A Natural Mechanism - Essay Example

Introduction: It’s a natural mechanism for plants to defend themselves through synthesis of chemically bioactive compounds which have been proven to have medicinal properties 5, (14). Tropical plants have served as a source of medicines for people for millennia 16. Naphthylisoquinoline is an unusual class of alkaloids which are grouped under the miscellaneous types of alkaloids as they are not easily assignable to the other classes of alkaloids 5. These alkaloids have medicinal, drug, systematic and biosynthetic properties. They are a group of 20 alkaloids, with special biosynthetic characteristics 1, 5. Structurally they originate, from the acetate polymalonate pathway and not from amino acids 1. They exist as monomeric and dimeric naphthylisoquinoline alkaloids. The monomeric alkaloids which possess a naphthalene-isoquinolene linkage and the dimeric naphthylisoquinolines include the michellamines. The Michellamines comprise two monomeric naphthylisoquinoline coupled together. The Michellamine dimers A, B and C are isolated from the leaves of Ancistrocladus korupensis 1,5,16, 18, 21. These dimeric alkaloids possess highly useful medicinal properties like antiviral and antiparasitic, which are distinct from their monomeric counterparts 13. In addition they also possess several pharmacological, toxicological and antiviral properties such as the inhibition of the cytopathic effects of the Human Immunodeficiency virus which is the major causative agent of AIDS 1, 9, 12, 19. Experiments show that Michellamines A and B give protection in vitro to human CEM-SS lymphocytes against the cytopathic effects of HIV virus (14)20, 21. Michellamine B has been shown to have inhibitory effect on drug-sensitive ,drug- Enzymatic assays showed Michellamine B to inhibit drug-sensitive, drug- resistant HIV-1and HIV-2 reverse transcriptases including the AZT resistant strains 16. Chemical Nature The naphthylisoquinoline alkaloids are exists as both monomers and dimmers. All the monomeric naphthylisoquinoline alkaloids are biaryls containing a naphthalene moiety and an isoquinoline moiety and these two are linked. They may contain a dihydrogenated (this is present in the said ref: 13,) or a tetra hydrogenated isoquinoline moiety 13. The linkage varies between the different alkaloids. There is also a restricted rotation about this linkage and hence these compounds exist as thermally stable atropisomers 1. This is an interesting characteristic feature among these alkaloids. These alkaloids are also unusual on account of the methyl group present at the 3- position and oxygenation at the 8 and/or 6 position of the isoquinoline ring, which suggest that they have a polyketide origin. These alkaloids can be grouped according to their biaryl linkages 1. Based on their sources the alkaloids are classified as Ancistrocladus alkaloids and Dioncophyllaceae alkaloids. The former is the largest group with the 5-1’ linkage. Another member of this group Ancistrocladidine possesses an uncommon 7-2’ linkage1. The dimeric forms are comprised of two monomeric units coupled together 1, 13. Michellamine A is a dimeric, 5-8’ linked alkaloid, from the same family, derived form the corresponding monomer Korupensamines A 1, 13. Its molecular formula is C46H48N2O8 14. It has a tetrahydroisoquinoline moiety coupled to a naphthalene moiety 13. The structure is as below 11, 12, 21 This not taken form ref. 12! (it is from11 and 12. I took the structure from the pdf ref that u had sent) Copied directly from “Bioorganic & Medicinal Chemistry Letters, Vol. 7, No. 20, pp. 2687-2690, 1997” ----- this diagram was taken from the pdf ref 12. the patent doc begins with these structures, hence I had written the ref as 12. The importance of the tetrahydroisoquinoline rings for the biological activity of the Michellamines were demonstrated in an experiment which used structurally analogues of Michellamines. From the study it was found that the analogues that had a dihydroxyphenyl substitution pattern similar to the naturally occurring michellamines were the only ones that elicited toxicity in the CEM-SS cells regardless of the substituents. These studies have indicated that the