass. The oily mass was dissolved in methanol (1.5 ml) and stirred for 2 min to obtain a clear solution. Water (ImI) was added dropwise to this solution to start the precipitation of artesunate and the suspension was stirred for 5 minutes. The solid was filtered, washed with cold water (2 x 2ml) and dried to yield 0.86 g of artesunate. The overall yield of artesunate was 86 % w/w.

Example 4

Reduction of artemisinin to dihydroartemisinin was carried out as described in Example 1. Dihydroartemisinin (0.92 g) was stirred in dichloromethane (10 ml) for 2 minutes at room temperature. Succinic anhydride (0.52 g) and imidazole (0.1 g) were added to this solution and stirred for 210 minutes. The pH of reaction mixture was adjusted to 5-6 and organic layer was washed with water, dried and concentrated to oily mass. The oily mass was dissolved in methanol (1.5 ml) and stirred for 2 min to obtain a clear solution. Water (ImI) was added dropwise to this solution to start the precipitation of artesunate and the suspension was stirred for 5 minutes. The solid was filtered, washed with cold water (2 x 2 ml) and dried to yield 0.9 g of artesunate. The overall yield of artesunate was 90 % w/w.

Example 5

Reduction of artemisinin to dihydroartemisinin was carried out as described in Example 1. Dihydroartemisinin (0.92 g) was stirred in dichloromethane (10 ml) for 2 minutes at room temperature. Succinic anhydride (0.44 g) and imidazole (0.2 g) were added to this solution and stirred for 60 minutes. The pH of reaction mixture was adjusted to 5-6 and organic layer was washed with water, dried and concentrated to oily mass. The oily mass was dissolved in methanol (1.5 ml) and stirred for 2 min to obtain a clear solution. Water (ImI) was added dropwise to this solution to start the precipitation of artesunate and the suspension was stirred for 5 minutes. The solid was filtered, washed with cold water (2 x 2 ml) and dried to yield 0.92 g of artesunate. The overall yield of artesunate was 92 % w/w. Example 6

Reduction of artemisinin to dihydroartemisinin was carried out as described in

Example 1. Dihydroartemisinin (0.92 g) was stirred in acetonitrile (10 ml) for 2 minutes at room temperature. Succinic anhydride (0.52 g) and imidazole (0.2gm) were added to this solution and stirred for 180 minutes. The pH of reaction mixture was adjusted to 5-6 and it was extracted with dichloromethane (10 ml). The organic layer was washed with water (20 ml), dried and concentrated to oily mass. The oily mass was dissolved in methanol (1.5 ml) and stirred for 2 min to obtain a clear solution. Water (ImI) was added dropwise to this solution to start the precipitation of artesunate and the suspension was stirred for 5 minutes. The solid was filtered, washed with cold water (2 x 2 ml) and dried to yield 0.92 g of artesunate. The overall yield of artesunate was 92 % w/w.

Mechanisms of action

In a hematin dependent manner, artesunate has been shown to potently inhibit the essential Plasmodium falciparum exported protein 1 (EXP1), a membrane glutathione S-transferase.[12]

Drug resistance

Clinical evidence of drug resistance has appeared in Western Cambodia, where artemisinin monotherapy is common.[13] There are as yet no reports of resistance emerging elsewhere.

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http://www.google.com/patents/WO2004050661A1?cl=en

Malaria is caused by protozoan parasites, notably Plasmodium falciparum. The range of drugs available in the market for prevention and treatment of malaria is limited, and there are problems of drag resistance. Artemisinin and its derivatives: artemether and arteether (oil soluble), artelinate and artesunate (water soluble), are a class of anti-malarial compounds derived from Artemisia annua which are now proving their promising activity and are being used for the treatment of uncomplicated/severe complicated/cerebral and multi drug resistant malaria. The chemistry and the anti-protozoal action of these compounds, described in the publications are listed as references cited.

The water-insoluble artesunic acid is customarily administered orally in the form of tablets or rectally in the form of suppositories, while the water- soluble artesunate is administered intravenously.

Artesunic acid together with a number of other Cio-ester and CiQ-ether” derivatives of dihydroartemisinin, were prepared for the first time by Chinese scientists at the end of 1979 to the beginning of 1980. Shaofeng et al., H Labeling of QHS Derivatives, Bull. Chin. Materia Medica 6 (4), 25-27 (1981) and Li et al, Synthesis of Ethers. Carboxylic esters and carbonates of Dihydroartemisinin, Acta Pharm. Sin 16(6), 429-39, 1981) describe the preparation of artesunic acid by acylation of dihydroartemisinin with succinic anhydride in pyridine. The above mentioned publications describe a general method for preparing various dihydroartemisinin Cι0-esters and also provide a process for preparing artesunic acid in a yield of 60% by means of warming dihydroartemisinin and succinic anhydride in pyridine at 30° C for 24 hours.

