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A new indole-alkaloid and a new phenolic-glycoside with cytotoxic activity from Strychnos fendleri a
a
b
Alírica I. Suárez , Monica Mancebo , Franco Delle Monache , María c
d
d
a
M. Tirri , Felipe Sojo , Francisco Arvelo & Stephen Tillett a
Facultad de Farmacia, Universidad Central de Venezuela, Caracas, Venezuela b
Dipartimento di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Università di Roma, Roma, Italy
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c
Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela d
IBE, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela Published online: 10 Mar 2015.
To cite this article: Alírica I. Suárez, Monica Mancebo, Franco Delle Monache, María M. Tirri, Felipe Sojo, Francisco Arvelo & Stephen Tillett (2015): A new indole-alkaloid and a new phenolic-glycoside with cytotoxic activity from Strychnos fendleri, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1019351 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1019351
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1019351
A new indole-alkaloid and a new phenolic-glycoside with cytotoxic activity from Strychnos fendleri Alı´rica I. Sua´reza*, Monica Manceboa, Franco Delle Monacheb, Marı´a M. Tirric, Felipe Sojod, Francisco Arvelod and Stephen Tilletta Facultad de Farmacia, Universidad Central de Venezuela, Caracas, Venezuela; bDipartimento di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita` di Roma, Roma, Italy; cFacultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela; dIBE, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela
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a
(Received 13 November 2014; final version received 10 February 2015)
N
H
H N H O
H
O
O H OH O
Strychnosinol
A new indole alkaloid strychnosinol (1) and a new phenolic-glycoside (2) were isolated from the bark and leaves of Strychnos fendleri Sprague & Sandwith, together with six known compounds reported for the first time in this species. The structures of these compounds were determined on the basis of spectroscopic data; mainly those obtained by using 1H and 13C NMR (1D and 2D) and mass spectrometry. Strychnosinol (1) and the phenolic glycoside (2) together with compounds 3 – 8 were evaluated for cytotoxicity against a panel of five tumour cell lines; IC50 values between 0.090 and 0.227 mM for the human tumour cell lines were observed for compound 2. Keywords: Strychnos fendleri; strychnosinol; phenolic glycoside; cytotoxicity
1. Introduction The genus Strychnos is well recognised for producing indole alkaloids (Zhao et al. 2012) which have been verified as the main biologically active constituent of its species (Tits et al. 1985; Rasoanaivo et al. 2001; Silva et al. 2005). Strychnos fendleri Sprague & Sandwith (Loganiaceae), a South American species is widespread in Venezuela, northeastern Colombia and in the Roraima region of Brazil. It is known as cruceto and palo de loro, and used as an ingredient in the preparation of curare (Steyermark et al. 2005). The stem bark has been used in local folk medicine for the treatment of malaria and general fevers (Quetin-Leclercq et al. 1990). A few decades ago, previous chemical investigation of this species collected in Brazil, led to the isolation of nine indole alkaloids (Galeffi et al. 1976; Galeffi & Marini-Bettolo 1980). As part of
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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one ongoing project directed towards the isolation of biologically active metabolites from plants used in Venezuelan traditional medicine, S. fendleri was reinvestigated. In this paper, we deal with the isolation and structural elucidation of a new indole alkaloid, namely strychnosinol (1), a new phenolic-glycoside (2) along with the known compounds N-acetylstrychnosplendine (3) (Galeffi & Marini-Bettolo 1980), vogeloside (4) (Sanchez et al. 2013), booneim (5) (MariniBettolo et al. 1983), 3-methoxyquercetin (6) (Peng et al. 2003), kaempferitrin (7) (Valente et al. 2009), and D -1-methyl-myo-inositol (8) (Angyal & Odier 1982), from bark and leaves. Compounds 4 – 8 (Figure S2) are reported for first time from this species. The cytotoxic activity of all these metabolites against five human cancer cell lines is also discussed.
