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Cytochalasins from static cultures of Rosellinia arcuata.

Introduction

The cytochalasins are a group of toxic fungal metabolites which show marked cytotoxic effects on mammalian cells in tissue culture. The isolation and structural elucidation of the first two members were achieved at about the same time independently at the ICI laboratories in Britain by Aldridge et al [1] and by Tamm [2,3] in Zurich. The compounds isolated from Phoma exigua were named phomin and dehydrophomin and those from Helminthosporium dematioideum were called cytochalasin A and B, dehydrophomin being identical to cytochalasin A and phomin to cytochalasin B. Soon afterwards, the ICI group isolated cytochalasins C and D from Metarrhizium spp. [3] since then several other groups of workers have isolated cytochalasins from various sources. Reports of cytochalasin C from Xylariaceous origin are confined to a few species. Edwards et al [4] isolated five new cytochalasins, N, O, P, Q and R alongside cytochalasins C and D from the fungus H. terricola. Dagne et al [5] in 1994, reported the isolation and characterisation of two new epoxycytochalasins; 19,20-epoxycytochalasin Q and 19,20 epoxydeacetylcytochalasin Q, from the fruiting bodies of Xylaria obovata. Another group of workers, Abate et al [6] isolated 19,20-epoxycytochalasin C and 19,20-epoxy-deacetylcytochalasin C from cultures of the same fungus. A Japanese strain of Rosellinia produced cytochalasin E and rosallichalasin [7]. Engleromyces goetzei, a peculiar tropical species that cannot be assigned with certainty to any of the large groups within the Xylariaceae, produced engleromycin [8]. Cytochalasins have mainly been found to be cytotoxic to mammalian cells. A recent report by Dombrowski et al shows that a cytochalasin L-696-474 isolated from H. fragiforme inhibits HIV-1 protease [9].

The study of the culture medium of R. arcuata which is reported in this paper produced an intimate mixture of four cytochalasins. The components in the mixture were identified by their NH resonance positions in the [sup.1]H NMR. Three of the components, cytochalasin C, 19,20-epoxycytochalasin C and a new cytochalasin, engleromycin acetate were isolated and characterised. The fourth component couldn't be resolved nor characterised. The mycelium produced relatively pure 19,20-epoxycytochalasin C, from which 19,20-dihydroxycytochalasin C was synthesised.

Results and Discussions

R arcuata was cultured in Thompson bottles containing 3% malt extract enriched with 6% glucose. The mycelium was removed after 8 weeks by filtering through a muslin. The filtrate was concentrated on a rotary evaporator and left overnight. A white solid was obtained which was filtered off. TLC studies indicated that the solid (6.52 g) was a mixture of cytochalasins. This was confirmed by XH NMR determined in [C.sub.5][D.sub.5]N. A sample of the solid 0.70 g was chromatographed over silica gel in 2.0 x 60.0 cm column. The column was eluted with chloroform-methanol (95:5) and the eluent collected in volumes of 2.0 ml. [sup.1]H NMR was used to monitor the contents of the tubes.

Characterisation of cytochalasin C

On evaporation, tubes (41-47) gave a white solid (13.0 mg). The solid was recrystallised from acetone to give white needles (7.0 mg) mp 262-263 [degrees]C (lit. [10], 262 [degrees]C), m/z 507 ([M.sup.+]), [[[alpha]].sup.D.sub.23]-13.4[degrees] (c = 0.82 in MeOH) (lit. (1) [[[alpha]].sup.D.sub.23]-14.7[degrees]c = 0.82, in dioxane), [R.sub.f] = 0.53 (toluene, ethyl acetate, acetic acid 50:49:1) required m/z 507 ([C.sub.30][H.sub.38]N[O.sub.6]), [[MH].sup.+] = 508, [[M + N[H.sub.4]].sup.+] = 525 and [[M + Na].sup.+] = 530. [sup.I]H NMR ([C.sub.5][D.sub.5]N) gave NH resonance position at [[delta].sub.H] 9.34 and indicated the presence of five methyl groups in this compound. Two of these are doublets at [[delta].sub.H] 0.97 and 1.88 and the rest are singlets at [[delta].sub.H] 1.33, 1.51 and 2.30. A methyl group at 2.30 might be a methyl of an acetate group (Table 1). A search through literature [11] revealed that this compound was cytochalasin C (1).

