Two New Sesquiterpenoids Including a Sesquiterpenoid Lactam from Curcuma wenyujin

Abstract

Two new sesquiterpenoids, namely elema-1,3,7(11),8-tetraen-8,12-lactam (1) and 7β,8α-dihydroxy-1α,4αH-guai-9,11-dien-5β,8β-endoxide (2), together with five known analogs were isolated from the EtOAc extract of the rhizomes of Curcuma wenyujin. Their structures and relative configurations were determined on the basis of spectroscopic methods including 2D-NMR techniques. All compounds were tested for the inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Compound 1 showed the significant inhibitory activity with IC₅₀ values of 9.4 µM.

Curcuma wenyujin, a Traditional Chinese Medicine distributed in the south of China. It is widely used in the treatment of jaundice, thoracic-abdominal pain, arthralgia, dysmenorrhea, epilepsy and psychataxia.^1–4) Recent pharmacological studies of this plant have been reported to possess various activities, including anti-inflammatory,⁵⁾ anticancer,⁶⁾ antioxidation,⁷⁾ and antidepressant.⁸⁾ Previous chemical studies on this species have attracted a great interest due to its diverse structures and significant biological activities. Herein, the investigation of bioactive constituents from the rhizomes of this species yielded two new sesquiterpenoids, along with five known analogs (Fig. 1). In the present paper, the isolation, structural elucidation, and the inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW264.7 macrophage cells of compounds 1–7 are reported.

Fig. 1. Chemical Structures of Compounds 1–7

Results and Discussion

The EtOAc-soluble fraction obtained from the EtOH extract of this specimen was subjected repeatedly to column chromatography on silica gel and LiChroprep RP-18 silica gel to afford compounds 1–7, including two new sesquiterpenoids, namely elema-1,3,7(11),8-tetraen-8,12-lactam (1) and 7β,8α-dihydroxy-1α,4αH-guai-9,11-dien-5β,8β-endoxide (2), together with five know compounds by comparison of their spectral data with those reported, hydroxyisogermafurenolide (3),⁹⁾ isogermafurenolide (4),¹⁰⁾ curcumenol (5),¹¹⁾ 4-epicurcumenol (6),¹²⁾ neocurcumenol (7).¹²⁾ The structures of the 1–7 were determined as shown in Fig. 1.

Compound 1 was obtained as white amorphous powder. The molecular formula, C₁₅H₁₉NO, consistent with seven degrees of unsaturation, was determined by high resolution-electrospray ionization-mass spectrum (HR-ESI-MS) (m/z 230.1548 [M+H]⁺, Calcd 230.1539) and NMR data. The ¹H-NMR spectrum of 1 exhibited the signals for three methyl groups including two olefinic methyls at δ_H 1.85 (3H, s, H₃-13) and 1.69 (3H, s, H₃-15), and a tertiary methyl at δ_H 1.12 (3H, s, H₃-14), while the ¹³C-NMR spectrum displayed 15 carbon signals including a carbonyl and eight olefinic carbons (Table 1), suggesting 1 was a sesquiterpene of high functionality. Three olefinic proton signals at δ_H 5.84 (1H, dd, J=10.8, 17.4 Hz, H-1), 5.04 (1H, d, J=10.8 Hz, H-2a), and 4.99 (1H, d, J=17.4 Hz, H-2b), in association with two carbon signals at δ_C 146.6 (CH, C-1), 113.0 (CH₂, C-2) were attributed to a monosubstituted terminal double bond. Two olefinic proton signals at δ_H 4.89 (1H, s, H-3a) and 4.75 (1H, s, H-3b), and two carbon signals at δ_C 146.1 (C, C-4), 114.3 (CH₂, C-3) indicated the presence of a disubstituted terminal double bond. A trisubstituted double bond was observed from the proton signal at δ_H 5.27 (1H, s, H-9) and carbon signals at δ_C 136.6 (C, C-8) and 116.1 (CH, C-9), while a tetrasubstituted double bond was confirmed by the signals at δ_C 141.3 (C, C-7) and 124.8 (C, C-11). Furthermore, the presence of a lactam moiety was disclosed by the NMR data at δ_H 7.46 (1H, br s, NH) and δ_C 173.5 (C-12), and IR absorptions at 3367 and 1741 cm⁻¹, in association with the fact that a nitrogen atom remained in the molecule according to the HR-ESI-MS data. Six degrees of unsaturation, accounted for by the functional groups from seven in the molecule, suggested the remaining of a cyclic structure in 1.

