- Email: [email protected]
Moandaensine, a dimeric indole alkaloid from Strychnos moandaensis (Loganiaceae)
Phytochemistry Letters 3 (2010) 100–103
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Moandaensine, a dimeric indole alkaloid from Strychnos moandaensis (Loganiaceae) Robert Verpoorte a, Michel Fre´de´rich b, Cle´ment Delaude b, Luc Angenot b, Georges Dive c, Philippe The´penier d, Marie-Jose´ Jacquier d, Monique Ze`ches-Hanrot d, Catherine Lavaud d, Jean-Marc Nuzillard d,* a
Department of Pharmacognosy, Section Metabolomics, IBL, Leiden University, PO Box 502, 2300RA Leiden, The Netherlands Universite´ de Lie`ge, Laboratoire de Pharmacognosie, Centre Interfacultaire de Recherche sur le Me´dicament (CIRM), De´partement de Pharmacie, Universite´ de Lie`ge, B36, B-4000 Lie`ge, Belgium c Centre d’Inge´ne´rie des Prote´ines, Universite´ de Lie`ge, Sart-Tilman, B-4000 Lie`ge, Belgium d Institut de Chimie Mole´culaire de Reims, CNRS UMR 6229, IFR 53 Biomole´cules, Universite´ de Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 02, France b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 January 2010 Received in revised form 16 February 2010 Accepted 17 February 2010 Available online 5 March 2010
The chemical investigation of the liana Strychnos moandaensis De Wild. led to the isolation of moandaensine, a novel dimeric indole alkaloid. The structure was elucidated by a thorough analysis of the spectroscopic data and by molecular modeling. Moandaensine, or methyl (2S)-2-[(2R,3E,12bS)-3ethylidene-2,4,6,7,12,12b-hexahydro-1H-indolo[2,3-a]quinolizin-2-yl]-3-[(2S,3R)-3-ethyl-2-[(1R)-1(hydroxymethyl)-2-methoxy-2-oxo-ethyl]-1,2,3,4-tetrahydroindolo[2,3-a]quinolizin-5-ium-7-yl]propanoate, contains a rare anhydronium base subunit. It presents a moderate antiplasmodial activity with IC50 values of 11.2 and 9.2 mM against, respectively, the chloroquine sensitive FCA 20 GHA and chloroquine resistant W2 strains of Plasmodium falciparum. ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Strychnos moandaensis Strychnos alkaloid Indole alkaloid Anhydronium base Anti-malarial activity
1. Introduction As a known source of anti-cancer indole alkaloids, the Strychnos genus (Loganiaceae) has been extensively studied during the last 40 years and has given access to numerous substances which encompass a wide diversity of structural types (Massiot and Delaude, 1988). The search for antiplasmodial activity of indole alkaloids is relatively recent (Fre´de´rich et al., 2008) and has led to the discovery of molecules that are active against chloroquine resistant Plasmodium falciparum strains. Among these indole alkaloids, some Strychnos alkaloids have revealed moderate to high antiplasmodial activity, even in vivo (Fre´de´rich et al., 2002, 2004). The study of Strychnos moandaensis is part of this effort toward the isolation of new bioactive molecules in the treatment of malaria and to the discovery of new alkaloid skeletal types. S. moandaensis De Wild. is a 2–10 m long liana growing along streams in the two Congos and Angola (Leeuwenberg, 1969). The species is rare, and consequently no traditional use is known. It has been studied neither for its constituents nor for its biological activity. We report here the first phytochemical study of this plant.
* Corresponding author. Tel.: +33 3 26918210; fax: +33 3 26913166. E-mail address: [email protected] (J.-M. Nuzillard).
It resulted in the isolation of a novel dimeric alkaloid from the root bark. 2. Results and discussion Leaves, stem bark and root bark were all extracted in the same way. Only small amounts of alkaloid could be observed on TLC, and all showed only one, and the same, major compound. This alkaloid was eventually isolated from the root bark and will be subsequently referred to as moandaensine, (1). The HR-ESI mass spectrum of 1 led to the molecular formula C42H48N4O5. The 1H, 13 C, COSY, ROESY, HSQC and HMBC NMR data and the botanical origin of 1 led to the planar structure of a dimeric indolomonoterpenic alkaloid with a serpentinine skeleton (Irie et al., 1972). NMR data is summarized in Table 1. The tricyclic anhydronium base-ring system is a feature that was already reported in Rauwolfia and Strychnos alkaloids (Irie et al., 1972; Fre´de´rich et al., 1998). Its presence was supported by the UV spectrum of 1 that showed maxima at 210, 221, 257, 308 and 373 nm and by the transformation of this chromophore to a purely indolic one after addition of NaBH4 (see Supporting Information). Moandaensine 1 may be considered as the assembly of the indolic ‘‘Northern’’ monomer and of the anhydronium base ‘‘Southern’’ monomer. The 3D structure of 1 is hereafter discussed by first considering the Northern part and then the Southern part.
1874-3900/$ – see front matter ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2010.02.005
R. Verpoorte et al. / Phytochemistry Letters 3 (2010) 100–103
101
Table 1 NMR data of moandaensine 1. Atom no.
