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Indole alkaloids fromTabernaemontana glandulosa
Pergamon
0031-9422(94)00513-3
INDOLE ALKALOIDS
Phytochemmy,
FROM TABERNAEMONTANA
Vol. 37, No. 6, pp. 1737-1743, 1994 Copy&t 8 1994 Elsener Saence Ltd Pm&d in Great Britain. All rights reserved CW-9422/94 $7.00 + 0.00
GLANDULOSA*
HANS ACHENBACH,?REINER WAIBEL and MANFREDZWANZGER Institute of Pharmacy and Food Chemistry, Department of Pharmaceutical Chemistry, University of Erlangen, D-91052 Erlangen, F.R.G. (Received 26 April 1994) Key Word Index-Tabernaemontana
lemenitine; 19-epi-difforlemenitine;
glandulosa; Apocynaceae; conophylline; tiorlemenine; diffor10,12-dimethoxynareline; structure revision of difforlemenine.
Abstract-Re-investigation of leaves and twigs from Tabernaemontana glundulosa yielded 11 indole alkaloids, besides six ubiquitous phenylpropanoids and pentacyclic triterpenes. Structures were determined by spectroscopic methods partly in combination with chemical modifications. Difforlemenitine, lg-epi-difforlemenitine and 10,12dimethoxynareline represent new indole alkaloids. Difforlemenine and the bisindole alkaloid, conophylline, were also among the isolated alkaloids; our results require a structural revision for difforlemenine.
INTRODUCTION During recent years, various Tabernaenwntana species have been the object of phytochemical investigations [2, 31. A couple of years ago from the African Tabernaemontana glandulosa, we described the isolation of the new indole alkaloids, tabernulosine and 19-hydroxycoronaridine, which exhibit significant antihypertensive and antibacterial activity, respectively [4,5]. This caused us to reexamine the methanolic twig and leaf extract of T. glandulosa, which now afforded the novel indole alkaloids difforlemenitine, 19-epi-difforlemenitine and 10,12dimethoxynareline. Furthermore, our results require a structural revision for difforlemenine [6, 71. RFSULTSAND DIBCUSSION The extract of the twigs and leaves of T. glundulosa collected in Ghana was separated chromatographically. This yielded from the petrol extract, acetyl esters of aamyrin, fl-amyrin and lupeol as the major constituents, and from the chloroform-soluble fraction of the methanol extract, 1-14 were isolated (Table 1). The structures were established mainly by spectroscopic studies, sometimes in combination with chemical derivatizations or interconversions and, as far as possible, by comparison with authentic substances. Compound 1 exhibited UV maxima and IR bands typical for a fi-anilinoacrylic acid ester. The FAB mass spectrum ([Ml’ m/z 794), together with information from ‘H and 13CNMR established the elemental com-
*Part 64 in the series ‘Constituents of Tropical Medicinal Plants’. For Part 63 see ref. [l]. tAuthor to whom corrcspondcnce should be addressed.
this suggested a bisindole alkalposition C,,H,,N,O,,; oid. All ‘H and 13C signals could be assigned by homoand hetezonuclear correlations to(i) a 10,l 1-disubstituted pachysiphine [21] and (ii) a 10,14a,lSfi-trihydroxy-11,12dimethoxy substituted vincadifformine [22,23]. The relative configuration and the details of the connection of the two units were established by NOE experiments and led to the presented structure 1, which has been attributed to conophylline, an alkaloid very recently isolated from T. diuaricata [S]. The absolute configuration of 1 was determined from CD measurements. In 7R-configurated (monomeric) aspidospermine-type alkaloids a negative Cotton effect is observed in the 270-320nm range and a positive one between 220 and 250 nm, whereas the 7S-configuration causes corresponding reverse Cotton effects [24]. Since no electronic interactions exist between the two chromophoric dihydroindole systems in 1, the CD curve of 1 results from addition of the curves of the chromophores of the two subunits [25]. The observed Cotton effects at 325 nm (de: - 16.3) and 244 nm (de: + 17.6) established the R-configuration for 1 at C-7, as well as at C-7’. Compound 3 in all properties agreed with the data published for difforlemenine [6], an alkaloid isolated from the leaves of Vinca d#ormis. However, HMBC [26] showed correlations between H-17 and C-21, as well as H-21 and C-17, which were not consistent with the claimed structure [6]. In addition, the ‘H-13C coupling constants for CH-19 and CH-21 were at 172.5 and 163 Hz, respectively, revealing that C-19 should be part of the oxirane ring system instead of C-21 [27, 281. Consequently, the oxirane was opened by HCl and this yielded 15 as the major product together with a minor compound, which was found to be identical to difforlemenitine (4). For 15, mass spectrometry indicated a chlorine 1737
1738
H. ACHENBACHet al.
R 2
H
10
OMc
CoBMe
atom, whose position at 019 was established by a sign~~nt downfield shift of the resonance signal of N-19 E63.17 (in 3)-r&4.56 (in 15)] and by a long range correlation observed in WMBC between the OH at C-20 and C21, Together with further information from 2D NMR and NOE measurements, all structura1 detaiis of 15 could be determined, except for the confi~ration at C-19. For difforlemenine, these results demand structure 3, which is in agreement with ail NMR data. NOE studies corroborated the depicted relative ~on~guration, which includes the stereo situation at C-19 (Fig. 1). Since the CD curve of
3 doseIy resembled that of 9, whose absolute contiguration is known [29], 3 also represents the absolute con@uration. Compa~son of the 13C NMR data of 4 with those of 3 and 15 (Table 2) showed close similarities for G-2 to C-14, but significant differences for the signals of C-15 to C-21 and aiso for the N-methyl group. On the basis of the s~tros~pic data, 4 contained two hydroxy groups and one of them must be at C-19, but there was no evidence for an oxirane ring This suggested a 19,2@dioi structure ori~nating from 3 by hydrolysis of
1739
Indole alkaloids from Tabemaemontana glandulosa COOMe
8
9
CHzOH
11
H
OMe HO
OMe
OH
15
OMe
Fig. 1. Important NOES observed in difforlemenine (3).
the oxirane. However, the lack of a tertiary OH and the shifts observed for H-19 and H-21 in the ‘H NMR of the acetylation product of 4, indicated hydroxy groups at C19 and C-21, but not at C-20. Further studies and particularly long range correlations observed in the HMBC (Fig. 2), which demanded corresponding structural vicinities between CH-17 and C-21, as well as between the NMe protons and C-20, led to structure 4. The fact, that besides 15 only 4 is formed in the course of the proton-catalysed oxirane ring opening of 3 reveals the stereospecificity of this reaction and determines the configuration of 4 in agreement with the results of NOE
Table 1. Compounds
isolated from the CHCI, extract of twigs and leaves of Tabernaemontana glandulosa
Classification
Compound
Content i%1*
Alkaloids
Conophylline (1) Coronaridine (2) DitTorlemenine (3) DitTorlemenitine (4) 19-eppi-Difforlemenitine (5) 10,lZDimethoxynareline (6) Tabemulosine (7) 5,10,12-Trimethoxystrictamine Vincaditline (9)
3.1 2.2 1.2 0.7 1.1 2.7 1.6 1.2 3.1
Phenylpropanoids Lignans
(8)
Voacangine (10)
0.5
Vobasine (11) Coumarin (12) Vanillin (13) Syringaresinol(l4)
1.2 0.15 1.2 1.8
*Dry weight of extract = looo%o.