dihydroxy substitution pattern may play an important role in cellular toxicities and also the heterocyclic portion of the ring is a significant factor in determining the biological activity of these compounds 21. Botanical Sources The plant families Ancistrocladaceae and Dioncophyllaceae are the only (is this true?) (yes pls refer source 1)sources identified from which these naphthylisoquinoline alkaloids can be extracted 1. The small paleotropical family, Ancistrocladaceae that comes under the order of Theales, contains a single genus Ancistrocladus. This genus I known to consist of about 20 species distributed in Africa and Asia 11, 12. Dioncophyllum and Triphyophyllum are the members of Dioncophyllaceae 15. Michellamine A was initially isolated form a Central African plant tentatively identified as Ancistrocladus abbreviatus. However subtle differences were identified between the authentic specimen of A.abbreviatus and the above source plant. In addition the chemical and bioactivity analysis showed the presence of Michellamines only in the source species and not in any other Ancistrocladus species. Thus it was confirmed that the original Michellamine A containing plant, now officially named as A. korupensis (previously referred as A. sp novum) is the only known source of michellamines 8 (the link for ref 8 is : http://pubs.acs.org/cgi-bin/abstract.cgi/jmcmar/1994/37/i12/f-pdf/f_jm00038a003.pdf) This plant is endemic to South west Cameroon and Nigeria 2 and it differs from all the other Ancistrocladus species in having petals slightly shorter than the sepals; the petals are about twice as long in the other species. It is the leaves of the plant which are a rich source of Michellamine A and are widely used for its extraction 11. The Michellamines thus extracted from A. korupensis has been shown to exhibit potent antiviral activity against HIV-11, 2, 9. Extraction and Isolation of Michellamine A The isolation of Michellamines from the Ancistrocladus sp. involves the following steps 11, 12. 1. Extraction: Air-dried plant material consisting of leaves, stems and twigs are used for the extraction. It is then coarsely powdered and extracted with 1:1 MeOH : CH2 Cl2, followed by a second extraction with methanol. This crude extract is then dissolved in 5% aqueous HCl and extracted with CHCl3. the aqueous is then made alkaline using concentrated NH4OH to a pH of 10-11. This is then extracted with 4:1 CHCl3 : MeOH and then with 1:1 MeOH : CHCl3 . 2. Solvent – Solvent partitioning and Centrifugal partition chromatography: The extract is then dissolved in the lower phase of a 5:5:3 biphasic solvent system (CHCl3 : MeOH : 0.5% aqueous HBr) and is then placed in a centrifugal partition chromatography system in the descending mode and the released effluent is monitored at 270 nm. The final peak to elute contains the HBr salts of both Michellamine A and B plus a trace of C. The solvent is then removed by passing the eluent through gel permeation chromatography. 3. HPLC: The mixture is further separated with the amino-bonded phase HPLC using 43: 7 CHCl3 : MeOH / 0.075% (NH4)2 CO3 as the solvent. 4. Quantitation of Michellamines A and B by high pressure liquid chromatography has yielded the following result 20: The above method however did not fully resolve Michellamines A and B 20. By the above procedure the overall yield of Michellamine A extract from the crude organic extract is about 0.5- 2%. Variations in the above process have resulted in a higher yield of Michellamines A. The isolation of the compounds can be monitored using UV, TLC and anti-HIV bioassays. Spectroscopic feature of Michellamine A: Mass spectral analysis of the isolated fraction revealed that Michellamines A and B had similar molecular weights (m/z 756) and the molecular formula was established using accurate- mass, fast atom bombardment mass spectroscopy and was established as C46H48N2O8 11, 12, 14. The dimeric nature of Michellamine A was further established using the NMR studies 11. They are derived from the corresponding monomers Korupensamines A, also isolated from A. korupensis. The NMR data for Michellamine A is provided below (source: Process of preparing Michellamine Compounds, Issued US Patent