Indole alkaloids from Muntafara sessilifolia with antiplasmodial and cytotoxic activities

Phytochemistry 73 (2012) 65–73

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Indole alkaloids from Muntafara sessilifolia with antiplasmodial and cytotoxic activities Marion Girardot a, Christiane Deregnaucourt a, Alexandre Deville a, Lionel Dubost a, Roger Joyeau a, Lucile Allorge b, Philippe Rasoanaivo c, Lengo Mambu a,⇑ a

UMR 7245 CNRS-MNHN Molécules de Communication et Adaptation des Micro-organismes, Département Régulations, Développement et Diversité Moléculaire, Muséum National d’Histoire Naturelle, 57 rue Cuvier (CP 54), 75231 Paris Cedex 05, France Département Systématique et Evolution, Muséum National d’Histoire Naturelle, 16 rue Buffon, 75231 Paris Cedex 05, France c Institut Malgache de Recherches Appliquées, BP 3833, 101-Antananarivo, Madagascar b

a r t i c l e

i n f o

Article history: Received 8 June 2011 Received in revised form 23 September 2011 Available online 25 October 2011 Keywords: Antiplasmodial activity Cytotoxic activity Monoterpene indole alkaloids Bisindole alkaloids Terpenoids Muntafara sessilifolia Apocynaceae

a b s t r a c t Four vobasinyl-iboga bisindole and one 2-acyl monomeric indole alkaloids were isolated from the stem bark of Muntafara sessilifolia along with eleven known compounds. Their structures and relative stereochemistry were elucidated on the basis of spectroscopic data including 1D and 2D NMR and mass spectrometry (MS). All isolated compounds were evaluated in vitro for antiplasmodial activity against the chloroquine-resistant strain FcB1 of Plasmodium falciparum, and for cytotoxicity against the human lung cell line MRC-5 and the rat skeletal muscle cell line L-6. 30 -Oxo-tabernaelegantine A exhibited antiplasmodial activity (4.4 lM IC50) associated with non-significant cytotoxicity (selectivity index of 48). Tabernaelegantine B and D displayed the highest cytotoxicity with IC50 values of 0.47 and 1.89 lM on MRC-5 cells, and 0.42 and 2.7 lM on L-6 cells, respectively. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The genus Tabernaemontana includes 170 species and is distributed in the tropical and subtropical regions mainly in lowland forest (Simões et al., 2010). These plants are widely used in traditional medicine for various pharmacological properties such as antimicrobial, antiparasitic, analgesic and febrifuge properties, but also for hormonal actions and activities on CNS (Van Beek et al., 1984). Pharmacological studies using extracts have confirmed these therapeutic potentialities, i.e. extracts were shown to exhibit antiprotozoal activities, in particular against Leishmania (Costa Soares et al., 2007; Delorenzi et al., 2001) and Plasmodium, (Federici et al., 2000), as well as antitumoral activities (De Almeida et al., 2004). They also displayed analgesic, anti-inflammatory (Taesotikul et al., 2003) or febrifuge (Van Beek et al., 1984) properties and cardiovascular activity (Taesotikul et al., 1998). They may be inductors of apoptosis (Mansoor et al., 2009), as well as inhibitors of acetylcholinesterase with potential use in the treatment of Alzheimer’s disease (Andrade et al., 2005; Chattikorn et al., 2007).

⇑ Corresponding author. Tel.: +33 1 40 79 56 07; fax: +33 1 40 79 31 35. E-mail address: [email protected] (L. Mambu). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.09.012

Previous studies have shown that the genus Tabernaemontana is a rich source of biologically active monoterpenoid indole alkaloids. More than three hundred indole and bisindole alkaloids have been isolated from Tabernaemontana species with various skeletal types including corynanthean or vobasinyl-type alkaloids, iboga typealkaloids, aspidospermatan-type alkaloids (Kisakürek et al., 1983). Most of bisindoles are of the vobasinyl-iboga, vobasinylaspidosperma, or iboga-plumeran types. An example of vobasinevobasine and vobasine-strychnan bisindoles has been also reported (Kam and Sim, 2002a). For the first time, a tetrakis monoterpene indole alkaloid, alasmontanine with an aspidosperma-type skeleton, was recently isolated from Tabernaemontana elegans (Hirasawa et al., 2009). Muntafara sessilifolia (Baker) Pichon, syn. Tabernaemontana sessilifolia (Baker) and Pagiantha sessilifolia (Baker) Markgr., is endemic to Madagascar, and belongs to the Apocynaceae family. The stem bark has been traditionally used as a stimulant, for circumcision and fever. In a previous study of M. sessilifolia, nineteen alkaloids were isolated of which nine monomeric indole alkaloids were described and ten bisindole alkaloids were mentioned (Panas et al., 1975). They were not identified, but their molecular weights were shown to be over 600 Da by mass spectrometry. M. sessilifolia has not been

66

M. Girardot et al. / Phytochemistry 73 (2012) 65–73

hydroxyindolenine (6) (Garnier et al., 1984), 3-oxocoronaridine or eglandulosine (7), 3(R/S)-hydroxycoronaridine or eglandine (8) (Panas et al., 1975; Le Men-Olivier et al., 1985), coronaridine (9) and tabernaemontanine (10) (Panas et al., 1975). Three bisindole alkaloids including tabernaelegantine B (11), tabernaelegantine D (12) and tabernaelegantine A (13) with vobasinyl-iboga skeletons were characterized (Bombardelli et al., 1976). The sterol was identified as sitosterol-b-D-glucoside (14), and the terpenoids were identified as lupeol acetate (15) and a-amyrin acetate (16). The known compounds were identified by direct comparison of their NMR and MS spectroscopic data with those reported in the literature. Compound 1 was isolated as a yellow amorphous powder, ½a20 D +14 (c 0.13, MeOH). HRESI-TOF exhibited a protonated molecular ion at 721.3950 [M+H]+ indicating a molecular formula of C43H52N4O6 (calcd. 721.3946) requiring twenty degrees of unsaturation. The UV spectrum showed absorption bands at 222 and 282 nm characteristic of an indole chromophore. The IR absorption bands at 3250, 1725 and 1665 cm1 suggested NH, ester and lactam carbonyl functionalities. The 13C spectrum revealed the presence of 39 signals consisting of three carbonyls, ten quaternary carbons, thirteen methines, seven methylenes, three methyls and three methoxy groups. The 1H spectrum performed at 298 K was very poorly resolved, in particular for the aromatic protons. A real improvement was observed at 273 K which was adopted for all 2D NMR experiments. It displayed the signals of two exchangeable protons at d 9.58 and 9.79; six aromatic protons of which four belong to an unsubstituted moiety characteristic of an indole unit at d 6.97 (2H), 7.11 and 7.68. The two singlets at d 6.87 and 6.90 indicated the presence of an aromatic ring substituted at C-10 and C-11. In addition, two intense singlets were observed at d 3.63 and 3.89 corresponding to methoxy groups, several aliphatic protons between d 1.20 and 5.09 and two methyl groups at d 0.87 (t, J = 7.2 Hz) and 0.93 (t, J = 7.2 Hz). The

investigated since this work and no pharmacological studies have been reported. In our continuous search for potential antimalarial compounds from medicinal plants, we evaluated the antiplasmodial activity of this routinely used species. A preliminary screening showed that the methanolic extract from M. sessilifolia stem bark exhibits substantial in vitro activity against a chloroquine-resistant strain of Plasmodium falciparum with an IC50 value of 1.24 lg/ml, whereas it displayed a moderate cytotoxicity against mammalian L-6 cells (IC50: 22.5 lg/ml). This paper deals with the isolation and structural elucidation of five indole alkaloids (1–5) of which four are derivatives of tabernaelegantine (Fig. 1). The antiplasmodial and cytotoxic activities of thirteen alkaloids and three terpenoids isolated from EtOAc soluble extract after bioassay guided fractionation on P. falciparum are evaluated.