[FIGURE 1 OMITTED]

Further proof of this was found in the [sup.13]C NMR and the DEPT 135 spectra. There are twelve carbon atoms between [[delta].sub.C] 120 and 140, six of which are aromatic and the rest olefinic carbon atoms. Additionally, there is a carbonyl peak at [[delta].sub.C] 210.05 which differentiates cytochalasin C from others such as S, G, H, [N.sub.PHO], [O.sub.PHO], [Q.sub.PHO], [P.sub.PHO], and [R.sub.PHO].

The identity of this compound was further confirmed by the [sup.13]C NMR Prediction software from Advanced Chemical Laboratory (ACDLabs). The calculated [sup.13]C NMR chemical shifts (II) were similar to the experimental carbon-13 chemical shift values obtained (III).

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Characterisation of 19,20-epoxycytochalasin C

Tubes (60 and 61) gave 9.3 mg of a white solid, which was recrystallised from ethyl acetate to give 8.1 mg of white needles mp 245-247[degrees]C [[[alpha]].sup.D.sub.23]-26.1[degrees] (c = 0.85 in MeOH), required m/z 523 ([C.sub.30][H.sub.38]N[O.sub.7]), [MH.sup.+] = 524, [[M + Na].sup.+] = 546, [[M + N[H.sub.4]].sup.+] = 541. [sup.1]H NMR in ([C.sub.5][D.sub.5]N) indicated a close similarity between this compound and cytochalasin C. The difference however lies in the region [[delta].sub.H] 6.57-6.72 where the olefinic protons at [[delta].sub.H] 5.58 and 6.51 in cytochalasin C are absent and instead there are two additional peaks upfield at [[delta].sub.H] 3.76 and 4.20. There is also a shift downfield of the NH proton from [[delta].sub.H] 9.34 in cytchalasin C to [[delta].sub.H] 9.46. In the [sup.13]C NMR of cytochalasin C, 10 resonance positions are observed between OC 79.42 and 38.53 and 12 resonance positions between [[delta].sub.C] 140 and 126. A reverse situation occurs in these regions in this compound where there is a shift upfield of carbon atoms from [[delta].sub.C] 132.90 to 54.04 and [[delta].sub.C] 133.36 to 61.39. This suggests a possible modification of a portion of cytochalasin C(A1) to (A2) in the compound under investigation.

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The [sup.1]H NMR and the [sup.13]C NMR assingments of A2 are consistant with the experimental data and indicates the structure (IV).

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Characterisation of Engleromycin acetate

Tubes (120-127); evaporation of the eluent gave a white solid which after recrystallisation from acetone afforded white needles (8.0 mg) mp 274-276 oC, [a] D +53[degrees] (c = 0.85, MeOH), found m/z 523 ([C.sub.30][H.sub.38]N[O.sub.7]), M[H.sup.+] = 524, [sup.13]C satellite M[H.sup.+] = 526, [[M + Na].sup.+] = 546, [[M + N[H.sub.4]].sup.+] = 541. [sup.1]H NMR spectrum ([C.sub.5][D.sub.5]N) indicated an NH resonance position at [[delta].sub.H] 9.22 which is different from the former compounds. Unlike the previous two compounds, this cytochalasin has four methyl groups; two singlets at [[delta].sub.H] 1.61 and 2.17 and two doublets at [[delta].sub.H] 0.68 and 1.00. In the [sup.13]C and DEPT 135 NMR spectra determined in pyridine,, an exocyclic methylene peak at [[delta].sub.C] 112.60 suggests that this cytochalasin is structurally related to cytochalasin D. A literature search revealed that a cytochalasin D derivative which closely fits the data of this compound is engleromycin (V).

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The difference however is the presence of a methyl group at [[delta].sub.H] 2.17 due to methyl protons of an acetoxy group. Also the peak at [[delta].sub.H] 7.14 due to the OH at C-21 in the proton NMR of engleromycin [8] is absent in this compound, showing that the OH is replaced by OAc. Confirming the structure of this compound as engleromycin acetate (VI).

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Engleromycin acetate is a new compound which has not yet been published. This is the first report of this compound.

