Synthesis of 6-, 7- and 8-carbon sugar analogues of potent anti-influenza 2,3-didehydro-2,3-dideoxy-N-acetylneuraminic acid derivatives

摘要

J. CHEM. SOC. PERKIN TRANS. 1 1995 Synthesis of 6-, 7-and 8-carbon sugar analogues of potentanti-influenza 2,3-didehydro-2,3-dideoxy-N-acetylneuraminic acid derivatives Mark J. Barnford," Julia Castro Pichel, Wahid Husman, Bina Patel, Richard Storer and Niall G. Weir Glaxo Research and Development Limited, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SGl2NY, UK Analogues of the potent anti-influenza A and B compound, 4-guanidino-NeuSAc2en, are described in which the stereochemically demanding C-6-glycerol side-chain is truncated. Syntheses of the one- and two-carbon side-chain analogues, of both 4-guanidino- and 4-amino-NeuSAc2en, are presented, as well as the syntheses of analogues lacking any side-chain. Whilst complete removal of the C-6 side-chain abolishes activity, a stepwise increase in inhibition of influenza neuraminidase and influenza A and B in cell culture with increasing C-6 chain length is observed.The one-carbon, hydroxymethyl derivative retains significant activity to represent a suitable lead in the search for neuraminidase inhibitors of reduced stereochemical demand and synthetic complexity. In the preceding paper' we describe the synthesis of 4- guanidino-Neu5Ac2en, a 4-substituted neuraminic acid deriv- ative which is currently under development as a potential drug for the prophylaxis and treatment of disease caused by the influenza A and B viruses. As part of our studies to understand the structure-activity relationships in this class we have prepared a series of analogues to examine the contribution made by the components of the glycerol side-chain to the overall profile.Molecular-modelling studies using GRID calculations suggested there were no specific interactions between the C-6 stereospecific triol side-chain of these molecules and the influenza neuraminidase (NA) enzyme. Analogues 1 and 2 were synthesized in order to probe this hypothesis. Removal of the triol side-chain may free the synthesis from the restrictions of using the expensive Neu5Ac as starting material, or from the stereospecific chemistry involved in building up the chiral side- chain from simpler and cheaper molecules. NeuSAc NeUSAcZen R=OH 4-Ami~o-NeUSAch R =NH2 ~-Gu~amp;o-Namp;Ac~~IBR =NHC(NH)NElz H2N-1 a X=H b X=CH20H c X = CH(OH)CH20H Results and discussion Analogues of 4-amino- and 4-guanidino-Neu5Ac2en lacking any C-6 side-chain were synthesized according to Scheme 1.N-Acetylglucosamine 3 was used to prepare the tri-0-acetyl-1- chloro derivative 4 by the procedure of H~rton.~ The main side- product was the 1-0-acetyl derivative formed in 4 (isolated) yield, but this was easily separated by flash chromatography on silica ge1.t Free-radical-initiated dehalogenation of chloride 4 furnished the crystalline tetrahydropyran compound 5 in good yield (80). The two 1-H protons resonated 1 ppm apart. After removal of acetyl protecting groups to give triol 6 by using 1 sodium in methanol, selective oxidation of the primary alcohol to give the acid 7 was successfully effected5 in almost quantitative yield by using oxygen gas with a catalytic amount of platinum in the presence of aq. sodium hydrogen carbonate in water.On repetition the reaction time varied from 2 h to 20 h presumably dependent on the rate of oxygen flow and the efficiency of stirring relative to the scale of reaction. No epimerisation at C-2 (adjacent to the newly formed acid) was apparent, a single compound being produced. Esterification of the acid 7 was subsequently carried out by treatment with Dowex (H+) resin in methanol. All attempts to obtain a crystalline form of the ester 8 were unsuccessful. Following acetylation of the two secondary hydroxy groups by using acetic anhydride in pyridine, elimination of the resulting protected compound 9 across the C-2-C-3 bond was achieved by refluxing with 1,8-diazabicyclo5.4.Oundec-7-ene (DBU) in chloroform. The a,P-unsaturated product 10 showed only one 0-acetyl resonance and a downfield shift in the 3-H resonance (from 6 5.23 to 6 6.20).This was converted by reaction' with tri- methylsilyl triflate (TMSOTf) into the oxazoline ll obtained as a gum in good yield (98). No attempt at further purification was made. The crude product was subjected to nucleophilic attack at C-4 by hydrazoic acid, generated in situ by the reaction of azidotrimethylsilane with tert-butyl alcohol at 80deg;C.The azide moiety was successfully incorporated in the pseudo-equatorial a-configuration evidenced by the H NMR spectrum. The small coupling constant (4 Hz) for 3-H due to the coupling with the pseudo-axial 4-H results from a dihedral angle close to 90".j3-Epimers have a larger coupling constant for 3-H due to an acute dihedral angle with the pseudo-equatorial 4-H.These characteristic coupling constants are inferred from t The I-chloro compound was found to be unstable when kept on silica for long periods. OH 3 4 5 HO--8 7 61vi ,OMe QMe 9 10 11 euro;I2 N3 13 12 la H2N 2a