Phytochemical and biological studies on rare and endangered plants endemic to China. Part XV. Structurally diverse diterpenoids and sesquiterpenoids from the vulnerable conifer Pseudotsuga sinensis

Phytochemistry 169 (2020) 112184

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Phytochemical and biological studies on rare and endangered plants endemic to China. Part XV. Structurally diverse diterpenoids and sesquiterpenoids from the vulnerable conifer Pseudotsuga sinensis

T

Ting Huanga, Sheng-Hui Yinga, Jing-Ya Lib, Hao-Wei Chena, Yi Zangb, Wen-Xuan Wangc, Jia Lib, Juan Xionga,∗∗, Jin-Feng Hua,∗ a b c

Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, PR China State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, PR China School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan, Hubei, 430074, PR China

ARTICLE INFO

ABSTRACT

Keywords: Pseudotsuga sinensis dode Pinaceae Rare and endangered plants (REPs) Degraded diterpenoids Pseudosinins ATP-Citrate lyase (ACL)

An extensive phytochemical investigation on the chemical constituents from the needles and twigs of the vulnerable conifer Pseudotsuga sinensis yielded 19 diterpenoids and 21 sesquiterpenoids with various carbocyclic skeletons. Among them, 13 (named pseudosinins A–M, resp.) were undescribed compounds. Their structures with absolute configurations were characterized by a combination of spectroscopic methods, calculated and experimental electronic circular dichroism (ECD) data, quantum chemical calculations of the chemical shifts, and single crystal X-ray diffraction analyses. In particular, an array of labdane-derived norditerpenoids with C19-, C18-, and C16-skeletons, and related drimane-type sesquitepenoids with C15- and C13-skeletons were found in the title plant. The possible biogenetic relationships of these degraded terpenoids were briefly discussed. Among the isolates, pseudosinin D, cis-communic acid, and 4β,15-dihydroxy-19-norabieta-8,11,13-trien-7-one showed moderate inhibitory activities against the enzyme ATP-citrate lyase (ACL), a potential drug target for the treatment of hyperlipidemia and hypercholesterolemia.

1. Introduction In recent years, increased attention has been paid to rare and endangered plants (REPs) due to their species fragility but higher potency for discovering new drugs than other botanic sources (Ibrahim et al., 2013; Xiong et al., 2018; Zhu et al., 2011). Since 2013, a special program has been launched to systematically identify structurally diverse bioactive/novel natural products from REPs endemic to China (Jiang et al., 2019; Xiong et al., 2018). In the process of project implementation, the rare and endangered conifers have aroused our special interest. The Pinaceae family ranks among the top 20 privileged drugprolific families on the one hand (Zhu et al., 2011). On the other hand, the fragile coniferous species are generally big trees, making the plant samples (normally the renewable needles and twigs) easier to harvest. Conifers (formally the Division Coniferopsida) are the largest group of living gymnosperms. There are more than 600 coniferous species belonging to six families (i.e., Araucariaceae, Cephalotaxaceae, Cupressaceae, Pinaceae, Podocarpaceae, and Taxaceae) worldwide, and

∗

ca. 190 species are distributed in China (Wu and Raven, 1999). The first volume of the China Plant Red Data Book (CPRDB) published in 1992 listed 388 endangered taxa warranting protection (Fu and Jin, 1992), among which 65 species are conifers, accounting for about one-sixth of the total species recorded in the CPRDB. These conifers are classified into five families (The number in the following parentheses indicates the number of the endangered species in each family): Pinaceae (39), Cupressaceae (12, this count includes the former Taxodiaceae), Taxaceae (7), Podocarpaceae (4), and Cephalotaxaceae (3). By far, three endangered species in the family Pinaceae (i.e., Abies beshanzuensis, Pinus dabeshanensis, and Pinus kwangtungensis) and one in the family Podocarpaceae (i.e., Podocarpus imbricatus) have been phytochemically investigated, which led to the discovery of a number of structurally diverse terpenoids with novel frameworks and interesting bioactivities, such as inhibitory effects against the protein tyrosine phosphatase 1B (PTP1B), neuroinflammation, and the influenza A virus H3N2 (Hu et al., 2016a, 2016b; 2017, 2018; Li et al., 2017). Most recently, a preliminary phytochemical investigation on the terpenoids from the

Corresponding author. Corresponding author. E-mail addresses: [email protected] (J. Xiong), [email protected] (J.-F. Hu).

∗∗

https://doi.org/10.1016/j.phytochem.2019.112184 Received 10 July 2019; Received in revised form 14 October 2019; Accepted 19 October 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.

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twigs and needles of another endangered Pinaceae species, Picea brachytyla, has just been carried out (Jiang et al., 2019). The Pinaceae family comprises ca. 235 species in 11 genera (e.g., Abies, Cathaya, Larix, Picea, Pseudotsuga, and Pinus) (The Plant List, 2013; Fu et al., 1999). Among them, Pseudotsuga is a small genus of evergreen coniferous trees with four or five recognized species distributed only in the northern hemisphere (Fu et al., 1999; Wei et al., 2010). The phytochemically and pharmacologically well-studied species is by far the widespread North American Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] (Barton, 1967, 1972; Dellus et al., 1997; Erdtman et al., 1968; Kimland and Norin, 1968; Krauze-Baranowska et al., 2013; Lai et al., 1992; Rogers et al., 1974). P. sinensis Dode, an endemic species to China, has been recorded as a ‘vulnerable’ plant both in the CPRDB (Fu and Jin, 1992) and the IUCN Red List of Threatened Species (Yang and Chirstian, 2013). This plant has been also categoriezed at the ‘second-grade’ for national protection in China since 1999 (The State Forestry Administration and the Ministry of Agriculature, 1999). Phytochemically, only several flavonoids from the bark of P. sinensis (Yi et al., 2002) have been reported. As part of our continuing research on the rare and endangered conifers endemic to China (Hu et al., 2016a, 2016b; 2017, 2018; Li et al., 2017), the terpenoids from the 90% MeOH extract of the needles and twigs of P. sinensis have been investigated herein for the first time. A total of 40 naturally occurring di- and sesqui-terpenoids were obtained (Fig. 1). Among the isolates, 13 were previously undescribed (pseudosinins A–M, 1–13, resp.). In this study, we present the isolation, characterization, biogenetic consideration, and bioactive evaluation of these isolates.

typical signals assignable for two tertiary [δ 0.61 (3H, s, Me-20), 1.26 (3H, s, Me-18)] and one vinyl [δ 1.77 (3H, br s, Me-16)] methyls, one oxymethine [δ 4.09 (1H, br d, J = 8.6 Hz, H-12)], and two terminal methylenes [δ 4.95/4.80, 4.89/4.50 (each 1H, br s)] (Table 2) were observed. These data showed general features similar to those of the cooccurring known C20 labdane-type diterpenoids 17–20, indicating a labd-8(17)-en-19-oic acid scaffold for 1. Differing from 17–20, the 13C NMR data of 1 (Table 1) showed only 19 carbon resonances comprising one carboxyl (δ 182.1), three methyls, eight methylenes (two olefinic at δ 109.8 and 106.7), three methines (one oxygenated at δ 73.9), and four quaternary carbons (two olefinic at δ 148.9 and 148.5). This, in accordance with the molecular formula, suggested that compound 1 is a C19 norlabdane-type diterpenoid. Detailed interpretation of its 1D and 2D (1H–1H COSY and HMBC, Fig. S1 in Supplementary material) NMR data verified the presence of an isopropenyl group [(δ 1.77 (br s, Me16), 4.95/4.80 (each br s, H2-14)] at the end of the long side chain, with C-15 being degraded in the structure of 1. In the HMBC spectrum of 1, both the vinyl methyl (Me-16) and the terminal methylene (H2-14) proton signals were found to be correlated with C-13 and C-12 (Fig. S1), allowing the location of a hydroxy group at C-12. The carboxylic acid unit was attached at C-19 as evidenced from the HMBC correlations of Me-18 with C-19, C-3, C-4, and C-5 (Fig. S1), as well as the diagnostic NOE correlations of Me-18 with H-5 and H-6α (Fig. 2). The relative configurations at C-5, C-9, and C-10 of 1 were consistent with those of 17−20 by analysis of the proton-proton coupling constants (Table 1) and NOE correlations (Fig. 2). The large magnitudes of JH-2β, 3α (12.7 Hz) and JH-5, 6β (11.8 Hz) indicated that both H-3α and H-5 were axially α-oriented. In the NOESY spectrum of 1, H-5 showed correlations with H-1α, H-3α, H-9, and Me-18, indicating the α-orientation for Me-18. As for C-12, the determination of its relative configuration is challenged due to the flexibility of the side chain. A literature survey revealed that the chemical shifts of the two olefinic protons at C-17 in 12S-isomers generally showed a smaller difference (Δ < 0.2 ppm) than that (Δ ~ 0.4 ppm) in 12R-isomers due to the deshielding effect of OH12 upon H-17b (Bell et al., 1975; Inoue et al., 1985; Fang et al., 1993; Wang et al., 2008). Such a difference (δH-17a − δH-17b = 0.39 ppm, see Table 2) of 1 suggested the configuration at C-12 to be R. The absolute configuration of 1 (4S,5R,9S,10R,12R) was finally confirmed by the GaKα X-ray crystallographic analysis (Fig. 3) with a perfect Flack parameter [0.04 (3)] (Flack and Bernardinelli, 2008). Thus, the structure of 1, pseudosinin A, was unequivocally established as (4S,5R,9S,10R,12R)-12-hydroxy-15-nor-labda-8(17),13-dien-19-oic acid. The molecular formula of pseudosinin B (2) was established to be C18H28O4 based on the HRESIMS ion at m/z 331.1877 ([M + Na]+, calcd for 331.1880). The 1H and 13C NMR data of 2 (Tables 1 and 2), with the aid of the HSQC NMR experiment, revealed signals attributed to two tertiary methyl groups [δH 1.25 (s, Me-18), 0.61 (s, Me-20); δC 28.9 (C-18), 13.0 (C-20)], an exocyclic methylene group [δH 4.57, 4.96, each br s; δC 106.2, 148.1], and a carboxyl group (δC 183.5), which were characteristics of a labda-8(17)-en-19-oic acid skeleton similar to compound 1. The 13C NMR spectrum of 2 displayed only 18 carbon resonances (Table 2). The above data implied that 2 is a dinor-diterpenoid, possessing a C18 backbone very similar to 13-oxo-14,15bisnor-labd-8(17)-en-19-oic acid (15) (Inoue et al., 1985). Differing from 15, compound 2 has an additional hydroxy group [δH 4.19 (br d, J = 11.0 Hz); δC 75.2], which was concluded to be at C-12 by the HMBC correlations from this oxymethine proton to C-9/C-11/C-13, and from Me-16 to C-12 (Fig. S1). By interpretation of the key proton-proton coupling constants (Table 2) and ROE correlations as shown in Fig. 2, the relative configurations at C-4, C-5, C-9, and C-10 in 2 were found to be the same with those of compound 1. Specially, the ROE correlations of H-5 with H-3α, H-9, and Me-18 required they all took the α-orientation. The absolute configuration of the bicyclic ring system of 2 was then determined by electronic circular dichroism (ECD) data. As