Synthesis of dihydroxycytochalasin C

The air-dried mycelium was pulverised (72.0 g) and soxhlet extracted with chloroform to give a brownish gum (6.73 g). The gum was dissolved in warm acetone (15 ml) and set aside for 24 h. A white solid (2.51 g) was obtained after the solution was filtered. TLC studies showed that the solid was an impure cytochalasin. 300 mg of the mixture was recrystallised from ethyl acetate to give white needles (120 mg), mp 245-247 [degrees]C. The compound gave a yellow spot with anisaldehyde spray reagent after heating at 110 [degrees]C for 2-3 min. [sup.1]H NMR and [sup.13]C NMR indicated that this compound is 19,20-epoxycytochalasin C (IV), which had already been isolated and characterised from the culture medium.

19,20-Epoxycytochalasin C (80.mg) was heated under reflux with dilute sulphuric acid (3 drops) in THF (4.0 ml) for an hour. The solution turned light green after heating for 15 min. It was allowed to cool and diluted with water (5.0 ml). The THF was removed under reduced pressure on a rotary evaporator and the resulting aqueous solution was extracted with chloroform (5.0 ml x 2). The chloroform extract was dried over anhydrous sodium sulphate. A light green gum (71 mg) was obtained when the chloroform was removed on a rotary evaporator. The gum was triturated with ethyl acetate to give off-white needles (57 mg) mp 251-253[degrees]C, [[[alpha]].sup.D.sub.23]-23[degrees] (c = 0.90, MeOH). Mixed TLC studies showed this compound had a lower [R.sub.f] value in toluene, ethyl acetate and acetic acid (50:49:1) compared with 19,20-epoxycytochalasin C. 1H and [sup.13]C NMR analysis however, could not give definitive evidence to differentiate between the starting material and the product. This is due to similar chemical structure of the two compounds. Mass spectrometry was used to establish the difference between the two compounds. The mass of the starting material, 19,20-epoxycytochalasin C was taken under the same conditions as the hydrolysed compound. The later gave M[H.sup.+] = 542, [[MH + Na].sup.+] = 565, [[M + [H.sub.2]O].sup.+] = 559 and a [sup.13]C satellite peak at 543. The scheme below illustrates the preparation.

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Giving the structure as 19,20-dihydroxycytochalasin C (VII).

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This is the first report of the synthesis of 19,20-dihydroxycytochalasin C.

Experimental

R arcuata was surface cultured in Thompson bottles containing 3% malt extract enriched with 6% glucose. The mycelium was removed after 8 weeks by filtering through a muslin. The filtrate was concentrated on a rotary evaporator and left overnight. A white solid was obtained which was filtered off. The weight was determined as 6.52 g. A TLC of this mixture showed four opaque spots when sprayed with p-nitroaniline spray reagent. This suggested that the solid was a mixture of four cytochalasins. This was confirmed by 1H NMR of the mixture determined in [C.sub.5][D.sub.5]N. 0.70 g of the solid was applied to silica gel in 2 x 60 cm column. The column was eluted with chloroform-methanol (95:5) and collected in volumes of 2.0 ml. [sup.1]H NMR was used to monitor the separation of the mixture of cytochalasins in the eluent. Two tubes in series of each ten tubes were analysed.

Isolation of cytochalasin C

Tubes (41-47) gave a white solid when the eluent was removed under reduced pressure on a rotary evaporator. The solid was recrystallised from acetone to give cytochalasin [C.sup.5] as white needles (7 mg) mp 262-263[degrees]C (lit. 262[degrees]C). Molecular formula [C.sub.30][H.sub.37]N[O.sub.6], m/z (EI) 507, [[[alpha]].sup.D.sub.23]-13.4[degrees] (c = 0.82 in MeOH). [R.sub.f] (Toluene, ethyl acetate, acetic acid (50:49:1) = 0.53. [[delta].sub.H] ([C.sub.5][D.sub.5]N) 0.97 (3H, d, J 6.0 Hz), 1.33 (3H, s), 1.51 (3H, s), 1.88 (3H, s), 2.11 (1H, dd, J 4.0, 5.0 Hz), 2.30 (3H, s), 2.63 (1H, dd, J 5.0, 10.0 Hz), 2.81 (1H, s), 3.02-3.20 (3H, m), 3.61 (1H, t, J 8.0 Hz), 4.41 (1H, m), 4.97 (H2O), 5.48 (1H, m), 5.58 (1H, dd, J 13.0, 2.0 Hz), 5.73 (1H, d, J 6.0 Hz), 6.00 (1H, s), 6.34 (1H, t, J 2.0 Hz), 6.51 (1H, dd, J 5.0, 10.0 Hz), 6.74 (1H, dd, J 4.0, 2.0 Hz) 7.16-7.26 (5H, m) and 9.34 (1H, s). [[delta].sub.C] ([C.sub.5][D.sub.5]N) gave resonance positions of the following carbon atom types at 14.78 (H-C[H.sub.3]), 17.14 (12-C[H.sub.3]), 19.31 (22-C[H.sub.3]), 20.54 (23-C[H.sub.3]), 24.60 (25-C[H.sub.3]), 38.53 (15-C[H.sub.2]), 42.33 (16-CH), 45.19 (10-C[H.sub.2]), 50.54 (3-C), 50.57 (4-CH), 60.84 (8-CH), 68.94 (7-CH), 76.17 (21-CH), 78.43 (18 CH), 126.47 (5-C), 126.49 (29-CH), 126.79 (14-CH), 128.56 (13-CH), 128.83 (28-CH, 30-CH), 129.77 (27-CH, 31-CH), 132.57 (6-CH), 132.90 (20-CH), 133.36 (19 CH), 138.78 (26-C), 170.81 (24-C=O), 175.30 (1-C=O) and 210.05 (17-C=O).

