New Acetophenone and Cardanol Derivatives From Knema pachycarpa

Abstract

Two new acetophenone derivatives named knepachycarpanone A (1) and knepachycarpanone B (2) together with a new cardanol derivative named knepachycarpanol C (3) were isolated from the EtOAc extract of Knema pachycarpa stems. Their chemical structures were established on the basis of spectral evidences. Compounds 1 and 2 exhibited moderate cytotoxicity against Hela cancer cell line with IC₅₀ values of 26.92 ± 1.46 and 30.20 ± 1.97 μM, respectively.

Keywords

knepachycarpanone A knepachycarpanone B knepachycarpanol C cytotoxicity

Knema is a genus of evergreen trees in the Myristicaceae family, comprising around 60 species distributed in tropical and subtropical regions of South East Asia.¹ Knema pachycarpa de Wilde is a native tree in Vietnam found in several central provinces.² Knema plants have been used in the traditional medicine for treatment of pimples, sores, and skin diseases.^3,4 Previous chemical studies of Knema species have reported the isolation of anacardic acids, cardanols, resorcinols, acetophenones, lignans, stilbene, and flavonoids.^5

-12 Knema plant exhibited antibacterial, anti-inflammatory, antioxidant, cytotoxic, and acetylcholinesterase inhibitory activities.^5

-14 Several lignans have been reported from the ethyl acetate extract of K. pachycarpa fruits¹⁵ but the chemical composition of the stems of this plant has not yet been investigated. Herein, we describe the isolation and structure elucidation of 2 new acetophenone derivatives named knepachycarpanone A (1) and knepachycarpanone B (2), together with a new cardanol derivative named knepachycarpanol C (3) from the ethyl acetate extract of the stems of K. pachycarpa. These isolated compounds were evaluated for the cytotoxicity against 3 human cancer cell lines including Hela (cervical adenocarcinoma), MCF7 (human breast adenocarcinoma), and Hep3B (human hepatoma cancer).

Compound 1 was isolated as a white amorphous powder. The molecular formula, C₂₃H₂₈O₅, was determined from the ion peak at m/z 385.2008 [M+H]⁺ in the positive high resolution electrospray ionization mass spectrometry (HR-ESI-MS) of 1. The infrared (IR) spectrum showed hydroxyl and carbonyl absorptions at 3363 and 1725 cm⁻¹. The ¹H and ¹³C nuclear magnetic resonance (NMR) data of 1 indicated signals of a resacetophenone moiety^6,7,9 including 2 aromatic protons in the meta position δ _H 6.26 (1H, d, J = 1.5 Hz, H-3), 6.25 (1H, d, J = 1.5 Hz, H-5), a hydrogen-bonded hydroxyl group at δ _H 13.02 (2-OH), and an acetyl group at δ _H 2.62. An additional 3,4-methylenedioxyphenyl moiety was observed with an ABX system protons at δ _H 6.66 (1H, s, J = 1.5 Hz, H-2″), 6.71 (1H, d, J = 8.0 Hz, H-5″), 6.61 (1H, d, J = 8.0 Hz, H-6″), and a singlet signal of a methylenedioxy group at δ _H 5.91. The correlations of methylenedioxy protons (δ _H 5.91) with C-3″ (δ _C 147.4) and C-4″ (145.3) were observed in the heteronuclear multiple bond correlation (HMBC) experiment. The ¹³C NMR and heteronuclear single quantum correlation (HSQC) spectra showed 23 carbon signals including 8 carbons of a resacetophenone ring (δ _C: 204.2 (C = O), 32.12 (CH₃), 115.1 (C-1), 165.9 (C-2), 101.6 (C-3), 161.5 (C-4), 110.8 (C-5), 147.7 (C-6)); 7 carbons of a methylenedioxyphenyl moiety (δ _C: 136.7 (C-1″), 108.8 (C-2′′), 147.4 (C-3′′), 145.3 (C-4″), 108.0 (C-5″), 121.0 (C-6″), 100.6 (OCH₂O)), and other 8 methylene carbons in the linker. The methylene protons of H-1′ (δ _H 2.80) correlated with C-1 (δ _C 115.1), C-5 (δ _C 110.8), C-6 (δ _C 147.7), and C-2′ (δ _C 32.1) while protons of H-8′ (δ _H 2.51) correlated with C-1″ (δ _C 136.7), C-2″ (δ _C 108.8), C-6″ (δ _C 121.0), and C-7′ (δ _C 31.6) in the HMBC spectrum (Figure 1). Based on these data, the structure of 1 was determined as 2,4-dihydroxy-6-[8′-(3″,4″-methylenedioxyphenyl)octyl]-acetophenone, that was named knepachycarpanone A (Figure 2).