2. Results and discussion By repeated chromatographic separations employing silica gel, MCI gel, Sephadex LH-20, and semipreparative HPLC, 13 new (1–13) and 27 known (14–40) terpenoids (Fig. 1) were isolated and identified from the 90% MeOH extract of the needles and twigs of P. sinensis. The known isolates, by comparing the spectroscopic data and physicochemical properties with those reported in the literature, were identified as (4S,5R,9S,10S)-15,16-bisnor-19-hydroxy-labda-8(17),11E-dien13-one (= metaglyptin I, 14) (Tu et al., 2019), 14,15-bisnor-13-oxolabda-8(17)-en-19-oic acid (15) (Inoue et al., 1985), 14,15-bisnor-13oxolabda-8(17),11(E)-dien-19-oic acid (16) (Muhammad et al., 1996), 12R,13R-dihydroxylabda-8(17),14-dien-19-oic acid (17) (Inoue et al., 1985; Fang et al., 1993; Wang et al., 2008), 12R,13S-dihydroxylabda8(17),14-dien-19-oic acid (18) (Inoue et al., 1985; Fang et al., 1993; Wang et al., 2008), 12S,13S-dihydroxylabda-8(17),14-dien-19-oic acid (19) (Fang et al., 1993; Wang et al., 2008), cis-communic acid (20) (Shimizu et al., 1988), 12R-labda-8(17),13-dien-15,12-olid-19-oic acid (21) (Iwamoto et al., 2001), methyl agathate (22) (Carman and Marty, 1966), methyl 15-hydroxy-7-oxo-dehydroabietate (23) (Ohmoto et al., 1987), 7-oxo-dehydroabietinol (24) (Tanaka et al., 1997), 4β,15-dihydroxy-19-norabieta-8,11,13-trien-7-one (25) (Kuo and Yeh, 1998), acrostalic acid (26) (Sato and Kakisawa, 1976), 11-hydroxydrim-8(12)en-14-oic acid (27) (Gan et al., 2009), cadin-10(14)-en-4β,5α-diol (28) (Kuo et al., 2003), oplopanone (29) (Takeda et al., 1965), teucladiol (30) (Bruno et al., 1993), litseachromolaevane A (31) (Zhang et al., 2003), 7R*-opposit-4(15)-en-1β,7-diol (32) (Iijima et al., 2003), opposit-4(15)-en-1β,11-diol (33) (Matsuoka et al., 2004), 7-epi-eudesma4(15)-en-1α,6α-diol (35) (Zhang et al., 2003), eudesma-4(15)-en-1β,6αdiol (36) (Sun et al., 2004), eudesma-4(15)-en-1β,7α-diol (37) (Sun et al., 2004), eudesma-4-en-1β,15-diol (38) (Zhao et al., 2014), eudesma-6-en-lβ,4β-diol (39) (San Feliciano et al., 1989), and oplodiol (40) (Minato and Ishikawa, 1967), respectively. Compound 1 was obtained as colorless crystals (in CDCl3). It has a molecular formula C19H30O3 as determined by the HRESIMS (m/z 307.2270 [M + H]+, calcd for C19H31O3, 307.2268), corresponding to five indices of hydrogen deficiency. In the 1H NMR spectrum of 1, 2

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Fig. 1. Chemical structures of terpenoids 1–40 from Pseudotsuga sinesis.

shown in Fig. 4, its ECD curve was comparable to those of 1 and related (9S)-labda-8(17)-ene derivatives (Sun et al., 2018). They all showed a negative Cotton effect around 190 nm arising from the exocyclic methylene group. Similar to 1, the chirality of C-12 in 2 could be also designated as R as a larger difference between the chemical shifts of H-

17a and H-17b (Δ = 0.39 ppm, see Table 2) was observed. Hence, compound 2 was determined to be (4S,5R,9S,10R,12R)-12-hydroxy-13oxo-14,15-dinor-labda-8(17)-en-19-oic acid. With a protonated molecular ion at m/z 291.1955 (calcd for C18H27O3, 291.1955), pseudosinin C (3) was found to have the same 3

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Table 1 13 C NMR data (δ in ppm, 150 MHz) for compounds 1–12.a No.

1b

2b

3b

4b

5b

6b

7c

8b

9b

10b

11ab

11bb

12b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe

39.0 19.8 38.0 44.1 56.2 26.0 38.6 148.5 51.8 40.0 30.4 73.9 148.9 109.8

38.8 19.8 37.8 44.2 56.0 26.0 38.6 148.1 51.3 40.1 29.0 75.2 210.5

39.6 19.5 38.0 43.9 55.1 24.9 37.1 148.5 53.5 40.0 145.7 129.1 199.4

25.4 106.2 28.9 183.5 13.0

31.8 107.6 28.8 180.8 12.8

39.3 19.8 37.8 44.1 56.2 25.9 38.5 147.3 51.8 40.7 27.5 84.3 169.2 116.8 173.1 14.7 107.8 28.9 182.3 12.4

35.9 18.1 37.1 46.4 43.3 25.9 129.2 133.2 142.6 35.8 114.8 33.8 73.5 73.8 32.0 15.9 15.8 181.8 16.8 20.8

38.6 21.6 38.9 44.5 53.4 20.5 33.9 132.2d 140.2d 38.2 175.0d 21.2 29.1 181.3 19.2

42.2e 19.4 38.1 44.1 53.1 24.5 42.4e 211.0 58.3 39.3

18.1 106.7 29.0 182.1 12.9

38.9 18.9 35.3 38.8 56.1 24.4 38.5 148.4 52.4 39.1 26.2 75.8 75.9 140.8 114.6 24.5 106.8 27.0 65.1 12.8

151.2 126.8 130.0 128.4 130.0 126.8 35.1 52.1f 211.3 53.7 69.6 29.3 29.1 21.7 166.9

153.3 127.4 130.5 127.0 130.5 127.4 36.6 45.9 67.8 47.5 24.6 23.1 22.2 23.0 169.9

47.8 26.9 27.5 38.6 27.5 26.9 161.6 122.2 201.8 53.6 25.1 22.6 22.6 17.9 179.8

39.6 26.8 27.5 38.6 27.5 26.8 162.8 123.9 200.9 53.6 25.1 22.6 22.6 21.0 179.8

45.9 21.6 31.9 134.3 124.1 34.6 37.5 27.6 74.9 73.6 26.3 15.4 21.4 25.7 23.6

28.6 182.0 18.0

52.0f

a

Assignments were made by a combination of 1D and 2D NMR experiments. Measured in CDCl3. c Measured in CD3OD. d Assigned by HMBC and HSQC data. e-f Signals with the same superscript might be interchangeable. b

molecular formula C18H26O3 as that of 14(15)-bisnor-13-oxolabda8(17),11(E)-dien-19-oic acid (16) (Muhammad et al., 1996). Its 1H and 13 C NMR data (Tables 1 and 2) also showed high similarity to those of 16, with appreciable differences only being observable for signals attributed to the Δ11,12 double bond [δH 6.14 (dd, J = 11.6, 10.8 Hz, H11), 6.31 (d, J = 11.6 Hz, H-12); δC 145.7 (C-11), 129.1 (C-12)]. Unlike 16, the stereochemistry of the Δ11,12 double bond in 3 was determined to be Z-rather than a E-configuration in 16, according to the large coupling constant of JH-11, 12 (11.6 Hz) and a significant ROE correlation between H-11 and H-12 (Fig. 2). The relative configuration of the bicyclic ring system in compound 3 was ascertained to be the same as 16 by analyses of the proton-proton coupling constants (Table 1) and the ROESY cross-peaks of H-5/Me-18, H-5/H-9, Me-18/H-6α, and Me20/H-6β (Fig. 2). The absolute configuration of 3 was then established as 4S,5R,9S,10S based on a negative Cotton effect at 197 nm arising from the exocyclic methylene group in its ECD curve, which is similar to those of compounds 1 and 2 (Fig. 4). Accordingly, compound 3 was defined as (4S,5R,9S,10S)-14,15-dinor-13-oxolabda-8(17),11(Z)-dien19-oic acid. Pseudosinin D (4) gave an [M + Na]+ ion at m/z 345.2385 in its positive mode HRESIMS, corresponding to the molecular formula of C20H34O3 when taken in conjunction with 13C NMR data (Table 1). From the 1H and 13C NMR spectroscopic data of 4 (Tables 1 and 2), signals assignable for three tertiary methyls [δH 1.34 (s, Me-16), 0.99 (s, Me-18), 0.65 (s, Me-20); δC 24.5, 27.0, 12.8], an oxymethine [δH 3.47 (br d, J = 10.8 Hz, H-12); δC 75.8], an exocyclic methylene group [δH 4.84, 4.45 (br s, H2-17); δC 148.4 (C-8), 106.8 (C-17)], and a terminal double bond [δH 5.95 (dd, J = 17.4, 10.8 Hz, H-14), 5.35 (br d, J = 17.4 Hz, H-15a), 5.23 (br d, J = 10.8 Hz, H-15b); δC 140.8 (C-14), 114.6 (C-15)] were readily distinguished. The aforementioned NMR data were similar to those of the known labdane-type diterpenoid 17, 12(R),13(R)-dihydroxylabda-8(17),14-dien-19-oic acid (Inoue et al., 1985), but differed in the absence of a carboxyl group at C-4. Instead, a resonance for an oxygenated methylene carbon at δC 65.1 with corresponding protons at δH 3.76 and 3.40 (ABq, J = 11.6 Hz) appeared, indicating a hydroxymethylene moiety attached to C-4. This group was

Table 2 1 H NMR data (δ in ppm, J in Hz, in CDCl3) for compounds 1–4.a No.

