Quassinoids and Alkaloids From the Roots of Eurycoma longifolia

Abstract

Five quassinoids and 2 alkaloids were isolated from the roots of Eurycoma longifolia. The structures of 3 new quassinoids, eurycomalide F, G, and H (1-3), were elucidated using a variety of spectroscopic methods. The known compounds were identified as eurycomalide A (4), laurycolactone B (5), 5-methoxycanthin-6-one (6), and canthin-6-one (7). Among the isolated compounds, canthin-6-one (7) exhibited the strongest inhibitory effect on nitric oxide production (IC₅₀ = 16.9 μM). 5-Methoxycanthin-6-one (6) and eurycomalide F (1) exhibited weak inhibition with IC₅₀ values of 23.4 and 32.7 μM, respectively.

Eurycoma longifolia Jack (Simaroubaceae) has been used in Southeast Asia to treat sexual dysfunction, constipation, exercise fatigue, fever, loss of libido, aging, stress, malaria, osteoporosis, diabetes, high blood pressure, cancer, leukemia, and glandular swelling. ¹ Quassinoids, canthin-6-one alkaloids, β-carboline alkaloids, biphenyl neolignans, and triterpenes have been identified as major phytochemical compounds in E. longifolia. ^2,3 This article describes the isolation and identification of 3 new quassinoids (1-3), together with eurycomalide A (4), ⁴ laurycolactone B (5), ⁵ 5-methoxycanthin-6-one (6), ⁶ and canthin-6-one (7) ⁷ from a methanol extract of E. longifolia roots.

Eurycomalide F (1) was obtained as colorless solid. The molecular formula C₁₉H₂₈O₆ for 1 was established from an ion peak at m/z 353.1968 [M + H]⁺ in its high resolution electrospray ionization mass spectrometry (HR-ESI-MS) spectrum. The ¹H nuclear magnetic resonance (NMR) spectrum showed 2 secondary methyl doublets at δ_H 1.03 (3H, br d, J = 7.5 Hz, CH₃-16) and 1.13 (3H, br d, J = 7.5 Hz, CH₃-19) and 2 tertiary methyl singlets at δ_H 1.56 (3H, br s, CH₃-18) and 1.46 (3H, br s, CH₃-17). The ¹³C NMR and heteronuclear single quantum correlation (HSQC) spectra indicated the presence of 4 methyl, 2 methylene, 9 methine, and 4 quaternary carbon atoms, including 1 carboxylic group and 1 carbonyl group (Table 1). These data were similar to those of eurycomalide A ⁴ with differences attributed to the presence of a methine group (δ_C 44.3, C-5) and a methylene group (δ_C 27.6, C-6) instead of double bonds. ⁴ Thus, compound 1 was considered to be a hydrogenated derivative of 4, which was confirmed by heteronuclear multiple bond correlation (HMBC) spectral analyses (Figure 1). The relative configurations of this compound were confirmed through nuclear Overhauser effect spectroscopy (NOESY) experiments. The nuclear Overhauser effect (NOE) cross peaks between H-1 (δ_H 3.14) and H-5 (δ_H 1.72), and between H-9 (δ_H 1.41) and H-12 (δ_H 4.29), indicated a β-orientation of hydroxyl groups at C-1 and C-12. An α-orientation at 2-OH was supported by a NOESY correlation between H-2 (δ_H 3.85) and CH₃-17 (δ_H 1.56) and a large coupling constant between H-1 and H-2 (J = 9.0 Hz). The couplings between CH₃-16 (δ_H 1.03) and CH₃-17, and H-13 (δ_H 2.93) and H-18 (δ_H 1.56), respectively, confirmed that CH₃-16 was β-oriented and CH₃-19 was α-oriented. Together, these data confirm the structure of eurycomalide F (1).

Table 1

NMR Spectroscopic Data of Compounds 1 to 3.