Figure 1

Key heteronuclear multiple bond correlations of 1 and 3.

Figure 2

Structures of new compounds 1 to 3 isolated from Knema pachycarpa.

Compound 2 was obtained as a white amorphous powder. The ¹H and ¹³C-NMR data of compound 2 were almost identical to those of compound 1 except for 2 more methylene groups in the linker in 2 relative to 1. This was further confirmed by HR-ESI-MS with a protonated ion peak at m/z 413.2302, corresponding to a molecular formula of C₂₅H₃₂O₅. Therefore, compound 2 was identified as 2,4-dihydroxy-6-[10′-(3″,4″-methylenedioxy-phenyl)decyl]acetophenone, which was named knepachycarpanone B (Figure 2).

Compound 3 was isolated as a white amorphous powder. The HR-ESI-MS spectrum showed the ion peak at m/z 417.2210 [M+Cl]^- corresponding to a molecular formula of C₂₅H₃₄O₃. The IR spectrum showed a hydroxyl absorption at 3381 cm⁻¹. The NMR data of compound 3 revealed proton signals of a cardanol ring at δ _H 6.65 (1H, s, H-2), 6.64 (1H, d, J = 8.0 Hz, H-4), 7.12 (1H, t, J = 8.0 Hz, H-5), and 6.75 (1H, d, J = 8.0 Hz, H-6).⁵ In addition, proton signals of a 3,4-methylenedioxyphenyl moiety at δ _H 6.66 (1H, d, J = 1.5 Hz, H-2″), 6.72 (1H, d, J = 8.0 Hz, H-5″), 6.61 (1H, d, J = 8.0 Hz, H-6″), and 5.91 (2H, s, OCH₂O) were also observed. The ¹³C-NMR and HSQC spectra showed 25 signals including 12 aromatic carbons, a methylenedioxy carbon at δ _C 100.9 and 12 methylene carbons in the linker. The HMBC spectrum showed correlations of H-1′ (δ _H 2.54) with C-1 (δ _C 144.9), C-2 (δ _C 115.3), C-6 (δ _C 120.8), and C-2′ (δ _C 31.2); correlations of proton H-12′ (δ _H 2.51) with C-1″ (δ _C 136.8), C-2″ (δ _C 108.8), C-6″ (δ _C 121.0), and C-11′ (δ _C 31.7). Based on these evidences, the structure of 3 was established as 3-hydroxy-[12′-(3″,4″-methylenedioxyphenyl)dodecyl]benzene, that was named knepachycarpanol C (Figure 2).

Acetophenone and cardanol derivatives have been isolated from Knema species such as the roots of Knema globularia and the stems of Knema glomerata and evaluated for cytotoxicity against several human cancer lines.^7,9 Therefore, in our study the cytotoxicity of these isolated compounds 1 to 3 against cancer cell lines including Hep3B (human hepatoma cancer), HeLa (human cervical adenocarcinoma), and MCF-7 (human breast adenocarcinoma) were tested in comparison with camptothecin. As shown in Table 1, compounds 1 and 2 showed similar cytotoxicity to Hela cancer cell line with IC₅₀ values of 26.92 ± 1.46 µM and 30.20 ± 1.97 µM, respectively. In the case of MCF-7 and Hep3B cell lines, compounds 1 and 2 were weakly active or inactive (MCF-7: IC₅₀ of 52.88 ± 2.97 µM and 46.22 ± 1.24 µM, respectively; Hep3B: IC₅₀ of >100 µM and 70.80 ± 2.06 µM, respectively). Cardanol 3 did not show cytotoxic activity against all tested cancer cell line (IC₅₀ >100 µM). The results suggested that the acetyl group in the acetophenone compounds essential for cytotoxicity was in agreement with the study of Sriphana et al.⁷

Table 1

Cytotoxicity of 1 to 3 Against the Hela, MCF-7, and Hep3B Cancer Cell Lines.

