Original Papers
Antiplasmodial Isoflavanes and Pterocarpans from Apoplanesia paniculata*
Authors
Qingxi Su 1, Priscilla Krai 2, Michael Goetz 3, Maria B. Cassera 2, David G. I. Kingston 1
Affiliations
1 2 3
Key words " antimalarial activity l " isoflavanes l " pterocarpans l " Apoplansia paniculata l (Fabaceae)
Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia, United States Department of Biochemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia, United States Natural Products Discovery Institute, Doylestown, Pennsylvania, United States
Abstract !
Bioassay-guided fractionation of an EtOH extract of the roots of the plant Apoplanesia paniculata (Fabaceae) led to the isolation of the three known compounds amorphaquinone (1), pendulone (2), and melilotocarpan C (3), and the two new pterocarpans 4 and 5. Compounds 1 and 2 exhibited good antiplasmodial activity with IC50 values of 5.7 ± 1.5 and 7.0 ± 0.8 µM, respectively. Compound
Introduction !
received revised accepted
February 24, 2015 April 2, 2015 April 7, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1546036 Published online May 27, 2015 Planta Med 2015; 81: 1128–1132 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. David G. I. Kingston Virginia Tech Davidson Hall Department of Chemistry and Virginia Tech Center for Drug Discovery 1040 Drillfield Drive Blacksburg, Virginia 24061 United States Phone: + 1 54 02 31 65 70 Fax: + 1 54 02 31 32 55 [email protected]
Malaria is one of the worldʼs most devastating diseases. It is caused by parasites of the genus Plasmodium, which are transmitted by mosquito vectors. About 3.2 billion people are at risk for contracting malaria, leading to an estimated 198 million malaria cases and nearly 600 000 deaths in 2013 [1]. Although experimental vaccines are now available, malaria control and eradication programs still rely mainly on vector control and the efficacy of chemotherapy. Artemisinin-based combination therapies (ACTs) are currently the preferred first-line antimalarials used against Plasmodium falciparum (the deadliest of the five species that infect humans), and ACTs have been adopted for first-line treatment in most of the countries where P. falciparum is endemic [1]. However, the rapid development of resistance to the partner drug and recent concerns about artemisinin resistance [2] underline the need for the development of new drugs to expand our repertoire of antimalarial agents to use in combination therapies [3]. Compounds isolated from plants have played a major role in the discovery and development of antimalarial drugs, as evidenced by quinine and * Dedicated to Professor Dr. Dr. h. c. mult. Adolf Nahrstedt on the occasion of his 75th birthday.
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3 exhibited weak antiplasmodial activity (41.8 ± 5.2 µM), while compounds 4 and 5 were inactive. Compound 6 was synthesized to confirm the structure of 5, and it showed enhanced antiplasmodial activity (15.8 ± 1.4 µM) compared to its analogues 3–5. Supporting information available online at http://www.thieme-connect.de/products
its many synthetic analogs and by artemisinin, and compounds with antimalarial activity continue to be found in plants [4–6]. The discovery of novel naturally occurring antimalarial agents is thus an important research objective. In pursuit of this objective, we obtained a MeOH extract from the roots of a plant identified as an Apoplanesia paniculata. The extract was selected for investigation based on its antiplasmodial activity against the Dd2 strain of the P. falciparum parasite and the fact that no previous chemical studies have been reported on the genus Apoplanesia.
Results and Discussion !
Bioassay-guided fractionation of the root extract of A. paniculata was carried out using a bloodbased bioassay for growth inhibition of P. falciparum. This yielded the three known compounds amorphaquinone (1), pendulone (2), and melilotocarpan C (3), and the two new pterocarpans named as apoplanesiacarpans A and B (4 and 5). The known compounds amorphaquinone (1) [7], pendulone (2) [8], and melilotocarpan C (3) [9] were identified by comparison of their spectroscopic and physical data with literature data. The 3R configuration of compounds 1 and 2 were con-
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1
Pos.