1

2

3

4

1α

1.10, m

1.80, m 1.44, m 1.82, m 1.11, ddd (13.6, 13.6, 4.1) 2.17, br d (13.6) 1.45, br d (10.1)

1.73, br d (12.9) 1.52, m 1.52, m 0.99, ddd, overlapped 1.84, br d (11.1) 1.35, br d (11.6)

6α 6β

2.00, m 1.88, m

1.15, ddd (13.0, 12.8, 3.1) 1.69, br d (13.0) 1.52, br d (14.8) 1.83, m 1.05, ddd (13.1, 12.8, 2.4) 2.15, br d (13.1) 1.43, dd (12.0, 4.2) 2.02, br d (11.6) 1.90, m

1.09, m

3β 5

1.16, ddd (13.0, 12.8, 4.0) 1.83, br d (13.0) 1.54, m 1.85, m 1.08, ddd (13.0, 12.7, 3.9) 2.17, br d (12.7) 1.42, br d (11.8)

1.85, br d (11.1) 1.35, m

7α 7β

1.99, br dd (13.0, 12.6) 2.44, br d (13.3)

1.99, br d (13.6) 1.88, ddd (13.6, 13.6, 4.4) 2.12, m

2.40, br d (12.1)

9α 11a

2.05, br d (10.5) 1.63, m

11b 12 14a

1.60, m 4.09, br d (8.6) 4.95, br s

2.44, ddd (13.2, 3.3, 3.2) 3.87, d (10.8) 6.14, dd (11.6, 10.8)

14b 15a 15b 16 17a 17b 18 19a 19b 20

4.80, br s

1β 2α 2β 3α

1.77, 4.89, 4.50, 1.26,

br s br s br s s

0.61, s

2.01, br dd (12.8, 11.6) 2.44, dd (12.8, 4.3) 2.17, br d (10.8) 1.85, m 1.41, m 4.19, br d (11.0)

2.26, 4.96, 4.57, 1.25,

s br s br s s

0.61, s

6.31, d (11.6)

2.21, 4.75, 4.41, 1.27,

s br s br s s

0.77, s

2.01, m

2.03, br d (11.6) 1.62, m 1.38, m 3.47, br d (10.8) 5.95, dd (17.4, 10.8) 5.35, 5.23, 1.34, 4.84, 4.45, 0.99, 3.76, 3.40, 0.65,

br d (17.4) br d (10.7) s br s br s s d (11.6) d (11.6) s

a

Recorded at 400 MHz, and assignments were made by a combination of 1D and 2D NMR experiments.

4

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Fig. 2. Observed key NOE or ROE correlations for indicated compounds.

Fig. 3. ORTEP drawings of compounds 1 and 17. 5

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(d, J = 7.4 Hz, Me-17), and 1.99 (m, H-15)], two tertiary methyl groups [δ 1.27 (s, Me-19), 1.02 (s, Me-20)], an oxymethine proton (δ 4.00, s, H14), and two olefinic protons [δ 5.87 (br d, J = 4.4 Hz, H-7), 5.41 (t, J = 2.8 Hz, H-11)] (Table 3). In its 13C NMR spectrum, resonance at δ 181.8 was attributable to a carboxylic acid moiety, whereas the resonances at δ 129.2, 133.2, 142.6, and 114.8 were ascribed to two double bonds (Table 1), which are supposed to construct a transoid Δ7,9(11)-diene system as evidenced by the UV absorption maximum at 246 nm (Herz and Wahlborg, 1965). These findings suggested that 6 is an abietic-type diterpenoid similar to Δ7,9(11)-abietadienoic acid (Herz and Wahlborg, 1965), except for the presence of a vicinal diol unit at C13 and C-14. This was supported by the HMBC correlations from Me-16 and Me-17 to C-13 (δ 73.5), from the oxymethine proton (H-14) to C-7, C-8, C-9, C-12, C-13, and C-15, and from H-7 to C-14 (δ 73.8) (Fig. S1). The relative configuration of 6 was assigned by interpretation of proton-proton coupling constants (Table 3) and ROESY spectrum (Fig. 2, recorded in C5D5N). The large coupling constant JH-5,6β (12.8 Hz) implied that both H-5 and H-6β adopted axial positions. The ROE correlations of Me-20 (δ 1.09) with Me-19 (δ 1.44) and H-6β (δ 2.62) suggested their β orientations, while the carboxyl group being αoriented. As for ring C, the observed ROE cross-peaks between Me-16 (δ 1.22) and the two protons at C-12 (δ 2.73/2.60) (Fig. 2) revealed that the isopropyl group is positioned equatorially on the cyclohexene ring, requiring 13-OH to be α-orientated. Further ROE correlations of H-14 (δ 4.46) with H-7 (δ 5.76) and Me-17 (δ 1.35) indicated the equatorial αorientation of H-14, otherwise H-14 would have strong ROE correlations with H2-12 (δ 2.73/2.60). This deduction was confirmed by quantum chemical calculations of the 13C NMR data of the four diastereoisomers at C-13 and C-14. As a result, (13R*,14S*)-6 was found to be the privileged structure with a DP4+ probability of 76.21% (for details see Supplementary material) (Grimblat et al., 2015). Finally, the absolute configuration (4R,5R,10S,13R,14S) of 6 was determined by the ECD computation method, from which the Boltzmann averaged ECD spectrum was well overlaid with the experimental one (Fig. 5). The structure of compound 6 was thus defined as (4R,5R,10S,13R,14S)13,14-dihydroxy-abieta-7,9(11)-dien-18-oic acid. Pseudosinin G (7) has a molecular formula C15H22O4 as deduced by the negative mode HRESIMS ion at m/z 265.1448 [M − H]− (calcd for C15H21O4, 265.1445). The fifteen well-resolved carbon resonances in the 13C NMR spectrum of 7, with the aid of HSQC NMR experiment, were attributable to two carboxyl carbons [δ 181.3 (C-14), 175.0 (C11)], three methyls, five methylenes, one methine, and four quaternary carbons (two olefinic at δ 140.2 and 132.2) (Table 1). The 1H NMR data (Table 3) of 7 suggested the presence of two tertiary methyls [δ 1.23 (s, Me-13), 1.12 (s, Me-15)] and one vinyl methyl (δ 1.67, br s, Me-12). These data were comparable to those of arecoic acid F, a known drimane-type sesquiterpenoid previously isolated from the fermented broth of Arecophila saccharicola YMJ96022401 (Lee et al., 2013). Comparison of the NMR data of 7 with those of arecoic acid F implied that the Δ7(8) double bond in the known structure was relocated at Δ8(9) in 7. The transfer of the double bond was deduced from the disappearance of olefinic proton signal and the HMBC correlations from Me-12 to C-7, C-8, C-9, and C-11, and from Me-15 to C-9 (Fig. S1). As depicted in Fig. 2, H-1α, H-2β, H-3α, H-5, and H-6β were all axially oriented as judged from the large coupling constants of H-1α/H-2β (12.0 Hz), H-2β/H-3α (13.0 Hz), and H-5α/H-6β (10.8 Hz). The crosspeaks of Me-15/H-2β, Me-15/H-6β, Me-13/H-5, and Me-13/H-6α in the NOSEY spectrum of 7 revealed that the decalin ring was trans-fused with the carboxyl group in the β orientation. Moreover, the ECD spectrum of 7 showed a positive Cotton effect at 236 nm due to the π→ π* transition of the α,β-unsaturated carboxyl group, indicative of an S configuration for C-10 (Weiss and Ziffer, 1963). Accordingly, 7 was identified as (4S,5R,10S)-drima-8-en-11,14-dioic acid. Pseudosinin H (8) was identified to be a dinor-drimane-type sesquiterpenoid based on its HRESIMS, 1D (Tables 1 and 3) and 2D (Fig. S1) NMR data. Its molecular formula, NMR spectroscopic data, and the

Fig. 4. Experimental ECD spectra of compounds 1–4 and 14 in MeCN.

located at C-19 based on the pronounced ROE correlations of Me-20 with H2-19, and of H-5 with Me-18 and H-9. As discussed for compounds 1 and 2, the chirality of C-12 was determined to be R as evidenced from the relatively larger difference (δH-17a − δH17b = 0.39 ppm for 4, Table 2) between the chemical shifts of the two olefinic protons at C-17. As for the configuration of C-13, appreciable difference could be observed for the proton resonances of Me-16 between the C-13 epimers [(13R)-17: δ 1.34; (13S)-18: δ 1.28; (13S)-19: δ 1.29] (Fang et al., 1993; Wang et al., 2008). Since the signal of Me-16 in 4 was observed at δ 1.34, the chirality at C-13 of 4 was thus supposed to be R. The (12R,13R) configuration of 4 was further confirmed by the fact that its 1H and 13C NMR data attributed to the side chain (from C11 to C-16) were fully congruent with those of 17. The absolute configuration (4S,5R,9S,10R,12R,13R) of 17 was unequivocally established by a single crystal X-ray diffraction analysis [Flack parameter: 0.04 (7)] in this study (Fig. 3). In addition, the absolute configuration of the trans-decalin core of 4 was the same as that in compounds 1–3 based on the observation of a similar negative Cotton effect at 192 nm in its ECD curve (Fig. 4). Therefore, compound 4 was defined as (4S,5R,9S,10R,12R,13R)-12,13-dihydroxylabda-8(17),14-dien-19-oic acid. Pseudosinin E (5) was found to possess the molecular formula C20H28O4 based on the HREISMS ion at m/z 333.2062 [M+H]+. The 1 H and 13C NMR spectroscopic data (Tables 1 and 3) of 5 resembled those of 12R-labda-8(17),13-dien-15,12-olid-19-oic acid (21) (Iwamoto et al., 2001). Only slight differences could be observed for H-12 [5: δ 4.83 (dd, J = 5.5, 5.1 Hz), but 21: δ 4.87 (br d, J = 11.0 Hz)] and for the exocyclic methylene proton H-17b (5: δ 4.68, but 21: δ 4.44), indicating these two compounds are the C-12 epimeric isomers. This inference was confirmed by the change in sign of the Cotton effect around 215 nm [(12S)-5: Δε 215 (+1.8) nm; (12R)-21: Δε 214 (−2.6) nm] aroused by the π−π* transition of the unsaturated lactone ring. (4S,5R,9S,10R,12S)-Labda-8(17),13-dien-15,12-olid-19-oic acid (5) was reported herein as a naturally occurring labdane-type diterpenoid for the first time. The asymmetric semi-synthesis of this compound had been previously accomplished, with the absolute configuration being well determined (Mack and Njardarson, 2013). The NMR data of 5 together with its ESI-MS data and the optical rotation value (see Experimental section) were all identical to those of the synthetic compound (Mack and Njardarson, 2013). Pseudosinin F (6) has a molecular formula of C20H30O4 as determined from the HRESIMS ion at m/z 357.2039 [M + Na]+ (calcd for C20H30O4Na, 357.2036). The 1H NMR spectrum displayed characteristic signals for an isopropyl group [δ 0.97 (d, J = 7.4 Hz, Me-16), 1.00 6

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Table 3 1 H NMR data (δ in ppm, J in Hz) for compounds 5–8.a No.