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2. Results and discussion Compound 1 was obtained as a light brown amorphous solid, mp 140 – 1428C, [a ]25 D þ 118 (c ¼ 1.00, CHCl3). The HR-EI-MS afforded the positive ion at m/z 435.4697 [M þ Na]þ, suggesting the molecular formula C23H28N2O5. The IR spectra displayed absorption bands for a hydroxyl (3383 cm21), for ester groups (1738 cm21), for an amide group (1654 cm21), and 1595, 1479 for an aromatic ring. The 1H NMR (Table S1) spectrum was closely related to that of Nacetylstrychnosplendine (3) (Galeffi & Marini-Bettolo 1980). The pattern of multiplicity of the four aromatic protons suggested a benzene aromatic ring ortho-disubstituted. A common feature of 1 with N-acetylstrichnosplendine (3) having the N-acetyl indole group. The effect of this group on the NMR signals had been well documented in N-acetyl-indole alkaloids, due the restricted rotation around the N-acetyl group. The two signals of three proton singlets at dH 2.38 and 2.36 ppm, clearly evidenced the two rotamer (cisoid and transoid) conformations of the N-acetyl group mentioned in the previous reports of Strychnos alkaloids (Galeffi et al. 1976; Tits et al. 1985). Another methyl group, belonging to O-acetyl was at d 2.01. The major differences found in the 1H NMR spectrum compared with 3 were, the absence of the signal corresponding to 18-Me group, instead of this methyl group the presence of two triplets was observed at 3.60/3.74 ppm with J ¼ 4.9 Hz due to methylene signals on 18-CH2. The absence of a hydroxyl group on C-3 (dC 58.8 vs 95.4) and the new O-acetyl group on C-17 were also evidenced as differences in 1. The comparison of 1H and 13C NMR spectral data of 1 and those of N-acetylstrychnosplendine (3) (Galeffi & Marini-Bettolo 1980) suggested that those compounds would have the same carbon skeleton. The HMQC experiment was necessary to assign the corresponding protons with each carbon (Table S1). 1H and 13C long correlations from the HMBC spectrum led to the unambiguous establishment of the planar structure of strychnosinol (1) (Figure S1). The relative configurations of the eight stereocentres in 1 were established by comparison with the Nacetylstrychnosplendine (3), and by the NOE correlations revealed in the NOESY spectra. The observed NOEs between H-3/H-16, H-17/H-15, H-17/H-20, and also between H-19/H-20, as well as the absence of NOEs between H/2 and H/16 suggested an a orientation of H-3, H-15, H-16, H19 and H-20. By a similar analysis, the configurations of C-2, C-20 and C-17 were assigned. Thus, the new alkaloid from Strycnos fendleri, was assigned the structure 2b, 7b, 20a, 3a, 15a, 16a, 17a, 20a, strychnosinol (1). It is possible that this compound was not identified in the previous phytochemical studies of this species, which were reported many years ago, due to the drastic conditions of acidity and basicity, usually used then, in the isolation of alkaloids. Compound 2 was isolated as a syrup from the methanolic extract of the bark of S. fendleri. The molecular formula C20H28O14 was indicated by the HR-EI-MS (m/z 493.4419 [M 2 H]2). Examination of the spectral data of 1H and 13C NMR indicated that part of the molecule was constituted by a benzene type aromatic ring system, 1, 2, 4 -trisubstituted, revealed by a welldefined ABX system. Three signals, each of which integrated for one proton in the zone between dH 7.58 and 6.71, indicated this substitution pattern. The signal at d 7.36 (d, J ¼ 2.5 Hz) was assigned to H-2, H-6 was observed at dH 6.90 (dd, J ¼ 2.5, 8.5 Hz), and at dH 6.54 (d,
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J ¼ 2.5 Hz) H-5 was identified. The described signals of NMR resembled those of the wellknown protocatechuic acid (Chang et al. 2009). In addition to the described signals, a group of resonances between 3.92 and 2 2.82 ppm was considered belonging to glycosylated groups. At d 4.60 (d, J ¼ 7.75 Hz), one anomeric proton was evidenced, and among the mentioned signals the resonance of one oxygenated methyl group at 3.28 ppm was also observed. 13C spectrum was analysed together with a DEPT 135, which, in accordance with the molecular formula obtained, allowed considering a structure composed of 20 carbon atoms of the following nature: four quaternary carbons (171.4, 157.5,148.4 and 120.4); one oxygenated methylene (60.3), one methoxy group (56.6) and the rest were all oxygenated methines belonging to the carbohydrate moieties. With the help of the 2D HMQC spectrum, all carbons were correlated with their corresponding protons. From the analysis of the constant coupling of the anomeric proton (J ¼ 7.75 Hz) and the rest of signals in NMR, it was evident that b-D -glucopyranose was part of the analysed molecule. The number of carbons and the substitution pattern clearly indicated the presence a carboxyl acid, in addition to the methoxy group and the glucose sugar. The remaining resonances of six oxygenated methine carbons were considered also belonging to a carbohydrate moiety, but it was also evident that only one anomeric proton was present in the structure. A careful analysis of the spectra showed that the analysed compound contained 1-O-methylmyo-inositol, which was also isolated and characterised from this extract (Table S2). A comparison of the NMR data confirmed this assumption. However, to corroborate the identity of the carbohydrate subunits, an acid hydrolysis using 1N HCl was performed on 2, and by comparative TLC with authentic samples of b-D -glucose and D -1-O-methyl-myo-inositol both compounds were confirmed. The COSY and HMBC were essential to establish the linkages and positions of the functional groups on the aromatic ring (Figure S1). The linkage of glucose on the phenyl ring was supported by the correlation between the anomeric proton (4.60 ppm) and C-4 (148 ppm). The interglucosidic linkage between the glucose and methyl-myo-inositol was also evidenced by the HMBC correlations between the proton at (3.15 ppm H-300 ) with the anomeric carbon (102.4 ppm), and with (71.8 C-20 ). Correlations between the protons of the methoxy group (3.28 ppm) with 81.7 (C-100 ) and 68.3 (C-200 ) help to establish the linkage between the sugar moieties. There are several other correlations in the COSY and HMBC experiments that agree with the proposed structure of 2 (Figure 1). The assigned configuration was obtained by positive NOEs in the NOESY experiments. Support for the structure was afforded by comparisons with the literature data (Angyal et al. 1983; Fujimatu et al. 2003; Lin et al. 2007; Lee et al. 2011). Therefore, after this comparison, and based on the analysis of all spectral data, we propose as a new natural product the 4-[-O-b-glucopyranosyl-(2 ! 3)-D -1-O-methyl-myoinositol]-3-hydroxy-benzoic acid (2). Phenolic glycoside and polysaccharides had previously been isolated from some Strychnos species (Bisset et al. 1989; Corsaro et al. 1995). The isolated compounds (1 – 8) were tested for cytotoxic activity against the following human tumour cell lines, PC-3 (prostate carcinoma), HeLa (cervix carcinoma), MCF-7 (breast carcinoma, without over-expression of the HER2/c-erb-2 gene), SKBr3 (breast carcinoma, in which the HER2/c-erb-2 gene is over-expressed, moreover HER-2 over-expressing mammary tumours are commonly indicative of a poor prognosis in patients), PANC-1 (pancreas carcinoma), and normal fibroblasts (dermis) as control cells using the MTT method. Among all compounds tested, only the new compounds 1 and 2 showed cytotoxic activity and more selectivity for tumour cell lines SKBr3 and PANC-1, with respect to control cells, but not for other tumour cell lines. Compound 3 presents the highest cytotoxicity for all cell lines, being less effective in the control cells (Figure S3). The remaining compounds tested showed cytotoxic activity for some cell lines. Interestingly, the IC50 values obtained with the normal dermis fibroblasts indicate that the tumour cells are more sensitive to the natural compounds, since the IC50 obtained with the control cell is higher than IC50 shown for the tumour cells. The evaluation of the new compounds against malaria is in progress in our laboratories.