Description) ______________________________________ NMR DATA FOR MICHELLAMINE A Carbon # δ (# attached H) 1 H δ (Multiplicity) J ______________________________________ (Hz) 1/1" 49.5 (1) 4.64 q 6.5 3/3" 45.2 (1) 3.54 ddq 11.8, 4.3, 6.5 4/4" 33.1 (2) (e)2.69 dd 18.6, 4.3; (a)2.05 dd 18.6, 11.8 4a/4a" 133.1 (0) 5/5" 120.3 (0) 6/6" 156.9 (0) 7/7" 102.0 (1) 6.40 s 8/8" 155.4 (0) 8a/8a" 113.1 (0) 1/1" 119.1 (1) 6.75 s 2/2" 137.6 (0) 3/3" 108.0 (0) 6.84 s 4/4" 158.1 (0) 4a/4a" 115.2 (0) 5/5" 152.2 (0) 6/6" 119.0 (0) 7/7" 134.8 (1) 7.30 s 8/8" 124.1 (0) 8a/8a" 136.6 (0) OMe/OMe 57.1 (3) 4.10 s Me-3/Me-3" 19.4 (3) 1.16 d 6.5 Me-1/Me-1" 18.4 (3) 1.57 d 6.5 Me-2/Me-2" 22.1 (3) 2.33 s ______________________________________ 13 C (125 MHz) and 1 H (500 MHz) NMR spectra of the HBr salt were recorded in d4methanol. # attached H determined from DEPT experiments. The designations "a" and "e" refer to axial and equatorial, respectively. Details of the spectral analysis: The C-NMR data of Michellamine A exhibits only 23 resonances. This indicates that the two naphthalene-isoquinoline moieties are equivalent. The linkage between the ring protons and the hydroxyl units and the linkage between the naphthalene and isoquinoline moieties were determined by Heteronuclear Multiple Quantum Coherance (HMQC) and by Heteronuclear Multiple Bond Coherance (HMBC). These data have suggested a 1, 3 linkage between the protons and the hydroxyl groups and the tetrahydroisoquinoline is linked to the naphthalene subunit by a bond C-5/C-5” to C-8/C-8”. The Naphthalene’s are therefore connected by C-6/C-6”.The methyl groups in the tetrahydroisoquinoline is attached to C-1/C-1” and C-3/C-3” 11, 12, 19. (there is no separate data for UV,IR and PKA of MA, all these reports only provide the NMR data) Biosynthesis of Michellamine A Michellamine A isolated from A.Korupensis has generated a lot of interest due to its cytopathic activity against HIV. It also possesses a high polarity due to the presence of free phenolic groups and two secondary amino groups in its structure, and the rare 5, 8’ coupling between the naphthalene and isoquinoline rings. The dimeric Michellamine A is synthezised from the monomer Korupensamine A ( structure 5 in figure below), which is also isolated from A.korupensis 1, 7. A selection of naphthylisoquinolines representing the biaryl coupling types detected in Dioncophyllaceae and Ancistrocladaceae Biomimetic synthesis of Michellamine A from Korupensamine A The process of synthesis was by biomimetic oxidative dimerization of a derivative of Korupensamine A. Korupensamine A and its atropisomer B were synthesized by an intermolecular strategy involving the Stille-type coupling. The synthesis begins with bromination of isoquinoline to provide tetrahydroisoquinoline moiety. This was follwed by the synthesis of Naphthalene by applying the Wittig surrogate to the Stobbe approach that involves converting an aldehyde into half ester which in turn underwent a ring closure in boiling acetic anhydride. Hydrolysis followed by methylation and reduction gave rise to naphthalene. This was deoxygenated to provide bromide.Metal-halogen exchange was followed by stannylation of the aryl lithium which gave stannane. This was coupled with the isoquinoline segment and under Pd(II) catalysis gave rise to atropisomers. These were deprotected with boron trichloride and then later separated. Michellamine A was synthesized from Korupensamine A through N-formylation followed by treating the formic anhydride with acetyl chloride that resulted in a diacetate 1. This was followed by dimerisation as described by Laatsch 1, 21 for simple naphthol dimerisation and this involved treatment with Ag2O with chloroform in the presence of NEt3. The resulting binaphylidendione was reduced with sodium borohydride and deprotection was done using methanolic HCl to provide Michellamine A 1. Michellamine A was also synthesized by the Convergent synthesis that involved the use of a binaphthyl and an intramolecular Suzuki-type coupling again mediated by palladium 1, 4. Pure Michellamine A was obtained by the method which was published in 1994 by Hoye and co-workers. The method involved a Suzuki-Type, intermolecular biaryl