5b

1α 1β 2α 2β 3α 3β 5 6α 6β 7α 7β 9α 9β 11a 11b 12 13 14 15a 15b 16 17a 17b 18 19 20

1.15, 1.78, 1.54, 1.86, 1.08, 2.18, 1.38, 2.01, 1.89, 1.93, 2.45, 1.84,

a b c

6b ddd (13.3, 12.7, 2.3) br d (13.3) m m ddd (13.7, 12.8, 2.8) br d (13.7) br d (11.1) m m m br d (10.2) m

1.51, 1.87, 1.69, 1.69, 1.71, 1.77, 2.13, 2.00, 2.20, 5.87,

7c m br d (11.9) m m m br d (12.5) br d (12.8) ddd, overlapped ddd (13.0, 12.8, 4.0) br d (4.4)

1.94, m 1.88, m 4.84, dd (5.5, 5.1)

5.41, t (2.8)

5.77, br s

4.00, s 1.99, m

2.09, 4.94, 4.68, 1.26,

0.97, d (7.4) 1.00, d (7.4)

s br s br s s

0.62, s

2.32, d (2H, 2.8)

1.36, 1.63, 1.48, 1.90, 1.05, 2.18, 1.34, 2.02, 1.96, 2.08, 2.08,

8b ddd (12.3, 12.0, 4.0) br d (12.3) br d (14.0) m ddd (14.0, 13.0, 3.8) br d (14.0) br d (10.8) m br dd (13.4, 13.0) m m

1.30, 1.53, 1.53, 1.90, 1.15, 2.24, 1.67, 2.28, 2.25, 2.28, 2.44. 2.05, 2.17,

ddd, overlapped br d (13.4) br d (13.1) q-like (13.1) br dd (13.4, 13.2) m br d (9.3) m m m brd (10.9) d (13.9) d (13.9)

1.67, br s 1.23, s

1.33, s

1.12, s

0.82, s

1.27, s 1.02, s

Assignments were made by a combination of 1D and 2D NMR experiments. Measured in CDCl3 at 400 MHz. Measured in CD3OD at 400 MHz.

(δ 3.91, s) (Table 4). In addition, two pairs of ortho-coupled olefinic protons (δ 7.97 and 7.28, each 2H, br d, J = 8.0 Hz) characteristic for a para-substituted benzene ring were observed. In addition to a methoxy carbon (δ 52.0), 15 resonances typical of a sesquiterpenoid skeleton were acquired in the 13C NMR spectrum of 9. These included a ketocarbonyl (δ 211.3, C-9), a conjugated ester carbonyl (δ 166.9, C-15), a para-substituted benzene ring (δ 151.2, 130.0 × 2, 126.8 × 2, 128.4), and an oxygenated tertiary carbon (δ 69.6, C-11) (Table 1). The above data implied compound 9 to be monocyclic, featuring a phenolic bisabolane sesquiterpenoid similar to (+)-(S)-ar-turmerone previously isolated from the rhizome of Curcuma longa (Huang et al., 2018). Differing from (+)-(S)-ar-turmerone, compound 9 not only has a methyl carboxylate substitute at C-4 rather than a methyl group, but also has a hydroxy group at C-11 along with the Δ10 double bond being hydrogenated. The planar structure of 9 was further ascertained by the HMBC experiment (Fig. S1), which demonstrated the correlations from Me-12 and Me-13 to C-10/C-11, from Me-14 to C-1/C-7/C-8, from H2-8 and H2-10 to C-9, and from the methoxy protons to C-15. The chirality of C7 was assigned to be S as evidenced from the positive specific rotation and a positive Cotton effect at 246 nm in its ECD spectrum, which are similar to those of (+)-(S)-ar-turmerone and relevant derivatives (Fujiwara et al., 2010; Golding and Pombo-Villar, 1992; Huang et al., 2018). Hence, compound 9 was established as methyl (S)-9-oxo-11hydroxy-bisabola-1,3,5-trien-15-oate [= (S)-4-(6-hydroxy-6-methyl-4oxoheptan-2-yl)benzoate]. The negative mode HRESIMS of pseudosinin J (10) gave a deprotonated ion at m/z 249.1497 [M − H]− (calcd for C15H21O3, 249.1496) in accordance with the molecular formula C15H22O3. Its NMR data were comparable to those of compound 9, with major differences occurring in the side chain. As evidenced from the NMR data (Tables 1 and 4, Fig. S1), C-9 was substituted with a hydroxy group in 10, along with the absence of the signals for the ketone at C-9 and tertiary hydroxy at C11. In addition, signals arising from the methoxy group were also absent, requiring a free carboxylic acid (δC 169.9) at C-4. The attempt to obtain the ECD data of 10 has failed. Nevertheless, based on a biogenetic relationship between 9 and 10, the chirality of C-7 could be

Fig. 5. Experimental and calculated ECD spectra of 6 in MeOH.

specific rotation were all identical to those of a synthetic compound, (1S,4aR)-perhydro-1,4a-dimethyl-6-oxonaphthalene-1- carboxylic acid (Vial et al., 1989). Compound 8 was isolated as a natural product for the first time. A diagnostic negative Cotton effect at 289 nm due to the n−π* transition of the C-8 carbonyl group was observed in the ECD spectrum of 8, from which the absolute configuration of C-10 could be readily established as R by application of the cyclohexanone octant rule (Moffitt et al., 1961). Accordingly, compound 8 was established as (4S,5R,10R)-8-oxo-11,12-dinor-drima-14-oic acid. The molecular formula of pseudosinin I (9) was determined as C16H22O4 by a sodium adduct ion at m/z 301.1415 [M + Na]+ (calcd for 301.1410) in its HRESIMS spectrum, corresponding to six degrees of unsaturation. The 1H NMR spectrum of 9 showed signals of one secondary (δ 1.28, d, J = 6.8 Hz, Me-14) and two tertiary [δ 1.20 (s, Me12), 1.16 (s, Me-13)] methyl groups, along with a methoxy substituent 7

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Table 4 1 H NMR data (δ in ppm, J in Hz, in CDCl3) for compounds 9–12. No.

9b

10b

1 2

7.28, br d (8.0)

7.33, br d (8.0)

3

7.97, br d (8.0)

8.04, br d (8.0)

4 5

7.97, br d (8.0)

8.04, br d (8.0)

6

7.28, br d (8.0)

7.33, br d (8.0)

7 8a 8b 9 10a 10b 11 12 13 14 15 OMe

3.39, m 2.78, dd (17.1, 6.6) 2.68, dd (17.1, 7.6)

3.12, 1.74, 1.67, 3.36, 1.35, 1.19, 1.72, 0.81, 0.80, 1.29,

a b c

2.56, d (17.4) 2.48, d (17.4) 1.20, s 1.16, s 1.28, d (6.8)

m m m m m m m d (6.5) d (6.5) d (6.9)

a

11ac

11bc

12c

1.99, dddd (11.6, 11.4, 3.8, 3.1) αH: 1.66, br d (12.7) βH: 1.48, ddd (14.1, 12.7, 3.1) βH: 2.25, dd (13.9, 2.2) αH: 1.59, dddd (13.2, 12.8, 4.0, 3.5) 2.75, br s βH: 2.25, dd (13.9, 2.2) αH: 1.59, dddd (13.2, 12.8, 4.0, 3.5) αH: 1.66, br d (12.7) βH: 1.48, ddd (14.1, 12.7, 3.1)

3.60, penta (7.5) 1.52, m 1.52, m βH: 2.23, br d (13.6) αH: 1.52, m 2.75, br s βH: 2.23, br d (13.6) αH: 1.52, m 1.52, m 1.52, m

1.60, dd, overlapped βH: 1.68, m αH: 1.88, m 1.95, m, 2H

6.03, s

5.98, s

2.30, br d (2H, 6.9)

2.27, br d (2H, 6.9)

2.13, 0.92, 0.92, 2.10,

2.13, 0.92, 0.92, 1.79,

m d (6.5) d (6.5) d (1.0)

3.91, s

m d (6.5) d (6.5) d (1.0)

5.55, d (4.1) 2.31, ddd (10.8, 5.3, 4.3) 1.63, m βH: 1.79, ddd (13.9, 13.4, 2.4) αH: 1.51, ddd (13.9, 3.0, 2.8) 3.69, br s 2.07, 0.87, 0.92, 1.37, 1.67,

m d (6.9) d (6.9) s br s

Assignments were made by a combination of 1D and 2D NMR experiments. Recorded at 400 MHz. Recorded at 600 MHz.