2. Results and discussion The stem bark of M. sessilifolia was extracted with MeOH and the resulting extract was partitioned between EtOAc and 2% aqueous acetic acid. Water soluble material was adjusted to pH 9 with saturated NaHCO3 and extracted with CH2Cl2 to provide an alkaloidal crude extract. The examination of the EtOAc-soluble material also showed the presence of alkaloids which may account for weakly or non basic compounds like amides. Furthermore, it displayed an antiplasmodial activity with an IC50 value of 6.5 lg/ml and weak cytotoxicity against a rat cell line L-6 cells with IC50 value of 35.2 lg/ml. Bioguided fractionation of the EtOAc soluble part led to the isolation of five alkaloids (1–5), including four derivatives of tabernaelegantine (1–4) and a 2-acylindole (5) along with eight known alkaloids, two terpenoids and a sterol. Among them, five were monomeric indole alkaloids, namely 3-oxocoronaridine

MeOOC

9 7 1

N H

12

5

H 2

9

13

4

21

12

20

14

1

2

N H

16

5

7

Me

N

3

MeOOC

H 16

H 3

H

Me

N

4

21 20

14 15

15 9'

7'

R2 R1

MeO

12'

N H

N

3' 16'

7'

H

21'

MeOOC

H

R1 MeO

20'

14'

17'

15'

9 7

13

16'

N 17'

MeOOC

3'

N H 21'

20'

14'

H

15'

2 : R1, R2 = O, β ethyl 3 : R1 = H, R2 = OH , β ethyl and R1 = OH, R2 = H , β ethyl 4 : R1 = OH, R2 = H , α ethyl 13 : R1 = H, R2 = H, β ethyl

10

12

12'

H

1 : R1, R2 = O, β ethyl 12 : R1 = H, R2 = H, α ethyl 11 : R1 = H, R2 = H, β ethyl

11

R2

1

OMe COOMe 16 H N 5

2

N H O

3

4

14 15

21

H

20

5 Fig. 1. Structures of alkaloids 1–5 and 11–13 from the stem bark of Muntafara sessilifolia.

M. Girardot et al. / Phytochemistry 73 (2012) 65–73

planar structure of 1 was further established by 2D NMR studies of the COSY, HSQC and HMBC spectra. Detailed analysis of the 1H–1H COSY spectrum revealed connectivities of seven partial structures : a (C-9/C-12), b (C-3/C-14), c (C-5/C-6, C-15/C-16), d (C-20/C-21), e (C-18/C-19), f (C-50 /C-60 ), g (C-170 /C-140 /C-150 /C-200 /C-180 /C-190 ). They were arranged into two monomeric halves. The connectivities of H-9 to C-13 and H-12 to C-8 along with those of the proton at d 9.55 attributed to N1-H, to C-2, C-6, C-7 and C-13 allowed building of the indole (partial structure a), and its attachment to the partial structure c was confirmed by the cross-peak of H-6 to C-2, C-7 and C-8. Furthermore, the correlation of H-14 with C-2 linked the partial structure b to the indole nucleus, while those of H-14 to C-15 established the connection between partial structure b and c. The connection of H-21 with C-5 and C-15 along with those of H-19 to C-20 allowed the linkage of C-21 to C5 through a nitrogen atom. The partial structure c was linked to d by the interaction of H-15 to C-20, whereas partial structure e was joined to the set by the correlation of H-19 to C-20. The cross-peak of the methyl at d 3.10 with C-5 suggested that it was attached to a nitrogen atom. The unusual shielded methoxy group at d 2.38 was attributed to methyl ester group on C-16 as indicated by the correlation of H-16 with the carbonyl group at d 169.8 and placed the ester function in the shielding zone of the aromatic ring. All of these data established this first monomeric half as a vobasinyl skeleton. The presence of the upfield aromatic carbon at d 93.2 suggested the proximity of an oxygenated carbon and was attributed to C-120 as confirmed by the connectivities of H-90 and the methoxy group at d 3.89 with the downfield aromatic carbon at d 153.8 corresponding to C-110 . The connections of H-120 to C-80 and C-130 , and with those of N10 –H to C-20 and C-70 permitted construction of the indole nucleus. The partial structure f was attached to this indole ring as demonstrated by the connection of protons H2-50 to C-70 . Further correlations of H-210 and H2-50 to the carbonyl at d 175.7 through the nitrogen indicated the presence of an amide function (lactam) at C-30 . Finally, correlations of H-210 with C-20 , C-30 , C-160 , C-170 , C-200 and the carbonyl at d 173.1 allowed assembly of the partial structure g to the rest of the second unit which showed characteristic features of an iboga skeleton. This unit was identified as 3-oxo-isovoacangine. Both indole units were linked between C-3 and C-100 as supported by the interaction of H-90 to C-3. The temperature dependence of the 1H spectrum could be explained by the presence of rotamers. Molecular modeling showed that rotation around C-3 and C-100 bond is not sterically hindered and leads to two conformers in equilibrium. At 303 or 298 K, coalescence of broad and non-resolved signals for aromatic and several aliphatic protons was observed. Lowering the temperature favored one conformer. The relative configuration of 1 was elucidated by NOESY experiment along with the analysis of vicinal 1H–1H coupling constants as shown in Fig. 2. A b-axial position was attributed to H-3 due to the trans-diaxial coupling constant (J = 9.1 Hz) with H-14ax. Hence the linkage of the iboga unit with the vobasinyl one at C-3 was deduced to have an a orientation. The NOE interaction between H-3 and H-90 along with those of N1-H to H-3/H-12/H-90 confirmed the proximity of these protons, while N10 –H correlated with H-120 . The proton H-6a correlated with H-21b, and their coupling constants indicated that they are both b-equatorial. Thus, the cross-peaks between H-5/H-16 and H-5/H6b supported their a-axial orientation. Moreover, H-5 correlated with N–CH3. Further interactions of H-15 equatorial with H-20 and H-14a, both axial, supported their a-orientation and corroborated the b-orientation of the ethyl chain at C20. The coupling constants of 6.2 and 12.2 Hz were consistent with a b-axial position for H-50 a. NOEs between H-50 b, H-60 a, H-200 and H-210 indicated that all these protons adopted an a-orientation and also confirmed the b-orientation of the ethyl chain at C-200 . The

