Note pubs.acs.org/jnp
Strychnobaillonine, an Unsymmetrical Bisindole Alkaloid with an Unprecedented Skeleton from Strychnos icaja Roots Alembert T. Tchinda,*,† Olivia Jansen,‡ Jean-Noel Nyemb,§ Monique Tits,‡ Georges Dive,⊥ Luc Angenot,‡ and Michel Frédérich‡ †
Center for Studies on Medicinal Plants and Traditional Medicine, Institute of Medical Research and Medicinal Plants Studies (IMPM), P.O. Box 6163, Yaoundé, Cameroon ‡ Laboratory of Pharmacognosy, Department of Pharmacy, CIRM, University of Liège, B36, 4000 Liège, Belgium § Department of Organic Chemistry, Faculty of Sciences, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon ⊥ Centre d’Ingénierie des Protéines, Université de Liège, B6, 3 Allée de la Chimie, 4000 Liège, Belgium S Supporting Information *
ABSTRACT: A reinvestigation of the roots of Strychnos icaja resulted in the isolation of a new bisindole alkaloid named strychnobaillonine (1) with original C-17−N-1′ and C-23−C17′ junctions, in addition to sungucine, bisnordihydrotoxiferine, and strychnohexamine (2). Compound 1 showed potent activity against the chloroquine-sensitive 3D7 strain of Plasmodium falciparum in vitro with an IC50 value of 1.1 μM. The structures of the compounds were defined by detailed spectroscopic analyses, especially 1H and 13C NMR, DEPT, HSQC, COSY, NOESY, HMBC, and HRESIMS. The proposed absolute configuration was based on biosynthetic considerations and spectroscopic data (CD, NMR) supported by molecular modeling.
named strychnobaillonine (1), was successively isolated together with sungucine, bisnordihydrotoxiferine, and strychnohexamine (2), previously isolated from the plant.6,9 Compounds 1 and 2 were tested in vitro against the chloroquine-sensitive 3D7 strain of P. falciparum. Moreover, their antiproliferative activity toward WI-38 fibroblasts was evaluated to determine their specific antiplasmodial activity. The cytotoxicity of compound 2 against a human fibroblast is reported here for the first time as well as its antiplasmodial activity against the 3D7 strain of P. falciparum. The alkaloid extract of the roots of S. icaja was subjected to a series of silica gel and Sephadex LH-20 chromatographic columns and preparative TLC to afford a new dimer named strychnobaillonine (1) and the known bisindole alkaloids sungucine and bisnordihydrotoxiferine and the trisindole strychnohexamine (2). The known compounds were identified by comparison of their NMR data with those in the literature6,9 and also with authentic samples available in the laboratory. The UV spectrum of 1 exhibited maxima at λmax 252 and 289 nm, with values very similar to those of retulinal and isoretulinal characteristic of an indolinic chromophore.10 The IR spectrum showed hydroxy and amide absorptions at 3390 and 1655 cm−1, respectively. On the basis of HRESIMS, C40H45N4O2 was
Strychnos icaja Baill. is a liana up to 100 m long, climbing with solitary tendrils and a stem up to 15 cm in diameter. The distinctively red-colored roots of the Strychnos icaja plant are mainly used by local populations of Africa (Congo, Cameroon, Gabon, Central African Republic, etc.) as an arrow or ordeal poison, but they are also used in the treatment of skin diseases and chronic, persistent malaria. Because of its toxicity due to the presence of strychnine and 12-hydroxystrychnine, it is usually administered under the supervision of a traditional medicine man.1,2 Several mono- and bisindole alkaloids have been isolated from different parts of the plant. Leaves contain mostly monoindole alkaloids. Stem and root bark contain both types of alkaloids with a high proportion of dimers present in the roots.3,4 The roots have attracted more interest because of the strong activity of their extracts and isolated bisindole alkaloids against various chloroquine-sensitive and -resistant strains of Plasmodium falciparum. Some bisindole alkaloids with IC50 values lower than 1 μM have been reported.5 During previous works on the roots and stem bark of S. icaja,6,7 a minor alkaloid giving a persistent red coloration after spraying with cerium sulfate reagent was identified, but could not be isolated due to a limited amount of the extracts. In a continuation of our search for potential antiplasmodial compounds from Strychnos species,6−8 we reinvestigated the tertiary alkaloids of S. icaja to isolate this minor alkaloid and evaluate its antiplasmodial activity. The targeted alkaloid, © 2014 American Chemical Society and American Society of Pharmacognosy
Received: October 28, 2013 Published: March 4, 2014 1078
dx.doi.org/10.1021/np400908u | J. Nat. Prod. 2014, 77, 1078−1082
Journal of Natural Products
Note
(C-22) and the deshielded signal H-12 (δH 8.23) due to the influence of the carbonyl.4 The remaining 19 carbons are suggestive of an Na-deacetylisoretuline moiety (lower part) characterized by the absence of the amide carbonyl and C-23. C-17 was shown by the 1H NMR and HSQC spectra to bear only one proton (δ 3.86). In compound 1, H-17 resonates at δH 3.86 (t, J = 4.5 Hz) and the carbon at δC 57.5, suggesting C-17 is probably attached to N-1′. This provided an indication that the attachment points from the upper indole unit are from C17 and C-23, as there was no evidence of substitution at any other carbon atom of the upper moiety. The HMBC spectrum was very useful for the establishment of bond connections between the two units of the dimer. It showed unambiguously correlations between H-23 and C-17, C-22, C-16′, and C-17′, H-17 and C-2, C-13′, C-15, C-16, and C-22, and also H-17′ and C-15′, C-17, C-22, and C-23. On the basis of HMBC correlations, C-17′ and N-1′ were deduced as connection points in the lower part of the molecule. C-17′ also bears a hydroxy group, as indicated by chemical shifts (δH 4.62, δC 69.5). The C-23−C-17′ and C-17−N-1′ bonds were identified as the linkage between the two moieties of the dimer. However, C-23 has been involved in the junction of the strychnan moieties in sungucine and derivatives previously isolated from S. icaja roots and is linked to C-5′.11 Although the C-17−N-1′ bond exists in curare derivatives,12 the C-23−C-17′ linkage is identified for the first time in the Strychnos genus. The COSY spectrum clearly disclosed the connectivities H-2/H-16/H-17/H-23/H-17′/H-16′/H-2′, thus confirming the linkage between the two monomers. The retuline and isoretuline skeletons are easily identified in the structure of 1, respectively at the upper and lower parts. The reported 13C NMR data of N-desacetylretuline and Ndesacetylisoretuline isolated from various Strychnos species including S. variabilis and S. matopensis3 show differences varying from 3.5 to 7.7 ppm around C-2, C-3, C-6, C-14, C-16, C-20, and C-21. Similar differences are observed in the corresponding carbon atoms of the upper and lower parts of compound 1. Moreover, the 13C NMR data of the two units are close to those of the respective monomers (Table S1, Supporting Information). This further observation confirms that compound 1 derives from retuline in the upper part and isoretuline in the lower part and is therefore a strychnan− strychnan-type dimer with an unprecedented C−C linkage. Because the lower part of the molecule belongs to the isoretuline series, H-2′ and H-16′ are antiperiplanar, and this implies a chair form of the piperidine ring. As a consequence, 17′-OH must be α-oriented to minimize steric hindrance.13,14 Moreover, the value of JH‑16′/H‑17′ (10.2 Hz) indicates also an antiperiplanar configuration, so H-17′ must be β. This H-17′β (17′R) configuration is similar to that of guianensine, strychnochrysine, and other derivatives.15 The orientation of the ethylidene groups is proposed to be E, because of the following NOESY correlations: H-19/H-21a, H3-18/H-17, H3-18/H-12′, H3-18′/H-17′, and H-19′/H-21′a. The relative configurations at the other stereogenic centers in the strychnan moieties were established from NOESY correlations and analysis of coupling constants, in the same manner as that carried out for the Strychnos alkaloids previously isolated from S. icaja.6 To the upper part was attributed the configuration of the retuline series and to the lower part the configuration of the isoretuline series. The configurations of H15α (15R), H-3α (3R), H-2β (2S), 7β (7R), H-15′α (R), H-3′α (R), H-2′β (2S), and 7′β (7R) are those commonly accepted
determined as the molecular formula. This was indicative of the presence of four nitrogen atoms, as expected for a bisindole alkaloid. The 13C NMR data (Table 1) accounted for 40 carbon resonances, comprising two methyl, eight methylene, 21 methine, one carbonyl, and eight quaternary carbons. The bisindole nature of compound 1 was further confirmed by the presence of eight aromatic protons in the region δH 6.52−8.23 characteristic of two disubstituted aromatic rings in addition to two ethylidene side chains with methyls at δH 1.72 (H3-18′) and 1.78 (H3-18) and vinyl protons at δH 5.43 (H-19) and 5.62 (H-19′). The NMR data (Table 1) evidenced the presence of a retuline moiety (upper part) comprising 21 carbon atoms. This skeleton was characterized by the amide carbonyl at δC 167.7 1079
dx.doi.org/10.1021/np400908u | J. Nat. Prod. 2014, 77, 1078−1082
Journal of Natural Products
Note
Table 1. NMR Spectroscopic Data for Strychnobaillonine (1) in MeOH-d4a pos.