the HRESIMS ion at m/z 261.1824 [M + Na]+ (calcd for C15H26O2Na, 261.1825). The 1H NMR data (Table 4) of 12 indicated the presence of an olefinic proton (δ 5.55, d, J = 4.1 Hz, H-5), an oxymethine proton (δ 3.69, br s, H-9), a vinyl methyl (δ 1.67, br s, Me-15), a tertiary methyl (δ 1.37, s, Me-14), and an isopropyl group [δ 0.87 (d, J = 6.9 Hz, Me-12), 0.92 (d, J = 6.9 Hz, Me-13), and 2.07 (m, H-11)]. Fifteen carbon signals (Table 1) were shown in the 13C NMR spectrum of 12, containing two olefinic [δ 134.3 (qC, C-4), 124.1 (CH, C-5)] and two oxygenated [δ 74.9 (CH, C-9), 73.6 (qC, C-10)] ones. The above data were comparable to those of 8α-hydroxy-T-muurolol, a T-muurolol-type sesquiterpenoid previously isolated from Chimonanthus salicifolius (Li et al., 2016). Differing from this known compound with an 8-OH group, the hydroxy group in 12 was relocated at C-9, which was established by 2D NMR experiments (Fig. S1). In particular, clear HMBC correlations from Me14 to C-1, C-9, and C-10 were observed for 12. Similar to the Tmuurolol sesquiterpenoids (Li et al., 2016), compound 12 also possesses a cis-fused bicyclic ring. The olefinic proton H-5 showed a significant coupling (J = 4.1 Hz) with its vicinal proton H-6, whereas in the transfused one, the olefinic proton appeared as a broad singlet (He et al., 1997; Li et al., 2016). This was further confirmed by the intense crosspeak between H-1 and H-6 in its ROESY spectrum (Fig. 2). The large magnitudes of coupling constants of H-6/H-7α (10.8 Hz), and H-7α/H8β (13.4 Hz), along with the ROE correlations of Me-12/H-5, Me-12/H6, and H-6/H-8β, indicated that H-6 and H-8β in 12 were axially βoriented while the isopropyl group was equatorial. The 9-OH group was deduced to adopt the axial α-position since H-9 showed small couplings (J values near zero) with the two neighboring protons at C-8. In addition, Me-14 was found to have a ROE correlation with H-2β but not with H-1, H-6 or H-8β, indicative of the equatorial α-configuration for Me14. Accordingly, the structure of 12 was characterized as 9α-hydroxy-Tmuurolol. Its absolute configuration was assigned as (1S,6R,7S,9R,10R) since its specific rotation ([α]D20 −68, MeOH) was comparable to that ([α]D20 −81, CHCl3) of 8α-hydroxy-T-muurolol, for which the absolute configuration was well established by single-crystal X-ray (Cu Ka) diffraction analysis (Li et al., 2016). The cadinane-type sesquiterpenoid, pseudosinin M (13), was found to have the same molecular formula (C15H24O2) and NMR spectroscopic data with those of 15-oxo-T-cadinol, a synthesized compound derived from T-cadinol (Wu et al., 2005). Correspondingly, the relative

deduced to be S. The stereochemistry at C-9 in the flexible chain of 10 is unascertained. Therefore, compound 10 was characterized as (7S)-9ξhydroxy-bisabola-1,3,5-trien-15-oic acid [ = 4-((2S)-4ξ-hydroxy-6-methylheptan-2-yl)benzoic acid]. Pseudosinin K (11) was obtained as an inseparable mixture of 11a and 11b, which could be separated by HPLC but quickly interconverted at room temperature to afford an equilibrium mixture in a ratio of 2:1 (11a:11b). This mixture showed a protonated molecular ion at m/z 253.1796 [M + H]+ (calcd for C15H25O3, 253.1798) in its HRESIMS. Taking the 13C NMR data (Table 1) into consideration, both 11a and 11b were found to have the same molecular formula of C15H24O3. The 1 H and 13C NMR data of 11a/11b (Tables 1 and 4) were similar to those of cis-dihydrotodomatuic acid, a known bisabolane-type sesquiterpenoid previously isolated from Douglas-fir (i.e., Pseudotsuga menziesii) (Rogers et al., 1974), except for the presence of a double bond situated between C-7 and C-8. This was evidenced from the absorption band at 1654 cm−1 (typical for a conjugated carbonyl group) in the IR spectrum of 11, and the key HMBC correlations from Me-14 to C-1, C-7, C-8, and C-9 (Fig. S1). Similar to cis-dihydrotodomatuic acid, the carboxyl group in both 11a and 11b occupied the axial orientation as implied by the slight magnitudes of the vicinal couplings (near to zero) observed for H4 (br s). Both 11a and 11b probably have a 1,4-cis configured cyclohexane ring with the bulky side chain taking the equatorial position. This inference was confirmed by the large magnitudes of the J values (11.6, 11.4 Hz) between H-1 and its vicinal axial protons in 11a. As for 11b, H-1 appeared as a quintet (J = 7.5 Hz). Considering that 11a and 11b could immediately interconvert at room temperature, the configuration at C-1 in these two isomers should be the same due to the large steric hindrance. Instead, the isomers possess different orientations across the Δ7 double bond, which was confirmed by NOESY data (Fig. 2 and Supplementary material). The significant NOE cross-peak between H-1 and H-8 demonstrated the trans-configuration in 11a (Fig. 2), while in 11b, the corresponding correlation was absent. Consistent with this, the H-1 signal in 11b shifted dramatically downfield to δ 3.60 (δ 1.99 in 11a) due to the deshielding effect by the C-8 carbonyl group in the cisconfiguration. Thus, the structures of 11a and 11b were established as 9-oxo-bisabola-7(E)-en-15-oic acid and 9-oxo-bisabola-7(Z)-en-15-oic acid, respectively. Pseudosinin L (12) has a molecular formula C15H26O2 according to 8

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configuration of 13 was consistent with that of 15-oxo-T-cadinol by analysis of the proton-proton coupling constants (see Experimental section) and the ROESY data (Fig. 2). Compound 13 is reported herein as a natural product for the first time. The assignments of the 13C chemical shifts of C-8 (δ 20.0, CH2), C-11 (δ 26.5, CH), and C-14 (δ 28.6, CH3) (Table 1) have been carefully revised in this work based on detailed analyses of the HSQC and HMBC data of 13 (Fig. S1 and Supplementary material). In addition, compound 34 was identified as eudesma-4(15)-en1α,6α-diol by interpretation of its 1D and 2D NMR spectroscopic data (see Experimental section and Supplementary material). This structure, with the trivial name polydactin B, has been previously reported (Zhang et al., 2008). However, 34 differed from polydactin B in the chemical shifts of Me-14 [δ 0.76 (s) for 34; 0.70 (s) for polydactin B] and particularly in the proton-proton coupling constants between H-1 and H2-2 [H-1: δ 3.39 (br s) for 34, 3.41 (dd, J = 11.5, 5.0 Hz) for polydactin B] (see Table S5 in Supplementary material). In compound 34, the H-1 signal appearing as a broad singlet clearly demonstrated its equatorial β-orientation. In the 13C NMR spectrum, significant differences were also noticeable for the chemical shifts of C-1 and C-14 (δ 74.6, 17.9 for 34; 78.9, 11.5 for polydactin B, respectively). In fact, the 1H and 13C NMR data of polydactin B were exactly the same as those of eudesma4(15)-en-1β,6α-diol (36) (Table S5 in Supplementary material), for which the structure with the relative configuration was unambiguously established by an X-ray crystallography study in 2004 (Sun et al., 2004). Thus, the structure of polydactin B (Zhang et al., 2008) should be revised as eudesma-4(15)-en-1β,6α-diol (36), and the 1H and 13C NMR data for eudesma-4(15)-en-1α,6α-diol (34) are accurately assigned herein. In the present study, an array of nor-labdane diterpenoids featuring C19 (1), C18 (2, 3, 14–16), and C16 (26) skeletons, along with structurally related drimane sesquiterpenoids with C15 (7, 27) or C13 (8) skeletons, were isolated and characterized. The putative biogenetic pathways towards these degraded labdane-derived diterpenoids and

sesquiterpnoids were herein proposed as shown in Scheme 1. Briefly, a series of oxidation reactions of (+)-copalyl PP (Dewick, 2009) would afford agathate (its methyl ester of C-19 was isolated, 22), which could suffer decarboxylization at C-15 followed by oxidation at C-12 to yield the 15-norlabdane-type diterpenoid 1 (C19 skeleton). The normal C20 labdane diterpenoid, cis-communic acid (20), was isolated as a major component in P. sinensis (5.21 g, 0.017% yield) and was thus considered as the biosynthetic precursor for the other related diterpenoids and sesquiterpenoids. The 14,15-dinorlabdane-type diterpenoid 2 (C18 skeleton) may be generated from 17, a dihydroxylated derivative of ciscommunic acid, by involving an oxidative cleavage between C-13 and C-14, with the loss of an ethylene moiety (Lin and Rosazza, 1998). Following dehydration of 2 could yield the isomers 3 and 16, the latter being reduced to account for the formation of 14 and 15. The oxidative cleavage between C-12 and C-13 in cis-communic acid (20) could furnish 26 (C16 skeleton) with the loss of 4 carbons (Kakisawa et al., 1973; Sato et al., 1974; Sato and Kakisawa, 1976); The drimane-type sesquiterpenoids 7 and 27 (C15 skeleton), would be tracked back to the dinorlabdenes 3 or 16 (C18 skeleton) via a further oxidative cleavage between C-11 and C-12. Alternatively, they could be also aroused from 26 (C16 skeleton) by a cascade of decarboxylization and oxidation reactions (Frija et al., 2011). The dinorsesqutierpenoid 8 (C13 skeleton) would be then constructed by the oxidative cleavage of the exocyclic double bond in the drimane-type sesquiterpenoid followed by a decarboxylization at C-11. Actually, the drimane-type sesquiterpenoids are usually recognized to be directly originated from the farnesyl pyrophosphate (FPP) via cyclization (Jansen and de Groot, 2004). However, in the present study, based on the structural similarity and the possible biogenetic relationships (as shown in Scheme 1) between drimanes and labdanes, the drimane-type sesquiterpenoids 7 and 27 could also be regarded as nor-labdanes with the loss of five carbons. Consistent with this, 14,15-bisnorlabdane-13-ones (e. g., 3, 16) were reported to be extremely suitable starting materials for the preparations of drimanes when Norrish II type photochemical degradation could be

Scheme 1. Putative biogenetic relationships of labdane-derived diterpenoids and sesquiterpenoids. 9

