New Antiplasmodial Diterpenes from Gutierrezia sarothrae

Abstract

Bioassay guided fractionation of the MeOH extract of the plant Gutierrezia sarothrae (Asteraceae) using an assay for antiplasmodial activity against the drug-resistant Dd2 strain of Plasmodium falciparum led to the isolation of the two new diterpenes 3α-angeloyloxy-15-hydroxylabda-7,13-dien-16,15-olid-18-oic acid (1) and 3α-angeloyloxy-15-methoxylabda-7,13-dien-16,15-olid-18-oic acid (2). The structures of 1 and 2 were elucidated by interpretation of 1D and 2D NMR spectroscopic data, mass spectrometry, and comparison with the data of related compounds reported in the literature. Compound 1 exhibited moderate antiplasmodial activity with an IC₅₀ values of 10.4 ± 4.3 µM.

The family Asteraceae (or Compositae) contains many perennial herbs as well as subshrubs that are important weeds on rangelands [1]. Gutierrezia sarothrae, a member of this family, is widely distributed in the rangelands of western North America [2]. The plant has been used externally to treat bee stings, snake bites, flesh wounds [3], and rheumatism [4]. Previous studies have indicated that the plant contains monoterpenes [5], volatile components such as cryptone and β-eudesmol [2], flavonoids [6], and labdane derivatives [7a,7b]. An antitumor proteinaceous substance has also been reported from the plant [8]. However, G. sarothrae has not previously been studied for potential antimalarial constituents.

In continuation of our investigation of antimalarial agents from plants [9a,9b], we obtained a sample of the methanolic extract of G. sarothrae. The crude extract was selected for investigation based on its antiplasmodial activity (IC50 between 5 and 10 µg/mL) against the Dd2 strain of the P. falciparum parasite. Bioassay-guided fractionation afforded the two new labdane derivatives 3α-angeloyloxy-15-hydroxylabda-7,13-dien-16,15-olid-18-oic acid (1) and 3α-angeloyloxy-15-methoxylabda-7,13-dien-16,15-olid-18-oic acid (2). We report herein the bioassay-guided isolation and structure elucidation of the two new compounds, as well as their antimalarial activity.

Liquid-liquid partition of the crude MeOH extract of G. sarothrae (800 mg) yielded antiplasmodial hexane and dichloromethane soluble fractions (IC₅₀ ca. 5 µg/mL each). These fractions were combined and subjected to size-exclusion column chromatography on Sephadex LH-20 followed by normal phase diol open column chromatography. The most active fraction from the diol column was subjected to semi-preparative C₁₈ HPLC to yield the two new labdane diterpenes (1–2).

Compound 1 was obtained as a colorless oil and had the molecular formula C₂₅H₃₄O₇ as indicated by HRESIMS analysis (m/z 469.2218 [M+Na]⁺, calcd. for C₂₅H₃₄O₇Na⁺, 469.2197; 915.4543 [2M+Na]⁺, calcd. for C₅₀H₆₈O₁₄Na⁺, 915.4501). Its ¹H NMR spectrum (Table 1) indicated the presence of five methyl groups, including three singlets (δ_H 0.82, 1.30 and 1.72, each 3H) and two multiplets (δ_H 1.93, dq, J = 7.0, 1.5 Hz, 3H and 1.83, m, 3H); five downfield protons between δ 5.23 and 6.88; and a number of multiplets between δ 1.30 and 2.55. The ¹³C NMR (Table 1) and HSQC spectra of 1 indicated the presence of 3 carbonyl groups, 5 quaternary, 7 methine, 5 methylene and 5 methyl carbons. The labdane diterpene skeleton was established based on HMBC correlations of Me-17 (δ_H 1.72, s, 3H) to C-7, C-8 and C-9 (δ_C 121.9, 134.7 and 54.0); Me-19 (δ_H 1.30, s, 3H) to C-3, C-4 and C-5 (δ_C 77.7, 51.2 and 46.1); Me-20 (δ_H 0.82, s, 3H) to C-1, C-5 and C-9 (δ_C 36.2, 46.1 and 54.0) (Figure 2). A carboxylic acid group was assigned to the C-3 position based on the HMBC correlation between Me-19 and C-18 (δ_C 179.2). Further examination of the ¹H NMR spectrum of 1 revealed characteristic signals of an angeloyl group. An olefinic proton (δ_H 6.03, qq, J = 7.0, 1.5 Hz, 1H), two methyl groups (δ_H 1.93, dq, J = 7.0, 1.5 Hz, 3H and 1.83, m, 3H), and the corresponding carbons (δ_C 167.3, 128.1, 138.3, 15.9 and 20.7) were assigned unambiguously to C-1' – C-5' based on HSQC and HMBC correlations. The HMBC spectrum also established correlations between the C-12 methylene protons (δ_H 2.55, m and 2.30, m, each 1H) to C-13, C-14 and C-16 (δ_C 138.4, 143.6 and 171.3), and from the oxygenated H-15 (δ_H 6.12, s, 1H) to C-13 and H-14 (δ_H 6.88, s, 1H), to C-15 (δ_C 96.7). These correlations suggested the presence of a hydroxy-furanone moiety, as shown in Figure 1. The relative configuration of compound 1 was confirmed by NOESY correlations between 3-H to 5-H, Me-19 to Me-20, and Me-20 to 11-H; the configuration at C-15 was not assigned (Figure 2). The spectroscopic data showed satisfactory agreement with those recorded for related labdane derivatives by Bohlmann et al. [7b].