Ying et al. in the Synthesis of some carboxylic esters and carbonates of Dihydroartemisinin by using 4-(N, N-Dimethylamino) pyridine as an active acylation catalyst, Acta Chim Sinica 40 (6), 557-561 982) proposed an improved version of the acylation of dihydroartemisinin. The said publication described in detail with the aid of the preparation of dihydroartemisinin – 10-valerate the aforesaid process. In this process dihydroartimisinin was dissolved in 1,2-dichloroethane and treated with valeric anhydride, 4-(N, N-dimethylamino) pyridine and triethylamine, and the mixture was stirred at room temperature until dihydroartemisinin had been used up. The reaction mixture was then acidified with dilute hydrochloric acid and the aqueous phase was separated off. The oily residue, obtained after washing and drying the organic phase and distilling off the solvent, was purified by chromatography on silica gel using petroleum ether 60-80° C degree/ethyl acetate (10:1) as an eluent. The use of this procedure for the preparation of the artesunic acid from dihydroartemisinin with succinic anhydride and 4-(N, N-dimethylamino) pyridine afforded artesunic acid in a yield of 65% in 5 hours.

U.S. Patent No. 5,654,446 granted to Ognyanov et al. titled “Process for preparation of Dihydroartemisinin Hemisuccinate (artesunic acid)”, dated August 5, 1997 teaches a process for preparing o α-artesunic acid by acylation of dihydroartemisinin with succinic anhydride, in the presence of trialkylamines and their mixture in a low boiling, neutral water miscible, inert organic solvent or solvent mixture at 20-60°C in 0.5 hours and the artesunic acid is then isolated directly at pH 5 to 8 in 91.8 to 97.2% yield.

The above mentioned methods carry some disadvantages being less cost effective and more time consuming as compared to the present invention it should be noted that all the above referenced methods require two separate steps to convert artemisinin into 10-esters of dihydroartemisinin i.e. (a) reduction of artemisinin into dihydroartemisinin in the first pot following by isolation of dihydroartemisinin, and (b) esterification of dihydroartemisinin into different esters in the second pot.

Further, solvent pyridine or 1,2 dichloroethane and catalyst, 4 (N, N-dimethylamino) pyridine used in these processes are not acceptable according to the health standard. Hence there is a need to provide a single step process that overcomes the above-mentioned disadvantages.

EXAMPLE 1

Artemisinin (500mg) and polyhydroxy compound (dextrose, 2.5g) are stirred in 1,4-dioxan (15ml) at room temperature for 5 minutes. Sodium borohydride (2.5g) is added slowly for 10 minutes and the reaction mixture is stirred for about 2 hours at room temperature (20- 30° C). After completion of the reaction (Checked by TLC), succinic anhydride (250 mg) and anion exchange (basic) resin (1.5g) are added at room temperature and the reaction mixture is stirred further for 2 hours at room temperature. Cold water (50 ml) is added to the reaction mixture and pH is adjusted between 6-7 with dilute acetic acid and extracted with 40% ethyl acetate in hexane (3 x 25 ml). The combined extract is washed with water (50 ml). The ethyl acetate π-hexane extract is dried over anhydrous sodium sulphate and evaporation of the solvent yield 655 mg of crude artesunic acid which upon purification over silica gel (1:5 ratio) with 20-30% ethyl acetate in hexane, furnish pure artesunic acid in 93% w/w (465 mg) yield (according to CO-TLC). After drying the pure α-artesunic acid, mp 140-142° C is characterized by spectral analysis.

EXAMPLE 2

Artemisinin (500 mg), polyhydroxy compound (dextrose, 2.0g) are stirred in 1,4-dixan (10 ml). Sodium borohydride (2.5g) is added slowly for 10 minutes and the reaction mixture is stirred for about 2 hours at room temperature (20-30° C). After completion of the reduction step, succinic anhydride (250 mg) and triethylamine (1ml) are added and the reaction mixture is further stirred for 2 hours at room temperature (20-30 degree C). After usual work up and purification of crude product (690mg) through column chromatography (1:4 ratio) 91.2%) pure artesunic acid is obtained.

