- Email: [email protected]
Antimalarial compounds from Hoslundia opposita
Phytochmistry, VOL 31, No. 11, pp. 3781- 3784, 1992
0031~9422/92 S5.00+0.00
Printedin Great Britain.
ANTIMALARIAL
0 1992Pagamon PressLtd
COMPOUNDS
FROM HOSLUAWA
HANS ACHENBACH, REINER WAIBEL, MAYUNGA
H. H. NKUNYA*
OPPOSITA
and HUGO WEEN&
Department of Pharmaceutical Chemistry, Institute of Pharmacy and Food Chemistry, University of Erlangen, Schuhstrasse 19, D-8520 Erlangen, Germany; *Department of Chemistry, University of Dar es Salaam, P-0. Box 35041, Dar es Salaam, Tanzania (Received 24 March
1992)
Key Word Index--Hoslundia opposita Vahl.; Lamiaceae; 3-0-benzoylhosloppone; benzoylhinokiol; 3-0-benzoylhosloquinone; antimalarial activity.
3-O-cinnamoylhosloppone;
3-O-
Abstmct-Four new abietane-type esters, 3-O-benzoylhosloppone, 3-O-cinnamoylhosloppone, 3-0-benzoylhinokiol and 3-0-benzoylhosloquinone were isolated from the root bark of the antimalarial plant HosZundia opposita Vahl. and their structures were determined by a combination of spectroscopic methods and chemical correlations. 3-0Benzoylhosloppone inhibits the growth of the multidrug resistant strain K, of Plusmodiumfalciparum in vitro with an X&value of 0.4 pg ml- ‘.
INTRODUCTION
RESULTS AND DISCUSSION
Hoslundia opposita Vahl. grows as a small shrub and is widely distributed in East and West Africa [ 11. Despite its known uses in African folk medicine and in perfumery [2-4) chemical studies on this plant have only recently been reported [5,6]. Three new Aavonoids were isolated from twigs collected in Cameroon [S], and it has been found that the essential oil from H. opposita consists mainly of sesquiterpene hydrocarbons [6]. Because of its traditional use in treating malaria, we have included this plant in our investigations of Tanzanian medicinal plants with antimalarial activity [7j. The crude n-hexane extract of the root bark was found to have signifjcant in vitro activity against Plasm+ dium fulciparum (I&,, = 5.6 pg ml- ’ ). The extract further exhibited a 26% inhibition of growth of P. berghei in mice, at a daily dose of 190 mg kg- ’ body weight, for four days, which was the highest dose tested. In order to evaluate the active principles, 1 and 2 were isolated and found to be esters of a new abietane-type quinomethane alcohol, which we named hosloppone (3). A third compound, 3-Obenzoylhinokiol(4), was isolated as a minor component. In addition to 1,2 and 4, the new o-quinone 5 was obtained in a small amount only from a second collection of the plant and named 3-O-benzoylhosloquinone. Compound 1 showed significant in vitro activity against the multidrug resistant K, strain of Phsmodium falciparum and against the chloroquine sensitive strain NF 54 (IC,, = 0.4 and 0.22 pg ml - ‘, respectively); compound 2 was also found to be active. Compounds 4 and 5 were obtained in quantities insufficient to be tested for antimalarial activity. This paper describes the structural determinations of 1, 2,4 and 5.
Compound 1 exhibited [M]’ at m/z 418.2144 ( =C2,HJOOQ) by HR-EIMS. The base peak at m/z 105.0340 (C7HSO) indicated a benzoyl group as part of the molecule. This was corroborated by the corresponding signals in the ‘H and 13C NMR spectra (Tables 1 and 2). Homo- and heteronuclear correlation NMR spectroscopy allowed the assignments of all ‘H and 13C NMR signals and established formula 1. The most important ‘H/l% long-range correlations of 1 are shown in Fig. 1. The relative configuration of 1 was determined by NOE studies (Fig. 2). To establish the absolute configuration, 1 was degraded to hosloppone (3) by reductive cleavage and subsequent oxidation by air [8]. The 3S-configuration followed from a Horeau experiment [9] with 3, and was corroborated by the CD curve of 3, which corresponds to that of 3@tcetoxyfuerstione (6) [S]. The spectroscopic properties of 2 closely resembled those of 1. The presence of a cinnamoyl moiety in 2, instead of the benzoyl group in 1, was demonstrated by comparison of the NMR and mass spectra (Tables 1 and 2). As in 1, cleavage of the ester group in 2 led to 3. In contrast to the red coloured compounds 1 and 2, compound 4 did not exhibit absorption in the visible part of the electron spectrum. The EI mass spectrum again indicated a benzoyl group and from the HR mass spectrum the elemental composition is C,,H,,O,. Interpretation of the NMR spectrum including homoand heteronuclear COSY studies resulted in structure 4, and this was corroborated by comparison of the 13CNMR data with those published for hinokiol(7) [lo]. The absolute configuration of 4 came from its methanolysis to 7, a compound with known absolute configuration [lo, 11). The ‘HNMR spectrum of 5 (Ca7H,,0,, by HR-MS) showed similarities with the spectra of 1 and 4. In particular, signals of three methyl3 one isopropyl and one benzoyl group were easily assigned. Homo- and hetero-
-i%esent address: Quest International, P.O. Box 2, 1400 CA Bussum, The Netherlands.