δC, type
2 3 5a 5b 6a 6b 7 8 9 10 11 12 13 14a 14b 15 16 17 18 19 20 21a 21b 22 23 2′ 3′ 5′
63.7, CH 66.3, CH 54.1, CH2
6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′a 14′b 15′ 16′ 17′ 18′ 19′ 20′ 21′a 21′b
36.5, CH2 52.8, 137.4, 122.3, 125.3, 128.0, 117.9, 141.4, 23.8,
C C CH CH CH CH C CH2
29.3, 39.0, 57.5, 13.7, 117.7, 140.5, 52.3,
CH CH CH CH3 CH C CH2
167.7, 47.8, 69.4, 61.7, 53.7,
C CH CH CH CH2
41.2, 51.8, 134.3, 122.2, 119.8, 128.2, 108.3, 150.0, 28.5,
CH2 C C CH CH CH CH C CH2
27.7, 47.9, 69.5, 13.0, 122.2, 133.8, 58.2, 58.2
CH CH CH CH3 CH C CH2
δH (J in Hz)
HMBCb
COSY
d (5.2) brd (2.9) m ov m m
3, 6, 2, 7, 3, 7, 21 3, 5, 3, 7,
7.26, 7.18, 7.29, 8.23,
ov ov ov d (7.9)
7, 8, 9, 8,
1.72, 1.73, 2.74, 2.57, 3.86, 1.78, 5.43,
ov ov brs m t (4.5) d (6.7) q (6.8)
3 17, 20 2, 7 2, 13′, C-15, 16, 22, 23 19, 20 15, 18, 21
H-5′a, H-5′b, H-6′a H-15 H-16, 2H-14 H-2, H-15, H-17 H-16, H-23 H-19 Me-18
3.62, d (14.5) 3.36, d (14.5)
3, 5, 15, 19, 20 3, 5, 15, 19, 20
H-21b H-21a
H-19
2.88, 3.06, 3.51, 3.18, 2.71, 2.30,
ov ov brs m dd (8.3, 3.7) m
16′, 17, 17′, 22 8′, 13′, 17′, 2′, 15′, 21′, 7′ 3′ 2′, 3′, 7′
H-17, H-17′ H-16′ H-14′a, H-14′b H-5′b, H-6′a, H-6′b H-5′a, H-6′a, H-6′b H-6′b, H−H-5′a, 5′b
H-17 H-17′ H-5′a, H-9′, H-14′a, H-16′ H-3′
7.09, 6.85, 7.17, 6.52,
d (7.0) t (7.3) ov d (7.9)
7′, 8′, 9′, 8′,
H-10′ H-9′, H-11′ H-10′, H-12′ H-11′
H-3′
2.04, 1.62, 3.03, 1.61, 4.62, 1.72, 5.62,
m m ov ov dd (10.2, 1.0) ov q (6.3)
3′ 3′ 21′
H-14′b, H-15′ H-14′a, H-15′ H-16′, H-14′a, H-14′b H-2′, H-15′, H-17, H-17′ H-16′, H-23 H-19′ Me-18′
H-3′
H-21″b H-21′a
H-19′
3.59, d (14.5) 2.91, d (14.5)
7, 15 8, 15 21
NOESY
4.45, 3.24, 3.18, 3.03, 2.46, 2.14,
H-16 H-14 H-5b, H-6a, H-6b H-5a, H-6a, H-6b H-5a, H-5b, H-6b H-5a, H-5b, H-6a
8 8
11, 13 12 13, 12 10, 13
H-6b, H-16
H-2
H-10 H-9, H-11 H-10, H-11 H-11
8′, 11′, 13′ 11′, 12′, 13′ 10′
15′, 17, 22, 23 19′, 20′ 15′, 18′, 21′ 3′, 15′, 19′, 20′
H-17 H-12′, H-17, H-21b H-12′, H-15, H-16, H-23, Me-18, H-16′ H-12′, H-17, H-19 H-21a, Me-18
H-16, H-17, Me-18
H-16′ H-3′, H-15′ H-2′, Me-18′ H-19′, H-17′ Me-18′, H-21′a
a
Spectra were recorded at 500 MHz for 1H and 125 MHz for 13C. bHMBC correlations are from proton(s) stated to the indicated carbon; ov: overlap.
H-17 was assigned the relative configuration α after examination of Dreiding stereomodels of the H-17α and β forms. In the 17β form, most NOESY couplings cannot be observed especially the correlation with H-12′. The larger dihedral angle observed in the Dreiding stereomodel than in molecular modeling accounts for the vicinal coupling observed between H-17 and H-23 and supports the proposed H-17α configuration. The NOESY correlations H-15/H-17, H-17/H16′, and H-17/H-12′ and the absence of the correlation
from biogenetic considerations.16 The configurations at H-16, H-17, H-23, H-17′, and H-16′ must still be considered. The small coupling constant between H-2 and H-16 (J = 5.2 Hz) confirms that compound 1 belongs to the retuline series with a 16β configuration.14 This was confirmed by a NOESY correlation between the two protons in question. Other key correlations observed in the NOESY spectrum included the ones between H-12′ and H-16, H-17 and H3-18, H-2′ and H17′, and also H-17 and H-12′, H-15, H-16, H-23, and H3-18. It was therefore concluded that H-23 and H-17′ were β-oriented. 1080
dx.doi.org/10.1021/np400908u | J. Nat. Prod. 2014, 77, 1078−1082
Journal of Natural Products
Note
Table 2. Antiplasmodial and Cytotoxicity Activities of the Isolated Compounds
a
compound
IC50 3D7, μg/mL
IC90 3D7, μg/mL
na
strychnobaillonine (1) strychnohexamine (2) pH 8 fraction chloroquine artemisinine camptothecin
0.7 ± 0.1 (1.1) 0.5 ± 0.1 (0.6) 0.8 0.02 ± 0.00 0.01 ± 0.00
2.6 1.6 7.5
2 2 1 3 3
b
IC50 WI-38, μg/mL >10 14 14). The antiplasmodial activity was more than 14 times higher than the cytotoxic activity. According to WHO, the specificity of a compound toward P. falciparum can be deduced if the SI value equals at least 10.13 Compound 2 was highly cytotoxic (IC50 < 5 μg/mL). Its SI was lower than 10 and decreases its chances of being considered as a potent antiplasmodial agent. However, this is the first study on its cytotoxicity.