67

Fig. 2. MM2 model and selected NOESY correlations in compound 1.

equatorial proton H170 b correlated with H-140 . Thus, the structure of 1 was determined to be a 30 -oxo derivative of tabernaelegantine B and was named 30 -oxo-tabernaelegantine B. The HRESI-TOF of alkaloid 2 gave the same molecular formula C43H52N4O6 as alkaloid 1, based on a pseudomolecular [M+H]+ ion peak at m/z 721.3952 (calcd for C43H53N4O6, 721.3946). This alkaloid also possesses an indole chromophore (219, 283 nm) as suggested by the UV spectrum. The IR absorption bands at 1732 and 1670 cm1 indicated the presence of an ester and a lactam carbonyl functions, respectively. The 1H and 13C NMR data for 2 (Tables 1 and 2) showed the characteristic pattern of a bisindole alkaloid. The 13C spectrum (CDCl3) showed the presence of 43 signals as indicated by the molecular formula. This spectrum was similar to that of alkaloid 1, however, the upfield shifted methine at d 93.2 was replaced by a signal at d 105.3. Comparison of the NMR data of 2 with those of 1 suggested that both alkaloids share the same vobasinyl and iboga moieties, except differences which suggested another linkage for the two monomeric units. The 1H spectrum was well resolved at 298 K, contrary to that of 1. Modifications were noticed for the aromatic protons which were typical of an unsubstituted indole ring and a disubstituted one. The connectivities observed in the HMBC spectrum allowed them to be distinguished. Two doublets observed at d 7.25 (H-90 , J = 8.7 Hz) and 6.84 (H-100 , J = 8.7 Hz) suggested an ortho coupling of these aromatic protons in the iboga moiety. The correlation of H100 with the quaternary carbon at d 112.8 (C-120 ) excluded the presence of a proton at C-120 and proved the iboga unit is engaged through its C-120 in a linkage with C-3 of the vobasinyl moiety. Molecular modeling of compound 2 showed that the steric hindrance around the junction C-3/C-120 would prevent rotation and by consequence the existence of conformers. Two singlets at d 7.57 and 7.82 were assigned to N1–H and N10 – H due to the absence of cross-peak on HSQC spectrum and longrange interactions observed in HMBC spectrum. The presence of

68

M. Girardot et al. / Phytochemistry 73 (2012) 65–73

Table 1 1 H NMR data for compounds 1–5. Position

1a

2b

3b 0

3 5 6a 6b 9 10 11 12 14a 14b 15 16 18 19a 19b 20 21a 21b 5-OMe 16-COOMe NH NMe 3’ 5’a 5’b 6’a 6’b 9’ 10’ 12’ 14’ 15’a 15’b 17’a 17’b 18’ 19’a 19’b 20’ 21’ 11’-OMe NH’ COOMe’ a b c

dd (3.3, 12.7) br t (8.5) dd (8.5, 14.8) dd (6.9, 14.8) d (6.7) m m m q (13.4) m m m t (7.3) m

4b

5c

– 4.71, 4.00, 3.64, 7.88, 7.16, 7.34, 7.43, 3.17, 2.66, 2.95, 3.80, 1.09, 1.41,

m dd (8.3, 14.4) m d (8.0) dd (8.0, 1.0) dd (8.3, 1.0) d (8.3) td (1.6, 14.5) m m s t (7.5) m

2.47, 4.14, 3.67, 3.80, 2.69,

m t (10.4) m s s

0

3S

3R

5.23, dd (3.2, 12.0) 4.10, m 3.30, m

5.23, dd (3.2,12.0) 4.10, m 3.30, m

5.22, dd (2.7, 12.8) 4.27, m 3.42, m 3.27, m

7.65, 7.10, 7.03, 6.98, 2.66,

m m m m m 1.90, m

7.64, 7.09, 7.04, 7.03, 2.66,

m m m m m 1.90, m

7.58, 7.11, 7.06, 7.02, 1.82,

d, (7.6) dd (6.8, 7.6) dd (6.8, 7.3) d (7.3) m

2.66, 2.96, 0.92, 1.72,

m m t, (7.3) m 1.50, m

2.66, 2.96, 0.92, 1.72,

m m t (7.3) m 1.50, m

1.36, 2.95, 0.91, 2.33,

m m t (7.3) m 1.16, m

5.09, br d (9.1) 4.54, m 3.82, dd (3.2, 13.9) 3.65, m 7.68, dd (1.8, 6.7) 6.97, m 6.97, m 7.11, dd (1.8, 6.7) 2.66, m 2.08, m 2.66, m 3.11, d (12.6) 0.93, t (7.2) 1.77, dq (7.2, 14.7) 1.57, m 1.68, m 3.82, m3.24, dd (3.3, 16.6)

5.22, 4.62, 3.55, 3.42, 7.52, 7.10, 7.10, 7.10, 2.51, 2.02, 2.09, 3.01, 0.98, 1.85,

1.51, m 3.01, m

1.29, m 2.86, m 2.38, m

1.29, m 2.86, m 2.38, m

3.08, m 2.36, m 2.33, m

2.38, s 9.58, s 3.10, s – 4.20, td (6.2, 12.2)2.99, m 2.91, m 2.66, m 6.87, s – 6.90, s 2.40, m 1.96, dt (2.9, 13.1) 1.20, m 2.66, m 2.14, ddd (2.5, 4.2, 13.5) 0.87, t (7.2) 1.39, dq (7.2, 13.6) 1.31, m 1.62, m 4.41, s 3.89, s 9.79, s 3.63, s

2.48, s 7.82, s 2.97, s – 4.31, dd (8.5, 12.4)3.01, m 2.92, m

2.49, 7.61, 2.61, 3.97, 3.27,

2.48, 7.61, 2.61, 3.69, 3.32,

2.49, 7.52, 2.78, 3.96, 3.27,

2.89, m

2.92, m

2.86, m

7.25, 6.80, – 1.15, 2.88,

7.24, 6.80, – 1.36, 1.31,

7.25, 6.81, – 1.49, 1.26,

7.26, 6.81, – 1.36, 1.14,

d (8.7) d (8.7) m m

s s s br d (8.9) m 3.06, dd (7.8, 13.5)

d (8.7) d (8.7) m m 1.11, m

s s s br d (8.4) m 2.93, m

d (8.7) d (8.7) m m 1.22, m

1.64, m 0.76, m

1.87, m 0.58, d (12.7)

0.86, 1.33, 1.26, 1.64, 4.28, 3.94, 7.57, 3.64,

0.81, t (7.4) 1.45, m 1.31, m

1.77,dd(1.87, 14.2)0.55, d, (14.2) 0.78, t (7.4) 1.45, m 1.31, m

1.31, 3.58, 3.95, 7.61, 3.67,

1.31, 3.61, 3.95, 7.61, 3.66,

t (7.3) m m m br s s s s

m m s s s

m m s s s

s s s br d (8.9) m 3.05, m

d (8.7) d (8.7) m m

1.77, d (14.1)0.64, d (14.1) 0.81, 1.42, 1.26, 1.10, 3.58, 3.95, 7.52, 3.61,

t (7.4) m m m br s s s s

Spectra were recorded in Acetone-d6. Spectra were recorded in CDCl3. Spectra were recorded in CD3OD.