Table 1. ¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz) Data of Compounds 1 and 2 (CDCl₃)

Position	1		2
	δH (J in Hz)	δC	δH (J in Hz)	δC
1	5.84 (1H, dd, 10.8, 17.4)	146.6 CH	1.94 (1H, m)	52.9 CH
2a	5.04 (1H, d, 10.8)	113.0 CH2	1.94 (1H, m)	27.2 CH2
2b	4.99 (1H, d, 17.4)		1.60 (1H, m)
3a	4.89 (1H, s)	114.3 CH2	1.93 (1H, m)	30.8 CH2
3b	4.75 (1H, s)		1.64 (1H, m)
4		146.1 qC	1.89 (1H, m)	40.2 CH
5	2.45 (1H, dd, 4.8, 9.0)	51.3 CH		85.4 qC
6a	2.68 (1H, dd, 4.8, 17.4)	25.3 CH2	2.31 (1H, m)	43.4 CH2
6b	2.58 (1H, dd, 9.0, 17.4)		1.96 (1H, m)
7		141.3 qC		85.1 qC
8		136.6 qC		102.5 qC
9	5.27 (1H, s)	116.1 CH	5.48 (1H, s)	123.2 CH
10		41.9 qC		141.0 qC
11		124.8 qC		143.1 qC
12a		173.5 qC	5.09 (1H, s)	114.5 CH2
12b			4.97 (1H, s)
13	1.85 (3H, s)	8.5 CH3	1.83 (3H, s)	20.7 CH3
14	1.12 (3H, s)	22.0 CH3	1.68 (3H, s)	20.9 CH3
15	1.69 (3H, s)	23.9 CH3	1.03 (3H, d, 6.0)	11.6 CH3
NH	7.46 (1H, br s)
8-OH			4.13 (1H, br s)

The heteronuclear multiple bond connectivity (HMBC) correlations from the tertiary methyl H₃-14 [δ_H 1.12 (3H, s)] to the aliphatic quaternary carbon C-10 (δ_C 41.9), the aliphatic methine carbon C-5 (δ_C 51.3), and two olefinic secondary carbons C-1 (δ_C 146.6) and C-9 (δ_C 116.1) suggested that both the monosubstituted terminal double bond and CH₃-14 were connected to C-10. Additional HMBC correlations observed from the olefinic methyl H₃-15 [δ_H 1.69 (3H, s)] to C-5 and two olefinic carbons C-4 (δ_C 146.1) and C-3 (δ_C 114.3) allowed to the assignment of an isopropenyl group located at C-5. Moreover, the ¹H–¹H correlation spectroscopy (COSY) correlations between H-5 [δ_H 2.45 (1H, dd, J=4.8, 9.0 Hz)] and H₂-6 [δ_H 2.68 (1H, dd, J=4.8, 17.4 Hz, H-6a), 2.58 (1H, dd, J=9.0, 17.4 Hz, H-6b)], in combination with the HMBC correlations from H-5 to C-6 [δ_C 25.3 (CH₂)], C-7 [δ_C 141.3 (C)], C-10, and C-9, from H₂-6 to C-7, C-8 [δ_C 136.6 (C)], C-11 [δ_C 124.8 (C)], from H-9 [δ_H 5.27 (1H, s)] to C-7, C-8, C-10 and C-11, and from H₃-13 [δ_H 1.85 (3H, s)] to C-7, C-11 and C-12 [δ_C 173.5 (C)] led to the establishment of an α,β-unsaturated butyrolactam moiety fused to a six-membered ring at C-7 and C-8. Thus, compound 1 was determined to be a sesquiterpene lactam possessing an elemane-type skeleton, established as elema-1,3,7(11),8-tetraen-8,12-lactam (Fig. 2). On biogenetic consider -ations, the relative configurations at C-5 and C-10 in 1 were assigned to be consistent with those of the co-occurring analogue hydroxyisogermafurenolide (3).⁹⁾ This assumption of stereochemistry for 1 was further confirmed by the nuclear Overhauser effect spectroscopy (NOESY) correlations (Fig. 3) between H₃-14/H-3b, H-3b/H-6b, and H-1/H-5.

