4-Methoxy-3-(3-methylbut-2-en-1-yl)-7-[(3-methylbut-2-en-1-yl)oxy]quinolin-2(1H)-one from Melicope Moluccana T.G. Hartley

Abstract

The crude alkaloid (5 g) was fractionated by column chromatography on silica gel eluted with mixtures of n-hexane-ethyl acetate (9:1 to 1:1) to give four major fractions A–D. Extraction and Isolation The dried and powdered leaves of M. moluccana T.G. Hartley (2.0 kg) were macerated in methanol at room temperature for 24 h two times, and the methanol extract was evaporated under reduced pressure to give methanol extract with dark brown residue (108 g). O′Donnell, F.; Ramachandran, V.N.; Smyth, T.J.P.; Brooks, P. An investigation of bioactive phytochemicals in the leaves Melicope vitiflora by electrospray ionisation trap mass spectrometry. Natural Products Chemistry Research Group, Organic Chemistry Division, Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Surabaya 60115, Indonesia *Author to whom correspondence should be addressed.

1. Introduction

Melicope is one of the genus of the family Rutaceae, consisting of about 320 species growing in the world [1]. Phytochemical studies have shown that the species produce a variety of alkaloids [2,3], flavonoids [4,5], coumarins [6], acetophenones [7], and lignans [8], which exhibit various biological activities, including antioxidant [8,9], anticancer [10,11], and antiinflammatory [12]. This study is part of our research on the chemical constituents of Melicope species found in Indonesia. In continuation of our research for alkaloid compounds in this medicinal plant, we report the isolation of 4-methoxy-3-(3-methylbut-2-en-1-yl)-7-[(3-methylbut-2-en-1-yl)oxy]quinolin-2(1H)-one (1) from the methanol extract of the leaves of Melicope moluccana T.G. Hartley. The chemical structure of compound 1 were established by UV, IR, HRESIMS, 1D and 2D-NMR, and by comparison with those related compounds previously reported. Cytotoxic and antiplasmodial activities of isolated compound from this species are also briefly described.

2. Result and Discussion

The dried and powdered leaves of M. moluccana (2.0 kg) were extracted with methanol. The residue was partitioned with n-hexane. The methanol extract was then adjusted to pH 3–4 with 3% sulfuric acid and partitioned with ethyl acetate to separate the non-alkaloid compound. Acid extracts were basified with ammonia solution (pH 8–9) and partitioned with ethyl acetate to give the crude alkaloid.

The crude alkaloid (5 g) was fractionated by column chromatography on silica gel eluted with mixtures of n-hexane-ethyl acetate (9:1 to 1:1) to give four major fractions A–D. Fraction A (325 mg) was further separated by radial chromatography to yield two sub-fractions (A₁, A₂). Sub-fraction A₁ was purified using radial chromatography eluted with n hexane-chloroform (from 1:1 to 3:7) to give compound 1 (Figure 1).

Figure 1. Structures of 4-methoxy-3-(3-methylbut-2-en-1-yl)-7-[(3-methylbut-2-en-1-yl)oxy]quinolin-2(1H)-one.

4-Methoxy-3-(3-methylbut-2-en-1-yl)-7-[(3-methylbut-2-en-1-yl)oxy]quinolin-2(1H)-one (1) was isolated as yellow solid, reacted positively with Dragendorff reagent to give reddish brown. The HRESIMS displayed a positive molecular ion peak at m/z 328.1928, indicating a molecular formula of C₂₀H₂₆NO₃ (see Figure S6, supporting material). The UV spectrum exhibited absorption maxima λ_max (nm) (log ε): 227 (4.57), 269 (3.86), 282 (3.85), 322 (4.09), and 336 (3.98) typical for a 2-quinolone skeleton [11]. The IR spectrum (see Figure S7, supporting information) indicated absorptions for hydroxyl (3421 cm⁻¹), conjugated carbonyl (1645 cm⁻¹), aromatic (1562, 1512, 1461 cm⁻¹) and ether (1093 cm⁻¹) groups [13]. The ¹H NMR spectrum of 1 (Table 1, Figures S1 and S2, supporting material)) showed the presence of ABX coupling aromatic systems at δ_H 7.65 (d, J = 8.8 Hz; H-5), 7.65 (dd, J = 8.8; 2.4 Hz; H-6), and 7.05 (d, J = 2.4 Hz; H-8) characteristic for 2-quinolone with three substituents. Furthermore, the ¹H NMR spectrum also showed an isoprenyl (3-methyl-2-butenyl) group [δ_H 1.83 (3H, s, H-4′), 1.79 (3H, s, H-5′), 3.39 (2H, d, J = 6.8 Hz, H-1′), 5.29 (1H, t, J = 6.8 Hz, H-2′)], an oxyisoprenyl (3-methylbut-2-en-1-yl)oxy) group [δ_H 1.83 (3H, s, H-4′′), 1.70 (3H, s, H-5′′), 4.59 (2H, d, J = 6.8 Hz, H-1′′), 5.52 (1H, t, J = 6.8 Hz, H-2′′)], and a singlet proton of methoxy group at δ_H 3.93. The ¹³C-NMR spectrum (Table 1) of 1 showed 20 carbon signals. The assignment of ¹³C-NMR spectrum was confirmed by HMQC and HMBC spectra. The placement of isoprenyl, oxyisoprenyl, and methoxy groups in 2-quinolone skeleton was established by HMQC and HMBC spectra. Long-range correlation was observed in the HMBC spectrum of 1 between the proton signal of a methoxy group at δ_H 3.93 with an oxyaryl atom (δ_C 162.5). The methylene proton signal of an isoprenyl group at δ_H 3.39 (H-1′) showed long-range correlations with a carbonyl carbon (δ_C 166.0; C-2), a oxyaryl carbon (δ_C 162.5), two quaternary carbons (δ_C 132.2, 119.3), and a methine carbon signal (δ_C 121.8), showing a methoxy group attached at C-4. The proton signal of an aromatic region at δ_H 7.65 (H-5) showed long-range correlations with an oxyaryl carbon signals (δ_C 160.6) and a quarternary carbon (δ_C 139.1). The result revealed δ_C 160.6 has position at C-7 and δ_C 139.1 at C-8a. Furthermore, the methylene proton signal at 4.59 (H-1′′) showed long-range correlations with an oxyaryl carbon (δ_C 160.6), a quarternary carbon (δ_C 139.0), and a methine carbon signal (δ_C 118.9), showing that the oxyisoprenyl group attached at C-7. See also Figure S3-S5, supporting information)