Refs PI c91 I31 -
il Cl01 Cll, 121 [13-161 [17-191 -
cw
H. ACHENBACHet al.
1740 Table 2. ‘“CNMR C 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 COOMe NMe COOMe
resonances of 3-5 and 15 (90 MHz, CDCI,)
3
15
4
5
134.6 189.7 59.6 30.7 120.8 128.1 120.9 120.7 127.1 112.0 136.5 41.8 38.6 49.5 72.8 17.2 59.3 63.5 90.1 171.0 40.8 50.9
134.2 189.9 59.8 31.0 119.7 127.8 120.5 120.2 126.8 111.6 136.4 40.7 44.8 49.0 72.0 20.4 61.8 75.7 88.1 171.8 41.0 50.6
134.6 191.8 65.5 33.3 120.8 128.9 121.18 120.3’ 126.6 111.7 136.4 40.5 41.10 56.8 70.6b 20.8 70.5b 69.1 93.4 171.0 34.6 50.6
134.6 192.1 65.8 33.3b 120.6 128.9 121.0 120.3 126.5 111.7 136.4 40.5’ 40.3” 56.5 70.8 19.2 68.3 69.5 93.1 170.8 33.8” 50.7
The UV spectrum of 6 indicated an indolenine-chromophore and NMR studies established the unusual basic ring system as described for nareline [30]. Two ‘additional’ OMe substituents were meta-positioned to each other and had to be placed at C-10 and C-12 rather than C-9 and C-l 1 due to a strong NOE interaction observed between H-9 and H-6, and in agreement with the 13CNMR resonances of the aromatic carbons. The agreement of the CD curves of nareline and 6 confirms the same absolute configuration. The monomeric alkaloids isolated from T. glandulosa belong
to the akuammilan-,
indole alkaloids,
ibogan-
or vobasan-type
which are widespread
of
in Tubernaemon-
tuna species [2, 31. Conophylline
(l), which only very recently has been described from T. divaricata [S], represents a bisindole alkaloid consisting of two aspidospermidine-type units, which are connected by a dihydrofuran ring. According to our knowledge, 10,12-dimethoxynareline (6) is the second example of an alkaloid with the unusual 6,21-cycle-45seco-akuammilan ring system. The occurrence of alkaloids with different ring systems containing the rare 10,lZdimethoxy substitution at the aromatic moiety emphasizes their close biogenetic relationship.
*“Assignments might be interchanged within a cohunn. EXPERIMENTAL
Fig. 2. Important ‘H-‘“C couplings observed in the HMBC of compound 4.
studies. For the HCl-induced reaction of 3 we discuss the mechanism depicted in Fig. 3. The spectral properties of 4 and 5 are very similar, with major differences for the NMR signals of atoms around C-19 only. NMR (2D) and NOE studies corroborated 5 as the 19-epimer of 4.
General. Mps: uncorr. Analytical TLC performed on precoated plates (Nano plates Sil-20 UV, MachreyNagel) using the solvent systems: S-l = cyclohexaneEtOAc (9: l), S-2 = cyclohexane-EtOAc (7: 3), S-3 = cyclohexane-EtOAc (2:3), S-4 = petrol-Me&O (2:3), S-6 = CHCl,-MeOH (19:1X S-S = CHCI,-MeOH (24: 1); detection: UV, anisaldehyde reagent [31] or ceric ammonium sulphate [32]. If not stated otherwise, [cl&, in CHCl, at room temp.; UV and CD in MeOH; IR in CHCl,. If not otherwise stated, MS and HRMS were obtained by EI at 70 eV. Unless key ions, ions are given with intensities > 10% and m/z > 100. If not indicated otherwise, ‘HNMR at 360 MHz and r3CNMR at 90 MHz in CDCl, with TMS as int. standard. Acetylation. The substance (3-5 mg) was dissolved in Ac,O-pyridine (1: 1, 0.5-l ml). After stirring at room temp. overnight, the solvents were removed by evapn. Only if necessary, purification was carried out by CC on silica gel.
