Phytochemistry, Vol. 34,No. 4, pp. 11471151,1993 Printedin Great Britain. ALKALOIDS 003l-9422/93 $6.00 + 0.00 0 1993PergamonPressLtd zyxwvutsrqpo OF zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ERYTHROXYLUM ZAMBESIACUM STEM-BARK* PHILIPPE CHRISTEN,tS MARGARET F. ROBERTS,~ J. DAVID PHILLIPSON$ and WILLIAM C. EVANS~~ TDepartment of Pharmacognosy, University of Geneva, Sciences II, 30, Quai E.-Ansennet, CH-1211 Geneva 4, Switzerland; @epartment of Pharmacognosy, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC 1N lAX, U.K.; IlDepartment of Pharmaceutical Sciences,University of Nottingham, Nottingham NG7 2RD, U.K. (Received in revised fom 14 April 1993) Key Word Index--Erythroxylum zambesiacum; Erythroxylaceae; stem-bark; tropane alkaloids; nico- tine; GC; GC-MS; chemotaxonomy. Abstract-Twenty-seven alkaloids were identified from the stem-bark of Erythroxylum zambesiacum by GC and GCMS. The characteristic alkaloids were tropane derivatives, together with nicotine. In addition to known substances previously isolated from the root-bark, a further 20 alkaloids were identified in the stem-bark. The principal base was 3a-(3’,4’,5’-trimethoxybenzoyloxy)tropane. Additionally, three new alkaloids were characterized as 6fibenzoyloxytropan-3-one and, tentatively, 6-isovaleryloxytropan-3-01 and 3-(2-methylbutyryloxy)tropan-6,7-diol. INTRODUCTION Erythroxylum zambesiacum is a small tree mainly confined to the Zambesi valley near the Victoria Falls. It was first described in 1962 [ 1J and is related to the W. African species E. mannii. The alkaloid analysis of the root-bark of E. zambesiacum has been reported previously [2]. However the stem-bark does not appear to have been investigated for alkaloids. Knowledge of the complete alkaloid pattern is of interest not only phytochemically, but also in relation to aspects of alkaloid biogenesis and metabolism. As a further contribution to our studies on the chemotaxonomy of this medicinally important genus, we now report a detailed phytochemical investigation of the alkaloidal pattern of the stem-bark of E. zambesiacum. RESULTS AND DISCUSSION A preliminary TLC examination of petrol and diethyl ether extracts of the powdered stem-bark revealed the presence of five major alkaloids as well as several other minor bases. During recent years, capillary GC has been used as a powerful method for the analysis of tropane alkaloids [3]. The combination of capillary GC with mass apectrometry is a sensitive tool which has shown that tropane alkaloid containing plants generally have a large number of alkaloids which were not detected by older, less sensitive methods [4]. A simple GC and GC-MS procedure has been developed for the identification of tropane alkaloids in crude plant extracts [S]; this revealed the presence of 27 *Part 11 in the series ‘Alkaloids of the genus Erythroxylwm’. For part 10 see. ref. [20]. IAuthor to whom correspondence should be addressed. compounds in the stem-bark of E. zambesiacum representing 0.1% of the dried plant material (Table 1). Of these, seven alkaloids have already been reported to occur in the root-bark. Twenty bases were unknown as constituents of E. zambesiacum. Furthermore, three novel alkaloids have been identified. The characteristic alkaloids are tropanol esters of a range of acids. The amino alcohols are tropine, 3,6dihydroxytropane and 3,6,7_trihydroxytropane and the acids involved in esterification are listed in Table 2. The alkaloid mixture was dominated by 3a-(3’,4’,5’-trimethoxybenzoyloxy)tropane (22), 3a-(3’,4’,5’-trimethoxycinnamoyloxy)tropane (24), 3a-(3’,4’,5’-trimethoxybenzoyloxy)tropan-6/I-01 (23), 68-benzoyloxytropan-3a-ol (19) and 3a-phenylacetoxytropane (17). Furthermore, one nor-derivative (15) and nicotine (7) were identified The latter compound has been isolated previously in cultivated coca, E. coca [6] as well as in the stem-bark of E. cuneatum [7]; it also co-occurs with tropane alkaloids in Wettsteins’s tribe Salpiglossideae of the