Semi-Synthesis and Evaluation of Sargahydroquinoic Acid Derivatives as Potential Antimalarial Agents

Abstract

Background: Malaria continues to present a major health problem, especially in developing countries. The development of new antimalarial drugs to counter drug resistance and ensure a steady supply of new treatment options is therefore an important area of research. Meroditerpenes have previously been shown to exhibit antiplasmodial activity against a chloroquinone sensitive strain of Plasmodium falciparum (D10). In this study we explored the antiplasmodial activity of several semi-synthetic analogs of sargahydroquinoic acid. Methods: Sargahydroquinoic acid was isolated from the marine brown alga, Sargassum incisifolium and converted, semi-synthetically, to several analogs. The natural products, together with their synthetic derivatives were evaluated for their activity against the FCR-3 strain of Plasmodium falciparum as well as MDA-MB-231 breast cancer cells. Results: Sarganaphthoquinoic acid and sargaquinoic acid showed the most promising antiplasmodial activity and low cytotoxicity. Conclusions: Synthetic modification of the natural product, sargahydroquinoic acid, resulted in the discovery of a highly selective antiplasmodial compound, sarganaphthoquinoic acid.

1. Introduction

Despite the impressive breakthroughs in the treatment of malaria [1], it remains a life-threatening disease. Southeast Asia and sub-Saharan Africa account for the vast majority of the estimated 219 million malaria cases reported worldwide, leading to 435,000 deaths [2]. More than 90% of malaria cases and deaths occur in Africa, of which more than 70% are children under five years of age [2]. The prospect of resistance to current drugs appears inevitable and paints a bleak picture indeed [3]. Although the reasons for this dire situation are complex, there is undoubtedly a need for the continued search for and development of new antimalarial drugs. Natural products have historically offered some of the most effective antimalarial drugs [4]. In a previous study, we reported on the antiplasmodial activity of natural products isolated from the South African brown seaweed, Sargassum incisifolium, against a chloroquine sensitive strain of Plasmodium falciparum (D10) [5]. S. incisifolium is relatively abundant along the South African coastline and produces sargahydroquinoic acid (1) as the major metabolite. The accessibility of 1 thus provided an opportunity to explore the structure activity relationships of analogs of this natural product. Herein we report on the antiplasmodial activity of semi-synthetic analogs of sargahydroquinoic acid (1) (Figure 1).

2. Materials and Methods

2.1. General Experimental

All solvents were of chromatographic grade (Merck, Darmstadt, Germany) and used without further purification. Column chromatography was performed on silica gel (40–63 μm particle size) from Merck, Darmstadt, Germany. Normal Phase HPLC was carried out using a Whatman Partisil 10 semi-preparative column (Sigma-Aldrich, Schnelldorf, Germany) (10 mm × 500 mm, 10 μm), while a Phenomenex Luna C₁₈ column (Sigma-Aldrich, Schnelldorf, Germany, 10 mm × 250 mm, 10 μm) was used for reversed phase HPLC. NMR spectra were recorded on Bruker Avance 400 and 600 MHz spectrometers (Bruker Biospin, Rheinstetten, Germany) and referenced to residual undeuterated CDCl₃ solvent signals (δ_H 7.26 ppm and δ_C 77.0 ppm). UV spectra were measured on a Perkin Elmer Lambda 25 UV/Vis spectrometer (Perkin-Elmer, Norwalk, CT, USA) while FT-IR data was obtained using a Perkin Elmer Spectrum 100 FT-IR spectrometer (Perkin-Elmer, Norwalk, CT, USA). High resolution electrospray ionization mass spectroscopy (HR-ESIMS) spectra were obtained on a Waters Synapt G2 mass spectrometer (Waters Corporation, Milford, MA, USA) at 20 V.

2.2. Extraction and Isolation of Natural Products

Specimens of Sargassum incisifolium were collected from Port Alfred (collection code PA071b) on the south east coast of South Africa on 21 September 2007 and stored at −20 °C. The samples were authenticated by comparison with voucher specimens from previous studies [5]. Voucher specimens are stored at the School of Pharmacy, University of the Western Cape.