3781
3182
H. ACHENBACH et al.
R,o#Ro...$$Y Ro& 19
ta R’
Rz
R
R
1
benmy
H
5 benaoyl
4 benwyl
2 3
chmoyl
H
H
H
6acetyl
Table 1. ‘HNMR
7H
OH
spectral data of compounds
1 and 2 (in
Table 2. ‘“CNMR
2
C
CD(&)
1 H
6
J WI
6
J
lax
1.69 ddd 3.41 ddd
14.0, 14.0, 5.0 14.0, 3.5, 3.5
1.65 3.39
* *
11.0, 5.5 7.0 7.0 0.5 7.0,0.5 7.0 7.0
4.76 6.43 6.73 6.94 3.17 1.2tY 1.19’ 1.25 1.40 1.62 7.40 (2H) 7.55 (2H) 7.40 6.48 d 7.71 d 7.74
teq 2ax 2cq 3ax 6 7 14 15 16 17 18 19 20 2’,6 3’,5 4’ ; OH
203
2.10 m (2H) 4.86 dd 6.45 d 6.74 d 6.95 d 3.17 s,d 1.20’ d 1.19’ d 1.28 s 1.47 s 1.63 s 8.07 m (2H) 7.47 tn (2H) 7.58 m 7.75 br s
* * l l * + * * * l
16.0 16.0 *
*Assignments may be intcrchangcd. *game couplings and mukiplicities as in 1.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 C=O 1’ 2 3’ 4 5 6
spectral data of compounds 1, 3.4 and 5 (in CDCI,)
1
3
4
5
30.5 23.9 79.1 42.7 163.1 119.5 137.9 127.8 127.1 42.6 146.3 178.5 141.8 133.0 27.0 21.6’ 21.8’ 24.7 27.9 24.5 165.9 130.5 129.6 128.4 133.1 128.4 129.6
30.8 27.3 65.4 43.8’ 165.0 119.2 138.3 127.7 127.0 42.8’ 146.4 178.4 141.7 133.1 26.9 21.7b 21.9b 23.1 27.7 24.4 --
36.6 24.4 81.3 38.3 49.9 19.0 29.9 126.9 147.4 37.3 111.0 150.9 131.9 126.6 26.8 22.5 22.7 28.3 16.8 24.9 166.3 130.9 129.6 128.4 132.8 128.4 129.6
129.7 30.3 76.6 37.9 43.6 146.3 123.6 125.4 132.3 137.5 180.5’ 180.4’ 147.8 136.5 27.6 21.4b 21.5b 26.8 23.8 20.2 166.5 130.8 129.7 128.5 132.9 128.5 129.7
-
‘+bAssignments within a column may be interchanged.
COSY, together with NOE studies (Fig. 3), established that 5 belongs to the rare class of 20(10+5)_ abeo-abietanes, from which only two members with the intact tricyclic skeleton have been reported in the literature [ 121. The stability of 5, particularly in the presence of acid, is limited and easy decomposition occurs. On the other hand, various experiments to convert 1 by a proton catalysed reaction and subsequent reoxidation into 5 failed [13,14]; therefore we do not regard 5 as an artifact. The observation that 5 could be isolated from the plant material only occasionally might be explained by the fact nuclear
Fig. 1. Important long-range correlations observed in the HMBC (= heteronuclear multiple bond correlation) spectrum of 1.
Antimalarial compounds from Hoslundia opposita
Fig. 2. Nuclear
Overhauser enhancements compound 1.
obsfmed
3783
in Fig 3. Nuclear
that the biosynthesis of 5 depends on the place or seasonal state of growth of the plant. Although plants of the family Lamiaceae am known to accumulate abietane-type quinomethides [8], this is the first isolation of the corresponding 3-benzoyloxy and cinnamoyloxy derivatives from a lamiaceous plant. The antimalarial activities of 1 and 2 are within the same range as that for the crude extract. Considering the low yield of the pure compounds and the activity results, it is possible that other active constituent(s) may be present. A corresponding re-examination for minor antimalarial constituents is in progress. Following our earlier observations [15], the antimalarial activities of 1 and 2,can be attributed to the presence of the a&unsaturated carbonyl moiety in the two compounds; this group is suspected to undergo a Michael reaction with nucleophilic sites in the parasite cell DNA molecules, thereby inhibiting the growth of the P. falcipamm parasite.
BXPERIlwENTAI. PIant material. The plant was collected from the Hill campus,
University of Dar es Salaam, in April 1991 and authenticated by Mr L. B. Mwasumbi at the Herbarium, Department of Botany, University of Dar es Salaam, where a voucher specimen is preserved. [a],-Values: CHCls; IR: KBr (unless othenv&e stated); UV/VIS: MeOH, NMR (360 MHz for ‘H, 90 MHz for 13C):CDCl, (TMS as int. standard); MS (EL 70 eV): m/z rel. int.
2 10% (unless key ions). Exaraction and isolation. DIM, powdered root bark (270 9) was subsequently soaked in n-hexane and then CHzCl, at room temp. (2 x 24 hr each). The coned n-hexane extract (1.72 g) was fractionated by VLC over silica gel (400 mesh, Merck), eluting with a stepwise gradient from n-hexane to n-hexane with increasing amounts of EtOAc, to give 6 frs (frs A-F). Fraction B (790mg) was further sepd by CC on silica gel (n-hexane-EtOAc, 19: 1) followed by CC on Sephadex LH-20 (MeOH) into the 2 main frs Bl (455 mg) and B2 (42 mg). From these frs l(410 mg) and 2 (23 mg) were obtained by crystallization (MeOH). Fraction C (282 rug) was subjected to repeated CC (silica gel, n-hexane-EtOAc, 4: 1) and then gel chromatography (Sephadex LH-20, MeOH) to give 4 (31 mg) as the major compound. From one batch of plant material, the coned CH,Cl, extract (3.0 g) by repeated CC and final purification on LH-20 (MeOH) gaveS(-5mg). 3-0-Benzoylhosloppon (1). Dark red needles from MeOH, mp 134-136”; [a];’ - 25” (c 0.12); UV/VIS d,, nm (log a): 423
Overhauser enhancements compound 5.