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Figure 1. Structure of new compounds 1 and 2.
3. Experimental 3.1. Plant material Aerial parts of Strychnos fendleri Sprague & Sandwith were collected in Maniapure, Bolı´var state, Venezuela, in December 2012. The plant was identified by one of us (S.T.). A voucher specimen (MYF 28423) has been deposited at the Herbarium ‘Dr Vı´ctor Manuel Ovalles’ of the Pharmacy Faculty, Universidad Central de Venezuela.
3.2. Chemicals and general experiments procedures Melting points were determined on a Kofler hot-stage melting point instrument and are uncorrected. Optical rotations were acquired with an ATAGO Polax-2L polarimeter (Atago USA, WA, USA). IR spectra were recorded on a Shimadzu 470 (Shimadzu Corp., Tokyo, Japan), EI-MS were obtained on a Varian Saturn 2000 (Varian, Walnut Creek, CA, USA), and HR-EI-MS were done with a Finnigan Trace mass spectrometer (Thermo Finnigan, West Chester, OH, USA). NMR spectra were measured on a JEOL 270 (Jeol, Tokyo, Japan) MHz, and a Bruker AMX-500 (Bruker, Rheinstetten, Germany). Chemical shifts are given in ppm referenced to the residual non-deuterated solvent signals. Column chromatography (CC) was performed using Si gel (70 – 230 mesh) from Scharlau, TLC analysis was carried out using plastic precoated plates (Merck AG, Darmstadt, Germany, Si gel plates GF254, 0.2 mm), and the spots were visualised using a UV lamp l ¼ 254 nm or by spraying with p-anisaldehyde. All solvents used were of analytical grade.
3.3. Extraction and isolation The air-dried and powdered stems and leaves (170 g) of S. fendleri were extracted with MeOH (2 L) for 12 h using a Soxhlet apparatus; the extract was concentrated in vacuo. The residue was
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dissolved in a mixture of MeOH:H2O (1:1), and then partitioned in turn with hexane, dichloromethane and EtOAc, to afford a hexane (1.95 g), CH2Cl2 (1.25 g), EtOAc (0.98 g) and a residual hydro-methanolic fraction (2.35 g). The CH2Cl2 fraction was chromatographed on silica gel using a mixture of hexane/EtOAc step-gradient ranging from 90:10 to 0:100 to yield 25 fractions. After purification of selected and combined fractions, N-acetylstrychnosplendine (3) (53.7 mg) was isolated. Other known metabolites such as vogeloside (4) (27.6 mg), booneim (5) (32.6 mg) and 3-methoxy quercetin (6) (47.3) were also isolated and identified. Column chromatography of the EtOAc fraction over Si gel, using a CHCl3 –EtOAc stepgradient ranging from 90:10 to 0:100 gave 15 fractions. Fraction 6 eluted with CHCl3 –EtOAc (50:50) furnished strychnosinol (1) (55.0 mg); fraction 9 eluted with EtOAc – MeOH (9:1) afforded kaempferitrin (7) (134 mg), fraction 12 eluted with EtOAc:MeOH (85:15) yielded phenyl-glycoside (2) (49.3 mg), and fraction 14 with EtOAc:MeOH (75:25) D -1-methylmyoinositol (8) (68.2 mg). Strychnosinol (1): Light brown amorphous solid mp 140 – 1428C [a ]25 D þ 118 (c ¼ 1.00, CHCl3); IR gmax film 3883, 2024, 1738, 1654, 1595, 1479 cm21; 1H NMR (500 MHz, CDCl3): d 4.34 (1H, t, J ¼ 10.7 Hz, H-2), 4.08 (1H, m, H-3), 3.87 (2H, m, H-5), 1.73/2.0 (2H, m, H-6), 2.83 (1H, m, H-7), 7.05 (1H, d, J ¼ 7.6 Hz, H-9), 7.13 (1H, t, J ¼ 3.15 Hz, H-10), 7.28 (1H, t, J ¼ 7.6 Hz, H-11), 7.14/7.97 (1H, d, J ¼ 7.8 Hz, H-12), 1.60/1.73 (2H, d, J ¼ 7.6 Hz, H-14), 2.0 (1H, m, H-15), 1.99 (1H, d, J ¼ 8.8 Hz, H-16), 5.82 (1H, brs, H-17), 3.60/3.74 (2H, d, J ¼ 4.9 Hz, H-18), 3.62 (1H, m, H-19), 2.83 (1H, d, J ¼ 