Fig. 3. Proposed mechanism for the reaction of 3 with HCI-MeOH
1742
H. ACHENBACH et al.
6), 5.22 (lH, br d, J = 4 Hz, H-21), 5.93 (lH, d, J = 8 Hz, OH-21), 7.09 (lH, m, H-lo), 7.28 (lH, m, H-l l), 7.47 (lH, br d, J = 8 Hz, H-12), 7.74 (lH, br d, J = 8 Hz, H-9), 10.3 (lH, br s, NH). 13CNMR: Table 2; 13CNMR (Me,COd,): 619.8 (C-18), 34.0 (C-6), 34.2 (NMe), 41.3 (C-14), 41.4 (C-15), 50.6 (OMe), 56.9 (C-16), 66.7 (C-5), 68.9 (C-19), 69.9 (C-20), 71.1 (C-17), 92.9 (C-21), 112.8 (C-12), 120.3 (Clo), 120.9 (C-7), 121.7 (C-9), 126.5 (C-11), 129.5 (C-8), 135.7 (C-2), 137.8 (C-13), 171.6 (CO), 192.2 (C-3). MS m/z (rel. int. > 20%): 414 [M]+ (75), 397 (21), 396 (lOO), 337 (21), 323 (23), 309 (45), 295 (22), 291 (20), 263 (22), 196 (44), 194 (22), 184 (30), 180 (26), 178 (22), 172 (26), 168 (27), 166 (39), 158 (34), 157 (38). 152 (33), 143 (23), 140 (64), 130 (39), 129 (21), 128 (49), 107 (21). 10,12-Dimethoxynareline (6). Crystals (35 mg). Mp 224 228” [CHCl,-MeOH (1: l)]. [~]o - 89” (~0.9). TLC: R, 0.28 (S-4); anisaldehyde: yellow. IR v”,f cm-‘: 3436,1738, 1627, 1594. UV i,,, (log&): 237 (4.22), 271 (4.01), 312 nm (3.93). CD A,,, (A&): 207 (+7.0), 227 (- 10.3), 249 (+ 13.6), 273 nm (- 1.2). ‘HNMR: 61.67 (3H, d, J=7 Hz, Me-18), 2.04 (lH, ddd, J,=13.5, J,=J3=3 Hz, H-14), 2.26 (lH, d, J=3 Hz, H-16), 2.29 (lH, ddd, J, = 13.5, J2 =J,=3Hz,H-14),3.30(1H,ddd,J1=J,=J,=3Hz,H15), 3.70 (lH, d, J=3 Hz, H-6), 3.70 (3H, s, OMe), 3.82 (3H, s, lo-OMe), 3.94(3H, s, 12-OMe), 3.99 (lH, br s, OH), 4.09 (lH, br d, J=3 Hz, H-21), 4.32 (lH, br s, H-5), 4.55 (lH,dd,J,=J,=3 Hz, H-3), 5.75 (lH,q,J=7 Hz,H-19), 6.51 (lH, d, J=2 Hz, H-11), 6.89 (lH, d, J=2 Hz, H-9). 13CNMR: 612.6 (Me-18), 31.5 (C-15), 35.1 (C-14), 51.7 (OMe), 53.8 (C-16), 55,6,55.7 (C-6, C-7), 55.7,55.8 (lo-OMe, 12-OMe), 62.6 (C-3), 65.5 (C-21), 99.2 (C-11), 100.2 (C-5), 102.4 (C-9), 122.7 (C-191, 130.6 (C-20), 139.3 (C-8). 142.3 (C13), 151.9 (C-12), 159.5 (C-lo), 170.8 (CO), 180.2 (C-2). MS m/z (rel. int): 412 CM]’ (70). 396 (13), 384 (13), 383 (27), 382 (32), 368 (14), 366 (ll), 353 (16), 326 (24), 325 (lOO), 322 (ll), 307 (15), 298 (12), 295 (13), 294 (18), 292 (ll), 264 (lo), 254 (12), 249 (lo), 248 (12), 230 (12), 218 (ll), 169 (10). Acetylation product of 6. Compound 6 (3 mg) was acetylated according to standard procedures. Crystals (2.5 mg), mp 171-173”. [a&, -54” (~0.8). TLC: R, 0.34 (S-4); anisaldehyde: yellow. IR v,, cm-‘: 1742, 1627, 1595. UV a,,,,, (log&): 237 (4.07), 272 (3.84), 314 nm (3.79). ‘HNMR: 61.70 (3H, d, J=7Hz, Me-18), 1.95 (3H, s, OCOMe), 2.09 (IH, ddd, J, = 13.5, J, = J, = 3 Hz, H14),2.30(1H,d, J = 3 Hz, H-16) 2.33 (lH,ddd, J, = 14, J, = J, = 3 Hz,H-14),3.33(1H,ddd, J, N J2 _ J, = 3 Hz, H-15), 3.71 (3H, s, COOMe), 3.79 (lH, d, J = 3 Hz, H-6), 3.82 (3H, s, OMe-lo), 3.98 (3H. s, OMe-12), 4.06 (lH, d, J = 3 Hz, H-21), 4.66 (1H. dd, J, ,., J, = 3 Hz, H-3), 5.18 (lH, s, H-5), 5.83 (lH, q. J = 7 Hz, H-19), 6.53 (lH, d, J = 2 Hz, H-11), 6.87 (lH, d, J = 2 Hz, H-9). 13CNMR: S 12.7(C-18), 21.0 (OCOMe), 31.6 (C-15), 35.3 (C-14), 51.8 (COOMe), 53.9 (C-16), 54.6 (C-6), 55.3 (C-7), 55.8 (loOMe or 12-OMe), 56.0 (lo-OMe or 12-OMe), 62.7 (C-3), 65.7 (C-21), 98.5 (C-5), 99.3 (C-l I), 102.7 (C-9), 123.3 (C19), 130.2 (C-20), 139.4 (C-8), 141.8 (C-13). 152.2 (C-12), 159.7 (C-lo), 169.1 (OCOMe), 170.9 (COOMe), 178.9 (C2). MS m/z (rel. int.): 454 CM] + (82), 413 (12), 412 (48), 395 (14). 384 (44), 383 (33), 382 (lOO), 367 (18), 326 (13). 325
(52), 322 (20), 307 (17), 295 (1 l), 294 (25), 292 (14), 264 (lo), 254 (1 l), 248 (14), 230 (11). Tubermdosine (7). Crystals (21 mg). Mp 190-192” (MeOH). {ref. [S] mp 190-191”). [a]o -20” (c 1.3). (ref. [4] [a]o -27”). TLC: R, 0.08 (S-4); ceric ammonium sulphate: yellow. Substance identical with an authentic sample. 5,10,12-Trimethoxystrictumine (8). Crystals (15 mg). Mp 170-175” (EtOAc) {ref. [lo] mp 183-185”). [cx]~ - 56” (~4.1). {ref. [IO] [a&, - 64”). TLC: R, 0.31 (S-4); anisaldehyde: yellow-orange; ceric ammonium sulphate: yellow. Substance identical with an authentic sample. Vincadijine (9). Crystals (40 mg). Mp 225-230”, dec. (Me,CO) {ref. [12] mp 230”). [a&, -81” (~0.1) {ref. [12] [aID - 121”). TLC: R, 0.38 (S-4); ceric ammonium sulphate: red. 13CNMR: 612.1 (C-18), 18.3 (C-6). 