between H-17 and H-17′ are in agreement with the H-17α absolute configuration. The CD spectrum showed a positive Cotton effect at λmax 257 nm, indicative of a 2β,7β configuration, as found in most Strychnos alkaloids such as protostrychnine and pseudostrychnine, with a positive Cotton effect at λmax 252 and 259 nm, respectively.4 Due to overlap in the 1H NMR spectrum, the H2′/H-16′ coupling constant could not be determined to confirm the configuration of H-16′. Nevertheless, 13C NMR data and NOESY correlations (H-16′/H-3′ and H-16′/H-15′) were consistent with an H-16′α configuration as in the isoretuline species. The absolute configuration at C-16′ and C-17 was verified by molecular modeling. Geometry optimization of the four possible diastereoisomers was performed depending on whether their respective protons were at α or β positions. For each conformation, all the degrees of freedom describing the geometry were fully optimized via energy minimization at the DFT level with the B3LYP functional 17 using the double-ζ basis set 6-31G(d) 18 implemented in the Gaussian suite of programs.13 The H16′α,H-17α and H-16′β,H-17α forms display the same energy and are more stable than the H-16′α,H-17β, with a difference of 3.91 kcal. The latter form is more stable (Δε = 2.61 kcal) than the H-16′β,H-17β diastereoisomer. On the basis of NOESY couplings, compound 1 was assigned with the H-16′α,H-17α absolute configuration. The full NMR data (Table 1) were consistent with the proposed structure of compound 1, for which the name strychnobaillonine was given in memory of Henri Ernest Baillon (1827−1895), a famous French botanist who identified S. icaja for the first time and reported the description in the first volume of the Dictionnaire de Botanique.19 Strychnobaillonine (1) represents a unique strychnan−strychnan-type structure with an unprecedented skeleton.20 The in vitro antiplasmodial activities of the pH 8 alkaloidic fraction and of compounds 1 and 2 were evaluated against the chloroquine-sensitive 3D7 strain of P. falciparum (Table 2). Compound 1 showed good activity [IC50 = 0.7 μg/mL (1.1 μM)]. According to WHO guidelines and basic criteria for drug discovery, a pure compound is defined as highly active if its IC50 is ≤1 μg/mL. This activity is close to that of other bisindole alkaloids such as matopensine and longicaudatine when tested on the FCA/Ghana chloroquino-sensitive strain. Previous studies have shown that monomers including strychnine and retuline were devoid of any antiplasmodial activity.5 One can easily conclude that the dimerization of monoindole alkaloids increases the antiplasmodial activity. In the course of the present work, strychnohexamine (2) also showed potent antiplasmodial activity [IC50 = 0.5 μg/mL (0.6 μM)] against the 3D7 strain of P. falciparum. It was more active compared to the FCA/Ghana strain during previous studies.4 Additional in vitro studies on the cytotoxic activity on normal human
■
EXPERIMENTAL SECTION
General Experimental Procedures. The optical rotation was measured on an ANALYS AA-10 automatic polarimeter. The UV spectrum was acquired using a U-2910 spectrophotometer. CD spectra were measured with a J-810 JASCO polarimeter. IR spectra were recorded on a Perkin-Elmer 1750 FTIR spectrometer. NMR spectra were recorded in CDCl3 or MeOH-d4 on a Bruker 500 MHz NMR AV II spectrometer equipped with a cryoprobe, with TMS as an internal reference. The HRESI mass spectra were recorded with a Bruker APEX-Qe 9.4T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer equipped with a hybrid quadrupole analyzer and using an electrospray source. Analytical TLC was performed on precoated Si gel F254 (Merck 1.05735) plates. After development, the dried plates were examined under short-wave (254 nm) or long-wave (366 nm) UV light and sprayed with 1% ceric sulfate in 10% sulfuric acid. LiChroprep Si 60 (43−60 μm, Merck 9336) was used for column chromatography. Si gel 60 PF254 (Art.1.07747, Merck) was used for purification of alkaloids by preparative TLC (1.25 mm thick, 20 × 20 cm Si gel plates). All solvents used were analytical grade (Merck). All calculations to determine the geometry of the molecule were performed with the Gaussian 03 program.13 Plant Material. The root bark of S. icaja was collected near Bertoua (East Region, Cameroon) in August 2012. The plant was authenticated at the herbarium of the Belgian National Botanical Garden at Meise, where a voucher specimen (BR0000005012251) was deposited. Extraction and Isolation of Compounds. The stem roots of S. icaja (400 g) were macerated with 300 mL of EtOAc−EtOH− NH4OH (96:3:1) and then percolated with EtOAc until complete extraction of alkaloids. The extract was concentrated under reduced pressure at 40 °C to yield 11 g of dry extract and then dissolved in EtOAc and extracted with 4% HOAc. The resulting acidic solution was basified to pH 8 with Na2CO3 and repeatedly extracted with CH2Cl2. The CH2Cl2 extracts obtained were dried over Na2SO4 and concentrated to yield an alkaloid extract (3 g), which was further fractionated first by column chromatography on 180 g of Merck LichroPrep Si 60 with CH2Cl2−MeOH mixtures. The 210 fractions of 20 mL were collected and pooled according to their TLC profile using the mixture EtOAc−iPrOH−NH4OH (60:35:15) as eluent. Sungucine (150 mg) crystallized from Frs. 45−90 collected from the column with the mixtures 96:4−92:8. Fractions 105−130 collected from the main column with the mixture CH2Cl2−MeOH (92:8) were further chromatographed on a silica gel column with CH2Cl2−MeOH, 0− 1081
dx.doi.org/10.1021/np400908u | J. Nat. Prod. 2014, 77, 1078−1082
Journal of Natural Products
Note