1 2 3 4 5 6 6a 6b 7 8 9 10 11 11a 11b 3-OMe 4-OMe 8-OMe 9-OMe 10-OMe
H and 13C NMR data (δ, ppm) for compounds 4–6 in CDCl3. 4
5
6
δH, (J in Hz)
δC, type
δH, (J in Hz)
δC, type
δH, (J in Hz)
7.21 d (8.6) 6.67 d (8.6) – – – 4.34 dd (11.0, 5.1) 3.63 dd (11.0, 11.0) 3.52 m – 6.47 s – – 6.83 s – 5.46 d (6.8) – 3.87 s 3.84 s 3.83 s – –
125.9 CH 106.3 CH 153.9 C 137.8 C 149.8 C 67.1 CH2
7.08 d (8.6) 6.67 d (8.6) – – – 4.36 dd (11.0, 5.1) 3.73 dd (11.0, 11.0) 3.53 m – 6.60 s – – – – 5.50 d (6.8) – 3.91 s – – 3.91 s 3.98 s
121.4 CH 105.4 CH 147.5 C 134.1 C 143.7 C 66.7 CH2
7.29 d (8.6) 6.68 d (8.6)
40.5 CH 118.2 C 110.7 CH 140.2 C 147.3 C 95.1 CH 153.1 C 78.4 CH 114.6 C 56.5 CH3 61.4 CH3 56.5 CH3 – –
40.7 CH 122.9 C 104.7 CH 140.0 C 138.9 C 137.8 C 143.4 C 78.3 CH 114.1 C 56.5 CH3 – – 60.5 CH3 61.5 CH3
Fig. 1
firmed by comparison of their circular dichroism spectra with those of previously reported data [10, 11]. Compound 4 (apoplanesiacarpan A) was obtained as a white powder. The quasi-molecular ion peak at m/z 331.1168 [M + H]+ (calcd. for C18H19O6+, 331.1176) corresponded to a molecular formula of C18H18O6. Its pterocarpan skeleton was confirmed by comparison of its 1H‑NMR data with that of compound 3 (melilo" Table 1) showed tocarpan C). The 1H and 13C‑NMR spectra of 4 (l signals for three methoxy groups (δH 3.83, 3.84, and 3.87, each 3H, s), an AB aromatic spin system [δH 7.21 (1H, d, J = 8.6 Hz) and 6.67 (1H, d, J = 8.6 Hz)] and two aromatic proton singlets (δH 6.83 and 6.47, each 1H, s). The positions of the sp2 protons and methoxy groups were confirmed by HSQC, HMBC, and NOE " Fig. 1). The HMBC correlations between H-11a (δ (l H 5.46, d, J = 6.8 Hz) and C-11b (δC 114.6) and between H-1 (δH 7.21, d, J = 6.8 Hz) and C-11b indicated that the AB spin system belongs to the A ring. HMBC correlations between H-2 (δH 6.67, d, J = 8.6 Hz) and C-3 (δC 153.9) and C-4 (δC 137.8), between 3-OMe
– – 4.38 dd (11.0, 5.1) 3.72 dd (11.0, 11.0) 3.53 m – 6.59 s – – – – 5.51 d (6.8) – 3.89 s 3.86 s 3.84 s 3.86 s 3.98 s
Structures of compounds 1–5.
(δH 3.87, s) and C-3, and between 4-OMe (δH 3.84, s) and C-4 confirmed the placement of two methoxy groups in the A ring. The position of MeO-8 was confirmed by the NOE correlation between 8-OMe (δH 3.83, s) and H-7 (δH 6.47, s). Other key HMBC " Fig. 2. A coupling constant and NOE correlations are shown in l of 6.8 Hz between H-6a and H-11a suggested these two protons were cis-diaxial; a larger coupling constant of 13–14 Hz would have been observed for the trans configuration [12]. The assigned relative configuration was in agreement with the fact that all natural pterocarpans found so far have the 6a,11a-cis configuration [13]. The absolute configurations of 6a and 11a were determined as R,R by comparison of the circular dichroism spectrum of 4 with that of known compounds [14, 15]. Compound 5 (apoplanesiacarpan B) was obtained as a white powder. The quasi-molecular ion peak at m/z 347.1127 [M + H]+ (calcd. for C18H19O7+, 347.1125) indicated a molecular formula of C18H18O7. Characteristic signals for H-6 (δH 4.36, dd, J = 11.0, 5.1 Hz and 3.73, dd, J = 11.0, 11.0 Hz), H-6a (δH 3.53, m), and HSu Q et al. Antiplasmodial Isoflavanes and …
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Table 1
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11a (δH 5.50, d, J = 6.8 Hz) in a pterocarpan skeleton were ob" Table 1). The 1H NMR specserved in its 1H‑NMR spectrum (l trum also showed the presence of three methoxy groups (δH 3.91, 3.91, and 3.98, each 3H, s), an AB aromatic proton spin system (δH 7.08, d, J = 8.6 Hz and 6.67, d, J = 8.6 Hz), and a single aromatic proton (δH 6.60, s). The HMBC correlation between H-11a and C-1 indicated that the AB spin system belonged to the A ring. The NOE correlation between H-2 (δH 6.67, d, J = 6.8 Hz) and MeO-3 (δH 3.91, s) and between H-2 and C-4 (δC 134.1) confirmed the placement of the methoxy and hydroxyl groups in " Fig. 2). The remaining assignment to be made was the A ring (l the position of the hydroxyl group and methoxy groups in ring D. NOE correlations between H-6a and H-7, and HMBC correlations between H-7 and C-8, C-9 were observed, indicating that carbons 8, 9, and 10 were oxygenated. No NOE correlation of H7 with any methoxy group was observed, suggesting the struc" Fig. 1 for compound 5. To confirm this proposed ture shown in l " Fig. 3). A key structure, compound 5 was methylated to give 6 (l NOE correlation between H-7 (δH 6.59, s) and MeO-8 (δH 3.84, s) was observed for 6, indicating that the HO-8 group in 5 was methylated, and thus confirming structure 5. Quinones 1 and 2 showed moderate inhibition of the growth of the drug-resistant Dd2 strain of P. falciparum, with IC50 values of 6 ± 2 and 7 ± 1 µM, respectively, and also displayed moderate antiproliferative activity against the A2780 human ovarian cancer cell line, with IC50 values of 6.6 and 19.6 µM. Quinones 1 and 2 have been reported to exhibit antileishmanial [16], antitumor [17], antiparasitic, and antimicrobial activities [11]. Melilotocarpan C (3) exhibited weak antiplasmodial activity (42 ± 5 µM), while apoplansiacarpan A (4) and apoplansiacarpan B (5) were inactive (IC50 > 20 µg/mL). The methylated derivative 6 was, how" Table ever, moderately active, with an IC50 value of 16 ± 1 µM (l 2). Pterocarpans are a large group of compounds derived from isoflavanoids and have been reported to display cytotoxic [18], antifungal [19], and antimycobacterial [20] activities. They might be interesting antimalarial agents as some pterocarpan derivatives have been reported to exhibit leishmanicidal activity [16]. The reduced activity of 3–6 compared with the activity of 1 and 2 suggested the quinone fraction of the molecules might be important for increased activity. Comparison of the IC50 values of 3–6 suggests the number and placement of the methoxyl and hydroxyl groups on the pterocarpan backbone can also affect the biological activity.