32.9 (C-15), 42.0 (NMe), 43.0 (C-14), 50.5 (OMe), 51.4 (C-21), 52.1 (C16), 57.8 (C-5), 69.6 (C-17), 111.9 (C-12), 119.8 (C-7), 120.3 (C-lo), 120.7 (C-9), 122.1 (C-19), 126.6 (C-11), 128.4 (C-8), 134.7 (C-20), 136.6 (C-13), 174.0 (COOMe), 189.6 (C-3). UV, IR, ‘H NMR, MS in agreement with published data c4, 341. Voacangine (10). Crystals (7 mg). Mp 134-136” (Me,CO) {ref. [IS] mp 137-138”}. [z& - 38” (~0.44) {ref. Cl51 C@l:: - 28”). TLC: R, 0.38 (S-2); cent ammonium sulphate: green-brown. UV, IR, ‘H NMR, MS in agreement with published data [33]. Substance identical with an authentic sample. vobasine(11). Crystals (15 mg). Mp 109-110” (Me,CO) {ref. [18] mp 111-113”). [a]u -140”(MeOH;c0.86) (ref. Cl81 Calk3 - 158.8”). TLC: R, 0.30 (S-4); anisaldehyde: yellow. UV, IR, ‘H NMR. 13C NMR, MS in agreement with published data [18. 34, 35). Coumarin (12). Crystals (2 mg). Mp 70” (Me&O). TLC: R, 0.54 (S-3); anisaldehyde: pink. Substance identical with an authentic sample. VaniIlin (13). Crystals (16 mg). Mp 76-79” (Me&O). TLC: R, 0.45 (S-6); ceric ammonium sulphate: orange. Substance identical with an authentic sample. (i-)-Syringaresinol(14). Crystals (23 mg). Mp 166-168” (Me&O). (ref. [20] mp 174”). [cl&, +O” (MeOH). TLC: R, 0.43 (S-4); anisaldehyde: first pink, then brown with a pink marginal zone. Properties in agreement with reported data [36] and identical with an authentic sample. 19-Chloro-19,0-seco-dlflorlemenine (15) and diybrlementine (4) by acidic hydrolysis of3. To 3 (5 mg) dissolved in 1 ml Me,CO, 0.2 ml 2 N HCI and 0.1 ml of a satd aq. soln of oxalic acid was added and the mixt. kept at 50” for 15 hr. After neutralization with K,CO, and evapn the residue was redissolved in H,O-MeOH (1: 1) and extracted with CHCI,. CC on silica gel yielded 19-chloro-19,0seco-difforlemenine (15) (2 mg) and difforlemenitine (4) (1 mg). 19-Chloro-19,0-seco-diflorlemenine (15). Crystals (2 mg). Mp 117-121” (Me&O). TLC: R, 0.43 (CHCl,MeOH (47:3)); anisaldehyde: pink. IR v,,, cm-‘: 3632, 3554,3450, 1712, 1647. UV A,,,,, (log&): 239 (4.09), 313 nm (4.16). ‘HNMR: 61.64 (3H, d, J=6.5 Hz, Me-18), 2.35 (3H, br s, OMe), 2.70 (3H, s, NMe), 2.83 (ifi, br dd, J,
Indole alkaloids from Tabernaemontana glandulosa =12,~z=6H~H-l5),2.95(lH,~rs,OH),3.14(lH,~~d~, J,=lS, J,=6SHz, H-6), 3.41 (lH, br d, J=7Hz, H-5), 3.41 (lH, dd, J,=J,=12.5 Hz, H-14), 3.66 (lH, br d, J =8 H~H-l7),3.73(lH,~~,~~ =12,5,=6 Hz,H-14),3.97 (lH, br d, J=7SHz, H-17), 4.02 (lH, dd, J,=lS, J, =9 Hz, H-6), 4.21 (lH, s, H-21), 4.56 (lH, q, .I=7 Hz, H19), 7.18 (lH, m, H-10), 7.35 (2H, m, H-l1 and H-12), 7.74 (lH, br d, J=BHz, H-9), 8.89 (lH, s, NH). 13CNMR: Table 2. MS m/z (rel. int.): 434 [Mi]’ (7), 433 (5), 432 [Mz]+ (18), 397 (19), 396 (41), 337 (11), 310 (17), 309 (34), 305 (1 1), 279 (1 1),278 (35), 250 (16), 238 (48), 208 (1 1), 199 (14), 196(13), 194(16), 192(12), 184(21), 183(15), 180(22), 178(11), 172(ll), 170(12), 169(11), 168(36), 167(22), 166 (13), 158(21), 156(33), l55(12), 154(29), 152(12), 144(12), 143 (ll), 140(26), 130 (42), 129(35), 128 (lo), 108 (12), 102 (12). Acknowledgements-We
are very thankful to Prof. Dr M.
Hesse, University of Ziirich, Switzerland, for his generous gift of authentic nareline [30]. Thanks are also due to the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie for financial support. REFFXE.NCES
1. Achenbach, H. and Schwinn, A. (1994) Arch. Pharm. (Weinheim) (in press). 2. Danieli, B. and Palmisano, G. (1986) in The Alkaloids (Brossi, A., ed.), Vol. 27, p. 1. Academic Press, New York. 3. van Beek, T. A. and Gessel, M. A. J. T. (1988) in Alkaloids: Chem. Biol. Perspecb. (Pelletier, S. W., ed.), Vol. 6, p. 75. Wiley, New York. 4. Achenbach, H., RalTelsberger, B. and Addae-Mensah, I. (1982) Liebigs Ann. Chem. 830. 5. Achenbach, H., Raffelsberger, B. and Brillinger, G.-U. (1980) Phytochemistry 19, 2185. 6. Gamier, J. and Mahuteau, J. (1985) Tetrahedron Lt?tters z&1513. 7. Achenbach, H., Waibel, R. and Zwanzger, M. (1993) Arch. Pharm. (Weinheim) 326, 724. 8. Kam, T.-S., Loh, K.-Y., Lim, L.-H., Loon& W.-L., Chuah, C.-H. and Wei, 6. (1992) Tetrahedron Letters 33, 969. 9. Gorman, M., Neuss, N., Cone, N. J. and Deyrup, J. A. (1960) J. Am Chem See. 82,1142.
10. Renner, C. (1983) Thesis, Universitiit Erlangen. 11. Falco, M., Gamier-Gosset, J., Fellion, E. and Le Men, J. (1964) Ann. Pharm. Fr. 22,455. 12. Das, B. C., Garnier-Gosset, J., Le Men, J. and Janot, M.-M. (1965) Bull. Sot. Chim. Ft. 1903.
1743
13. Janot, M.-M. and Goutarel, R. (1955) C. R. Acad. Sci. 240,180O. 14. La Barre, J. and Gillo, L. (1955) Bull. Acad. ML;d. Belg. 20, 194. 15. Walls, F., Collera, 0. and Sandoval, L. A. (1958) Tetrahedron 2, 173. 16. Bartlett, M. F., Dickel, D. F. and Taylor, W, I. (1958) J. Am. Chem. Sot. 80, 126. 17. Renner, U. (1959) Experientia 15, 185. 18. Renner, U., Prins, D. A., Burlingame, A. L. and Biemann, K. (1963) Helv. Chim. Acta 46, 2186. 19. Cava, M. P., Talapatra, S. K., Weisbach, J. A., Douglas, B. and Dudek, G. 0. (1963) Tetrahedron Letters 53. 20. Freudenberg, K. and Dietrich, H. (1953) Chem. Ber. 86, 4.