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realized (Vlad et al., 2006). Interestingly, as the drimane-type sesquiterpenoids possess the decalin core just like many diterpenoids, they have been considered as the missing biogenetic linkage between the lower and the higher terpenoids (Jansen and de Groot, 2004; Toyota et al., 1994). This study would stimulate further biogenetic considerations on this type of sesquiterpenoid. In our ongoing research program on chemical constituents from the rare and endangered conifers endemic to China, great attention has been paid to evaluating the potential of the isolates in the treatment of metabolic disorders associated with the abnormal glucose and lipid metabolism, such as type 2 diabetes mellitus (T2DM) (Hu et al., 2016a, 2016b, 2017) and hypercholesterolemia (Jiang et al., 2019). The adenosine triphosphate (ATP)-citrate lyase (ACL), mainly expressed in lipogenic tissues such as liver and adipose, has been considered to serve as a critical enzyme linking glucose catabolism to lipogenesis by providing acetyl-CoA from mitochondrial citrate for both fatty acid and cholesterol biosynthesis (Pinkosky et al., 2017; Verschueren et al., 2019; Wellen et al., 2009). ACL inhibitors were reported to suppress the synthesis of fatty acids and cholesterol and decrease plasma lipids, and also have positive results in human clinical trials as LDL cholesterol (LDL-C) lowering drugs (Burker and Huff, 2017). ACL has been therefore considered as a potential drug target for the treatment of T2DM, hyperlipidemia, and hypercholesterolemia (Pinkosky et al., 2017). In this study, all the isolates were evaluated for their ACL inhibitory effects. Among them, diterpenoids 4, 20, and 25 were found to show moderate activities, with IC50 values of 59.25 ± 8.82, 11.06 ± 0.33, and 52.78 ± 7.12 μM, respectively. The rest were judged inactive (IC50 > 100 μM). The known inhibitor BMS 303141 (Koerner et al., 2017) was used as the positive control (IC50: 0.37 ± 0.03 μM). In addition, the isolates were also tested for their inhibitory activities against nuclear factor kappa B (NF-κB), a potential target for the regulation of the dysfunction of immunity and inflammation (Kim et al., 2005; Pikarsky et al., 2004). As a result, only compound 24 displayed inhibitory effect with an IC50 value of 18.38 ± 1.95 μM. PS-341 (Sunwoo et al., 2001) was used as the positive control (IC50: 0.06 ± 0.01 μM).

4. Experimental 4.1. General experimental procedures Optical rotations were measured on a Rudolf Autopol IV automatic polarimeter. UV absorptions and IR spectra were obtained respectively on a Hitachi U-2900E and an Avatar 360 ESP FTIR spectrometers. ECD spectra were recorded on a JASCO-810 spectropolarimeter. NMR spectra were recorded on a Bruker Avance III 400 MHz or 600 MHz spectrometer. Chemical shifts are expressed in δ (ppm) and referenced to the residual solvent signals. HRESIMS were recorded on an AB SCIEX Triple TOF 5600 spectrometer. X-ray data were collected on a Bruker D8 Venture diffractometer. Semipreparative HPLC was performed on a Shimadzu LH-20AT system with a SPD-M20A prominence diode array (PDA) detector and an ODS column (Waters XBridge: 250 × 10 mm, 5 μm; flow rate: 3.0 mL/min). Column chromatography (CC) was performed using silica gel (100–200 or 200–300 mesh, Qingdao Marine Chemical Co. Ltd., China), MCI gel CHP20P (75–150 μm, Mitsubishi Chemical Industries, Tokyo, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Silica gel precoated plates (GF254, 0.25 mm, Qingdao Marine Chemical Co. Ltd., China) were used for TLC detection. Spots were visualized using UV light (254 and/or 365 nm) and by spraying with 10% (v/v) H2SO4/EtOH reagent. 4.2. Plant material The needles and twigs of Pseudotsuga sinensis Dode (Pinaceae) were collected from Jinfo Mountains (29°02′N, 107°10′E), Chongqing, PR China, in September 2014 (wet season). The plant was identified by Prof. Si-Rong Yi (Chongqing Institute of Medicinal Plant Cultivation). A voucher specimen (No. 20140901) was deposited at the Herbarium of the Department of Natural Products Chemistry, School of Pharmacy at Fudan University. 4.3. Extraction and isolation Air-dried and powdered needles and twigs of P. sinensis (30.0 kg) were extracted with 90% MeOH (4 × 30 L) at room temperature. After evaporation in vacuo, the crude residue (3.1 kg) was suspended in H2O (6 L) and partitioned successfully with petroleum ether (PE, 3 × 6 L), EtOAc (3 × 6 L), and n-BuOH (3 × 6 L). The EtOAc fraction (500 g) was subjected to silica gel CC (100–200 mesh) with PE-EtOAC gradients (50:1 → 20:1 → 10:1 → 7:1 → 5:1 → 2:1 → 1:1 → 1:2 → 1:5→neat EtOAC, each 36.0 L) to afford ten fractions (Fr.1–Fr.10) as monitored by TLC. Compound 20 (5.21 g) was recrystallized from Fr. 2. Fr. 6 (18.1 g) was chromatographed over an MCI gel column eluted with gradients of MeOH in H2O (30%→50%→60%→ 80%→100% MeOH, each 3.6 L) to obtain eight sub-fractions (Fr.6aFr.6h). Fr.6f (450 mg) was then subjected to silica gel CC (200–300 mesh) eluted with a gradient of PE/acetone (30:1 → 20:1 → 10:1, each 0.6 L) and further purified by semi-preparative HPLC (MeOH–H2O 65:35) to afford compounds 15 (1.5 mg, tR = 24.7 min) and 31 (3.6 mg, tR = 18.4 min). Fr. 7 (26.9 g) was fractionated into eight sub-fractions (Fr.7a–Fr.7h) by an MCI gel column with MeOH/H2O (30%→50%→ 60%→80%→90%→100% MeOH, each 5.4 L). Fr. 7d was repeatedly purified by Sephadex LH-20 (MeOH, 1.0 L) followed by semi-preparative HPLC to yield compounds 8 (1.7 mg, tR = 12.0 min), 10 (0.7 mg, tR = 20.0 min), 26 (2.1 mg, tR = 19.2 min), 27 (1.5 mg, tR = 14.0 min), and 36 (2.5 mg, tR = 19.4 min) using a mobile phase consisting of MeOH–H2O (60:40), and to afford compounds 17 (10.2 mg, tR = 12.0 min) and 40 (2.3 mg, tR = 18.1 min) with a mobile phase of 60% MeCN in H2O. Fr. 7e was chromatographed repeatedly over silica gel (PE-acetone, 30:1 → 20:1 → 10:1 → 5:1, each 1.1 L) and Sephadex LH-20 (MeOH, 0.5 L), with final purifications on semi-preparative HPLC (MeCN–H2O 40:60) to furnish 2 (6.7 mg, tR=13.0 min), 12 (0.8 mg, tR = 23.9 min), 18 (10.1 mg, tR = 12.0 min), 19 (2.0 mg,

3. Concluding remarks Over the past few years, natural products chemists in China have made great achievements (Yang et al., 2018). These include a special program of systematically identifying structurally diverse bioactive/ novel natural products from the precious and fragile rare and endangered plants (Xiong et al., 2018). In particular, the rare and endangered conifers have attracted great attentions due to their high drug-productive potency and relatively easier access of plant materials. The work reported herein is the Part XV in a series of phytochemical and biological studies on REPs endemic to China (Part I - Part XIII, see Xiong et al., 2018; for Part XIV, see Jiang et al., 2019). In this study, 19 diterpenoids (1–6, 14–26) and 21 sesquiterpenoids (7–13, 27–40) with diverse structures were obtained from the needles and twigs of Pseudotsuga sinensis, a vulnerable Pinaceae species in China and has been nationally protected at the second-grade. Differing from our previous findings on the rare and endangered conifer species (Hu et al., 2016a, 2016b; 2017, 2018; Li et al., 2017), the title plant was especially abundant in labdane-derived norditerpenoids (C19-, C18-, and C16-skeletons) and sesquiterpenoids (C15- and C13-skeletons). Their possible biogenetic relationships were briefly discussed. Our findings would enlighten further systematic investigations on the biogenetic relationships between the lower and the higher terpenoids. Moreover, some isolated compounds exhibited moderate inhibitory effects against ACL and NF-κB, which could provide new clues for the treatment of ACL and NF-κB related diseases.

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tR = 13.5 min), 28 (2.4 mg, tR = 23.8 min), 29 (7.1 mg, tR = 14.1 min), 32 (0.9 mg, tR = 17.6 min), 34 (1.0 mg, tR = 18.9 min), 35 (2.3 mg, tR = 18.5 min), and 38 (1.0 mg, tR = 14.7 min). Fr. 7f was subjected to Sephadex LH-20 (MeOH, 0.8 L) and further purified by semi-preparative HPLC with a mobile phase of MeCN–H2O (43:57) to give 9 (0.6 mg, tR = 12.5 min), 14 (0.7 mg, tR = 23.2 min), 16 (6.4 mg, tR = 22.5 min), 23 (11.9 mg, tR = 25.5 min), and 30 (4.3 mg, tR = 30.2 min). Fr. 8 was subjected to an MCI gel column eluted with a gradient of MeOH–H2O (50%→60%→70%→80%→100% MeOH, each 4.5 L) to give seven sub-fractions (Fr.8a–Fr.8g). From Fr. 8e (899 mg), compounds 5 (4.5 mg, tR = 29.2 min) and 21 (5.4 mg, tR = 32.2 min) were isolated by CC over Sephadex LH-20 (MeOH, 0.8 L) followed by semi-preparative HPLC (MeOH–H2O 60:40). Fr. 9 (33.2 g) was fractioned into eleven sub-fractions (Fr.9a–Fr.9k) by CC over MCI gel with a gradient of MeOH–H2O (50%→60%→70%→80%→100% MeOH, each 6.0 L). Fr.9f was further purified repeatedly by silica gel CC (PE/ acetone, 30:1 → 20:1 → 10:1 → 5:1 → 3:1 → 1:1, each 0.6 L), Sephadex LH-20 (MeOH, 0.5 L), and semi-preparative HPLC, furnishing 4 (0.8 mg, tR = 12.3 min, MeCN–H2O, 35:65), 6 (0.8 mg, tR = 17.0 min, MeCN–H2O 38:62), 25 (1.1 mg, tR = 12.9 min, MeOH–H2O 68:32), 33 (0.9 mg, tR = 21.0 min, MeCN–H2O, 35:65), 37 (2.3 mg, tR = 15.7 min, MeCN–H2O, 35:65), and 39 (2.4 mg, tR = 23.3 min, MeCN–H2O, 35:65). In a similar way, compounds 1 (1.5 mg, tR = 27.4 min, MeCN–H2O 42:58), 3 (0.8 mg, tR = 19.1 min, MeOH–H2O 65:35), 7 (2.1 mg, tR = 13.7 min, MeOH–H2O 58:42), 13 (1.8 mg, tR = 25.5 min, MeCN–H2O 42:58), and a mixture of 11a and 11b (in a ratio of 2:1, 2.5 mg, tR = 21.8 min, MeOH–H2O 65:35) were isolated from Fr. 9h. The PE fraction (43.5 g) was subjected to silica gel CC (100–200 mesh) with PE/EtOAC gradients (50:1 → 20:1 → 10:1 → 7:1 → 5:1 → 2:1 → 1:1→neat EtOAC, each 12.0 L) to afford ten fractions (Fr. P1-Fr. P10). By using the LC-MS-based detection and dereplication approach, only Fr. P8 (723 mg) was found to contain terpenoids different from the above isolated compounds and thus subjected to further separations. Finally, compounds 22 (2.7 mg, tR = 17.4 min) and 24 (3.1 mg, tR = 12.1 min) were obtained from this fraction by repeated chromatography over silica gel CC (PE/acetone, 50:1 → 30:1 → 20:1 → 10:1 → 5:1 → 1:1, each 0.8 L) and semi-preparative HPLC (MeOH–H2O 80:20). Trifluoroacetic acid (TFA) was added in water with concentration 0.05% (v/v) as modifier in all HPLC purifications.

for C20H34O3Na, 345.2400, Δ = −4.3 ppm).