No	1		2		3
	δ H (J in Hz)	δ C	δ H (J in Hz)	δ C	δ H(J in Hz)	δ C
1	3.14 (1H, d, 9.0)	85.4	3.13 (1H, d, 9.0)	83.5	4.17 (1H, d, 2.0)	77.3
2	3.85 (1H, m)	69.2	3.78 (1H, m)	69.7	5.42 (1H, dd, 2.0, 10.0)	131.6
3	1.87 (1H, m)	40.4	1.86 (1H, m)	40.9	6.22 (1H, d, 10.0)	132.4
	1.58 (1H, m)		1.62 (1H, m)
4	1.78 (1H, m)	34.7	1.76 (1H, m)	34.9	-	142.3
5	1.72 (1H, m)	50.7	1.65 (1H, m)	44.3	2.32 (1H, m)	50.4
6	3.16 (1H, m)	40.4	2.18 (1H, m)	27.6	4.18 (1H, dd, 4.0, 11.0)	65.8
	2.07 (1H, dd, 3.5, 15.5)		1.77 (1H, m)
7	-	210.6	4.04 (1H, dd, 2.5, 3.0)	85.5	4.10 (1H, d, 4.0)	88.1
8	-	52.7	-	44.3	-	44.1
9	1.41 (1H, d, 3.5)	52.4	1.59 (1H, d, 2.5)	45.7	1.83 (1H, d, 1.5)	42.5
10	-	44.5	-	44.0	-	44.5
11	4.67 (1H, dd, 3.5, 5.0)	70.9	4.67 (1H, dd, 3.0, 2.5)	74.3	4.68 (1H, dd, 1.5, 3.0)	73.5
12	4.29 (1H, dd, 1.5, 5.0)	85.1	3.59 (1H, t, 3.0)	76.0	3.61 (1H, t, 3.0)	76.1
13	2.93 (1H, m)	33.6	2.32 (1H, m)	28.4	2.35 (1H, m)	28.1
14	2.83 (1H, d, 1.5)	54.4	2.24 (1H, d, 5.5)	56.9	2.31 (1H, d, 5.5)	56.7
15	-	179.0	-	180.1	-	179.4
16	1.03 (3H, d, 7.5)	14.9	1.01 (3H, d, 7.5)	15.2	5.41 and 5.12 (each 1 H, s)	114.4
17	1.46 (3H, br s)	16.1	1.24 (3H, br s)	16.3	1.05 (3H, br s)	12.5
18	1.56 (3H, br s)	23.8	1.53 (3H, br s)	21.6	1.56 (3H, br s)	20.8
19	1.13 (3H, br d, 7.5)	16.9	1.37 (3H, br d, 7.5)	14.8	1.39 (3H, br d, 7.5)	14.8

Figure 1

Structures, and key HMBC (→) and NOESY (↔) correlations of 1 to 3.

Compound 2 was obtained as a colorless solid with the molecular formula C₁₉H₃₀O₆, as deduced from a molecular ion peak at m/z 355.2129 [M + H]⁺ in its HR-ESI-MS spectrum. The NMR data of 2 closely resembled those of 1 with 19 carbon signals, including 2 secondary methyl and 2 tertiary methyl groups, which indicate the same quassinoid skeleton. The replacement of the carbonyl signal in 1 by an oxymethine signal (δ_C 85.5) and a 2 Da difference in molecular weight suggest hydroxylation of the ketone group at C-7. Heteronuclear multiple bond correlation correlations between H-6, H-9, and CH₃-18 to C-7 confirm the location of hydroxylation (Figure 1). However, a larger coupling constant of H-14 (5.5 Hz) compared to that of 1 (1.5 Hz), and an HMBC correlation between H-7 (δ_H 4.04) and C-15 (δ_C 180.1), suggest lactone cyclization at C-15 and C-7. ⁸ Similarities in the NOESY correlation patterns of 2 and 1 were used to determine the relative configurations of 2. Furthermore, NOE coupling between H-7, H-14 (δ_H 2.24), and CH₃-18 (δ_H 1.53) suggest β-orientations for H-7 and H-14. Therefore, the structure of 2 was determined as shown and the compound was named eurycomalide G.

Eurycomalide H (3) was obtained as a white solid. The HR-ESI-MS spectrum of 3 revealed a molecular ion peak at m/z 351.1813 [M + H]⁺, corresponding to the molecular formula C₁₉H₂₇O₆. The ¹H NMR spectrum showed 1 secondary methyl doublet at δ_H 1.39 (3H, br d, J = 7.5 Hz, CH₃-19), 2 tertiary methyl singlets at δ_H 1.56 (3H, br s, CH₃-18) and 1.05 (3H, br s, CH₃-17), and 4 olefinic proton signals at δ_H 6.22 (1H, d, J = 10.0 Hz, H-3), 5.42 (1H, dd, J = 2.0, 10.0 Hz, H-2), 5.41, and 5.12 (each 1 H, s, H-16a and 16b). ¹³C NMR and HSQC spectra showed 19 signals including 1 carboxylic group (δ_C 179.4), 1 exomethylene (δ_C 142.4/114.4), 1 double bond (δ_C 131.6/132.4), 5 oxygenated methines at δ_C 77.3 (C-1), 65.8 (C-6), 88.1 (C-7), 73.5 (C-11), and 76.1 (C-12), 4 sp³ methines, and 2 sp³ quaternary carbon atoms. By comparing these NMR data with those reported for E. longifolia, the structure of 3 resembled that of the Δ⁴⁽¹⁸⁾-isomer of longilactone for rings B, C, and D. ^8,9 They differed in the presence of a double bond instead of a C-2 oxygenated methine and C-3 methylene, which was confirmed by HMBC correlations between H-2 (δ_H 5.42) and C-3 (δ_C 132.4), C-4 (δ_C 142.4), and C-10 (δ_C 44.5), as well as H-3 (δ_H 6.22) and C-1 (δ_C 77.3), C-4, C-5 (δ_C 50.4), and C-18 (δ_C 114.4) (Figure 1). The relative configurations of 3 was deduced from NOESY correlations shown in Figure 1. Accordingly, OH-1, H-7, CH₃-17, CH₃-18, OH-11, H-13, and H-14 were β-oriented, while H-5, OH-6, H-9, and CH₃-19 were α-oriented.

