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Sunday 2 February 2014

Zanamivir, Relenza spectral data



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
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
mass

1H NMR
HydrogenChemical 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
CarbonShift /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
 ref 12
IR 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:
1: N-acetyl-neuraminic acid
2: 5- Acetamido- 3,5- dideoxy- D- glycero- �- D- galacto- 2- nonulo- pyranosonic acid methyl ester
3: Methyl (3aR, 4R, 7aR)- 2- Methyl- 4- [(1'S, 2'R)- 1', 2', 3' - triacet- oxypropyl]- 3a, 7a- dihydro- 4H- pyrano [3, 4-d] oxazole- 6- carboxlate.
4: 5- Acetamido- 7, 8, 9- tri- O- acetyl- 2, 6- anhydro- 4- azido- 3, 4, 5- trideoxy- D- glycero- D- galacto- non- 2- enonic acid methyl ester.
5: 5- Acetamido- 2, 6- anhydro- 4- azido- 3, 4, 5- trideoxy- D- glycero- D- galacto- non- 2- enonic acid methyl ester.
6: 5- Acetamido- 4- amino- 2, 6- anhydro- 3, 4, 5- trideoxy- D- glycero- D- galacto- non- 2- enonic acid.
Part one of reaction scheme

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

Reaction scheme part 2:
Reaction scheme 3 shows the conversion of compound 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 over an eight hour period. A yield of about 48% of the crystalline product 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:
8. 5- Acetamido- 2, 6- anhydro- 3, 4, 5- trideoxy- 4- guanidino- D- glycero- D- galacto- non- 2- enonic acid. (Relenza)
9. 5- Acetylamino- 2, 6- anhydro- 4- cyanoamino- 3, 4, 5- trideoxy- D- glycero- D- galacto- non- 2- enonic acid
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 1 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:
1: N-acetyl-neuraminic acid
2: 5- Acetamido- 3,5- dideoxy- D- glycero- �- D- galacto- 2- nonulo- pyranosonic acid methyl ester
3: Methyl (3aR, 4R, 7aR)- 2- Methyl- 4- [(1'S, 2'R)- 1', 2', 3' - triacet- oxypropyl]- 3a, 7a- dihydro- 4H- pyrano [3, 4-d] oxazole- 6- carboxlate.
4: 5- Acetamido- 7, 8, 9- tri- O- acetyl- 2, 6- anhydro- 4- azido- 3, 4, 5- trideoxy- D- glycero- D- galacto- non- 2- enonic acid methyl ester.
5: 5- Acetamido- 2, 6- anhydro- 4- azido- 3, 4, 5- trideoxy- D- glycero- D- galacto- non- 2- enonic acid methyl ester.
6: 5- Acetamido- 4- amino- 2, 6- anhydro- 3, 4, 5- trideoxy- D- glycero- D- galacto- non- 2- enonic acid.
Part one of reaction scheme

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. The 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

Reaction scheme part 2:
Reaction scheme 3 shows the conversion of compound 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 over an eight hour period. A yield of about 48% of the crystalline product 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:
8. 5- Acetamido- 2, 6- anhydro- 3, 4, 5- trideoxy- 4- guanidino- D- glycero- D- galacto- non- 2- enonic acid. (Relenza)
9. 5- Acetylamino- 2, 6- anhydro- 4- cyanoamino- 3, 4, 5- trideoxy- D- glycero- D- galacto- non- 2- enonic acid
Part 2 of reaction scheme
Part 2 of reaction scheme
ref are 13 and 14