an upfield doublet of doublets at d 0.76, which is coupled with the proton at d 1.64 and correlated with carbon at d 33.8, corresponding to an inequivalent methylene H2-170 was noticed. These protons are significantly shielded compared to those of alkaloid 1 due to the anisotropic effect of the indole ring on H-170 . Indeed, the occurrence of this upfield signal is characteristic of vobasinyl-iboga bisindoles linked at C-3 and C-120 isolated from several species of Tabernaemontana (Kam et al., 2003; Takayama et al., 1994). In comparison to 1, the signals of carbons C-14, C-100 , C-140 and C0 17 were shifted upfield (4.1, 23.4, 8.2 and 2.6 ppm), whereas C-15 and C-120 were shifted downfield (+3.7 and +9.6 ppm). Analysis of the NOESY spectrum showed that alkaloid 2 shares the same relative configuration as 1. From the above evidence, the structure of 2 was established to be 30 -oxo-tabernaelegantine A. Alkaloid 3 was isolated as an amorphous powder, ½a20 D 36.8 (c 0.08, MeOH). Its UV spectrum displayed two maxima characteristic of an indole chromophore (224 and 285 nm) and the IR spectrum revealed the presence of OH and NH groups at 3375 cm1 and a carbonyl group at 1732 cm1. The 1H and 13C spectra were characterized by peak splitting with a 1:2 ratio of intensity, and this alkaloid

was isolated as an inseparable mixture of two isomers. The 1H spectrum was similar to that of alkaloid 2 with the pattern of a vobasinyl-iboga bisindole in which two units are linked at C-3/C-120 . Analysis of the 13C spectrum showed the appearance of an intense peak at d 85.9, a small one at d 93.7, and the disappearance of one carbonyl group at d 175.7 compared to 2, suggesting reduction of the carbonyl to an hydroxyl group at C-30 . The HSQC spectrum revealed that the oxymethine proton at d 3.69 correlated with the carbon at d 93.7, while the one at d3.97 was connected with the carbon at d 85.9. They are characteristic of a hydroxyl group at an a position to a nitrogen atom. A weak signal at d 2.61 integrating for three protons which correlated with carbon at d 43.0 and corresponding to the N-CH3 group was observed. The reduction of the lactam during biosynthesis would lead to the carbinolamine and in comparison to 2, C-50 and C-200 were downfield, whereas C-150 was upfield for the two epimers of 3. However, C-140 was shifted downfield only for the major epimer. Indeed, the planar structure was established as 30 -hydroxytabernaelegantine A. A similar alkaloid with a vobasinyl-iboga linkage on 3–100 (30 (R/S)-hydroxytabernaelegantine B), along with

M. Girardot et al. / Phytochemistry 73 (2012) 65–73 Table 2 13 C NMR data for compounds 1–5. Position

1a

2b

3b 0

2 3 5 6 7 8 9 10 11 12 13 14 15 16 18 19 20 21 5-OMe CO 16 OMe NMe 2’ 3’ 5’ 6’ 7’ 8’ 9’ 10’ 11’ 12’ 13’ 14’ 15’ 16’ 17’ 18’ 19’ 20’ 21’ 11-OMe’ CO OMe a b c

140.0 38.1 60.3 19.5 107.3 130.1 118.3 119.1 122.1 110.9 137.2 39.7 34.2 42.1 12.6 25.2 42.9 47.5 – 169.8 50.2 40.9 134.2 175.7 42.6 21.7 109.8 122.5 118.2 128.7 153.8 93.2 136.3 39.0 32.0 56.2 36.4 11.6 28.3 36.1 56.7 56.0 173.1 53.6

137.0 35.0 59.2 19.7 105.3 128.4 117.0 119.5 122.3 110.4 136.0 35.6 37.9 42.5 12.9 25.7 42.9 46.8 – 169.4 50.3 41.5 133.0 175.7 42.5 20.8 108.0 123.5 117.7 105.3 152.1 112.8 135.1 30.8 32.0 54.0 33.8 11.3 27.6 35.3 55.8 56.8 172.5 52.7

4b

5c

136.2 35.5 59.3 19.7 110.2 128.7 117.6 119.7 122.5 110.0 136.0 27.5 34.8 49.1 10.8 22.4 40.2 49.3 – 170.0 50.2 41.0 135.1 86.0 51.0 21.5 109.0 123.8 117.1 105.1 152.0 114.5 135.1 34.3 24.6 53.9 33.7 11.5 26.7 37.5 55.3 56.7 175.4 52.3

135.2 192.1 73.5 25.1 116.2 129.2 121.7 121.8 127.9 113.5 138.2 39.4 30.2 43.6 11.3 22.9 39.2 61.9 55.8 170.2 51.5

0

3S

3R

136.8 35.3 59.2 17.7 109.7 129.4 117.8 119.4 122.1 109.8 135.9 36.7 34.8 43.7 12.8 25.6 42.8 46.8 – 172.4 49.9 43.0 135.9 85.9 51.0 21.5 108.6 123.8 116.9 105.0 151.9 114.6 135.2 34.2 24.5 53.6 33.6 11.5 26.7 37.5 55.8 56.8 174.2 52.3

136.8 35.3 59.2 17.7 109.7 129.4 117.8 119.4 122.1 109.8 135.9 36.7 34.8 43.7 12.8 25.6 42.8 46.8 – 172.4 49.9 43.0 135.9 93.7 51.9 21.6 108.6 123.8 116.9 105.0 151.8 114.6 135.2 30.6 24.8 53.9 33.6 11.5 26.5 37.6 55.2 56.8 174.0 52.3

Spectra were recorded in Acetone-d6. Spectra were recorded in CDCl3. Spectra were recorded in CD3OD.

30 (R/S)-methoxytabernaelegantine C was isolated from T. elegans, but detailed NMR data were not published (Van der Heijden et al., 1986). The configuration at C-30 of the major epimer was determined to be 30 S, as the chemical shift at d 85.9 is compatible with that of 30 S-hydroxyconodurine, while the minor 30 R epimer is in agreement with 30 R-hydroxyconodurine (Van Beek et al., 1985). Proton H-5 was on the same side as the N–CH3 group, as indicated by their NOE correlation. The interactions between H-20/H-15, H-200 /H-210 and H-200 /160 COOMe suggested their a -orientation as observed from the NOESY spectrum. The mass spectrum performed by EI-MS showed an intense ion at m/z 706 [M16]+, while an ion at m/z 705 [M+H18]+ was observed in the ESI-TOF spectrum along with a diprotonated ion at m/z 353.2020 [M+2HH2O]2+. The expected pseudomolecular ion at m/z 722 corresponding to the molecular formula of C43H54N4O6 was not detected. This mass spectral phenomenon has been well described from 3-hydroxy substituted iboga alkaloids (Van Beek et al., 1985). The formation of the ion at m/z 705 [M+HH2O]+ suggested the loss of H2O.