The following isolation protocol is representative and was repeated several times in order to generate sufficient quantities of 1 for synthetic modification. The frozen alga (38.77 g, extracted dry weight) was allowed to thaw at room temperature after which it was soaked in methanol for one hour. The methanol was removed and the alga extracted three times with MeOH-CH₂Cl₂ (1:2) at 40 °C for 30 min. Extracts were pooled and separated into aqueous and organic phases by the addition of distilled water. Concentration of the organic phase under reduced pressure gave a dark green residue (3.87 g). A portion of the organic fraction (1.09 g) was fractionated by step-gradient elution on a silica gel column (10 g) using solvents of increasing polarity (n-hexane-EtOAc) to give seven fractions as follows: Fr A (H-E, 10:0, 8.6 mg), Fr B (H-E, 9:1, 27 mg), Fr C (H-E, 8:2, 132 mg), Fr D (H-E, 6:4, 218 mg), Fr E (H-E, 4:6, 65 mg), Fr F (H-E, 2:8, 9.7 mg) and Fr G (H-E, 0:10, 50 mg) followed by MeOH-EtOAc (1:1), Fr 7H (238 mg). Fraction B (19 mg) was further purified by silica gel column chromatography using a mobile phase of n-hexane-EtOAc (9:1) to give 1.7 mg of sargaquinal (9). Fraction C (40 mg) was purified by normal phase HPLC using n-hexane-EtOAc as mobile phase (8:2) to give 15 mg of sargaquinoic acid (3). Fraction D (20 mg) was purified by reversed phase HPLC using MeOH-H₂O phase (90:10) as the mobile phase to give sargahydroquinoic acid (1) (6.8 mg) and sargachromenol (7) (2.4 mg), respectively. The isolation of compounds 1, 3, 7 and 9 is summarised in Scheme S1 and their structures were confirmed by spectroscopic methods, which were in agreement with literature data (Table S1) [5]. The NMR spectra for compounds 1 (Figures S1 and S2), 3 (Figures S3 and S4), 7 (Figures S5 and S6) and 9 (Figures S7 and S8) are presented in the Supplementary Materials.

2.3. Sargaquinoic Acid (3) and Sarganaphthoquinoic Acid (10)

To a solution of 1 (154.0 mg, 0.36 mmol) in a mixture of CHCl₃ (8 mL) and MeOH (7 mL) was added Ag₂O (100 mg, 0.43 mmol). The reaction mixture was stirred at room temperature for 24 h, after which the resulting suspension was filtered through diatomaceous earth and concentrated under reduced pressure. The crude product was filtered through a plug of charcoal (n-hexane-EtOAc, 4:6) to give a yellow mixture of compounds which was separated by silica gel column chromatography (n-hexane-EtOAc, 7:3) to give sargaquinoic acid (3) (80 mg, 70%) and compound 10 (9.8 mg, 6%) as light yellow oils. NMR spectra for compound 10 (Figures S9–S14) can be found in the Supplementary Materials.

Sarganaphthoquinonoic acid (10): IR (film) ν_max (cm⁻¹): 1600, 1663, 2850, 2924; ¹H and ¹³C NMR data see

Table 1. NMR spectroscopic data for sarganaphthoquinoic aicd (10) (600 and 125 MHz, CDCl₃).