13 a
C
1 2 3 5
133.8 53.1 51.5
6
18.1
7 8 9 10 11 12 13 14 15 16 17
108.4 128.0 118.7 119.9 122.0 111.4 136.4 30.4 36.0 46.2 30.6
18 19 20 21
14.1 125.10 134.2 52.7
22 22-OMe 20 30 50 60 70 80 90 100 110 120 130 140
176.1 52.2 135.3 140.8 127.4 129.9 130.1 120.2 123.6 121.8 131.0 115.5 146.0 25.5
150 160 170
34.6 50.4 60.2
180 190
11.4 26.2
200 210
39.39 58.9
220 220 -OMe
173.5 52.9
a b
1
Hb
COSYc
9.06 br s
HMBCd
ROESYc
2, 7, 8, 13
3f, 12, 14, 16, 22-OMe 1f, 5b, 14, 15f, 21bf 5b, 6a, 6b, 21af, 21b 3, 5a, 6b 5a, 6b, 21a, 9 5a, 5b, 6a, 9
4.41 br s a: 3.30 br d (11)e b: 3.18 br d (11)e a: 3.01 t (15)e b: 2.65 dd (15.7, 4.7)
14 5b, 6a, 6bf 5a, 6a, 6b 5a, 5b, 6b 5af, 5b, 6a
2, 15f 3, 7 3, 7f 2f, 5f, 7f 2, 5f, 7
7.47 7.07 7.11 7.38
10 9, 11 10, 12 11
7, 8, 9, 8,
2.30 m 3.26 m 2.91 td (11.5, 3) a: 3.41 dd (16, 12.8) b: 3.17 bd (15.1) 1.90 d (6.6) 5.87 q (6.7)
3, 15 16 15, 17a, 17b 16, 17b 16, 17a 19, 21af 18
2, 3, 15, 16, 20 3, 16, 17, 19, 20, 22 15f, 22f, 60 f 15f, 16, 22, 50 ,60 ,70 16, 50 f, 22, 60 f 19, 20 15, 18, 21
1, 3, 15, 16 3f, 14, 17a, 18 1, 14, 17b, 21a, 50 , 90 15, 17b, 18f, 50 f, 90 16, 17a, 90 15, 17af, 100 f, 90 f 21b
a: 3.58 br d (13) b: 3.10 d (13)
18f, 21b 21a
19, 20 3, 15, 19, 20
5af, 6a, 16, 21b, 90 f 3f, 5af, 19, 21a
3.51 s
22
1f
6.83 s
30 , 70 , 210 , 17f
210 af, 210 b, 16, 17af
100 90 , 110 100 , 120 110
70 , 80 , 90 , 80 ,
16, 17a, 17b, 18f, 21af 18f
140 b, 150 f 150 140 af, 140 b, 200 170 a, 170 b 160 160 190 a, 190 b 180 , 200 f 180 , 200 f 150 , 190 af, 90 bf, 210 af, 210 bf 200 f, 210 b 200 f, 210 a
30 20 d 140 , 160 , 190 , 220 f 140 , 150 , 170 , 220 150 f 150 , 220 190 , 200 150 f, 200 f, 210 f 150 f, 18, 200 f, 210 f 150 f, 160 , 180 f, 190 f 30 f 150
7.90 7.19 7.56 7,88
d (7.5) t (7.3) t (7.3) d (7.7)
d (7.7) t (7.6) t (7.6) d (7.7)
a: 4.58 br d (15.3)e b: 3.37 dd (15, 12)e 2.16 m 3.02 m a: 4.08 dd (11.3, 9.6) b: 3.99 dd (11.3, 5) 0.81 t (7) a: 0.96 m b: 0.87 m 2.04 m a: 4.28 dd (12.8, 3.5) b: 3.54 br d (14)e 3.66 s
8f, 11, 13 9, 12 13 10
110 , 130 110 f, 120 130 100
f
6a, 6b
1
140 b 140 a, 210 a 160 , 180 f, 190 af 150 , 170 af, 170 b, 180 f, 200 160 f, 170 b 160 , 170 a 150 f, 160 f, 200 f, 210 bf 150 f, 200 f 200 f 160 , 180 f, 190 af, 190 bf, 210 af, 210 bf 50 f, 140 b, 200 f, 210 b 50 , 180 f, 200 f, 210 a
220
d in ppm. d in ppm, mult (J in Hz).
c 1
H number. C number; From HSQC row; Weak signal.
d 13 e f
The configuration of H-15 was assumed, for biogenetic reasons, to be a in both monomeric units. A ROESY correlation between H-21b and H-19 and another one between the H-18 methyl protons and H-15 proved that the C-19/C-20 double bond had an E geometry. The high chemical shift value of H-3 (d 4.41) was only compatible with an H-3a cis (S) conformation of the C/D quinolizidine ring system and a H-16b configuration, as pointed out in Lounasmaa et al. (1994). The assigned configuration was further supported by the use of molecular modeling with NOESY and J-coupling data. For this purpose, a model of the Northern moiety of 1 was built using the Macromodel software (Macromodel). A benzene ring was attached to C-17 in order to simulate the presence of the Southern moiety. The configurations were set to H-3a, H-15a, H-16b, with
an E double bond. The initial conformation showed a flat ring system. A set of 1000 conformations was randomly drawn and each one was allowed to evolve to a minimum energy state. The lowest energy conformation presented a bent shape that was stabilized by an intramolecular hydrogen bond between the oxygen of the carbonyl group at C-22 and the indolic hydrogen atom. In this conformation, the proximity of the N-4 lone pair with H-3 accounts for the high chemical shift of the latter. H-3 appears in the 1H NMR spectrum as a broad singlet due to its two poorly resolved small couplings with both H-14a and H-14b. The initial flat conformer has H-3 and one of the H-14 hydrogens in a trans diaxial arrangement and would lead to a coupling constant of about 10 Hz that is not observed. The H-3/H-5b ROESY correlation
102
R. Verpoorte et al. / Phytochemistry Letters 3 (2010) 100–103
indicated that H-5b was in the a position in ring C. The H-5a/H-21b ROESY correlation set H-21b in the a position in ring D. The H-15/ H-17a and H-16/H-17b ROESY correlations clearly distinguished H-17b from H-17a. The latter appeared as a large doublet of doublets in the 1H NMR spectrum because it coupled with H-17b and H-16 with two strong coupling constants. The stereospecific assignment was completed by the H-21a/H-6a ROESY correlation. The bent shape of the Northern moiety was also proved by the NH/ H-16 and H-16/H-21a ROESY correlations (see Supporting Information). The 3D structure of the Southern moiety depended on the configurations at C-200 and C-160 . The four corresponding stereoisomers of 1 were modeled, using the method that was described above. The shape of the Northern part was fixed as previously determined and only the rotations between the C-15/C16, C-16/C-17, C-17/C-6 and C-16/C-22 bonds were allowed in this moiety. The likelihood