Antiplasmodial Isoflavanes and Pterocarpans from Apoplanesia paniculata

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 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.

Introduction

Malaria is one of the world’s 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 P. 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 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 blood-based 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 – 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 confirmed 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 (melilotocarpan C). The ¹H and ¹³C-NMR spectra of 4 (Table 1) showed signals for three methoxy groups (δ_H 3.83, 3.84 and 3.87, each 3H, s), an AB aromatic spin system [δ_H 7.21 (1H, d, J = 8.6 Hz) and 6.67 (1H, d, J = 8.6 Hz)] and two aromatic proton singlets (δ_H 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_H 5.46, d, J = 6.8 Hz) and C-11b (δ_C 114.6) and between H-1 (δ_H 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 (δ_H 6.67, d, J = 8.6 Hz) and C-3 (δ_C 153.9) and C-4 (δ_C 137.8), between 3-OMe (δ_H 3.87, s) and C-3, and between 4-OMe (δ_H 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 (δ_H 3.83, s) and H-7 (δ_H 6.47, s). Other key HMBC and NOE correlations are shown in Fig. 1. A coupling constant 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].

Table 1.

¹H and ¹³C NMR Data (δ, ppm) for compounds 4–6 in CDCl₃

Pos.	4		5		6
	δH, (J in Hz)	δC, type	δH, (J in Hz)	δC, type	δH, (J in Hz)
1	7.21 d (8.6)	125.9 CH	7.08 d (8.6)	121.4 CH	7.29 d (8.6)
2	6.67 d (8.6)	106.3 CH	6.67 d (8.6)	105.4 CH	6.68 d (8.6)
3	-	153.9 C	-	147.5 C
4	-	137.8 C	-	134.1 C	-
5	-	149.8 C	-	143.7 C	-
6	4.34 dd (11.0, 5.1) 3.63 dd (11.0, 11.0)	67.1 CH2	4.36 dd (11.0, 5.1) 3.73 dd (11.0, 11.0)	66.7 CH2	4.38 dd (11.0, 5.1) 3.72 dd (11.0, 11.0)
6a	3.52 m	40.5 CH	3.53 m	40.7 CH	3.53 m
6b	-	118.2 C	-	122.9 C	-
7	6.47 s	110.7 CH	6.60 s	104.7 CH	6.59 s
8	-	140.2 C	-	140.0 C	-
9	-	147.3 C	-	138.9 C	-
10	6.83 s	95.1 CH	-	137.8 C	-
11	-	153.1 C	-	143.4 C	-
11a	5.46 d (6.8)	78.4 CH	5.50 d (6.8)	78.3 CH	5.51 d (6.8)
11b	-	114.6 C	-	114.1 C	-
3-OMe	3.87 s	56.5 CH3	3.91 s	56.5 CH3	3.89 s
4-OMe	3.84 s	61.4 CH3	-	-	3.86 s
8-OMe	3.83 s	56.5 CH3	-	-	3.84 s
9-OMe	-	-	3.91 s	60.5 CH3	3.86 s
10-OMe	-	-	3.98 s	61.5 CH3	3.98 s

Fig. 1.

Structures of compounds 1 – 5.

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 (δ_H 4.36, dd, J = 11.0, 5.1 Hz and 3.73, dd, J = 11.0, 11.0 Hz), H-6a (δ_H 3.53, m) and H-11a (δ_H 5.50, d, J = 6.8 Hz) in a pterocarpan skeleton were observed in its ¹H-NMR spectrum (Table 1). The ¹H NMR spectrum also showed the presence of three methoxy groups (δ_H 3.91, 3.91 and 3.98, each 3H, s), an AB aromatic proton spin system (δ_H 7.08, d, J = 8.6 Hz and 6.67, d, J = 8.6 Hz), and a single aromatic proton (δ_H 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 (δ_H 6.67, d, J = 6.8 Hz) and MeO-3 (δ_H 3.91, s) and between H-2 and C-4 (δ_C 134.1) confirmed the placement of the methoxy and hydroxyl groups in the A ring. The remaining assignment to be made was 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 H-7 with any methoxy group was observed, suggesting thestructure shown in Fig. 1 for compound 5. To confirm this proposed structure, compound 5 was methylated to give 6 (Fig. 2). A key NOE correlation between H-7 (δ_H 6.59, s) and MeO-8 (δ_H 3.84, s) was observed for 6, indicating that the HO-8 group in 5 was methylated, and thus confirming structure 5.

Fig. 2.

Key HMBC and NOE correlations of compounds 4 and 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 however moderately active, with an IC₅₀ value of 16 ± 1 µM (Table 2).

Table 2.

Antiplasmodial and antiproliferative activity data of compounds 1 – 6.

Compound	P. falciparum Dd2 strain	A2780 ovarian cancer cells
	IC50 (µM)	IC50 (µM)
Amorphaquinone (1)	6 ± 2	6.6 ± 0.6
Pendulone (2)	7 ± 1	20.6 ± 4.1
Melilotocarpan C (3)	42 ± 5	> 61
Apoplanesiacarpan A (4)	> 60	> 60
Apoplanesiacarpan B (5)	> 58	> 58
Methylapoplanesiacarpan B (6)	16 ± 1	> 56

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). Semi-preparative HPLC was performed on a Shimadzu LC-10AT instrument with a semipreparative C18 Varian Dynamax column (5 µm, 250 ×10 mm).

Intraerythrocytic stage antimalarial bioassays

The effect of each fraction and pure compounds on in vitro parasite growth of Dd2 strain was measured in a 72 h growth assay in the presence of 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. Voucher specimens of the plant are on deposit under the accession number JJC02914a, b and c (specimen ID 41281) at the NYBG.

Extraction and isolation

Dried, powdered plant material was exhaustively extracted with EtOH at room temperature to give an extract designated 60029-6C; a total of 600 mg of this extract was made available to Virginia Tech. Extract 60029-6C (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, t_R 35 min, 2.5 mL), 2 (2.8 mg, t_R 36 min, 2 mL), 3 (3.0 mg, t_R 42 min, 2.5 mL), 4 (4.2 mg, t_R 39 min, 2 mL), and 5 (6.2 mg, t_R 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 be 95% purity or better by NMR spectroscopy before determining bioactivity.

Apoplanesiacarpan A (4)

white powder; ${[\alpha ]}_D^{23}$ − 54.8 (c 0.023, MeOH); UV (MeOH) λ_max (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) ν_max 1773, 1523, 1216 and 1080 cm⁻¹; ¹H and ¹³C NMR data, see Table 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; ${[\alpha ]}_D^{23}$ − 80.2 (c 0.12, MeOH); UV (MeOH) λ_max (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) ν_max 1775, 1613, 1523, 1218 and 1080 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS (pos.): m/z 347.1127 [M+H]⁺ (calcd 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 was based on its ¹H NMR data (Table 1); HRESIMS (pos.): m/z 375.1421 [M+H]⁺ (calcd for C₂₀H₂₃O₇⁺, 375.1439), 397.1237 [M+Na]⁺ (calcd for C₂₀H₂₂NaO₇⁺, 397.1258).

Fig. 3.

Methylation of 5 to 6 and key NOE correlations observed of 6.