observed
in
(3.98), 330 (sh), 279 (3.72), 272 (3.74), 244 (sh), 229 (4.523; IRv _ cm-r: 3436,2961,2928,2872,1717,1598,1523,1273,712; MS m/z (rel. int.): 418.2146 (9, [Ml’, calcd. for Cz,H,,O,: 418.2144), 296 (31), 228 (19), 227 (50), 105.0342 (100, calcd for C,H,O: 105.0340),77 (17); ‘HNMR: see Table 1; i3CNMR: see Table 2. 3-0-Cinnamoylhosloppone (2). Dark red needles from MeOH, mp 156-158”; [a];, -90” (~0.15); UV/vIS 1, nm (logs): 423 (3.95), 334 (sh), 277 (4.19); IR v,, cm- ‘: 3432, 2920, 1696, 1342, 1270; MS m/z (tel. int.): 444 (2, [Ml’), 297 (15), 296 (71), 228 (21), 227 (lOO),221 (12), 131 (46), 103 (18); IHNMR: see Table 1. 3-0-BenzoyfbinokioI (4). Pale yellow powder; [a];’ +68” (~0.2); UV I,,, nm (logs): 292 (sh), 281(3.52), 273 (sh), 226 (4.20); IR vcnc’3 MI cm-‘: 3423, 3019,2%5, 2932, 1712, 1280; MS m/z (rel. int.): 406.2504 (55, [Ml+, calcd for C2,H3403: 406.2508), 270 (21), 269.1901 (100, calcd for C,,H,,O: 269.1905), 227 (17), 199 (12), 175 (16), 147 (13). 105.0339 (84 calcd for C,H,O: 105.0340), 77 (29); ‘HNMR: 81.02 (3H, s, Me-20), 1.13 (3H, s, Me-18), 1.24 (3H, d, J=7.0 Hz, Me-16 or Me-17), 1.25 (3H, d, J=7.0 Hx, Me-17 or Me-16), 1.25 (3H, s, Me-19), 1.47 (lH, dd, J, =12.0, J,=2.5Hx, H-5), 1.68 (lH, ddd, J,-Jz-13.5, J, =4.0HgH-lax),l77(1H,dddd,J,-Jz-J,*12.0,J,=6.5Hg H&ax), 1.85-2.07 (3H, H-Zax, H&q, H&q), 2.26 (lH, ddd, J, -13.5, J, -J,-3.5Hx, H-leq), 2.80 (lH, ddd, J,=17.0, J,=11.5, J,=7.5Hz, H-7ax), 2.91 (lH, ddd, J,=17.0, J2=7.0, 5,=1.5Hz,H-7eq),3.13(1H,s,,J=7.0Hz,H-15),4.68(1H,brs, -OH), 4.79 (lH, dd, J, = 11.5, J,= 5.0 Hx, H-3), 6.63 (lH, s, Hll), 6.85 (lH, s, H-14), 7.45 (2H, m, H-3, H-S), 7.57 (lH, tn, H-4’), 8.07 (2H, m, H-2’, H-6’); i3C NMR: see Table 2. 3-O-Benzoylkosloquiaone (5). Purple needles from MeOH, mp 122-123”; [a]$’ i-251” (CHCl,; ~0.1); UV/VIS I,,, nm (logs): 538 (3.13), 326 (3.76), 300 (sh), 290 (sh), 242 (sh), 230 (4.44); IRv,,cm-‘: 3442, 2964, 2930, 2874, 1715, 1678, 1656, 1638, 1602, 1276, 1109, 712; MS m/z (ml. int.): 416.1981 (3, [Ml’, calcd for C,,H,sO,: 416.1987), 296 (22), 295 (15), 294 (69), 266 (15), 251 (26), 227 (33), 211 (lo), 105 (lOO), 77 (24); 1HNMR:81.02(3H,s,Me-19),1.14(3H,d,J=7.0H~Me-160r Me-17), 1.15 (3H, d, J=7.0 Hz, Me-17 or Me-16), 1.19 (3H, s, Me18), 144(3H, s, Me-20), 2.56(1H, dd, J,=21.5, J,=5.0Hz, H-2A), 2.87 (lH, &id, Jl=21.5, J,=5.5, J,=3.5 Hz, H-2,), 2.98 (lH,s,d,J,=7.~J,=l.OHz,~-15),5.07(1H,d,J=5.5Hz,H-3), 6.04 (lH, d, J= 10.0 Hz, H-7), 6.51 (lH, d, J = 10.0 Hz, H-6), 6.62 (lH,d, J= 1.0 Hx, H-14),6.71 (lH,dd, J1 =5.0, J,=3.5 Hz, H-l), 7.45 (2H, m, H-3, H-5’), 7.56 (lH, m, H-4’), 8.01 (2H, m, H-2’, H6’)i i3C NMR: see Table 2. Hosloppone (3). According to ref. [S], 10 mg 1, dissolved in 2 ml EtOEt (abs.) were stirred with 40 mg LiAlH, (1 hr). After