10%, and then purified through preparative TLC using EtOAc− iPrOH−NH4OH (60:35:15) to give bisnordihydrotoxiferine (10 mg) and strychnohexamine (18 mg). Fractions 137−165 obtained from the main column with the mixtures 92:8−90:10 were passed through a Sephadex LH-20 column using MeOH as eluent and then purified by preparative TLC using EtOAc−iPrOH−NH4OH (60:35:15) to give compound 1 (6 mg) and additional strychnohexamine (7 mg). Strychnobaillonine (1): amorphous, off-white solid; spraying with CeSO4−H2SO4 reagent on TLC immediately gave the compound a persistent red coloration (Supporting Information); [α]25 D −10 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 205 (2.5), 252 (1.7), 289 (1.4) nm; CD (c 0.33 × 10−3, MeOH) λmax (Δε) 230.5 (+0.56), 244 (−1.86), 257 (+3.66), 277 (−1.87), 295 (−0.04), 313 (−2.0) nm; IR (KBr) 3390, 2913, 2858, 1650, 1586, 1478, 1444, 1388, 1339, 1003, 730 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 613.35415 [M + H]+ (calcd for C40H45N4O2, m/z 613.3542); HRESIMS-MS m/z 613.35307.1832, 595.29545, 552.30058, 486.25375, 460.23797, 409.22712, 319.18031, 307.68205, 308.18380, 295.18031, 277.16981. In Vitro Antiplasmodial Assay. Activity against P. falciparum chloroquine-sensitive 3D7 strains was assessed following the procedure already described in Frederich et al.5 The parasites were obtained from Prof. Grellier (Museum d’Histoire Naturelle, Paris, France). Each alkaloid and extract was applied in a series of eight 3fold dilutions (final concentrations ranging from 0.09 to 200 μg/mL for an extract and from 0.02 to 50 μg/mL for a pure substance) on two rows of a 96-well microplate and were tested in duplicate (n = 2) or triplicate (n = 3). Parasite growth was estimated by determination of lactate dehydrogenase activity as described previously in Jonville et al.13 Artemisinin (98%, Sigma−Aldrich) and chloroquine were used as positive controls. In Vitro Cytotoxic Assay. Human normal fetal lung fibroblasts, WI-38, were maintained in continuous culture in a humid atmosphere at 37 °C and 5% CO2 in DMEM medium (Bio-Whittaker-LONZA), supplemented with 10% heat-inactivated fetal bovine serum, 1% Lglutamine (200 mM), and penicillin (100 IU/mL)−streptomycin (100 μg/mL) (Pen-Strep) (Bio-Whittaker-LONZA). The assays followed the procedure already described in Jonville et al.13 Dead cells were found to be smaller and to have a round shape. The viability of the cells was determined by adding WST-1 (Roche) tetrazolium salt as a cytotoxicity indicator and by reading the absorbance at 450 nm with a scanning multiwell spectrophotometer after about 1 h. Camptothecin (95% HPLC, Sigma−Aldrich) was used as a positive control. The selectivity index (SI) value allows the evaluation of the selective activity of the extracts/pure compound against the parasite compared to its toxicity for human cells. The SI value is calculated as the ratio between cytotoxic IC50 values and 3D7 parasitic IC50 values.
■
fellowship to A.T.T. at the Department of Pharmacognosy, University of Liège, Belgium. G.D. is research associate of the FRS-FNRS. G.D. acknowledges the Céci consortium for access to the HPC facilities installed in Liège and Louvain-la-Neuve. The authors are grateful to Ms. J. Widart, Laboratory of Mass Spectrometry, Giga Center, University of Liège, for the recording of mass spectra.
■
ASSOCIATED CONTENT
S Supporting Information *
Stereostructures of the H-16′β,H-17β; H-16′β,H-17α; H16′α,H-17α; and H-16′α,H-17β forms; NMR and mass spectra; possible biosynthesis pathway; TLC coloration behavior of strychnobaillonine (1); comparison of selected 13C NMR data of strychnobaillonine (1), N-desacetylretuline, and N-desacetylisoretuline. This material is available free of charge via the Internet at http://pubs.acs.org.
■
REFERENCES
(1) Philippe, G.; Angenot, L.; Tits, M.; Frederich, M. Toxicon 2004, 44, 405−416. (2) Mosango, D. M. 2008. Strychnos icaja Baill. In Plant Resources of Tropical Africa 11(1). Medicinal Plants 11(1); Schmelzer, G. H.; GuribFakim, A., Eds.; Foundation PROTA, Wageningen, The Netherlands; Backhuys Publishers: Leiden, The Netherlands, pp 640−642. (3) Delaude, C.; Delaude, L. Bull. Soc. R. Liège 1997, 66, 183−286. (4) Philippe, G.; De Mol, P.; Zèches-Hanrot, M.; Nuzillard, J.-M.; Tits, M.-H.; Angenot, L.; Frédérich, M. Phytochemistry 2003, 62, 623− 629. (5) Frédérich, M.; Jacquier, M. J.; Thepenier, P.; De Mol, P.; Tits, M.; Philippe, G.; Delaude, C.; Angenot, L.; Zeches-Hanrot, M. J. Nat. Prod. 2002, 65, 1381−1386. (6) Frédérich, M.; De Pauw, M.-C.; Llabrès, G.; Tits, M.; Hayette, M.-P.; Brandt, V.; Penelle, J.; De Mol, P.; Angenot, L. Planta Med. 2000, 66, 262−269. (7) Tchinda, A. T.; Tamze, V.; Ngono, A. R. N.; Ayimele, G. A.; Cao, M.; Angenot, L.; Frédérich, M. Phytochem. Lett. 2012a, 5, 108−113. (8) Tchinda, A. T.; Ngono, A. R. N.; Tamze, V.; Jonville, M.-C.; Cao, M.; Angenot, L.; Frédérich, M. Planta Med. 2012b, 78, 377−382. (9) Philippe, G.; Prost, E.; Nuzillard, J.-M.; Zèches-Hanrot, M.; Tits, M.; Angenot, L.; Frédérich, M. Tetrahedron Lett. 2002, 43, 3387− 3390. (10) Tits, M.; Angenot, L. Tetrahedron Lett. 1980, 21, 2439−2442. (11) Frederich, M.; De Pauw, M. C.; Prosperi, C.; Tits, M.; Brandt, V.; Penelle, J.; Hayette, M. P.; De Mol, P.; Angenot, L. J. Nat. Prod. 2001, 64, 12−16. (12) Zlotos, D. P. J. Nat. Prod. 2000, 63, 864−865. (13) Jonville, M.-C.; Dive, G.; Angenot, L.; Bero, J.; Tits, M.; Ollivier, E.; Frédérich, M.. Phytochemistry 2013, 87, 156−163. (14) Tavernier, D.; Anteunis, M. J. O.; Tits, M. J. G.; Angenot, L. J. G. Bull. Soc. Chim. Belg. 1978, 78, 595−607. (15) Gadi-Biala, R.; Tits, M.; Penelle, J.; Frederich, M.; Brandt, V.; Prosperi, C.; Llabres, G.; Angenot, L. J. Nat. Prod. 1998, 61, 139−141. (16) Klyne, W.; Buckingham, J. Atlas of Stereochemistry. Absolute Coǹuration of Organic Molecules; Chapman & Hall, London, 1974. (17) Becke, A. D. J. Chem. Phys. 1993, 98, 5648−5652. (18) Hehre, W. J.; Ditchfield, R.; Pople, J. A. J. Chem. Phys. 1972, 56, 2257−2261. (19) Baillon, H. Dictionnaire de Botanique; Tome, I., Ed.; Libraire Hachette et Cie: Paris, 1876. (20) Fu, Y.; Zhang, Y.; He, H.; Hou, L.; Di, Y.; Li, S.; Luo, X.; Hao, X. J. Nat. Prod. 2012, 75, 1987−1990.