Materials and Methods !
General experimental procedures Optical rotations were recorded on a JASCO P-2000 polarimeter. UV spectra were measured on a Shimadzu UV-1201 spectrophotometer. IR spectra were measured on a MIDAC M-series FTIR spectrometer. NMR spectra were recorded in CDCl3 on a Bruker Avance 500 spectrometer. Chemical shifts are given in δ (ppm) and coupling constants are reported in Hz. Mass spectra were obtained on an Agilent 6220 LC‑TOF‑MS in the positive ion mode. Open column chromatographies were performed using Sephadex LH-20 (3 × 50 cm, 150 g) and C18 (2.5 × 10 cm, 40–63 µm, 45 g). Semipreparative HPLC was performed on a Shimadzu LC-10AT instrument with a semipreparative C18 Varian Dynamax column (5 µm, 250 × 10 mm).
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Planta Med 2015; 81: 1128–1132
Fig. 2
Key HMBC and NOE correlations of compounds 4 and 5.
Fig. 3
Methylation of 5 to 6 and key NOE correlations of 6.
Table 2 Antiplasmodial and antiproliferative activity data of compounds 1–6. Compound Amorphaquinone (1) Pendulone (2) Melilotocarpan C (3) Apoplanesiacarpan A (4) Apoplanesiacarpan B (5) Methylapoplanesiacarpan B (6)
P. falciparum Dd2
A2780 ovarian can-
strain IC50 (µM)
cer cells IC50 (µM)
6±2 7±1 42 ± 5 > 60 > 58 16 ± 1
6.6 ± 0.6 20.6 ± 4.1 > 61 > 60 > 58 > 56
Intraerythrocytic stage antimalarial bioassays The effect of each fraction and the pure compounds on in vitro parasite growth of the Dd2 strain was measured in a 72-h growth assay in the presence of an inhibitor, as described previously with minor modifications [21, 22]. Ring stage parasite cultures (100 µL per well, with 1 % hematocrit and 1 % parasitaemia) were grown for 72 h in the presence of increasing concentrations of the inhibitor in a 5.05 % CO2, 4.93 % O2, and 90.2 % N2 gas mixture at 37 °C. After 72 h in culture, parasite viability was determined by DNA quantitation using SYBR Green I, as described previously [22]. The half-maximum inhibitory concentration (IC50) values are the average of three independent determinations with each determination in duplicate and are expressed ± S. E. M.
Antiproliferative bioassays Antiproliferative activities were obtained at Virginia Polytechnic Institute and State University against the drug-sensitive A2780 human ovarian cancer cell lines, as previously described [23, 24].
Plant material Roots of A. paniculata C. Presl. were collected from plants along a river edge in Guatemala, close to Zapaca, Teculutan by Juan Jose Castillo under the auspices of the New York Botanical Garden.
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Extraction and isolation Dried, powdered plant material was exhaustively extracted with EtOH at room temperature to give an extract designated 60 029– 6C; a total of 600 mg of this extract was made available to Virginia Tech. Extract 60 029–6C (600 mg, IC50 > 20 µg/mL in the P. falciparum Dd2 antimalarial assay) was suspended in aqueous MeOH (MeOH‑H2O, 9 : 1, 100 mL) and extracted with hexane (5 × 100 mL portions) to give 34 mg of hexane-soluble material. The aqueous fraction was then diluted to MeOH‑H2O, 6 : 4 (150 mL) and further extracted with CH2Cl2 (5 × 100 mL portions) to give a CH2Cl2-soluble fraction (306 mg) with an IC50 value of > 10 µg/ mL; the residual MeOH‑H2O-soluble fraction (257 mg) was inactive in the P. falciparum Dd2 antimalarial assay. The CH2Cl2 fraction was subjected to size exclusion open column chromatography on a Sephadex LH-20 column (3 × 50 cm, 150 g) eluted with CH2Cl2 : MeOH (1 : 1). A total of 60 fractions of 4 mL each were collected, concentrated, and combined on the basis of TLC to yield the four fractions F1 (29 mg, 128 mL), F2 (51 mg, 24 mL), F3 (193 mg, 48 mL), and F4 (21 mg, 40 mL). The most active fraction, F3, exhibited an IC50 between 5 and 10 µg/mL, and was then subjected to column chromatography on C18 silica gel (2.5 × 10 cm, 40–63 µm, 45 g). Elution