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The occurrence of mixtures of 3R and 3S epimers has been reported in the literature for 3-hydroxyiboga alkaloids isolated from several species of Tabernaemontana, notably with Tabernaemontana calcarea (Chaturvedula et al., 2003; Kam et al., 2003). Two epimers (30 S and 30 R) were obtained as for 3-hydroxy bisindoles isolated from Tabernaemontana chippii which are in equilibrium and are interconvertible (Van Beek et al., 1985). Since this alkaloid was purified from SiO2 layers, the interconversion was probably catalyzed by this acidic medium. Carbinolamines are particularly unstable in the presence of acids and can undergo dehydration, leading to an iminium ion intermediate which can react with alcohols to give the corresponding carbinolamine ethers (Le MenOlivier et al., 1985). For example, 3(R/S) ethoxy-iboga alkaloids were reported from Tabernaemontana glandulosa (Achenbach and Raffelsberger, 1980), Tabernaemontana corymbosa (Kam and Sim, 2002b) and Tabernaemontana divaricata (Kam et al., 2004). As a consequence, they are often considered as artifacts of extraction. On the other hand, 3R-methoxycoronaridine was claimed to be a genuine product of Tabernaemontana markgrafiana, since it was isolated from petrol extract, without contact with methanol (Nielsen et al., 1994). Finally, the structure of alkaloid 3 was determined to be 30 (R/S)-hydroxytabernaelegantine A. Alkaloid 4 was isolated as an amorphous powder. Three absorption bands at 221, 285 and 294 nm were observed in the UV spectrum. This alkaloid was closely related to alkaloid 3 with the same planar structure and molecular formula of C43H54N4O6. They share similar behavior on ESI-TOF and a diprotonated ion was observed at m/z 353.2068. Examination of the 1H and 13C spectra revealed a single epimer as no peak splitting was observed. Moreover, the oxymethine proton at d 3.96 correlated with the carbon at d 86.0 in the HSQC experiment. This chemical shift is compatible with a 30 S epimer. The coupling constant of H-30 with H-140 (J = 8.9 Hz) indicated that these protons have a trans-diaxial relationship. Differences were noticed for the proton H-20 which was shifted downfield (+1.79), while H-15 was shifted upfield (1.30) with regard to compound 3. From the vobasinyl unit, the upfield chemical shift of C-14, C18, C-19 and C-20, along with the downfield shift of C-16 in comparison with 1–3 suggested an inversion of configuration at C-20 (Ahond et al., 1976). The proton H-20 adopted a b-orientation and the ethyl chain was a -oriented. NOEs between H-3 and N1–H, H60 and H-90 , H-100 and 110 -OMe established their proximity. Obtaining alkaloid 4 as a single epimer could be explained by the mildest conditions used for the chromatography procedure. It was purified by normal phase on HPLC, thus shortening the time of contact of the product with the stationary phase (silica gel), and acid-free CDCl3 was used for the NMR experiments thus preventing interconversion. Alkaloid 4 was identified as 30 (S)-hydroxytabernaelegantine C. Alkaloid 5 was isolated as an amorphous powder and was optically active ½a20 D 31.3 (c 0.21, MeOH). A molecular formula of C21H26N2O4 was assigned for 5 on the basis of its HRESI-TOF which exhibited a pseudomolecular ion at m/z 371.1955 (calcd for C21H27N2O4 371.1960) corresponding to ten degrees of unsaturation. Its UV spectrum displayed three maxima bands at 218, 237 and 312 nm characteristic of a 2-acyl indole chromophore (Brunell and Medina, 1971; Kogure et al., 2005). The IR spectrum revealed the presence of a broad band at 3298 cm1 and two strong bands in the region of carbonyl functions. The band at 1651 cm1 was assignable to a ketone at C-3 conjugated to the indole moiety, and the other at 1732 cm1 was typical of an ester. The 13C spectrum (CD3OD) displayed 21 signals consisting of two carbonyls at d 192.2 and 170.2, four sp2 quaternary carbons, four sp2 methines, four sp3 methines, including an oxymethine, four sp2 methylenes, two methoxy and one methyl groups. The 1H NMR spectrum showed the presence of four aromatic protons characteristic of an unsubstituted indole moiety at

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Table 3 In vitro antiplasmodial and cytotoxic activities of compounds 1–16. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Chloroquine Camptothecin

10.4 ± 0.9 4.4 ± 0.23 6.2 ± 0.3 6.1 ± 0.4 137.8 ± 3.7 118.2 ± 2.5 10.8 ± 0.6 5.9 ± 0.8 6.21 ± 0.3 12.0 ± 0.8 2.70 ± 0.14 1.20 ± 0.1 13.3 ± 0.7 115 365.4 ± 3.0 92.9 ± 13.4 0.097 ± 0.002 –

37.5 ± 5.0 >138 7.2 ± 0.8 24.9 ± 1.4 >250 226.4 ± 11.5 99.9 ± 14.3 118.6 ± 8.4 63.1 ± 3.4 100.7 ± 9.1 2.7 ± 0.1 0.42 ± 0.04 22.2 ± 2.2 ND 244 213.7 20.1 ± 0.1 –

3.6 >31 1.16 4.1 >1.8 1.9 9.2 20.1 10.2 8.4 1.0 0.35 1.7 – – – 270.2 –

25.2 ± 3.4 215 ± 10.8 7.0 ± 0.3 51.6 ± 4.3 >250 >250 58.9 ± 5.5 34.1 ± 2.3 22.9 ± 2.0 61.9 ± 5.3 1.89 ± 0.16 0.47 ± 0.02 12.9 ± 0.7 ND 158.0 ± 42.9 189.2 ± 27.0 ND 0.0035 ± 0.0009

2.4 48.8 1.1 8.5 1.8 >2.1 5.5 5.8 3.7 5.2 0.7 0.39 0.96 – – – –

Results are expressed as IC50 values (lM) ± standard deviations. All experiments were realized in triplicate. Chloroquine was used as positive control for antiplamodial activity and camptothecin for cytotoxicity. ND: not determined.