Fig. 2. Selected ¹H–¹H COSY (—) and HMBC (→) Correlations of Compounds 1 and 2

Fig. 3. Key NOE Correlations and Computer-Generated Models Using MM2 Force Field Calculations for Compounds 1 and 2

Compound 2 was obtained as a colorless oil. The molecular formula of 2 was determined to be C₁₅H₂₂O₃ on the basis of HR-ESI-MS data (m/z 273.1474 [M+Na]⁺, Calcd 273.1461), implying five degrees of unsaturation. The ¹H-NMR spectra of 2 showed three methyl group signals, including two olefinic methyl singlets at δ_H 1.83 (3H, s, H₃-13) and 1.68 (3H, s, H₃-14), and a methyl doublet at δ_H 1.03 (3H, d, J=6.0 Hz, H₃-15). The ¹³C-NMR spectrum presented a total of fifteen carbon resonances, including four olefinic carbons (Table 1), implying that 2 was a sesquiterpene possessing two double bonds in a tricyclic backbone according to the units of unsaturation in the molecule. The ¹H-NMR signal at δ_H 5.48 (1H, s, H-9) and ¹³C- NMR signals at δ_C 141.0 (C, C-10) and δ_C 123.2 (CH, C-9) were attributed to a trisubstituted double bond. The establishment of an isopropenyl group was suggested by the NMR data for a disubstituted terminal double bond [δ_C 143.1 (C, C-11), 114.5 (CH₂, C-12); δ_H 5.09 (1H, s, H-12a), 4.97 (1H, s, H-12b)] and an olefinic methyl group [δ_C 20.7 (CH₃, C-13); δ_H 1.83 (3H, s, H₃-13)], in association with the HMBC correlations observed from the olefinic methyl protons to the two carbons of the terminal double bond. In addition, the presence of a cyclic hemiketal was indicated by the diagnostic carbon signals at δ_C 102.5 (C, C-8) and 85.4 (C, C-5), in accordance with those reported for curcumenol (5), a principal guaiane-type sesquiterpene originated from C. wenyujin.¹¹⁾ Moreover, an additional tertiary hydroxyl group was observed from an oxygenated quaternary carbon signals at δ_C 85.1 (C-7). The ¹H–¹H COSY correlations allowed to establish the partial structure of consecutive proton system extending from H-1 [δ_H 1.94 (1H, m)] to H₃-15 [δ_H 1.03 (3H, d, J=6.0 Hz)] via H₂-2 [δ_H 1.94 (1H, m), 1.60 (1H, m)], H₂-3 [δ_H 1.93 (1H, m), 1.64 (1H, m)], and H-4 [δ_H 1.89 (1H, m)]. Detailed HMBC spectrum analysis disclosed that the gross structure of 2 was closely related to curcumenol (5), with the exception that C-7 was linked to a hydroxyl and an isopropenyl group instead of forming a double bond with C-11 in the latter. This was supported by the presence of an isopropenyl group as mentioned above, and its protons H₃-13, H₂-12, and OH-8 [δ_H 4.13 (1H, br s)] exhibiting HMBC correlations to an additional oxygenated quaternary carbon C-7 (δ_C 85.1). The relative configurations at C-1, C-4, and C-5 in 2 were assigned to be consistent with those of curcumenol (5) based on the similar NOE relationships and NMR data, while the obvious NOE correlations between H-1/H-4, H-1/H-6a [δ_H 2.31 (1H, m)], H-6a/H₃-13 supported the α-orientation of isopropenyl group at C-7. Consequently, compound 2 was established as 7β,8α-dihydroxy-1α,4αH-guai-9,11-dien-5β,8β-endoxide.

Table 2. The IC₅₀ of Compounds 1–7 on NO Production in LPS-Activated RAW264.7 Macrophages Were Evaluated

Compound	Inhibition %				IC50(µM)
	3 µM	10 µM	30 µM	100 µM
1	21.7±1.8	51.5±1.7	68.1±2.4	98.1±1.4	9.4±1.6
2	−1.7±2.9	−1.3±2.4	9.5±3.8	39.6±3.6	>100
3	26.3±1.6	44.2±1.5	53.1±2.2	76.1±2.3	17.5±2.4
4	6.9±1.7	11.5±1.8	24.9±2.4	38.5±1.7	>100
5	1.4±1.8	6.8±1.5	31.6±1.6	72.4±1.1	52.3±3.6
6	1.6±2.1	2.9±2.7	11.5±2.4	42.2±1.9	>100
7	2.2±2.7	3.5±3.6	21.8±2.1	53.6±3.9	97.9±2.8
Hydrocortisone	1.9±2.0	12.3±2.3	26.5±2.3	82.2±3.9	42.7±3.1

Positive control: hydrocortisone. n=3, mean±S.D.