3784
H. ACHENBACH et al.
work-up the combined organic extracts were evapd and purified by CC on silica gel (cyclohexane-EtOAc, 9: 1) to give 5 mg 3 as a dark red powder. [a]g - 164” (c 0.2); UV,VIS I,,, nm (logs): 249 (3.69), 261 (sh). 431 (4.06); IR v:::‘~ cm-‘: 3621, 3019, 2929, 159.5,1521; ‘HNMR: 6 1.18 (3H, d, J=7.0 Hz, Me-16 or Me-17), 1.19(3H,d,J=7.0 Hz,Me-170r Me-16), 1.24(3H,s,Me-18), 1.32 (3H, s, Me-19), 1.53 (lH, ddd. J, -J, - 14.0, J, -4.0 Hz, H-lax), 1,56(3H,s,Me-20), 1.88(1H,m,H-2eq), 1.96(1H,m,H-2axk3.16 (1H. s,d, J,=7.0, J,=l.OHz, H-15) 3.35 (lH, ddd, J,=14.0, J,-J,-3.5 Hz, H-leq), 3.37 (lH, dd, J,=12.0, J,=5.0Hz, H-3), 6 42 (lH, d, J= 7.0 Hz, H-6), 6.73 (lH, d. J=7.0 Hz, H-7), 6.93(1H,d,J=1.0Hz,H-14),7.75(1H,brs,-OH);”CNMR:see Table 2; MS m/z (rel. int.): 314 (48, [Ml’), 296 (36), 253 (lo), 242 (17), 229 (97). 228 (100). 227 (69), 213 (45), 201 (21), 199 (19), 165 (23). 153 (17), 115 (19) 108 (36), 107 (22), 105 (22), 79 (42), 77 (39), 57 (40) 43 (51); CD nm (A&):253 (+2.26), 356 (-3.01), almost identical with the CD curve published for 3-b-acetoxyfuerstion (6) C81. Horeau experiment with 3 [9]. Compound 3 (5 nmol), dissolved in 30 ~1 pyridine (abs.), was mixed with 1Opmol (f )-a-phenylbutyric anhydride. The mixture was kept at 40” for 3 hr. (+)-(R)-a-Phenylethyl amine (2.9 ~1) was added and the mixture held at 40” for a further 15 min. After dilution with 300 ~1 EtOAc the relation of (-)-(R)and (+)-(S)-aphenylbutyramide was determined by GC (25 m SE 54, 4: 0.3 mm; 190” isothermal). The results were corrected accordmg to a simultaneous experiment with cyclohexanol. R,s (10 psi He): (R,R)-amide 20.02 min. (E,S)-amide 22.07 min. For 3 a corrected relation of (R,S)-amide/(R,R)-amide of 1: 1.086 was measured; for lupeol 1: 1.446. Methanolysis ofcompound 4. Compound 4 (1.5 mg), dissolved in 1 ml MeOH, was treated with 1 mg NaOMe (10 mm/room temp.). After neutralization with NH,Cl, the mixture was filtered and evapd. Purification by CC (LH-20, MeOH) afforded 0.8 mg hinokiol (7) as an oil; [u];’ + 52’ (c 0.08) {lit. [ 1l] [a&, + 66.2” (EtOH)}: all spectroscopic properties in agreement with the data published for 7 [lo. 111. Antimalarial rests. The in vitro antimalarial activity of the crude extracts and pure compounds was assayed at the Swiss Tropical Institute, Base], Switzerland, and at the London School of Hygiene and Tropical Medicine, U.K., as previously described [7, 16, 171. The in t:iuo antimalarial test of the crude extract of H. oppostta was carried out at the London School of Hygiene and Tropical Medicine, usmg the 4-day suppressive test against P. berghei infection in mice [ 181. Acknowledgements--Financial support by The Norwegian Agency for International Development (NORAD) and The Netherlands Government through the Dar es Salaam/
Nijmwegen Organic Chemistry Project is gratefully acknowledged. Thanks are also due to the Fonds der Chemischen Industrie, Germany. Furthermore we thank Mrs Monika Gessler, Swiss Tropical Institute in Basel, Switzerland, and Dr D. H. Bray, then at the London School of Hygiene and Tropical Medicine, for the antimalarial tests, the London School for offering the testmg facilities, and Mr F. Sung’hwa, Universtty of Dar es Salaam, for technical assistance.
REFERENCES 1. Hutchinson, J. and Dalziel, J. M. (1963) Flora of West Tropical Africa 2nd Edn. Vol. 2, p. 456. Crown Agents, London. 2. Ayensu, E. S. (1978) Medicinal Plants of West Africa, p. 162. Reference Publications, Algonac, Michigan, U.S.A. 3. Kokwaro, J. 0. (1976) Medicinal Plants ofEast Africa, p. 108. East African Literature Bureau, Nairobi. 4. Uphof, J. C. T. (1959) Dicttonary ofEconomic Plants, p. 188, Wheldon and Wesley, Codicote, U.K. 5. Ngadjui, B. T., Ayafor, J. F., Sondengam, B. L., Connolly, J. D. and Rycroft, D. S. (1991) Tetrahedron 47, 3555. 6. Onayade, 0. A., Ntezurubanza, L.. Scheffer, J. J. C. and Baerheim-Svendsen, A. (1990) Sci. Pharm. 58, 311. 7. Weenen, H., Nkunya, M. H. H., Bray, D. H., Mwasumbi, L. B., Kinabo, L. S. and Kilimali, V. A. E. B. (1990) Planta Med. 56, 368. 8. Miyase, T., Ruedi, P. and Eugster, C. H. (1977) Helv. Chim. Acta 60, 2789. 9. Brooks, C. J. W. and Gilbert, J. D. (1973) .I. Chem. Sot., Chem. Commun. 194. 10. Harrison, L. J. and Asakawa, Y. (1987) Phytochemistry 26, 1211.
11. Matsumoto, T., Usui, S., Kawashima, H. and Mitsuki, M. (1981) Bull. Chem. Sot. Jpn 54, 581. 12. Meng, Q. and Hesse, M. (1990) Heir. Chim. Acta 73, 455. 13. Tahara, A., Mizuno, H. and Ohsawa, T. (1972) Chem. Letters 1163. 14. Tahara, A., Akita, H., Takizawa, T. and Mizuno, H. (1974) Tetrahedron Letters 2837. 15. Weenen, H., Nkunya, M. H. H., Bray, D. H., Mwasumbi,
L. B., Kinabo, L. S., Kilimali, V. A. E. B. and Wijnberg, J. B. P. A. (1990) Planta Med. 56, 371. 16. Desjardins, R. E., Cantield, C. J., Haynes, J. D. and Chulay, J. D. (1979) Antimicrob. Agents Chemother. 16, 710. 17. O’Neill, M. J., Bray, D. H., Boardman, P., Phillipson, J. D. and Warhurst, D. C. (1985) Ptanta Med. 394. 18. Peters, W., Portus, J. H. and Robinson, B. L. (1975) Ann. Trop. Med. Parasitol. 69, 155.