21. Kunesch, N., Cav&, A., Hagaman, E. W. and Wenkert, E. (1980) Tetrahedron Letters 1727. 22. Bui, A.-M., Das, B. C. and Potier, P. (1980) Phyto~~rnis~y 19,1473. 23. Kan-Fan, C., Massiot, G., Das, B. C. and Potier, P. (1981) J. Org. Chem. 46, 1481. 24. Klyne, W., Swan, R. J., Bycroft, B. W., ~humann, D. and Schmid, H. (1965) Helo. Chim. Acta 48, 443. 25. Stiickigt, J., Pawelka, K.-H., Tanahashi, T., Danieli, B. and Hull, W. E. (1983) Helv. Chim. Acta 66,2525. 26. Bax, A. and Summers, M. F. (1986) J. Am. Chem. Sot. lOg2093. 27. Ellis, P. D. and Ditchfield, R. (1976) Top. Carbon13 NMR Spectrosc. 2,433. 28. Kalinowski, H.-O., Berger, S. and Braun, S. (1984) in 13C NMR Spektroskopie, p. 449. Thieme, Stuttgart, 29. Braekman, J. C., Kaisin, M., Pecher, J. and Martin, R. H. (1966) Bull. Sot. Chim. Belg. 75, 465. 30. Morita, Y., Hesse, M., Schmid, H., Bane+, A., Bane@, J., Chatterjee, A. and Oberhksli, W. E. (1977) Helu. Chim. Acta 60, 1419. 31. Stahl, E. (1967) in Diinnschichtchromatographie, 2nd Edn, p. 817. Springer, Berlin. 32. Stahl, E. (1967) in Diinnschiehtchromatographie, 2nd Edn, p. 820. Springer, Berlin. 33. Gunasekera, S. P, Cordell, G. A. and Farnsworth, N. R. (1980) Phylochemistry 19, 1213. 34. Ahond, A., Bui, A.-M., Potier, P., Hagaman, E. W. and Wenkert, E. (1976) J. Org. Chem. 41, 1878. 35. Van Beek, T. A., Kuijlaars, F. L. C., Thomassen, P. H. A. M., Verpoorte, R. and Baerheim Svendsen, A. (1984) Phy~ochemistry 23, 1771. 36. Nawwar, M. A. M., Buddrus, J. and Bauer, H. (1982) Phytochemistry 21, 1755.
0031-9422(94)00513-3
INDOLE ALKALOIDS
Phytochemmy,
FROM TABERNAEMONTANA
Vol. 37, No. 6, pp. 1737-1743, 1994 Copy&t 8 1994 Elsener Saence Ltd Pm&d in Great Britain. All rights reserved CW-9422/94 $7.00 + 0.00
GLANDULOSA*
HANS ACHENBACH,?REINER WAIBEL and MANFREDZWANZGER Institute of Pharmacy and Food Chemistry, Department of Pharmaceutical Chemistry, University of Erlangen, D-91052 Erlangen, F.R.G. (Received 26 April 1994) Key Word Index-Tabernaemontana
lemenitine; 19-epi-difforlemenitine;
glandulosa; Apocynaceae; conophylline; tiorlemenine; diffor10,12-dimethoxynareline; structure revision of difforlemenine.
Abstract-Re-investigation of leaves and twigs from Tabernaemontana glundulosa yielded 11 indole alkaloids, besides six ubiquitous phenylpropanoids and pentacyclic triterpenes. Structures were determined by spectroscopic methods partly in combination with chemical modifications. Difforlemenitine, lg-epi-difforlemenitine and 10,12dimethoxynareline represent new indole alkaloids. Difforlemenine and the bisindole alkaloid, conophylline, were also among the isolated alkaloids; our results require a structural revision for difforlemenine.
INTRODUCTION During recent years, various Tabernaenwntana species have been the object of phytochemical investigations [2, 31. A couple of years ago from the African Tabernaemontana glandulosa, we described the isolation of the new indole alkaloids, tabernulosine and 19-hydroxycoronaridine, which exhibit significant antihypertensive and antibacterial activity, respectively [4,5]. This caused us to reexamine the methanolic twig and leaf extract of T. glandulosa, which now afforded the novel indole alkaloids difforlemenitine, 19-epi-difforlemenitine and 10,12dimethoxynareline. Furthermore, our results require a structural revision for difforlemenine [6, 71. RFSULTSAND DIBCUSSION The extract of the twigs and leaves of T. glundulosa collected in Ghana was separated chromatographically. This yielded from the petrol extract, acetyl esters of aamyrin, fl-amyrin and lupeol as the major constituents, and from the chloroform-soluble fraction of the methanol extract, 1-14 were isolated (Table 1). The structures were established mainly by spectroscopic studies, sometimes in combination with chemical derivatizations or interconversions and, as far as possible, by comparison with authentic substances. Compound 1 exhibited UV maxima and IR bands typical for a fi-anilinoacrylic acid ester. The FAB mass spectrum ([Ml’ m/z 794), together with information from ‘H and 13CNMR established the elemental com-
*Part 64 in the series ‘Constituents of Tropical Medicinal Plants’. For Part 63 see ref. [l]. tAuthor to whom corrcspondcnce should be addressed.
this suggested a bisindole alkalposition C,,H,,N,O,,; oid. All ‘H and 13C signals could be assigned by homoand hetezonuclear correlations to(i) a 10,l 1-disubstituted pachysiphine [21] and (ii) a 10,14a,lSfi-trihydroxy-11,12dimethoxy substituted vincadifformine [22,23]. The relative configuration and the details of the connection of the two units were established by NOE experiments and led to the presented structure 1, which has been attributed to conophylline, an alkaloid very recently isolated from T. diuaricata [S]. The absolute configuration of 1 was determined from CD measurements. In 7R-configurated (monomeric) aspidospermine-type alkaloids a negative Cotton effect is observed in the 270-320nm range and a positive one between 220 and 250 nm, whereas the 7S-configuration causes corresponding reverse Cotton effects [24]. Since no electronic interactions exist between the two chromophoric dihydroindole systems in 1, the CD curve of 1 results from addition of the curves of the chromophores of the two subunits [25]. The observed Cotton effects at 325 nm (de: - 16.3) and 244 nm (de: + 17.6) established the R-configuration for 1 at C-7, as well as at C-7’. Compound 3 in all properties agreed with the data published for difforlemenine [6], an alkaloid isolated from the leaves of Vinca d#ormis. However, HMBC [26] showed correlations between H-17 and C-21, as well as H-21 and C-17, which were not consistent with the claimed structure [6]. In addition, the ‘H-13C coupling constants for CH-19 and CH-21 were at 172.5 and 163 Hz, respectively, revealing that C-19 should be part of the oxirane ring system instead of C-21 [27, 281. Consequently, the oxirane was opened by HCl and this yielded 15 as the major product together with a minor compound, which was found to be identical to difforlemenitine (4). For 15, mass spectrometry indicated a chlorine 1737