Solanaceae. Intermediates and products of side-reactions of the tropane alkaloid biosynthetic pathway, e.g. hygrine (l), hygrolines (2,3), tropinone (4), tropine (5) and pseudotropine (6) were identified by GC-MS as constituents of E. zambesiacum stem-bark. The hygrolines are found as two diastereoisomen which have not been detected previously in this species, but their trivial derivation from hygrine makes them wmmon side-products of the biosynthetic route leading to the tropane alkaloids. The El mass spectrum of8 exhibited a [M]’ at m/z 155 (corresponding to the formula CsH,,NO,) and a peak at m/z 111 [M-C(6)HOH-C(7)H,]+ indicating a 6hydroxy-3-one derivative. Furthermore, a strong peak at 1147 P. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF CHRISTEN et al. 1148 Table 1. Alkaloids identified in stem-bark of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON Erythroxylum zambesiacum Compound no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 CM1+ Reference* material Reference? mass spectrum + -. + + + + + + + + + + + Previously identifiedt in the root-bark zyxwvutsrqpo Alkaloid zin) m/z Hygrine Hygroline A Hygroline B Tropinone Tropine Pseudotropine Nicotine Tropan-68-ol-3-one Tropan-3a,6/?diol 3-(2-Methylbutyryloxy)tropane 3cc-Isovaleryloxytropan-6/Z-o] (valeroidine) C,,H,,NO, 6-Isovaleryloxytropan-3-o& 6/I-Tigloyloxytropan-3a-ol 3-Phenylacetoxynortropane 3-(2-Methylbutyryloxy)tropan-6,7-dials 3a-Phenylacetoxytropane 6/?-Benzoyloxytropan-3-o@ 6/LBenzoyloxytropan-3a-ol 3a-(3’,4’-Dimethoxybenzoyloxy)tropane (convolamine) 3-Cinnamoyloxytropan-6-01 b-(3’,4’,5’-Trimethoxybenzoyloxy)tropane 3a-(3’,4’,5’-Trimethoxybenzoyloxy)tropan-6/?-o] 3a-(3’,4’,5’-Trimethoxycinnamoyloxy)tropane 3a-(3’,4’,5’-Trimethoxybenzoyloxy)tropan68,7B-diol 3a-(3’,4’,5’-Trimethoxycinnamoyloxy~6/3benzoyloxytropane 3a-(3’,4’,5’-Trimethoxycinnamoyloxy)-6/Ibenzoyloxy-7jGacetoxytropane 2.62 2.85 3.07 3.96 4.49 5.45 8.07 8.68 10.12 12.68 22.91 23.03 23.11 23.33 23.66 23.80 24.53 26.95 29.00 141 143 143 139 141 141 162 155 157 225 241 239 241 239 245 257 259 259 261 34.79 35.88 37.85 41.38 43.95 305 287 335 351 361 47.15 367 + + + 57.76 481 + + + 59.03 539 + + + + + + + + + + _ + + -I- .+ + + c *Sample available from W. C. Evans. PMS available or in literature. $Alkaloid present in root-bark. $New alkaloid. Table 2. Acids recorded as ester components of tropanols in E. zambesiacum stem-bark Acetic (1) 2-Methylbutyric (2) Isovaleric (2) Tight (1) Benzoic (4) Phenylacetic (2) Cinnamic (1) 3,4-Dimethoxybenzoic (1) 3,4,5-Trimethoxybenzoic (3) 3,4,5-Trimethoxycinnamic (3) Numbers in parenthesis indicate number of alkaloids found involving this acid. m/z 83 is due to further decomposition of the m/z 111 ion fragment by loss of carbon monoxide. This expulsion is diagnostic for 6- and 7-oxygenated tropinones [S]. Other typical fragments at m/z 97,82,57 and 42 were consistent with an alkaloid having the structure, tropan-6@-ol-3one. This compound had identical properties (TLC, GC, mass spectrum) with those of the synthesized compound run under the same conditions. This is the first report of the natural occurrence of tropan-68-ol-3-one. Tropan3a,6/&diol (9) was also identified as a constituent of the stem-bark. It has not been reported previously as a natural alkaloid from the genus Erythroxylum although it has been already isolated from species of Datura [9] and Schizanth [lo]. 3-(2-Methylbutyryloxy)tropane (10) is reported to occur in the genus Erythroxyhm for the first time. However, in the absence of other spectroscopic data, it was not possible to assign the configuration of the substituent at C-3. This alkaloid was isolated from Duboisia kichhardtii [ 1 l] and recently identified by GC-MS in Datura innoxia c41. The CM] + of alkaloid 12 at m/z 239, together with prominent ions at m/z 156,139, 122,96 and 94 suggested a disubstituted tropane nucleus of molecular formula C13HZ1N0, with an esterifying acid C,H,O,. The base peak at m/z 113 indicated that the free hydroxyl