Scheme 1 Reagents: i, AcCI; ii, Bu,SnH, AIBN, toluene; iii, Na, MeOH; iv, Pt, O,, water; v, Dowex H', MeOH; vi, Ac,O, pyridine; vii, DBU, CHCl,; viii, TMSOTf, MeCN; ix, TMSN,, Bu'OH; x, H,, Pd/C, 1,Cdioxane-water; xi, aq. Et,N; xii, AIMSA, aq.K,CO, similar observations for the parent NeuSAc2en analogues. Hydrogenation of the azide 12 to give the amine 13 proved problematic. Under the conditions employed, hydrogen with a catalytic amount of 10 Pd/C, there was a competitive reduction of the double bond. Hence, the reaction was halted well before all the azide had reacted and the amine 13 was obtained in only 19 yield after chromatography. This was then deprotected to give the target amino acid la as the partial triethylamine salt, requiring no further purification. Reaction of amine la with aminoiminomethanesulfonic acid (AIMSA) under basic conditions effected introduction of the guanidino functionality. Recrystallisation of the product obtained from Dowex (H ) ion-exchange chromatography was + unsuccessful when using water, propan-2-01, ethanol and methanol as solvents, even though the compound with a triol side-chain at C-6 is crystalline under similar conditions.Analytically pure guanidino compound 2a was obtained as the trifluoroacetate salt after preparative HPLC. The stereo- chemistry at C-4 of amino compound la and guanidino compound 2a were again inferred from the 'H NMR coupling constants of the vinylic 3-H proton. The 4-amino and 4-guanidino compounds (lb and 2b, respectively) possessing a hydroxymethyl group at the C-6 position were also synthesized. In order to simplify the chemistry as much as possible, the approach taken was to cleave two carbons from the appropriate parent neuraminic J.CHEM. soc. PERKIN TRANS. 1 1995 acid derivative, preferably without any protection required. Such a strategy has been used' in looking at the binding of the C-6-truncated forms of sialylglycoproteins to influenza virus haemagglutinin. Thus, the C-6 side-chain of 4-amino- NeuSAc2en was cleaved with two mole equivalents of sodium periodate and the resulting aldehyde was reduced with excess of sodium boranuide to the hydroxymethyl derivative 14, which was purified by Dowex (OH-) ion-exchange chromatography. Interestingly, in this compound the amine group was con- comitantly formylated. The formyl group was cleaved by using HCl in methanol and, owing to the resulting formation of the methyl ester of the carboxylic acid group (as observed by 'H NMR examination of the crude acid-treated product), this was subsequently treated with aq.triethylamine. The product was purified by Dowex (H+ ) ion-exchange chromatography. This, and the formyl intermediate, both contained an impurity from which the desired product could not be freed. Similar aldehydes have been isolated and used previously without such problems, * but it could not be ruled out' that this impurity was due to epimerisation at C-6 of the intermediate aldehyde under the basic reducing conditions. In an attempt to control the sequence of reactions more closely and thereby avoid formation of the impurity, the amine functionality was protected to give compound 15 prior to the oxidation/reduction step. In this way the C-6-truncated protected amino derivative 16 was obtained as a single compound in good yield and in crystalline form without the need for chromatography.This compound showed none of the contaminant previously observed without amine protection, and represents a versatile synthetic intermediate for the exploration of additional analogues. It was found that the tert-butoxycarbonyl (Boc) group is cleaved on heating of compound 16 in water to give pure crystalline truncated amino derivative lb. Attempts to convert the amino derivative lb into the 4- guanidino derivative 2b by using the AIMSA methodology (Scheme 2) gave impure material which resisted attempts at purification by ion-exchange chromatography and crystal- lisation. This target was obtained most conveniently (Scheme 2) directly from the parent guanidino compound 2a by the oxidation/reduction protocol, analytically pure compound being obtained in modest (47) yield following ion-exchange Dowex (H ) chromatography and crystallisation.+ The 4-amino and 4-guanidino compounds possessing the intermediary length 2-carbon C-6-side-chain, analogues lc and 2c, respectively, were obtained (Scheme 3) by using a slightly modified route; Boc protection was again used to mask the 4-amino group whilst, in order to simplify handling of inter- mediates, the acid functionality was protected as its diphenyl- methyl (DPM) ester. Thus, Boc-protected intermediate 15 was treated with diazodiphenylmethane at 21 "C for 3 days to give the fully protected DPM ester 17.This was subjected to the oxidation/reduction methodology in which only 1.1 mole equivalents each of NaIO, and NaBH, were used. The presence of protecting groups allowed purification by chromatography on silica to give the 2-carbon truncated intermediate 18. Concomitant removal of both the DPM ester and Boc protecting groups with aq. trifluoroacetic