Seco-tabersonine alkaloids from Tabernaemontana corymbosa
Kuan-Hon Lim, Noel F. Thomas, Zanariah Abdullah, Toh-Seok Kam *
Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia
Abstract
Two seco-tabersonine alkaloids, jerantiphyllines A and B, in addition to a tabersonine hydroxyindolenine, jerantinine H, and a recently reported vincamine alkaloid 7, were isolated from the leaf extract of the Malayan Tabernaemontana corymbosa and the structures were established using NMR and MS analysis. Biomimetic conversion of jerantinines A and E to their respective vincamine and 16-epivincamine derivatives were also carried out.
1. Introduction
Plants of the genus Tabernaemontana comprising about 110 spe- cies and widely distributed in the pantropical regions are rich in alkaloids (Leeuwenberg, 1991; Van Beek et al., 1984; Danieli and Palmisano, 1986; Kam, 1999). In our systematic study of the Malaysian representatives of this genus, we have reported many examples of new alkaloids which are distinguished by their struc- tural novelty, as well as useful bioactivity (Kam et al., 2004a,b, 2003a,b, 2001, 2000, 1999, 1998, 1993; Kam and Sim, 2003a,b, 2002a). The Malayan T. corymbosa Roxb. ex Wall for instance pro- vided several new alkaloids which are characterized by novel molecular skeletons such as the hexacyclic alkaloid, tronoharine (Kam et al., 1999), the pentacyclic indole tronocarpine (Kam et al., 2000), and the quinolinic alkaloid, voastrictine (Kam et al., 2001). The same plant also yielded a number of new indole and bisindole alkaloids (Kam et al., 2003b; Kam and Sim, 2003a,b, 2002a,b,c; Zhang et al., 2007; Zèches et al., 1994), including several vobasinyl-iboga bisindoles which reverse multidrug-resistance in vincristine resistant KB cells (Kam et al., 1998). In continuation of our studies of biologically active alkaloids from Malaysian Tab- ernaemontana (Kam et al., 2004a,b, 2003a,b, 2001, 2000, 1999, 1998, 1993, 1992; Kam and Sim, 2003a,b, 2002a,b,c, 2001, 1999), we recently reported the isolation of several new cytotoxic Aspido- sperma-type alkaloids, jerantinines A G, from the leaf extract of the same species, but involving plant material collected from a dif- ferent location (Lim et al., 2008). We now wish to report the fur- ther isolation of additional new alkaloids from the leaf extract of the same plant.
2. Results and discussion
Jerantiphylline A (1) was obtained from the leaf extract of T. cor- ymbosa as a colorless oil, with [a]D 214 (c 0.08, CHCl3). The EIMS of 1 showed a molecular ion at m/z 414, which analyzed for C22H26N2O6, requiring 11° of unsaturation, while the fragment ion observed at m/z 382 is due to loss of MeOH. The UV spectrum showed absorption maxima at 247, 318 and 342 nm (log e 3.89, 4.11 and 4.01, respectively), consistent with a b-anilinoacrylate chromophore and reminiscent of tabersonine alkaloids. In addition to the absorption band due to the presence of the b-anilinoacrylate chromophore (1606 cm—1), the IR spectrum showed bands at 3529, 3377, 1710, 1660 and 1624 cm—1 due to OH, NH, aldehyde, conju- gated ester and amide functions, respectively. The 13C NMR spectrum (Table 1) showed a total of 22 separate carbon resonances (four methyls, four methylenes, four methines and ten quaternary carbons), in agreement with the molecular formula established from HREIMS measurements. The presence of the b-anilinoacrylate chromophore was also indicated by the 13C NMR spectrum which showed the characteristic carbon resonances for C-2 at d 168.5, C-16 at d 89.3 and CO2Me at d 168.5 and 51.2. In addition to the conjugated methyl ester carbonyl resonance, two other carbonyl resonances observed at d 205.3 and 170.6 were assigned to alde- hyde and amide functions, respectively. The 1H NMR spectrum of 1 (Table 1) showed the presence of an isolated aldehyde group, two methoxy groups (one aromatic methoxy and one belonging to an ester CO2Me group), two isolated aromatic hydrogens, an iso- lated methyl, methylene and methine, an ethyl side chain, a pheno- lic OH and an indolic NH. The aromatic methoxy substituent and phenolic OH were deduced to be at C-10 and C-11, respectively, from examination of the aromatic carbon resonances and from the HMBC data (three bond correlations from OH to C-9, C-11 and from 11-OMe to C-11). The COSY and HMQC data revealed the presence of an NCH2CH2 fragment which corresponds to NC(5)– C(6).