with aqueous MeOH with the MeOH : H2O ratios of 30 : 70, 50 : 50, 70 : 30, and 100 : 0 (200 mL each) gave four subfractions indexed F3-1 (48 mg, 200 mL), F3-2 (68 mg, 200 mL), F3-3 (42 mg, 200 mL), and F3–4 (31 mg, 200 mL). Fractions F3-2 (68 mg, IC50 between 5 and 10 µg/mL) and F3-3 (42 mg, IC50 between 2.5 and 5 µg/mL) were combined. Further separation of the combined fractions was carried out by HPLC on a semipreparative C18 Varian Dynamax column (5 µm, 10 × 250 mm) eluted with a solvent gradient from CH3CN : H2O, 30 : 70 to 70 : 30 from 0 to 30 min, to 100 : 0 from 30 to 40 min, ending with 100 % CH3CN from 40 to 50 min at a flow rate of 2.5 mL/min. This process gave 1 (2.9 mg, tR 35 min, 2.5 mL), 2 (2.8 mg, tR 36 min, 2 mL), 3 (3.0 mg, tR 42 min, 2.5 mL), 4 (4.2 mg, tR 39 min, 2 mL), and 5 (6.2 mg, tR 25 min, 2.5 mL). All isolated compounds were purified to 95% purity or better by HPLC using an aqueous MeOH solvent system and were judged to have a purity of 95 % or better by NMR spectroscopy before determining the bioactivity. Apoplanesiacarpan A (4): white powder; [α]23 D − 54.8 (c 0.023, MeOH); UV (MeOH) λmax (log ε) 208 (4.7), 234 (4.1), 297 (3.9) nm; CD (MeOH) [Δε]297 nm + 1.9, [Δε]234 nm − 14.1; IR (film) νmax " Ta1773, 1523, 1216, and 1080 cm−1; 1H and 13C NMR data, see l ble 1; HRESIMS (pos.): m/z 331.1188 [M + H]+ (calcd. for C18H19O6+, 331.1177), 353.0990 [M + Na]+ (calcd. for C18H18 NaO6+, 353.0996). Apoplanesiacarpan B (5): white powder; [α]23 D − 80.2 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 229 (5.1), 238 (5.0), 297 (4.6) nm; CD (MeOH) [Δε]297 nm + 3.9, [Δε]234 nm − 65.5; IR (film) νmax 1775, 1613, 1523, 1218, and 1080 cm−1; 1H and 13C NMR data, " Table 1; HRESIMS (pos.): m/z 347.1127 [M + H]+ (calcd. see l for C18H19O7+, 347.1125), 369.0948 [M + Na]+ (calcd. for C18H18NaO7+, 369.0945).
Methylation of compound 5 Apoplanesiacarpan B (5, 2.5 mg) was dissolved in anhydrous acetone (2 mL) and treated with K2CO3 (120 mg) and methyl iodide (200 µL). The mixture was stirred at room temperature for 24 h. The reaction mixture was evaporated and dissolved in water.
The aqueous solution was then extracted with EtOAc to yield compound 6, which was further purified on C18 HPLC eluted by 80–100 % CH3CN in H2O. Structure elucidation of compound 6 " Table 1); HRESIMS (pos.): m/z was based on its 1H NMR data (l 375.1421 [M + H]+ (calcd. for C20H23O7+, 375.1439), 397.1237 [M + Na]+ (calcd. for C20H22NaO7+, 397.1258).
Supporting information 1 H NMR and 13C NMR spectra for compounds 4 and 5, the 1H NMR spectrum of 6, and the final HPLC purification of compounds 4–6 are available as Supporting Information.
Acknowledgements !
This project was supported by the National Center for Complementary and Integrative Health (NCCIH) under awards 1 R01 AT008088–01 3U01 and TW000313–19S1, the latter as a supplement to Cooperative Agreement U01 TW000313–19 from the Fogarty International Center, the National Cancer Institute, the National Science Foundation, the National Heart, Lung and Blood Institute, the National Institute of Mental Health, the Office of Dietary Supplements, and the Office of the Director of NIH, with the International Cooperative Biodiversity Groups. These supports are gratefully acknowledged. This work was also supported by the National Science Foundation under Grant No. CHE-0619382 for purchase of the Bruker Avance 500 NMR spectrometer and Grant No. CHE-0722638 for the purchase of the Agilent 6220 mass spectrometer. We thank Mr. B. Bebout for obtaining the mass spectra. We gratefully acknowledge Juan Jose Castillo of the New York Botanical Garden for the provision of plant material.
Conflict of Interest !
The authors declare no conflict of interest.
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Voucher specimens of the plant are on deposit under the accession number JJC02914a, b, and c (specimen ID 41 281) at the NYBG.
