Selaginella pulvinata (Hook. et Grev.) Maxim. (Selaginellaceae),is a perennial herb widely distributed in China. As one of the two qualified species listed in Chinese Pharmacopoeia (2010 edition)^[1],it has been widely used in traditional Chinese medicine for the treatment of cancer,dysmenorrhea,asthma,and traumatic injury^[2]. The pharmacological investigations of the genus Selaginella revealed that it has numerous biological activities,including anticancer^[3,4],antivirus^[5],anti-inflammation^[6,7,8],anti-diabetes^[9],and vascular endothelial protective effect^[10]. In addition to biflavonoids,a series of interesting selaginellin derivatives^{[11,12,13,14]} featuring unique alkynyl and p-quinone methide functionalities have been discovered from this genus. Continued investigation of the title plant led to the isolation of a new selaginellin derivative named as selaginellin S (1),together with a known compound (2,selaginellin M) (Figure 1). Compound 2 is first reported in this plant. Herein,we report the isolation and structural elucidation of the new compound by extensive spectroscopic analyses.

Figure 1 The structures of compounds 1-2

Selaginellin S (1) was obtained as a red powder; its molecular formula was determined as C₂₈H₂₀O₅ by HR-ESI-MS with an ion peak at m/z 437.138 15 [M+ H]⁺ (calcd. 436.687 14),which indicated 19 degrees of unsaturation. The UV absorption at 282 nm was indicative of an aromatic system. The ¹H NMR spectral data (Table 1) indicated the presence of two ortho- coupled aromatic signals δ_H 7.67 and δ_H 7.41 (1H,d,J = 8.4 Hz),attributed to the tetra-substituted A-ring; protons of three AA'XX' systems representing the respective p- substituted rings B,C,and D,respectively. Differentiation between the above spin systems,were achieved via ¹H-¹H COSY spectrum. The ¹³C NMR spectrum showed a total of 28 carbon signals,including one carbonyl,one oxygenated methylene,two acetylenic and twenty-four aromatic carbons. The NMR spectra of 1 were similar to those of selaginellin; instead of a semi-quinone unit,there was a carbonyl carbon.

Table 1

Table 1 1H and 13C NMR (DMSO-d6) data for 1. J in Hz No. δH δC HMBC 1 - 162.3 - 2,6 6.77 (d,8.5) 115.3 C-4 3,5 7.53 (d,8.5) 131.7 C-1,C-7 4 - 128.8 - 7 - 195.3 - 8 - 117.7 - 9 - 142.0 - 10 7.67 (d,8.4) 126.6 C-8,C-12,C-28 11 7.41 (d,8.4) 129.2 C-9,C-13,C-19 12 - 137.5 - 13 - 141.0 - 14,18 7.08 (d,8.0) 129.7 C-12,C-16 15,17 6.63 (d,8.0) 115.0 C-16,C-19 16 - 156.7 - 19 - 130.0 - 20 - 83.2 - 21 - 98.7 - 22,26 6.93 (dd,7.5,2.5 ) 132.5 C-21,C-24 23,25 6.69 (dd,7.5,2.5 ) 115.6 C-24,C-27 24 - 158.1 - 27 - 112.1 - 28 4.76 (s) (2H) 60.9 C-8,C-9,C-10

Table 1 1H and 13C NMR (DMSO-d6) data for 1. J in Hz

The apparent HMBC correlations from H-14/18 to C-12,and from H-11 to C-19,linked the A-ring to C- 12,which was further supported by ROESY correlation between H-11 and H-18. The location of a hydroxy- methyl [δ_H/δ_C 4.76 (2H,s)/60.9,C-28] group at C-9 (A) was confirmed using a combination of HMBC and ROESY experiments as depicted in Figure 2. Two characteristic signals at δ_C 98.7 and 83.2 in the ¹³C NMR (DEPT) spectrum implied the presence of an alkynyl,which was further confirmed by the absorption at 2 202 cm^-1 inthe IR spectrum. HMBC correlations from H-22/26 to C-21 (δ_C 98.7) connected the B-ring to the alkynyl,whereas those from H-3/5 to C-7 (δ_C 195.3) placed the ketone group at C-4. In particular,the chemical shifts for C-8 (δ_C 117.7) and C-13 (δ_C 141.0) established the connections of C-8/C-20 and C-13/C-7,respectively,as is the case for reported selaginellins,thus defining the planar structure of 1 as drawn.

Figure 2 1H-1H COSY (—),key HMBC (→),and ROESY (↔) of 1

Despite extensive effort,attempts to produce suitable crystals of 1 for X-ray diffraction were unsuccessful. Alternatively,electronic circular dichroism (ECD) analysis was utilized as an indirect determination of absolute configuration. The theoretical ECD was calculated with a time-dependent density functional theory (TD-DFT) method at the B3LYP/6-311+G(d) level. The calculated ECD spectrum was then produced by SpecDis software^[15]. As shown in Figure 3,the calculated ECD curve matched very well with the experimental CD curve (determined in CH₃CN),indicating that 1 has an R configuration. Apparently,selaginellin S is a key intermediate in the biosynthesis pathway of selaginellins.

