Diterpenes from the Trunk of Abies holophylla and Their Potential Neuroprotective and Anti-inflammatory Activities Chung Sub Kim, † Lalita Subedi, ‡,§ Sun Yeou Kim, ‡,§ Sang Un Choi, ⊥ Ki Hyun Kim, † and Kang Ro Lee* ,† † Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea ‡ Gachon Institute of Pharmaceutical Science, Gachon University, Incheon 21936, Republic of Korea § College of Pharmacy, Gachon University, #191, Hambakmoero, Yeonsu-gu, Incheon 21936, Republic of Korea ⊥ Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea * S Supporting Information ABSTRACT: Eleven new abietane-type diterpenes, holophyllins D-N(1-11), and 17 known analogues (12-28), were isolated from a MeOH extract of the trunk of Abies holophylla. The chemical structures of 1-11 were determined through spectroscopic data analysis, including NMR ( 1 H and 13 C NMR, DEPT, 1 H- 1 H COSY, HMQC, HMBC, and NOESY) and HRFABMS methods. All isolated compounds (1-28) were evaluated for their cytotoxicity against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-116), for their potential neuroprotective effects through induction of nerve growth factor in C6 glioma cells, and for their effects on nitric oxide levels in lipopolysaccharide-stimulated murine microglia BV2 cells. T here are approximately 50 species in the genus Abies (Pinaceae), and some have been used in Korean traditional medicine for treating vascular and pulmonary diseases, indigestion, rheumatic diseases, and stomachache. 1,2 A. holophylla Maxim. is a coniferous tree distributed in the mountainous areas of Korea, mainland China, and Russia. Several bioactive monoterpenes, sesquiterpenes, triterpenes, and phenolic compounds have been isolated from this plant. 2,3 In a continuing search for bioactive constituents from Korean medicinal plants, it was found that the n-hexane and CHCl 3 layers of an A. holophylla MeOH extract were cytotoxic against A549, SK-OV-3, SK-MEL-2, and HCT-116 cells in a sulforhod- amine B (SRB) bioassay (11.3-13.8 μg/mL for the n-hexane layer, 11.2->20 μg/mL for the CHCl 3 layer) and reduced nitric oxide (NO) levels in lipopolysaccharide (LPS)-stimulated murine microglia BV2 cells. We have previously reported lignans and rearranged diterpenes from the n-hexane and EtOAc layers of an A. holophylla MeOH extract. 1,4 Herein, 11 further new diterpenes have been isolated, namely, holophyllins D-N(1-11), and 17 known diterpenes (12-28) from the active n-hexane and CHCl 3 layers. All isolated compounds (1- 28) were evaluated for their cytotoxicity, potential neuro- protective activity, and effects on nitric oxide levels. In this paper, the isolation and structural elucidation of compounds 1-28 and their biological activities are presented. ■ RESULTS AND DISCUSSION Holophyllin D (1) was isolated as a colorless gum. The molecular formula was C 21 H 32 O 5 from the [M + Na] + ion peak obtained by positive-ion high-resolution fast-atom bombard- ment mass spectrometry (HRFABMS). The 1 H and 13 C NMR spectra of 1 were similar to those of 7α,8α,13β,14β- diepoxyabietan-18-oic acid, 5 except for the presence of a carbonyl signal (δ C 209.1), instead of an epoxide [δ C 58.7; δ H 3.19 (1H, brs)] signal at C-7 (Tables 1 and 2). Additionally, a methoxy group signal was observed [δ C 52.3; δ H 3.34 (3H, s)], for which the location was confirmed to be at C-8 based on the HMBC cross-peak of OCH 3 -8 with C-8 (Figure 1). The planar structure of 1 was elucidated through two-dimensional (2D) NMR analysis, including the DEPT, COSY, HMQC, and HMBC spectra. A trans -fused A/B ring junction was corroborated from the NOESY cross-peaks of H-5/H-9 and H-19/H-20 (Figure 2). The relative configurations of the OCH 3 -8 and 13-isopropyl groups were both α-oriented, based on the distinct NOESY correlations from OCH 3 -8 to H-9 and H-16 (Figure 2). Thus, the structure of 1 was established as 13β,14β-epoxy-8α-methoxy-7-oxoabietan-18-oic acid. Received: November 26, 2015 Article pubs.acs.org/jnp © XXXX American Chemical Society and American Society of Pharmacognosy A DOI: 10.1021/acs.jnatprod.5b01053 J. Nat. Prod. XXXX, XXX, XXX-XXX