HETEROCYCLES

Short Paper | Regular issue | Vol. 89, No. 3, 2014, pp. 747-752
Received, 30th December, 2013, Accepted, 29th January, 2014, Published online, 3rd February, 2014.
DOI: 10.3987/COM-13-12928

■ Serralongamine A, a New Lycopodium Alkaloid from Lycopodium serratum var. longipetiolatum

Wen-Ping Jiang, Kan'ichiro Ishiuchi, Jin-Bin Wu,* and Susumu Kitanaka*

Organic and Material Synthesis Laboratory, Graduate Institute of Pharmaceutical Chemistry, China Medical University, No. 91 Hsueh-Shih Rd. Taichung 40402, Taiwan

Abstract

A new Lycopodium alkaloid, serralongamine A (1), has been isolated from the club moss Lycopodium serratum var. longipetiolatum, and the structure was elucidated on the basis of spectroscopic data.

Club moss (Lycopodiaceae) are known to be a rich source of Lycopodium alkaloids1 possessing unique heterocyclic ring system such as C16N1, C16N2, and C27N3, which have attracted great interest from biogenetic,2 synthetic,3 and biological4 points of view. In our continuing efforts to find new Lycopodium alkaloids,5 a new phlegmarine-type alkaloid, serralongamine A (1), was isolated from the club moss Lycopodium serratum var. longipetiolatum. In this paper, we describe the isolation and structure elucidation of 1.

The club moss L. serratum var. longipetiolatum collected in Taiwan, was extracted with MeOH, and the MeOH extract was partitioned between EtOAc and 3% tartaric acid. Water-soluble materials, which were adjusted at pH 10 with saturated Na2CO3, were partitioned with CHCl3. CHCl3-soluble materials were subjected to an amino silica gel column (n-hexane/EtOAc, then CHCl3/MeOH), followed by a silica gel column (CHCl3/MeOH, then CHCl3/MeOH/H2O/TFA). The fraction eluted with CHCl3/MeOH/H2O/TFA (6/4/1/0.01) was purified by C18 HPLC (MeOH/H2O/TFA, 14/86/0.1) to afford serralongamine A (1, 0.0008% yield), huperzine A,4 huperzine B,4 and lycoposerramine-V.6

Serralongamine A {1, [α]D18 -9.1 (c 0.6, MeOH)} was revealed to have the molecular formula, C17H26N2, by HRESIMS data [m/z 259.2176, [M+H]+, Δ +0.2 mmu]. The 1H, 13C NMR (Table 1), and HMQC spectra of 1 showed signals due to one sp2 quaternary carbon, four sp2 methines, four sp3 methines, six sp3 methylenes, and two methyls. Among them, one sp3 methine (δC 66.0), one sp3 methylene (δC 57.4), and one methyl carbon (δC 41.4) were ascribed to those bearing a nitrogen atom. Also one sp2 methine (δC 143.8) and one sp2 quaternary carbon (δC 158.1) were assigned to those bearing the other nitrogen atom.
The gross structure of 1 was elucidated by analysis of 2D NMR data including the 1H-1H COSY, HMQC, and HMBC spectra (Figure 1). The 1H-1H COSY spectrum disclosed three structural units a (C-1 to C-4), b (C-6 to C-8), and c (C-9 to C-16). Connectivities of C-9, C-13, and C-17 through N-9 were revealed by HMBC cross-peaks of H3-17 (δH 2.87) to C-9 (δC 57.4) and C-13 (δC 66.0). An HMBC cross-peak of H-3 (δH 8.44) to C-5 (δC 158.1) revealed that C-4 connected with C-5. An HMBC correlation for H-1 (δH 8.73) to C-5 suggested the connectivity between C-1 and C-5 via N-1 to form the monosubstituted pyridine ring (C-1 to C-5, N-1). HMBC cross-peaks of H-6a, b (δH 3.51, 2.66) to C-4 (δC 128.8) and C-5 revealed that C-6 was attached to C-5. HMBC correlations for H-16 (δH 0.96) to C-8 (δC 36.8) suggested that C-8 connected with C-15. The connectivity of C-7 and C-12 was revealed by HMBC cross-peaks of H-6b and H-8a, b (δH 1.38, 1.15) to C-12 (δC 46.4). Thus, the gross structure of serralongamine A was elucidated to be 1 (Figure 1).