acid (TFA) afforded the amino acid derivative lc in very high yield (94 from 18) as its TFA salt. This was converted in the usual manner into the 4-guanidino derivative 2c which was purified by preparative HPLC. The target compounds 1 and 2 were tested in vitro against isolated neuraminidase enzyme and influenza virus, and their IC,,-values were compared with those of the parent neuraminic acid analogues (Table 1).The data for compounds la and 2a indicate that complete removal of the triol side-chain results in loss of virtually all J. CHEM. SOC. PERKIN TRANS. I 1995 viii ' H2N 4-Amino-NeuSAc2en AcNH 0C02H -iii, iv AcNH +CO2H '-HN Hamp; LO 14 lb v, vi I AcNH C02H -i, ii AcNH GCO2H Ho2k m' m' =NH kNH H2N H2N 4-Guanidino-NeuSAc2en 2b Scheme 2 Reagents and conditions: i, aq. NaIO, (2 rnol equiv.); ii, NaBH, (9 rnol equiv.); iii, POCI, (2 rnol equiv.) MeOH; iv, aq. Et,N (10 rnol equiv.); v, NaOH (pH 9); vi, AIMSA (3.4 rnol equiv.), aq. K,CO, (3.3 mol equiv.); vii, (Boc),O, aq. Na2C0,; viii, water, reflux HO AcNH -AcNH 0 B~NH' BOCNH' 17 18 iii AcNH C02H -AcNH COzH H2N 2c lc Scheme 3 Reagents: i, Diazodiphenylmethane, aq.1 ,Cdioxane-CH,C12;ii, (a) aq. NaIO,, MeOH; (b) aq. NaBH,, MeOH; iii, aq. TFA; iv, AIMSA, aq. K,CO, neuraminidase inhibitory, and influenza A and B plaque-reducing activity. Thus, whilst our computational studies suggest no major spec@ interaction of this moiety with the enzyme, it must fulfil a vital role in binding. It is possible that the hydroxy groups undergo a water-mediated interaction with the enzyme in this region of the active site or play an entropic role Table 1 Enzyme inhibition and anti-viral data IC,,NA" IC,,FluAb IC,,FluB' Compound (pmol drn-,) (pg ern-,) (pg ~m-~) Neu5 Aden 8.6 12 4.8 4-Amino-NeuSAc2en 0.32 1.5 0.065 4-Guanidino-NeuSAc2en 0.005 0.023 0.005 la 1000 d d 2a 130 100 48 lb 270 100 19 2b 9.2 17 1.9 lc 13 3.8 24 2c 0.55 0.1 2.1 " NA = isolated neuraminidase enzyme inhibitory assay.b.c Flu A and B = in vitro influenza virus A and B inhibitory assay as determined by plaque reduction. lo Not determined. through the displacement of water. Compounds lb and 2b possessing a simple hydroxymethyl functionality at C-6 also show much reduced activity relative to the parents, but compound 2b is still a potent inhibitor (equivalent in activity to NeuSAc2en) of both isolated NA enzyme and influenza viruses A and B in vitro. It therefore represents a suitable lead in the search for NA inhibitors of reduced stereochemical demand and synthetic complexity.Compounds lc and 2c show inhibitory activity intermediary to that of the simple CHzOH side-chain compounds and the parent Neu5Ac2en analogues; thus, each hydroxymethylene unit contributes significantly to the binding of 4-amino- and 4-guanidino-NeuSAc2en to the enzyme active site. Experimental Mps were determined in open capillaries and those using a Mettler FP5 1 automatic melting point apparatus are expressed in "C as M"y where x = rate of temperature rise ("C min-') and y = the starting temperature. 'H NMR spectra were run on a Bruker 250 MHz spectrometer with Me,Si as internal standard; coupling constants (J) are quoted in Hz. IR spectra were obtained using a Nicolet 5SxC FT-IR spectrometer.Optical rotations were measured (alD-units are 10 ' deg cm2 g-') using apparatus supplied by Optical Activity Ltd., England. Preparative silica column chromatography was performed on Merck 9385 silica under flash conditions. GLC analysis was performed by using a column derivatised to 5 with methyl- phenyl silicone (temperature: 50-325 "C; gradient: 10 "C min-'; carrier: He gas). HPLC analysis was conducted as follows: Column 1: S5-ODS2, with acetonitrile in water as eluent and a flow 0f~2.0 cm3 min-'. Detection was by UV at 210 nm; column 2: Hypersil SAS, with 10 acetonitrile-0.05 mol dm-3 NH4- H2P0, as eluent and a flow of 1.0 cm3 min-'. Detection was by UV at 235 nm; column 3: Dynamax C18 + guard with 5 acetonitrile + 0.1 TFA-water + 0.1 TFA and a flow of 1 cm3 min-'.Detection was by UV at 230 nm. Preparative HPLC used (unless otherwise stated) column type 1, solvent aceto- nitrile-water with 5-25 TFA, flow 40 cm3 min-' detection UV, 230 nm. Capillary zone electrophoresis (CZE) analysis was performed using a 50 pm fused silica column, 72 cm total length, UV detector (210 nm), 50 cm distant, 20 kV at 3OoC, pH 7 (attained by 50 mmol dm-3 phosphate + 50 mmol dm-3 borate buffer). Enzyme and virus inhibitory assays were conducted as previously described. lo 2-Acetamido-3,4,6-tri-O-acetyl-2deoxy-a-~-glucopyranosyl chloride 4 Dried (1 mm Hg, 22 "C, 24 h) 2-acetamido-2-deoxy-~-glucose (60 g, 0.27 mol) was added to vigorously stirred acetyl chloride (120 cm3, excess).The resulting suspension was stirred under dry conditions (calcium chloride tube) for 16 h, at 22 "C. The amber solution obtained was diluted with dry chloroform (480 cm3) and poured into a stirred mixture of ice (480 g) and water (120 cm3). The organic layer was separated, and neutralised by being stirred with ice in saturated aq. sodium hydrogen carbonate (490 cm3) before