Long-chain alkyl-substituted gentisic acid and benzoquinone derivatives from the root of Micronychia tsiramiramy (Anacardiaceae)

1 Introduction

Micronychia is a genus of the Anacardiaceae family, and the species Micronychia tsiramiramy is endemic to Madagascar, where it is found mainly in the south-eastern region, in forests up to 1500 m altitude [1, 2]. According to our ethnobotanical survey the root decoction of this plant is used in traditional medicine to treat several ailments such as malaria, diabetes and hypertension. Plants of the Anacardiaceae family have been extensively investigated from phytochemical and biological points of view. Antifungal, antimicrobial, antioxidant, antimalaria, antiprotozoal, leishmanicidal, hypoglycemic and insecticidal activities have been reported for extracts of different species of the family [3–5]. Several bioactive secondary metabolites, e.g. terpenoids, flavonoids, resorcinols and xanthones, have been isolated from several genera of this family [6–10]. To the best of our knowledge, neither phytochemical nor pharmacological studies have been done on plants of the genus Micronychia. In a continuing search for bioactive compounds from Madagascarian medicinal plants, we have investigated the ethyl acetate soluble fraction of the methanol extract of the root of M. tsiramiramy. We report here the isolation and structure elucidation of two new compounds, micronyc acid (1) and micronone (2) together with cytotoxicity data against the human cervix carcinoma cell line KB-3-1 and the antiplasmodial activity of compound 1.

2 Results and discussion

The investigation of the ethyl acetate soluble fraction of the methanol extract of the root of M. tsiramiramy (Anacardiaceae) led to the isolation of a new gentisic acid derivative, named micronyc acid (1), and a new 1,4-benzoquinone derivative, named micronone (2) (Fig. 1), together with the known compounds gallic acid (3), methyl gallate (4) [11], moronic acid (5) [12, 13], masticadienolic acid (6) [14, 15], and masticadienediol (7) [16, 17].

Fig. 1:

Structures of compounds 1, 1a, and 2.

Compound 1 was obtained as white amorphous powder. Its molecular formula C₂₄H₃₈O₄ was deduced from the HR-ESI-MS which showed the pseudo-molecular ion [M+Na]⁺ at m/z = 413.26689 (calcd. 413.26623) containing six degrees of unsaturation. Its IR spectrum displayed the vibration of one or more hydroxy groups at 3380 cm⁻¹. The ¹H NMR spectrum of 1 (Table 1) showed two ortho-coupled aromatic protons at δ = 6.92 ppm (1H, J = 8.9 Hz, H-4) and 6.72 ppm (1H, J = 8.9 Hz, H-5); a multiplet assigned to two olefinic methine protons at δ = 5.28 ppm (H-10′, H-11′); another multiplet assigned to the protons of a methylene group attached to an aromatic ring at δ = 2.90 ppm (H-1′), signals of two sets of allylic methylene protons at δ = 1.94 ppm (overlapping, H-9′, H-12′), multiplets of 22 overlapping methylene protons between δ = 1.52–1.18 ppm and a terminal methyl group at δ = 0.81 ppm (t, H-17′). The ¹³C NMR (Table 1) and DEPT spectra of compound 1 displayed resonances characteristic for a carboxylic group (δ = 173.2 ppm) and an oxygenated aromatic ring (δ = 156.5, 145.4, 130.3, 122.9, 114.9 and 110.0 ppm). The ¹³C NMR also indicated the presence of an alkyl chain [21–30.9 (CH₂)_n, 13.1 (CH₃)] with a single double bond (δ = 128.8 ppm, 2C). The chemical shift of the carbons at 158.5 ppm and 145.4 ppm indicated that they are linked to hydroxyl groups. All these data indicated that compound 1 possesses a phenolic moiety linked to an unsaturated side chain (Fig. 1). The location of the substituents on the aromatic ring was deduced from the correlations observed in the HMBC spectrum between H-4 (δ = 6.92 ppm) and C-6 (δ = 156.5 ppm), C-3 (δ = 145.4 ppm) and C-2 (δ = 130.3 ppm), as well as between H-5 (δ = 6.72 ppm) and C-6 (δ = 156.5 ppm), C-3 (δ = 145.4 ppm), C-1 (δ = 110.0 ppm), and the carboxylate carbon (δ = 173.2 ppm), suggesting that the two hydroxyl groups are located on the aromatic ring at C-3 and C-6 and the carboxylic group at C-1. The attachment of the alkenyl side chain to C-2 of the aromatic ring was deduced from the HMBC spectrum where cross peaks were observed between H-1′ (δ = 2.90 ppm) and C-3 (δ = 145.4 ppm), C-2 (δ = 130.3 ppm), and C-1 (δ = 110.0 ppm). The presence of the carboxylic group was also confirmed by the peak observed on the EI-MS spectrum at m/z = 346 corresponding to a loss of CO₂ [18]. The peak at m/z = 167 corresponds to benzyl cation forming fragmentation. Likewise, a similar benzyl cation giving rise to a peak at m/z = 123 is formed from the decarboxylated compound (m/z = 346). Both cations prove the presence of a C₁₇H₃₃ side chain. The location of the double bond within the C₁₇ side chain between C-10′ and C-11′ was established using the fragmentation patterns of the EIMS with the diagnostic fragment at m/z = 320 ppm corresponding to the formation of [M–C₅H₁₀]⁺ by McLafferty rearrangement (Fig. 2). The cis (Z) configuration of the double bond was concluded from the chemical shifts of the olefinic carbons (below 130 ppm) and of the carbons next to the double bond at δ = 25.9 ppm (C-9′, C-12′). Both the olefinic and the allylic protons in Z-configured double bonds usually are shifted towards higher field compared to E-configured double bonds (128–129 ppm vs. 130–131 ppm and 25–28 ppm vs. 31–33 ppm [19–23]. Hence, the structure of compound 1 was unambiguously assigned to (Z)-2-(heptadec-10-en-1-yl)-3,6-dihydroxybenzoic acid and the compound was given the name micronyc acid.