EXAMPLE 3 Artemisinin (500 mg), polyhydroxy compound (dextrose, 2.0g) are stirred in tetrahydrofuran (10 ml). Sodium borohydride (2.5g) is added slowly for 10 minutes and the reaction mixture is stirred for about 2 hours at room temperature. After completion of the reduction step succinic anhydride (250 mg) and triethylamine (1ml) are added and the reaction mixture is further stirred for 2 hours at room temperature. After usual work up and purification of the crude product (615mg) through column chromatography 87.4% pure artesunic acid is obtained.

EXAMPLE 4

Artemisinin (500 mg) and polyhydroxy compound (dextrose, 2g) are stirred in dioxan (15 ml) for 5 minutes. Sodium borohydride (2.4gm) is added slowly and the reaction mixture is stirred for 2 hours at room temperature (20-30 degree C). After completion of the reduction step succinic anhydride (250 mg) and sodium bicarbonate (3.5g) are added and the reaction mixture is further stirred for 2 hours. After usual workup and purification of impure reaction product (650 mg), 89.6%w/w pure artesunic acid is obtained.

EXAMPLE 5

Artemisinin (500mg) and cation exchange resin (lg) are stirred in tetrahydrofuran (10ml) at room temperature for 5 minutes. Sodium borohydride (250mg) is added slowly for 10 minutes and the reaction mixture is stirred for about 30 minutes at room temperature (20- 35 degree C). After completion of the reaction succinic anhydride (250mg) and triethylamine (0.7ml) are added at room temperature and the reaction mixture is stirred further for 1 hours at room temperature. The resin is filtered. After usual workup and column chromatography of the crude product (710mg), 480mg of pure artesunic acid (yield

= 96%w/w) is obtained.

EXAMPLE 6

Artemisinin (500mg) and cation exchange resin (lg) are stirred in 1,4 dioxan (10ml) at room temperature for 5 minutes. Sodium borohydride (250mg) is added slowly for 10 minutes and the reaction mixture is stirred for about 30 minutes at room temperature (20-35 degree C). After completion of the reaction succinic anhydride (250mg) and triethylamine (0.7ml) are added slowly at room temperature and the reaction mixture is stirred further for 1.25 hours at room temperature. After usual work up and purification of the crude artesunic acid (680mg) pure product in 91.7% w/w is obtained.

EXAMPLE 7

Artemisinin (500 mg), cation exchange resin (lOg) are stirred in 1,4 dioxan (10 ml). Sodium borohydride (250mg) is added slowly for 10 minutes and the reaction mixture is stirred for about 45minutes at room temperature (20-35 degree C). After completion of the reduction step succinic anhydride (250 mg) and sodium bicarbonate (2.5g) are added and the reaction mixture is further stirred for 1.5 hours at room temperature (20-35 degree C). After usual work up and purification of the crude artesunic acid (630mg) pure product in 85%o w/w yield is obtained.

EXAMPLE 8 Artemisinin (500 mg) and cation exchange resin (lg) are stirred in tetrahydrofuran (15 ml) for 5 minutes. Sodium borohydride (2.4gm) is added slowly and the reaction mixture is stirred for 45 minutes at room temperature (20-35 degree C). After completion of the reduction reaction, succinic anhydride (245 mg) and sodium bicarbonate (3.5g) are added and the reaction mixture is further stirred for 1.25 hours. After usual workup and purification of impure reaction product (650 mg), pure artesunic acid in 93%w/w yield is obtained.

EXAMPLE 9

Artemisinin (lOOmg) and cation exchange resin (200mg) are stirred in tetrahydrofuran (3ml) at room temperature for 5 minutes. Sodium borohydride (50mg) is added slowly for 10 minutes and the reaction mixture is stirred for about 30 minutes at room temperature (20-35 degree C). After completion of the reaction propionic anhydride (0.5ml) and triethylamine (0.2ml) are added at room temperature and the reaction mixture is stirred further for 1.5 hours at room temperature. After usual workup and purification of the crude products through preparative TLC 44 mg of pure dihydroartemisinin 10- propionate characterized by its spectral analysis is obtained.

EXAMPLE 10

Artemisinin (lOOmg) and cation exchange resin (200mg) are stirred in tetrahydrofuran (3ml) at room temperature for 5 minutes. Sodium borohydride (50mg) is added slowly for

10 minutes and the reaction mixture is stirred for about 30 minutes at room temperature (20-35 degree C). After completion of the reaction chloroacetic anhydride (50mg) and triethylamine (0.2ml) are added at room temperature and the reaction mixture is stirred further for 1.5 hours at room temperature. After usual workup and purification of the crude products through preparative TLC 35mg of pure dihydroartemisinin 10- chloroacetate characterized by its spectral analysis is obtained.