10.8 Hz, H-20), 3.50/4.0 (2H, m, H-21), 2.36/2.38 (3H, s, CH3-CO-N), 1.97/2.01 (3H, s, CH3CO-O); 13C NMR (125 MHz, CDCl3): 63.5 (d, C-2), 58.9 (d, C-3), 53.2 (t, C-5), 37.7 (t, C-6), 53.9 (d, C-7), 132.9 (s, C-8), 124.7 (d, C-9), 121.6 (d, C-10), 128.7 (d, C-11), 115.9/119.0 (d, C-12), 142.3 (s, C-13), 25.1 (t, C-14), 36.1 (d, C-15), 44.7 (d, C-16), 102.2 (d, C-17), 72.3 (t, C-18), 75.3 (d, C-19), 33.1 (d, C-20), 51.4 (t, C21), 168.9/170.1 (s, CO-N), 21.7/24.1 (C, CH3-CO-N), 160.4/168.8 (s, CO-O), 18.1/21.0 (c, CH3-CO-O); HR-EI-MS m/z 435.4697 [M þ Na]þ (calcd for C23H27N2O5Na, 435.4695). 4-[-O-b-glucopyranosyl-(2 ! 3)-D -1-O-methyl-myo-inositol]-3-hydroxy-benzoic acid (2): Syrup, [a ]25 D 2 67.4 (c ¼ 0.1, MeOH); IR gmax film 3405, 1737, 1612, 1651, 1510, 1456, 1073 cm21; 1H and 13C NMR data: see Table S2; 1H NMR (500 MHz, DMSO): d 7.36 (1H, d, J ¼ 2.15 Hz, H-2), 6.54 (1H, d, J ¼ 8.5 Hz, H-5), 6.90 (1H, dd, J ¼ 8.5, 2.15 Hz, H6), 4.60 (1H, d, J ¼ 7.75 Hz, H-10 ), 3.44 (1H, m, H-20 ), 3.45 (1H, m, H-30 ), 3.13 (1H, dd, J ¼ 13.5, 2.6 Hz, H40 ), 3.46 (1H, m, H-50 ), 3.43/3.60 (2H, m, H-60 ), 2.82 (1H, dd, 9.6, 2.5 Hz, H-100 ), 3.91 (1H, t, J ¼ 2.5 Hz, H-200 ), 3.15 (1H, d, J ¼ 5.9 Hz, H-300 ), 3.20 (1H, dd, J ¼ 13.1, 3.1 Hz, H-400 ), 2.90 (1H, t, J ¼ 9.6 Hz, H-500 ), 3.18 (1H, m, H-600 ), 3.28 (3H, s, OCH3). 13C NMR (125 MHz, DMSO): d 120.5 (s, C-1), 118.2 (d, C-2), 157.5 (s, C-3), 148.4 (s, C-4), 115.7 (d, C-5), 121.0 (d, C-6), 102.4 (d, C-10 ), 73.8 (d, C-20 ), 76.6 (d, C-30 ), 69.7 (d, C-40 ), 76.9 (d, C-50 ), 60.3 (d, C-60 ), 81.7 (d, C-100 ), 68.3 (d, C-200 ), 71.7 (d, C-300 ), 73.4 (d, C-400 ), 75.3 (d, C-500 ), 72.5 (d, C-600 ), 56.6 (C, CH3O), 171.4 (s, COOH); HR-EI-MS m/z 493.4493 [M 2 H]2 (calcd for C20H30O14, 494.4498). 3.4. Cytotoxicity assay Cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide) assay, which is based on the ability of viable cells to metabolically reduce a yellow tetrazolium salt (MTT; Sigma, St. Louis, MO, USA) to purple crystals of formazan (Mosmann 1983). This reaction takes place when mitochondrial reductases are active. Cells were grown in 96-well plates (5 £ 105cells/well) and incubated at 378C for 72 h with the natural compounds at concentrations of 0, 0.001, 0.01, 0.1, 1, 5, 10, 15, 25 and 100 mg/mL, respectively, in a humidified atmosphere with 5% CO2. The final concentration of the DMSO in culture medium was always lower than 1% (v/v), a concentration that has neither cytotoxic effect nor causes any interference with the colorimetric detection methods. After incubation, the medium
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was removed and the cells were treated with 100 mL MTT for 3 h at 378C. Subsequently, 100 mL DMSO was added to the mixture. The solubilised formazan product was quantified with the help of a microplate reader TECAN-Sunrisee at 570 nm (Tecan Group Ltd, Ma¨nnedorf, Switzerland). Taxol (Bristol-Myers Squibb, Brystol-Myers, Syracuse, NY, USA) was used as a positive control in the assay. The experiment was carried out in triplicate. The IC50 value was defined as the concentration of compound required to induce a 50% reduction of absorbance as compared with control, non-treated cells.
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Supplementary material Supplementary material relating to this paper is available online, alongside Tables S1 and S2 and Figures S1 –S8.
Funding This work was supported by FONACIT [grant number PEII-705], [grant number CFI-00293].