Carbon Number	δC	Type	δH, mult, J (Hz)	COSY	HMBC
1	185.4	C	-
2	130.2	C	-
3	132.2	C	-
4	185.4	C	-
5	136.0	CH	6.81, s	H-7
6	149.0	C	-
7	16.4	CH3	2.18, s,	H-5	C-6, C-5
1’	126.7	CH	8.00, d, 7.9	H-2’	C-2, C-1
2’	133.8	CH	7.51, d, 7.9	H-4’, H-20’	C-1, C-20’
3’	148.2	C	-
4’	36.2	CH2	2.77, t, 7.6	H-5’	C-5’, C-3’
5’	29.1	CH2	2.36, m	H-4’, H-6’	C-4’, C-6’, C-7’
6’	123.2	CH	5.17, m
7’	136.0	C	-
8’	39.0	CH2	2.08, m	H-9’	C-6’
9’	28.2	CH2	2.57, m	H-10’	C-8’, C-10’
10’	145.0	CH	5.96, t, 7.3	H-9’	C-8’
11’	130.6	C	-
12’	27.8	CH2	2.26, m	H-13’, H-14’ (lr)	C-13’, C-14’
13’	28.2	CH2	2.11, m	H-14’	C-15’, C-11’
14’	123.4	CH	5.17, t, 7.0	H-13’, H-14’ (lr)
15’	132.3	C	-
16’	25.6	CH3	1.68, s	H-14’ (lr)	C-15’, C-14’
17’	17.7	CH3	1.59, s	H-14’ (lr)	C-16’
18’	171.9	C	-
19’	15.9	CH3	1.58, s	H-6’	C-6’
20’	125.9	CH	7.86, s	H-4’	C-4’, C-4

COSY: ¹H-¹H Correlation spectroscopy; HMBC: ¹H-¹³C Heteronuclear multiple-bond correlation spectroscopy.

; HRESIMS m/z 419.2222 [M-H] (calcd. for C₂₇H₃₅O₃, 419.2221)

2.4. Sargaquinoic Acid Methyl ester (5)

To a solution of 1 (122.4 mg, 0.29 mmol) dissolved in 2 mL acetone, was added K₂CO₃ (207.4 mg, 1.50 mmol) in 5 mL acetone and dimethylsulphate (250 µL, 2.63 mmol). The mixture was heated at 40 °C for 8 h followed by stirring at room temperature for 16 h. The reaction mixture was filtered, concentrated under reduced pressure and separated by silica gel column chromatography (n-hexane- EtOAc, 8:2) to give the methyl ester of 1, which, upon exposure to air was completely oxidized to 5. NMR spectra for compound 5 (Figures S15–S16) can be found in the Supplementary Materials.

Yellow oil, ¹H NMR (400 MHz, CDCl₃) δ 6.52 (1H, s, H-3), 6.44 (1H, s, H-5), 5.83 (1H, t, J = 7.0 Hz, H-10’), 5.10 (3H, m, H-2’, H-6’, H-14’), 3.71 (3H, s, OMe), 3.11 (2H, d, J = 6.8 Hz, H-1’), 2.49 (2H, m, H-9’), 2.22 (2H, m, H-12’), 2.05 (2H, m, H-4’) 2.03-2.05 (6H, m, H-5’, H-8’, H-13’), 1.65 (6H, s, H-7, H-19’), 1.61 (3H, s, H-20’), 1.58 (3H, s, H-16’), 1.55 (3H, s, H-17’); 188.0 (C-1, C-4), 168.4 (C-18’), 148.4 (C-6), 145.8 (C-2), 142.1 (C-10’), 140.0 (C-3’) 134.8 (C-7’) 133.1 (C-3, C-15’), 132.1 (C-5), 131.4 (C-11’), 124.4 (C-6’), 123.5 (C-14’), 118.0 (C-2’), 51.0 (OMe), 39.8 (C-4’), 39.1 (C-8’), 34.7 (C-12’), 28.0 (C-9’), 27.8 (C13’), 27.5 (C-1’), 26.4 (C-5’), 25.6 (C-16’), 17.6 (C-17’), 16.1 (C-7), 15.9 (C-19’); HRESIMS m/z 437.2710 [M-H] (calcd. for C₂₈H₃₇O₄, 437.2692).