group is at the C-3 position. In the absence of other spectroscopic 1149 Alkaloids from zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK Erythroxylum The EI mass spectral fragmentation of 18 was condata, it was not possible to define which isomer (angeloyl, tigloyl or senecioyl) was attached to C-6. sistent with that for a benzoic acid ester (m/z 122, 105,77) The mass spectra of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 11 and 13 showed identical [M]’ of a hydroxytropinone (m/z 111,110,83). The occurrence at m/z 241 corresponding to the elemental composition of fragment ion peaks at m/z 154 [M-C6H$0]+ and 138 [M -C,H$Oz]+ confirmed the presence of benzoic Ci3Hz,N03. The fragmentation patterns are characteracid as the esterifying acid. The position of the ester group istic of monoesters of dihydroxytropane. The alkaloid within the molecule was established from the mass spec11 shows fragment ion peaks at m/z 197 [M-C(6) trum in which loss of the C-6 and C-7 atoms gave rise to HOH-C(7)HJ +, 156 [M-CSH90]+ and 140 [M the base peak at m/z 111 [M - C(7)H,C&)HOCOC,H,]‘. -CSH902]+ with a base peak at m/z 94. This confirms the attachment of the ester function at C-3 and a free Such cleavage has been shown to occur most readily when the C-6 and/or C-7 atoms of the tropane ring bear a hydroxyl group at C-6 of the tropane nucleus. On the other hand, alkaloid 13 had a base peak at m/z 113 and a substituent [S]. The [Ml+ corresponded with the formula C,,Hi,NOs. The strong peak at m/z 83 supports prominent ion at m/z 96, indicating that the free hydroxyl is at the C-3 position and the esterifying acid at C-6. The the 3-one assignment due to elimination of the carbonyl assignments of alkaloids 11 and 13 as isovaleryl esters. group [13]. Other typical fragments at m/z 96,95,94,82 could be deduced from the fragmentation pattern of and 42 are characteristic of the fragmentation of the tropane nucleus. The structure of the new alkaloid was the acid moieties. The presence of [M - 151’ and [M -43]+ ions and the absence of a [M - 29]+ ion are confirmed at 6/I-benzoyloxytropan-3-one by comparison (TLC, GC, mass spectrum) with the semi-synthetic comassigned to the loss of an isovaleryl moiety. Valeroidine and (11) was first isolated as a by-product in the manufacture of pound prepared from 6fl-hydroxytropan-3-one benzoyl chloride [ 163. cocaine from Peruvian coca leaves and from the rootbark of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA E. dekindtii [12]. Compound 13, tentatively 6/I-Benzoyloxytropan-3a-ol(19) was identified by comidentified as 6-isovaleryloxytropan-3-01 has not been parison (TLC, GC, mass spectrum) with an authentic sample isolated from the leaves of E. cuneatum [7]. This previously recorded. However, lack of material precluded alkaloid has also been isolated from E. zambesiacum rootthe making of any stereochemical observations. bark [2]. 6fi-Tigloyloxytropan-3a-ol(14), was identified by comFrom a biogenetic point of view, it is of interest to point parison (TLC, GC, mass spectrum) with an authentic out for the Solanaceae that it is usually considered that sample. The alkaloid was first isolated from Datura hydroxylation and esterification of the tropane ring at Ccornigera [14] and has also been detected in E. cuneatum [7]. 6 and C-7 take place after the reduction of tropinone to tropine and Y-tropine. Biosynthetic experiments originAlkaloid 15 gave a mass spectrum consistent with that ally indicated the reductions to be non-reversible [17], predicted for 3-phenylacetoxynortropane, the base peak but recently Hashimoto et al. [18] have demonstrated, for at m/z 110 being typical of a substituted nortropan-3-ok the presence of m/z 91 (C,H,) and the absence of m/z 119 Hyoscyamus root cultures, the reversibility of the mediated tropinone to tropine conversion. The identification (ArCO) suggested phenylacetic acid as esterifying acid. of alkaloids S, 9,18 and 19 suggests that the biosynthesis This alkaloid has been