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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new indole-alkaloid and a new phenolic-glycoside with cytotoxic activity from Strychnos fendleri a
a
b
Alírica I. Suárez , Monica Mancebo , Franco Delle Monache , María c
d
d
a
M. Tirri , Felipe Sojo , Francisco Arvelo & Stephen Tillett a
Facultad de Farmacia, Universidad Central de Venezuela, Caracas, Venezuela b
Dipartimento di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Università di Roma, Roma, Italy
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Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela d
IBE, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela Published online: 10 Mar 2015.
To cite this article: Alírica I. Suárez, Monica Mancebo, Franco Delle Monache, María M. Tirri, Felipe Sojo, Francisco Arvelo & Stephen Tillett (2015): A new indole-alkaloid and a new phenolic-glycoside with cytotoxic activity from Strychnos fendleri, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1019351 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1019351
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1019351
A new indole-alkaloid and a new phenolic-glycoside with cytotoxic activity from Strychnos fendleri Alı´rica I. Sua´reza*, Monica Manceboa, Franco Delle Monacheb, Marı´a M. Tirric, Felipe Sojod, Francisco Arvelod and Stephen Tilletta Facultad de Farmacia, Universidad Central de Venezuela, Caracas, Venezuela; bDipartimento di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita` di Roma, Roma, Italy; cFacultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela; dIBE, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela
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a
(Received 13 November 2014; final version received 10 February 2015)
N
H
H N H O
H
O
O H OH O
Strychnosinol
A new indole alkaloid strychnosinol (1) and a new phenolic-glycoside (2) were isolated from the bark and leaves of Strychnos fendleri Sprague & Sandwith, together with six known compounds reported for the first time in this species. The structures of these compounds were determined on the basis of spectroscopic data; mainly those obtained by using 1H and 13C NMR (1D and 2D) and mass spectrometry. Strychnosinol (1) and the phenolic glycoside (2) together with compounds 3 – 8 were evaluated for cytotoxicity against a panel of five tumour cell lines; IC50 values between 0.090 and 0.227 mM for the human tumour cell lines were observed for compound 2. Keywords: Strychnos fendleri; strychnosinol; phenolic glycoside; cytotoxicity
1. Introduction The genus Strychnos is well recognised for producing indole alkaloids (Zhao et al. 2012) which have been verified as the main biologically active constituent of its species (Tits et al. 1985; Rasoanaivo et al. 2001; Silva et al. 2005). Strychnos fendleri Sprague & Sandwith (Loganiaceae), a South American species is widespread in Venezuela, northeastern Colombia and in the Roraima region of Brazil. It is known as cruceto and palo de loro, and used as an ingredient in the preparation of curare (Steyermark et al. 2005). The stem bark has been used in local folk medicine for the treatment of malaria and general fevers (Quetin-Leclercq et al. 1990). A few decades ago, previous chemical investigation of this species collected in Brazil, led to the isolation of nine indole alkaloids (Galeffi et al. 1976; Galeffi & Marini-Bettolo 1980). As part of
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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one ongoing project directed towards the isolation of biologically active metabolites from plants used in Venezuelan traditional medicine, S. fendleri was reinvestigated. In this paper, we deal with the isolation and structural elucidation of a new indole alkaloid, namely strychnosinol (1), a new phenolic-glycoside (2) along with the known compounds N-acetylstrychnosplendine (3) (Galeffi & Marini-Bettolo 1980), vogeloside (4) (Sanchez et al. 2013), booneim (5) (MariniBettolo et al. 1983), 3-methoxyquercetin (6) (Peng et al. 2003), kaempferitrin (7) (Valente et al. 2009), and D -1-methyl-myo-inositol (8) (Angyal & Odier 1982), from bark and leaves. Compounds 4 – 8 (Figure S2) are reported for first time from this species. The cytotoxic activity of all these metabolites against five human cancer cell lines is also discussed.