4.3.1. Pseudosinin A (1) Colorless crystals (from CDCl3); [α]D20 +44 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.02) nm; ECD (c 4.25 × 10−3 M, CH3CN) λmax (Δε) 200 (−2.2) nm; 1H and 13C NMR data, see Tables 1 and 2; (+) ESIMS m/z 307.2 [M+H]+; (+) HRESIMS m/z 307.2270 [M +H]+ (calcd for C19H31O3, 307.2268, Δ = 0.6 ppm).

4.3.9. Pseudosinin I (9) Colorless, amorphous power; [α]D20 +13 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.22), 245 (3.74) nm; ECD (c 3.60 × 10−3 M, CH3CN) λmax (Δε) 246 (+1.2), 232(−0.2), 209 (+0.2) nm; 1H and 13C NMR data, see Tables 1 and 4; (+) ESIMS m/z 301.1 [M+Na]+; (+) HRESIMS m/z 301.1415 [M+Na]+ (calcd for C16H22O4Na, 301.1410, Δ = 1.4 ppm).

4.3.5. Pseudosinin E (5) White, amorphous power; [α]D20 +75 (c 0.2, CHCl3) [ref: [α]D23 +33 (c 0.08, CHCl3)] (Mack and Njardarson, 2013); UV (MeOH) λmax (log ε) 203 (4.33) nm; ECD (c 6.02 × 10−3 M, CH3CN) λmax (Δε) 215 (+1.8) nm; 1H and 13C NMR data, see Tables 1 and 3; (+) HRESIMS m/ z 333.2062 [M+H]+ (calcd for C20H29O4, 333.2060, Δ = 0.4 ppm). 4.3.6. Pseudosinin F (6) White, amorphous power; [α]D20 -28 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 245 (4.65) nm; ECD (c 2.40 × 10−3 M, MeOH) λmax (Δε) 234 (−2.4) nm; 1H and 13C NMR data in CDCl3, see Tables 1 and 3; 1H NMR (C5D5N, 600 MHz): δ 5.76 (1H, br s, H-7), 5.50 (1H, br s, H-11), 4.46 (1H, s, H-14), 2.73 (1H, br d, J = 17.8 Hz, H-12a), 2.62 (1H, m, H6β), 2.60 (1H, m, H-12b), 2.52 (1H, sept., J = 6.7 Hz, H-15), 2.27 (1H, m, H-6α), 2.23 (1H, dd, overlapped, H-5), 2.01 (1H, dd, J = 13.1, 12.9, 4.0 Hz, H-3α), 1.84 (1H, br d, J = 12.8 Hz, H-1β), 1.78 (1H, br d, J = 13.1 Hz, H-3β), 1.64 (1H, q-like, J = 12.4 Hz, H-2β), 1.53 (1H, m, H-2α), 1.44 (1H, dd, overlapped, H-1α), 1.44 (3H, s, Me-19), 1.35 (3H, d, J = 6.7 Hz, Me-17), 1.22 (3H, d, J = 6.7 Hz, Me-16), 1.09 (3H, s, Me20); (+) ESIMS m/z 357.2 [M+Na]+; (+) HRESIMS m/z 357.2039 [M +Na]+ (calcd for C20H30O4Na, 357.2036, Δ = 0.8 ppm). 4.3.7. Pseudosinin G (7) White, amorphous power; [α]D20 +60 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 203 (3.62) nm; ECD (c 3.01 × 10−3 M, CH3CN) λmax (Δε) 207 (−0.5), 236 (+1.1) nm; 1H and 13C NMR data, see Tables 1 and 3; (−) ESIMS m/z 265.1 [M−H]−; (−) HRESIMS m/z 265.1448 [M−H]− (calcd for C15H21O4, 265.1445, Δ = 1.0 ppm). 4.3.8. Pseudosinin H (8) Yellow oil; [α]D20 −12 (c 0.1, MeOH) [ref: [α]D20 −14.7 (c 2.07, MeOH)] (Vial et al., 1989); UV (MeOH) λmax (log ε) 203 (4.10) nm; ECD (c 3.57 × 10−3 M, MeOH) λmax(Δε) 289 (−2.7) nm; 1H and 13C NMR data, see Tables 1 and 3; (+) ESIMS m/z 247.1 [M+Na]+; (+) HRESIMS m/z 247.1302 [M+Na]+ (calcd for C13H20O3Na, 247.1305, Δ = −1.2 ppm).

4.3.2. Pseudosinin B (2) White, amorphous power; [α]D20 +90 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 207 (4.55) nm; ECD (c 6.49 × 10−3 M, CH3CN) λmax (Δε) 276 (−0.5), 190 (−1.2) nm; 1H and 13C NMR data, see Tables 1 and 2; (+) ESIMS m/z 331.2 [M+Na]+; (+) HRESIMS m/z 331.1877 [M +Na]+ (calcd for C18H28O4Na, 331.1880, Δ = −0.7 ppm).

4.3.10. Pseudosinin J (10) Colorless, amorphous power; [α]D20 −12 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 233 (3.92), 203 (3.92) nm; 1H and 13C NMR data, see Tables 1 and 4; (−) ESIMS m/z 249.1 [M−H]−; (−) HRESIMS m/z 249.1497 [M−H]− (calcd for C15H21O3, 249.1496, Δ = 0.5 ppm).

4.3.3. Pseudosinin C (3) White, amorphous power; [α]D20 −4 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 203 (3.41), 225 (3.31) nm; ECD (c 2.41 × 10−3 M, CH3CN) λmax (Δε) 229 (+1.1) nm, 197 (−5.0) nm; 1H and 13C NMR data, see Tables 1 and 2; (+) ESIMS m/z 291.1 [M+H]+; (+) HRESIMS m/z 291.1955 [M+H]+ (calcd for C18H27O3, 291.1955, Δ = 0 ppm).

4.3.11. Pseudosinin K (an inseparable mixture of 11a and 11b) Yellow oil; [α]D20 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 241 (3.80) nm; 1H and 13C NMR data, see Tables 1 and 4; (+) ESIMS m/z 253.2 [M+H]+; (+) HRESIMS m/z 253.1796 [M+H]+ (calcd for C15H25O3, 253.1798, Δ = −0.9 ppm).

4.3.4. Pseudosinin D (4) White, amorphous power; [α]D20 +14 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 203 (3.23) nm; ECD (c 2.48 × 10−3 M, CH3CN) λmax (Δε) 192 (−3.0) nm; 1H and 13C NMR data, see Tables 1 and 2; (+) ESIMS m/z 345.2 [M+Na]+; (+) HRESIMS m/z 345.2385 [M+Na]+, (calcd

4.3.12. Pseudosinin L (12) Colorless, amorphous power; [α]D20 −68 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 203 (3.45) nm; 1H and 13C NMR data, see Tables 1 and 4; (+) ESIMS m/z 261.2 [M+Na]+; (+) HRESIMS m/z 261.1824 [M+Na]+ (calcd for C15H26O2Na, 261.1825, Δ = −0.3 ppm). 11

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4.3.13. Pseudosinin M (13) Yellow oil; [α]D20 −3 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 230 (3.21) nm; 1H NMR (C5D5N, 600 MHz): δ 9.65 (1H, s, H-15), 6.98 (1H, br s, H-5), 2.66 (1H, dd, J = 11.0, 11.0 Hz, H-6), 2.60 (1H, dd, J = 17.8, 4.6 Hz, H-3β), 2.19 (1H, m, H-11), 2.15 (1H, m, H-2β), 2.10 (1H, br d, J = 12.1 Hz, H-1), 1.93 (1H, br d, J = 12.6 Hz, H-9β), 1.81 (1H, ddd, J = 13.1, 13.1, 13.1, 5.4 Hz, H-8β), 1.65 (1H, dd, J = 12.5, 12.1, 5.4 Hz, H-3α), 1.50 (1H, br d, J = 12.6 Hz, H-8α), 1.43 (1H, dd, J = 13.1, 13.1, 4.4 Hz, H-9α), 1.36 (3H, s, H3-14), 1.16 (1H, m, H-2α), 0.94 (3H, d, J = 6.8 Hz, H3-12), 0.83 (3H, d, J = 6.8 Hz, H3-13); 1H NMR (CDCl3, 400 MHz): δ 9.44 (1H, s, H-15), 6.95 (1H, br s, H-5), 2.47 (1H, br d, J = 16.4 Hz, H-3β), 2.36 (1H, m, H-6), 2.26 (1H, m, H-11), 2.06 (2H, m, H-2β, H-3α), 1.78 (1H, m, H-9a), 1.56 (1H, m, H-8a), 1.49 (1H, m, H-8b), 1.47 (1H, m, H-9b), 1.34 (1H, m, H-2α), 1.27 (3H, s, H314), 1.21 (1H, m, H-7), 0.99 (3H, d, J = 6.6 Hz, H3-12), 0.88 (3H, d, J = 6.6 Hz, H3-13); 13C NMR (CDCl3, 150 MHz): δ 194.6 (C-15), 152.8 (C-5), 141.2 (C-4), 70.7 (C-10), 47.8 (C-1), 45.6 (C-7), 40.1 (C-9), 39.4 (C-6), 28.6 (C-14), 26.5 (C-11), 22.0 (C-3), 21.3 (C-2), 21.3 (C-12), 20.0 (C-8), 15.2 (C-13); (+) ESIMS m/z 259.2 [M+Na]+; (+) HRESIMS m/z (calcd for C15H24O2Na, 259.1669, 259.1667 [M+Na]+ Δ = −0.5 ppm).