Compounds	IC₅₀ (µM)
Compounds	Hela	MCF-7	Hep3B
1	26.92 ± 1.46	52.88 ± 2.97	>100
2	30.20 ± 1.97	46.22 ± 1.24	70.80 ± 2.06
3	>100	>100	>100
Camptothecin	0.43 ± 0.14	0.57 ± 0.17	0.57 ± 0.17

In conclusion, chemical investigation of the stems of K. pachycarpa resulted in the isolation and identification 2 new acetophenone derivatives, knepachycarpanone A (1) and knepachycarpanone B (2) and a new cardanol derivative named knepachycarpanol C (3). Compounds 1 and 2 showed moderate cytotoxicity against Hela cancer cell lines with IC₅₀ values of 26.92 ± 1.46 and 30.20 ± 1.97 µM, respectively.

General Experimental Procedures

The UV spectra were recorded on a Shimadzu UV-240 spectrophotometer. IR spectra were obtained on a Bruker TENSOR 37 FT-IR spectrometer using KBr pellets. The HR-ESI-MS were obtained using an Agilent 6530 Accurate mass quadrupole time of flight liquid chromatography mass spectrometry system. One-dimensional and 2D NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer, chemical shifts (δ) are expressed in ppm with reference to the tetramethyl silane signals. Column chromatography (CC) was performed with silica gel (230-400 mesh, Merck). Fractions were monitored by thin-layer chromatography (TLC), spots were visualized by UV light (254 and 365 nm) or by heating silica gel plates sprayed with Ce-Mo stain. All solvents used in CC were redistilled.

Plant Materials

The stems of K. pachycarpa de Wilde (Myristicaceae) were collected in A-Luoi, Hue city, Viet Nam, in October, 2015, and identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. A voucher specimen (VN-1527) was deposited at the Herbarium of Institute of Marine Biochemistry and Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology.

Extraction and Isolation

The air-dried and powdered stems of K. pachycarpa (1.2 kg) were extracted with MeOH (10 L × 4 times) at room temperature for 1 day each time. The MeOH extracts were concentrated in vacuo, suspended in H₂O (500 mL), and partitioned successively with n-hexane and ethyl acetate (1 L × 3). The EtOAc extract (160 g) was subjected to silica gel CC and eluted with solvent gradient of n-hexane/EtOAc to afford 8 fractions (E1-E8). Fraction E1 (2.5 g) was chromatographed on silica gel column (n-hexane/acetone, 98:2) to give 4 subfractions E1.1-E1.4. Subfraction E1.4 (0.65 g) was separated by silica gel CC and eluted with n-hexane/acetone, 95:5 to yield 4 smaller fractions E1.4.1-E1.4.4. Fraction E1.4.3 (0.5 g) was purified by Sephadex^® LH-20 CC, eluted with MeOH-CH₂Cl₂ (9:1) to yield 3 (4 mg). Fraction E3 (3.5 g) was separated on Sephadex^® LH-20 CC eluted with MeOH/CH₂Cl₂ (9:1) to give 4 subfractions E3.1-E3.4. Subfraction E3.3 (0.1 g) was further separated by CC on silica gel and eluted with CH₂Cl₂/EtOAc (9:1) to yield 5 fractions E3.3.1-E3.3.5. Fraction E3.3.2 (48 mg) was purified by Sephadex^® LH-20 CC, eluted with MeOH-CH₂Cl₂ (9:1) to give a mixture of acetophenones (35 mg). The acetophenone mixture was separated by reversed-phase silica gel Pre-TLC using MeCN-water (9:1) as eluent to yield 1 (22 mg) and 2 (8 mg).

Knepachycarpanone a (1)

White amorphous powder.

MP: 66-67°C.

IR ν_max (KBr): 3363, 2925, 2854, 1725, 1587, 1503, 1488, 1244, 1039 cm^–1.

UV (CHCl₃) λ max (log ε) 281 (1.04), 322 (0.37).

¹H NMR (CDCl₃, 500 MHz) and ¹³C NMR (CDCl₃, 125 MHz): Table 2.

Table 2

¹H and ¹³C NMR of Compounds 1, 2, and 3 (CDCl₃).