of lowest energy conformers was first judged from the spatial arrangement of the H-21’a/b pair and of H200 . The HSQC row at the chemical shift of C-210 showed that the two H-210 signal were doublets, thus indicating that the H-210 protons had negligible couplings with H-20. The two stereoisomers having H-200 in a b position are the only ones to respect this constraint. The D’ ring in these stereoisomers have a bent shape in which C-140 , C-150 , C-200 and C-210 are coplanar. This geometry imposes very different environments on the H-140 protons and the two H-210 protons as well, as reflected by a strong chemical shift difference within each pair (1.2 ppm for H-140 a/b and 0.7 ppm for the H-210 a/b). H-150 , being in an axial position, coupled with H140 b with a strong constant (in a b position, J = 11.6 Hz) and with a smaller one with H-14’a (in an a position). The ROESY correlation between H-14’b and H-210 a assigned them a b position in the D0 ring. The trans arrangement of H-150 and H-200 was also present in the anhydronium base moiety of serpentinine. There was no clear clue that would lead to the configuration at position 160 . Hydrogen H-160 was assumed to be in the b position, again assuming that the Northern and the Southern moieties shared the same biogenetic origin (see Supporting Information). Therefore, we proposed a 3S, 15R, 16S, 150 S, 160 R, 200 R, 19-20E configuration for moandaensine 1 (Fig. 1). The relative orientation of the Northern and Southern moieties could be tentatively defined by means of inter-monomer ROESY correlations between H-50 and H-16 and those between H-90 and H-16, H-17a, H-17b, H-18 and H-21a. The weak H-100 /H-18 ROESY correlation also provided some hints about the global molecular geometry. However, the relatively important breadth of the H-50
and of the C-50 NMR resonances could be interpreted by the existence of some flexibility in the inter-unit linkage. The geometry of moandaensine was fully optimized at the B3LYP functional level (Becke, 1993) using the polarized double z basis set 6-31G(d) (Haharan and Pople, 1973). All calculations were performed with the Gaussian 03 program (Gaussian, 2004). This geometry provided an explanation for the chemical shift value of H-50 (6.83 ppm), that was unusually low for a pyridinium proton: H-50 was close to the shielding zone of the indolic aromatic nucleus. In comparison, the chemical shift of H-50 in 30 ,40 ,50 ,60 tetrahydrolongicaudatine Y is 8.46 ppm (Fre´de´rich et al., 1998). Moandaensine presented a moderate antiplasmodial activity with IC50 values of 11.2 and 9.2 mM against, respectively, the chloroquine sensitive FCA 20 GHA and chloroquine resistant W2 strains of P. falciparum. IC50 references values for chloroquine and quinine are reported in the experimental part. 3. Experimental 3.1. General UV spectra were obtained using a Philips PU 8720 spectrophotometer. IR spectra were recorded on a Nicolet Avatar 320 FT-IR spectrometer. Optical rotation was measured on a PerkinElmer model 341 polarimeter. NMR spectra were recorded on a Bruker DRX-500 spectrometer at 500 MHz for 1H NMR and 125 MHz for 13 C NMR. The 2D NMR experiments were performed using standard Bruker microprograms. NMR spectra were recorded in CDCl3, using solvent signals for calibration (13CDCl3 at 77.4 ppm and residual C1HCl3 at 7.26 ppm). Mass spectrometry was carried out on a Micromass ESI-Q-TOF apparatus using ESI ionization in the positive mode. Column chromatography (CC) was performed on Silica gel 60 (63–200 mm, Merck), TLC analysis was run on 60 F254 precoated silica gel plates (Merck). 3.2. Plant material The plant was collected by Pr. C. Delaude in Congo (Kinshasa) in the Mbanza Ngungu area, near the village of Wombe, along the Congo river. Voucher specimens (H. Breyne 3976) were kept in the Herbarium of the Botanical Garden of Belgium at Meise (Brussels). 3.3. Extraction and isolation Root bark (2.18 kg) was ground, wet with 1.3 l of NH3 soln. (7.3 M) and extracted by maceration for 48 h with 40 l of EtOAc. The EtOAc soln. was exhaustively extracted with 2% (v/v) H2SO4. After alkalizing the aqueous phase with 14.6 M NH3 soln., the alkaloids were extracted by CHCl3. The yield was 2.9 g. The extract was separated on a 150 g silica gel column (Merck H-60, Darmstadt), with a CHCl3–MeOH gradient under 10 bar pressure. The alkaloid containing fraction (93 mg) was further purified on a silica gel column (CHCl3–MeOH gradient) at atmospheric pressure, resulting in the isolation of 30 mg of the almost pure alkaloid. In a further purification step by preparative TLC on silica gel (K6F Merck 60, Darmstadt) with CHCl3–MeOH (4:1) as eluent, 14 mg of the pure alkaloid was obtained. 3.4. Moandaensine 1
Fig. 1. The structure of moandaensine 1.
Yellow solid; [a]D 278 (c 0.08, MeOH); UV (MeOH) lmax 210, 221, 257, 308, 373 nm; IR (KBr) nmax 3173 (br), 3072, 2936, 1730, 1643, 1441, 1340, 1265, 1165, 1103, 1028, 802, 750 cm1; 1H and 13 C NMR data, see Table 1; ESIMS m/z 689.3701 [M+H]+, calc. 689.3703 for [C42H48N4O5+H]+.