EXAMPLE 11

Artemisinin (lOOmg) and cation exchange resin (200mg) are stirred in tetrahydrofuran (3ml) at room temperature for 5 minutes. Sodium borohydride (50mg) is added slowly for 10 minutes and the reaction mixture is stirred for about 30 minutes at room temperature (20-35 degree C). After completion of the reaction acetic anhydride (50mg) and triethylamine (0.2ml) are added at room temperature and the reaction mixture is stirred further for 1.5 hours at room temperature. After usual workup and purification of the crude products through preparative TLC 42mg of pure dihydroartemisinin 10-acetate identified by its spectral analysis is obtained.

EXAMPLE 12

Artemisinin (5g) and cation exchange resin (lOg) are stirred in tetrahydrofuran (60ml) at room temperature for 5 minutes. Sodium borohydride (2.5g) is added slowly for 20 minutes and the reaction mixture is stirred for about 1 hour at room temperature (20-35 degree C). After completion of the reaction succinic anhydride (2.5g) and triethylamine (6ml) are added at room temperature and the reaction mixture is stirred further for 1.5 hours at room temperature. After usual workup and purification of the crude product

(6.92g) through CC pure artesunic acid in 94.6%w/w yield is obtained.

ADVANTAGES OF THE PRESENT INVENTION

1. The two pot reactions: reduction of artemisinin into dihydroartemismin and esterification of dihydroartemisinin to artesunic acid carried out in one pot avoids the process of isolation of dihydroartemisinin is avoided which saves chemicals, labour and losses of dihydroartemisinin in isolating it.

2. Conversion of artemisinin into artesunic acid in one pot takes place in about 2-5 hours and is a less time consuming method as compared to previously reported methods in which conversion of artemisinin into dihydroartemisinin in first pot followed by isolation of dihydroartemisinin and its esterification into artesunic acid in the second pot is also a long process. 3. The conversion of artemisinin into artesunic acid in one pot is carried out at room temperature (20-35 degree C) and thereby avoids use of cooling unit.

4. The solvent used to carry out the reduction reaction is also being used in esterification and thus enabling the process cost effective.

5. The catalysts, polyhydroxy compound or cation exchange resin used to carry out the reduction of artemisinin into dihydroartemisinin at room temperature (20-35°C) are cost effective.

6. The conversion of artemisinin into crude artesunic acid followed by workup and purification to yield pure product takes 6-10 hours as compared to previously reported methods (about 20-40 hours) and thus the process is less time consuming.

7. The yield of final product in the present invention i.e. pure artesunic acid is upto 96%, w/w.

8. Thus, this improved process which avoids the disadvantages of previously known process is suitable for the preparation of artesunic acid in large scale.