being dried over MgS04 (30 g). The entire washing procedure was completed within 15 min at 0 "C. The solution was concentrated under reduced pressure at 50 "C to -75 cm3, and dry diethyl ether (600 cm3) was added to initiate crystallisation. After 60 h the solid was filtered off, washed with dry diethyl ether (150 cm3) and dried to give a crystalline product (77.66 g).This was purified by flash chromatography dichloromethane-ethyl acetate (2 :1, then 1 :l). Evaporation of fractions containing the first eluted species gave the title compound 4as a foam (41 -85 g, 43.7); mp (M2,,) 114.6; a;' + 107 (c 1.1, CHCl,) (Found: C, 46.0; H, 6.3; H, 3.8. C14H20ClN08 requires C, 45.97; H, 5.51; N, 3.83); amp;(CDCl,) 6.20 (1 H, d, J3, 1-H), 5.90 (I H, d, J8, NH), 5.38- 5.19(2H,m,3-and4-H),4.53(1 H,m,2-H),4.32-4.10(3H,m, 5-H and 6-H2), 2.12 (3 H, s, OAc), 2.06 (6 H, s, OAc) and 2.0 (3 H, s, NHAc). Evaporation of fractions containing the second eluted product gave a foam, consistent with the 1-0-acetyl compound: GH(CDCI3) 6.20 (1 H, d, J 3.75, l-H), 5.77 (1 H, d, NH), 5.23 (2 H, m, 3- and 4-H), 4.50 (1 H, m, 2-H), 4.25 and 4.05 (2 H, m, 6-H2), 4.00 (1 H, m, 5-H), 2.20, 2.1 1, 2.10 and 2.05 (12 H, 4 s, 4 x 0Ac)and 1.95 (3 H, s,NAc).2-Acetamido-3,4,6-tri-O-acetyl-l,5-anhydro-2-deoxy-~-glucitol5 A stirred solution of chloride 4(9.1 g, 0.025 mol) in dry toluene (150 cm3) was degassed with nitrogen for 30 min. Tributyltin hydride (8.7 g, 0.03 mol) was added followed by azoisobutyro- nitrile (ATBN) (81 5 mg) and'the solution was heated at reflux under nitrogen for 1 h 20 min. The reaction mixture was evaporated to dryness and purified by flash chromatography. Elution with dichloromethane-ethyl acetate (1 : 1, then 1 :3), then ethyl acetate, followed by ethyl acetate-methanol (10: 1) furnished the title compound 5as a foam (6.59 g, 80); mp 158- 160 "C; a;' +4.9 (c 1.03, CHCl,) (Found: C, 50.0; H, 6.3; N, 3.8.Cl4H2,NO, requires C, 50.75; H, 6.39; N, 4.23); umax(CHBr3)/cm-' 3418 (NH), 1738, 1678 (CG) and 1237; amp;(CDCl,) 5.70 (I H, d, J7.5, NH), 5.09 (1 H, d, J 10, 3-H), 4.95(1 H,d, JlO,4-H),4.25-4.10(4H,m, l-Hb,2-Hand6-H,), 3.55 (1 H, m, 5-H), 3.16 (1 H, dd, J 12.5, 1-Ha), 2.13-2.03 (9 H, 3 s, 3 x OAc) and 1.95 (3 H, s, NHAc). 2-Acetamido-l,5-anhydro-2-deoxy-~-glucitol6 A solution of compound 5 (24.17 g, 0.073 mol) in methanol (100 cm3) was stirred with exclusion of moisture with 1 sodium in methanol (36.80 cm3). A solid immediately pre- cipitated out; this was filtered off, washed with the minimum amount of methanol, and dried in vacuo to give the title compound6 as a crystalline solid (1 1.67 g, 78); a further crop was obtained from the mother liquors (1.33 g, 9); mp 206.3 "C; a;' + 10.3 (c 0.58, water) (Found: C, 46.7; H, 6.9; N, 6.5.C,H,,NO, requires C, 46.83; H, 6.88; N, 6.83); vmax-(Me,SO)/cm-' 3334 and 3232 (OH, NH), 1668 (C=O), 1549, 1371 and 1303; dH(D2O) 4.00-3.20 (8 H, m) and 2.01 (3 H, s, NHAc); m/z206 (MH'). 5-Acetamido-2,6-anhydro-5deoxy-~-gulo-hexonicacid 7 A solution of compound 6 (3.722 g, 0.018 mol) and sodium hydrogen carbonate (2.59 g, 0.031 mol) in water (180 cm3) was treated with platinum obtained by hydrogenation of platinum(rv) oxide (0.6 g) for 5 h. Oxygen was bubbled through the vigorously stirred mixture for 6 h at 90 "C.The catalyst was J.CHEM. SOC. PERKIN TRANS. I 1995 removed by filtration through Kieselguhr and the filtrate was acidified using Dowex 50W x 8 (1WO mesh) resin, H+-form. After 0.5 h the resin was filtered off and the filtrate was con- centrated under reduced pressure to furnish the title compound 7 as a foam (3.86 g, 97); a;' -8.41 (c 0.36, water); ~,~,(Me~S0)/cm-~3293 (OH, NH), 1728 and 1640 (C=O), 1557 and 1097; dH(D2O) 4.00-3.50 (5 H, m), 3.30 (1 H, t, J 10, 6-Ha) and 2.0 (3 H, s, NHAc) Found: (M' + l), 220.081 743. C,H,,NO, requires m/z,220.082 1121; GLC 96 pure. 5-Acetamido-2,6-anhydro-5deoxy-~-gufo-hexonicacid methyl ester 8 A solution of compound 7 (12.27 g, 56 mmol) in methanol (250 cm3) was stirred with Dowex 50W x 8 (1 640 mesh) resin (1 2 g; H+-form, methanol-washed) at ambient temperature for 66 h.The reaction mixture was filtered and the filtrate was evaporated under reduced pressure to give the title compound 8as an off-white foam (10.02 g, 77); dH(D20) 4.03-3.85 (3 H, m, 2- and 5-H, 6-Hb), 3.82 (3 H, s, C02Me), 3.64-3.56 (2 H, m, 3- and 4-H), 3.38-3.28 (1 H, m, 6-Ha) and 2.01 (3 H, s, NHAc) Found: (M+ + l), 234.097 773. C,,HI6NO, requires mlz, 234.097 762); HPLC 90 pure. 5-Acetamido-3,4-di-O-acetyl-2,6-anhydro-5deoxy-~-gufo-hexonic acid methyl ester 9 A solution of compound 8 (10.02 g, 0.043 mol) in dry pyridine (40 cm3)-acetic anhydride (20 cm3) was stirred with exclusion of moisture at 21 "C for 16 h. The reaction mixture was then evaporated to dryness and the residue was purified by flash chromatography ethyl acetate-cyclohexane (4 :1) followed by ethyl acetate.The appropriate fractions were combined and concentrated to 30 cm'. The title compound 9slowly crystallised at 21 "C, as a solid (4.62 g, 3373, mp 152.4 "C; a;' + 16 (c 1.1, CHCl,) (Found: C, 49.2; H, 6.3; N, 4.5. C13H19NOs requires C, 49.20; H, 6.03; N, 4.41); d,(CDCl,) 5.82 (1 H, J7.5, NH), 5.23 (1 H, d, J8, 3-H), 4.98 (1 H, d, J8, 4-H), 4.31 (1 H, dd, J, 5, J28, 6-Hb),4.20(1 H, m, 5-H),4.02(1 H, d, J7.5, 2-H), 3.76 (3 H, s, C02Me), 3.30 (1 H, dd, J, 8, J22.5, 6-H"), 2.07 (6 H, 2 s, 2 x OAc) and 1.95 (3 H, s, NHAc); m/z 318 (MH+) and 335 (MNH,+). 