N-Containing sesquiterpenes are quite rarely found in nature. We have the strong interest to view its biosynthesis. We assume that the lactone oxygen atom of isogermafurenolide (4) possesses an α,β-unsaturated ester and a vinyl hemiketal and may be rather active, as a result, it can easily be replaced by the nitrogen atom¹³⁾ in plant tissue.

Compounds 1–7 were evaluated for their inhibitory effects on NO production. Unfortunately, four guaiane-type sesquiterpenoids showed weak inhibitory activities. Among the elemane-type sesquiterpenoids, compounds 1, 3 showed the stronger activities than compound 4 with IC₅₀ values of 9.4, 17.5 µM. We presumed that the existing of nitrogen atom and hydroxyl group could affect the inhibitory activities.

Experimental

General Experimental Procedures

Melting points were measured on an X-4 digital display micro-melting point apparatus. Optical rotations were recorded on a Perkin-Elmer 341 polarimeter at room temperature. UV spectra were measured on a TU1901 spectrophotometer. IR spectra were measured on a Nicolet FTIR-670 instrument. HR-ESI-MS were recorded on a Bruker APEX II. ESI-MS were obtained on an HP-5988 MS spectrometer. 1D (¹H, ¹³C) and 2D (COSY, NOESY, heteronuclear multiple quantum coherence (HMQC), HMBC) NMR spectra were taken on a Bruker Avance III-600 spectrometer (600.13 MHz for ¹H and 150.90 MHz for ¹³C) in CDCl₃ with tetramethylsilane (TMS) as internal standard. Chemical shifts (δ) were expressed in parts per million (ppm), and coupling constants (J) were reported in Hertz (Hz). The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. Column chromatography (CC) was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and Lichroprep RP₁₈ gel (40–63 µm, Merck, Darmstadt, Germany). TLC was carried out with glass precoated silica gel GF₂₅₄ plates. Spots were visualized under UV light or by spraying with 10% H₂SO₄ in EtOH followed by heating at 105°C.

Plant Material

The rhizomes of C. wenyujin was collected in Ruian County of Wenzhou, P. R. China, in September 2008. The identity of the plant material was verified by Prof. Guanyang Lin at Wenzhou Medical University, and a voucher specimen (No.　2008B15) was deposited at School of Pharmacy, Wenzhou Medical University, Wenzhou 325035, P. R. China.

Extraction and Isolation

Dried rhizomes of C. wenyujin (20 kg) were powdered and extracted with 95% EtOH at room temperature (3×10 L, 7 d, each) to yield an EtOH extract (1800 g). A portion of which (1700 g) was suspended in distilled water and then partitioned successively with petroleum ether (bp 60–90°C), EtOAc, and n-BuOH to give three corresponding portions. The EtOAc extract (600 g) was chromatographed on a silica gel column, by eluting with a stepped gradient of petroleum ether–EtOAc (100 : 0 to 0 : 1) to afford eight fractions (1–8) according to the TLC analysis. Fraction 2 (48.4 g) was subjected to CC on silica gel eluting with petroleum ether–EtOAc (40 : 1 to 0 : 1) to give six subfractions (2a–f). Subfraction 2b (3.2 g) was extensively subjected to CC over silica gel eluting with petroleum ether–Me₂CO (40 : 1) to yield compound 5 (23 mg). Subfraction 2d (4.7 g) was separated on LiChroprep RP-18 silica gel CC with elution of decreasing polarity (20–100% methanol) to obtain compound 4 (51 mg). Fraction 3 (13 g) was subjected to silica gel CC with eluting with petroleum ether–EtOAc (30 : 1 to 0 : 1) to give seven subfractions (3a–g). Compound 1 (89 mg) was purified from subfraction 3c (1.2 g) by repeated column chromatography over silica gel with petroleum ether–EtOAc (15 : 1). Subfraction 3d was treated with the same procedures as subfraction 3c to obtain compound 3 (26 mg) and compound 7 (14 mg). Subfraction 3e (3.9 g) was performed on LiChroprep RP-18 silica gel CC eluted with MeOH–H₂O (60 : 40) to afford compound 2 (21 mg) and compound 6 (9 mg).