19,20-epoxycytochalasin C

Tubes (60 and 61) yielded 9.3 mg of white solid, which was recrystallised from ethyl acetate to give 19,20-epoxycytochalasin C, white needles (8.1 mg), mp 245-247[degrees]C [[[alpha]].sup.D.sub.23]-26.1[degrees] (c = 0.85 in MeOH), m/z (EI) 523, Molecular formula [C.sub.30][H.sub.37]N[O.sub.7], [[delta].sub.C] ([C.sub.5][D.sub.5]N) 0.99 (3H, d, J 7.0 Hz), 1.24 (3H, s), 1.60 (3H, s), 1.84 (3H, s), 1.97 (1H, dd, J 6.0, 6.0 Hz), 2..11 (3H, s), 2.30 (1H, s), 2.63 (1H, dd, J 5.0 10.0 Hz), 2.81 (1H, m) 3.19 (1H, d, J 8.0 Hz), 3.59 (1H, t, J 7.0, 2.0 Hz), 3.76 (1H, d, J 2.0 Hz), 4.20 (1H, d, J 2.0 Hz), 4.40 (1H, m), 4.99 ([H.sub.2]O), 5.80 (1H, d, J 4.0 Hz), 5.88 (1H, m), 6.27 (1H, s), 6.76 (1H, dd, J 6.0, 10.0 Hz), 7.20-7.32 (5H, m) and 9.46 (1H, s). [[delta].sub.C] ([C.sub.5][D.sub.5]N) 14.74 (H-C[H.sub.3]), 17.02 (12-C[H.sub.3]), 19.02 (22-C[H.sub.3]), 20.32 (23-C[H.sub.3]), 22.50 (25-C[H.sub.3]), 38.39 (15-C[H.sub.2]), 41.95 (16-CH), 45.22 (10-CH), 50.01 (3-CH), 50.95 (4-CH), 52.75 (8-CH), 54.04 (19-CH), 60.64 (9-C), 61.39 (20-CH), 69.01 (7-CH), 73.30 (21-CH), 77.09 (18-C), 126.58 (5-C), 126.61 (29-C), 126.84 (14-CH), 127.21 (13-Ch), 128.79 (28,30-CH), 129.53 (27, 31-CH),133.10 (6-C), 138.72 (26-C), 170.80 (24-C=O), 175.29 (1-C=O) and 216.14 (17-C=O).

