Zanamivir
Chemical name:
| 5- Acetamido- 2, 6- anhydro- 3, 4, 5- trideoxy- 4- guanidino- D- glycero- D- galacto- non- 2- enonic acid |
Synonyms: | Zanamivir, GG167, 4-guanidino-Neu5Ac2en and 2,3- Didehydro- 2, 4- dideoxy- 4- guanidino- N- acetyl- D- neuraminic acid |
Empirical formula: |
C12H20N4O7
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Structural formula: | |
Molecular weight: | 332.31g |
Beilstein number: | 7083099 |
Normal State: | Powder |
Colour: | White to 'off white' |
Melting point: | 325oC |
Optical rotary power: | Type [�]Conc: 0.9g/100ml Solvent: H2O Optical rotary power: 41 deg Wavelength: 589nm Temp: 20oC |
CAS number: | 139110-80-8 |
Solubility: | 18mg/mL in water at 20oC |
1H NMR |
Hydrogen | Chemical shift /ppm |
(1H, d, 3-H) | 5.53 |
(2H, 2dd, 4- and 6-H) | 4.50 - 4.38 |
(1H, dd, 5-H) | 4.21 |
(2H, dd+ddd, 9-Ha and 8-H) | 4.00-3.88 |
(2H, 2dd, 9-Hb and 7-H) | 3.70-3.62 |
(3H, s, Ac) | 2.05 |
13C NMR |
Carbon | Shift /ppm |
(C=O, Ac) | 177.3 |
(C-1) | 172.1 |
(guanidino) | 159.9 |
(C-2) | 152.1 |
(C-3) | 106.8 |
(C-6) | 78.3 |
(C-8) | 72.6 |
(C-7) | 71.0 |
(C-9) | 65.9 |
(C-4) | 54.0 |
(C-5) | 50.6 |
(Me) | 24.8 |
IR spectra:
UV spectra
The following peaks are present in the IR spectra of Relenza: 3332cm-1, 1676cm-1, 1600cm-1, 1560cm-1, 1394cm-1, 1322cm-1 and 1281cm-1. |
UV spectra
The maximum peak is 235nm giving E = 199 dm-3 mol-1cm-1 |
ref 13
Synthesis of AIMSA |
Reaction scheme part 1: |
The commercially available N-acetyl-neuraminic acid 1 is the starting reagent for the most direct approach to the synthesis of 4-guanidino-Neu5Ac2en (Relenza). In reaction scheme 113 the steps for the conversion of N-acetyl-neuraminic acid 1 to its 4-amino analogue is shown. Step 1 is the addition of methanolic HCl (MeOH and HCl gas), which produces the methyl ester of 1, followed by acetic anhydride in pyridine with 4-(dimethylamino)pyridine catalysis, which produces the penta-acetoxy compound, 2. In step 2, 2 is converted into the oxazoline 3 at high yield using trimethylsilyl trifluoromethanesulfonate (TMSOTf) in ethyl acetate at 52oC. In step 3, the azido compound, 4, is produced by the reaction of 3 with trimethylsilyl azide in tert-butyl alcohol at 80oC. In step 4 catalytic sodium methoxide in methanol was used to remove the acetate protecting groups from 4 to give triol 5. The 4-amino analogue, 6 was made in step 5, by hydrolysis using triethylamine in water, hydrogenolysis with a Lindlar catalyst and finally the addition of Dowex 2 * 8 resin. The triethylamine salt of the 6 was made during hydrogenolysis and the purpose of the Dowex 2 * 8 resin was to desalt this intermediate. The chemical names of the compounds are:
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Part one of reaction scheme
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Synthesis of reactant necessary for part 2 of reaction: |
Aminoiminomethane-sulfonic acid (AIMSA), 7, which is necessary for the conversion of compound 6 into Relenza, 9, is synthesised in Reaction scheme 2 ref 14The oxidizing solution necessary for the reaction is prepared by the addition of peracetic acid to 30% hydrogen peroxide and then conc. sulfuric acid. This is followed by acetic anhydride and, once the reaction has completed, methanol. Thiourea is dissolved in methanol and added slowly to the oxidizing solution.to produce compound 7. Note that any crystals that form are removed and that the reaction needs to be carried out under cooled conditions. See the reference source for more experimental details. |
Synthesis of AIMSA
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Reaction scheme part 2: |
Reaction scheme 3 shows the conversion of compound 6 into Relenza. For route A, 3 mol equivalent of AIMSA, 7, and 3 mol equivalent of potassium carbonate are added in a portionwise manner to compound 6 over an eight hour period. A yield of about 48% of the crystalline product 8 should be obtained for this method. An alternative route is to treat compound 6 with 1.1 mol equivalent of cyanogen bromide in the presence of sodium acetate in methanol. Route B step 1 gives compound 9, which can be converted into the final product 8 by treating it with ammonium hydroxide and ammonium formate at 85oC. A 36% yield of the purified product can be obtained after purification with ion-exchange chromatography and crystallisation. The chemical names of the compounds in this scheme are:
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Immunology
Fig 4: The influenza viruses as seen under the electron microscope. Neuraminidase and haemagglutin spikes are visible.