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2. Results and discussion Compound 1 was obtained as a light brown amorphous solid, mp 140 – 1428C, [a ]25 D þ 118 (c ¼ 1.00, CHCl3). The HR-EI-MS afforded the positive ion at m/z 435.4697 [M þ Na]þ, suggesting the molecular formula C23H28N2O5. The IR spectra displayed absorption bands for a hydroxyl (3383 cm21), for ester groups (1738 cm21), for an amide group (1654 cm21), and 1595, 1479 for an aromatic ring. The 1H NMR (Table S1) spectrum was closely related to that of Nacetylstrychnosplendine (3) (Galeffi & Marini-Bettolo 1980). The pattern of multiplicity of the four aromatic protons suggested a benzene aromatic ring ortho-disubstituted. A common feature of 1 with N-acetylstrichnosplendine (3) having the N-acetyl indole group. The effect of this group on the NMR signals had been well documented in N-acetyl-indole alkaloids, due the restricted rotation around the N-acetyl group. The two signals of three proton singlets at dH 2.38 and 2.36 ppm, clearly evidenced the two rotamer (cisoid and transoid) conformations of the N-acetyl group mentioned in the previous reports of Strychnos alkaloids (Galeffi et al. 1976; Tits et al. 1985). Another methyl group, belonging to O-acetyl was at d 2.01. The major differences found in the 1H NMR spectrum compared with 3 were, the absence of the signal corresponding to 18-Me group, instead of this methyl group the presence of two triplets was observed at 3.60/3.74 ppm with J ¼ 4.9 Hz due to methylene signals on 18-CH2. The absence of a hydroxyl group on C-3 (dC 58.8 vs 95.4) and the new O-acetyl group on C-17 were also evidenced as differences in 1. The comparison of 1H and 13C NMR spectral data of 1 and those of N-acetylstrychnosplendine (3) (Galeffi & Marini-Bettolo 1980) suggested that those compounds would have the same carbon skeleton. The HMQC experiment was necessary to assign the corresponding protons with each carbon (Table S1). 1H and 13C long correlations from the HMBC spectrum led to the unambiguous establishment of the planar structure of strychnosinol (1) (Figure S1). The relative configurations of the eight stereocentres in 1 were established by comparison with the Nacetylstrychnosplendine (3), and by the NOE correlations revealed in the NOESY spectra. The observed NOEs between H-3/H-16, H-17/H-15, H-17/H-20, and also between H-19/H-20, as well as the absence of NOEs between H/2 and H/16 suggested an a orientation of H-3, H-15, H-16, H19 and H-20. By a similar analysis, the configurations of C-2, C-20 and C-17 were assigned. Thus, the new alkaloid from Strycnos fendleri, was assigned the structure 2b, 7b, 20a, 3a, 15a, 16a, 17a, 20a, strychnosinol (1). It is possible that this compound was not identified in the previous phytochemical studies of this species, which were reported many years ago, due to the drastic conditions of acidity and basicity, usually used then, in the isolation of alkaloids. Compound 2 was isolated as a syrup from the methanolic extract of the bark of S. fendleri. The molecular formula C20H28O14 was indicated by the HR-EI-MS (m/z 493.4419 [M 2 H]2). Examination of the spectral data of 1H and 13C NMR indicated that part of the molecule was constituted by a benzene type aromatic ring system, 1, 2, 4 -trisubstituted, revealed by a welldefined ABX system. Three signals, each of which integrated for one proton in the zone between dH 7.58 and 6.71, indicated this substitution pattern. The signal at d 7.36 (d, J ¼ 2.5 Hz) was assigned to H-2, H-6 was observed at dH 6.90 (dd, J ¼ 2.5, 8.5 Hz), and at dH 6.54 (d,
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J ¼ 2.5 Hz) H-5 was identified. The described signals of NMR resembled those of the wellknown protocatechuic acid (Chang et al. 2009). In addition to the described signals, a group of resonances between 3.92 and 2 2.82 ppm was considered belonging to glycosylated groups. At d 4.60 (d, J ¼ 7.75 Hz), one anomeric proton was evidenced, and among the mentioned signals the resonance of one oxygenated methyl group at 3.28 ppm was also observed. 13C spectrum was analysed together with a DEPT 135, which, in accordance with the molecular formula obtained, allowed considering a structure composed of 20 carbon atoms of the following nature: four quaternary carbons (171.4, 157.5,148.4 and 120.4); one oxygenated methylene (60.3), one methoxy group (56.6) and the rest were all oxygenated methines belonging to the carbohydrate moieties. With the help of the 2D HMQC spectrum, all carbons were correlated with their corresponding protons. From the analysis of the constant coupling of the anomeric proton (J ¼ 7.75 Hz) and the rest of signals in NMR, it was evident that b-D -glucopyranose was part of the analysed molecule. The number of carbons and the substitution pattern clearly indicated the presence a carboxyl acid, in addition to the methoxy group and the glucose sugar. The remaining resonances of six oxygenated methine carbons were considered also belonging to a carbohydrate moiety, but it was also evident that only one anomeric proton was present in the structure. A careful analysis of the spectra showed that the analysed compound contained 1-O-methylmyo-inositol, which was also isolated and characterised from this extract (Table S2). A comparison of the NMR data confirmed this assumption. However, to corroborate the identity of the carbohydrate subunits, an acid hydrolysis using 1N HCl was performed on 2, and by comparative TLC with authentic samples of b-D -glucose and D -1-O-methyl-myo-inositol both compounds were confirmed. The COSY and HMBC were essential to establish the linkages and positions of the functional groups on the aromatic ring (Figure S1). The linkage of glucose on the phenyl ring was supported by the correlation between the anomeric proton (4.60 ppm) and C-4 (148 ppm). The interglucosidic linkage between the glucose and methyl-myo-inositol was also evidenced by the HMBC correlations between the proton at (3.15 ppm H-300 ) with the anomeric carbon (102.4 ppm), and with (71.8 C-20 ). Correlations between the protons of the methoxy group (3.28 ppm) with 81.7 (C-100 ) and 68.3 (C-200 ) help to establish the linkage between the sugar moieties. There are several other correlations in the COSY and HMBC experiments that agree with the proposed structure of 2 (Figure 1). The assigned configuration was obtained by positive NOEs in the NOESY experiments. Support for the structure was afforded by comparisons with the literature data (Angyal et al. 1983; Fujimatu et al. 2003; Lin et al. 2007; Lee et al. 2011). Therefore, after this comparison, and based on the analysis of all spectral data, we propose as a new natural product the 4-[-O-b-glucopyranosyl-(2 ! 3)-D -1-O-methyl-myoinositol]-3-hydroxy-benzoic acid (2). Phenolic glycoside and polysaccharides had previously been isolated from some Strychnos species (Bisset et al. 1989; Corsaro et al. 1995). The isolated compounds (1 – 8) were tested for cytotoxic activity against the following human tumour cell lines, PC-3 (prostate carcinoma), HeLa (cervix carcinoma), MCF-7 (breast carcinoma, without over-expression of the HER2/c-erb-2 gene), SKBr3 (breast carcinoma, in which the HER2/c-erb-2 gene is over-expressed, moreover HER-2 over-expressing mammary tumours are commonly indicative of a poor prognosis in patients), PANC-1 (pancreas carcinoma), and normal fibroblasts (dermis) as control cells using the MTT method. Among all compounds tested, only the new compounds 1 and 2 showed cytotoxic activity and more selectivity for tumour cell lines SKBr3 and PANC-1, with respect to control cells, but not for other tumour cell lines. Compound 3 presents the highest cytotoxicity for all cell lines, being less effective in the control cells (Figure S3). The remaining compounds tested showed cytotoxic activity for some cell lines. Interestingly, the IC50 values obtained with the normal dermis fibroblasts indicate that the tumour cells are more sensitive to the natural compounds, since the IC50 obtained with the control cell is higher than IC50 shown for the tumour cells. The evaluation of the new compounds against malaria is in progress in our laboratories.