previously reported as a minor of 6-hydroxytropane esters, at least in Erythroxylum constituent of the root-bark of E. hypericijiofolium[15]. The N-methyl analogue (17) was unambiguously identified by species, takes place before the reduction of the ketone at C-3. This hypothesis has no chemical or biochemical comparison (TLC, GC, mass spectrum) with the base precedence and leads us to propose the possible new prepared from tropine and phenylacetyl chloride. Compound 17 has been previously isolated from E. biosynthetic relationships which are illustrated in Fig. 1. Furthermore, it is noteworthy that no 6,7dihydroxytropandekindtii [ 121. Compound 16 is a new alkaloid identified as 3-(2- 3-one esters have been detected in the alkaloid mixture methylbutyryloxy)tropan-6,7-diol. Its mass spectrum is indicating that esterification at C-7 occurs after the stereospecific reduction of the ketone. This hypothesis characteristic of a monoester of 3,6,7_trihydroxytropane. requires experimental verification and at this stage we The [M]’ at m/z 257 corresponds to the molecular cannot rule out the possibility that tropane alkaloids formula C,sHz3N0,. Further indication of the 1w, came from the CI mass spectrum which displayed a [M + H] + formed in the roots are hydrolysed and oxidized in the peak at m/z 258. Since the base peak was at m/z 94 it was transpiration stream from roots to aerial parts. The EI mass spectrum of alkaloid 20 exhibited a [M] + considered that the alkaloid had the ester function attachThe ed to C-3. The occurrence of a prominent peak at m/z 197 at m/z 305 consistent with the formula C,,H,,NO,. fragmentation pattern [m/z 124 (base peak), 140, 94, 83, [M-60]+ confirmed the attachment of two hydroxyl 82, 67, 421 corresponded to that of a 3-substituted groups to C-6 and C-7. Lack of material prevented the tropane nucleus. Ions at m/z 124 and 140 are consistent application of other analytical techniques to assign the stereochemical orientation of the substituent attached to with the loss of a dimethoxybenzoic acid from the [M] +. The presence of this acid is further indicated by ions at C-3. The esterifying acid was identified as 2-methylbum/z 165 [(MeO),C,H,CO]+ and 182 [(MeO), tyric acid by the presence of ions corresponding to [M C,H,C02H] +. In the absence of other spectroscopic -Me]+ and [M -CzHJ+. The absence of an ion corresdata, it was not possible to establish the configuration of ponding to [M - C(Me),] + is evidence for the 2-methylbutyryl ester and ruled out the possibility of other acids. the substituent at C-3 and to define the position of the 1150 P. CHRISTENet al. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON EXPERIMENTAL Stem-bark of E. zambesiacum N. Robson was collected in Zambia from the same plants as examined in ref. [Z]. 2,3 TLC. Carried out on silica gel F,,, with (i) Me&OH,O-conc. NH, (80:15:2) and (ii) CHCl,-Me,COcont. NH, (50: 50: l), and on aluminium oxide 60 F254 with (iii) Et,O-EtOH (95: 1). GC and GC- M S. Alkaloids were identified by GC on the basis of the similarity of R,s with authentic compounds and by their GC-MS fragmentation patterns. An instrument equipped with two detectors (NPD and FID) was used in the present study as described previously [S]. A 15m x 0.252mm i.d. fused-silica capillary column coated with the methylsilicone phase DB-1 (film thickness 0.25 pm) and with He as carrier gas at 0.5 bar pressure was used. The temp. prog. was, isothermal 80” for 1 min, 80-310” at 4” min-‘, isothermal 310” for 5 min. Both inj. and det. temps were maintained at 360”. GC-MS was performed in the EI mode at 70 eV. Operating conditions were (i) isothermal 35” for 2 min, 35-100” at 30” min-‘, 100-300” at 10” min-‘, isothermal 300” for 5 min. (ii) isothermal 35” for 2 min, 35-300” at 30” min-I, isoth0” zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDC OH OH OR ermal 300” for 5 min. CI mass spectra were recorded on 19 9 5 6 the same instrument using NH, as reagent gas. Fig. 1. Suggested