C	1		2
C	δ_C ^a	δ_H ^b (m, J, Hz)	δ_C ^a	δ_H ^b (m, J, Hz)	δ_C ^a	δ_H ^b (m, J, Hz)
1	115.1	-	115.2	-	144.9	-
2	165.9	-	166.0	-	115.3	6.65 (s)
3	101.6	6.26 (d,1.5)	101.6	6.26 (d,1.5)	155.5	-
4	161.5	-	161.2	-	112.4	6.64 (d, 8.0)
5	110.8	6.25 (d,1.5)	110.6	6.25 (d,1.5)	129.3	7.12 (t, 8.0)
6	147.7	-	147.7	-	120.8	6.75 (d, 8.0)
1′	36.3	2.80 (t, 7.5)	36.3	2.82 (t, 8.0)	35.8	2.54 (t, 7.5)
2′	32.19	1.57 (m)	32.2	1.57 (m)	31.2	1.56 (m)
3′	29.6	1.35-1.29 (8H, m)	29.6	1.36-1.27 (12H, m)	29.6 29.6 29.6 29.5 29.5 29.5 29.2 29.1	1.29-1.25 (16H, m)
4′	29.3		29.48
5′	29.3		29.48
6′	29.0		29.43
7′	31.6	1.57 (m)	29.3
8′	35.6	2.51(t, 7.0)	29.1
9′	-	-	31.6	1.56 (m)
10′	-	-	35.6	2.51 (t, 7.5)
11′	-	-	-	-	31.7	1.56 (m)
12′	-	-	-	-	35.6	2.51 (t, 8.0)
1″	136.7	-	136.8		136.8	-
2″	108.8	6.68 (d,1.5)	108.8	6.66 (d,1.0)	108.8	6.66 (d,1.5)
3″	147.4	-	147.4		147.4	-
4″	145.3	-	145.3		145.3	-
5″	108.0	6.71 (d,8.0)	108.0	6.71 (d,8.0)	108.0	6.72 (d, 8.0)
6″	121.0	6.61 (d,8.0)	121.0	6.61 (d,8.0)	121.0	6.61 (d, 8.0)
OCH₂O	100.6	5.91, s	100.6	5.90, s	100.6	5.90, s
C = O	204.2		204.1			-
CH₃	32.12	2.62 (s)	32.1	2.63 (s)		-
OH		13.02 (s)		12.98 (s)		-

Recorded at ^a) 125 MHz, ^b) 500 MHz

HR-ESI-MS (positive-ion mode) m /z 385.2008 [M + H]⁺, calcd for C₂₃H₂₉O₅, 385.2015.

Knepachycarpanone B (2)

White amorphous powder.

MP: 67-68°C.

IR ν_max (KBr): 3170, 2922, 2849, 1731, 1589, 1504, 1488, 1235, 1032 cm^–1.

UV (CHCl₃) λ max (log ε) 281 (1.49), 322 (0.48).

¹H NMR (CDCl₃, 500 MHz) and ¹³C NMR (CDCl₃, 125 MHz): Table 2.

HR-ESI-MS (positive-ion mode): m/z 413.2302 [M+H]⁺, calcd for C₂₅H₃₃O₅, 413.2328.

Knepachycarpanol C (3)

White amorphous powder.

MP: 56-57°C.

IR ν_max (KBr) 3381, 2923, 2852, 1588, 1503, 1488, 1442, 1243, 1155, 1039 cm^–1.

UV (CHCl₃) λ max (log ε) 280 (0.49).

¹H NMR (CDCl₃, 500 MHz) and ¹³C NMR (CDCl₃, 125 MHz): Table 2.

HR-ESI-MS (negative-ion mode): m/z 417.2210 [M+Cl]^-, calcd for C₂₅H₃₄O₃Cl, 417.2196.

Cytotoxicity Assay

The cancer cell lines (Hela, MCF-7, and Hep3B) were maintained in RPMI 1640 or DMEM medium, supplemented with 10% FBS, l-glutamine (2 mM), penicillin G (100 UI/mL), and streptomycin (100 µg/mL). Stock solutions of compounds were prepared in DMSO, and cytotoxicity assays were carried out in 96-well microtiter plates against Hela, MCF-7, and Hep3B cancer cell lines (2 × 10⁵ cells/mL) using MTT method.¹⁶ After 72 hours of incubation at 37^o C in air/CO₂ (95:5) with or without test compounds, cell growth was estimated by colorimetric measurement of formazan. Optical density was determined at 540 nm with a Titertek Multiskan photometer. The IC₅₀ value was defined as the concentration of a sample necessary to inhibit the cell growth to 50% of the control. Camptothecin was used as a reference compound.

Supplemental Material

Supplementary - Supplemental material for New Acetophenone and Cardanol Derivatives From Knema pachycarpa

Supplemental material, Supplementary, for New Acetophenone and Cardanol Derivatives From Knema pachycarpa by Tran Huu Giap, Ha Thi Thoa, Vu Thi Kim Oanh, Nguyen Thi Minh Hang, Nguyen Hai Dang, Dinh Ngoc Thuc, Nguyen Van Hung, and Le Nguyen Thanh in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.01-2017.47.

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