HETEROCYCLES

Note | Special issue | Vol. 79, No. 1, 2009, pp. 1101-1105
Received, 3rd October, 2008, Accepted, 1st December, 2008, Published online, 5th December, 2008.
DOI: 10.3987/COM-08-S(D)62

■ Nupharic Acid, a New Sesquiterpene Alkaloid from Nuphar japonicum

Noriyuki Kogure, Akiko Nozoe, Mariko Kitajima, and Hiromitsu Takayama*

Laboratory of Molecular Structure and Biological Function, Graduate School of Pharmaceutical Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan

Abstract

A new sesquiterpene alkaloid named nupharic acid (1) was isolated from the aerial part of Nuphar japonicum together with known alkaloids nupharidine (2) and deoxynupharidine (3). Alkaloid 1 is the first example of Nuphar alkaloids that lack the common furan moiety.

INTRODUCTION
Nuphar japonicum DC. is widely distributed in Japan and Korea and is a well-known plant used in Chinese traditional medicine. Nuphar rhizoma, the dried rhizome of Nuphar japonicum DC. and Nuphar pumilum (Timm.) DC., has been prescribed for its tonic, hemostatic, and diuretic effects in Japanese and Chinese traditional medicine.1 Chemical studies of N. japonicum have been carried out and a number of sesquiterpene alkaloids, such as nupharidine (2) and deoxynupharidine (3)2, as well as thio-containing dimeric alkaloids3 have been identified (Figure 1). We investigated the chemical constituents in the aerial part of this plant for the first time and isolated a new alkaloid called nupharic acid (1).

RESULTS AND DISCUSSION
The molecular formula of new alkaloid 1 was established as C15H23NO3 from HRFABMS [m/z 266.1743 (MH+)], which showed that 1 possessed two oxygens more than deoxynupharidine (3).
The 1H-NMR data (Table 1) showed significant signals characteristic of an aldehyde group [(δ 9.51, H-14)], an olefinic or an aromatic proton [(δ 6.96, H-15)], and two tertiary methyl groups [δ 1.19 (3H, d, H3-12), δ 0.99 (3H, d, H3-11)]. The 13C-NMR spectrum revealed the presence of two carbonyl carbons [δC 193.3 (C-14), δC 167.6 (C-16)], two sp2 carbons [δC 150.5 (C-15), δC 142.4 (C-13)], three low-field sp3 carbons bearing an oxygen or a nitrogen atom [δC 70.2 (C-10), δC 58.8 (C-4), δC 58.1 (C-6)], and eight sp3 carbons. Comparing the NMR data of compound 1 with those of 2 and 3 (Table 1), 1 possessed a quinolizidine ring but lacked a furan residue that commonly exists in Nuphar alkaloids.

1H-1H COSY and HMQC analyses (Figure 2) indicated the presence of a long carbon chain consisting of eleven carbons. The three low-field carbons (C-4, C-6, and C-10) are all connected to a common nitrogen atom and form a quinolizidine ring. This idea was supported by HMBC correlations from H-6 to C-4 and C-10. The IR spectrum indicates Bohlmann absorption at 2850-3000 cm-1 due to the trans-quinolizidine ring.4 HMBC correlations from a low-field olefin proton (δH 6.96, H-15) to two carbonyl carbons [δC 193.3 (C-14), δC 167.6 (C-16)] and from an aldehyde proton (δH 9.51, H-14) to an olefin carbon (δC 142.4 C-13) revealed the existence of a β-formyl acrylic acid residue. Furthermore, HMBC crosspeaks from H-14 and H-15 to C-4 demonstrated that the β-formyl acrylic acid residue was attached to C-4 on the quinolizidine ring at β-position (C-13).
NOE correlations of H-10/H-4, H-4/H-6, and H-6/H-7 suggested that H-4, H-6, H-7, and H-10 have syn configuration (Figure 3). NOE correlation between H-1 and H-3α indicated that the methyl group at C-1 should have equatorial orientation. The geometry of the C13-15 double bond was deduced to be E by NOE experiments.