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Structure of the flu virus:Influenza (Fig 4) is an RNA virus which may exist as any shape from round balls to long, spaghetti-like filaments. The genome of this virus is associated with five different viral proteins and is surrounded by a lipid membrane, which means that influenza belongs to the "enveloped" group of viruses. Eight separate pieces of ribonucleic acid (RNA) make up the influenza virus genome and each piece of RNA specifies the amino acid sequence of one and sometimes two of the virus's proteins. The segmented nature of the RNA allows differenet flu viruses to easily "mate" with each other to form hybrid progeny viruses with bits of RNA from each parent virus. Two glycoprotein molecules, known as hemagglutinin (HA) and neuraminidase (NA) (Fig 5) are stuck onto the lipid envelope of the virus and both play a crucial role in the infection of the epithelial cells of the upper respiratory tract. HA is a rod-shaped triangular molecule.and NA exists as a mushroom shaped spike with a box-like head on top of a long stalk, containing a hydrophobic region by which it is embedded in the viral membrane.. |
Fig 5: The Neuraminidase enzyme
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The enzyme Neuraminidase, also known as sialidase, is a tetramer with C-4 symmetry and an approximate molecular weight of 250 000. It contains a symmetrical folding pattern of six four-stranded antiparallel �-sheets arranged like propeller blades. Nine types of neuraminidase have been identified for influenza A and only one subtype for influenza B, and only 30% of the overall amino acid sequence is conserved between all known types of neuraminidase8 - these are the amino acids which line and surround the walls of the binding pocket. If they mutate, the enzyme is inactivated, so the virus could not mutate to escape from a drug which interfered with this site. So neuraminidase offers an attractive site for therapeutic intervention in influenza infections. | ||
How the influenza virus works:The influenza virus (like all viruses) can only replicate after invading selected living cells and growing inside them. It makes thousands of new virus particles from the cellular machinery and then goes on to infect other cells.. Hemagglutinin allows the virus to infect the epithelial cells of the upper respiratory tract by attaching it to cells through receptors on the cell containing sialic acid, it fuses the cell membrane with the membrane of the virus, allowing the RNA of the virus to get inside the cell and thus instruct the cell to make thousands of new virus particles. After this viral replication, the progeny virions must be released from the cell to repeat the cell cycle of infection. Neuraminidase removes the sialic acid receptors from the host cell and other newly made virus particles by cleavage of -glycosidic bonds. |
Fig. 6: The life cycle of the influenza virus. Click once on this image to see a larger version | ||
The life cycle of the influenza virusG begins with the individual virus entering the cell lining of the respiratory tract (letter a in Fig. 6), and the cell being induced to take up the virus because hemagglutinin on the virus binds to the sialic acid (b and c in Fig 6). The virus then dispatches its genetic material (made up of RNA) and its internal proteins to the nucleus of the cell (e and f). Messenger RNA is produced when some of the internal proteins duplicate the RNA (f). This messenger RNA is used by the cell as a template for making viral proteins (g and h) and genes which become new viral particles and leave the cell covered in sialic acid. This sialic acid needs to be removed so that the hemagglutinin molecules on one particle don't attach to the sialic acid on other ones, thus causing the new viruses to clump together and stick to the cell. The sialic acid is removed from the surface of the new viral particle by neuraminidase (j) and the new viral particles are able to travel and invade other cells (k).