The 1H and 13C NMR data of 1 are somewhat similar to those of tabersonine alkaloids, particularly jerantinines A E (Lim et al., 2008).
However, the N(4)–C(3)–C(14)–C(15) fragment usually present in the tabersonine/Aspidosperma alkaloids was conspicu- ously absent in 1, being replaced instead by an N-acetyl and an isolated aldehyde group. Since the structural elements associated with rings A, B, C and E of 1 remained intact when compared with those of jerantinines A E (Lim et al., 2008), the N-acetyl and aldehyde groups in 1 must be associated with an altered ring-D. Furthermore, since the degree of unsaturation for 1 is 11, a tetracyclic carbon skeleton with the lost of ring-D was indi- cated. Further clues to the structure of 1 were provided by the observed heteronuclear correlations from the HMBC spectrum (Fig. 1). The observed correlation from H-21 to the acetyl car- bonyl indicated attachment of the acetyl group to N-4, while correlations from the aldehyde hydrogen to C-17 and C-19, as well as from H-17 and H-19 to the aldehyde carbon, indicated that the aldehyde group is branched from the quaternary C-20. The structure deduced is entirely consistent with the rest of the HMBC data (Fig. 1) as well as with the NOESY/DNOE data (Fig. 1). The latter also revealed the relative stereochemistry at all the stereogenic centers in 1. Thus, irradiation of H-21 caused enhancement of H-9 and H-19, indicating that the orientation of the aldehyde group at C-20 is b. Irradiation of H-9 on the other hand resulted in enhancement of H-5, the acetyl CH3 and H-21. These observations allowed the orientation of the N-4 lone pair to be assigned as b. Irradiation of H-12 resulted in enhancement of NH and 11-OMe, providing additional confirmation for the substitution pattern of the aromatic ring. Jerantiphylline A (1) represents the first example of a ring-D-seco-tabersonine alkaloid. A possible origin of this ring-opened alkaloid is from a 3-oxo-tabersonine derivative such as jerantinine C (2), via a retro-Aldol reaction.
Jerantiphylline B (3) was isolated as a colorless oil, with [a]D 182 (c 0.07, CHCl3). The UV spectrum was characteristic of an oxindole chromophore with absorption maxima at 213, 267 and 303 nm, while the IR spectrum showed bands at 3538, 3281 and 1706 cm—1 due to OH, NH and carbonyl functions, respectively.
The 1H NMR spectrum of 3 (Table 1) revealed some similarities with those of the jerantinines. Firstly, the aromatic substitution pattern in 3 is similar to that in the jerantinine alkaloids (Lim et al., 2008). In addition, 3 resembles jerantinine E (4) in having in common, an unfunctionalized piperidine ring-D. The presence of the oxindole moiety, which was also indicated in the 13C NMR spectrum (d 184.1, Table 1), suggested that the main change in 3 when compared to 4, is the loss of ring C, giving rise to a 2,16-seco-tabersonine alkaloid. The 1H, 13C and 2D NMR data indicated the presence of a trisubstituted double bond at C-16 and C-17, with the latter being an olefinic methine from the ob- served three-bond correlations from H-17 to C-15, C-19, C-21 and CO2Me in the HMBC spectrum. The molecular formula of 3 (m/z 416, C22H28N2O6) differs from that of jerantinine E (4) by 32 mass units, suggesting that 3 possesses two additional oxygen atoms compared to jerantinine E (4). Since the oxindole moiety accounted for one of the additional oxygen atoms, the other is due to an OH group which is in turn linked to the ester bearing, olefinic C-16. This is consistent with the observed carbon reso- nance of C-16 at dC 143.4, as well as the HMBC data, which showed a three-bond correlation from the downfield enol OH sig- nal at dH 15.1 to the carbonyl carbon of the methyl ester group. The unusual deshielding experienced by the enol OH is likely due to intramolecular hydrogen bonding between the enol hydro- gen and the proximate ester carbonyl oxygen. The occurrence of such intramolecular hydrogen bonding probably accounts for the stability of the enol moiety in 3. Jerantiphylline B (3) is therefore the 2,16-seco-derivative of jerantinine E (4) and is characterized by the presence of an unusually stable enol moiety. Only one example (vincatine) with a similar carbon skeleton is known as a natural product (Dopke et al., 1969), while several 2,16-seco- derivatives similar to 3 and incorporating a similar enol function, have been obtained in some instances on further oxidation of various vincadifformine 16-hydroxindolenine derivatives (Danieli recently isolated together with its C(16)-epimer 8, from Ervatamia divaricata occurring in China (Zhang et al., 2007).