Table 1.

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

	1		2
Posn	δHb	δCc	δHb	δCc
1	1.97 m, 1.36 m	36.7, CH2	1.93m, 1.31m	36.7, CH2
2	1.93 m, 1.68 m	25.1, CH2	2.05 m, 1.65 m	25.2, CH2
3	5,23 dd (11.9, 4.0)	77.7, CH	5.27 dd (12.0, 3.7)	77.7, CH
4	-	51.2, C	-	51.1, C
5	2.05 m	46.1, CH	2.02 m	46.1, CH
6	1.95 m, 2.07 m	23.6, CH2	1.84 m, 1.68 m	23.8, CH2
7	5.36 brs	121.9, CH	5.35 brs	122.1, CH
8	-	134.7, C	-	134.6, C
9	1.79 m	54.0, CH	1.75 m	54.2, CH
10	-	36.2, C	-	36.2, C
11	1.74 m, 1.49 m	25.0, CH2	1.69 m, 1.48 m	25.0, CH2
12	2.55 m, 2.30 m	27.4, CH2	2.54 m, 2.27 m	27.5, CH2
13	-	138.4, C	-	138.7/ 138.8, C
14	6.88 s	143.6, CH	6.80 s	142.1/ 142.2, CH
15	6.12 s	96.7, CH	5.75 d (1.3)	102.6/ 102.7, CH
16	-	171.3, C	-	171.4, C
17	1.72 s	22.1, CH3	1.71 s	22.1, CH3
18	-	179.2, C	-	180.2, C
19	1.30 s	12.0, CH3	1.27 s	12.1, CH3
20	0.82 s	14.1, CH3	0.81 s	14.0, CH3
1'	-	167.3, C	-	167.5, C
2'	-	128.1, C	-	128.1, C
3'	6.03 qq (7.0, 1.5)	138.3, CH	6.02 qq (7.0, 1.5)	138.3, CH
4'	1.93 dq (7.0, 1.5)	15.9, CH3	1.93, brd (7.5)	15.9, CH3
5'	1.83 m	20.7, CH3	1.82 brs	20.8, CH3
OMe			3.59 s	57.35/57.32, CH3

^aAssignments based on analysis of 2D NMR spectra.

^bData (δ) measured at 500 MHz; s =singlet, br s =broad singlet, d =doublet, dd =doublet of doublets, dq =doublet of quartets, qq =quartet of quartets, m =multiplet. J values are in Hz and are omitted if the signals overlapped as multiplets. The overlapped signals were assigned from HSQC and HMBC spectra without designating multiplicity.

^cData (δ) measured at 125 MHz; CH₃, CH₂, CH, and C multiplicities were determined by an HSQC experiment.

Figure 2.

HMBC (left) and NOESY (right) correlations of compound 1.

Figure 1.

Structures of compounds 1–2 isolated from G. sarothrae

Compound 2 was obtained as a colorless oil and had the molecular formula C₂₆H₃₆O₇ as indicated by HRESIMS analysis (m/z 461.2560 [M+H]⁺, calcd. for C₂₆H₃₇O₇⁺, 461.2534; 483.2361 [M+Na]⁺, calcd. for C₂₆H₃₆NaO₇⁺, 483.2354). Examination of its 1H-NMR spectroscopic data indicated that compound 2 was similar to compound 1, except for the presence of a methoxy group in place of a hydroxy group. Placement of the methoxy group was determined by an HMBC correlation between the methoxy protons and C-15. Examination of the NOESY correlations of compound 2 suggested that its relative configuration was identical to that of compound 1. Compound 2 was obtained as a mixture of C-15 epimers that were not separable by HPLC, resulting in two sets of signals for C-13, C-14, C-15 and –OMe [10]. Compound 2 might possibly be an artefact formed by reaction of 1 with MeOH during the extraction of the plant material. However, we were unable to collect fresh plant material and extract it with a different solvent to confirm this hypothesis.

Compounds 1 and 2 were evaluated for their antimalarial activity against the chloroquine/mefloquine-resistant Dd2 strain of P. falciparum. Compound 1 exhibited moderate antiplasmodial activity with an IC₅₀ value of 10.4 ± 4.3 µM in this assay, while compound 2 was inactive at doses below 20 µg/mL. The activity of compound 1 is thus most likely associated with its ring-opened α,β-unsaturated aldehyde form, since 2 is incapable of ring opening under mild conditions.