The NOESY spectrum of 1 showed cross-peaks as shown in Figure 2. NOESY correlations for H-13/H-7 and H-13/H3-16 revealed that a cyclohexane ring (C-7-C-8 and C-12-C-15) was chair form and C-6 and C-16 were in an equatorial and an axial position of the cyclohexane ring, respectively. The chair form of the piperidine ring (C-9-C-13, N-9) was implied from NOESY cross-peaks of H-13/H-11b, H-17/H-13, and H-17/H-14a, which indicated a trans-fused ring junction between the piperidine ring and the cyclohexane ring (C-7-C-8 and C-12-C-15). Thus, the relative stereochemistry of serralongamine A (1) was elucidated to be shown in Figure 2.
Serralongamine A (1) is a new phlegmarine-type Lycopodium alkaloid possessing a monosubstituted pyridine ring and a trans decahydroquinoline ring. There are many variations of decahydroquinoline ring system, stereochemistry of C-7, C-12, C-13, and C-15, in phlegmarine-type alkaloids isolated from Lycopodium spp., such as phlegmarine,7 carinatumin C,8 cermizine B,9 lycoposerramine-X and Z,10 huperzine M,11 and serratezomine E.12 The stereochemistry of the decahydroquinoline moiety of 1 is rare in phlegmarine-type alkaloids (Scheme 1). Serralongamine A (1) did not show acetylcholinesterase inhibitory activity13 (IC50 > 100 µM).

EXPERIMENTAL
Optical rotation was recorded on a JASCO P-1020 polarimeter. UV spectrum was recorded on a HITACHI U-1800 spectrophotometer. IR spectrum was recorded on a JASCO FT/IR-4100 spectrometer. NMR spectra were recorded on a JEOL JNM-ECX500 spectrometer using 3.0 mm microcells (Shigemi Co., Ltd.). Chemical shifts (ppm) in CD3OD are reported using residual CD2HOD and CD3OD (δH 3.31 and δC 49.0, respectively) as internal references. Positive-mode ESITOFMS was obtained on a Xevo G2-S QTof spectrometer (Waters Co., Ltd.) using a samle dissolved in MeOH.

Plant Material
The club moss Lycopodium serratum var. longipetiolatum was collected at Miaoli County in Taiwan. The botanical identification was made by Dr. Y.-C. Chen, Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University. A voucher specimen has been deposited in the herbarium of China Medical University.

Extraction and Isolation
The club moss Lycopodium serratum var. longipetiolatum (190 g, dry weight) was crushed and extracted with MeOH. The MeOH extract was treated with 3% tartaric acid (pH 3) and then partitioned with EtOAc. The aqueous layer was treated with Na2CO3 (aq) to pH 10 and extracted with CHCl3 to give a crude alkaloidal fraction. The alkaloidal fraction was subjected to an amino silica gel column (n-hexane/EtOAc, then CHCl3/MeOH), in which a fraction eluted with n-hexane/EtOAc (10:1) was purified by silica gel columns (CHCl3/MeOH, 1:0 to 1:1 and then CHCl3/MeOH/H2O/TFA, 6:4:1:0 to 6:4:1:0.01). The fraction eluted with CHCl3/MeOH/H2O/TFA (6:4:1:0.01) was further purified by C18 HPLC (CAPCELL PAK C18 AQ (SHISEIDO), 5 µm, 10 mm I.D. x 250 mm, solvent MeOH/H2O/TFA, 14:86:0.1, flow rate 2.5 ml/min, detection 254 nm) to afford serralongamine A (1, 0.0008% yield), huperzine A (0.06% yield),4 huperzine B (0.03% yield),4 and lycoposerramine-V (0.001% yield).6

Serralongamine A (1): A colorless amorphous solid; [α]D18 -9.1 (c 0.6, MeOH); UV (MeOH) λmax 261 (ε 1828); IR (KBr) νmax 2928, 1594, and 1476 cm-1; 1H and 13C NMR data (Table 1); ESITOFMS m/z 259 [M+H]+; HRESITOFMS m/z 259.2176 [M+H]+; calcd for C17H27N2, 259.2174).

ACKNOWLEDGEMENTS
The authors thank Dr. K. Metori, Analytical Center, School of Pharmacy, Nihon University, for measurements of ESITOFMS, Mr. Fu-Shun Ho for collecting the plant, and Dr. Y.-C. Chen, Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University for botanical identifications of the plant. This work was partly supported by Grant-in-Aid for Young Scientists B (to K.I.), The Research Foundation for Pharmaceutical Sciences (to K.I.), and Grant-in-Aid for Scientific Research C (to S.K.).

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