0031~9422/92 S5.00+0.00
Printedin Great Britain.
ANTIMALARIAL
0 1992Pagamon PressLtd
COMPOUNDS
FROM HOSLUAWA
HANS ACHENBACH, REINER WAIBEL, MAYUNGA
H. H. NKUNYA*
OPPOSITA
and HUGO WEEN&
Department of Pharmaceutical Chemistry, Institute of Pharmacy and Food Chemistry, University of Erlangen, Schuhstrasse 19, D-8520 Erlangen, Germany; *Department of Chemistry, University of Dar es Salaam, P-0. Box 35041, Dar es Salaam, Tanzania (Received 24 March
1992)
Key Word Index--Hoslundia opposita Vahl.; Lamiaceae; 3-0-benzoylhosloppone; benzoylhinokiol; 3-0-benzoylhosloquinone; antimalarial activity.
3-O-cinnamoylhosloppone;
3-O-
Abstmct-Four new abietane-type esters, 3-O-benzoylhosloppone, 3-O-cinnamoylhosloppone, 3-0-benzoylhinokiol and 3-0-benzoylhosloquinone were isolated from the root bark of the antimalarial plant HosZundia opposita Vahl. and their structures were determined by a combination of spectroscopic methods and chemical correlations. 3-0Benzoylhosloppone inhibits the growth of the multidrug resistant strain K, of Plusmodiumfalciparum in vitro with an X&value of 0.4 pg ml- ‘.
INTRODUCTION
RESULTS AND DISCUSSION
Hoslundia opposita Vahl. grows as a small shrub and is widely distributed in East and West Africa [ 11. Despite its known uses in African folk medicine and in perfumery [2-4) chemical studies on this plant have only recently been reported [5,6]. Three new Aavonoids were isolated from twigs collected in Cameroon [S], and it has been found that the essential oil from H. opposita consists mainly of sesquiterpene hydrocarbons [6]. Because of its traditional use in treating malaria, we have included this plant in our investigations of Tanzanian medicinal plants with antimalarial activity [7j. The crude n-hexane extract of the root bark was found to have signifjcant in vitro activity against Plasm+ dium fulciparum (I&,, = 5.6 pg ml- ’ ). The extract further exhibited a 26% inhibition of growth of P. berghei in mice, at a daily dose of 190 mg kg- ’ body weight, for four days, which was the highest dose tested. In order to evaluate the active principles, 1 and 2 were isolated and found to be esters of a new abietane-type quinomethane alcohol, which we named hosloppone (3). A third compound, 3-Obenzoylhinokiol(4), was isolated as a minor component. In addition to 1,2 and 4, the new o-quinone 5 was obtained in a small amount only from a second collection of the plant and named 3-O-benzoylhosloquinone. Compound 1 showed significant in vitro activity against the multidrug resistant K, strain of Phsmodium falciparum and against the chloroquine sensitive strain NF 54 (IC,, = 0.4 and 0.22 pg ml - ‘, respectively); compound 2 was also found to be active. Compounds 4 and 5 were obtained in quantities insufficient to be tested for antimalarial activity. This paper describes the structural determinations of 1, 2,4 and 5.
Compound 1 exhibited [M]’ at m/z 418.2144 ( =C2,HJOOQ) by HR-EIMS. The base peak at m/z 105.0340 (C7HSO) indicated a benzoyl group as part of the molecule. This was corroborated by the corresponding signals in the ‘H and 13C NMR spectra (Tables 1 and 2). Homo- and heteronuclear correlation NMR spectroscopy allowed the assignments of all ‘H and 13C NMR signals and established formula 1. The most important ‘H/l% long-range correlations of 1 are shown in Fig. 1. The relative configuration of 1 was determined by NOE studies (Fig. 2). To establish the absolute configuration, 1 was degraded to hosloppone (3) by reductive cleavage and subsequent oxidation by air [8]. The 3S-configuration followed from a Horeau experiment [9] with 3, and was corroborated by the CD curve of 3, which corresponds to that of 3@tcetoxyfuerstione (6) [S]. The spectroscopic properties of 2 closely resembled those of 1. The presence of a cinnamoyl moiety in 2, instead of the benzoyl group in 1, was demonstrated by comparison of the NMR and mass spectra (Tables 1 and 2). As in 1, cleavage of the ester group in 2 led to 3. In contrast to the red coloured compounds 1 and 2, compound 4 did not exhibit absorption in the visible part of the electron spectrum. The EI mass spectrum again indicated a benzoyl group and from the HR mass spectrum the elemental composition is C,,H,,O,. Interpretation of the NMR spectrum including homoand heteronuclear COSY studies resulted in structure 4, and this was corroborated by comparison of the 13CNMR data with those published for hinokiol(7) [lo]. The absolute configuration of 4 came from its methanolysis to 7, a compound with known absolute configuration [lo, 11). The ‘HNMR spectrum of 5 (Ca7H,,0,, by HR-MS) showed similarities with the spectra of 1 and 4. In particular, signals of three methyl3 one isopropyl and one benzoyl group were easily assigned. Homo- and hetero-
-i%esent address: Quest International, P.O. Box 2, 1400 CA Bussum, The Netherlands.