Nupharic acid (1) is the first example of Nuphar alkaloids lacking a furan residue. Biogenetically, 2 and 3 were derived from trans, cis-farnesyl pyrophosphate via the formation of a quinolizidine ring and a furan ring. In contrast, 1 would be derived from cis, cis-farnesyl pyrophosphate via the formation of a quinolizidine ring and oxidation at C-14 and C-16 positions (Figure 4).
The MeOH extract and its alkaloid fraction from the rhizomes of Nuphar pumilum are known to show cytotoxic effects on human leukemia cell (U937), mouse melanoma cell (B16F10), and human fibroblast (HT1080)1e, however, compound 1 did not show cytotoxic activity against tumor cell lines (A549 and HT29).

EXPERIMENTAL
General Procedure
1H- and 13C-NMR spectra: JEOL JNM A-500 at 500 MHz (1H-NMR) and at 125 MHz (13C-NMR). UV: JASCO V-560. IR: JASCO FT/IR-230. FAB-MS: JEOL JMS-AX500. HR-FAB-MS: JEOL JMS-HX110. Optical rotation: JASCO P-1020. CD: JASCO J-720WI. TLC: Precoated silica gel 60 F254 plates (Merck, 0.25 mm thick). Column chromatography: Silica gel 60 (Merck, 70-230 mesh). Flash column chromatography: Silica gel 60N (Kanto Chemical, 40-50 μm).

Plant Material
Nuphar japonicum DC. was harvested from the medicinal plant garden of Chiba University, Japan. A voucher specimen (No. 20060501) was deposited at the Faculty of Pharmaceutical Sciences, Chiba University, Japan. The plant was identified by Professor Dr. F. Ikegami and a voucher specimen was deposited in the herbarium of our department.

Extraction and Isolation of Nupharic Acid (1)
The aerial part of N. japonicum DC. (2751 g, dry weight) was extracted with MeOH (5 L, three times at room temperature and three times under reflux) to give the extract (152.9 g). The MeOH extract (152.9 g) was dissolved in H2O (0.66 L) containing a small amount of MeOH (20 mL) and extracted with n-hexane (0.27 L x 4) to give the n-hexane extract (20.15 g). The aqueous layer was successively extracted with AcOEt (0.35 L x 3), 5% MeOH/CHCl3 (0.4 L x 4), and n-BuOH (0.35 L x 4) to give the AcOEt extract (3.23 g), the 5% MeOH/CHCl3 extract (1.75 g), and the n-BuOH extract (10.95 g), respectively. The 5% MeOH-CHCl3 extract (1.75 g) was separated by SiO2 flash column chromatography with MeOH/CHCl3 gradient to give 6 fractions: fr. A CHCl3 (143.4 mg); fr. B 1% MeOH/CHCl3 (33.7 mg); fr. C 5% MeOH/CHCl3 (627.6 mg); fr. D 10-20% MeOH/CHCl3 (578.3 mg); fr. E 20-30% MeOH/CHCl3 and MeOH (466.4 mg); and fr. F 5% AcOH/MeOH (3.9 mg). Fr. C was purified successively by SiO2 flash column chromatography (MeOH/AcOEt gradient and MeOH/CHCl3 gradient) to afford nupharic acid (1, 34.3 mg). Nupharic acid (1): FAB-MS (NBA) m/z: 266 (M+H+), HR-FAB-MS (NBA/PEG) m/z: 266.1743 (M+H+, Calcd for C15H24NO3 : 266.1756). 1H-NMR: see Table 1. 13C-NMR: see Table 1. UV (MeOH) λmax nm: 206.0. CD (c = 0.358 mmol/L, MeOH, 24 °C) Δε (nm): 0 (316), +2.3 (259), 0 (234), -2.0 (217). IR (CHCl3, cm-1): 3384, 3021, 2931, 2857, 1760, 1689, 1608. [α]D24 -5.3° (c = 0.256, MeOH).
Cell Culture.
Human lung and colorectal cancer cell lines, A549 and HT29, respectively, were obtained from ATCC (Rockville, MD, USA). A549 and HT29 cells were maintained in Dulbecco’s modified eagle’s medium (D-MEM) (D6046, D6046) and D-MEM/F-12 medium (D8062, Sigma) with 10% heat-inactivated fetal bovine serum (FBS) and 5 mg/mL gentamicin, respectively, at 37 °C in a humidified atmosphere containing 5% CO2.

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