d 7.88 (d, J = 8.0 Hz, H-9), 7.43 (d, J = 8.0 Hz, H-12), 7.34 (dd, J = 8.0, 1.0 Hz, H-11) and 7.16 (dd, J = 8.0, 1.0 Hz, H-10). Two strong singlets corresponding each to three protons were observed at d 3.80 (OCH3) and 2.69 (COOCH3). The protons of an ethyl chain were observed as a triplet at d 1.09 (J = 7.5 Hz, CH3-18) coupled to a multiplet at d 1.41 (H2-19). The 1H NMR spectrum of 5 showed some similarity with that of the 2-acyl indole alkaloid, tabernaemontanine, also isolated from this extract, except for the absence of a signal characteristic of a N–CH3 group. Moreover, comparison of their 13 C spectra demonstrated some differences. The presence of an oxymethine carbon at d 73.5 which correlated with a proton at d 4.71 in the HSQC spectrum was noted. Another carbon which correlated with the nonequivalent methylene protons at d 3.67 and 4.14 was observed at d 61.9. The 1H-1H COSY spectrum allowed for the identification of five spin systems: C-9/C-10/C-11/C-12; C-5/C-6/N-4/C-16; C-14/C-15; C-20/C-21 and C-18/C-19. However, the absence of a cross-peak between H-5 and H-16 excluded the direct linkage of C-5 and C-16. The indole nucleus was built considering correlations of H-9 with C-7, C-8 and C-13, and confirmed by connectivities of H-6 with C-2, C-7 and C-8. On the other hand, the strong correlation of the methyl group at d 3.80 with C-5 and C-21 suggested that it was located in position 5. Finally, connectivities of H-14 with C-2, C-3, C-15 and C-20, and those of H-20 with C-19 and C-21, completed the structural assignment. HMBC data showed the presence of a methoxy group at C-5 and a bridge at N-4/C-16/C-15. This is unlike of other monoterpene indole alkaloids with a vobasinyl unit or a corynanthean skeleton, such as tabernaemontanine in which the bridge is observed at C-5/C-16/C-15. Nevertheless, the methyl ester remained under the influence of the shielding zone of the aromatic ring and resonated at d 2.69. The relative configuration of 5 was determined by an analysis of the 1H–1H NOESY correlations. As the protons H-16 and 5-OCH3 were overlapped at d 3.8, the NOE correlation observed with H-5 permitted the deduction that this latter proton was on at the same side of the molecule as H-16. Likewise, the correlations H-5/H-6a, H-6a/H-16, H-15/H-14b, H-15/H-16, H-15/H-20 and H-16/H-20 indicated their a -orientation. These observations suggested a b-orientation for the methoxy group at C-5 and the ethyl chain at C-20. The structure of 5 is therefore a reduced derivative of eleganine isolated from T. elegans (Mansoor et al., 2009). They differ by the existence of a b-ethyl chain at C-20 for 5 resulting from the reduction of the double bond at C-19/C-20 of the ethylidene chain of

eleganine. They share the same relative stereochemistry as shown by their NOE correlations. Thus, the structure of 5 was established as 19,20a-dihydroeleganine A. All of the isolated compounds were evaluated for their ability to inhibit the in vitro intraerythrocytic development of the chloroquine-resistant strain FcB1 of P. falciparum. Additionally, the cytotoxicity of the isolated compounds was tested against the human MRC-5 and rat L-6 cell lines. Antiplasmodial and cytotoxic activities with the selectivity index (SI) are reported in Table 3. SI is defined as the ratio of the IC50 value on mammalian cells to the IC50 value on P. falciparum (Zirihi et al., 2005). Antiplasmodial activity of monoindole alkaloids ranged from 5.9 to 137.8 lM (IC50 values) and the bisindoles seemed more active with IC50 values ranging from 1.2 to 13.3 lM. Hydroxylated (8) and oxidized (7) derivatives at C-3 of the iboga-type indole coronaridine (9) exhibited comparable activities (5.9 and 10.8 lM, respectively). Thus, the nucleus indole seems to play an important role for biological activities as shown by the drastic diminution of the activities for alkaloid 6 which has an indolenine nucleus. All these monomeric indoles displayed moderate cytotoxic activity. Alkaloids 7, 8 and 9 displayed higher selective index on L6 cells than on MRC-5 cells. Concerning the acyl indoles, tabernaemontanine (10) displayed mild activity against P. falciparum, but hindrance of the nitrogen N4, which is found in the ring junction of alkaloid 5 causes a loss of activity. They are both very weakly toxic against mammalian cells. Among the bisindole alkaloids, 11 and 12 were the most active against P. falciparum, with IC50 values of 2.7 and 1.2 lM, respectively, whereas alkaloid 13 exhibited mild activity (IC50: 13.3 lM). Their oxidized derivatives 1 and 2 displayed moderate but significant activity with IC50 values of 10.4 and 4.4 lM, respectively. Alkaloids 3 and 4 showed similar activities, with IC50 values around 6 lM. Although alkaloids 1, 3, 4, 11, 12 and 13 showed moderate to mild efficiencies at inhibiting the growth of P. falciparum, their toxicity towards mammalian cells differed significantly, as exemplified by selectivity indexes ranging from 0.35 to 4.1 regarding L6 cells and from 0.39 to 8.5 regarding MRC-5 cells. Among all of the alkaloids, tabernaelegantine B (11) and D (12) were the most toxic against the mammalian cell lines. Their toxicity and that of tabernaelegantine A (13) to monkey Vero cells had been previously reported (Tizzoni et al., 1993). Besides, the influence of oxidation at C-30 on cytotoxicity was remarkable for 11 compared to 1, for which it was decreased

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significantly. Therefore, alkaloid 2 with SI of 48.8 (MRC-5 cells/P. falciparum) and >31 (L6 cells/P. falciparum) appeared to be the most selective antiplasmodial alkaloid isolated here, while the corresponding non-oxidized alkaloid 13 (tabernaelegantine A) was devoid of selectivity. It must be noticed however that these bisindoles exhibited low antiplasmodial activity in comparison with voacamine, a related vobasinyl-iboga bisindole containing a voacangine moiety linked between C-3 and C-110 with a vobasinyl unit (Federici et al., 2000). This alkaloid was found to display in vitro activity in the nanomolar range and was also potent in vivo (Ramanitrahasimbola et al., 2001). The terpenoids and sterol demonstrated no significant antiplasmodial activity with an IC50 value >100 lM. Lupeol acetate (16) was the less active with an IC50 of 365 lM, unlike lupane triterpenoids, betulinic acid and lupeol, which have been reported to exhibit moderate antiplasmodial activity in vitro (Bringmann et al., 1997; Ziegler et al., 2002). On the other hand, the influence of a ketone at C-3 and an aldehyde at C-30 on the activity for lupane derivatives, such as oxolupenal, hydroxylupenal and oxolupenol has been demonstrated (Mambu et al., 2006). 3. Conclusion In this study, thirteen indole alkaloids and three terpenoids were isolated by bioassay-guided fractionation from the EtOAc extract of the stem bark of M. sessilifolia. The structures were elucidated by NMR at low or at room temperature. Two bisindoles alkaloids, tabernaelegantines B and D, were toxic to human and rat cells, which confirms their cytotoxic properties toward mammalian cells. Additionally, they exhibit anti-protozoan properties, as exemplified through their anti-Plasmodium activity, which suggests that those alkaloids might target widely-shared mechanisms among living organisms. Interestingly, the 30 -oxo-derivative of tabernaelegantine A (alkaloid 2) was found to be more potent against Plasmodium and displayed only weak cytotoxicity against mammalian cells (SI  50), which makes of it a potential anti-malarial drug candidate. Further investigations are needed to elucidate the mechanism that underlies the anti-Plasmodium activity of this alkaloid. Further investigation of the crude alkaloid extract is ongoing. 4. Experimental section 4.1. General experimental procedures Optical rotations were measured on a Perkin Elmer model 341 polarimeter at 20 °C. IR spectra were taken on a Shimadzu FTIR8400S spectrophotometer. Mass spectra data were recorded using an electrospray time of flight mass spectrometer (ESI-TOF-MS) operating in the positive mode (QSTAR Pulsar I of Applied Biosystems). 13C NMR spectra were recorded on an AC 300 BRUKER spectrometer operating at 75.47 MHz (for 13C). 1H and 2D-NMR spectra were recorded on an Avance-400 BRUKER spectrometer operating at 400.13 MHz, equipped with 1H-broad-band reverse gradient probe head. Temperature was controlled by a Bruker BCU-05 refrigeration unit and a BVT 3000 control unit. The 1H and 13C NMR chemical shifts are given in ppm relative to TMS, with coupling constants (J) reported in Hz. For the HMBC experiments, the delay (1/2 J) was 70 ms and for the NOESY experiments the mixing time was 150 ms. Analytical and preparative TLC were carried out on precoated Si gel 60 F254 plates (Merck). Spots were detected under UV (254 and 366 nm) before spraying with Dragendorff solution. Column chromatography was performed on 200–400 mesh silica gel 60 (Merck). Preparative medium-pressure