coupling followed by the biomimetic phenolic oxidative coupling mediated by Ag2O. A mixture of Michellamines A, B and C was obtained and a HPLC separation of a portion of this mixture gave a pure sample of Michellamine A 1. Methods have also been described for the interconversion of Michellmines A, B and C 12. Derivatives obtained from Michellamine A Structural modifications can be made to the naturally occurring Michellamines to get derivatives which also possess anti- HIV activity. Based on the amount of the reactant Michellamine A can be substituted at one or more positions. When reacted with CH3COCl acetate, substitution can occur at R2, R3, R4, R7, R8, R9. When it is reacted with benzene sulfoniyl chloride, one or both of R1 and R6 can form benzene sulphonamide derivatives. For preparation of esters they can be treated with acid halide or an acid. For preparation of ethers or tertiary amines they can be reacted with alkyl halides. Derivatives can also be obtained by substituting one or more of the hydrogen substituents on the aryl systems by halogen, nitro, hydroxyl, cyano groups etc. Bromine substituted derivatives are obtained by treating Michellamine A with Br2 and H2O. Likewise treatment with HNO3 will provide nitro derivatives, which in turn can be reduced to an amino derivative11, 12. Pharmacological properties and pharmaceuticals derived from Michellamine A Natural products, owing to their broad chemical structural diversity, help to deliver significant therapeutic advances in the treatment of diseases such as HIV 22. Many effective antimicrobial, antifungal and antiviral agents have been isolated from various plant species. The well known being, the antimalarial drug quinine, isolated form the bark of the tree Cinchona Officianalis, following which several anti-HIV and anti-cancer drugs have been isolated from various plant species. About one hundred and twenty pharmaceutical products currently in use are plant derived of which a major portion are from tropical plants 16. The number of compounds exhibiting anti-HIV activity isolated from natural sources is increasing steadily 18.Plant secondary metabolites like terpenes, phenolics, peptides, alkaloids and carbohydrates are inhibitors of HIV reverse transcriptase 22. The dimeric michellamine alkaloids A, B and C isolated from A.korupensis, have been shown to possess several pharmacologically important properties. 1. Anti- HIV acitivity: The most pharmacologically useful property of Michellamine A is its antiviral property, especially against HIV-1 1, 6, 9, 11. All the three michellamines exhibit antiviral activity against HIV-1 and HIV-2 in cell culture 17.Studies have been carried out using the dimers against a number of biologically diverse HIV-1 strains and also against AZT resistant strains and also HIV-2 3, 11.In vitro studies done on the most abundant member of the series Michellamine B have showed that it acts at an early stage in the HIV- life cycle by inhibiting the reverse transcriptase from both HIV-1 and HIV-2, as well as in the later stages by inhibiting cellular fusion and synctium formation 3, 10, 17, 18, 21. Studies have been carried out to determine the anti-HIV activity of Michellamine A in both the free base and as Michellamine HBr salt. The relative numbers of viable human CEM-SS lymphoblastoid target cells, either uninfected or infected with the HIV virus were determined at various concentrations of Michellamine A present either as free base or as their HBr salts. At Michellamine A concentrations between 20 to 200 m the viability assays showed essentially complete protection of the target cells from the killing effects of the virus. Similar assays carried out 6 days after introduction of various concentrations of Michellamine A showed a complete inhibition of p24 viral core antigen production and viral reverse transcriptase activity 12. 