AUTHOR INFORMATION
Corresponding Author
*Tel: +/237/76925929. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The Fund for Scientific Research (FNRS) is acknowledged for its support through grant no. 3.4533.10 and a research 1082
dx.doi.org/10.1021/np400908u | J. Nat. Prod. 2014, 77, 1078−1082
Strychnobaillonine, an Unsymmetrical Bisindole Alkaloid with an Unprecedented Skeleton from Strychnos icaja Roots Alembert T. Tchinda,*,† Olivia Jansen,‡ Jean-Noel Nyemb,§ Monique Tits,‡ Georges Dive,⊥ Luc Angenot,‡ and Michel Frédérich‡ †
Center for Studies on Medicinal Plants and Traditional Medicine, Institute of Medical Research and Medicinal Plants Studies (IMPM), P.O. Box 6163, Yaoundé, Cameroon ‡ Laboratory of Pharmacognosy, Department of Pharmacy, CIRM, University of Liège, B36, 4000 Liège, Belgium § Department of Organic Chemistry, Faculty of Sciences, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon ⊥ Centre d’Ingénierie des Protéines, Université de Liège, B6, 3 Allée de la Chimie, 4000 Liège, Belgium S Supporting Information *
ABSTRACT: A reinvestigation of the roots of Strychnos icaja resulted in the isolation of a new bisindole alkaloid named strychnobaillonine (1) with original C-17−N-1′ and C-23−C17′ junctions, in addition to sungucine, bisnordihydrotoxiferine, and strychnohexamine (2). Compound 1 showed potent activity against the chloroquine-sensitive 3D7 strain of Plasmodium falciparum in vitro with an IC50 value of 1.1 μM. The structures of the compounds were defined by detailed spectroscopic analyses, especially 1H and 13C NMR, DEPT, HSQC, COSY, NOESY, HMBC, and HRESIMS. The proposed absolute configuration was based on biosynthetic considerations and spectroscopic data (CD, NMR) supported by molecular modeling.
named strychnobaillonine (1), was successively isolated together with sungucine, bisnordihydrotoxiferine, and strychnohexamine (2), previously isolated from the plant.6,9 Compounds 1 and 2 were tested in vitro against the chloroquine-sensitive 3D7 strain of P. falciparum. Moreover, their antiproliferative activity toward WI-38 fibroblasts was evaluated to determine their specific antiplasmodial activity. The cytotoxicity of compound 2 against a human fibroblast is reported here for the first time as well as its antiplasmodial activity against the 3D7 strain of P. falciparum. The alkaloid extract of the roots of S. icaja was subjected to a series of silica gel and Sephadex LH-20 chromatographic columns and preparative TLC to afford a new dimer named strychnobaillonine (1) and the known bisindole alkaloids sungucine and bisnordihydrotoxiferine and the trisindole strychnohexamine (2). The known compounds were identified by comparison of their NMR data with those in the literature6,9 and also with authentic samples available in the laboratory. The UV spectrum of 1 exhibited maxima at λmax 252 and 289 nm, with values very similar to those of retulinal and isoretulinal characteristic of an indolinic chromophore.10 The IR spectrum showed hydroxy and amide absorptions at 3390 and 1655 cm−1, respectively. On the basis of HRESIMS, C40H45N4O2 was
Strychnos icaja Baill. is a liana up to 100 m long, climbing with solitary tendrils and a stem up to 15 cm in diameter. The distinctively red-colored roots of the Strychnos icaja plant are mainly used by local populations of Africa (Congo, Cameroon, Gabon, Central African Republic, etc.) as an arrow or ordeal poison, but they are also used in the treatment of skin diseases and chronic, persistent malaria. Because of its toxicity due to the presence of strychnine and 12-hydroxystrychnine, it is usually administered under the supervision of a traditional medicine man.1,2 Several mono- and bisindole alkaloids have been isolated from different parts of the plant. Leaves contain mostly monoindole alkaloids. Stem and root bark contain both types of alkaloids with a high proportion of dimers present in the roots.3,4 The roots have attracted more interest because of the strong activity of their extracts and isolated bisindole alkaloids against various chloroquine-sensitive and -resistant strains of Plasmodium falciparum. Some bisindole alkaloids with IC50 values lower than 1 μM have been reported.5 During previous works on the roots and stem bark of S. icaja,6,7 a minor alkaloid giving a persistent red coloration after spraying with cerium sulfate reagent was identified, but could not be isolated due to a limited amount of the extracts. In a continuation of our search for potential antiplasmodial compounds from Strychnos species,6−8 we reinvestigated the tertiary alkaloids of S. icaja to isolate this minor alkaloid and evaluate its antiplasmodial activity. The targeted alkaloid, © 2014 American Chemical Society and American Society of Pharmacognosy
Received: October 28, 2013 Published: March 4, 2014 1078
dx.doi.org/10.1021/np400908u | J. Nat. Prod. 2014, 77, 1078−1082
Journal of Natural Products
Note
(C-22) and the deshielded signal H-12 (δH 8.23) due to the influence of the carbonyl.4 The remaining 19 carbons are suggestive of an Na-deacetylisoretuline moiety (lower part) characterized by the absence of the amide carbonyl and C-23. C-17 was shown by the 1H NMR and HSQC spectra to bear only one proton (δ 3.86). In compound 1, H-17 resonates at δH 3.86 (t, J = 4.5 Hz) and the carbon at δC 57.5, suggesting C-17 is probably attached to N-1′. This provided an indication that the attachment points from the upper indole unit are from C17 and C-23, as there was no evidence of substitution at any other carbon atom of the upper moiety. The HMBC spectrum was very useful for the establishment of bond connections between the two units of the dimer. It showed unambiguously correlations between H-23 and C-17, C-22, C-16′, and C-17′, H-17 and C-2, C-13′, C-15, C-16, and C-22, and also H-17′ and C-15′, C-17, C-22, and C-23. On the basis of HMBC correlations, C-17′ and N-1′ were deduced as connection points in the lower part of the molecule. C-17′ also bears a hydroxy group, as indicated by chemical shifts (δH 4.62, δC 69.5). The C-23−C-17′ and C-17−N-1′ bonds were identified as the linkage between the two moieties of the dimer. However, C-23 has been involved in the junction of the strychnan moieties in sungucine and derivatives previously isolated from S. icaja roots and is linked to C-5′.11 Although the C-17−N-1′ bond exists in curare derivatives,12 the C-23−C-17′ linkage is identified for the first time in the Strychnos genus. The COSY spectrum clearly disclosed the connectivities H-2/H-16/H-17/H-23/H-17′/H-16′/H-2′, thus confirming the linkage between the two monomers. The retuline and isoretuline skeletons are easily identified in the structure of 1, respectively at the upper and lower parts. The reported 13C NMR data of N-desacetylretuline and Ndesacetylisoretuline isolated from various Strychnos species including S. variabilis and S. matopensis3 show differences varying from 3.5 to 7.7 ppm around C-2, C-3, C-6, C-14, C-16, C-20, and C-21. Similar differences are observed in the corresponding carbon atoms of the upper and lower parts of compound 1. Moreover, the 13C NMR data of the two units are close to those of the respective monomers (Table S1, Supporting Information). This further observation confirms that compound 1 derives from retuline in the upper part and isoretuline in the lower part and is therefore a strychnan− strychnan-type dimer with an unprecedented C−C linkage. Because the lower part of the molecule belongs to the isoretuline series, H-2′ and H-16′ are antiperiplanar, and this implies a chair form of the piperidine ring. As a consequence, 17′-OH must be α-oriented to minimize steric hindrance.13,14 Moreover, the value of JH‑16′/H‑17′ (10.2 Hz) indicates also an antiperiplanar configuration, so H-17′ must be β. This H-17′β (17′R) configuration is similar to that of guianensine, strychnochrysine, and other derivatives.15 The orientation of the ethylidene groups is proposed to be E, because of the following NOESY correlations: H-19/H-21a, H3-18/H-17, H3-18/H-12′, H3-18′/H-17′, and H-19′/H-21′a. The relative configurations at the other stereogenic centers in the strychnan moieties were established from NOESY correlations and analysis of coupling constants, in the same manner as that carried out for the Strychnos alkaloids previously isolated from S. icaja.6 To the upper part was attributed the configuration of the retuline series and to the lower part the configuration of the isoretuline series. The configurations of H15α (15R), H-3α (3R), H-2β (2S), 7β (7R), H-15′α (R), H-3′α (R), H-2′β (2S), and 7′β (7R) are those commonly accepted
determined as the molecular formula. This was indicative of the presence of four nitrogen atoms, as expected for a bisindole alkaloid. The 13C NMR data (Table 1) accounted for 40 carbon resonances, comprising two methyl, eight methylene, 21 methine, one carbonyl, and eight quaternary carbons. The bisindole nature of compound 1 was further confirmed by the presence of eight aromatic protons in the region δH 6.52−8.23 characteristic of two disubstituted aromatic rings in addition to two ethylidene side chains with methyls at δH 1.72 (H3-18′) and 1.78 (H3-18) and vinyl protons at δH 5.43 (H-19) and 5.62 (H-19′). The NMR data (Table 1) evidenced the presence of a retuline moiety (upper part) comprising 21 carbon atoms. This skeleton was characterized by the amide carbonyl at δC 167.7 1079
dx.doi.org/10.1021/np400908u | J. Nat. Prod. 2014, 77, 1078−1082
Journal of Natural Products
Note
Table 1. NMR Spectroscopic Data for Strychnobaillonine (1) in MeOH-d4a pos.