All of the isolated compounds were evaluated for their inhibition of nitric oxide (NO) production. The most active compound was canthin-6-one (7), followed by 5-methoxycanthin-6-one (6), with the IC₅₀ values of 16.9 and 23.4 µM, respectively. Among the isolated quassinoids, only eurycomalide F (1) exhibited a weak effect (IC₅₀ = 32.7 µM).

Experimental

General

Optical rotation, JASCO P-2000 digital polarimeter; IR, Tensor 37 FT-IR spectrometer. NMR, Bruker AM500 FT-NMR Spectrometer. HR-ESI-MS, API Q-STAR PULSAR I of Applied Biosystem. Absorbance of bioassay solutions, xMark microplate spectrophotometer (Molecular Devices, CA). Thin-layer chromatography was performed using precoated Kiesel gel 60 F254 (Merck) and visualized by UV light 254 nm and 10% H₂SO₄ reagent with heat.

Plant Material

The roots of E. longifolia were collected at Quang Ninh province, Vietnam in March, 2017. The sample was identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. The voucher specimens (BB-01) were deposited at the Advanced Center for Bio-organic Chemistry.

Extraction and Isolation

The air-dried and powdered roots of E. longifolia (2 kg) were extracted with methanol (5 L × 3 times) at room temperature for 24 hours. The combined extracts were evaporated under reduced pressure to give the crude extract (82.5 g), which was then resuspended in water (2 L) and successively with n-hexane and ethyl acetate (each 1 L × 3 times) to obtain n-hexane (17.5 g) and ethyl acetate (47.4 g) extracts, respectively. The later was chromatographed on a silica gel column using mobile phase of a gradient of 0% to 100% methanol in dichloromethane to obtain 6 fractions EL1-6. Fraction EL2 was subjected to a silica gel column eluted by dichloromethane-methanol 30:1 (v/v) to obtain 6 (2.6 mg) and 7 (4.4 mg). Compound 5 (7.8 mg) was purified from EL3 by a Sephadex LH20 column eluted by dichloromethane-methanol 2:1 (v/v). Fraction EL4 was passed through a silica gel column using dichloromethane-acetone 4:1 (v/v) as eluent to obtain 1 (13.3 mg) and 4 (4.0 mg). Repeated silica gel column (eluted with dichloromethane-methanol 5:1) and C-18 column (eluted with methanol-water 1:1) applied for EL5 to give 3 (4.1 mg). Compounds 2 (5.5 mg) was obtained from EL6 by a C-18 column eluted by methanol-water 1:1 (v/v).

Eurycomalide F (1)

Rf: 0.32 (dichloromethane - acetone, 4:1).

[α]_D ²⁴: +43.4 (c 0.2, MeOH).

IR ν_max(KBr): 3390, 1729, 1120, 1016 cm⁻¹.

¹H NMR (500 MHz, CD₃OD) and ¹³C NMR (125 MHz, CD₃OD): Table 1.

HR-ESI-MS: m/z [M + H]⁺ calcd for C₁₉H₂₉O₆: 353.1964; found: 353.1968.

13.3 mg (6.6 × 10⁻⁶% of dried weight).

Eurycomalide G (2)

Rf: 0.65 (dichloromethane - methanol, 10:1).

[α]_D ²⁴: +32.1 (c 0.1, MeOH).

IR ν_max(KBr): 3435, 1715, 1143, 1035 cm⁻¹.

¹H NMR (500 MHz, CD₃OD) and ¹³C NMR (125 MHz, CD₃OD): see Table 1.

HR-ESI-MS: m/z [M + H]⁺ calcd for C₁₉H₃₁O₆: 355.2121; found: 355.2129.

5.5 mg (2.7 × 10⁻⁶% of dried weight).

Eurycomalide H (3)

Rf: 0.45 (dichloromethane - methanol, 10:1).

[α]_D ²⁴: +12.5 (c 0.1, MeOH).

IR ν_max(KBr): 3450, 1736, 1648, 1125, 1034 cm⁻¹.

¹H NMR (500 MHz, CD₃OD) and ¹³C NMR (125 MHz, CD₃OD): see Table 1.

HR-ESI-MS: m/z [M + H]⁺ calcd for C₁₉H₂₇O₆: 351.1808; found: 351.1813.

4.1 mg (2.0 × 10⁻⁶% of dried weight).

Assay for Inhibition of NO Production

The effects of compounds on the NO production in LPS-stimulated RAW264.7 cells were evaluated as previously described. ¹⁰ Cardamonin was used as a positive control (IC₅₀ = 2.80 µM). The MTT assay showed that all compounds had no significant toxicity at their effective doses for the NO inhibition (data not shown).