Experimental section

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 (I.D.×L 3×50 cm) and Si-diol (I.D.×L 2.5 ×35 cm, 40–63 µm). HPLC separation was performed on a Shimadzu LC-10AT instrument with a semipreparative C18 Phenomenex Luna column (5 µm, 250 × 10 mm).

Antimalarial bioassay

The effect of each fraction and pure compound 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 [11a,11b]. 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 [11b]. The half-maximum inhibitory concentration (IC₅₀) values were calculated with KaleidaGraph software using a nonlinear regression curve fitting. IC₅₀ values are the average of three independent determinations with each determination in duplicate, and are expressed ± S.E.M. Artemisinin was used as the positive control with an IC₅₀ of 6 ± 1 nM.

Plant material

Leaves and stems of G. sarothrae Kuntze were collected on the edge of a lake near Saskatoon, Canada (52.10N, 106.40W) by Cori M. Morenberg under the auspices of the New York Botanical Garden. A voucher specimen is on deposit under accession number CM00068a.

Extraction and isolation

Dried, powdered plant material was exhaustively extracted with MeOH to give a MeOH-soluble extract designated X-5689; a total of 800 mg of this extract was made available to Virginia Tech. This extract (IC₅₀ between 5 and 10 µg/mL aganist P. falciparum Dd2 strain) was suspended in aqueous MeOH (MeOH:H₂O, 9:1, 100 mL) and extracted with hexane (5×100 mL portions). Evaporation of the solvent afforded 155.2 mg of hexane soluble materials. The aqueous fraction was then diluted to MeOH-H₂O, 6:4 (150 mL) and further extracted with CH₂Cl₂ (5×100 mL portions) to yield 212.3 mg of CH₂Cl₂ fraction and 429.9 mg of aqueous fraction. The active hexane and CH₂Cl₂ fractions (IC₅₀ around 5 µg/mL) were combined and subjected to size exclusion open column chromatography (I.D.×L 3×50 cm) on Sephadex LH-20 eluted with CH₂Cl₂:MeOH, 1:1. Four fractions (F1: 155.7 mg, F2: 52.9 mg, F3: 111.0 mg, F4: 43.3 mg) were collected based on TLC profile. The most active fraction F3 (IC₅₀ between 2.5~5 µg/mL) was then subjected to Si-diol open column chromatography (I.D.×L 2.5 ×35 cm, 40–63 µm) eluted with EtOAc:hexane (1:4) to give five subfractions indexed F3-1 (16.6 mg), F3-2 (23.5 mg), F3-3 (19.7 mg), F3–4 (16.8 mg) and F3-5 (28.6 mg). Fractions F3-3 (IC₅₀ around 2.5 µg/mL) and F3–4 (IC₅₀ between 2.5~5 µg/mL) were combined and further separated by HPLC on a semipreparative C₁₈ column (Phenomenex Luna column, 5 µm, 250 × 10 mm) eluted with a solvent gradient from CH₃CN:H₂O, 50:50 to 60:40 from 0 to 10 min, to 100:0 from 10 to 30 min, ending with 100% CH₃CN from 30 to 40 min at a flow rate of 2.5 mL/min. This process gave compounds 1 (2.3 mg, t_R 12.5 min) and 2 (1.2 mg, t_R 22.0 min).

3α-angeloyloxy-15-hydroxylabda-7,13-dien-16,15-olid-18-oic acid (1)

Colorless oil, ${[\alpha ]}_D^{23}$: +21.7 (c 0.037, MeOH);

UV (MeOH) λ_max (log ε) 221 (4.83);

IR (film) ν_max 3332, 1657, 1451, 1024 cm⁻¹;

¹H NMR (500 MHz, CDCl₃): Table 1.

¹³C NMR (125 MHz, CDCl₃): Table 1.

HRESIMS (pos.): m/z [M+Na]⁺, calcd. for C₂₅H₃₄O₇Na⁺: 469.2197, found: 469.2218; [2M+Na]⁺, calcd. for C₅₀H₆₈O₁₄Na⁺, 915.4501, found: 915.4543.

3α-angeloyloxy-15-methoxylabda-7,13-dien-16,15-olid-18-oic acid (2)

Colorless oil. ${[\alpha ]}_D^{23}$: +16.5(c 0.030, MeOH);

UV (MeOH) λ_max (log ε) 218 (4.89);

IR (film) ν_max 3324, 1641, 1452, 1017 cm⁻¹;

¹H NMR (500 MHz, CDCl₃): Table 1.

¹³C NMR (125 MHz, CDCl₃): Table 1.

HRESIMS (pos.): m/z [M+H]⁺, calcd. for C₂₆H₃₇O₇⁺, 461.2534, found: 461.2560; [M+Na]⁺, calcd. for C₂₆H₃₆NaO₇⁺, 483.2354, found: 483.2361.