δC, type
2 3 5a 5b 6a 6b 7 8 9 10 11 12 13 14a 14b 15 16 17 18 19 20 21a 21b 22 23 2′ 3′ 5′
63.7, CH 66.3, CH 54.1, CH2
6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′a 14′b 15′ 16′ 17′ 18′ 19′ 20′ 21′a 21′b
36.5, CH2 52.8, 137.4, 122.3, 125.3, 128.0, 117.9, 141.4, 23.8,
C C CH CH CH CH C CH2
29.3, 39.0, 57.5, 13.7, 117.7, 140.5, 52.3,
CH CH CH CH3 CH C CH2
167.7, 47.8, 69.4, 61.7, 53.7,
C CH CH CH CH2
41.2, 51.8, 134.3, 122.2, 119.8, 128.2, 108.3, 150.0, 28.5,
CH2 C C CH CH CH CH C CH2
27.7, 47.9, 69.5, 13.0, 122.2, 133.8, 58.2, 58.2
CH CH CH CH3 CH C CH2
δH (J in Hz)
HMBCb
COSY
d (5.2) brd (2.9) m ov m m
3, 6, 2, 7, 3, 7, 21 3, 5, 3, 7,
7.26, 7.18, 7.29, 8.23,
ov ov ov d (7.9)
7, 8, 9, 8,
1.72, 1.73, 2.74, 2.57, 3.86, 1.78, 5.43,
ov ov brs m t (4.5) d (6.7) q (6.8)
3 17, 20 2, 7 2, 13′, C-15, 16, 22, 23 19, 20 15, 18, 21
H-5′a, H-5′b, H-6′a H-15 H-16, 2H-14 H-2, H-15, H-17 H-16, H-23 H-19 Me-18
3.62, d (14.5) 3.36, d (14.5)
3, 5, 15, 19, 20 3, 5, 15, 19, 20
H-21b H-21a
H-19
2.88, 3.06, 3.51, 3.18, 2.71, 2.30,
ov ov brs m dd (8.3, 3.7) m
16′, 17, 17′, 22 8′, 13′, 17′, 2′, 15′, 21′, 7′ 3′ 2′, 3′, 7′
H-17, H-17′ H-16′ H-14′a, H-14′b H-5′b, H-6′a, H-6′b H-5′a, H-6′a, H-6′b H-6′b, H−H-5′a, 5′b
H-17 H-17′ H-5′a, H-9′, H-14′a, H-16′ H-3′
7.09, 6.85, 7.17, 6.52,
d (7.0) t (7.3) ov d (7.9)
7′, 8′, 9′, 8′,
H-10′ H-9′, H-11′ H-10′, H-12′ H-11′
H-3′
2.04, 1.62, 3.03, 1.61, 4.62, 1.72, 5.62,
m m ov ov dd (10.2, 1.0) ov q (6.3)
3′ 3′ 21′
H-14′b, H-15′ H-14′a, H-15′ H-16′, H-14′a, H-14′b H-2′, H-15′, H-17, H-17′ H-16′, H-23 H-19′ Me-18′
H-3′
H-21″b H-21′a
H-19′
3.59, d (14.5) 2.91, d (14.5)
7, 15 8, 15 21
NOESY
4.45, 3.24, 3.18, 3.03, 2.46, 2.14,
H-16 H-14 H-5b, H-6a, H-6b H-5a, H-6a, H-6b H-5a, H-5b, H-6b H-5a, H-5b, H-6a
8 8
11, 13 12 13, 12 10, 13
H-6b, H-16
H-2
H-10 H-9, H-11 H-10, H-11 H-11
8′, 11′, 13′ 11′, 12′, 13′ 10′
15′, 17, 22, 23 19′, 20′ 15′, 18′, 21′ 3′, 15′, 19′, 20′
H-17 H-12′, H-17, H-21b H-12′, H-15, H-16, H-23, Me-18, H-16′ H-12′, H-17, H-19 H-21a, Me-18
H-16, H-17, Me-18
H-16′ H-3′, H-15′ H-2′, Me-18′ H-19′, H-17′ Me-18′, H-21′a
a
Spectra were recorded at 500 MHz for 1H and 125 MHz for 13C. bHMBC correlations are from proton(s) stated to the indicated carbon; ov: overlap.
H-17 was assigned the relative configuration α after examination of Dreiding stereomodels of the H-17α and β forms. In the 17β form, most NOESY couplings cannot be observed especially the correlation with H-12′. The larger dihedral angle observed in the Dreiding stereomodel than in molecular modeling accounts for the vicinal coupling observed between H-17 and H-23 and supports the proposed H-17α configuration. The NOESY correlations H-15/H-17, H-17/H16′, and H-17/H-12′ and the absence of the correlation
from biogenetic considerations.16 The configurations at H-16, H-17, H-23, H-17′, and H-16′ must still be considered. The small coupling constant between H-2 and H-16 (J = 5.2 Hz) confirms that compound 1 belongs to the retuline series with a 16β configuration.14 This was confirmed by a NOESY correlation between the two protons in question. Other key correlations observed in the NOESY spectrum included the ones between H-12′ and H-16, H-17 and H3-18, H-2′ and H17′, and also H-17 and H-12′, H-15, H-16, H-23, and H3-18. It was therefore concluded that H-23 and H-17′ were β-oriented. 1080
dx.doi.org/10.1021/np400908u | J. Nat. Prod. 2014, 77, 1078−1082
Journal of Natural Products
Note
Table 2. Antiplasmodial and Cytotoxicity Activities of the Isolated Compounds
a
compound
IC50 3D7, μg/mL
IC90 3D7, μg/mL
na
strychnobaillonine (1) strychnohexamine (2) pH 8 fraction chloroquine artemisinine camptothecin
0.7 ± 0.1 (1.1) 0.5 ± 0.1 (0.6) 0.8 0.02 ± 0.00 0.01 ± 0.00
2.6 1.6 7.5
2 2 1 3 3
b
IC50 WI-38, μg/mL >10 14 14). The antiplasmodial activity was more than 14 times higher than the cytotoxic activity. According to WHO, the specificity of a compound toward P. falciparum can be deduced if the SI value equals at least 10.13 Compound 2 was highly cytotoxic (IC50 < 5 μg/mL). Its SI was lower than 10 and decreases its chances of being considered as a potent antiplasmodial agent. However, this is the first study on its cytotoxicity.