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Figure 1. Structure of new compounds 1 and 2.
3. Experimental 3.1. Plant material Aerial parts of Strychnos fendleri Sprague & Sandwith were collected in Maniapure, Bolı´var state, Venezuela, in December 2012. The plant was identified by one of us (S.T.). A voucher specimen (MYF 28423) has been deposited at the Herbarium ‘Dr Vı´ctor Manuel Ovalles’ of the Pharmacy Faculty, Universidad Central de Venezuela.
3.2. Chemicals and general experiments procedures Melting points were determined on a Kofler hot-stage melting point instrument and are uncorrected. Optical rotations were acquired with an ATAGO Polax-2L polarimeter (Atago USA, WA, USA). IR spectra were recorded on a Shimadzu 470 (Shimadzu Corp., Tokyo, Japan), EI-MS were obtained on a Varian Saturn 2000 (Varian, Walnut Creek, CA, USA), and HR-EI-MS were done with a Finnigan Trace mass spectrometer (Thermo Finnigan, West Chester, OH, USA). NMR spectra were measured on a JEOL 270 (Jeol, Tokyo, Japan) MHz, and a Bruker AMX-500 (Bruker, Rheinstetten, Germany). Chemical shifts are given in ppm referenced to the residual non-deuterated solvent signals. Column chromatography (CC) was performed using Si gel (70 – 230 mesh) from Scharlau, TLC analysis was carried out using plastic precoated plates (Merck AG, Darmstadt, Germany, Si gel plates GF254, 0.2 mm), and the spots were visualised using a UV lamp l ¼ 254 nm or by spraying with p-anisaldehyde. All solvents used were of analytical grade.
3.3. Extraction and isolation The air-dried and powdered stems and leaves (170 g) of S. fendleri were extracted with MeOH (2 L) for 12 h using a Soxhlet apparatus; the extract was concentrated in vacuo. The residue was
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dissolved in a mixture of MeOH:H2O (1:1), and then partitioned in turn with hexane, dichloromethane and EtOAc, to afford a hexane (1.95 g), CH2Cl2 (1.25 g), EtOAc (0.98 g) and a residual hydro-methanolic fraction (2.35 g). The CH2Cl2 fraction was chromatographed on silica gel using a mixture of hexane/EtOAc step-gradient ranging from 90:10 to 0:100 to yield 25 fractions. After purification of selected and combined fractions, N-acetylstrychnosplendine (3) (53.7 mg) was isolated. Other known metabolites such as vogeloside (4) (27.6 mg), booneim (5) (32.6 mg) and 3-methoxy quercetin (6) (47.3) were also isolated and identified. Column chromatography of the EtOAc fraction over Si gel, using a CHCl3 –EtOAc stepgradient ranging from 90:10 to 0:100 gave 15 fractions. Fraction 6 eluted with CHCl3 –EtOAc (50:50) furnished strychnosinol (1) (55.0 mg); fraction 9 eluted with EtOAc – MeOH (9:1) afforded kaempferitrin (7) (134 mg), fraction 12 eluted with EtOAc:MeOH (85:15) yielded phenyl-glycoside (2) (49.3 mg), and fraction 14 with EtOAc:MeOH (75:25) D -1-methylmyoinositol (8) (68.2 mg). Strychnosinol (1): Light brown amorphous solid mp 140 – 1428C [a ]25 D þ 118 (c ¼ 1.00, CHCl3); IR gmax film 3883, 2024, 1738, 1654, 1595, 1479 cm21; 1H NMR (500 MHz, CDCl3): d 4.34 (1H, t, J ¼ 10.7 Hz, H-2), 4.08 (1H, m, H-3), 3.87 (2H, m, H-5), 1.73/2.0 (2H, m, H-6), 2.83 (1H, m, H-7), 7.05 (1H, d, J ¼ 7.6 Hz, H-9), 7.13 (1H, t, J ¼ 3.15 Hz, H-10), 7.28 (1H, t, J ¼ 7.6 Hz, H-11), 7.14/7.97 (1H, d, J ¼ 7.8 Hz, H-12), 1.60/1.73 (2H, d, J ¼ 7.6 Hz, H-14), 2.0 (1H, m, H-15), 1.99 (1H, d, J ¼ 8.8 Hz, H-16), 5.82 (1H, brs, H-17), 3.60/3.74 (2H, d, J ¼ 4.9 Hz, H-18), 3.62 (1H, m, H-19), 2.83 (1H, d, J ¼ 10.8 Hz, H-20), 3.50/4.0 (2H, m, H-21), 2.36/2.38 (3H, s, CH3-CO-N), 1.97/2.01 (3H, s, CH3CO-O); 13C NMR (125 MHz, CDCl3): 63.5 (d, C-2), 58.9 (d, C-3), 53.2 (t, C-5), 37.7 (t, C-6), 53.9 (d, C-7), 132.9 (s, C-8), 124.7 (d, C-9), 121.6 (d, C-10), 128.7 (d, C-11), 115.9/119.0 (d, C-12), 142.3 (s, C-13), 25.1 (t, C-14), 36.1 (d, C-15), 44.7 (d, C-16), 102.2 (d, C-17), 72.3 (t, C-18), 75.3 (d, C-19), 33.1 (d, C-20), 51.4 (t, C21), 168.9/170.1 (s, CO-N), 21.7/24.1 (C, CH3-CO-N), 160.4/168.8 (s, CO-O), 18.1/21.0 (c, CH3-CO-O); HR-EI-MS m/z 435.4697 [M þ Na]þ (calcd for C23H27N2O5Na, 435.4695). 4-[-O-b-glucopyranosyl-(2 ! 3)-D -1-O-methyl-myo-inositol]-3-hydroxy-benzoic acid (2): Syrup, [a ]25 D 2 67.4 (c ¼ 0.1, MeOH); IR gmax film 3405, 1737, 1612, 1651, 1510, 1456, 1073 cm21; 1H and 13C NMR data: see Table S2; 1H NMR (500 MHz, DMSO): d 7.36 (1H, d, J ¼ 2.15 Hz, H-2), 6.54 (1H, d, J ¼ 8.5 Hz, H-5), 6.90 (1H, dd, J ¼ 8.5, 2.15 Hz, H6), 4.60 (1H, d, J ¼ 7.75 Hz, H-10 ), 3.44 (1H, m, H-20 ), 3.45 (1H, m, H-30 ), 3.13 (1H, dd, J ¼ 13.5, 2.6 Hz, H40 ), 3.46 (1H, m, H-50 ), 3.43/3.60 (2H, m, H-60 ), 2.82 (1H, dd, 9.6, 2.5 Hz, H-100 ), 3.91 (1H, t, J ¼ 2.5 Hz, H-200 ), 3.15 (1H, d, J ¼ 5.9 Hz, H-300 ), 3.20 (1H, dd, J ¼ 13.1, 3.1 Hz, H-400 ), 2.90 (1H, t, J ¼ 9.6 Hz, H-500 ), 3.18 (1H, m, H-600 ), 3.28 (3H, s, OCH3). 13C NMR (125 MHz, DMSO): d 120.5 (s, C-1), 118.2 (d, C-2), 157.5 (s, C-3), 148.4 (s, C-4), 115.7 (d, C-5), 121.0 (d, C-6), 102.4 (d, C-10 ), 73.8 (d, C-20 ), 76.6 (d, C-30 ), 69.7 (d, C-40 ), 76.9 (d, C-50 ), 60.3 (d, C-60 ), 81.7 (d, C-100 ), 68.3 (d, C-200 ), 71.7 (d, C-300 ), 73.4 (d, C-400 ), 75.3 (d, C-500 ), 72.5 (d, C-600 ), 56.6 (C, CH3O), 171.4 (s, COOH); HR-EI-MS m/z 493.4493 [M 2 H]2 (calcd for C20H30O14, 494.4498). 3.4. Cytotoxicity assay Cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide) assay, which is based on the ability of viable cells to metabolically reduce a yellow tetrazolium salt (MTT; Sigma, St. Louis, MO, USA) to purple crystals of formazan (Mosmann 1983). This reaction takes place when mitochondrial reductases are active. Cells were grown in 96-well plates (5 £ 105cells/well) and incubated at 378C for 72 h with the natural compounds at concentrations of 0, 0.001, 0.01, 0.1, 1, 5, 10, 15, 25 and 100 mg/mL, respectively, in a humidified atmosphere with 5% CO2. The final concentration of the DMSO in culture medium was always lower than 1% (v/v), a concentration that has neither cytotoxic effect nor causes any interference with the colorimetric detection methods. After incubation, the medium
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was removed and the cells were treated with 100 mL MTT for 3 h at 378C. Subsequently, 100 mL DMSO was added to the mixture. The solubilised formazan product was quantified with the help of a microplate reader TECAN-Sunrisee at 570 nm (Tecan Group Ltd, Ma¨nnedorf, Switzerland). Taxol (Bristol-Myers Squibb, Brystol-Myers, Syracuse, NY, USA) was used as a positive control in the assay. The experiment was carried out in triplicate. The IC50 value was defined as the concentration of compound required to induce a 50% reduction of absorbance as compared with control, non-treated cells.
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Supplementary material Supplementary material relating to this paper is available online, alongside Tables S1 and S2 and Figures S1 –S8.
Funding This work was supported by FONACIT [grant number PEII-705], [grant number CFI-00293].
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