R. Verpoorte et al. / Phytochemistry Letters 3 (2010) 100–103
3.5. Biological assay Parasite viability was measured using parasite lactate dehydrogenase (pLDH) activity according to the method that is reported in Makler et al. (1993) and modified according to Jonville et al. (2008). Quinine (Aldrich) and chloroquine (Sigma) were used as positive controls in all experiments with an initial concentration of 100 ng/ml. FCA and W2 IC50 values were respectively 0.011 0.005 mM (n = 6) and 0.284 0.017 mM (n = 5) for chloroquine and 0.269 0.006 (n = 3) and 0.413 0.011 (n = 3) for quinine. Stock solns. of the alkaloid were prepared in DMSO at 20 mg/ml. They were then further diluted in medium to give 2 mg/ml solns. The highest concentration of DMSO to which the parasites were exposed was 1%, which was shown to have no measurable effect on parasite viability. All tests were performed in triplicate. Acknowledgements We thank Dr. Karen Ple´ for linguistic advice, Dr. G. Massiot for stimulating discussions and D. Harakat for MS analysis. G.D. and M.F. are respectively Research Associate and Senior Research Associate of the FRS-FNRS. G.D. thanks the FRS-FNRS for the financial support of the high performance computing systems installed in Lie`ge and Louvain-la-Neuve. M.F. and L.A. thank the FRS-FNRS for the financial support of antiplasmodial assays (FNRS grant no. 3452005). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytol.2010.02.005. References Becke, A.D., 1993. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652.
103
Fre´de´rich, M., Quentin-Leclercq, J., Gadi Biala, R., Brandt, V., Penelle, J., Tits, M., Angenot, L., 1998. 30 ,40 ,50 ,60 Tetradehydrolongicaudatine Y, an anhydronium base from Strychnos usambarensis. Phytochemistry 48, 1263–1266. Fre´de´rich, M., Jacquier, M.-J., The´penier, P., De Mol, P., Tits, M., Philippe, G., Delaude, C., Angenot, L., Ze`ches-Hanrot, M., 2002. Antiplasmodial activity of alkaloids from various Strychnos species. J. Nat. Prod. 65, 1381–1386. Fre´de´rich, M., Tits, M., Goffin, E., Philippe, G., Grellier, P., De Mol, P., Hayette, M.-P., Angenot, L., 2004. In vitro and in vivo antimalarial properties of isostrychnopentamine, an indolomonoterpenic alkaloid from Strychnos usambarensis. Planta Med. 70, 520–525. Fre´de´rich, M., Tits, M., Angenot, L., 2008. Potential antimalarial activity of indole alkaloids. Trans. R. Soc. Trop. Med. Hyg. 102, 11–19. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Montgomery Jr., J.A., Vreven, T., Kudin, K.N., Burant, J.C., Millam, J.M., Iyengar, S.S., Tomasi, J., Barone, V., Mennucci, B., Cossi, M., Scalmani, G., Rega, N., Petersson, G.A., Nakatsuji, H., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Klene, M., Li, X., Knox, J.E., Hratchian, H.P., Cross, J.B., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R.E., Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C., Ochterski, J.W., Ayala, P.Y., Morokuma, K., Voth, G.A., Salvador, P., Dannenberg, J.J., Zakrzewski, V.G., Dapprich, S., Daniels, A.D., Strain, M.C., Farkas, O., Malick, D.K., Rabuck, A.D., Raghavachari, K., Foresman, J.B., Ortiz, J.V., Cui, Q., Baboul, A.G., Clifford, S., Cioslowski, J., Stefanov, B.B., Liu, G., Liashenko, A., Piskorz, P., Komaromi, I., Martin, R.L., Fox, D.J., Keith, T., Al-Laham, M.A., Peng, C.Y., Nanayakkara, A., Challacombe, M., Gill, P.M.W., Johnson, B., Chen, W., Wong, M.W., Gonzalez, C., Pople, J.A., 2004. Gaussian 03, Revision C.02. Gaussian, Inc., Wallingford, CT. Haharan, P.C., Pople, J.A., 1973. The influence of polarization functions on molecular orbital hydrogenation energies. Theor. Chim. Acta 28, 213–217. Irie, H., Ishizuka, K., Kawashima, S., Masaki, N., Osaki, K., Shingu, T., Uyeo, S., 1972. Xray crystallographic determination of the structure of the alkaloid serpentinine. J. Chem. Soc. Chem. Commun. 871. Jonville, M.C., Kodja, H., Humeau, L., Fournel, J., De Mol, P., Cao, M., Angenot, L., Fre´de´rich, M., 2008. Screening of medicinal plants from Reunion Island for antimalarial and cytotoxic activity. J. Ethnopharmacol. 120, 382–386. Leeuwenberg, A., 1969. The Loganiaceae of Africa VIII. Strychnos III. Revision of the African species with notes on the extra – African, Veenman, H., Zonen N.V. (Eds.), Wageningen, pp. 133–138, 267–272. Lounasmaa, M., Jokela, R., Hanhinen, P., Miettinen, J., Salo, J., 1994. Preparation and conformational study of Z- and E-isositsirikine epimers and model compounds. Determination of their C-16 configurations. Tetrahedron 50, 9207–9222. MacroModel, version 5.5, Department of Chemistry, Columbia University, New York, NY 10027. Makler, M.T., Ries, J.M., Williams, J.A., Bancroft, J.E., Piper, R.C., Gibbins, B.L., Hinrichs, D.J., 1993. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. Am. J. Trop. Med. Hyg. 48, 739–741. Massiot, G., Delaude, C., 1988. African Strychnos alkaloids. In: Brossi, A. (Ed.), The Alkaloids: Chemistry and Pharmacology, vol. 34, Academic Press, San Diego, pp. 211–329, Chapter 5.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Moandaensine, a dimeric indole alkaloid from Strychnos moandaensis (Loganiaceae) Robert Verpoorte a, Michel Fre´de´rich b, Cle´ment Delaude b, Luc Angenot b, Georges Dive c, Philippe The´penier d, Marie-Jose´ Jacquier d, Monique Ze`ches-Hanrot d, Catherine Lavaud d, Jean-Marc Nuzillard d,* a