3781
3182
H. ACHENBACH et al.
R,o#Ro...$$Y Ro& 19
ta R’
Rz
R
R
1
benmy
H
5 benaoyl
4 benwyl
2 3
chmoyl
H
H
H
6acetyl
Table 1. ‘HNMR
7H
OH
spectral data of compounds
1 and 2 (in
Table 2. ‘“CNMR
2
C
CD(&)
1 H
6
J WI
6
J
lax
1.69 ddd 3.41 ddd
14.0, 14.0, 5.0 14.0, 3.5, 3.5
1.65 3.39
* *
11.0, 5.5 7.0 7.0 0.5 7.0,0.5 7.0 7.0
4.76 6.43 6.73 6.94 3.17 1.2tY 1.19’ 1.25 1.40 1.62 7.40 (2H) 7.55 (2H) 7.40 6.48 d 7.71 d 7.74
teq 2ax 2cq 3ax 6 7 14 15 16 17 18 19 20 2’,6 3’,5 4’ ; OH
203
2.10 m (2H) 4.86 dd 6.45 d 6.74 d 6.95 d 3.17 s,d 1.20’ d 1.19’ d 1.28 s 1.47 s 1.63 s 8.07 m (2H) 7.47 tn (2H) 7.58 m 7.75 br s
* * l l * + * * * l
16.0 16.0 *
*Assignments may be intcrchangcd. *game couplings and mukiplicities as in 1.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 C=O 1’ 2 3’ 4 5 6
spectral data of compounds 1, 3.4 and 5 (in CDCI,)
1
3
4
5
30.5 23.9 79.1 42.7 163.1 119.5 137.9 127.8 127.1 42.6 146.3 178.5 141.8 133.0 27.0 21.6’ 21.8’ 24.7 27.9 24.5 165.9 130.5 129.6 128.4 133.1 128.4 129.6
30.8 27.3 65.4 43.8’ 165.0 119.2 138.3 127.7 127.0 42.8’ 146.4 178.4 141.7 133.1 26.9 21.7b 21.9b 23.1 27.7 24.4 --
36.6 24.4 81.3 38.3 49.9 19.0 29.9 126.9 147.4 37.3 111.0 150.9 131.9 126.6 26.8 22.5 22.7 28.3 16.8 24.9 166.3 130.9 129.6 128.4 132.8 128.4 129.6
129.7 30.3 76.6 37.9 43.6 146.3 123.6 125.4 132.3 137.5 180.5’ 180.4’ 147.8 136.5 27.6 21.4b 21.5b 26.8 23.8 20.2 166.5 130.8 129.7 128.5 132.9 128.5 129.7
-
‘+bAssignments within a column may be interchanged.
COSY, together with NOE studies (Fig. 3), established that 5 belongs to the rare class of 20(10+5)_ abeo-abietanes, from which only two members with the intact tricyclic skeleton have been reported in the literature [ 121. The stability of 5, particularly in the presence of acid, is limited and easy decomposition occurs. On the other hand, various experiments to convert 1 by a proton catalysed reaction and subsequent reoxidation into 5 failed [13,14]; therefore we do not regard 5 as an artifact. The observation that 5 could be isolated from the plant material only occasionally might be explained by the fact nuclear
Fig. 1. Important long-range correlations observed in the HMBC (= heteronuclear multiple bond correlation) spectrum of 1.
Antimalarial compounds from Hoslundia opposita
Fig. 2. Nuclear
Overhauser enhancements compound 1.
obsfmed
3783
in Fig 3. Nuclear
that the biosynthesis of 5 depends on the place or seasonal state of growth of the plant. Although plants of the family Lamiaceae am known to accumulate abietane-type quinomethides [8], this is the first isolation of the corresponding 3-benzoyloxy and cinnamoyloxy derivatives from a lamiaceous plant. The antimalarial activities of 1 and 2 are within the same range as that for the crude extract. Considering the low yield of the pure compounds and the activity results, it is possible that other active constituent(s) may be present. A corresponding re-examination for minor antimalarial constituents is in progress. Following our earlier observations [15], the antimalarial activities of 1 and 2,can be attributed to the presence of the a&unsaturated carbonyl moiety in the two compounds; this group is suspected to undergo a Michael reaction with nucleophilic sites in the parasite cell DNA molecules, thereby inhibiting the growth of the P. falcipamm parasite.
BXPERIlwENTAI. PIant material. The plant was collected from the Hill campus,
University of Dar es Salaam, in April 1991 and authenticated by Mr L. B. Mwasumbi at the Herbarium, Department of Botany, University of Dar es Salaam, where a voucher specimen is preserved. [a],-Values: CHCls; IR: KBr (unless othenv&e stated); UV/VIS: MeOH, NMR (360 MHz for ‘H, 90 MHz for 13C):CDCl, (TMS as int. standard); MS (EL 70 eV): m/z rel. int.
2 10% (unless key ions). Exaraction and isolation. DIM, powdered root bark (270 9) was subsequently soaked in n-hexane and then CHzCl, at room temp. (2 x 24 hr each). The coned n-hexane extract (1.72 g) was fractionated by VLC over silica gel (400 mesh, Merck), eluting with a stepwise gradient from n-hexane to n-hexane with increasing amounts of EtOAc, to give 6 frs (frs A-F). Fraction B (790mg) was further sepd by CC on silica gel (n-hexane-EtOAc, 19: 1) followed by CC on Sephadex LH-20 (MeOH) into the 2 main frs Bl (455 mg) and B2 (42 mg). From these frs l(410 mg) and 2 (23 mg) were obtained by crystallization (MeOH). Fraction C (282 rug) was subjected to repeated CC (silica gel, n-hexane-EtOAc, 4: 1) and then gel chromatography (Sephadex LH-20, MeOH) to give 4 (31 mg) as the major compound. From one batch of plant material, the coned CH,Cl, extract (3.0 g) by repeated CC and final purification on LH-20 (MeOH) gaveS(-5mg). 3-0-Benzoylhosloppon (1). Dark red needles from MeOH, mp 134-136”; [a];’ - 25” (c 0.12); UV/VIS d,, nm (log a): 423
Overhauser enhancements compound 5.