Fig. 2:

Mass fragmentation pattern of compound 1. The benzyl cation at m/z = 167 is derived from the molecular ion, while the benzyl cation at m/z = 123 is derived from [M–CO₂]⁺.

Compound 2 was isolated as a yellow oil and its molecular formula C₂₃H₃₆O₂ with six double bond equivalents was deduced from the HR-ESI-MS that showed the pseudo-molecular ion [M+Na]⁺ at m/z = 367.26097 (calcd. 367.26075). The ¹H NMR spectrum of compound 2 showed the triplet of one proton at δ = 6.56 ppm (H-3, J = 1.2 Hz) and a multiplet of two ortho-coupled protons between δ = 6.69–6.76 ppm (H-5 and H-6). The spectrum also exhibited characteristic signals of an alkenyl moiety in the side chain; a multiplet corresponding to two olefinic protons between δ = 5.33–5.36 ppm (H-4′, H-5′), a double triplet corresponding to the protons of a methylene group attached to an aromatic ring at δ = 2.41 ppm (J = 1.2, 7.9 Hz, H-1′) with an allyl coupling to H-3, and two allylic methylene groups at δ = 2.00–2.03 ppm (H-3′ and H-6′), together with 22 other overlapping methylene protons in the range δ = 1.32–1.25 ppm and a terminal methyl group at δ = 0.88 ppm (t, H-17′). The ¹³C NMR spectrum of compound 2 (Table 2) exhibited signals of 23 carbon atoms that where sorted by DEPT and HSQC into three quaternary carbons, among them two carbonyl groups of benzoquinone at δ = 187.8 ppm (C-1) and 188.1 ppm (C-4), five methine groups, among them three benzoquinone carbons at δ = 132.6 ppm (C-3), 136.5 ppm (C-5), 137.0 ppm (C-6), and two olefinic methine carbons at δ = 129.9 ppm (C-4′) and δ = 130.3 ppm (C-5′); 14 methylene groups (δ = 22.9–32.1 ppm) and one methyl group (δ = 14.3 ppm). All these data suggested that compound 2 is an alkenyl benzoquinone (Fig. 1). The correlations observed in the HMBC spectrum between H-1′ (δ = 2.41 ppm) and C-1 (δ = 187.8 ppm), C-2 (δ = 149.9 ppm), and C-3 (δ = 132.6 ppm), showed that the alkenyl chain was connected to the benzoquinone at C-2. The EI mass spectrum features a molecular ion peak at m/z = 346 [M+2H], while the exact mass of the pseudomolecular ion [M+Na]⁺ was determined to be m/z = 367.26097 according to HR-ESI-MS. Such hydroquinone formation in the MS inlet is a well-known artifact in EI-MS of benzoquinones. The benzyl cation [M+2H–C₁₆H₃₁] giving rise to the base peak at m/z = 123 proves the presence of a C₁₇H₃₃ side chain. The peak at m/z = 124 corresponds to the product of an arene-based McLafferty rearrangement. The location of the double bond within the C₁₇ side chain between C-4′ and C-5′ was established using the fragmentation patterns of the EIMS with the diagnostic fragment at m/z = 136 corresponding to a McLafferty rearrangement leading to the formation of the vinyl hydroquinone [C₃H₈O₂]⁺.

The configuration of the double bond was assigned as Z like discussed for 1 (vide supra) based on the close similarity of the ¹³C NMR values of the two olefinic carbons and, in addition, of the allylic carbons [24]. Compound 2 was assigned to be (Z)-2-(heptadec-4-en-1-yl)cyclohexa-2,5-diene-1,4-dione, and it was given the name micronone.

Acetylation of the compound 1 gave the monoacetylated compound 1a characterized by an MS peak [M–H]⁻ at m/z = 431.3 (Fig. 3), corresponding to the introduction of only one acetyl group. This agrees with the fact that compound 1 contains a chelated hydroxyl group at position 6, which is it less prone towards acetylation.

Fig. 3:

Acetylation of compound 1.

Compound 1 was tested for antiplasmodial activity in vitro against the Plasmodium falciparum chloroquine-resistant strain W2. The compound showed moderate antiplasmodial activity with an IC₅₀ value of 25.6 μm.

Compounds 1 and 1a were tested for cytotoxicity against the human cervix carcinoma cell line KB-3-1, and displayed moderate cytotoxicity with IC₅₀ values of 13.5 μm for 1 and 14.4 μm for 1a, respectively. Although cytotoxicity is observed at a similar concentration as antiplasmodial activity, the compound might still provide useful information regarding parasite biology.

3 Conclusion

The investigation of the root extracts of M. tsiramiramy led to the isolation of new long-chain alkyl substituted gentisic acid and benzoquinone derivatives together with previously described compounds belonging to different classes of natural products. The structural diversity and the previously reported biological activities of some of the isolates highlight the rich potential of this Madagascarian medicinal plant as a source of biologically active ingredients.

4 Experimental section