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19 Meragelman TL, Tucker KD, McCloud TG, Cardellina JH, Shoemaker RH. Antifungal flavonoids from Hildegardia barteri. J Nat Prod 2005; 68: 1790–1792 20 Rukachaisirikul T, Innok P, Suksamrarn A. Erythrina alkaloids and a pterocarpan from the bark of Erythrina subumbrans. J Nat Prod 2008; 71: 156–158 21 Bennett TN, Paguio M, Gligorijevic B, Seudieu C, Kosar AD, Davidson E, Roepe PD. Novel, rapid, and inexpensive cell-based quantification of antimalarial drug efficacy. Antimicrob Agents Chemother 2004; 48: 1807–1810 22 Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M. Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrob Agents Chemother 2004; 48: 1803–1806 23 Cao S, Brodie PJ, Miller JS, Randrianaivo R, Ratovoson F, Birkinshaw C, Andriantsiferana R, Rasamison VE, Kingston DGI. Antiproliferative xanthones of Terminalia calcicola from the Madagascar rain forest. J Nat Prod 2007; 70: 679–681 24 Pan E, Harinantenaina L, Brodie PJ, Miller JS, Callmander MW, Rakotonandrasana S, Rakotobe E, Rasamison VE, Kingston DGI. Four diphenylpropanes and a cycloheptadibenzofuran from Bussea sakalava from the Madagascar dry forest. J Nat Prod 2010; 73: 1792–1795
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Antiplasmodial Isoflavanes and Pterocarpans from Apoplanesia paniculata*
Authors
Qingxi Su 1, Priscilla Krai 2, Michael Goetz 3, Maria B. Cassera 2, David G. I. Kingston 1
Affiliations
1 2 3
Key words " antimalarial activity l " isoflavanes l " pterocarpans l " Apoplansia paniculata l (Fabaceae)
Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia, United States Department of Biochemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia, United States Natural Products Discovery Institute, Doylestown, Pennsylvania, United States
Abstract !
Bioassay-guided fractionation of an EtOH extract of the roots of the plant Apoplanesia paniculata (Fabaceae) led to the isolation of the three known compounds amorphaquinone (1), pendulone (2), and melilotocarpan C (3), and the two new pterocarpans 4 and 5. Compounds 1 and 2 exhibited good antiplasmodial activity with IC50 values of 5.7 ± 1.5 and 7.0 ± 0.8 µM, respectively. Compound
Introduction !
received revised accepted
February 24, 2015 April 2, 2015 April 7, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1546036 Published online May 27, 2015 Planta Med 2015; 81: 1128–1132 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. David G. I. Kingston Virginia Tech Davidson Hall Department of Chemistry and Virginia Tech Center for Drug Discovery 1040 Drillfield Drive Blacksburg, Virginia 24061 United States Phone: + 1 54 02 31 65 70 Fax: + 1 54 02 31 32 55 [email protected]
Malaria is one of the worldʼs most devastating diseases. It is caused by parasites of the genus Plasmodium, which are transmitted by mosquito vectors. About 3.2 billion people are at risk for contracting malaria, leading to an estimated 198 million malaria cases and nearly 600 000 deaths in 2013 [1]. Although experimental vaccines are now available, malaria control and eradication programs still rely mainly on vector control and the efficacy of chemotherapy. Artemisinin-based combination therapies (ACTs) are currently the preferred first-line antimalarials used against Plasmodium falciparum (the deadliest of the five species that infect humans), and ACTs have been adopted for first-line treatment in most of the countries where P. falciparum is endemic [1]. However, the rapid development of resistance to the partner drug and recent concerns about artemisinin resistance [2] underline the need for the development of new drugs to expand our repertoire of antimalarial agents to use in combination therapies [3]. Compounds isolated from plants have played a major role in the discovery and development of antimalarial drugs, as evidenced by quinine and * Dedicated to Professor Dr. Dr. h. c. mult. Adolf Nahrstedt on the occasion of his 75th birthday.
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3 exhibited weak antiplasmodial activity (41.8 ± 5.2 µM), while compounds 4 and 5 were inactive. Compound 6 was synthesized to confirm the structure of 5, and it showed enhanced antiplasmodial activity (15.8 ± 1.4 µM) compared to its analogues 3–5. Supporting information available online at http://www.thieme-connect.de/products
its many synthetic analogs and by artemisinin, and compounds with antimalarial activity continue to be found in plants [4–6]. The discovery of novel naturally occurring antimalarial agents is thus an important research objective. In pursuit of this objective, we obtained a MeOH extract from the roots of a plant identified as an Apoplanesia paniculata. The extract was selected for investigation based on its antiplasmodial activity against the Dd2 strain of the P. falciparum parasite and the fact that no previous chemical studies have been reported on the genus Apoplanesia.
Results and Discussion !
Bioassay-guided fractionation of the root extract of A. paniculata was carried out using a bloodbased bioassay for growth inhibition of P. falciparum. This yielded the three known compounds amorphaquinone (1), pendulone (2), and melilotocarpan C (3), and the two new pterocarpans named as apoplanesiacarpans A and B (4 and 5). The known compounds amorphaquinone (1) [7], pendulone (2) [8], and melilotocarpan C (3) [9] were identified by comparison of their spectroscopic and physical data with literature data. The 3R configuration of compounds 1 and 2 were con-
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1
Pos.