Elema-1,3,7(11),8-tetraen-8,12-lactam (1): White amorphous powder; mp greater than 290°C; [α]_D²⁰ +39.0 (c=0.01, MeOH); UV (MeOH) λ_max (log ε) 236 (4.98) nm; IR (KBr) ν_max 3367, 1741, 1690 cm⁻¹; ¹H-NMR (CDCl₃, 600 MHz) and ¹³C-NMR (CDCl₃, 150 MHz), see Table 1; ESI-MS m/z 229.8 [M+H]⁺; HR-ESI-MS m/z 230.1548 [M+H]⁺ (Calcd for C₁₅H₂₀NO, 230.1539).

7β,8α-Dihydroxy-1α,4αH-guai-9,11-dien-5β,8β-endoxide (2): Colorless oil; [α]_D²⁰ +19.5 (c=0.02, MeOH); UV (MeOH) λ_max (log ε) 240 (4.1) nm; IR (KBr) ν_max 3434, 1701, 1644, and 1158 cm⁻¹; ¹H-NMR (CDCl₃, 600 MHz), and ¹³C-NMR (CDCl₃, 150 MHz), see Table 1; ESI-MS m/z 272.8 [M+Na]⁺, 230.8 (50), 188.8 (60); HR-ESI-MS m/z 273.1474 [M+Na]⁺ (Calcd for C₁₅H₂₂O₃Na, 273.1461).

Bioassay for NO Production

Inhibitory effects on NO production was evaluated according to the methods described in previous literature.¹⁴⁾ RAW264.7 macrophages were seeded at 1×10⁵ (cells/well) in 96-well plates and treated with or without different concentrations of compounds for 30 min. The control groups only received dimethyl sulfoxide (DMSO), LPS groups only received LPS. Then cells were treated with 1 µg/mL of LPS for 24 h. NO production in each well was assessed by measuring the accumulation of nitrite in the cell culture supernatant using Griess reagent. The absorbance of nitrite production at 540 nm was determined by a microplate reader and the inhibitory rate was calculated.

Acknowledgment

This work was supported by Key project of Traditional Chinese Medicine Science and Technology of Zhejiang Province (2013ZA087), Zhejiang Provincial Natural Science Foundation of China (LY13H280003).

References

• 1)  The State Pharmacopoeia Commission of P. R. China, “Pharmacopoeia of the P. R. China,” 2010 ed., Vol. I, China Medical Science Press, Beijing, 2010, pp. 193–194.
• 2)  Lou Y., Zhao F., He H., Peng K. F., Chen L. X., Qiu F., Chem. Biodivers., 7, 1245–1253 (2010).
• 3)  Ma Z. J., Meng Z. K., Zhang P., Fitoterapia, 80, 374–376 (2009).
• 4)  Wang S. S., Zhang J. M., Guo X. H., Song Q. L., Zhao W. J., Yao Xue Xue Bao, 42, 1062–1065 (2007).
• 5)  Liu A. L., Yang F., Zhu M., Zhou D., Lin M., Lee S. M., Wang Y. T., Du G. H., Planta Med., 76, 1874–1876 (2010).
• 6)  Lim C. B., Ky N., Ng H. M., Hamza M. S., Zhao Y., Integr. Cancer Ther., 9, 36–49 (2010).
• 7)  Sasaki Y., Goto H., Tohda C., Hatanaka F., Shibahara N., Shimada Y., Terasawa K., Komatsu K., Biol. Pharm. Bull., 26, 1135–1143 (2003).
• 8)  Zhao Z. R., Zhang P., Wu Y. G., Liu H., China Journal of Traditional Chinese Medicine and Pharmacy, 26, 1868–1869 (2011).
• 9)  Liu X. Y., Lou Y., Hu D., Chen L. X., Bu G. M., Qiu F., Journal of Shenyang Pharmaceutical University, 24, 682–686 (2007).
• 10)  Friedrich D., Bohlmann F., Tetrahedron, 44, 1369–1392 (1988).
• 11)  Hikino H., Sakurai Y., Numabe S., Takemoto T., Chem. Pharm. Bull., 16, 39–42 (1968).
• 12)  Matsuda H., Morikawa T., Toguchida I., Ninomiya K., Yoshikawa M., Chem. Pharm. Bull., 49, 1558–1566 (2001).
• 13)  Chen Z. L., Cao W. Y., Zhou G. X., Wichtl M., Phytochemistry, 45, 765–767 (1997).
• 14)  Qin J. J., Zhu J. X., Zeng Q., Cheng X. R., Zhang S. D., Jin H. Z., Zhang W. D., Planta Med., 78, 1002–1009 (2012).