Fig. 1. Selected HMBCs and NOEs of 1.
Scrutiny of the structures of jerantinines A (9) and E (4), jerant- inine H (5), and the vincamine alkaloid 7, indicated that they cor- respond to the precursors, oxidized intermediate and final products, respectively, of the Aspidosperma?eburnea transforma- tion, originally proposed by Wenkert to account for the origin of the eburnane/vincamine alkaloids (Wenkert and Wickberg, 1965). Accordingly, such a transformation was attempted as shown in Scheme 1 (Hugel et al., 1972). Protection of the labile phenolic OH of both 4 and 9 as the acetates 10 and 11, respectively, were first carried out to prevent conversion of these alkaloids into their respective iminoquinones. Peracid oxidation of the acetates (10 and 11) followed by an unexpected OH deprotection with 10% Although 16-hydroxindolenines similar to 5 and 2,16-seco- derivatives similar to 3 have been obtained in studies related to the Aspidosperma?eburnea transformation, these compounds have not been previously isolated from any natural source, except for a 16-hydroxyindolenine 6 isolated from the seeds of Amsonia elliptica (Aimi et al., 1978). The authors then noted that the possi- bility that 6 was an artifact of the isolation procedure cannot be ru- led out. In the present study, it was observed that the solutions of jerantinine E (4) in dichloromethane when stored over long peri- ods, resulted in decomposition yielding a complex mixture of com- pounds, from which the 2,16-seco-compound, jerantiphylline B (3), and the 16-hydroxyindolenine, jerantinine H (5), were isolated in trace amounts. In the light of this observation, as well as from the results of the oxidative transformations carried out on 4 and 9 above, the possibility that 3 and 5 may also be artifacts derived from jerantinine E (4) cannot be completely discounted.
In contrast to jerantinines A–E which were previously found to display pronounced cytotoxicity towards both drug-sensitive as well as vincristine-resistant KB cells (Lim et al., 2008), the alkaloids 3, 5 and 7 were found to be ineffective. In the case of 3 and 5, it would appear that a drastic departure from the vincadifformine structure (loss of ring C or the anilinoacrylate chromophore) abol- ished the biological activity altogether.
A comparison of the present results (plant material collected from Tekam Forest, Pahang, Malaysia) with those of the previous one based on samples collected from a different location (plant material collected from Chenderiang, Perak, Malaysia), revealed a variation in the alkaloidal composition. The leaf material from the present study (this report and Lim et al., 2008) yielded only alkaloids of the Aspidosperma-type with the exception of one vinca- mine alkaloid, while leaf samples from the previous study gave predominantly ibogan alkaloids and iboga-vobasinyl bisindoles (Kam et al., 2003b, 1992; Kam and Loh, 1993; Kam and Sim, 2003a,b, 2002a,b, 2001, 1999).
3. Experimental
3.1. General
Optical rotations were determined on a JASCO P-1020 digital polarimeter. IR spectra were recorded on a Perkin–Elmer RX1 FT- IR spectrophotometer. UV spectra were obtained on a Shimadzu UV-3101PC spectrophotometer. 1H and 13C NMR spectra were re- corded in CDCl3 using TMS as internal standard on a JEOL JNM- LA 400 spectrometer at 400 and 100 MHz, respectively. EIMS and HREIMS and HR-FT-APCIMS were obtained at Organic Mass Spec- trometry, Central Science Laboratory, University of Tasmania, Tas- mania, Australia.
3.2. Plant material
Plant material was collected in Pahang, Malaysia, and identifica- tion was confirmed by Dr. K. M. Wong, Institute of Biological Sci- ences, University of Malaya, Kuala Lumpur, Malaysia. Herbarium voucher specimens (K 667) are deposited at the Herbarium, Uni- versity of Malaya.
3.3. Extraction and isolation
Extraction of the ground leaf material was carried out in the usual manner by partitioning the concentrated EtOH extract with dilute acid as has been described in detail elsewhere (Kam and Tan, 1990) to provide a basic fraction (Lim et al., 2008). The alkaloids were isolated by initial column chromatography of the basic fraction on silica gel using CH2Cl2 with increasing proportions of MeOH, followed by rechromatography of the appropriate partially resolved fractions using centrifugal TLC. Solvent systems used for centrifugal TLC were Et2O/hexane (2:1), Et2O/MeOH (50:1), EtOAc/hexane (1:6), EtOAc/hexane (1:3), EtOAc/hexane (1:2), EtOAc/hexane (1:1), CH2Cl2/hexane (2:1), CH2Cl2/hexane (5:1), CH2Cl2/hexane (6:1), CH2Cl2, CH2Cl2/MeOH (100:1) and CHCl3/ MeOH (50:1). The yields (g Kg—1) of the alkaloids were as follows: jerantiphylline A (1) (0.0008), jerantiphylline B (3) (0.002), jerantinine H (5) (0.001) and 14,15-didehydro-10-hydroxy-11-methoxyvincamine (7) (0.010).