Purification of Engleromycin acetate

Tubes (120-127) gave white needles that crystallised from the eluent. The solvent was removed under reduced pressure on a rotary evaporator and the solid recrystallised from acetone to give engleromycin acetate, white needles (8.0 mg) mp 274-276 oC, [[[alpha]].sup.23.sub.D]+53[degrees] (c = 0.85, MeOH), m/z (EI) 523, [C.sub.30][H.sub.37]N[O.sub.7]. Oh ([C.sub.5][D.sub.5]N) 0.68 (3H, d, J 7 Hz), 1.00 (3H, d, J 7.0 Hz), 1.61 (3H, s), 2.00 (1H, dd, 4.0, 8.0 Hz), 2.17 (3H, s), 2.53 (1H, m), 2.75 (1H, m), 2.86 (2H, m), 3.20 (2H, m), 3.57 (1H, t, J 2.0 Hz), 3.74 (1H, d, J 2.0 Hz), 4.33 (1H, d, J 2.0 Hz), 4.42 (1H, m), 4.49 ([H.sub.2]O), 5.01 ([1H.sub.a], s), 5.37 (1He, s), 5.69 (1H, s), 5.94 (1H, m), 6.03 (1H, s), 6.26 (1H, d, J 5.0 Hz), 6.57 (1H, dd, J 6.0, 10.0 Hz), 7.26-7.51 (5H, m) and 9.22 (1H, s). [[delta].sub.C] ([C.sub.5][D.sub.5]N) 13.32 (22-C[H.sub.3]), 18.93 (23C[H.sub.3]), 20.33 (H-C[H.sub.3]), 22.68 (25-C[H.sub.3]), 32.89 (5-CH), 39.01 (15-C[H.sub.2]), 42.41 (16-CH), 45.40 (3-CH), 47.30 (10-C[H.sub.2]), 50.52 (4-CH), 53.63 (9-C), 53.92 (19-CH), 54.34 (20-CH), 60.66 (8-CH), 71.34 (7-CH), 75.07 (21-CH), 77.27 (18-C), 112.00 (12 C[H.sub.2]), 126.75 (29-Ar-CH), 128.74 (28, 30-Ar-CH), 129.95 (27, 31-Ar-CH), 132.01 (13-CH), 132.05 (14-CH), 138.34 (26-C), 149.89 (6-C), 170.59 (24-C=O), 175.01 (1C=O) and 216.34 (17-C=O).

Synthesis of 19,20-dihydroxycytochalasin C

19,20-epoxycytochalasin C (80 mg) was heated under reflux with dilute sulphuric acid (3 drops) in THF (4.0 ml) for 1 h. The solution turned light green after boiling for 15 min. It was allowed to cool and diluted with water (5.0 ml). The THF was removed under reduced pressure on a rotary evaporator and the resulting aqueous solution was extracted with chloroform (5.0 ml x 2). Anhydrous sodium sulphate was used to dry the combined chloroform extracts. A light green gum (71 mg) was obtained when the chloroform was removed on a rotary evaporator. The gum was triturated with ethyl acetate to give 19,20-dihydroxycytochalasin C off-white needles (57 mg), mp 251-253 [degrees]C, [[[alpha]].sup.D.sub.23]-23[degrees] (c = 0.90, MeOH) [[M + Na].sup.+] 564, [R.sub.f] (chloroform methanol 95:5) = 0.23.

Conclusion

Static cultures of Rosellinia arcuata were extracted with ethyl acetate to give intimate mixtures of cytochalasins. Four cytochalasins were identified in the mixture. Isolation and purification of the cytochalasins gave cytochalasin C, 19,20-epoxycytochalasin C and a new cytochalasin engleromycin acetate. Soxhlets extraction of the pulverized mycelium gave pure 19,20-epoxycytochalasin C, from which another new cytochalasin 19,20-dihydroxycytochalasin C was synthesized.

Reference

[1] D. C. Aldridge, J. J. Amstrong, R. N. Speake and W. B. Turner: The structures of cytochalasins A and B. J. Chem. Soc. C , 1967, 1667.

[2] W. Rothwesler and C. Tamm: Experientia, 1966, 22, 750.

[3] D. C. Aldridge and W. B. Turner: Structures of cytochalasins C and D. J. Chem. Soc. C, 1969, 923.

[4] R. L. Edwards, D. J. Maitland and A. J. S. Whalley: J. Chem. Soc., Perkin Trans. 1, 1989, 57.

[5] E. Dagne, A. A. L. Gunatilaka, S. Asmellash, D. Abate, D. G. I. Kingston: Phytochemistry, 1997, 42(6), 1343-1348.

[6] D. Abate, W. Abraham and H. Meyer: Phytochemistry, 1997, 44(8), 14431448.

[7] Sivanesam and P. Holliday: Description of pathogenic fungi and bacteria, Commonwealth Myc. Inst. 1973.

[8] E. J. Pedersen, P. Larsen, and M. Boll, Tetrahedron Letters, 1980, 21, 50795082.

[9] W. Dombrowski, G. F. Bills, G. Sabnis, L. R. Koupal, R. Meyer, J. G. Ondeyka, R. A Giacobbe, R. L. Monaghan and R. B. Lingham: J. Antibio., 1992, 45(5), 671-678.