1 2 3 4 5 6 6a 6b 7 8 9 10 11 11a 11b 3-OMe 4-OMe 8-OMe 9-OMe 10-OMe
H and 13C NMR data (δ, ppm) for compounds 4–6 in CDCl3. 4
5
6
δH, (J in Hz)
δC, type
δH, (J in Hz)
δC, type
δH, (J in Hz)
7.21 d (8.6) 6.67 d (8.6) – – – 4.34 dd (11.0, 5.1) 3.63 dd (11.0, 11.0) 3.52 m – 6.47 s – – 6.83 s – 5.46 d (6.8) – 3.87 s 3.84 s 3.83 s – –
125.9 CH 106.3 CH 153.9 C 137.8 C 149.8 C 67.1 CH2
7.08 d (8.6) 6.67 d (8.6) – – – 4.36 dd (11.0, 5.1) 3.73 dd (11.0, 11.0) 3.53 m – 6.60 s – – – – 5.50 d (6.8) – 3.91 s – – 3.91 s 3.98 s
121.4 CH 105.4 CH 147.5 C 134.1 C 143.7 C 66.7 CH2
7.29 d (8.6) 6.68 d (8.6)
40.5 CH 118.2 C 110.7 CH 140.2 C 147.3 C 95.1 CH 153.1 C 78.4 CH 114.6 C 56.5 CH3 61.4 CH3 56.5 CH3 – –
40.7 CH 122.9 C 104.7 CH 140.0 C 138.9 C 137.8 C 143.4 C 78.3 CH 114.1 C 56.5 CH3 – – 60.5 CH3 61.5 CH3
Fig. 1
firmed by comparison of their circular dichroism spectra with those of previously reported data [10, 11]. Compound 4 (apoplanesiacarpan A) was obtained as a white powder. The quasi-molecular ion peak at m/z 331.1168 [M + H]+ (calcd. for C18H19O6+, 331.1176) corresponded to a molecular formula of C18H18O6. Its pterocarpan skeleton was confirmed by comparison of its 1H‑NMR data with that of compound 3 (melilo" Table 1) showed tocarpan C). The 1H and 13C‑NMR spectra of 4 (l signals for three methoxy groups (δH 3.83, 3.84, and 3.87, each 3H, s), an AB aromatic spin system [δH 7.21 (1H, d, J = 8.6 Hz) and 6.67 (1H, d, J = 8.6 Hz)] and two aromatic proton singlets (δH 6.83 and 6.47, each 1H, s). The positions of the sp2 protons and methoxy groups were confirmed by HSQC, HMBC, and NOE " Fig. 1). The HMBC correlations between H-11a (δ (l H 5.46, d, J = 6.8 Hz) and C-11b (δC 114.6) and between H-1 (δH 7.21, d, J = 6.8 Hz) and C-11b indicated that the AB spin system belongs to the A ring. HMBC correlations between H-2 (δH 6.67, d, J = 8.6 Hz) and C-3 (δC 153.9) and C-4 (δC 137.8), between 3-OMe
– – 4.38 dd (11.0, 5.1) 3.72 dd (11.0, 11.0) 3.53 m – 6.59 s – – – – 5.51 d (6.8) – 3.89 s 3.86 s 3.84 s 3.86 s 3.98 s
Structures of compounds 1–5.
(δH 3.87, s) and C-3, and between 4-OMe (δH 3.84, s) and C-4 confirmed the placement of two methoxy groups in the A ring. The position of MeO-8 was confirmed by the NOE correlation between 8-OMe (δH 3.83, s) and H-7 (δH 6.47, s). Other key HMBC " Fig. 2. A coupling constant and NOE correlations are shown in l of 6.8 Hz between H-6a and H-11a suggested these two protons were cis-diaxial; a larger coupling constant of 13–14 Hz would have been observed for the trans configuration [12]. The assigned relative configuration was in agreement with the fact that all natural pterocarpans found so far have the 6a,11a-cis configuration [13]. The absolute configurations of 6a and 11a were determined as R,R by comparison of the circular dichroism spectrum of 4 with that of known compounds [14, 15]. Compound 5 (apoplanesiacarpan B) was obtained as a white powder. The quasi-molecular ion peak at m/z 347.1127 [M + H]+ (calcd. for C18H19O7+, 347.1125) indicated a molecular formula of C18H18O7. Characteristic signals for H-6 (δH 4.36, dd, J = 11.0, 5.1 Hz and 3.73, dd, J = 11.0, 11.0 Hz), H-6a (δH 3.53, m), and HSu Q et al. Antiplasmodial Isoflavanes and …
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Table 1
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11a (δH 5.50, d, J = 6.8 Hz) in a pterocarpan skeleton were ob" Table 1). The 1H NMR specserved in its 1H‑NMR spectrum (l trum also showed the presence of three methoxy groups (δH 3.91, 3.91, and 3.98, each 3H, s), an AB aromatic proton spin system (δH 7.08, d, J = 8.6 Hz and 6.67, d, J = 8.6 Hz), and a single aromatic proton (δH 6.60, s). The HMBC correlation between H-11a and C-1 indicated that the AB spin system belonged to the A ring. The NOE correlation between H-2 (δH 6.67, d, J = 6.8 Hz) and MeO-3 (δH 3.91, s) and between H-2 and C-4 (δC 134.1) confirmed the placement of the methoxy and hydroxyl groups in " Fig. 2). The remaining assignment to be made was the A ring (l the position of the hydroxyl group and methoxy groups in ring D. NOE correlations between H-6a and H-7, and HMBC correlations between H-7 and C-8, C-9 were observed, indicating that carbons 8, 9, and 10 were oxygenated. No NOE correlation