biosynthetic Inter-relationships between Extraction of alkaloids. Powdered stem-bark (64g) was alkaloids of the 3.6-dihydroxytropane-type. extracted with 250 ml n-hexane. The dried defatted plant material was mixed with Ca(OH), (12 g) and H,O (50 ml), allowed to stand overnight and then exhaustively exmtthoxy groups on the aromatic ring. However, TLC, tracted with EtzO. The Et,0 extract was coned to a small GC retention time and mass spectral data were consistent vol., acidified with 0.1 M HCl (10 ml) and extracted with with the structure of convolamine [3o+‘$dimethoxybenEt,0 (3 x 5 ml). The aq. layer, basified by adding 17% zoyloxy)tropane]. This alkaloid has been isolated preNH, soln was repeatedly extracted with petrol (bp viously from Conuoloulus [ 191 but has not been reported as a component of Erythroxylum. 4&60”) and Et,O. The petrol and ether extracts were Compound 21 has been tentatively identified as 3dried (Na,SO,) to afford 2 residues of 29.7 mg and cinnamoyloxytropan-6-01: m/z 287 [M] +; 243 [M 66.9 mg, respectively. - C(7)H,C(6)HOH] +; 156 [M - PhCH=CHCO] +; 140 Identijication of alkaloids. Retention data and mass [M -PhCH=CHCO,] +; 131 [PhCH=CHCO] +; 148 spectra of known bases were compared with those of [PhCH=CHCO,H] +. The 3cr,6/?-isomer was isolated as authentic material or from lit. values (Table 1). The the principal alkaloid from the leaves of E. hypericijiilium stereochemistry of substituents at C-3, C-6 and C-7 could [20] and as a minor constituent from the root-bark of E. only be determined by GC with alkaloids for which ref. australe [7]. However, it has not been reported in E. material was available. The 3 new alkaloids have the zambesiacum. following mass spectra. A characteristic feature of E. zambesiacum is the abund6- Isoualery loxy tropan- 3- 01 (13). EIMS, m/z (rel. int.): ance of 3’,4’,5’-trimethoxybenzoic (22,23,25) and 3’,4’,5’- 241 [M]’ (ascribable to C,,H,,NO,) (7), 226 [M trimethoxycinnamic (24,26,27) derivatives in the alkaloid -Me]+ (I), 224 [M-OH]’ (l), 198 [M-C3H7]+ (I), mixture (Table 2). The compounds identified in the stem156 [M-C,H,O]+ (4), 140 [M-C,H,O,]+ (15), 113 bark are typical of those isolated in the root-bark [2]. [M - C(7)H,C(6)HOCOC,H,] + (lOO), 96 (46), 94 (26), 82 Compounds 22-27 were compared (TLC, GC, mass (16), 57 (24), 42 (27). spectra) with authentic compounds and were consistent 3- (2- M ethy lbuty ry loxy )tropan- 6,7- diol(l6). EIMS, m/z with the assigned structures. The principal alkaloid iden(rel. int.): 257 [M]’ (ascribable to C,,H,,NO,) (I), 228 tified in the stem-bark is 3c(-(3’,4’,5’-trimethoxybenz[M -CIHS]+ (l), 197 [M-C(6)HOHC(‘T)HOH]+ (3), oyloxy)tropane (22). However, the corresponding nor172 CM-C,H,O]+ (l), 156 [M-C,H,OJ+ (9), 102 derivatives of 22 and 23, as well as 6#?-benzoyloxytropan[C,H&O,H]+ (l), 95 (65), 94 (lOO), 57 (17), 42 (20). 3c(,7/?-diol, 6/?-benzoyloxy-3a-(3’,4’,5’-trimethoxycinna6P- Benzoy loxy tropan- 3- one. (18). EIMS, m/z (rel. moyloxy)tropan-78-01, 3a-phenylacetoxytropan-68-01 int.): 259 [M]’ (ascribable to C,SH,,NO,) (9), 201 (5), and 6/?-benzoyloxytropan-3a-ol have not been detected 154 [M-C6HSCO]+ (27), 138 [M-C,H&O,-J+ in the stem-bark, but they have been reported from the (18), 137 (15). 122 [C,H,CO,H]+ (3), 111 [M root-bark 121. -C(7)H,C(6)HOCOC,H,] + (lOO), 110 [C,H,NO] + Plant material. Alkaloidsfrom Erythroxylum (39), 105 [C6HSCO]+ (52), 96 (20), 95 (28), 94 (74), 83 (94), 82 (42), 77 (56), 42 (93). The EIMS was identical with that of the synthetic compound conditions. [16] run under the same 1151 zyxwvutsrqp 8. Blossey, E. C., Budzikiewicz, H., Ohashi, M., Fodor, G. and Djerassi, C. (1964) Tetrahedron 20, 585. 9. Evans, W. C. and Than, M. P. (1962) J. Phurm. Pharmac. 14, 147. 10. Gambaro, V., Lab&, C. and Castillo, M. (1983) authors are indebted to Dr G. J. Phytochemistry 22, 1838. Langley and Dr M. E. Harrison (ULIRS Mass Spectro11. Rosenblum, E. I. and Taylor, W. S. (1954) J. 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