Department of Pharmacognosy, Section Metabolomics, IBL, Leiden University, PO Box 502, 2300RA Leiden, The Netherlands Universite´ de Lie`ge, Laboratoire de Pharmacognosie, Centre Interfacultaire de Recherche sur le Me´dicament (CIRM), De´partement de Pharmacie, Universite´ de Lie`ge, B36, B-4000 Lie`ge, Belgium c Centre d’Inge´ne´rie des Prote´ines, Universite´ de Lie`ge, Sart-Tilman, B-4000 Lie`ge, Belgium d Institut de Chimie Mole´culaire de Reims, CNRS UMR 6229, IFR 53 Biomole´cules, Universite´ de Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 02, France b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 January 2010 Received in revised form 16 February 2010 Accepted 17 February 2010 Available online 5 March 2010
The chemical investigation of the liana Strychnos moandaensis De Wild. led to the isolation of moandaensine, a novel dimeric indole alkaloid. The structure was elucidated by a thorough analysis of the spectroscopic data and by molecular modeling. Moandaensine, or methyl (2S)-2-[(2R,3E,12bS)-3ethylidene-2,4,6,7,12,12b-hexahydro-1H-indolo[2,3-a]quinolizin-2-yl]-3-[(2S,3R)-3-ethyl-2-[(1R)-1(hydroxymethyl)-2-methoxy-2-oxo-ethyl]-1,2,3,4-tetrahydroindolo[2,3-a]quinolizin-5-ium-7-yl]propanoate, contains a rare anhydronium base subunit. It presents a moderate antiplasmodial activity with IC50 values of 11.2 and 9.2 mM against, respectively, the chloroquine sensitive FCA 20 GHA and chloroquine resistant W2 strains of Plasmodium falciparum. ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Strychnos moandaensis Strychnos alkaloid Indole alkaloid Anhydronium base Anti-malarial activity
1. Introduction As a known source of anti-cancer indole alkaloids, the Strychnos genus (Loganiaceae) has been extensively studied during the last 40 years and has given access to numerous substances which encompass a wide diversity of structural types (Massiot and Delaude, 1988). The search for antiplasmodial activity of indole alkaloids is relatively recent (Fre´de´rich et al., 2008) and has led to the discovery of molecules that are active against chloroquine resistant Plasmodium falciparum strains. Among these indole alkaloids, some Strychnos alkaloids have revealed moderate to high antiplasmodial activity, even in vivo (Fre´de´rich et al., 2002, 2004). The study of Strychnos moandaensis is part of this effort toward the isolation of new bioactive molecules in the treatment of malaria and to the discovery of new alkaloid skeletal types. S. moandaensis De Wild. is a 2–10 m long liana growing along streams in the two Congos and Angola (Leeuwenberg, 1969). The species is rare, and consequently no traditional use is known. It has been studied neither for its constituents nor for its biological activity. We report here the first phytochemical study of this plant.
* Corresponding author. Tel.: +33 3 26918210; fax: +33 3 26913166. E-mail address: [email protected] (J.-M. Nuzillard).
It resulted in the isolation of a novel dimeric alkaloid from the root bark. 2. Results and discussion Leaves, stem bark and root bark were all extracted in the same way. Only small amounts of alkaloid could be observed on TLC, and all showed only one, and the same, major compound. This alkaloid was eventually isolated from the root bark and will be subsequently referred to as moandaensine, (1). The HR-ESI mass spectrum of 1 led to the molecular formula C42H48N4O5. The 1H, 13 C, COSY, ROESY, HSQC and HMBC NMR data and the botanical origin of 1 led to the planar structure of a dimeric indolomonoterpenic alkaloid with a serpentinine skeleton (Irie et al., 1972). NMR data is summarized in Table 1. The tricyclic anhydronium base-ring system is a feature that was already reported in Rauwolfia and Strychnos alkaloids (Irie et al., 1972; Fre´de´rich et al., 1998). Its presence was supported by the UV spectrum of 1 that showed maxima at 210, 221, 257, 308 and 373 nm and by the transformation of this chromophore to a purely indolic one after addition of NaBH4 (see Supporting Information). Moandaensine 1 may be considered as the assembly of the indolic ‘‘Northern’’ monomer and of the anhydronium base ‘‘Southern’’ monomer. The 3D structure of 1 is hereafter discussed by first considering the Northern part and then the Southern part.
1874-3900/$ – see front matter ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2010.02.005
R. Verpoorte et al. / Phytochemistry Letters 3 (2010) 100–103
101
Table 1 NMR data of moandaensine 1. Atom no.