Figure 3 Experimental and calculated CD of (R)-1

General experimental procedures NMR spectra were recorded on BRUKER AV-500 spectrometers. UV/Vis spectra were recorded using a Shimadzu UV-2500PC spectrophotometer equipped with a 1 cm pathlength cell. IR spectra were obtained using a BRUKER Tensor 27 spectrometer with KBr disks. MS and HR-ESI-MS were measured on a VG Auto Spec-3000 mass spectrometer and an LTQ Orbitrap XL mass spectrometer (Thermo Scientific). Optical rotations were measured with a Jasco P-1020 polarimeter equipped with a 1 dm pathlength cell. CD spectra were recorded with a Jasco J-810 spectrometer with a 1 mm pathlength cell. Column chromatography was performed over silica gel H (200-300 mesh and 10-40 μm),and Sephadex LH-20 (50 μm; Uppsala,Sweden). TLC was conducted on precoated silica gel plates GF₂₅₄ (Qingdao Marine Chemical Ltd.,Qingdao,People’s Republic of China).

Plant material The whole plants of Selaginella pulvinata were collected from Yunnan Province,P.R.China in October 2011 and identified by Dr. Prof. Jin-ao Duan. A voucher specimen (2011-10-018) was deposited at Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization,Nanjing University of Chinese Medicine.

Extraction and isolation The air-dried and powdered plants of S. pulvinata (5 kg) were extracted by 95% EtOH at room temperature for three times (50 L × 3),once per week. The EtOH solution was collected together and evaporated under reduced pressure to give a residue (65 g). The residue was suspended in water,and partitioned by n-hexane (10 L × 3),EtOAc (10 L × 3),and n-BuOH (10 L × 3). The EtOAc layer was chromatographed over silica gel eluted with CHCl₃- CH₃OH gradients (from 99∶1 to 1∶1) to afford five fractions (A-E),based on their TLC characteristics. Fraction C was further separated b y silica gel column eluted with CHCl₃-CH₃OH gradients (from 9∶1 to 1∶1) to afford five subfractions (C1-C5). Fraction C2 was isolated repeatedly by Sephadex LH-20 gel column (CH₃OH,CH₃OH-H₂O) to yield compounds 1 (7 mg) and 2 (3 mg).

Selaginellin S (1): Red powder; [α]_D²⁵= +40.4 (c 0.1,CH₃CN); UV nm (logε): 282 (4.83); IR cm^-1: 3 396,2 806,2 202,1 642,1 606,1 588,1 512,1 219,1 151,829; HR-ESI-MS m/z 437.138 15 [M+H]⁺ (calcd. for C₂₈H₂₀O₅,436.687 14); ¹H NMR (500 MHz,DMSO- d₆) and ¹³C NMR (125 MHz,DMSO-d₆) see Table 1; CD 332 nm.

Selaginellin M (2): C₃₅H₂₆O₅,red powder; [α]_D²⁵= 0 ( c 0.01,CH₃OH); ESI-MS m/z 527 [M+H]⁺; ¹H NMR (DMSO-d₆,500 MHz) δ_H: 6.37 (1H,dd,J = 10.0,2.0 Hz,H-2),7.43 (1H,dd,J = 10.0,2.0 Hz,H-3),7.20 (1H,dd,J = 10.0,2.0 Hz,H-5),6.31 (1H,dd,J = 10.0,2.0 Hz,H-6),6.83 (4H,m,H-8,9,11,12),7.69 (1H,d,J = 8.0 Hz,H-16),7.34 (1H,d,J = 8.0 Hz,H-17),6.78 (2H,d,J = 8.4 Hz,H-20/24),6.59 (2H,d,J = 8.4 Hz,H-21/ 23),6.95 (2H,d,J = 8.4 Hz,H-28/32),6.71 (2H,d,J = 8.4 Hz,H-29/31),4.78 (2H,d,J = 5.4 Hz,H-34),3.71 (3H,s,H-35),5.59 (1H,br s),9.62 (1H,br s),10.16 (1H,br s); ¹³C NMR (DMSO-d₆,125 MHz) δ_C: 185.0 (C-1),128.1 (C-2),138.5 (C-3),130.8 (C-4),139.5 (C-5),128.1 (C-6),159.0 (C-7),132.7 (C-8/12),113.9 (C-9/11),160.4 (C-10),130.7 (C-13),121.5 (C-14),142.9 (C-15),127.5 (C-16),129.7 (C-17),140.6 (C-18),140.1 (C-19),129.5 (C-20/24),114.6 (C-21/23),156.9 (C-22),130.7 (C-25),83.8 (C-26),98.7 (C-27),132.8 (C-28/32),115.8 (C-29/31),158.3 (C-30),112.8 (C-33),61.4 (C-34),55.4 (C-35). These data were consistent with those reported^[14].