2.5. Diacetyl Sargahydroquinoic Acid (2)

To sargahydroquinoic acid (1) (110.0 mg, 0.26 mmol) was added acetic anhydride (3 mL, 31.8 mmol) and pyridine (2 mL, 24.8 mmol). The reaction mixture was stirred at room temperature for 30 h. The crude product was acidified with 1 M HCl (10 mL) and extracted with EtOAc (5 mL × 3). The organic layer was collected and concentrated under reduced pressure to give a crude product which was further purified by silica gel column chromatography (n-hexane:EtOAc, 7:3) to give compound 2 (12.8 mg, 12%) as a yellow oil. The structure of compound 2 was confirmed by spectroscopic methods, which were in agreement with literature data [6,7]. NMR spectra for compound 2 (Figures S17 and S18) can be found in the Supplementary Materials.

2.6. Sargaquinol (6) and Sargachromendiol (8)

To a solution of sargahydroquinoic acid (1) (140.7 mg, 0.33 mmol) dissolved in anhydrous THF (5 mL), was added LiAlH₄ (0.104 g, 2.74 mmol). The reaction mixture was stirred at room temperature, under a nitrogen atmosphere for 1.25 h. The reaction was quenched with a few drops of EtOAc, concentrated and partitioned between EtOAc (10 mL⁻²) and H₂O (5 mL). The organic layer was concentrated under reduced pressure to give a crude product which was purified by silica gel chromatography (n-hexane:EtOAc, 8:2) to give sargaquinol (6) (12.2 mg, 30%) and the alcohol derivative of sargachromenol (8) (2.8 mg, 3.5%). The structure of compound 6 was confirmed by spectroscopic methods, which were in agreement with literature data (Table S1) [7]. NMR spectra for compounds 6 (Figures S19 and S20) and 8 (Figures S21 and S22) can be found in the Supplementary Materials.

Sargaquinol (6) yellow oil; ¹H NMR (400 MHz, CDCl₃) δ 6.54 (s, 1H) (H-3), 6.46 (s, 1H) (H-5), 5.15 (dd, J = 21.0, 13.2 Hz) (H- 2’, 6’, 14’), 4.11 (s) (H-18’), 3.63 (t, J = 6.5 Hz) (H-10’), 3.12 (d, J = 7.1 Hz) (H-1’), 2.12 (s) (H-5’, 9’, 13’), 2.05 (s) (H-4’, 8’), 1.67 (s) (H- 7, 16’), 1.60 (s) (H-19’, 20’), 1.57 (s) (H-17’). ¹³C NMR (100 MHz, CDCl₃) δ 188.0 (C-1), 187.97 (C-4), 148.5 (C-6), 145.9 (C-2), 139.7 (C-3’), 135.0 (C-7’), 133.1 (C-3), 131,2 (C-11’), 132.24 (C-5), 133.7 (C-15’), 124.7 (C-6’), 124.2 (C-14’), 118.1 (C-2’), 71.8 (C-18’), 62.8 (C-10’), 39.8 (C-4’), 39.5 (C-8’), 35.2 (C-12’), 27.1 (C-13’),27.5 (C-1’), 26.2 (C-5’), 26.3 (C-9’), 25.6 (C-16’), 17.7 (C-7), 16.11 (C-17’), 16.07 (C-19’), 16.0 (C-20’).

Sargachromendiol (8) yellow oil; ¹H NMR (400 MHz, CDCl₃) δ 6.47 (d, J = 2.4 Hz) (H-5), 6.32 (d, J = 2.5 Hz) (H-2), 6.26 (s) (H-2’), 5.98 (t, J = 7.2 Hz) (H-9’), 5.57 (d, J = 9.8 Hz) (H-3’), 5.12 (dt, J = 19.0, 6.4 Hz) (H-5’, 14’), 2.59 (q, J = 7.3 Hz (H-8’), 2.27 (t, J = 7.4 Hz), 2.13 (s) (H-12’), 2.09–2.04 (m) (H-4’, 7’), 1.68 (s) (H-8), 1.58 (d, J = 3.5 Hz) (H-17’, 19’), 1.36 (s) (20’); ¹³C NMR (100 MHz, CDCl₃) δ 148.6 (C-5), 145.0 (C-10’), 144.8 (C-8), 134.7 (C-7’), 131.8 (C-15’), 130.6 (C-11’), 126.3 (C-3), 124.7 (C-6’), 124.1 (C-1’), 122.9 (C-14’), 121.3 (C-2), 117.0 (C-4), 110.3 (C-6), 77.8 (C-3’), 60.3 (C-18’), 40.7 (C-4’), 39.8 (C-8’), 35.1 (C-12’), 35.3 (C-12’), 27.0 (C-9’), 26.1 (C-13’), 25.9 (C-20’), 25.7 (C-16’), 22.6 (C-5’), 17.7 (17’), 15.9 (C-7), 15.5 (C-19’).