References

  1.  “WHO Model List of EssentialMedicines”. World Health Organization. October 2013. Retrieved 22 April 2014.
  2.  World Health Organization. “Guidelines for the treatment of malaria; Second edition 2010”. World Health Organization. Retrieved 10 January 2014.
  3.  Dondorp AL, et al. (2010). “Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): an open-label, randomised trial”.The Lancet 376 (9753): 1647–1657. doi:10.1016/S0140-6736(10)61924-1.PMC 3033534. PMID 21062666.
  4.  South East Asian Quinine Artesunate Malaria Trial (SEAQUAMAT) (2005). “Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial”. The Lancet 366 (9487): 717–725. doi:10.1016/S0140-6736(05)67176-0. PMID 16125588.
  5. Jump up^ Sinclair, D; Donegan, S; Isba, R; Lalloo, DG (Jun 13, 2012). “Artesunate versus quinine for treating severe malaria.”. The Cochrane database of systematic reviews 6: CD005967.doi:10.1002/14651858.CD005967.pub4. PMID 22696354.
  6. Jump up^ WHO (2007). Assessment of the safety of artemisinin compounds in pregnancy. World Health Organization, Geneva.
  7. Jump up^ Boulangier D, Dieng Y, Cisse B, et al. (2007). “Antischistosomal efficacy of artesunate combination therapies administered as curative treatments for malaria attacks”. Trans R Soc Trop Med Hyg 101 (2): 113–16. doi:10.1016/j.trstmh.2006.03.003.PMID 16765398.
  8. Jump up^ Clark RL (2012). “Effects of artemisinins on reticulocyte count and relationship to possible embryotoxicity in confirmed and unconfirmed malarial patients”. Birth defects research. Part A, Clinical and molecular teratology 94 (2): 61–75.doi:10.1002/bdra.22868.
  9.  Rolling T, Agbenyega T, Issifou S, et al. (2013). “Delayed hemolysis after treatment with parenteral artesunate in African children with severe malaria—a double-center prospective study.”. J Infect Dis 209 (12): 1921–8. doi:10.1093/infdis/jit841.PMID 24376273.
  10.  Clark RL (2013). “Hypothesized cause of delayed hemolysis associated with intravenous artesunate.”. Med Hypotheses 82 (2): 167–70.doi:10.1016/j.mehy.2013.11.027. PMID 24370269.
  11.  Clark RL (2009). “Embryotoxicity of the artemisinin antimalarials and potential consequences for use in women in the first trimester.”. Reprod Toxicol 28 (3): 285–96.doi:10.1016/j.reprotox.2009.05.002. PMID 19447170.
  12.  Lisewski, A. M.; Quiros, J. P.; Ng, C. L.; Adikesavan, A. K.; Miura, K; Putluri, N; Eastman, R. T.; Scanfeld, D; Regenbogen, S. J.; Altenhofen, L; Llinás, M; Sreekumar, A; Long, C; Fidock, D. A.; Lichtarge, O (2014). “Supergenomic Network Compression and the Discovery of EXP1 as a Glutathione Transferase Inhibited by Artesunate”. Cell 158(4): 916–28. doi:10.1016/j.cell.2014.07.011. PMID 25126794. edit
  13. White NJ (2008). “Qinghaosu (Artemisinin): The price of success”. Science 320 (5874): 330–334. doi:10.1126/science.1155165. PMID 18420924.

Literature References:

Derivative of artemisinin, q.v. Prepn: China Cooperative Research Group on Qinghaosu, J. Tradit. Chin. Med. 2, 9 (1982).

Absolute configuration: X.-D. Luo et al., Helv. Chim. Acta 67, 1515 (1984).

GC/MS determn.: A. D. Theoharideset al., Anal. Chem. 60, 115 (1988);

HPLC determn in plasma: H. Naik et al., J. Chromatogr. B 816, 233 (2005).

Pharmacology: Y. Zhao, J. Trop. Med. Hyg. 88, 391 (1985). Antimalarial activity: W. Peters et al., Ann. Trop. Med. Parasitol. 80, 483 (1986); A. J. Linet al., J. Med. Chem. 30, 2147 (1987).

Inhibition of cytochrome oxidase: Y. Zhao et al., J. Nat. Prod. 49, 139 (1986).

Toxicology: China Cooperative Research Group on Qinghaosu, J. Tradit. Chin. Med. 2, 31 (1982).

Series of articles on chemistry, pharmacology, and antimalarial efficacy: ibid. 3-50.

Clinical trial as add-on therapy in pediatric malaria: L. von Seidlein et al.,Lancet 355, 352 (2000).

Review: R. N. Price, Expert Opin. Invest. Drugs 9, 1815-1827 (2000).

THE CHEMISTRY AND SYNTHESIS OF QINGHAOSU DERIVATIVES” JOURNAL OF TRADITIONAL CHINESE MEDICINE, BEIJING, CN, vol. 2, no. 1, 1982, pages 9-16, XP008019918 ISSN: 0255-2922
2 * DATABASE CAPLUS [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; PHAN DINH CHAU ET AL: “Semisynthesis of an antimalarial artesunate” retrieved from STN Database accession no. 119:249761 XP002250029 -& TAP CHI DUOC HOC (1992), (5), 10-12 , XP001162710
3 * HAYNES, RICHARD K. ET AL: “C-10 ester and ether derivatives of dihydroartemisinin – 10-.alpha. artesunate, preparation of authentic 10-.beta. artesunate, and of other ester and ether derivatives bearing potential aromatic intercalating groups at C-10” EUROPEAN JOURNAL OF ORGANIC CHEMISTRY (2002), (1), 113-132 , XP002250027
4 * LI Y ET AL: “Studies on artemisinine analogs. I. Synthesis of ethers, carboxylates and carbonates of dihydroartemisinine” YAO HSUEH HSUEH PAO – ACTA PHARMACEUTICA SINICA, BEIJING, CN, vol. 16, no. 6, June 1981 (1981-06), pages 429-439, XP002119789 ISSN: 0513-4870

Artesunate

How does Artesunate kill cancer?