5-Acetamido-4-O-acetyl-2,6-anhydro-3,5dideoxy-~-threo-hex-2-enonic acid methyl ester 10 A solution of compound 9 (2.704g, 8.5 mmol) in dry chloroform (40 cm3), under nitrogen, was treated with DBU (5.1 cm3, 34 mmol).The resulting mixture was heated at reflux for 1.5 h and was then allowed to cool to 21 "C. The solution was washed sequentially with 2 rnol dm-3 hydrochloric acid (2 x 40 cm3), water (50 cm3) and brine (60 cm3), dried over MgSO,, filtered, and concentrated under reduced pressure to give an off-white foam. The crude product was purified by flashchromatography (gradient elution with 0-3 methanol in chloroform) to afford the title compound 10 as a gum (2.35 g, quant.), a? +212.2 (C 0.49, CHCl,); d~(cDCl3) 6.20 (I H, dd, J, 5, J2 1, 3-H), 5.69 (1 H, br, NH), 5.03 (1 H, m, 4-H), 4.33-4.10 (3 H, m, 5-H and 6-H2), 3.83 (3 H, s, C02Me), 2.07 (3 H, s, OAc) and 1.98 (3 H, s, NHAc) Found: (M' + l), 258.097 015.C,,H,,NO, requires m/z,258.097 7621; HPLC 93 pure. Methyl (3aS,7aR)-2-methyl-3a,7a-dihydro-4H-pyrano3,4-6-oxazole-6-carboxylate11 A solution of compound 10 (4.68 g, 17.9 mmol) in dry acetonitrile (80 cm3) under nitrogen was treated with TMSOTf (3.63 cm3, 18.8 mmol). The resulting solution was heated at 50 "C for 1 h. The solution was allowed to cool to 21 "C and then was poured into ice-cold, saturated aq. sodium hydrogen carbonate (60 cm3) containing extra sodium hydrogen carbonate (6 g). After being stirred for 5 min the mixture was J. CHEM. SOC. PERKIN TRANS. I 1995 extracted with cold ethyl acetate (2 x 100 cm3).The combined organic extracts were washed with brine (120 cm3), dried over MgSO,, filtered, and evaporated to give the title compound 11 as a pale yellow gum (3.47 g, 98); vma,(Me2SO)/cm-' 1736 (C=O), 1668 (C=C,C=N), 1439, 1388, 1300, 1278 and 1153; amp;(CDC13) 6.23 (1 H, d, J 3.75, 3-H), 4.94 (1 H, dd, J, 3.75, J, 5, 4-H), 4.31-4.18 (2 H, m, 6-Hb and 5-H), 3.88-3.78 (4 H, m, C0,Me and 6-Ha) and 2.0 (3 H, s, Me). 5-Acetamido-2,6-anhydro-4-azido-3,4,5-trideoxy-~-fhreo-hex-2-enonic acid methyl ester 12 Azidotrimethylsilane (3.4 cm3, 24 mmol) was added dropwise (during 1 h) to a stirred solution of compound 11 (1.581 g, 8 mmol) in dry 2-methylpropan-2-01 at 80deg;C. TLC analysis showed incomplete reaction and so further azidotrimethyl- silane (1 cm3) was added dropwise after 4 h and again (2.4 cm3, 48 mmol total) an hour later.The mixture was stirred at 80 "C for a further 3 h and then at 21 "C for 16 h, then was poured into ice-cold, saturated aq. sodium hydrogen carbonate (100 cm3) containing extra sodium hydrogen carbonate (7 g) and was stirred for 15 min. The mixture was extracted with cold ethyl acetate (3 x 50 cm3), and the combined organic extracts were washed with brine (70 cm3), dried over MgSO,, filtered, and evaporated to dryness. The crude product was purified by flash chromatography ethyl acetate-cyclohexane (3 :2) followed by ethyl acetate-cyclohexane (2 : 1) and then neat ethyl acetate, changing to ethyl acetate-methanol (10: l) to furnish the title compound 12 as a gum (1.02 g, 53); a;' +304.9 (c 1.06, CHCI,); v,,,(CHBr3)/cm-' 3422 (NH), 2100 (N3), 1733 and 1676 (C=O), 1507, 1263 and 1236; GH(CDC13) 6.14 (1 H, dd, J, 2, J, 4, 3-Hj, 5.83 (1 H, d, J7,NH), 4.29-3.98 (4 H, m, 4- and 5-H and 6-H,), 3.86 (3 H, s, C0,Me) and 1.90 (3 H, s, NHAc); m/z 241 (MH'), 213 (MH -N,)' and 198 (MH -N3)' Found: (M' + l), 241.092 800.C,H,,N404 requires m/z 241.093 680); HPLC (column 2) 96 pure. 5-Acetamido-4-amino-2,6-anhydro-3,4,5-trideoxy-~-threo-hex-2-enonic acid methyl ester 13 A solution of compound 12 (843 mg, 3.5 mmol) in 1,4-dioxane (15 cm3)-water (15 cm3) was hydrogenated over 10 Pd/C (90 mg) at ambient temperature for 1.6 h. The reaction mixture was filtered through Kieselguhr and the filtrate was evaporated to dryness under reduced pressure.The crude product was purified by flash chromatography (gradient elution with amp;15 methanol in chloroform) to obtain the title compound 13 as a pale yellow solid (146 mg, 19) (Found: C, 49.8; H, 6.7; N, 13.0. C9H14N204 requires C, 50.05; H, 6.59; N, 13.08); v,,,(CHBr,)/cm-' 3423 (NH), 1729 (C=O ester), 1669 (C=O amide), I5 1 1,1437,1309 and 1262; GH(CDC13) 6.12 (1 H, d, J 5, 3-H), 5.72 (1 H, br, NH), 4.15 (2 H, s, 6-H2), 3.95 (1 H, m, 5-H), 3.85 (3 H, s, CO,Me), 3.40 (1 H, m, 4-H) and 1.98 (3 H, s, NHAc): m/z 215 (MH)'. 