Acknowledgment
We would like to thank the University of Malaya and MOSTI, Malaysia (Science Fund), for financial support.
References
Aimi, N., Asada, Y., Sakai, S.I., Haginiwa, J., 1978. Studies on plants containing indole alkaloids VII. Isolation of several Aspidosperma- and vincamine-type alkaloids from the seeds of Amsonia elliptica Roem. et Schult. Chem. Pharm. Bull. 26, 1182–1187.
Calabi, L., Danieli, B., Lesma, G., Palmisano, G., 1982. Dye-sensitized photo- oxygenation of the Aspidosperma alkaloids vincadifformine and tabersonine. A Kam, T.S., Loh, K.Y., Chen, W., 1993. Conophylline and conophyllidine: new dimeric alkaloids from Tabernaemontana divaricata. J. Nat. Prod. 56, 1865–1871.
Kam, T.S., Loh, K.Y., Lim, L.H., Loong, W.L., Chuah, C.H., Chen, W., 1992. New
alkaloids from the leaves of Tabernaemontana divaricata. Tetrahedron Lett. 33, 969–972.
Kam, T.S., Sim, K.M., 2003a. Conodurine, conoduramine, and ervahanine derivatives from Tabernaemontana corymbosa. Phytochemistry 63, 625–629.
Kam, T.S., Sim, K.M., 2003b. Conodirinines A and B, novel vobasine-iboga bisindoles incorporating an additional tetrahydro-1,3-oxazine unit on the vobasinyl moiety. Helv. Chim. Acta 86, 122–126.
Kam, T.S., Sim, K.M., 2002a. Vobasonidine and vobatrcine, novel bisindole alkaloids from a Malayan Tabernaemontana. Helv. Chim. Acta 85, 1027–1032.
Kam, T.S., Sim, K.M., 2002b. Five new iboga alkaloids from Tabernaemontana corymbosa. J. Nat. Prod. 65, 669–672.
Kam, T.S., Sim, K.M., 2002c. New tabernamine derivatives from Tabernaemontana.
Heterocycles 57, 2137–2143.
Kam, T.S., Sim, K.M., 2001. Dippinines A D, new iboga-derived indole alkaloids from Tabernaemontana. Heterocycles 55, 2405–2412.
Kam, T.S., Sim, K.M., 1999. Dippinine A, a new alkaloids of the chippine-type from a Malayan Tabernaemontana. Nat. Prod. Lett. 13, 143–146.
Kam, T.S., Loh, K.Y., 1993. 5-Oxo-19,20-dehydroervatamine from leaves of
Tabernaemontana corymbosa. Phytochemistry 32, 1357–1358.
Kam, T.S., Tan, P.S., 1990. Plumeran alkaloids from Kopsia profunda. Phytochemistry 29, 2321–2322.
Leeuwenberg, A.J.M., 1991. Tabernaemontana: The Old World Species, Royal Botanic Gardens, Kew.
Lim, K.H., Hiraku, O., Komiyama, K., Kam, T.S., 2008. Jerantinines A G, cytotoxic Aspidosperma alkaloids from Tabernaemontana corymbosa. J. Nat. Prod. 71, 1591–1594.
Van Beek, T.A., Verpoorte, R., Baerheim Svendsen, A., Leeuwenberg, A.J.M., Bisset, N.G., 1984. Tabernaemontana L. (Apocynaceae): a review of its taxonomy, phytochemistry, ethnobotany and pharmacology. J. Ethnopharmacol. 10, 1–156.
Wenkert, E., Wickberg, B., 1965. General methods of synthesis of indole alkaloids.
IV. A synthesis of dl-eburnamonine. J. Am. Chem. Soc. 87, 1580–1589.
Zèches, M., Mesbah, K., Loukaci, A., Richard, B., Schaller, H., Sévenet, T., Le Men- Olivier, L., 1994. Alkaloids from leaves and stem bark of Ervatamia corymbosa. Planta Med. 61, 96–97.
Zhang, H., Wang, X.N., Lin, L.P., Ding, J., Yue, J.M., 2007. Indole alkaloids from three species of the Ervatamia Genus: E. officinalis, E. divaricata, and E. divaricata Gouyahua. J. Nat. Prod. 70, 54–59.