between H-17 and H-17′ are in agreement with the H-17α absolute configuration. The CD spectrum showed a positive Cotton effect at λmax 257 nm, indicative of a 2β,7β configuration, as found in most Strychnos alkaloids such as protostrychnine and pseudostrychnine, with a positive Cotton effect at λmax 252 and 259 nm, respectively.4 Due to overlap in the 1H NMR spectrum, the H2′/H-16′ coupling constant could not be determined to confirm the configuration of H-16′. Nevertheless, 13C NMR data and NOESY correlations (H-16′/H-3′ and H-16′/H-15′) were consistent with an H-16′α configuration as in the isoretuline species. The absolute configuration at C-16′ and C-17 was verified by molecular modeling. Geometry optimization of the four possible diastereoisomers was performed depending on whether their respective protons were at α or β positions. For each conformation, all the degrees of freedom describing the geometry were fully optimized via energy minimization at the DFT level with the B3LYP functional 17 using the double-ζ basis set 6-31G(d) 18 implemented in the Gaussian suite of programs.13 The H16′α,H-17α and H-16′β,H-17α forms display the same energy and are more stable than the H-16′α,H-17β, with a difference of 3.91 kcal. The latter form is more stable (Δε = 2.61 kcal) than the H-16′β,H-17β diastereoisomer. On the basis of NOESY couplings, compound 1 was assigned with the H-16′α,H-17α absolute configuration. The full NMR data (Table 1) were consistent with the proposed structure of compound 1, for which the name strychnobaillonine was given in memory of Henri Ernest Baillon (1827−1895), a famous French botanist who identified S. icaja for the first time and reported the description in the first volume of the Dictionnaire de Botanique.19 Strychnobaillonine (1) represents a unique strychnan−strychnan-type structure with an unprecedented skeleton.20 The in vitro antiplasmodial activities of the pH 8 alkaloidic fraction and of compounds 1 and 2 were evaluated against the chloroquine-sensitive 3D7 strain of P. falciparum (Table 2). Compound 1 showed good activity [IC50 = 0.7 μg/mL (1.1 μM)]. According to WHO guidelines and basic criteria for drug discovery, a pure compound is defined as highly active if its IC50 is ≤1 μg/mL. This activity is close to that of other bisindole alkaloids such as matopensine and longicaudatine when tested on the FCA/Ghana chloroquino-sensitive strain. Previous studies have shown that monomers including strychnine and retuline were devoid of any antiplasmodial activity.5 One can easily conclude that the dimerization of monoindole alkaloids increases the antiplasmodial activity. In the course of the present work, strychnohexamine (2) also showed potent antiplasmodial activity [IC50 = 0.5 μg/mL (0.6 μM)] against the 3D7 strain of P. falciparum. It was more active compared to the FCA/Ghana strain during previous studies.4 Additional in vitro studies on the cytotoxic activity on normal human
■
EXPERIMENTAL SECTION
General Experimental Procedures. The optical rotation was measured on an ANALYS AA-10 automatic polarimeter. The UV spectrum was acquired using a U-2910 spectrophotometer. CD spectra were measured with a J-810 JASCO polarimeter. IR spectra were recorded on a Perkin-Elmer 1750 FTIR spectrometer. NMR spectra were recorded in CDCl3 or MeOH-d4 on a Bruker 500 MHz NMR AV II spectrometer equipped with a cryoprobe, with TMS as an internal reference. The HRESI mass spectra were recorded with a Bruker APEX-Qe 9.4T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer equipped with a hybrid quadrupole analyzer and using an electrospray source. Analytical TLC was performed on precoated Si gel F254 (Merck 1.05735) plates. After development, the dried plates were examined under short-wave (254 nm) or long-wave (366 nm) UV light and sprayed with 1% ceric sulfate in 10% sulfuric acid. LiChroprep Si 60 (43−60 μm, Merck 9336) was used for column chromatography. Si gel 60 PF254 (Art.1.07747, Merck) was used for purification of alkaloids by preparative TLC (1.25 mm thick, 20 × 20 cm Si gel plates). All solvents used were analytical grade (Merck). All calculations to determine the geometry of the molecule were performed with the Gaussian 03 program.13 Plant Material. The root bark of S. icaja was collected near Bertoua (East Region, Cameroon) in August 2012. The plant was authenticated at the herbarium of the Belgian National Botanical Garden at Meise, where a voucher specimen (BR0000005012251) was deposited. Extraction and Isolation of Compounds. The stem roots of S. icaja (400 g) were macerated with 300 mL of EtOAc−EtOH− NH4OH (96:3:1) and then percolated with EtOAc until complete extraction of alkaloids. The extract was concentrated under reduced pressure at 40 °C to yield 11 g of dry extract and then dissolved in EtOAc and extracted with 4% HOAc. The resulting acidic solution was basified to pH 8 with Na2CO3 and repeatedly extracted with CH2Cl2. The CH2Cl2 extracts obtained were dried over Na2SO4 and concentrated to yield an alkaloid extract (3 g), which was further fractionated first by column chromatography on 180 g of Merck LichroPrep Si 60 with CH2Cl2−MeOH mixtures. The 210 fractions of 20 mL were collected and pooled according to their TLC profile using the mixture EtOAc−iPrOH−NH4OH (60:35:15) as eluent. Sungucine (150 mg) crystallized from Frs. 45−90 collected from the column with the mixtures 96:4−92:8. Fractions 105−130 collected from the main column with the mixture CH2Cl2−MeOH (92:8) were further chromatographed on a silica gel column with CH2Cl2−MeOH, 0− 1081
dx.doi.org/10.1021/np400908u | J. Nat. Prod. 2014, 77, 1078−1082
Journal of Natural Products
Note