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How Relenza works:
Relenza adopts a position within the active site of the enzyme and copies the geometry of the sialoside hydrolysis transition state9. It can achieve very good binding through appropriate presentation of its four pendent substituents and contains a hydrogen bonding glycerol sidechain. The guanidino group in Relenza is believed to form salt bridges with Glu 119 in the neuraminidase active site and add a strong charge interaction with Glu 2278.
Two hydroxyl groups of the 6-glycerol side chain are hydrogen bonded to Glu276 and the 4-hydroxyl is oriented towards Glu119. The NH group of the 5-N acetyl side chain interacts with a bound water molecule on the floor of the active site. The carbonyl oxygen of the same side chain is hydrogen bonded to Arg152 and the methyl group enters a hydrophobic pocket lined by Ile222 and Trp178. The glycosidic oxygen projects into bulk solvent.
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Fig 7. Relenza bound to neuraminidase
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The binding involved in Fig 7 is shown more clearly in Fig 8 below. Neuraminidase can no longer remove the sialic acid receptors from the host cell and newly made virus particles because of this binding. Therefore the virsuse 'clump' together or to the host cell and cannot go on to effect new cells.
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Fig 8: Depiction of interaction of Relenza (GG 167) in the neuraminidase binding site6
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References
1): K. J. Lui and A. P. Kendal, Am. J. Public Health, 1987, 77, 712 |
2): Scheiget, Zambonis, Bernstein and Roy, Org. Prep. Proced. Int., 1995, 27, 637- 644 |
3): Glaxo Wellcome Inc. Relenza� (zanamivir for inhalation) [package insert]. Research Triangle Park, NC: Glaxo Wellcome, Inc., 1999 |
4): N Seppa, Scientific American, July 10th 1999, Volume 156 |
5): L. Gubareva, Lancet, March 4th 2000, 355: 827-35 |
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7):P Smith, S Sollis, P Howes, P Cherry, I Starkey, K Cobley, H Weston, J Scicinski, A Merritt, A Whittington, P Wyatt, N Taylor, D Green, R Bethall, S Madar, R Fenton, P Morley, T Pateman, A Beresford. A. J. Med. Chem, 41, 1998, 787-797 |
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10): A. J. Hay, A. J. Wolstenholme, J. J. Skehel and M. H. Smith. EMBO J,. 1985, 4, 3021: L. J. Holsinger and R. A. Lamb, Cell, 1992, 69, 517 |
11): J. C. Stoof, J. Booij, B. Drukarch and E. C. Wolters, Eur. J. Pharmacol., 1992, 213, 439 |
12): W. Graeme Laver, Norbert Bischofberger, and Robert G. Webster, Perspectives in Biology and Medicine 43.2 (2000) 173-192. This can be seen by visitinghttp://www.press.jhu.edu/journals/perspectives_in_biology_and_medicine/v043/43.2laver.html nmr |
13): M. Chandler, M. J. Bamford, R. Conroy, B. Lamont, B. Patel, V. K. Patel, I. P. Steeples, R. Storer, N. G. Weir, M. Wright, C. Williamson, J. Chem. Soc. Perkin Trans. 1, 1995, 1173- 1180 nmr synth |
14): A. E. Miller, J. J. Bischoff, Synthesis, 1986, 777- 779 |
15): G. D. Allena, S. T. Brookesa, A. Barrow, b, J. A. Dunnc and C. M. Grossec, Journal of Chromatography B: 1999, 732, 383-393 |
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