2.7. Z-sargaquinal (4)

To a solution of sargaquinol (6) (37.2 mg, 0.09 mmol) dissolved in anhydrous CH₂Cl₂ (8 mL), Dess-Martin Periodinane (107 mg, 0.26 mmol) was added. The reaction mixture was stirred at room temperature for 2 h after which it was quenched with CH₂Cl₂ (10 mL) and de-ionized water (10 mL). The organic phase was separated and washed with saturated solutions of NaHCO₃ (10 mL × 3) and Na₂S₂O₃ (10 mL × 3), dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (n-hexane:EtOAc, 8:2) to give compound 4 (42%, 14.9 mg), as a yellow oil. The structure of compound 4 was confirmed by spectroscopic methods, which were in agreement with literature data (Table S1) [7]. NMR spectra for compound 4 (Figures S23–S24) can be found in the Supplementary Materials.

2.8. Antiplasmodial Assays

All compounds were tested in triplicate against the chloroquine-resistant Gambian FCR-3 strain of P. falciparum. The in vitro erythrocytic stage of the parasite was maintained using the method outlined by Trager and Jensen [8]. The antimalarial activity of the compounds was determined using the tritiated hypoxanthine incorporation assay using a 0.5% parasitaemia and 1% haematocrit [9]. All assays were carried out using untreated parasites and uninfected red blood cells as controls. The concentration that inhibited 50% parasite growth (IC₅₀ value) was determined from the log sigmoid dose response curve using GraphPad Prism. Quinine was used as the reference antiplasmodial agent. The selectivity index for the compounds was determined from the ratio of cytotoxicity IC₅₀ to antimalarial IC₅₀.

2.9. Cytotoxicity Assay

All compounds were tested in triplicate against MDA-MB-231 breast carcinoma cells, which were purchased from the ATCC (Catalogue number HTB-26, Manassas, VA, USA). The cytotoxicity of the compounds was determined using the WST-1 assay method (Roche). The cells were treated with a range of concentrations of the test compounds or vehicle control (DMSO). Cells treated with DMSO were considered to represent 100% viability and the viability of cells at each dose was represented relative to this value. The concentration resulting in a decrease of cell viability to 50% was calculated from the linear portion of the dose response curve.

3. Results and Discussion

3.1. Isolation and Synthetic Modification of Sargahydroquinoic Acid Derivatives

Sargahydroquinoic acid (1) is the major component of the CH₂Cl₂-MeOH extract of Sargassum incisifolium and has also been reported from several other Sargassum spp. [5,6,7]. This compound slowly converts to sargaquinoic acid (3) and sargachromenol (7) on storage of the seaweed, the extract and during purification. Specimens of S. incisifolium (PA071b) were collected from Port Alfred on the south eastern coast of South Africa and extracted with CH₂Cl₂-MeOH. The crude extract was first fractionated by silica gel column chromatography, followed by normal or reversed phase HPLC to give compounds 1, 3, 7 and 9. The identities of all isolated compounds were confirmed by comparison of their NMR spectroscopic data (Table S1) to literature values [5]. The above protocol yielded sufficient quantities of 1 to perform structural modifications and biological assays.