Artesunate is a drug that was initially designed for combating malaria, however, recently it has shown great promise as a cancer therapy1,2,3. It has been used in combination with some chemotherapies to improve outcomes in advanced cancer patients5. When fighting cancer it is important to use every tool at your disposal to weaken the cancer and strengthen your own cells. Artesunate is another weapon in the arsenal of natural remedies that can make a significant difference in the fight against cancer.

The mechanism of action for artesunate in the context of cancer therapy is very well defined. Cancer cells have a tendency to absorb iron at high levels and this is thought to accelerate the mutation rate within these cells. Iron reacts with oxygen to form free radicals, which are reactive molecules that damage DNA. In normal cells this reaction is a problem; in cancer cells it allows them to mutate and develop resistance to therapies. Artesunate activates mitochondrial apoptosis by iron catalyzed lysosomal reactive oxygen species production4. In other words, this drug will use the iron within the cancer cells against them.

http://yaletownnaturopathic.com/how-does-artesunate-kill-cancer/

Dr. Adam McLeod is a Naturopathic Doctor (ND), BSc. (Hon) Molecular biology, First Nations Healer, Motivational Speaker and International Best Selling Author. He currently practices at his clinic in Vancouver, British Columbia where he focuses on integrative oncology. http://www.yaletownnaturopathic.com
References:

1) MIYACHI, HAYATO, and CHRISTOPHER R. CHITAMBAR. “The anti-malarial artesunate is also active against cancer.” International journal of oncology 18 (2001): 767-773.

2) Michaelis, Martin, et al. “Anti-cancer effects of artesunate in a panel of chemoresistant neuroblastoma cell lines.” Biochemical pharmacology 79.2 (2010): 130-136.

3) Du, Ji-Hui, et al. “Artesunate induces oncosis-like cell death in vitro and has antitumor activity against pancreatic cancer xenografts in vivo.” Cancer chemotherapy and pharmacology65.5 (2010): 895-902.

4) Efferth, Thomas, et al. “Enhancement of cytotoxicity of artemisinins toward cancer cells by ferrous iron.” Free Radical Biology and Medicine 37.7 (2004): 998-1009.

5) Zhang, Z. Y., et al. “[Artesunate combined with vinorelbine plus cisplatin in treatment of advanced non-small cell lung cancer: a randomized controlled trial].” Zhong xi yi jie he xue bao= Journal of Chinese integrative medicine 6.2 (2008): 134-138.

WormwoodArtesunate is…

a water-soluble ‘artemesinin’ drug derived from the ‘sweet wormwood’ plant, Artemsia annua, an herb used to treat infections and other illnesses in China for centuries. Interestingly, according to Wikipedia artemsia was lost as an herbal remedy in China until 1970 when an ancient Chinese medical manual dating back to 340 AD was found. The active ingredient in the plant – artemesinin – was isolated by scientists and it anti-malarial properties were quickly noted (1972). It is now used to treat malaria and schistosoma infections.

Artesunate also reduces anti-oxidant activity in the red blood cell thus exposing the cell to high free radical levels. Artesunate is currently being studied as an adjunct to chemotherapeutic agents because of its ability to induce cancerous cells to commit suicide (apoptosis) by inducing high rates of oxidative stress. The ability of the antioxidant NAC to thwart Artesunate’s effects in one study substantiated the important role increased free radical production plays in the drugs effect.

Malaria – Artemesia annua is native to China but has become naturalized around the world including the eastern United States. Artesunate was recently approved for emergency use in patients with severe malaria in the United States.

In April 2009 the FDA approved CoArtem which contains a derivative of artemesinin and a broad spectrum antibiotic called lumefantrine. Upon binding to infected red blood cells artesunate triggers the release of oxygen and carbon-based free radicals that attack proteins in the parasites.

Herpesviruses – Recent culture cell experiments indicated Artesunate was effective at significantly reducing viral protein production in HHV-6A infected cells. A 2005 in vitro study suggested Artesunate significantly reduced cytomegalovirus replication in cells. Because Artesunate effects HHV-6 early in its life cycle it may hold special promise in the kind of smoldering infections that may occur in chronic fatigue syndrome (ME/CFS).

Artesunate’s effects on herpesviruses, however, have not been well studied with just five studies published to date. Interest in this drug appears to be increasing, however, three of the five studies were published in 2008.

Artesunate May Work in Chronic Fatigue Syndrome (ME/CFS) Because..

it may be able to reduce herpesvirus activity in some patients. It’s use, however, is highly experimental.

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