13 a
C
1 2 3 5
133.8 53.1 51.5
6
18.1
7 8 9 10 11 12 13 14 15 16 17
108.4 128.0 118.7 119.9 122.0 111.4 136.4 30.4 36.0 46.2 30.6
18 19 20 21
14.1 125.10 134.2 52.7
22 22-OMe 20 30 50 60 70 80 90 100 110 120 130 140
176.1 52.2 135.3 140.8 127.4 129.9 130.1 120.2 123.6 121.8 131.0 115.5 146.0 25.5
150 160 170
34.6 50.4 60.2
180 190
11.4 26.2
200 210
39.39 58.9
220 220 -OMe
173.5 52.9
a b
1
Hb
COSYc
9.06 br s
HMBCd
ROESYc
2, 7, 8, 13
3f, 12, 14, 16, 22-OMe 1f, 5b, 14, 15f, 21bf 5b, 6a, 6b, 21af, 21b 3, 5a, 6b 5a, 6b, 21a, 9 5a, 5b, 6a, 9
4.41 br s a: 3.30 br d (11)e b: 3.18 br d (11)e a: 3.01 t (15)e b: 2.65 dd (15.7, 4.7)
14 5b, 6a, 6bf 5a, 6a, 6b 5a, 5b, 6b 5af, 5b, 6a
2, 15f 3, 7 3, 7f 2f, 5f, 7f 2, 5f, 7
7.47 7.07 7.11 7.38
10 9, 11 10, 12 11
7, 8, 9, 8,
2.30 m 3.26 m 2.91 td (11.5, 3) a: 3.41 dd (16, 12.8) b: 3.17 bd (15.1) 1.90 d (6.6) 5.87 q (6.7)
3, 15 16 15, 17a, 17b 16, 17b 16, 17a 19, 21af 18
2, 3, 15, 16, 20 3, 16, 17, 19, 20, 22 15f, 22f, 60 f 15f, 16, 22, 50 ,60 ,70 16, 50 f, 22, 60 f 19, 20 15, 18, 21
1, 3, 15, 16 3f, 14, 17a, 18 1, 14, 17b, 21a, 50 , 90 15, 17b, 18f, 50 f, 90 16, 17a, 90 15, 17af, 100 f, 90 f 21b
a: 3.58 br d (13) b: 3.10 d (13)
18f, 21b 21a
19, 20 3, 15, 19, 20
5af, 6a, 16, 21b, 90 f 3f, 5af, 19, 21a
3.51 s
22
1f
6.83 s
30 , 70 , 210 , 17f
210 af, 210 b, 16, 17af
100 90 , 110 100 , 120 110
70 , 80 , 90 , 80 ,
16, 17a, 17b, 18f, 21af 18f
140 b, 150 f 150 140 af, 140 b, 200 170 a, 170 b 160 160 190 a, 190 b 180 , 200 f 180 , 200 f 150 , 190 af, 90 bf, 210 af, 210 bf 200 f, 210 b 200 f, 210 a
30 20 d 140 , 160 , 190 , 220 f 140 , 150 , 170 , 220 150 f 150 , 220 190 , 200 150 f, 200 f, 210 f 150 f, 18, 200 f, 210 f 150 f, 160 , 180 f, 190 f 30 f 150
7.90 7.19 7.56 7,88
d (7.5) t (7.3) t (7.3) d (7.7)
d (7.7) t (7.6) t (7.6) d (7.7)
a: 4.58 br d (15.3)e b: 3.37 dd (15, 12)e 2.16 m 3.02 m a: 4.08 dd (11.3, 9.6) b: 3.99 dd (11.3, 5) 0.81 t (7) a: 0.96 m b: 0.87 m 2.04 m a: 4.28 dd (12.8, 3.5) b: 3.54 br d (14)e 3.66 s
8f, 11, 13 9, 12 13 10
110 , 130 110 f, 120 130 100
f
6a, 6b
1
140 b 140 a, 210 a 160 , 180 f, 190 af 150 , 170 af, 170 b, 180 f, 200 160 f, 170 b 160 , 170 a 150 f, 160 f, 200 f, 210 bf 150 f, 200 f 200 f 160 , 180 f, 190 af, 190 bf, 210 af, 210 bf 50 f, 140 b, 200 f, 210 b 50 , 180 f, 200 f, 210 a
220
d in ppm. d in ppm, mult (J in Hz).
c 1
H number. C number; From HSQC row; Weak signal.
d 13 e f
The configuration of H-15 was assumed, for biogenetic reasons, to be a in both monomeric units. A ROESY correlation between H-21b and H-19 and another one between the H-18 methyl protons and H-15 proved that the C-19/C-20 double bond had an E geometry. The high chemical shift value of H-3 (d 4.41) was only compatible with an H-3a cis (S) conformation of the C/D quinolizidine ring system and a H-16b configuration, as pointed out in Lounasmaa et al. (1994). The assigned configuration was further supported by the use of molecular modeling with NOESY and J-coupling data. For this purpose, a model of the Northern moiety of 1 was built using the Macromodel software (Macromodel). A benzene ring was attached to C-17 in order to simulate the presence of the Southern moiety. The configurations were set to H-3a, H-15a, H-16b, with
an E double bond. The initial conformation showed a flat ring system. A set of 1000 conformations was randomly drawn and each one was allowed to evolve to a minimum energy state. The lowest energy conformation presented a bent shape that was stabilized by an intramolecular hydrogen bond between the oxygen of the carbonyl group at C-22 and the indolic hydrogen atom. In this conformation, the proximity of the N-4 lone pair with H-3 accounts for the high chemical shift of the latter. H-3 appears in the 1H NMR spectrum as a broad singlet due to its two poorly resolved small couplings with both H-14a and H-14b. The initial flat conformer has H-3 and one of the H-14 hydrogens in a trans diaxial arrangement and would lead to a coupling constant of about 10 Hz that is not observed. The H-3/H-5b ROESY correlation
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R. Verpoorte et al. / Phytochemistry Letters 3 (2010) 100–103
indicated that H-5b was in the a position in ring C. The H-5a/H-21b ROESY correlation set H-21b in the a position in ring D. The H-15/ H-17a and H-16/H-17b ROESY correlations clearly distinguished H-17b from H-17a. The latter appeared as a large doublet of doublets in the 1H NMR spectrum because it coupled with H-17b and H-16 with two strong coupling constants. The stereospecific assignment was completed by the H-21a/H-6a ROESY correlation. The bent shape of the Northern moiety was also proved by the NH/ H-16 and H-16/H-21a ROESY correlations (see Supporting Information). The 3D structure of the Southern moiety depended on the configurations at C-200 and C-160 . The four corresponding stereoisomers of 1 were modeled, using the method that was described above. The shape of the Northern part was fixed as previously determined and only the rotations between the C-15/C16, C-16/C-17, C-17/C-6 and C-16/C-22 bonds were allowed in this