Sargaquinoic acid (3) is normally isolated from fresh seaweed in relatively small quantities; however, it can be produced more efficiently by the oxidation of 1 [7,10]. Thus, treatment of 1 with Ag₂O gave 3 in moderate to good yields. Interestingly, although this conversion is facile, we consistently observed a series of unusual peaks between δ_H 7 and 8 in the ¹H NMR spectrum of the crude reaction product. The compound (10) responsible for these peaks was isolated and its structure was elucidated by NMR spectroscopy and mass spectrometry.

The HRESIMS spectrum of compound 10 showed a molecular ion peak at m/z 419.2222 [M-H] which corresponds to a molecular formula of C₂₇H₃₁O₄. Characteristic deshielded methine resonances at δ_H 8.00 (d, J = 7.9), δ 7.98 (s) and δ 7.51 (d, J = 7.9) were evident in its ¹H NMR spectrum. In addition, one of the aromatic singlets had shifted downfield from δ_H 6.46 in 3 to δ_H 6.81 in 10. Data from the ¹³C NMR spectrum of compound 10 revealed no change in the number of carbon atoms when compared to the starting material (1). It revealed the presence of two quinone carbonyls signals at (δ_C 185.4 and δ 185.4) and a carboxylic acid moiety (δ_C 171.9). In addition, the DEPT-135 NMR spectrum indicated the loss of one methyl signal (δ_C 16.1, C-20’) and a methylene signal (δ_C 27.5, C-1’) when compared to 3, together with the appearance of two additional olefinic methine signals at δ_C 126.7 (C-1’) and 125.9 (C-20’). HMBC correlations (Figure 2) from the doublet at δ_H 8.00 (H-1’) to carbon signals at δ_C 126.7 (C-1’) and δ_C 133.8 (C-2’); the methine signal at δ_H 7.51 (H-2’) to the carbon signal at δ_C 125.9 (C-20’) and from δ_H 7.86 (H-20’) to carbon signals at δ_C 36.2 (C-4’) and δ_C 185.4 (C-4), allowed for the assignment of the naphthoquinone moiety. All other spectroscopic data are consistent with a polyprenyl side chain with a 6’E,10’Z-double bond geometry (as in 3). We assigned the name sarganaphthoquinoic acid to this new compound. A related compound, chabrolonaphthoquinone, had previously been reported from the Taiwanese soft coral, Nephthea chabrolii [11]. The main differences between the two compounds are the methyl substituent at C-6 and the 10-double bond geometry in compound 10.

The direct conversion of prenylated hydroquinones to naphthoquinones is uncommon and presents a novel approach to the synthesis of this important group of compounds. To the best of our knowledge there is only a single report describing the formation of a naphthoquinone as a side-product in the synthesis of chromenes from prenylated quinones [12]. Naphthoquinones are typically synthesized by Diels-Alder reactions between p-benzoquinones and dienes or by the prenylation of halogenated naphthoquinone moieties [13,14,15]. Compound 10 is proposed to form via tautomerism and oxidation of the intermediate quinone (3) followed by 6πelectrocyclization and further oxidation (Scheme 1).

In order to establish preliminary structure-antiplasmodial activity relationships for this series of sargahydroquinoic acid derivatives, we focused our attention on modification of the carboxylic acid and quinone moieties. Acetylation of 1 with acetic anhydride/pyridine gave the diacetate (2), while its reduction with lithium aluminium hydride gave a mixture of sargaquinol (6) and sargachromendiol (8). The facile conversion of the hydroquinone to a mixture of the quinone and chromene on exposure to air is often seen in this series of compounds [6,7]. Spectroscopic evidence for the identity of alcohols 6 and 8 were provided by the disappearance of the ¹³C NMR signal due to the carboxylic acid group at δ_C 172 ppm and the appearance of an oxymethylene carbon signal at δ_C 60.3 ppm in both compounds.