5-Acetamido-4-amino-2,6-a~ydro-3,4,5-h.ideoxy-~-~hre~-hex-2-enonic acid la A suspension of compound 13 (123 mg, 0.57 mmol) in water (1.2 cm3j was treated with triethylamine (0.42 cm3, excess).The resulting solution was stirred at ambient temperature for 4 h and was then concentrated under reduced pressure to give a gum. This was taken up in water (5 cm3) and the mixture was stirred with charcoal (Norrits ultra, 2 mg) for 10 min. The now colourless solution was filtered through Kieselguhr, the Kieselguhr was washed thoroughly, and the combined filtrate and washings were freeze-dried to give the title compound la as a pale yellow solid (106 mg, 88) (Found: C, 42.9; H, 6.7; N, 12.3. C8H1,N2o4 requires C, 43.2; H, 7.0; N, 12.3); vmaX(Me,SO)/ cm-' 3248 (OH, NH), 1583 (M),1463, 1377 and 1281; d,(D,O) 5.75 (1 H, d, J4.0, 3-H), 4.30-4.10 (3 H, m, 5-H and 6-H2), 3.91 (1 H, t, J4.0, 4-H) and 2.02 (3 H, s, NHAc); CZE 100 pure.5-Acetamido-2,6-anhydro-3,4,5-trideoxy-4-guanidin~L-~hreo-hex-2-enonic acid 2a A solution of compound la (70 mg, 0.345 mmol) in water (0.5 cm3) was treated with 0.1 mol dmP3 sodium hydroxide (3.45 cm3) at 21 "C. The solution was then warmed to 35 "C and treated with potassium carbonate (10.4 mg) followed by AIMSA (9.3 mg) every 0.5 h for 8 h (total 16 additions, 1.2 mmol each). The reaction mixture was then stirred at ambient temperature for 16 h, diluted with water (1 cm3) filtered, and the solid was washed with water (1 cm3). The filtrate and washings were combined and applied to a column of Dowex 50W x 8 (16-40 mesh, H+-form) (37 cm3). Elution with water (270 cm3) followed by 0.6 mol dmP3 aq. triethylamine (550 cm3) and then evaporation of the appropriate fractions gave the crude product as an off-white powder.Purification was achieved by pre- parative HPLC on a c18 'Dynamax microsorb' column and elution with 5 acetonitrile containing 0.1 TFA at a flow rate of 1 cm3 min-', UV detection at 230 nm. The required fractions were concentrated under reduced pressure and then freeze dried to furnish the title compound 2a as a powder (40 mg, 27) (Found: C, 35.4; H, 4.2; N, 13.75. C,Hl,N,04 requires C, 35.26; H, 4.22; N, 13.76); vma,(Me2SO)/cm-' 1692 and 1653 (C=O) 1549 and 1199; G,(D,O) 5.95 (1 H, dd, J1 5, J, 1.25, 3-H), 4.35 (1 H, m, 5-H), 4.15-3.80 (3 H, m, 4-H, 6-H2) and 2.03 (3 H, s, NHAc); HPLC (column 3) 99 pure. 5-Acetamido-2,6-anhydro-3,4,5-trideoxy-4-formamido-L-arabino-hept-2-enonic acid 14 4-Amino-NeuSAc2en (871 mg, 3.0 mmol) was dissolved in water (45 cm3) and the solution was treated with sodium metaperiodate (1.41 g, 6.59 mmol).This mixture was stirred in the dark at 20 "C for 1 h, diluted with water (45 cm3), and then treated with aq. sodium boranuide (1.04 g, 27 mmol in 45 cm3) by dropwise addition over a period of 10 min. This mixture was stirred at 2OoC for 1 h. pH was adjusted to 4 by addition of glacial acetic acid ( -2 cm3). The whole was then freeze-dried and the solid was redissolved in water (170 cm3) and loaded onto a column of ion-exchange resin (Dowex 2 x 8,OH--form) (30 g). This was washed with water (850 cm3), then eluted with 1 mol dm-3 AcOH. Fractions containing the UV-active species R, 0.25 on silica, butan-1-01-AcOH-water (3 :1 :l) were combined, evaporated under reduced pressure (T 50 "C) to a volume of 100 cm3, then freeze-dried to give the title compound 14 as a crispy solid (551 mg, 71) (Found: C, 43.6; H, 6.0; N, 9.65.CloH14N20,~l .5H20.0. 15Et3N requires C, 43.57; H, 6.45; N, 10.02); ;l,,,(water)/nm 234; ~,,,(Me,S0)/cm~~ 3500, 1714,1675 and 1546; d,(D,O) 8.12 l H, s, HC(O)NH, 5.85 (1 H, d, J2.5, 3-H), 4.85 (1 H, m, 4-H), 4.20-4.00 (2 H, m, 5- and 6-H), 3.9Ck3.70 (2 H, m, 7-H,) and 2.00 (3 H, s, Ac); G,(Me,SO) 171.6 (MeCO), 164.8 (C-1), 162.7 (HCONH), 147.5 (C-2), 109.9 (C-3), 80.7 (C-4), 61.8 (C-7), 48.6 (C-6), 46.8 (C-5) and 24.1 (Me); m/z (CI) 259 (MH') and 276 (MNH,'); m/z (FAB) 259 (MH'); HPLC (column 1) 90 pure.5-Acetamido-4-amino-2,6-anhydro-3,4,5-~id~xy-~-arabino-hept-2-enonic acid lb Compound 14 (500 mg, 1.80 mmol) was dissolved in methanol (46 cm3), and treated dropwise over a period of 5 min with phosphoryl trichloride (34 cm3, 3.67 mmol). This mixture was stirred at 20 "C for 3 h. Solvent was removed under reduced pressure to give a brown gum, which was dissolved in water (10 cm3) and the mixture was treated with triethylamine (2.6 cm', 10.5 mequiv.); this mixture was stirred at 20 "C for 3.5 h. Solvent was evaporated under reduced pressure and the resulting gum was dissolved in water (50 cm3). This solution was applied to a column of ion-exchange resin Dowex 50W x 8 (H'), 190 cm3 and washed with water (1.4 dm3).Elution with 0.6 mol dm-3 Et3N (-1.5 dm3) and evaporation under reduced pressure of the appropriate fractions gave the title compound lb as an off-white, crispy foam (320 mg, 77); vm,,(Me,SO)/cm-' 3400,1714,1666,1602,1551,1373 and 1263; dH(D20) 5.70 (1 H, d, J 2.5, 3-H), 4.25-4.10 (3 H, m, 4-, 5- and 6-H), 3.95-3.75 (2 H, m, 7-H,) and 2.10 (3 H, s, Ac); dc(D,O) 188.6 (GO Ac), 174.8 (COZH), 150.5 (C-2), 100.0 (C-3), 77.0 (C-4), 60.0 (C-7), 49.9 (C-6), 46.0 (C-5) and 22.0 (Me); m/z 187 (MH' -CO,), 231 (MH') and 248 (MNH,'); CZE 89.6 pure, contains 10.4 impurity. 