[10] R. L. Edwards, D. J. Maitland and A. J. S. Whalley: J. Chem. Soc., Perkin Trans. 1, 1989, 57.

[11] L. Schipper: J. Phytology, 1978, 68, 868.

E.K. Oppong *, R.L. Edwards **, D.J. Maitland **, A.J.S. Whalley *** and Y. Ameyaw *

* Dept. of Science Education, University of Education, P. O. Box 25, Winneba, Ghana

** Dept. of Chemistry and Forensic Science, University of Bradford, Richmond Road, West Yorkshire, BD7 1DP. United Kingdom

*** Dept. of Biology, Liverpool Polytechnique, Liverpool, L3 3AF, United Kingdom
Table 1: 1H and 13C NMR data of cytochalasin C

[[delta].sub.H] (J values (Hz)) [sup.1]H assignment

0.97 (3H, d, J 6.0 Hz) 22-[H.sub.3]
1.33 (3H, s) 12-[H.sub.3]
1.51 (3H, s) 23-[H.sub.3]
1.88 (3H, s) 11-[H.sub.3]
2.11 (1H,dd, J 4.0,5.0 Hz) 15-[H.sub.a]
2.30 (3H, s) 25-[H.sub.3]
2.63(1H,dd,J 5.0,10.0 Hz) 15-[H.sub.e]
2.81 (1H, s) 4-H
3.02-3.20 (3H, m) 8, 10, 16-H
3.61 (1H, t, J 7.0, 8.0 Hz) 3-H
4.41 (1 H, m) 7-H
4.97 ([H.sub.2]O) [H.sub.2]O peak
5.48 (1H, m) 14-H
5.58 (1H, d, J 6.0, Hz) 19-H
6.00 (1H, s) 21-H
6.34 (1H, t, J 2.0 Hz) 13-H
6.51(1H,dd,J 5.0,10.0 Hz) 20-H
7.16-7.26 (5H, m) 5(Ar-H)
9.34 (1H, s) NH

[[delta].sub.H] (J values (Hz)) [[delta].sub.C] (ppm)

0.97 (3H, d, J 6.0 Hz) 19.31
1.33 (3H, s) 17.14
1.51 (3H, s) 20.54
1.88 (3H, s) 14.78
2.11 (1H,dd, J 4.0,5.0 Hz) 38.53
2.30 (3H, s) 24.60
2.63(1H,dd,J 5.0,10.0 Hz) 38.53
2.81 (1H, s) 50,57
3.02-3.20 (3H, m) 60.84,45.19,42.33
3.61 (1H, t, J 7.0, 8.0 Hz) 50.54
4.41 (1 H, m) 68.94
4.97 ([H.sub.2]O) 5.48
(1H, m) 126.79
5.58 (1H, d, J 6.0, Hz) 132.90
6.00 (1H, s) 76.17
6.34 (1H, t, J 2.0 Hz) 126.81
6.51(1H,dd,J 5.0,10.0 Hz) 133.36
7.16-7.26 (5H, m) 126.49,128.83,129.71
9.34 (1H, s) 78.43

 126.47
 132.57
 138.78
 170.81
 175.30
 210.05

[[delta].sub.H] (J values (Hz)) [sup.13]C assignment

0.97 (3H, d, J 6.0 Hz) 22
1.33 (3H, s) 12
1.51 (3H, s) 23
1.88 (3H, s) 11
2.11 (1H,dd, J 4.0,5.0 Hz) 15
2.30 (3H, s) 25
2.63(1H,dd,J 5.0,10.0 Hz) 15
2.81 (1H, s) 4
3.02-3.20 (3H, m) 8,10,16
3.61 (1H, t, J 7.0, 8.0 Hz) 3
4.41 (1 H, m) 7
4.97 ([H.sub.2]O) 5.48
(1H, m) 14
5.58 (1H, d, J 6.0, Hz) 19
6.00 (1H, s) 21
6.34 (1H, t, J 2.0 Hz) 13
6.51(1H,dd,J 5.0,10.0 Hz) 20
7.16-7.26 (5H, m) 27,28,29,30,31
9.34 (1H, s) 18

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Author:Oppong, E.K.; Edwards, R.L.; Maitland, D.J.; Whalley, A.J.S.; Ameyaw, Y.
Publication:International Journal of Applied Environmental Sciences
Date:Feb 1, 2010
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