of H7 with any methoxy group was observed, suggesting the struc" Fig. 1 for compound 5. To confirm this proposed ture shown in l " Fig. 3). A key structure, compound 5 was methylated to give 6 (l NOE correlation between H-7 (δH 6.59, s) and MeO-8 (δH 3.84, s) was observed for 6, indicating that the HO-8 group in 5 was methylated, and thus confirming structure 5. Quinones 1 and 2 showed moderate inhibition of the growth of the drug-resistant Dd2 strain of P. falciparum, with IC50 values of 6 ± 2 and 7 ± 1 µM, respectively, and also displayed moderate antiproliferative activity against the A2780 human ovarian cancer cell line, with IC50 values of 6.6 and 19.6 µM. Quinones 1 and 2 have been reported to exhibit antileishmanial [16], antitumor [17], antiparasitic, and antimicrobial activities [11]. Melilotocarpan C (3) exhibited weak antiplasmodial activity (42 ± 5 µM), while apoplansiacarpan A (4) and apoplansiacarpan B (5) were inactive (IC50 > 20 µg/mL). The methylated derivative 6 was, how" Table ever, moderately active, with an IC50 value of 16 ± 1 µM (l 2). Pterocarpans are a large group of compounds derived from isoflavanoids and have been reported to display cytotoxic [18], antifungal [19], and antimycobacterial [20] activities. They might be interesting antimalarial agents as some pterocarpan derivatives have been reported to exhibit leishmanicidal activity [16]. The reduced activity of 3–6 compared with the activity of 1 and 2 suggested the quinone fraction of the molecules might be important for increased activity. Comparison of the IC50 values of 3–6 suggests the number and placement of the methoxyl and hydroxyl groups on the pterocarpan backbone can also affect the biological activity.
Materials and Methods !
General experimental procedures Optical rotations were recorded on a JASCO P-2000 polarimeter. UV spectra were measured on a Shimadzu UV-1201 spectrophotometer. IR spectra were measured on a MIDAC M-series FTIR spectrometer. NMR spectra were recorded in CDCl3 on a Bruker Avance 500 spectrometer. Chemical shifts are given in δ (ppm) and coupling constants are reported in Hz. Mass spectra were obtained on an Agilent 6220 LC‑TOF‑MS in the positive ion mode. Open column chromatographies were performed using Sephadex LH-20 (3 × 50 cm, 150 g) and C18 (2.5 × 10 cm, 40–63 µm, 45 g). Semipreparative HPLC was performed on a Shimadzu LC-10AT instrument with a semipreparative C18 Varian Dynamax column (5 µm, 250 × 10 mm).
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Fig. 2
Key HMBC and NOE correlations of compounds 4 and 5.
Fig. 3
Methylation of 5 to 6 and key NOE correlations of 6.
Table 2 Antiplasmodial and antiproliferative activity data of compounds 1–6. Compound Amorphaquinone (1) Pendulone (2) Melilotocarpan C (3) Apoplanesiacarpan A (4) Apoplanesiacarpan B (5) Methylapoplanesiacarpan B (6)
P. falciparum Dd2
A2780 ovarian can-
strain IC50 (µM)
cer cells IC50 (µM)
6±2 7±1 42 ± 5 > 60 > 58 16 ± 1
6.6 ± 0.6 20.6 ± 4.1 > 61 > 60 > 58 > 56
Intraerythrocytic stage antimalarial bioassays The effect of each fraction and the pure compounds on in vitro parasite growth of the Dd2 strain was measured in a 72-h growth assay in the presence of an inhibitor, as described previously with minor modifications [21, 22]. Ring stage parasite cultures (100 µL per well, with 1 % hematocrit and 1 % parasitaemia) were grown for 72 h in the presence of increasing concentrations of the inhibitor in a 5.05 % CO2, 4.93 % O2, and 90.2 % N2 gas mixture at 37 °C. After 72 h in culture, parasite viability was determined by DNA quantitation using SYBR Green I, as described previously [22]. The half-maximum inhibitory concentration (IC50) values are the average of three independent determinations with each determination in duplicate and are expressed ± S. E. M.
Antiproliferative bioassays Antiproliferative activities were obtained at Virginia Polytechnic Institute and State University against the drug-sensitive A2780 human ovarian cancer cell lines, as previously described [23, 24].
Plant material Roots of A. paniculata C. Presl. were collected from plants along a river edge in Guatemala, close to Zapaca, Teculutan by Juan Jose Castillo under the auspices of the New York Botanical Garden.