1738
H. ACHENBACHet al.
R 2
H
10
OMc
CoBMe
atom, whose position at 019 was established by a sign~~nt downfield shift of the resonance signal of N-19 E63.17 (in 3)-r&4.56 (in 15)] and by a long range correlation observed in WMBC between the OH at C-20 and C21, Together with further information from 2D NMR and NOE measurements, all structura1 detaiis of 15 could be determined, except for the confi~ration at C-19. For difforlemenine, these results demand structure 3, which is in agreement with ail NMR data. NOE studies corroborated the depicted relative ~on~guration, which includes the stereo situation at C-19 (Fig. 1). Since the CD curve of
3 doseIy resembled that of 9, whose absolute contiguration is known [29], 3 also represents the absolute con@uration. Compa~son of the 13C NMR data of 4 with those of 3 and 15 (Table 2) showed close similarities for G-2 to C-14, but significant differences for the signals of C-15 to C-21 and aiso for the N-methyl group. On the basis of the s~tros~pic data, 4 contained two hydroxy groups and one of them must be at C-19, but there was no evidence for an oxirane ring This suggested a 19,2@dioi structure ori~nating from 3 by hydrolysis of
1739
Indole alkaloids from Tabemaemontana glandulosa COOMe
8
9
CHzOH
11
H
OMe HO
OMe
OH
15
OMe
Fig. 1. Important NOES observed in difforlemenine (3).
the oxirane. However, the lack of a tertiary OH and the shifts observed for H-19 and H-21 in the ‘H NMR of the acetylation product of 4, indicated hydroxy groups at C19 and C-21, but not at C-20. Further studies and particularly long range correlations observed in the HMBC (Fig. 2), which demanded corresponding structural vicinities between CH-17 and C-21, as well as between the NMe protons and C-20, led to structure 4. The fact, that besides 15 only 4 is formed in the course of the proton-catalysed oxirane ring opening of 3 reveals the stereospecificity of this reaction and determines the configuration of 4 in agreement with the results of NOE
Table 1. Compounds
isolated from the CHCI, extract of twigs and leaves of Tabernaemontana glandulosa
Classification
Compound
Content i%1*
Alkaloids
Conophylline (1) Coronaridine (2) DitTorlemenine (3) DitTorlemenitine (4) 19-eppi-Difforlemenitine (5) 10,lZDimethoxynareline (6) Tabemulosine (7) 5,10,12-Trimethoxystrictamine Vincaditline (9)
3.1 2.2 1.2 0.7 1.1 2.7 1.6 1.2 3.1
Phenylpropanoids Lignans
(8)
Voacangine (10)
0.5
Vobasine (11) Coumarin (12) Vanillin (13) Syringaresinol(l4)
1.2 0.15 1.2 1.8
*Dry weight of extract = looo%o.
Refs PI c91 I31 -
il Cl01 Cll, 121 [13-161 [17-191 -
cw
H. ACHENBACHet al.
1740 Table 2. ‘“CNMR C 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 COOMe NMe COOMe
resonances of 3-5 and 15 (90 MHz, CDCI,)
3
15
4
5
134.6 189.7 59.6 30.7 120.8 128.1 120.9 120.7 127.1 112.0 136.5 41.8 38.6 49.5 72.8 17.2 59.3 63.5 90.1 171.0 40.8 50.9
134.2 189.9 59.8 31.0 119.7 127.8 120.5 120.2 126.8 111.6 136.4 40.7 44.8 49.0 72.0 20.4 61.8 75.7 88.1 171.8 41.0 50.6
134.6 191.8 65.5 33.3 120.8 128.9 121.18 120.3’ 126.6 111.7 136.4 40.5 41.10 56.8 70.6b 20.8 70.5b 69.1 93.4 171.0 34.6 50.6
134.6 192.1 65.8 33.3b 120.6 128.9 121.0 120.3 126.5 111.7 136.4 40.5’ 40.3” 56.5 70.8 19.2 68.3 69.5 93.1 170.8 33.8” 50.7
The UV spectrum of 6 indicated an indolenine-chromophore and NMR studies established the unusual basic ring system as described for nareline [30]. Two ‘additional’ OMe substituents were meta-positioned to each other and had to be placed at C-10 and C-12 rather than C-9 and C-l 1 due to a strong NOE interaction observed between H-9 and H-6, and in agreement with the 13CNMR resonances of the aromatic carbons. The agreement of the CD curves of nareline and 6 confirms the same absolute configuration. The monomeric alkaloids isolated from T. glandulosa belong
to the akuammilan-,
indole alkaloids,
ibogan-
or vobasan-type
which are widespread
of
in Tubernaemon-
tuna species [2, 31. Conophylline
(l), which only very recently has been described from T. divaricata [S], represents a bisindole alkaloid consisting of two aspidospermidine-type units, which are connected by a dihydrofuran ring. According to our knowledge, 10,12-dimethoxynareline (6) is the second example of an alkaloid with the unusual 6,21-cycle-45seco-akuammilan ring system. The occurrence of alkaloids with different ring systems containing the rare 10,lZdimethoxy substitution at the aromatic moiety emphasizes their close biogenetic relationship.
*“Assignments might be interchanged within a cohunn. EXPERIMENTAL
Fig. 2. Important ‘H-‘“C couplings observed in the HMBC of compound 4.
studies. For the HCl-induced reaction of 3 we discuss the mechanism depicted in Fig. 3. The spectral properties of 4 and 5 are very similar, with major differences for the NMR signals of atoms around C-19 only. NMR (2D) and NOE studies corroborated 5 as the 19-epimer of 4.