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liquid chromatography (MPLC) was performed with a pump K-120 (Knauer) and Flashsmart cartridges (Si and C-18 gels 20–40 lm, AIT, France). HPLC was carried out using Gilson 321 pump equipped with a 170 DAD and Kromasil C-18 columns (for analytical HPLC, 250  4.6 mm, 5 lm particle size, and for preparative HPLC, 250  7.5 mm, 5 lm particle size).

4.2. Biological activities 4.2.1. In vitro antiplasmodial test The P. falciparum strain FcB1 (Colombia) was maintained continuously in culture on human erythrocytes as described by Trager and Jensen (1976). In vitro antiplasmodial activity of plant extracts and purified molecules was determined using a modification of the semi-automated microdilution technique of Desjardins et al. (1979). Stock solutions of chloroquine diphosphate and test compounds were prepared in sterile distilled water and DMSO, respectively. Drug solutions were serially diluted with culture medium and added to asynchronous parasite cultures (1% parasitemia and 2% final hematocrite) in 96-well plates for 24 h, at 37 °C, prior to the addition of 0.5 lCi of [3H]hypoxanthine (1–5 Ci/mmol) per well, for 24 h. Parasite growth inhibition by each drug concentration was determined by comparison of the radioactivity incorporated into the treated culture with that in the control culture (without drug) maintained on the same plate. The concentration of the drug that inhibits growth to 50% (IC50) was determined graphically (from the drug concentration–response curve). Final IC50 value for each compound was expressed as the mean ± standard deviation of values determined from independent experiments. Chloroquine diphosphate (Sigma–Aldrich Chimie SARL, St. Quentin Fallavier, France) was used to determine the level of resistance of the FcB1 strain in our culture and test conditions, and as positive control of antiplasmodial activity.

4.2.2. In vitro cytotoxicity test on mammalian cells The human diploid embryonic lung cell line (MRC-5) and the rat myoblast-derived cell line (L-6) were used to assess the cytotoxicity of plant extracts and purified compounds toward mammalian cells. MRC-5 and L-6 cells were seeded into 96-well microplates at 5000 cells per well in RPMI supplemented with 10% fetal calf serum. After 24 h, the cells were washed and maintained with different concentrations of extracts for 5 days, at 37 °C under a 5% CO2 atmosphere. Cytotoxicity was determined using the colorimetric MTT assay (Mossman, 1983) according to the manufacturer’s recommendations (cell proliferation kit I, Roche Applied Science, France), and scored as the percentage of absorbance reduction at 540 nm of the treated cultures versus untreated control cultures. IC50 value (concentration causing 50% cytotoxicity) was deduced from the drug concentration–response curve. Final IC50 value of each compound was expressed as the mean ± standard deviation of IC50 values issued from independent experiments. Cells were obtained from ATCC (Rockville, Maryland, USA). Camptothecin was used as positive control of cytotoxicity.

4.3. Plant material M. sessilifolia was collected in November 1999, at Anjozorobe (Madagascar) and was identified by Lucile Allorge by comparison with an authentic specimens held in the Department of Botany, Parc Botanique et Zoologique de Tshimbazaza, Antananarivo. A voucher specimen MAD-0693 has been kept at the Institut Malgache de Recherches Appliquées.

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4.4. Extraction and isolation The ground stem-bark of M. sessilifolia (485 g) was defatted in cyclohexane (1.5 L) and extracted with MeOH (1.2 L) at room temperature and concentrated to dryness under reduced pressure. The crude MeOH extract (60 g) was acidified with an aqueous solution of CH3COOH (2%) and extracted with EtOAc to afford 25 g of extract. The aqueous layer was then neutralized with a solution of NaHCO3 (5%) and extracted with CH2Cl2 to furnish the crude tertiary alkaloid extract (14 g). The evaluation of antiplasmodial activity showed that the EtOAc and alkaloid extracts inhibited the growth of P. falciparum with IC50 values of 6.5 and 1.6 lg/ml, respectively. However, it was noticed the presence of alkaloids in the EtOAc extract which was then partitioned with water and extracted successively with EtOAc and n-BuOH. An acid–base extraction at pH differential was performed on EtOAc extract-1. The EtOAc-2 soluble extract was obtained at pH 3 and a CH2Cl2-2 soluble extract was obtained after treatment of aqueous layer with Na2CO3 at pH 8. Sub-fraction CH2Cl2-3 was obtained in the same manner by neutralization at pH 10 with NH4OH. TLC analysis showed presence of alkaloids in these subfractions and purification was attempted on actives subfractions (EtOAc-2, CH2Cl2-2 and nBuOH). The subfraction EtOAc-2 (9.5 g) was subjected to fractionation using a Silica gel column eluted with cyclohexane–EtOAc 90/10 to 80/20, CH2Cl2/MeOH/ NH4OH 100/0/0 to 50/50/5 to give six fractions (F1-F6). Three fractions showed activity between 3.6 and 6 lg/mL. From fraction F2, alkaloid 3-oxocoronaridine-7-hydroxyindolenine 6 (2 mg) was obtained by repeated purification on MPLC followed by Silica gel column eluted with CH2Cl2/MeOH 99/1 and 98/2. Purification of fraction F4 (1.6 g) by MPLC afforded 16 sub– fractions, 3 oxocoronaridine 7 (6 mg) and 3-hydroxycoronaridine 8 (4 mg) were obtained from sub-fraction F4-4 through silica gel with CH2Cl2/MeOH 95/5 followed by Sephadex LH-20 eluted with MeOH. Chromatography of subfraction F4-7 by repeated Sephadex LH-20 yielded 30 -oxotabernaelegantine A 2 (2 mg) after a passage on MPLC. Fraction F-5 was also subjected to Sephadex LH-20 column to afford 12 subfractions. Subfraction 5-5 was separated further by MPLC using CH2Cl2/MeOH 95/5 and preparative TLC to furnish 30 oxotabernaelegantine B 1 (5 mg). Subfraction 5-10 was fractionated by reversed-phase MPLC (MeOH–H2O 0.1 % TFA 60/40 to 30/ 70) to give 1 (28 mg), and a mixture which was purified by Sephadex LH-20 eluted with MeOH to give tabernaelegantine B 11 (6 mg). Tabernaelegantine D 12 (10 mg) was obtained from Subfraction 5-11 by purification on Sephadex LH-20. The CH2Cl2-2 soluble extract (Subfraction alkaloidal B-2) was subjected to passage over Sephadex LH20 eluted with MeOH to give four fractions of which two were active. Fraction B2–F2 was separated by Sephadex LH20 yielding five subfractions. Subfraction B2–F2-2 was subjected to MPLC chromatography on silica gel eluted with gradient CH2Cl2/MeOH (100/1 to 95/5) to furnish of tabernaelegantine A 13 (7 mg) and 30 (R/S)-hydroxytabernaelegantine A 3 (30 mg). Subfraction B2–F2-3 was subjected successively on MPLC chromatography (CH2Cl2/MeOH) then to ODS HPLC (30 to 35% ACN – 0.1% TFA (aq), 30 min) to afford 2 (2 mg). 30 SHydroxytabernaelegantine A 4 (4 mg) was isolated from subfraction B2–F2-4 by MPLC chromatography on silica gel eluted with gradient CH2Cl2/MeOH (100/0 to 92/8). Subfraction B2-F3 was chromatographed on MPLC RP-18 reversed phase (20/80 to 30/70 MeOH-0.1% TFA (aq)) followed by Sephadex LH-20 to give coronaridine 9 (20 mg) and tabernaemontanine 10 (15 mg). The chromatography of subfraction n-BuOH was carried out over Sephadex LH20 eluted with MeOH to give five fractions. Purification of F3 was achieved by MPLC with reversed phase on a