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Extraction and isolation Dried, powdered plant material was exhaustively extracted with EtOH at room temperature to give an extract designated 60 029– 6C; a total of 600 mg of this extract was made available to Virginia Tech. Extract 60 029–6C (600 mg, IC50 > 20 µg/mL in the P. falciparum Dd2 antimalarial assay) was suspended in aqueous MeOH (MeOH‑H2O, 9 : 1, 100 mL) and extracted with hexane (5 × 100 mL portions) to give 34 mg of hexane-soluble material. The aqueous fraction was then diluted to MeOH‑H2O, 6 : 4 (150 mL) and further extracted with CH2Cl2 (5 × 100 mL portions) to give a CH2Cl2-soluble fraction (306 mg) with an IC50 value of > 10 µg/ mL; the residual MeOH‑H2O-soluble fraction (257 mg) was inactive in the P. falciparum Dd2 antimalarial assay. The CH2Cl2 fraction was subjected to size exclusion open column chromatography on a Sephadex LH-20 column (3 × 50 cm, 150 g) eluted with CH2Cl2 : MeOH (1 : 1). A total of 60 fractions of 4 mL each were collected, concentrated, and combined on the basis of TLC to yield the four fractions F1 (29 mg, 128 mL), F2 (51 mg, 24 mL), F3 (193 mg, 48 mL), and F4 (21 mg, 40 mL). The most active fraction, F3, exhibited an IC50 between 5 and 10 µg/mL, and was then subjected to column chromatography on C18 silica gel (2.5 × 10 cm, 40–63 µm, 45 g). Elution with aqueous MeOH with the MeOH : H2O ratios of 30 : 70, 50 : 50, 70 : 30, and 100 : 0 (200 mL each) gave four subfractions indexed F3-1 (48 mg, 200 mL), F3-2 (68 mg, 200 mL), F3-3 (42 mg, 200 mL), and F3–4 (31 mg, 200 mL). Fractions F3-2 (68 mg, IC50 between 5 and 10 µg/mL) and F3-3 (42 mg, IC50 between 2.5 and 5 µg/mL) were combined. Further separation of the combined fractions was carried out by HPLC on a semipreparative C18 Varian Dynamax column (5 µm, 10 × 250 mm) eluted with a solvent gradient from CH3CN : H2O, 30 : 70 to 70 : 30 from 0 to 30 min, to 100 : 0 from 30 to 40 min, ending with 100 % CH3CN from 40 to 50 min at a flow rate of 2.5 mL/min. This process gave 1 (2.9 mg, tR 35 min, 2.5 mL), 2 (2.8 mg, tR 36 min, 2 mL), 3 (3.0 mg, tR 42 min, 2.5 mL), 4 (4.2 mg, tR 39 min, 2 mL), and 5 (6.2 mg, tR 25 min, 2.5 mL). All isolated compounds were purified to 95% purity or better by HPLC using an aqueous MeOH solvent system and were judged to have a purity of 95 % or better by NMR spectroscopy before determining the bioactivity. Apoplanesiacarpan A (4): white powder; [α]23 D − 54.8 (c 0.023, MeOH); UV (MeOH) λmax (log ε) 208 (4.7), 234 (4.1), 297 (3.9) nm; CD (MeOH) [Δε]297 nm + 1.9, [Δε]234 nm − 14.1; IR (film) νmax " Ta1773, 1523, 1216, and 1080 cm−1; 1H and 13C NMR data, see l ble 1; HRESIMS (pos.): m/z 331.1188 [M + H]+ (calcd. for C18H19O6+, 331.1177), 353.0990 [M + Na]+ (calcd. for C18H18 NaO6+, 353.0996). Apoplanesiacarpan B (5): white powder; [α]23 D − 80.2 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 229 (5.1), 238 (5.0), 297 (4.6) nm; CD (MeOH) [Δε]297 nm + 3.9, [Δε]234 nm − 65.5; IR (film) νmax 1775, 1613, 1523, 1218, and 1080 cm−1; 1H and 13C NMR data, " Table 1; HRESIMS (pos.): m/z 347.1127 [M + H]+ (calcd. see l for C18H19O7+, 347.1125), 369.0948 [M + Na]+ (calcd. for C18H18NaO7+, 369.0945).
Methylation of compound 5 Apoplanesiacarpan B (5, 2.5 mg) was dissolved in anhydrous acetone (2 mL) and treated with K2CO3 (120 mg) and methyl iodide (200 µL). The mixture was stirred at room temperature for 24 h. The reaction mixture was evaporated and dissolved in water.
The aqueous solution was then extracted with EtOAc to yield compound 6, which was further purified on C18 HPLC eluted by 80–100 % CH3CN in H2O. Structure elucidation of compound 6 " Table 1); HRESIMS (pos.): m/z was based on its 1H NMR data (l 375.1421 [M + H]+ (calcd. for C20H23O7+, 375.1439), 397.1237 [M + Na]+ (calcd. for C20H22NaO7+, 397.1258).
Supporting information 1 H NMR and 13C NMR spectra for compounds 4 and 5, the 1H NMR spectrum of 6, and the final HPLC purification of compounds 4–6 are available as Supporting Information.
Acknowledgements !
This project was supported by the National Center for Complementary and Integrative Health (NCCIH) under awards 1 R01 AT008088–01 3U01 and TW000313–19S1, the latter as a supplement to Cooperative Agreement U01 TW000313–19 from the Fogarty International Center, the National Cancer Institute, the National Science Foundation, the National Heart, Lung and Blood Institute, the National Institute of Mental Health, the Office of Dietary Supplements, and the Office of the Director of NIH, with the International Cooperative Biodiversity Groups. These supports are gratefully acknowledged. This work was also supported by the National Science Foundation under Grant No. CHE-0619382 for purchase of the Bruker Avance 500 NMR spectrometer and Grant No. CHE-0722638 for the purchase of the Agilent 6220 mass spectrometer. We thank Mr. B. Bebout for obtaining the mass spectra. We gratefully acknowledge Juan Jose Castillo of the New York Botanical Garden for the provision of plant material.
Conflict of Interest !
The authors declare no conflict of interest.
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