General. Mps: uncorr. Analytical TLC performed on precoated plates (Nano plates Sil-20 UV, MachreyNagel) using the solvent systems: S-l = cyclohexaneEtOAc (9: l), S-2 = cyclohexane-EtOAc (7: 3), S-3 = cyclohexane-EtOAc (2:3), S-4 = petrol-Me&O (2:3), S-6 = CHCl,-MeOH (19:1X S-S = CHCI,-MeOH (24: 1); detection: UV, anisaldehyde reagent [31] or ceric ammonium sulphate [32]. If not stated otherwise, [cl&, in CHCl, at room temp.; UV and CD in MeOH; IR in CHCl,. If not otherwise stated, MS and HRMS were obtained by EI at 70 eV. Unless key ions, ions are given with intensities > 10% and m/z > 100. If not indicated otherwise, ‘HNMR at 360 MHz and r3CNMR at 90 MHz in CDCl, with TMS as int. standard. Acetylation. The substance (3-5 mg) was dissolved in Ac,O-pyridine (1: 1, 0.5-l ml). After stirring at room temp. overnight, the solvents were removed by evapn. Only if necessary, purification was carried out by CC on silica gel.
Fig. 3. Proposed mechanism for the reaction of 3 with HCI-MeOH
1742
H. ACHENBACH et al.
6), 5.22 (lH, br d, J = 4 Hz, H-21), 5.93 (lH, d, J = 8 Hz, OH-21), 7.09 (lH, m, H-lo), 7.28 (lH, m, H-l l), 7.47 (lH, br d, J = 8 Hz, H-12), 7.74 (lH, br d, J = 8 Hz, H-9), 10.3 (lH, br s, NH). 13CNMR: Table 2; 13CNMR (Me,COd,): 619.8 (C-18), 34.0 (C-6), 34.2 (NMe), 41.3 (C-14), 41.4 (C-15), 50.6 (OMe), 56.9 (C-16), 66.7 (C-5), 68.9 (C-19), 69.9 (C-20), 71.1 (C-17), 92.9 (C-21), 112.8 (C-12), 120.3 (Clo), 120.9 (C-7), 121.7 (C-9), 126.5 (C-11), 129.5 (C-8), 135.7 (C-2), 137.8 (C-13), 171.6 (CO), 192.2 (C-3). MS m/z (rel. int. > 20%): 414 [M]+ (75), 397 (21), 396 (lOO), 337 (21), 323 (23), 309 (45), 295 (22), 291 (20), 263 (22), 196 (44), 194 (22), 184 (30), 180 (26), 178 (22), 172 (26), 168 (27), 166 (39), 158 (34), 157 (38). 152 (33), 143 (23), 140 (64), 130 (39), 129 (21), 128 (49), 107 (21). 10,12-Dimethoxynareline (6). Crystals (35 mg). Mp 224 228” [CHCl,-MeOH (1: l)]. [~]o - 89” (~0.9). TLC: R, 0.28 (S-4); anisaldehyde: yellow. IR v”,f cm-‘: 3436,1738, 1627, 1594. UV i,,, (log&): 237 (4.22), 271 (4.01), 312 nm (3.93). CD A,,, (A&): 207 (+7.0), 227 (- 10.3), 249 (+ 13.6), 273 nm (- 1.2). ‘HNMR: 61.67 (3H, d, J=7 Hz, Me-18), 2.04 (lH, ddd, J,=13.5, J,=J3=3 Hz, H-14), 2.26 (lH, d, J=3 Hz, H-16), 2.29 (lH, ddd, J, = 13.5, J2 =J,=3Hz,H-14),3.30(1H,ddd,J1=J,=J,=3Hz,H15), 3.70 (lH, d, J=3 Hz, H-6), 3.70 (3H, s, OMe), 3.82 (3H, s, lo-OMe), 3.94(3H, s, 12-OMe), 3.99 (lH, br s, OH), 4.09 (lH, br d, J=3 Hz, H-21), 4.32 (lH, br s, H-5), 4.55 (lH,dd,J,=J,=3 Hz, H-3), 5.75 (lH,q,J=7 Hz,H-19), 6.51 (lH, d, J=2 Hz, H-11), 6.89 (lH, d, J=2 Hz, H-9). 13CNMR: 612.6 (Me-18), 31.5 (C-15), 35.1 (C-14), 51.7 (OMe), 53.8 (C-16), 55,6,55.7 (C-6, C-7), 55.7,55.8 (lo-OMe, 12-OMe), 62.6 (C-3), 65.5 (C-21), 99.2 (C-11), 100.2 (C-5), 102.4 (C-9), 122.7 (C-191, 130.6 (C-20), 139.3 (C-8). 142.3 (C13), 151.9 (C-12), 159.5 (C-lo), 170.8 (CO), 180.2 (C-2). MS m/z (rel. int): 412 CM]’ (70). 396 (13), 384 (13), 383 (27), 382 (32), 368 (14), 366 (ll), 353 (16), 326 (24), 325 (lOO), 322 (ll), 307 (15), 298 (12), 295 (13), 294 (18), 292 (ll), 264 (lo), 254 (12), 249 (lo), 248 (12), 230 (12), 218 (ll), 169 (10). Acetylation product of 6. Compound 6 (3 mg) was acetylated according to standard procedures. Crystals (2.5 mg), mp 171-173”. [a&, -54” (~0.8). TLC: R, 0.34 (S-4); anisaldehyde: yellow. IR v,, cm-‘: 1742, 1627, 1595. UV a,,,,, (log&): 237 (4.07), 272 (3.84), 314 nm (3.79). ‘HNMR: 61.70 (3H, d, J=7Hz, Me-18), 1.95 (3H, s, OCOMe), 2.09 (IH, ddd, J, = 13.5, J, = J, = 3 Hz, H14),2.30(1H,d, J = 3 Hz, H-16) 2.33 (lH,ddd, J, = 14, J, = J, = 3 Hz,H-14),3.33(1H,ddd, J, N J2 _ J, = 3 Hz, H-15), 3.71 (3H, s, COOMe), 3.79 (lH, d, J = 3 Hz, H-6), 3.82 (3H, s, OMe-lo), 3.98 (3H. s, OMe-12), 4.06 (lH, d, J = 3 Hz, H-21), 4.66 (1H. dd, J, ,., J, = 3 Hz, H-3), 5.18 (lH, s, H-5), 5.83 (lH, q. J = 7 Hz, H-19), 6.53 (lH, d, J = 2 Hz, H-11), 6.87 (lH, d, J = 2 Hz, H-9). 13CNMR: S 12.7(C-18), 21.0 (OCOMe), 31.6 (C-15), 35.3 (C-14), 51.8 (COOMe), 53.9 (C-16), 54.6 (C-6), 55.3 (C-7), 55.8 (loOMe or 12-OMe), 56.0 (lo-OMe or 12-OMe), 62.7 (C-3), 65.7 (C-21), 98.5 (C-5), 99.3 (C-l I), 102.7 (C-9), 123.3 (C19), 130.2 (C-20), 139.4 (C-8), 141.8 (C-13). 152.2 (C-12), 159.7 (C-lo), 169.1 (OCOMe), 170.9 (COOMe), 178.9 (C2). MS m/z (rel. int.): 454 CM] + (82), 413 (12), 412 (48), 395 (14). 384 (44), 383 (33), 382 (lOO), 367 (18), 326 (13). 325