[ Table omitted. See PDF. ]

On the basis of above spectral evidence, the structure of 1 was elucidated as 4-methoxy-3-(3-methylbut-2-en-1-yl)-7-[(3-methylbut-2-en-1-yl)oxy]quinolin-2(1H)-one. Other HMBC correlations consistent with structure 1 are shown in Table 1 and Figure 2. The structure of 1 has not been previously reported, and it is a novel compound. On cytotoxic evaluation against P-388 cells, compound 1 exhibited IC₅₀ values of 0.63 μg/mL. That cytotoxic data suggested that compound 1 has high activity. The antiplasmodial activity of 1 showed IC₅₀ values of 4.28 μg/mL, which categorized as moderate activity. The data on cytotoxic and antiplasmodial activity are presented in Table 2.

Figure 2. Selected HMBC correlations for 1.

[ Table omitted. See PDF. ]

3. Experimental Section

3.1. General

The UV and IR spectra were measured with a Shimadzu 1800 spectrophotometer (Kyoto, Japan) and Perkin Elmer Spectrum One FTIR spectrophotometer (Waltham, MA, USA). NMR spectra were recorded on a JEOL ECA 400 spectrometer (Tokyo, Japan) in CDCl₃ at 400 (¹H) and 100 (¹³C) MHz using TMS as the internal standard. The mass spectra were recorded using a Waters LCT Premier XE (Santa Clara, CA, USA). Column chromatography and radial chromatography were carried out using Si gel 60 G 1.07734.1000 and Si gel 60 PF₂₅₄ 1.07749.1000 (Merck, Darmstadt, Germany).

3.2. Plant Material

The leaves of M. moluccana T.G. Hartley were collected in July 2015 from the conserved forest of Weda, Central Halmahera, North Maluku, Indonesia. The plant was identified at the Herbarium Bogoriense, and a voucher specimen number 46889 was deposited in the Bogor Botanical Garden, Bogor, Indonesia.

3.3. Extraction and Isolation

The dried and powdered leaves of M. moluccana T.G. Hartley (2.0 kg) were macerated in methanol at room temperature for 24 h two times, and the methanol extract was evaporated under reduced pressure to give methanol extract with dark brown residue (108 g). The methanol extract was partitioned with n-hexane (1:1 v/v) and then evaporated by rotavapor to give n-hexane extract (15 g). To the methanol extract was then added 3% sulfuric acid and adjusted to pH 3–4. Furthermore, the acid extracts were partitioned with ethyl acetate and then evaporated by rotavapor, yielding non-alkaloid (phenolic compound) extract (8 g). The acid extracts were basified with ammonia solution and adjusted to pH 8–9 and then partitioned with ethyl acetate to give crude alkaloid. The crude alkaloid (5 g) of M. moluccana was separated by column chromatography on silica gel. Elution with n-hexane-ethyl acetate mixture by increasing amount of ethyl acetate (9:1 to 1:1) to give four major fractions A–D. On thin layer chromatographic (TLC) analysis, fraction A (325 mg) showed two major spots. Separation of fraction A using planar radial chromatography and eluted with n-hexane-acetone mixture (from 9:1 to 7:3) to give two subfractions (A₁, A₂). Purification of subfraction A₁ by planar radial chromatography with eluent n-hexane-chloroform (from 1:1 to 3:7) yielded compound 1 (14 mg).

3.4. Cytotoxic Assay

Preliminary cytotoxic evaluation of compound 1 was carried out against murine leukemia P-388 cells according to the MTT assay, as previously described [14,15].

3.5. Antiplasmodial Analysis

The antiplasmodial activity of compound 1 was expressed by the 50% inhibitory concentrations (IC₅₀), representing the concentration of chloroquine that induced a 50% parasitaemia decrease compared to the positive control culture referred as 100% parasitaemia [16,17].

Supplementary Materials

1H-NMR, 13C-NMR, HMQC, HMBC, and HRESIMS spectra are reported in the supplementary materials as Figure S1–S7 and structure refinement parameters as Table S1.

Supplementary File 1Supplementary File 2Supplementary File 3Supplementary File 4

Acknowledgments

This research was funded by Universitas Airlangga, Ministry of Research, Technology and Higher Education, 2017.

Author Contributions

M.T. and T.S.T. designed the whole experiment. R.D.S. and R.A.W. executed the experiment. All authors interpreted data and prepare the manuscript in the same contribution.

Conflicts of Interest

The authors declare no conflict of interest.

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