Introduction

Relenza (Zanamivir for oral inhalation) is the first in a new generation of influenza virus-specific drugs known as neuraminidase inhibitors, which work by interferring with the life cycles of influenza viruses A and B. It prevents the virus spreading infection to other cells by blocking the neuraminidase enzyme present on the surface of the virus. Relenza is available as a powder that is administered by inhalation of 2 blisters from the rotadisk inside the diskhaler (Fig. 1) twice daily for five daysThis means that 20mg of Relenza is delivered to the principal site of viral replication each day.The main method for preventing influenza since the 1960s is by vaccination and although this and anti-viral drugs such as amantadine and its analogue rimantadine have long been available (since 1976 and 1993 respectively), they are only of limited use because of the constant mutation of the virus. This chameleon-like nature also means that the virus can become unrecognizable to the human immune system and thus repeatedly infect millions of people year after year. 
Fig 1: The diskhaler used to administer Relenza. Each blister in the Rotadisk contains 5mg of the drug
Why there is a need for a more effective influenza treatment: At present influenza is basically an uncontrolled disease and an effective method is needed for both the prevention and treatment of it. In the 20th century there were some major pandemics such as the 1918-1919 Spanish 'flu which killed 20 million people world wide, the 1957 Asian 'flu, the 1968 Hong Kong 'flu and the 1977 Russian 'flu12 These viruses also affect different animals, especially domesticated chickens and turkeys and in Hong Kong in 1997 a virulent bird flu virus, started infecting and killing people for the first time ever. Of the 18 people affected 6 died, although there was no evidence that the virus was able to spread between people. Given the antigenic properties of the influenza virus, in the future the virus may be passed from person to person, and because human immune systems are not prepared for avian viruses the effects on the population could be grave. It would not be possible to prepare vaccines in time and anti-viral drugs are not always adequate.

Advantages of Relenza over previous treatments:
Relenza has a number of advantages over the existing treatments for influenza. It does not cause significant side effects and the development of zanamivir-resistant viruses is not expected to occur readily in patients. This is because selection of drug-resistant mutants characterized by changes in neuraminidase requires prolonged passage in tissue culture and may be a biological cripple. If started within two days of the onset of influenza symptoms and if a fever is present, the duration of illness is decreased by an average of 1.5 days. It appears to decrease the severity of flu symptoms for the remainder of the illness, as well as decreasing the number of complications from the flu. It is also possible that Relenza could be used as a method of 'flu prevention although it has not yet been approved for this use.

Comparison of the symptoms of the 'flu with that of a common cold:
People infected by an influenza virus suffer a lot more than those with a cold. As you can see from the table below, some of the symptoms are similar, but with a cold they are less severe.Influenza also becomes more serious when it leads to secondary bacterial pneumonia or primary influenza viral pneumonia or when it exacerbates underlying medical conditions such as pulmonary or cardiac disease. In children, the symptoms are similar to those observed in adults, however children often have higher fevers and younger ones may develop gastrointestinal manifestations. It should be noted that Relenza is not effective on people with colds or other viral illnesses.
InfluenzaCold
Sore throatMild sore throat
  High fever and chillsLow-grade fever
Non-productive coughCough
Severe muscle aches  Congestion
Headache
Intense fatigue. 
The effect of Relenza on patients with respiratory diseases:Relenza is not generally recommended for the treatment of patients with respiratory dieseases such as asthma or chronic obstructive pulmonary disease (COPD) and has carried an approval since its approval in July 1999. Some patients with underlying airway diseases have experienced serious adverse events following treatment, with some fatal outcomes although causality has been difficult to establish. It has been recommended that patients with asthma have a fast-acting bronchodilator inhaler available and use it about 15 minutes before taking Relenza.

Successfulness of Relenza:The sialidase inhibitory activities (determined by methods described in reference 7) of Relenza compared to the more recent neuraminidase inhibitor Oseltamivir are shown in the table below9.IC50 is the concentration that reduces enzyme activity by 50%.
CompoundInfluenza A IC50 (�M)Influenza B IC50 (�M)
Relenza0.0050.004
Oseltamivir0.0020.032
 The results demonstrate that both compounds are good inhibitors of influenza A and B, with Oseltamivir being more selective towards Influenza A and Relenza showing a better overall performance. In phase I and II tests reported by the Lancet5, no important adverse effects were found in healthy patients or those reported to have mild to moderate asthma following an inhaled administration of 40mg/day of Relenza. There was a significant improvement of the symptoms of people taking Relenza compared to those taking the placebo.
1940s: Discovery that the influenza virus's enzyme was destroying receptors on red blood cellsF.This was discovered by George Hirst, who noticed that when red blood cells were mixed with fluids from influenza infected chicken embryos in cold conditions the cells were very heavily agglutinated by the virus. These red cells dispersed when warmed up and could not be re-agglutinated in the cold with fresh virus. This led him to the conclusion that the influenza virus's enzyme was destroying receptors on red blood cells.
The finding of sialidase (also known as neuraminidase):Alfred Gottschalk heard of Hirst's experiment and interpretation of results, and this led him to believe that there was a "split product". He discovered sialic or neuraminic acid (Fig 2), a type of sugar, and the enzyme on the virus was called neuraminidase (or sialidase). At this time it was thought that it was the neuraminidase which was responsible for the observations made by Hirst, but it was later shown by Robin Valentine, W. Graeme Laver, Norbert Bischofberger and Robert G. Webster that the hemagglutinin (receptor-binding) and neuraminidase (receptor-destroying) activities of the virus resided in two quite different spikes on the surface of the virus.