observed
in
(3.98), 330 (sh), 279 (3.72), 272 (3.74), 244 (sh), 229 (4.523; IRv _ cm-r: 3436,2961,2928,2872,1717,1598,1523,1273,712; MS m/z (rel. int.): 418.2146 (9, [Ml’, calcd. for Cz,H,,O,: 418.2144), 296 (31), 228 (19), 227 (50), 105.0342 (100, calcd for C,H,O: 105.0340),77 (17); ‘HNMR: see Table 1; i3CNMR: see Table 2. 3-0-Cinnamoylhosloppone (2). Dark red needles from MeOH, mp 156-158”; [a];, -90” (~0.15); UV/vIS 1, nm (logs): 423 (3.95), 334 (sh), 277 (4.19); IR v,, cm- ‘: 3432, 2920, 1696, 1342, 1270; MS m/z (tel. int.): 444 (2, [Ml’), 297 (15), 296 (71), 228 (21), 227 (lOO),221 (12), 131 (46), 103 (18); IHNMR: see Table 1. 3-0-BenzoyfbinokioI (4). Pale yellow powder; [a];’ +68” (~0.2); UV I,,, nm (logs): 292 (sh), 281(3.52), 273 (sh), 226 (4.20); IR vcnc’3 MI cm-‘: 3423, 3019,2%5, 2932, 1712, 1280; MS m/z (rel. int.): 406.2504 (55, [Ml+, calcd for C2,H3403: 406.2508), 270 (21), 269.1901 (100, calcd for C,,H,,O: 269.1905), 227 (17), 199 (12), 175 (16), 147 (13). 105.0339 (84 calcd for C,H,O: 105.0340), 77 (29); ‘HNMR: 81.02 (3H, s, Me-20), 1.13 (3H, s, Me-18), 1.24 (3H, d, J=7.0 Hz, Me-16 or Me-17), 1.25 (3H, d, J=7.0 Hx, Me-17 or Me-16), 1.25 (3H, s, Me-19), 1.47 (lH, dd, J, =12.0, J,=2.5Hx, H-5), 1.68 (lH, ddd, J,-Jz-13.5, J, =4.0HgH-lax),l77(1H,dddd,J,-Jz-J,*12.0,J,=6.5Hg H&ax), 1.85-2.07 (3H, H-Zax, H&q, H&q), 2.26 (lH, ddd, J, -13.5, J, -J,-3.5Hx, H-leq), 2.80 (lH, ddd, J,=17.0, J,=11.5, J,=7.5Hz, H-7ax), 2.91 (lH, ddd, J,=17.0, J2=7.0, 5,=1.5Hz,H-7eq),3.13(1H,s,,J=7.0Hz,H-15),4.68(1H,brs, -OH), 4.79 (lH, dd, J, = 11.5, J,= 5.0 Hx, H-3), 6.63 (lH, s, Hll), 6.85 (lH, s, H-14), 7.45 (2H, m, H-3, H-S), 7.57 (lH, tn, H-4’), 8.07 (2H, m, H-2’, H-6’); i3C NMR: see Table 2. 3-O-Benzoylkosloquiaone (5). Purple needles from MeOH, mp 122-123”; [a]$’ i-251” (CHCl,; ~0.1); UV/VIS I,,, nm (logs): 538 (3.13), 326 (3.76), 300 (sh), 290 (sh), 242 (sh), 230 (4.44); IRv,,cm-‘: 3442, 2964, 2930, 2874, 1715, 1678, 1656, 1638, 1602, 1276, 1109, 712; MS m/z (ml. int.): 416.1981 (3, [Ml’, calcd for C,,H,sO,: 416.1987), 296 (22), 295 (15), 294 (69), 266 (15), 251 (26), 227 (33), 211 (lo), 105 (lOO), 77 (24); 1HNMR:81.02(3H,s,Me-19),1.14(3H,d,J=7.0H~Me-160r Me-17), 1.15 (3H, d, J=7.0 Hz, Me-17 or Me-16), 1.19 (3H, s, Me18), 144(3H, s, Me-20), 2.56(1H, dd, J,=21.5, J,=5.0Hz, H-2A), 2.87 (lH, &id, Jl=21.5, J,=5.5, J,=3.5 Hz, H-2,), 2.98 (lH,s,d,J,=7.~J,=l.OHz,~-15),5.07(1H,d,J=5.5Hz,H-3), 6.04 (lH, d, J= 10.0 Hz, H-7), 6.51 (lH, d, J = 10.0 Hz, H-6), 6.62 (lH,d, J= 1.0 Hx, H-14),6.71 (lH,dd, J1 =5.0, J,=3.5 Hz, H-l), 7.45 (2H, m, H-3, H-5’), 7.56 (lH, m, H-4’), 8.01 (2H, m, H-2’, H6’)i i3C NMR: see Table 2. Hosloppone (3). According to ref. [S], 10 mg 1, dissolved in 2 ml EtOEt (abs.) were stirred with 40 mg LiAlH, (1 hr). After