(52), 322 (20), 307 (17), 295 (1 l), 294 (25), 292 (14), 264 (lo), 254 (1 l), 248 (14), 230 (11). Tubermdosine (7). Crystals (21 mg). Mp 190-192” (MeOH). {ref. [S] mp 190-191”). [a]o -20” (c 1.3). (ref. [4] [a]o -27”). TLC: R, 0.08 (S-4); ceric ammonium sulphate: yellow. Substance identical with an authentic sample. 5,10,12-Trimethoxystrictumine (8). Crystals (15 mg). Mp 170-175” (EtOAc) {ref. [lo] mp 183-185”). [cx]~ - 56” (~4.1). {ref. [IO] [a&, - 64”). TLC: R, 0.31 (S-4); anisaldehyde: yellow-orange; ceric ammonium sulphate: yellow. Substance identical with an authentic sample. Vincadijine (9). Crystals (40 mg). Mp 225-230”, dec. (Me,CO) {ref. [12] mp 230”). [a&, -81” (~0.1) {ref. [12] [aID - 121”). TLC: R, 0.38 (S-4); ceric ammonium sulphate: red. 13CNMR: 612.1 (C-18), 18.3 (C-6). 32.9 (C-15), 42.0 (NMe), 43.0 (C-14), 50.5 (OMe), 51.4 (C-21), 52.1 (C16), 57.8 (C-5), 69.6 (C-17), 111.9 (C-12), 119.8 (C-7), 120.3 (C-lo), 120.7 (C-9), 122.1 (C-19), 126.6 (C-11), 128.4 (C-8), 134.7 (C-20), 136.6 (C-13), 174.0 (COOMe), 189.6 (C-3). UV, IR, ‘H NMR, MS in agreement with published data c4, 341. Voacangine (10). Crystals (7 mg). Mp 134-136” (Me,CO) {ref. [IS] mp 137-138”}. [z& - 38” (~0.44) {ref. Cl51 C@l:: - 28”). TLC: R, 0.38 (S-2); cent ammonium sulphate: green-brown. UV, IR, ‘H NMR, MS in agreement with published data [33]. Substance identical with an authentic sample. vobasine(11). Crystals (15 mg). Mp 109-110” (Me,CO) {ref. [18] mp 111-113”). [a]u -140”(MeOH;c0.86) (ref. Cl81 Calk3 - 158.8”). TLC: R, 0.30 (S-4); anisaldehyde: yellow. UV, IR, ‘H NMR. 13C NMR, MS in agreement with published data [18. 34, 35). Coumarin (12). Crystals (2 mg). Mp 70” (Me&O). TLC: R, 0.54 (S-3); anisaldehyde: pink. Substance identical with an authentic sample. VaniIlin (13). Crystals (16 mg). Mp 76-79” (Me&O). TLC: R, 0.45 (S-6); ceric ammonium sulphate: orange. Substance identical with an authentic sample. (i-)-Syringaresinol(14). Crystals (23 mg). Mp 166-168” (Me&O). (ref. [20] mp 174”). [cl&, +O” (MeOH). TLC: R, 0.43 (S-4); anisaldehyde: first pink, then brown with a pink marginal zone. Properties in agreement with reported data [36] and identical with an authentic sample. 19-Chloro-19,0-seco-dlflorlemenine (15) and diybrlementine (4) by acidic hydrolysis of3. To 3 (5 mg) dissolved in 1 ml Me,CO, 0.2 ml 2 N HCI and 0.1 ml of a satd aq. soln of oxalic acid was added and the mixt. kept at 50” for 15 hr. After neutralization with K,CO, and evapn the residue was redissolved in H,O-MeOH (1: 1) and extracted with CHCI,. CC on silica gel yielded 19-chloro-19,0seco-difforlemenine (15) (2 mg) and difforlemenitine (4) (1 mg). 19-Chloro-19,0-seco-diflorlemenine (15). Crystals (2 mg). Mp 117-121” (Me&O). TLC: R, 0.43 (CHCl,MeOH (47:3)); anisaldehyde: pink. IR v,,, cm-‘: 3632, 3554,3450, 1712, 1647. UV A,,,,, (log&): 239 (4.09), 313 nm (4.16). ‘HNMR: 61.64 (3H, d, J=6.5 Hz, Me-18), 2.35 (3H, br s, OMe), 2.70 (3H, s, NMe), 2.83 (ifi, br dd, J,
Indole alkaloids from Tabernaemontana glandulosa =12,~z=6H~H-l5),2.95(lH,~rs,OH),3.14(lH,~~d~, J,=lS, J,=6SHz, H-6), 3.41 (lH, br d, J=7Hz, H-5), 3.41 (lH, dd, J,=J,=12.5 Hz, H-14), 3.66 (lH, br d, J =8 H~H-l7),3.73(lH,~~,~~ =12,5,=6 Hz,H-14),3.97 (lH, br d, J=7SHz, H-17), 4.02 (lH, dd, J,=lS, J, =9 Hz, H-6), 4.21 (lH, s, H-21), 4.56 (lH, q, .I=7 Hz, H19), 7.18 (lH, m, H-10), 7.35 (2H, m, H-l1 and H-12), 7.74 (lH, br d, J=BHz, H-9), 8.89 (lH, s, NH). 13CNMR: Table 2. MS m/z (rel. int.): 434 [Mi]’ (7), 433 (5), 432 [Mz]+ (18), 397 (19), 396 (41), 337 (11), 310 (17), 309 (34), 305 (1 1), 279 (1 1),278 (35), 250 (16), 238 (48), 208 (1 1), 199 (14), 196(13), 194(16), 192(12), 184(21), 183(15), 180(22), 178(11), 172(ll), 170(12), 169(11), 168(36), 167(22), 166 (13), 158(21), 156(33), l55(12), 154(29), 152(12), 144(12), 143 (ll), 140(26), 130 (42), 129(35), 128 (lo), 108 (12), 102 (12). Acknowledgements-We
are very thankful to Prof. Dr M.
Hesse, University of Ziirich, Switzerland, for his generous gift of authentic nareline [30]. Thanks are also due to the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie for financial support. REFFXE.NCES
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