Fig 2: Sialic Acid

Discovery of how new pandemic strains of 'flu A occured.
Ed Kilbourne, W. Graeme Laver, Norbert Bischofberger and Robert G. Webster realised that hybrid viruses could be formed by infecting cells simultaneously with two different Type A flu viruses. This was because the RNA pieces coding the various virus proteins reassorted, some of the viruses contained the hemagglutinin from one parent and the neuraminidase from the other. This "mating" of two parent viruses to give a hybrid virus explained how new pandemic strains of 'flu A occurred, and led to a very good way of producing influenza viruses with any desired combination of hemagglutinin and neuraminidase spikes. This helped towards finding a way of producing pure neuraminidase which was later essential for crystal growth and drug design experiments.


The crystallization of neuraminidase:
Laver, Bischofberger and Webster isolated one type of influenza virus by sucking off the allantoic fluid surrounding the embryo of infected chicken eggs and purifying this. The virus particles were incubated with an enzyme capable of digesting proteins. This enzyme was selected to split the "heads" of the neuraminidase spikes off the virus particle without destroying them and to leave behind or destroy the hemagglutinin spike. The neuraminidase "heads" obtained were concentrated using high-speed centrifugation. The tiny pellet of neuraminidase heads examined had a crystalline appearance, and X-ray diffraction analysis of larger crystals showed that they were made of protein.

Neu5Ac2en (DANA) was shown to inhibit influenza neuraminidase:
Different variants of 'flu neuraminidase were known to exist, each containing an amino acid sequence that varies between types of neuraminidase apart from one small sequence.It was seen that the conserved amino acids came together when the neuraminidase polypeptide folded up to form the active enzyme. This formed a well conserved cavity which was the active catalytic site of the neuraminidase enzyme. It became apparent that a plug-drug could be made to exactly fit into the active site and inhibit the neuraminidase activity from other influenza viruses. A synthetic analog of sialic acid called Neu5Ac2en (DANA) (Fig 3) was shown to inhibit the influenza virus neuraminidase, but not sufficiently enough to be used treatment for the 'flu in humans.

Fig 3: Neu5Ac2en (DANA)
The plug drug.Mark von Itzstein and colleagues discovered that replacing the OH at the 4 position of sialic acid with a positively charged amino group made a better inhibitor than sialic acid or its analogue, DANA. Replacing the OH at the 4 position of sialic acid with a guanidino group led to a potent inhibitor of 'flu neuraminidase. This compound was given the names GG167 and Zanamivir and is now more commonly known as Relenza. Peter Colman soaked the substrate for sialic acid in neuraminidase crystals and used X-ray crystallography to determine the three-dimensional structure of the crystals. The strong binding of Relenza by 'flu neuraminidase which was seen is due to the positively charged guanidino group being anchored by the negatively charged glutamic acids. More details about this are provided in the immunology section.

Immunology


Fig 4: The influenza viruses as seen under the electron microscope. Neuraminidase and haemagglutin spikes are visible.
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
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. This enables the virus to escape from the cell in which it grew and spread in the body to infect other cells. The action of NA may also facilitate viral mobility through the mucus of the respiratory tract. 
virusattack.gif (46720 bytes)
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).
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.
Fig 7. Relenza bound to neuraminidase
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.
Fig 8: Depiction of interaction of Relenza (GG 167) in the neuraminidase binding site6

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
6): J. Medicinal Chemistry. 1999, 42, 2332-2343
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
8): C Kim, W Lew, M Williams, H Liu, L Zhang, S Swaminathan, N Bischofberger, M Chen, D Mendel, C Tai, G Laver, R Stevens, J Am Chem Soc, 1997119, 681-690
9): P Smith, J Robinson, D Evans, S Sollis, P Howes, N Trivedi and R Bethell, Bioorganic and Medicinal Chemistry Letters 9, 1999, 601-604
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
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