5-Acetamido-2,6-anhydrO-4-( tert-butoxycarbonylamino)-3,4,5-trideoxy-D-gamp;cevo-D-gdacto-non-~-enonicacid 15 A suspension of 4-amino-NeuSAc2en (0.29 g, 1 mmol) in methanol (1.5 cm3) was treated with a solution of di-tert-butyl dicarbonate (240 mg, 1.1 mmol) in methanol (0.5 cm3), followed by aq.sodium hydrogen carbonate (176 mg, 2.1 mmol in 1.5 cm3). The resulting solution was stirred at 20 "C for 45 min, then was evaporated under reduced pressure. The residue was taken up in water and washed with diethyl ether; the aqueous solution was stirred with Dowex 50W x 8 (H+) until acidic. This mixture was filtered and freeze-dried to give the title compound 15 as a low-density solid (289 mg, 7473, mp 210 (decomp); a;' +21.6 (c 0.83, MeOH) (Found: C, 46.9; H, 6.9; N, 6.7. C16H,,N,09~H,0 requires C, 47.06; H, 6.91; N, 6.86); h,,,(EtOH)/nm 233 (E 6309); vmaX(Nujol)/cm1 3331, 2949, 2854, 1689 and 1461; amp;(D,O) 5.95 (1 H, s, 3-H), 4.50- 4.35 (2 H, m, 5- and 6-H), 4.15 (1 H, t, J 10,4-H), 3.90 and 3.65 (4 H, m, 7- and 8-H and 9-H,), 2.02 (3 H, s, Ac) and 1.40 (9 H, s, Bu'); m/z (TSP +ve) 391 (MH'), 335 (MH' -Bur)and 291 (MH+ -Bu'OCO); CZE 98.6 pure.5-Acetamido-2,6-anhydrO-4-(tert-butoxycarbony~amino)-3,4,5-trideoxy-~-urubino-hept-2-enonicacid 16 Compound 15 (7.02 g, 18 mmol) was dissolved in water (300 cm3), and the solution was treated portionwise with sodium metaperiodate (8.46 g, 39.5 mmol) and stirred at 20 "C for 45 min. Aq. sodium boranuide (6.24 g, 162 mmol in 240 cm3) was added dropwise during 20 min and the mixture was stirred for 1 h. Acetic acid was added to adjust the pH to 4, and the resulting solution was concentrated to 300 cm3 and the pH was adjusted to 1 with 2 mol dm-3 HCl. This mixture was stored at 2 "C for 16 h and the resulting crystals were filtered off, washed with a little water, and dried over P,O,.The filtrate was concentrated to -70 cm3 to give a second crop (combined yield of title compound 16,5.78 g, 97); a;' +43.95 (c 0.5, MeOH) (Found: C, 48.1; H, 6.8; N, 7.9. CI4H,,N,O7-H,O requires C, 48.27; H, 6.94; N, 8.04); amp;,,,(EtOH)/nm 236 (E 6670); vmax(Nujol)/cm-' 3613,3487,3343,3300,2926,2853, 1714,1687, J. CHEM. SOC. PERKIN TRANS. I 1995 5-Acetamido-2,6-anhydro-3,4,5-trideoxy4guanidino-~-arabino-hept-2-enonic acid 2b 4-Guanidino-Neu5Ac2en (507 mg, 1.75 mmol) was dissolved in water (25 cm3) and the solution was treated with sodium metaperiodate (0.824 g, 3.84 mmol) at 20 "C.After being stirred in the dark for 1 h, the solution was diluted with water (25 cm3) and treated with aq. sodium boranuide (0.608 g, 15.7 mmol in 25 cm3) in a dropwise manner over a period of 10 min. This mixture was then stirred at 20 "C for 1 h. Adjustment to pH 4 with acetic acid (glacial) was followed by freeze-drying to give a solid. This was taken up in water (30 cm3) and applied to a column of ion-exchange resin Dowex 50W x 8 (H'), 30 cm3. The column was washed with water (200 cm3), then was eluted with 0.6 mol dmP3 aq. triethylamine. Appropriate fractions were combined, and evaporated under reduced pressure. The residue was repeatedly co-evaporated with water to give a solid (0.33 g), which was dissolved in water (6 cm3), the solution was warmed (45 "C) and propan-2-01 (15 cm3) was added to the swirled mixture.Crystallisation was observed on cooling of the solution. The crystals were filtered off, washed with (3: 1) propan-2-ol-water, then were dried at 60 "C under vacuum, to give the title compound 2b as fine needles (0.226 g, 4779, mp 240 "C (decomp.); a;' +15.1 (c 0.53, water) (Found: C, 41.1; H, 5.9; N, 19.2. CloHI6N,O,-H,O requires C, 41.37; H, 6.25; N, 19.30); A,,,(EtOH)/nm 233 (E 4964); v,,,(Me,- SO)/cm-' 3327,3214,3127,3046,2983, 1673, 1648, 1600, 1596 and 1392; dH(D2O) 5.68 (1 H, d, J 2, 3-H), 4.32 (1 H, m, 4-H), 4.22 (1 H, m, 6-H), 4.12 (1 H, m, 5-H), 3.80 (2 H, m, 7-H,) and 2.02(3H,s, Me);amp;(D,O) 177.2(C=OAc), 171.6(COZH), 159.5 NHC(=NH)NHJ, 151.2 (C-2), 105.6 (C-3), 79.3 (C-6), 62.8 (C-7), 52.2 (C-4), 50.5 (C-5) and 24.3 (Me); CZE 99.1 pure. 5-Acetamido-2,6-anhydro-4-(tert-butoxycarbonylamino)-3,4,5-t~deoxy-D-gamp;cero-~-gufuc~o-non-2-enon~cacid benzhydryl ester 17 Compound 15 (10 g, 34.4 mmol) was dissolved in 1,4-dioxane (60 cm3)-water (30 cm3).To this at 21 "Cwas added a solution of diazodiphenylmethane in dichloromethane (45 cm3; 0.965 mol dm-3). This mixture was stirred at 21 "C for 3 days. The organic phase was separated and evaporated to dryness. The resulting solid was treated with anhydrous diethyl ether and the mixture filtered. The solid was washed with diethyl ether several times, then dried to give the title compound 17 (13.8 g, 72) (Found: C, 59.2; H, 6.5; N, 4.7.C,,H36N,09*1 .6H,O requires C, 59.49; H, 6.75; N, 4.78); v,,,(Nujol)/cm-' 3322br, 2955, 2924,2853, 1716, 1689, 1653, 1521, 1456, 1367, 1249 and 1164; dH(CD3),SO 8.10 (1 H, br d, NH), 7.25-7.50 (10 H, m, Ph), 7.15 (1 H, br d, NH), 6.90 (1 H, s, CHPh,), 5.90 (1 H, s, 3-H), 4.74.6 (1 H, m, 6-H), 4.50 (1 H, t, J9, OH), 4.35 (1 H, t, J5.8, 4-H), 4.10 (1 H, d, J 11,5-H), 3.95 and 3.70 (4 H, m, 7- and 8-H, and 9-H,), 1.90 (3 H, s, Ac) and 1.40 (9 H, s, Bu'); HPLC 99.2 1462 and 1377; dH(CD,),SO 12.85 (1 H, br d, CO,H), 8.00 pure.(~H,~,J~.~,NHCO,)~.O~(~H,~,J~,NHCOAC),~.