3784
H. ACHENBACH et al.
work-up the combined organic extracts were evapd and purified by CC on silica gel (cyclohexane-EtOAc, 9: 1) to give 5 mg 3 as a dark red powder. [a]g - 164” (c 0.2); UV,VIS I,,, nm (logs): 249 (3.69), 261 (sh). 431 (4.06); IR v:::‘~ cm-‘: 3621, 3019, 2929, 159.5,1521; ‘HNMR: 6 1.18 (3H, d, J=7.0 Hz, Me-16 or Me-17), 1.19(3H,d,J=7.0 Hz,Me-170r Me-16), 1.24(3H,s,Me-18), 1.32 (3H, s, Me-19), 1.53 (lH, ddd. J, -J, - 14.0, J, -4.0 Hz, H-lax), 1,56(3H,s,Me-20), 1.88(1H,m,H-2eq), 1.96(1H,m,H-2axk3.16 (1H. s,d, J,=7.0, J,=l.OHz, H-15) 3.35 (lH, ddd, J,=14.0, J,-J,-3.5 Hz, H-leq), 3.37 (lH, dd, J,=12.0, J,=5.0Hz, H-3), 6 42 (lH, d, J= 7.0 Hz, H-6), 6.73 (lH, d. J=7.0 Hz, H-7), 6.93(1H,d,J=1.0Hz,H-14),7.75(1H,brs,-OH);”CNMR:see Table 2; MS m/z (rel. int.): 314 (48, [Ml’), 296 (36), 253 (lo), 242 (17), 229 (97). 228 (100). 227 (69), 213 (45), 201 (21), 199 (19), 165 (23). 153 (17), 115 (19) 108 (36), 107 (22), 105 (22), 79 (42), 77 (39), 57 (40) 43 (51); CD nm (A&):253 (+2.26), 356 (-3.01), almost identical with the CD curve published for 3-b-acetoxyfuerstion (6) C81. Horeau experiment with 3 [9]. Compound 3 (5 nmol), dissolved in 30 ~1 pyridine (abs.), was mixed with 1Opmol (f )-a-phenylbutyric anhydride. The mixture was kept at 40” for 3 hr. (+)-(R)-a-Phenylethyl amine (2.9 ~1) was added and the mixture held at 40” for a further 15 min. After dilution with 300 ~1 EtOAc the relation of (-)-(R)and (+)-(S)-aphenylbutyramide was determined by GC (25 m SE 54, 4: 0.3 mm; 190” isothermal). The results were corrected accordmg to a simultaneous experiment with cyclohexanol. R,s (10 psi He): (R,R)-amide 20.02 min. (E,S)-amide 22.07 min. For 3 a corrected relation of (R,S)-amide/(R,R)-amide of 1: 1.086 was measured; for lupeol 1: 1.446. Methanolysis ofcompound 4. Compound 4 (1.5 mg), dissolved in 1 ml MeOH, was treated with 1 mg NaOMe (10 mm/room temp.). After neutralization with NH,Cl, the mixture was filtered and evapd. Purification by CC (LH-20, MeOH) afforded 0.8 mg hinokiol (7) as an oil; [u];’ + 52’ (c 0.08) {lit. [ 1l] [a&, + 66.2” (EtOH)}: all spectroscopic properties in agreement with the data published for 7 [lo. 111. Antimalarial rests. The in vitro antimalarial activity of the crude extracts and pure compounds was assayed at the Swiss Tropical Institute, Base], Switzerland, and at the London School of Hygiene and Tropical Medicine, U.K., as previously described [7, 16, 171. The in t:iuo antimalarial test of the crude extract of H. oppostta was carried out at the London School of Hygiene and Tropical Medicine, usmg the 4-day suppressive test against P. berghei infection in mice [ 181. Acknowledgements--Financial support by The Norwegian Agency for International Development (NORAD) and The Netherlands Government through the Dar es Salaam/
Nijmwegen Organic Chemistry Project is gratefully acknowledged. Thanks are also due to the Fonds der Chemischen Industrie, Germany. Furthermore we thank Mrs Monika Gessler, Swiss Tropical Institute in Basel, Switzerland, and Dr D. H. Bray, then at the London School of Hygiene and Tropical Medicine, for the antimalarial tests, the London School for offering the testmg facilities, and Mr F. Sung’hwa, Universtty of Dar es Salaam, for technical assistance.
REFERENCES 1. Hutchinson, J. and Dalziel, J. M. (1963) Flora of West Tropical Africa 2nd Edn. Vol. 2, p. 456. Crown Agents, London. 2. Ayensu, E. S. (1978) Medicinal Plants of West Africa, p. 162. Reference Publications, Algonac, Michigan, U.S.A. 3. Kokwaro, J. 0. (1976) Medicinal Plants ofEast Africa, p. 108. East African Literature Bureau, Nairobi. 4. Uphof, J. C. T. (1959) Dicttonary ofEconomic Plants, p. 188, Wheldon and Wesley, Codicote, U.K. 5. Ngadjui, B. T., Ayafor, J. F., Sondengam, B. L., Connolly, J. D. and Rycroft, D. S. (1991) Tetrahedron 47, 3555. 6. Onayade, 0. A., Ntezurubanza, L.. Scheffer, J. J. C. and Baerheim-Svendsen, A. (1990) Sci. Pharm. 58, 311. 7. Weenen, H., Nkunya, M. H. H., Bray, D. H., Mwasumbi, L. B., Kinabo, L. S. and Kilimali, V. A. E. B. (1990) Planta Med. 56, 368. 8. Miyase, T., Ruedi, P. and Eugster, C. H. (1977) Helv. Chim. Acta 60, 2789. 9. Brooks, C. J. W. and Gilbert, J. D. (1973) .I. Chem. Sot., Chem. Commun. 194. 10. Harrison, L. J. and Asakawa, Y. (1987) Phytochemistry 26, 1211.
11. Matsumoto, T., Usui, S., Kawashima, H. and Mitsuki, M. (1981) Bull. Chem. Sot. Jpn 54, 581. 12. Meng, Q. and Hesse, M. (1990) Heir. Chim. Acta 73, 455. 13. Tahara, A., Mizuno, H. and Ohsawa, T. (1972) Chem. Letters 1163. 14. Tahara, A., Akita, H., Takizawa, T. and Mizuno, H. (1974) Tetrahedron Letters 2837. 15. Weenen, H., Nkunya, M. H. H., Bray, D. H., Mwasumbi,
L. B., Kinabo, L. S., Kilimali, V. A. E. B. and Wijnberg, J. B. P. A. (1990) Planta Med. 56, 371. 16. Desjardins, R. E., Cantield, C. J., Haynes, J. D. and Chulay, J. D. (1979) Antimicrob. Agents Chemother. 16, 710. 17. O’Neill, M. J., Bray, D. H., Boardman, P., Phillipson, J. D. and Warhurst, D. C. (1985) Ptanta Med. 394. 18. Peters, W., Portus, J. H. and Robinson, B. L. (1975) Ann. Trop. Med. Parasitol. 69, 155.