Flashsmart cartridge (gradient MeOH-0.1%TFA (aq)) to yield 19,20a-dihydroeleganine A 5 (4.7 mg), sitosterol-b-D-glucoside 14 (25 mg), and lupeol acetate 15 (3.5 mg). Purification of F4 on MPLC with reversed phase afforded a-amyrin acetate 16 (4 mg). 4.4.1. 30 -Oxotabernaelegantine B (1) Amorphous powder; ½a20 D +14 (c 0.13, MeOH); UV (MeOH) kmax (log e) 222 (4.49), 282 (4.02) nm; IR (CHCl3) mmax 3250, 2918, 1725, 1665, 1459, 1200 cm1; 1H NMR and 13C NMR data, see Tables 1 and 2; HRESI-MS m/z: 721.3957 [M+H]+ (calcd for C43H53N4O6 721.3946). 4.4.2. 30 -Oxotabernaelegantine A (2) Amorphous powder; ½a20 D 26.9 (c 0.11, MeOH); UV (MeOH) kmax (log e) 219 (4.0), 283 (3.31) nm; IR (CHCl3) mmax 2924, 2854, 1732, 1670, 1465, 1199 cm1; 1H NMR and 13C NMR data, see Tables 1 and 2; HRESI-MS m/z: 721.3952 [M+H]+ (calcd for C43H53N4O6 721.3946). 4.4.3. 30 (R/S)-Hydroxytabernaelegantine A (3) Amorphous powder; ½a20 D 36.8 (c 0.08, MeOH); UV (MeOH) kmax (log e) 224 (4.56), 285 (4.09) nm; IR (CHCl3) mmax 3375, 1732, 1654, 1458, 1238; 1080 cm1; 1H NMR and 13C NMR data, see Tables 1 and 2; HRESI-MS m/z: 704.3884 [M+HH2O]+ (calcd for C43H53N4O5 704.3920). 4.4.4. 30 (S)-Hydroxytabernaelegantine C (4) Amorphous powder; ½a20 D 32.2 (c 0.08, MeOH); UV (MeOH) kmax (log e) 221 (4.36), 285 (3.92) nm; IR (CHCl3) mmax 3360; 2920, 1732, 1462, 1265, 736 cm1; HRESI-MS m/z: 704.3980 [M+HH2O]+ (calcd for C43H53N4O5 704.3920). 4.4.5. 19,20a-Dihydroeleganine A (5) Amorphous powder; ½a20 D 31.3 (c 0.21, MeOH); UV (MeOH) kmax (log e) 218 (3.70), 237 (3.68), 312 (3.73) nm; IR (CHCl3) mmax 3298; 3059, 2962, 1732, 1685, 1651, 1458, 1199 cm1; 1H NMR and 13C NMR data, see Table 2. HRESI-MS m/z: 371.1955 [M+H]+ (calcd for C21H27N2O4 371.1960). Acknowledgments This work is dedicated to the memory of Armand Rakotozafy also called ‘‘Papa Armand’’. It was supported by a grant from the French ‘‘Ministère de la recherche’’ for Marion Girardot. Dr. Bastien Nay is thanked for discussions and comments on the manuscript. References Achenbach, H., Raffelsberger, B., 1980. 19-Ethoxycoronaridine, a novel alkaloid from Tabernaemontana glandulosa. Phytochemistry 19, 716–717. Ahond, A., Bui, A.-M., Potier, P., Hagaman, E.W., Wenkert, E., 1976. Carbon-13 nuclear magnetic resonance analysis of vobasine-like indole alkaloids. J. Org. Chem. 41, 1878–1879. Andrade, M.T., Lima, J.A., Pinto, A.C., Rezende, C.M., Larvalho, M.P., Epifanio, R.A., 2005. Indole alkaloids from Tabernaemontana australis (Müell. Arg) Miers that inhibit acetylcholine-sterase enzyme. Bioorg. Med. Chem. 13, 4092–4095. Bombardelli, E., Bonati, A., Gabetta, B., Martinelli, E.M., Mustich, G., Danieli, B., 1976. Structures of tabernaelegantines A–D and tabernaelegantinines A and B, new indole alkaloids from Tabernaemontana elegans. J. Chem. Soc. Perkin I, 1432– 1438. Bringmann, G., Saeb, W., Aké Assi, L., François, G., Narayanan, A.S., Peters, K., Peters, E.-M., 1997. Betulinic acid. Isolation from Triphyophyllum peltatum and Ancistrocladus heyneanus, antimalarial activity and crystal structure of the benzyl ester. Planta Med. 63, 255–257. Brunell, R.H., Medina, J.D., 1971. Alkaloids of Tabernaemontana psychotrifolia. Can. J. Chem. 49, 307–316. Chattikorn, S., Pongpanparadorn, A., Pratchayasakul, W., Pongchaidacha, A., Ingkaninan, K., Chattipakorn, N., 2007. Tabernaemontana divaricata extract inhibits neuronal acetylcholi-nesterase activity in rats. J. Ethnopharmacol. 110, 61–68.

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