moiety. The likelihood of lowest energy conformers was first judged from the spatial arrangement of the H-21’a/b pair and of H200 . The HSQC row at the chemical shift of C-210 showed that the two H-210 signal were doublets, thus indicating that the H-210 protons had negligible couplings with H-20. The two stereoisomers having H-200 in a b position are the only ones to respect this constraint. The D’ ring in these stereoisomers have a bent shape in which C-140 , C-150 , C-200 and C-210 are coplanar. This geometry imposes very different environments on the H-140 protons and the two H-210 protons as well, as reflected by a strong chemical shift difference within each pair (1.2 ppm for H-140 a/b and 0.7 ppm for the H-210 a/b). H-150 , being in an axial position, coupled with H140 b with a strong constant (in a b position, J = 11.6 Hz) and with a smaller one with H-14’a (in an a position). The ROESY correlation between H-14’b and H-210 a assigned them a b position in the D0 ring. The trans arrangement of H-150 and H-200 was also present in the anhydronium base moiety of serpentinine. There was no clear clue that would lead to the configuration at position 160 . Hydrogen H-160 was assumed to be in the b position, again assuming that the Northern and the Southern moieties shared the same biogenetic origin (see Supporting Information). Therefore, we proposed a 3S, 15R, 16S, 150 S, 160 R, 200 R, 19-20E configuration for moandaensine 1 (Fig. 1). The relative orientation of the Northern and Southern moieties could be tentatively defined by means of inter-monomer ROESY correlations between H-50 and H-16 and those between H-90 and H-16, H-17a, H-17b, H-18 and H-21a. The weak H-100 /H-18 ROESY correlation also provided some hints about the global molecular geometry. However, the relatively important breadth of the H-50
and of the C-50 NMR resonances could be interpreted by the existence of some flexibility in the inter-unit linkage. The geometry of moandaensine was fully optimized at the B3LYP functional level (Becke, 1993) using the polarized double z basis set 6-31G(d) (Haharan and Pople, 1973). All calculations were performed with the Gaussian 03 program (Gaussian, 2004). This geometry provided an explanation for the chemical shift value of H-50 (6.83 ppm), that was unusually low for a pyridinium proton: H-50 was close to the shielding zone of the indolic aromatic nucleus. In comparison, the chemical shift of H-50 in 30 ,40 ,50 ,60 tetrahydrolongicaudatine Y is 8.46 ppm (Fre´de´rich et al., 1998). Moandaensine presented a moderate antiplasmodial activity with IC50 values of 11.2 and 9.2 mM against, respectively, the chloroquine sensitive FCA 20 GHA and chloroquine resistant W2 strains of P. falciparum. IC50 references values for chloroquine and quinine are reported in the experimental part. 3. Experimental 3.1. General UV spectra were obtained using a Philips PU 8720 spectrophotometer. IR spectra were recorded on a Nicolet Avatar 320 FT-IR spectrometer. Optical rotation was measured on a PerkinElmer model 341 polarimeter. NMR spectra were recorded on a Bruker DRX-500 spectrometer at 500 MHz for 1H NMR and 125 MHz for 13 C NMR. The 2D NMR experiments were performed using standard Bruker microprograms. NMR spectra were recorded in CDCl3, using solvent signals for calibration (13CDCl3 at 77.4 ppm and residual C1HCl3 at 7.26 ppm). Mass spectrometry was carried out on a Micromass ESI-Q-TOF apparatus using ESI ionization in the positive mode. Column chromatography (CC) was performed on Silica gel 60 (63–200 mm, Merck), TLC analysis was run on 60 F254 precoated silica gel plates (Merck). 3.2. Plant material The plant was collected by Pr. C. Delaude in Congo (Kinshasa) in the Mbanza Ngungu area, near the village of Wombe, along the Congo river. Voucher specimens (H. Breyne 3976) were kept in the Herbarium of the Botanical Garden of Belgium at Meise (Brussels). 3.3. Extraction and isolation Root bark (2.18 kg) was ground, wet with 1.3 l of NH3 soln. (7.3 M) and extracted by maceration for 48 h with 40 l of EtOAc. The EtOAc soln. was exhaustively extracted with 2% (v/v) H2SO4. After alkalizing the aqueous phase with 14.6 M NH3 soln., the alkaloids were extracted by CHCl3. The yield was 2.9 g. The extract was separated on a 150 g silica gel column (Merck H-60, Darmstadt), with a CHCl3–MeOH gradient under 10 bar pressure. The alkaloid containing fraction (93 mg) was further purified on a silica gel column (CHCl3–MeOH gradient) at atmospheric pressure, resulting in the isolation of 30 mg of the almost pure alkaloid. In a further purification step by preparative TLC on silica gel (K6F Merck 60, Darmstadt) with CHCl3–MeOH (4:1) as eluent, 14 mg of the pure alkaloid was obtained. 3.4. Moandaensine 1
Fig. 1. The structure of moandaensine 1.
Yellow solid; [a]D 278 (c 0.08, MeOH); UV (MeOH) lmax 210, 221, 257, 308, 373 nm; IR (KBr) nmax 3173 (br), 3072, 2936, 1730, 1643, 1441, 1340, 1265, 1165, 1103, 1028, 802, 750 cm1; 1H and 13 C NMR data, see Table 1; ESIMS m/z 689.3701 [M+H]+, calc. 689.3703 for [C42H48N4O5+H]+.