~~(~H,~, J2.25, 3-H), 4.70 (1 H, br d, OH), 4.32 (1 H, t, J7.5,4-H), 3.80 (2 H, m, 5- and 6-H), 3.50 (2 H, m, 7-H,), 1.85 (3 H, s, Ac) and 1.40 (9 H, s, Bu'); m/z (LSIMS +ve) 331 (MH'), 353 (MNa'), 275 (MH' -Bu'), 231 (MH+ -Bu'OCO) and 214 (MH' -NHC0,Bu'); CZE 97.6 pure. Amine lb from carbamate 16 Compound 16 (821 mg, 2.48 mmol) was boiled in water (130 cm3) for 1 h.On cooling, crystallisation gave some recovered starting material as large needles (216 mg, 26). Freeze-drying of the mother liquors followed by trituration of the resulting solid with methanol gave the title compound lb as small needles (196 mg, 33); mp 250 "C (decomp.) (Found: C, 46.4; H, 5.9; N, 11.9. C,H,,N,O, requires C, 46.95; H, 6.13; N, 12.17); CZE 97 pure. 5-Acetamido-2,6-anhydro-4-(tert-butoxycarbonylamino)-3,4,5-trideoxy-~gufucto-ot-2-enonic'acid benzhydryl ester 18 Compound 17 (2.0g, 3.6 mmol) was dissolved in methanol (80 cm3), and water (5 cm3) was added until precipitation was observed.To this mixture at 0 "C was added sodium metaperiodate (770 mg) and the mixture was stirred for 1 h before being filtered, and the filtrate was evaporated under reduced pressure. The solid residue was treated with methanol (60 cm3) and to this suspension was added water (10 cm3), followed by sodium boranuide (150 mg, 4.0 mmol). After 3 h the mixture was acidified with 1 mol dm-3 HCl and evaporated. The solid residue was purified by flash chromatography (4 methanol in dichloromethane) to give the title compound 18 as a foam (1.38 g, 73) (Found: C, 60.8; H, 6.7; N, 5.01.Cz8H34N,08-0.67H20 requires c, 60.75; H, 6.74; N, 5.06); J. CHEM. SOC. PERKIN TRANS. 1 1995 v,,,(Me,SO)/cm-' 3326,3232,2977,1732,1704,1652 and 1589; dH(CD3),SO 8.10(1 H,d, J7.7,NH), 7.25-7.50(10H,m,Ph), 7.12(1 H,d, J9.5,NH),6.90(1 H,s,Ph,CH), 5.90(1 H,d, J2, 3-H), 4.65 (2 H, m, 5-and 6-H), 4.45 (1 H, br, OH), 3.95 (2 H, m, 4-H and OH), 3.5 (3 H, m, 7-H and 8-H), 2.90 (3 H, s, Ac) and 1.4 (9 H, s, Bu'); m/z 527 (MH+) and 549 (MNH4+). 5-Acetamido-4-amino-2,6-anhydro-3,4,5-trideoxy-~-gducto-oct-2-enonic acid-trifluoroacetic acid complex lc Compound 18 (150 mg, 0.576 mmol) was dissolved at 0 "C in a TFA-water mixture (4 cm3 of a 5:2 mixture) and the mixture was then allowed to warm to 21 "C over a period of 4 h.The mixture was treated with diethyl ether (25 cm3) and the organic phase was extracted with water. The combined aqueous solutions were freeze-dried to give the title compound lc as a pale yellow solid (70 mg, 94) (Found: C, 36.5; H, 4.7; N, 6.7. Cl,H,,N20,~C,HF302~H2~requires C, 36.74; H, 4.88; N, 7.14); v,,,(MeOH)/cm-l 3500-3100br, 3079, 2947, 1607, 1556, 1203 and 1139;d,(D,O) 5.95 (1 H, s, 3-H), 4.35 (1 H, t, J 8.5,5-H),4.25(2H,m,4-and6-H),3.90(1 H, t, J6.5,7-H), 3.75 (2 H, d, J 6.5, 8-H2) and 2.10 (3 H, s, Ac); HPLC 93 pure. (1 H,t, J6.5,7-H),3.75(2H,d, J6.5,8-H2)and2.05(3H,s,Ac); m/z 303 (MH+). Acknowledgements We thank the Structural Chemistry Department, Glaxo Research and Development Ltd., Stevenage, UK, for provision of spectral and analytical data, and Jackie Woods and Richard Bethell of the Virology Department, for provision of biological data.References 1 M. J. Bamford, M. Chandler, R. Conroy, B. Lamont, B. Patel, V. K. Patel, 1. P. Steeples, R. Storer, N. G. Weir, M. J. Wright and C. Williamson, J. Chem.Soc.,Perkin Trans. I, 1994, precedingpaper. 2 P. McMeekin, Computational Chemistry Department, Glaxo R amp;D, Greenford, UK, unpublished results. 3 P. J. Goodford, J. Med. Chem., 1985,243,847. 4 D. Horton, Org. Synrh., 1973, Coll. Vol., 5, 1. 5 R. Julina, I. Muller, A. Vasella and R. Wyler, Carbohydr. Res., 1987, 164,415. 6 A. E. Miller and J. J. Bischoff, Synthesis, 1986, 777. 5-Acetamido-2,6-anhydro-3,4,5-trideoxy4~anidin~~gu~cf~-oct-2-enonic acid-trifluoroacetic acid complex 2c A solution of compound lc (104 mg, 0.4 mmol) in water (5 cm3) was treated with aq. potassium carbonate (0.4cm3 of a 110 mg cm-3 solution). To this were then added AIMSA (20 mg) and aq. potassium carbonate (0.1 cm3) to a total of 16 additions over a period of 2 days. This aqueous solution was then purified by preparative HPLC (SODS2 column, 5-25 TFA in aceto- nitrile-water gradient, 40 cm3 min-' ) to give, after evaporation and freeze-drying of appropriate fractions, the title compound 2c as a solid (67 mg, 36) (Found: C, 33.9; H, 4.55; N, 12.1. C, ,H1,N40,~l .2C,HF30,~l.6H,0 requires C, 34.40; H, 4.83; N, 11.97); v,,,(MeOH)/cm-l 3079, 2822, 2448, 2301, 1919, 1702, 1415, 1299, 1186, 1115 and 1093; 6,(D20) 6.00 (1 H, d, J2.5, 3-H), 4.5 (1 H, m, 5-H), 4.30 (2 H, m, 4-and 6-H), 3.95 7 M. N. Matrosovich, A. S. Gambaryan, F. N. Reizin and M. P. Chumakov, Virology, 1991,182,879. 8 For example: K. E. Pfitzner and J. G. Moffatt, J. Am. Chem. Soc., 1963,85,3027. 9 M. J. Bamford, P. L. Coe and R. T. Walker, J. Med. Chem., 1990, 33, 2494. 10 M. von Itzstein, W.-Y. Wu, G. B. Kok, M. S. Pegg, J. C. Dyason, B. Jin, T. V. Phan, M. L. Smythe, H. F. White, S. W. Oliver, P. M. Colman, J. N. Varghese, D. M. Ryan, J. M. Woods, R. C. Bethell, V. J. Hotham, J. M. Cameron and C. R. Penn, Nature, 1993,363,418. Paper 4/06 170E Received 10th October 1994 Accepted 22nd December 1994