2. Antioxidant property: It has also been recently discovered that Michellamines also behave as potent antioxidants. The inhibitory effect of Michellamines A, B and C on the enzymatic activity of Protein Kinase C was postulated. The modelling and kinetic study results suggested that michellamines bind in such a way as to overlap both the ATP and peptide substrate subsites of PKC and other protein kinases, thus inhibiting its activity 17. Conclusion Michellamine A obtained from Ancistrocladus korupensis, has immense pharmacological applications due to its potent anti-HIV property, established through cell-culture. Structural analogues of Michellamines have also been studied for anti-HIV properties. Analogues that bore the dihydroxyphenyl substitution as occurring in natural michellamines where shown to elicit toxicity in cell culture 21. This coupled with the recently found anti-oxidant property has resulted in a considerable pharmaceutical interest towards this compound. Further clinical studies are required to pave the way for its use as a potent antiviral and anti-oxidant compound. Reference: 1. Rahman. A, Fatima, Z.B, 1988, Studies in Natural Products Chemistry. Elsevier Science. 2. Kubitzki. K, Kramer. K.U, Green. P.S, Rohwer. J.G, Bittrich. V, Huber. H, Kadereit. J.W, Jeffery. C. The families and genera of Vascular plants. Springer Vol.5. 3. Tringali. C, 2001, Bioactive compounds from Natural sources: Isolation, Characterization and Biological properties, CRC Press. 4. Wirth.T, 2003, Hypervalent Iodine Chemistry: Modern developments in Organic Synthesis, Springer. 5. Seigler. D.S, 1998, Plant Secondary Metabolism, Springer. 6. Ancistrocladus: Potential Anti-AIDS source, 1994, New Crops News, Vol.4, no 1. Spring. 7. Bringmann. G, Feineis. D, 2001, Stress-related polyketide metabolism of Dioncophyllaceae and Ancistrocladaceae. Journal of Experimental Botany. October 1.. Vol 52, no 363. Oxford University Press. http://jxb.oxfordjournals.org/cgi/content/full/52/363/2015#F4 8. Boyd. M.R et al, 1994, Anti – HIV Michellamines from Ancistrocladus korupensis. J.Med.Chem, Vol 37. 9. Mcmahon J.B. et al, 1995, Michellamine B, a novel plant alkaloid, inhibits Human Immunodeficiency virus- induced cell killing by at least two distinct mechanisms. Antimicrobial Agents and Chemotherapy, Feb issue. 10. Supko J.G, Malspies L, 1995, Pharmakokinetics of Michellamine B, a Naphthylisoquinoline alkaloid with in vitro activity against Human Immunodeficiency Virus types 1 and 2, in the mouse and Dog. Antimicrobial Agents and Chemotherapy. Jan issue. 11. Boyd et al, 1997, Process of preparing Michellamine compounds, US Patent number 5, 654, 432, United States Patent 19. http://www.patentstorm.us/patents/5654432-description.html 12. Boyd et al, 1995, Michellamine antiviral agents, compositions and treatment methods, US Patent number 5, 455,451, United States Patent 19. http://www.freepatentsonline.com/5455251.html 13. Monomeric and dimeric Naphthylisoquinoline alkaloids and synthesis methods therof. http://www.freepatentsonline.com/EP1325915.html 14. Natural Product with Anti-HIV activity. http://home.ncifcrf.gov/mtdp/Catalog/compounds/650898.html 15. Fairlamb. A.H, Ridley. R.G, Vial. H.J. Drugs against parasitic diseases: R&D methodologies and issues. Discoveries and drug development. 16. Bukuru. J 2003, Ioslation and Structural Elucidation of Natural Products from Pentas Bussei K. Krause, Pentas lanceolata (Forsk) Deflers and Pentas Parvifolia Hiern Rubiaceae, Doctoral Thesis. https://archive.ugent.be/retrieve/705/19989621.pdf 17. White. E.L et al, 1999, Michellamine alkaloids inhibit protein Kinase C, Archives of Biochemistry and Biophysics, Vol 365, No http://www.ideallibrary.com 18. Singh I.P, Bharate S.B, Bhutani K.K, 2005, Anti-HIV natural products, Current Science, Vol 89, No 2. http://www.iisc.ernet.in/~currsci/jul252005/269.pdf 19. Boyd et al, 1997, Antiviral Naphthoquinone compounds, compositions and uses thereof, US patent Number 5, 672,607, United States Patent 19. http://www.patentstorm.us/patents/5672607-fulltext.html 20. McCloud T.G et al, 1997, Quantitation of the anti-HIV alkaloids Michellamines A and B BY High Pressure Liquid Chromatography, Phytochemical analysis, Vol 8. 21. Zhang H, Zembower D.E, Chen Z, 1997, Structural analogues of the Michellamine anti- HIV agents. Importance of the Tetrahydroisoquinoline rings for biological activity, Bioorganic and Medicinal chemistry letters, Vol 7, No 20. 22. Gad S.C, 2005, Natural Products, Drug discovery Handbook, John Wiley & Sons Inc. Read More