Mild oxidation of 6 with Dess-Martin periodinane, gave 10Z-sargaquinal (4). The structures of aldehydes 4 and 9 were confirmed by comparison of their spectroscopic data with literature values [5,7]. A comparison of the ¹H NMR spectra of the natural and semi-synthetic aldehydes revealed differences in chemical shifts of both proton and carbon atoms associated with the aldehyde group. The ¹H and ¹³C NMR spectra of the semi-synthetic aldehyde (4) showed signals at δ_H 10.1 and δ_C 190.9 ppm compared to δ_H 9.55 and δ_C 205.4 ppm in the natural aldehyde (9). ¹H-¹H NOESY correlations in both compounds confirmed the difference in the geometry of the Δ¹⁰ double bond with the semi-synthetic aldehyde (4) bearing a 10Z-geometry and the natural aldehyde (9) a 10E-geometry. The formation of 2’E,6’E,10’Z-sargaquinal (4) from 2’E,6’E,10’Z-sargahydroquinoic acid (1) has been reported in the literature [7]. However, this is the first report of its ¹³C and 2D NMR data.

Interestingly, methylation of 1 with dimethylsulphate/potassium carbonate did not produce the dimethyl ether, but instead produced sargaquinoic acid methyl ester (5). This was confirmed by the appearance of an additional methyl signal at δ_C 51.0 and an upfield shift of the C-18’ carbonyl signal from δ_C 172 to δ 168.4 ppm in the ¹³C NMR spectrum of 5 (Table S1).

3.2. Biological Assays

The ten sargahydroquinoic acid derivatives were assessed for both antiplasmodial and cytotoxic activity against the chloroquine-resistant Gambian FCR-3 strain of P. falciparum and MDA-MB-231 breast cells, respectively (

Table 2. Bioassay results for compounds 1–10.

Compound	IC50 (μM)			Selectivity Index
	D10 1	FCR-3	MDA-MB-231
Sargahydroquinoic acid (1)	15.2	38.6	70	1.8
Sargahydroquinoic acid di-acetate (2)	-	84.3	286	3.4
Sargaquinoic acid (3)	12.0	10.8	658	60.9
10Z-sargaquinal (4)	-	72.6	211	2.9
Sargaquinoic acid methyl ester (5)	-	8.2	70	8.6
Sargaquinol (6)	-	93.1	99	1.1
Sargachromenol (7)	-	114.8	56	0.5
Sargachromendiol (8)	-	34.2	187	5.5
10E-sargaquinal (9)	2.0	104.4	69	0.7
Sarganaphthoquinone (10)	-	5.4	2410	443
Quinine		0.17	-	-

¹ From reference [5].

). All compounds showed moderate to good antiplasmodial activity. However, the most promising compound in this series is the naphthoquinone 10 which not only revealed good antiplasmodial activity (IC₅₀ 5.4 μM), but also very low cytotoxicity (IC₅₀ 2410 μM), resulting in a high selectivity index of 443. Sargaquinoic acid (3) also shows promising antiplasmodial activity (IC₅₀ 10.8 μM), but is slightly more toxic (IC₅₀ 658 μM) than 10. It appears that the carboxylic acid in the prenyl side chain is important for activity since both aldehydes (4) and (9) and the alcohol (6) showed decreased antiplasmodial activity. The quinone/naphthoquinone scaffold is present in several antimalarial natural products and drugs [16]. It is therefore likely that the mode of action of the compounds reported here is related to this important pharmacophore [16,17,18,19,20].

4. Conclusions

In this study we isolated the relatively abundant antiplasmodial natural product, sargahydroquinoic acid (1) and converted it to several analogs which were evaluated for antiplasmodial and cytotoxic activity. The serendipitous formation of sarganaphthoquinoic acid (10) gave a compound with good antiplasmodial activity while being almost non-toxic. Due to the small number of compounds no clear structure activity relationships can be established, however it appears that the presence of a quinone and carboxylic acid are important for selective activity against P. falciparum. Further studies are warranted to explore the mode of action of these compounds and to further improve on its antiplasmodial activity.