10%, and then purified through preparative TLC using EtOAc− iPrOH−NH4OH (60:35:15) to give bisnordihydrotoxiferine (10 mg) and strychnohexamine (18 mg). Fractions 137−165 obtained from the main column with the mixtures 92:8−90:10 were passed through a Sephadex LH-20 column using MeOH as eluent and then purified by preparative TLC using EtOAc−iPrOH−NH4OH (60:35:15) to give compound 1 (6 mg) and additional strychnohexamine (7 mg). Strychnobaillonine (1): amorphous, off-white solid; spraying with CeSO4−H2SO4 reagent on TLC immediately gave the compound a persistent red coloration (Supporting Information); [α]25 D −10 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 205 (2.5), 252 (1.7), 289 (1.4) nm; CD (c 0.33 × 10−3, MeOH) λmax (Δε) 230.5 (+0.56), 244 (−1.86), 257 (+3.66), 277 (−1.87), 295 (−0.04), 313 (−2.0) nm; IR (KBr) 3390, 2913, 2858, 1650, 1586, 1478, 1444, 1388, 1339, 1003, 730 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 613.35415 [M + H]+ (calcd for C40H45N4O2, m/z 613.3542); HRESIMS-MS m/z 613.35307.1832, 595.29545, 552.30058, 486.25375, 460.23797, 409.22712, 319.18031, 307.68205, 308.18380, 295.18031, 277.16981. In Vitro Antiplasmodial Assay. Activity against P. falciparum chloroquine-sensitive 3D7 strains was assessed following the procedure already described in Frederich et al.5 The parasites were obtained from Prof. Grellier (Museum d’Histoire Naturelle, Paris, France). Each alkaloid and extract was applied in a series of eight 3fold dilutions (final concentrations ranging from 0.09 to 200 μg/mL for an extract and from 0.02 to 50 μg/mL for a pure substance) on two rows of a 96-well microplate and were tested in duplicate (n = 2) or triplicate (n = 3). Parasite growth was estimated by determination of lactate dehydrogenase activity as described previously in Jonville et al.13 Artemisinin (98%, Sigma−Aldrich) and chloroquine were used as positive controls. In Vitro Cytotoxic Assay. Human normal fetal lung fibroblasts, WI-38, were maintained in continuous culture in a humid atmosphere at 37 °C and 5% CO2 in DMEM medium (Bio-Whittaker-LONZA), supplemented with 10% heat-inactivated fetal bovine serum, 1% Lglutamine (200 mM), and penicillin (100 IU/mL)−streptomycin (100 μg/mL) (Pen-Strep) (Bio-Whittaker-LONZA). The assays followed the procedure already described in Jonville et al.13 Dead cells were found to be smaller and to have a round shape. The viability of the cells was determined by adding WST-1 (Roche) tetrazolium salt as a cytotoxicity indicator and by reading the absorbance at 450 nm with a scanning multiwell spectrophotometer after about 1 h. Camptothecin (95% HPLC, Sigma−Aldrich) was used as a positive control. The selectivity index (SI) value allows the evaluation of the selective activity of the extracts/pure compound against the parasite compared to its toxicity for human cells. The SI value is calculated as the ratio between cytotoxic IC50 values and 3D7 parasitic IC50 values.
■
fellowship to A.T.T. at the Department of Pharmacognosy, University of Liège, Belgium. G.D. is research associate of the FRS-FNRS. G.D. acknowledges the Céci consortium for access to the HPC facilities installed in Liège and Louvain-la-Neuve. The authors are grateful to Ms. J. Widart, Laboratory of Mass Spectrometry, Giga Center, University of Liège, for the recording of mass spectra.
■
ASSOCIATED CONTENT
S Supporting Information *
Stereostructures of the H-16′β,H-17β; H-16′β,H-17α; H16′α,H-17α; and H-16′α,H-17β forms; NMR and mass spectra; possible biosynthesis pathway; TLC coloration behavior of strychnobaillonine (1); comparison of selected 13C NMR data of strychnobaillonine (1), N-desacetylretuline, and N-desacetylisoretuline. This material is available free of charge via the Internet at http://pubs.acs.org.
■
REFERENCES
(1) Philippe, G.; Angenot, L.; Tits, M.; Frederich, M. Toxicon 2004, 44, 405−416. (2) Mosango, D. M. 2008. Strychnos icaja Baill. In Plant Resources of Tropical Africa 11(1). Medicinal Plants 11(1); Schmelzer, G. H.; GuribFakim, A., Eds.; Foundation PROTA, Wageningen, The Netherlands; Backhuys Publishers: Leiden, The Netherlands, pp 640−642. (3) Delaude, C.; Delaude, L. Bull. Soc. R. Liège 1997, 66, 183−286. (4) Philippe, G.; De Mol, P.; Zèches-Hanrot, M.; Nuzillard, J.-M.; Tits, M.-H.; Angenot, L.; Frédérich, M. Phytochemistry 2003, 62, 623− 629. (5) Frédérich, M.; Jacquier, M. J.; Thepenier, P.; De Mol, P.; Tits, M.; Philippe, G.; Delaude, C.; Angenot, L.; Zeches-Hanrot, M. J. Nat. Prod. 2002, 65, 1381−1386. (6) Frédérich, M.; De Pauw, M.-C.; Llabrès, G.; Tits, M.; Hayette, M.-P.; Brandt, V.; Penelle, J.; De Mol, P.; Angenot, L. Planta Med. 2000, 66, 262−269. (7) Tchinda, A. T.; Tamze, V.; Ngono, A. R. N.; Ayimele, G. A.; Cao, M.; Angenot, L.; Frédérich, M. Phytochem. Lett. 2012a, 5, 108−113. (8) Tchinda, A. T.; Ngono, A. R. N.; Tamze, V.; Jonville, M.-C.; Cao, M.; Angenot, L.; Frédérich, M. Planta Med. 2012b, 78, 377−382. (9) Philippe, G.; Prost, E.; Nuzillard, J.-M.; Zèches-Hanrot, M.; Tits, M.; Angenot, L.; Frédérich, M. Tetrahedron Lett. 2002, 43, 3387− 3390. (10) Tits, M.; Angenot, L. Tetrahedron Lett. 1980, 21, 2439−2442. (11) Frederich, M.; De Pauw, M. C.; Prosperi, C.; Tits, M.; Brandt, V.; Penelle, J.; Hayette, M. P.; De Mol, P.; Angenot, L. J. Nat. Prod. 2001, 64, 12−16. (12) Zlotos, D. P. J. Nat. Prod. 2000, 63, 864−865. (13) Jonville, M.-C.; Dive, G.; Angenot, L.; Bero, J.; Tits, M.; Ollivier, E.; Frédérich, M.. Phytochemistry 2013, 87, 156−163. (14) Tavernier, D.; Anteunis, M. J. O.; Tits, M. J. G.; Angenot, L. J. G. Bull. Soc. Chim. Belg. 1978, 78, 595−607. (15) Gadi-Biala, R.; Tits, M.; Penelle, J.; Frederich, M.; Brandt, V.; Prosperi, C.; Llabres, G.; Angenot, L. J. Nat. Prod. 1998, 61, 139−141. (16) Klyne, W.; Buckingham, J. Atlas of Stereochemistry. Absolute Coǹuration of Organic Molecules; Chapman & Hall, London, 1974. (17) Becke, A. D. J. Chem. Phys. 1993, 98, 5648−5652. (18) Hehre, W. J.; Ditchfield, R.; Pople, J. A. J. Chem. Phys. 1972, 56, 2257−2261. (19) Baillon, H. Dictionnaire de Botanique; Tome, I., Ed.; Libraire Hachette et Cie: Paris, 1876. (20) Fu, Y.; Zhang, Y.; He, H.; Hou, L.; Di, Y.; Li, S.; Luo, X.; Hao, X. J. Nat. Prod. 2012, 75, 1987−1990.
AUTHOR INFORMATION
Corresponding Author
*Tel: +/237/76925929. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The Fund for Scientific Research (FNRS) is acknowledged for its support through grant no. 3.4533.10 and a research 1082
dx.doi.org/10.1021/np400908u | J. Nat. Prod. 2014, 77, 1078−1082