R. Verpoorte et al. / Phytochemistry Letters 3 (2010) 100–103
3.5. Biological assay Parasite viability was measured using parasite lactate dehydrogenase (pLDH) activity according to the method that is reported in Makler et al. (1993) and modified according to Jonville et al. (2008). Quinine (Aldrich) and chloroquine (Sigma) were used as positive controls in all experiments with an initial concentration of 100 ng/ml. FCA and W2 IC50 values were respectively 0.011 0.005 mM (n = 6) and 0.284 0.017 mM (n = 5) for chloroquine and 0.269 0.006 (n = 3) and 0.413 0.011 (n = 3) for quinine. Stock solns. of the alkaloid were prepared in DMSO at 20 mg/ml. They were then further diluted in medium to give 2 mg/ml solns. The highest concentration of DMSO to which the parasites were exposed was 1%, which was shown to have no measurable effect on parasite viability. All tests were performed in triplicate. Acknowledgements We thank Dr. Karen Ple´ for linguistic advice, Dr. G. Massiot for stimulating discussions and D. Harakat for MS analysis. G.D. and M.F. are respectively Research Associate and Senior Research Associate of the FRS-FNRS. G.D. thanks the FRS-FNRS for the financial support of the high performance computing systems installed in Lie`ge and Louvain-la-Neuve. M.F. and L.A. thank the FRS-FNRS for the financial support of antiplasmodial assays (FNRS grant no. 3452005). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytol.2010.02.005. References Becke, A.D., 1993. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652.
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Fre´de´rich, M., Quentin-Leclercq, J., Gadi Biala, R., Brandt, V., Penelle, J., Tits, M., Angenot, L., 1998. 30 ,40 ,50 ,60 Tetradehydrolongicaudatine Y, an anhydronium base from Strychnos usambarensis. Phytochemistry 48, 1263–1266. Fre´de´rich, M., Jacquier, M.-J., The´penier, P., De Mol, P., Tits, M., Philippe, G., Delaude, C., Angenot, L., Ze`ches-Hanrot, M., 2002. Antiplasmodial activity of alkaloids from various Strychnos species. J. Nat. Prod. 65, 1381–1386. Fre´de´rich, M., Tits, M., Goffin, E., Philippe, G., Grellier, P., De Mol, P., Hayette, M.-P., Angenot, L., 2004. In vitro and in vivo antimalarial properties of isostrychnopentamine, an indolomonoterpenic alkaloid from Strychnos usambarensis. Planta Med. 70, 520–525. Fre´de´rich, M., Tits, M., Angenot, L., 2008. Potential antimalarial activity of indole alkaloids. Trans. R. Soc. Trop. Med. Hyg. 102, 11–19. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Montgomery Jr., J.A., Vreven, T., Kudin, K.N., Burant, J.C., Millam, J.M., Iyengar, S.S., Tomasi, J., Barone, V., Mennucci, B., Cossi, M., Scalmani, G., Rega, N., Petersson, G.A., Nakatsuji, H., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Klene, M., Li, X., Knox, J.E., Hratchian, H.P., Cross, J.B., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R.E., Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C., Ochterski, J.W., Ayala, P.Y., Morokuma, K., Voth, G.A., Salvador, P., Dannenberg, J.J., Zakrzewski, V.G., Dapprich, S., Daniels, A.D., Strain, M.C., Farkas, O., Malick, D.K., Rabuck, A.D., Raghavachari, K., Foresman, J.B., Ortiz, J.V., Cui, Q., Baboul, A.G., Clifford, S., Cioslowski, J., Stefanov, B.B., Liu, G., Liashenko, A., Piskorz, P., Komaromi, I., Martin, R.L., Fox, D.J., Keith, T., Al-Laham, M.A., Peng, C.Y., Nanayakkara, A., Challacombe, M., Gill, P.M.W., Johnson, B., Chen, W., Wong, M.W., Gonzalez, C., Pople, J.A., 2004. Gaussian 03, Revision C.02. Gaussian, Inc., Wallingford, CT. Haharan, P.C., Pople, J.A., 1973. The influence of polarization functions on molecular orbital hydrogenation energies. Theor. Chim. Acta 28, 213–217. Irie, H., Ishizuka, K., Kawashima, S., Masaki, N., Osaki, K., Shingu, T., Uyeo, S., 1972. Xray crystallographic determination of the structure of the alkaloid serpentinine. J. Chem. Soc. Chem. Commun. 871. Jonville, M.C., Kodja, H., Humeau, L., Fournel, J., De Mol, P., Cao, M., Angenot, L., Fre´de´rich, M., 2008. Screening of medicinal plants from Reunion Island for antimalarial and cytotoxic activity. J. Ethnopharmacol. 120, 382–386. Leeuwenberg, A., 1969. The Loganiaceae of Africa VIII. Strychnos III. Revision of the African species with notes on the extra – African, Veenman, H., Zonen N.V. (Eds.), Wageningen, pp. 133–138, 267–272. Lounasmaa, M., Jokela, R., Hanhinen, P., Miettinen, J., Salo, J., 1994. Preparation and conformational study of Z- and E-isositsirikine epimers and model compounds. Determination of their C-16 configurations. Tetrahedron 50, 9207–9222. MacroModel, version 5.5, Department of Chemistry, Columbia University, New York, NY 10027. Makler, M.T., Ries, J.M., Williams, J.A., Bancroft, J.E., Piper, R.C., Gibbins, B.L., Hinrichs, D.J., 1993. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. Am. J. Trop. Med. Hyg. 48, 739–741. Massiot, G., Delaude, C., 1988. African Strychnos alkaloids. In: Brossi, A. (Ed.), The Alkaloids: Chemistry and Pharmacology, vol. 34, Academic Press, San Diego, pp. 211–329, Chapter 5.