MeOH, M = 336.45, orthorhombic, space group P212121, a = 9.7174(2) Å, b = 10.2420(2) Å, c = 18.5477(3) Å, α = β = γ = 90°, V = 1845.97(6) Å3, Z = 4, Dcalcd = 1.211 Mg/m3, μ (Ga Kα) = 0.423 mm−1, crystal size 0.15 × 0.08 × 0.06 mm3, F (000) = 736, 16,727 reflections collected, 3459 independent reflections (Rint = 0.0416), R1 = 0.0325 [I > 2σ(I)], wR2 = 0.0889 [I > 2σ(I)], R1 = 0.0335 (all data), wR2 = 0.0889 (all data), Goodness of fit = 1.067, Flack parameter = 0.04(7). The crystal structures of compound 1 and 17 were solved with the SHELXT. Refinements were performed with SHELXL-2015 using fullmatrix least-squares calculations on F2, with anisotropic displacement parameters for all the non-hydrogen atoms. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. CCDC 1902831 (1) and CCDC 1902834 (17) contain the supplementary crystallographic data, which can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam. ac.uk. 4.5. ECD calculation of compound 6 Confab was used to generate conformers. All the generated conformers were optimized by MOPAC2016 with PM7 semiempirical method. The conformers with 4 kcal/mol higher energy than lowest conformer were subjected to further optimization and frequency calculations on B3PW91-D3/TZVP level of theory with IEFPCM solvent model (MeOH). The TDDFT calculation of each conformer was performed on mPW1PW91/6-311G(d) level of theory with IEFPCM solvent model (MeOH). All the DFT calculations were performed by Guassian09 software. ECD spectra were simulated by SpecDis v1.71 with bandwidth σ of 0.3 eV (Bruhn et al., 2013). The contribution of each conformer to the final ECD spectrum was Boltzmann weighted according to their Gibbs free energy.

4.3.14. 12R,13R-dihydroxylabda-8(17),14-dien-19-oic acid (17) Colorless crystals (MeOH); [α]D18 +40.0 (c 0.16, MeOH); 1H NMR (400 MHz, CDCl3): δ 5.94 (1H, dd, J = 16.9, 11.2 Hz, H-14), 5.35 (1H, d, J = 16.9 Hz, H-15a), 5.24 (1H, d, J = 11.2 Hz, H-15b), 4.87 (1H, br s, H-17a), 4.44 (1H, br s, H-17b), 3.51 (1H, d, J = 10.6 Hz, H-12), 2.41 (1H, br d, J = 11.3 Hz, H-7β), 2.15 (1H, br d, J = 13.0 Hz, H-3β), 2.01 (1H, br d, J = 11.5 Hz, H-9), 1.73–1.98 (5H, m, H-2β, 6α, 6β, 7α, 11a), 1.61 (1H, m, H-1β), 1.52 (1H, br d, J = 13.7 Hz, H-2α), 1.41 (1H, br d, J = 11.4 Hz, H-5), 1.40 (1H, m, H-11b), 1.34 (3H, s, Me-16), 1.25 (3H, s, Me-18), 1.10 (1H, dd, J = 13.3, 12.7 Hz, H-1α), 1.07 (1H, dd, J = 13.8, 13.0 Hz, H-3α), 0.59 (3H, s, Me-20); 13C NMR (150 MHz, CDCl3): δ 182.4 (C-19), 148.3 (C-8), 140.8 (C-14), 114.6 (C-15), 106.7 (C-17), 75.9 (C-13), 75.8 (C-12), 56.2 (C-5), 51.6 (C-9), 44.1 (C-4), 40.1 (C-10), 39.0 (C-1), 38.6 (C-7), 37.9 (C-3), 29.0 (C-18), 26.3 (C-11), 26.0 (C-6), 24.5 (C-16), 19.8 (C-2), 12.9 (C-20); (+) ESIMS m/z 359 [M +Na]+, (−) ESIMS m/z 335 [M−H]−.

4.6. ACL inhibitory activity assay The ADP-Glo™ Kinase Assay (Promega, Madison, WI) was performed to assess the activity of ACL by quantification of the amount of ADP generated by the enzymatic reaction (Koerner et al., 2017). The kinase assay was carried out in a 384-well plate (ProxiPlateTM-384 Plus, PerkinElmer) in a volume of 5 μL reaction mixture containing 2.0 μL of ACL, 2.0 μL of ATP, and 1.0 μL of test compounds with different concentrations. Suitable negative control wells without the protein kinase, substrate and ATP were also included in the kinase assay. Reactions in each well were kept going for 30 min under 37 °C. After the enzymatic reaction, 2.5 μL of ADP-Glo™ reagent was added to each well to terminate the kinase reaction and deplete the unconsumed ATP within 60 min at room temperature. In the end, 5.0 μL of kinase detection reagent was added to each well and incubated for 1 h to simultaneously convert ADP to ATP and allow the newly synthesized ATP to be measured using a luciferase/luciferin reaction. The luminescent signal generated, which is proportional to the ADP concentration produced and is correlated with the kinase activity, was measured using an EnVision multilabel plate reader (PerkinElmer, MA, USA) and. The data presented here are the mean ± standard error (SE) of n ≥ 3 of two independent experiments. Statistical analyses were performed using GraphPad Prism 5 statistical software program (GraphPad Software, CA, USA). The known inhibitor BMS 303141 (Koerner et al., 2017) was used as the positive control.

4.3.15. Eudesma-4(15)-en-1α,6α-diol (34) Yellow oil; [α]D20 +40.0 (c 0.05, CH3OH). 1H NMR (400 MHz, CDCl3): δ 0.76 (3H, s, Me-14), 0.87 (3H, d, J = 6.9 Hz, Me-12), 0.96 (3H, d, J = 6.9 Hz, Me-13), 1.21 (1H, br d, J = 13.3 Hz, H-9β), 1.24 (1H, br d, J = 12.4 Hz, H-8α), 1.30 (1H, dd, J = 12.4, 10.4, 2.6 Hz, H8β), 1.33 (1H, dd, J = 13.0, 12.7, 5.0 Hz, H-3α), 1.54 (1H, m, H-7), 1.73 (1H, m, H-2α), 1.84 (1H, dd, J = 13.3, 13.0, 2.8 Hz, H-9α), 1.93 (1H, dd, J = 13.3, 13.0 Hz, H-2β), 2.17 (1H, dd, J = 12.7, 3.4 Hz, H-3β), 2.26 (1H, sept, J = 7.1 Hz, H-11), 2.35 (1H, br d, J = 11.1 Hz, H-5), 3.39 (1H, br s, H-1), 3.71 (1H, dd, J = 10.1, 10.0 Hz, H-6), 4.73 (1H, br s, H-15a), 5.02 (1H, br s, H-15b); 13C NMR (150 MHz, CDCl3): δ 74.6 (C1), 31.1 (C-2), 31.9 (C-3), 147.8 (C-4), 50.6 (C-5), 67.4 (C-6), 49.5 (C7), 18.1 (C-8), 33.1 (C-9), 42.0 (C-10), 25.9 (C-11), 16.1 (C-12), 21.1 (C-13), 17.9 (C-14), 106.9 (C-15); (+) ESIMS m/z 261 [M+Na]+. 4.4. X-ray crystallographic data of compounds 1 and 17 X-ray crystal data of 1: C19H30O3·1/4 CH2Cl2, colorless crystals obtained from CDCl3, M = 327.66, tetragonal, space group I4, a = 19.8988(5) Å, b = 19.8988(5) Å, c = 9.5476(2) Å, α = β = γ = 90°, V = 3780.5(2) Å3, Z = 8, Dcalcd = 1.151 Mg/m3, μ (Ga Kα) = 0.795 mm−1, crystal size 0.15 × 0.1 × 0.08 mm3, F (000) = 1428, 10,495 reflections collected, 3567 independent reflections (Rint = 0.0443), R1 = 0.0799 [I > 2σ (I)], wR2 = 0.2188 [I > 2σ (I)], R1 = 0.1005 (all data), wR2 = 0.2406 (all data), Goodness of fit = 1.047, Flack parameter = 0.04(3). X-ray crystal data of 17: C20H34O4, colorless crystals obtained from

4.7. NF-κB inhibitory activity assay HEK293 with stable NF-κB expression cell line was used for the luciferase assay (Peng et al., 2013). Cells were seeded into 96-well plates and incubated for 24 h, and then treated with different concentrations of compounds followed by stimulation with 20 ng/mL TNFα (50 μL). The luciferase substrate was added to each well after 12

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incubation for 6 h, then the released luciferin signal was detected using an EnVision microplate reader. IC50 values were derived from a nonlinear regression model (curve-fit) based on a sigmoidal dose-response curve (variable slope) and computed using Graphpad Prism 5 (Graphpad Software). PS-341 (Sunwoo et al., 2001) was used as the positive control.

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Declaration of competing interest The authors confirm that this article content has no conflict of interest. Acknowledgements This work was supported by NSFC grants (No. 81773599, 21937002, 21772025) and a MOST grant (2019ZX09735002-005). The authors are grateful to the distinguished botanist Prof. Si-Rong Yi (Chongqing Institute of Medicinal Plant Cultivation, Chongqing, China) for his carefully collection of the plant material. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112184. References Barton, G.M., 1967. A new C-methyl flavanone from diseased (Poria weirii Murr.) Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) roots. Can. J. Chem. 45, 1020–1022. Barton, G.M., 1972. New C-methylflavanones from Douglas-fir. Phytochemistry 11, 426–429. Bell, R.A., Gravestock, M.B., Taguchi, V.Y., 1975. Synthesis of methyl 12S- and 12Rhydroxylabd-8(17)-en-19-oates. Can. J. Chem. 53, 2869–2873. Bruhn, T., Schaumloffel, A., Hemberger, Y., Bringmann, G., 2013. SpecDis: quantifying the comparison of calculated and experimental electronic circular dichroism spectra. Chirality 25, 243–249. Bruno, M., de la Torre, M.C., Rodríguez, B., Omar, A.A., 1993. Guaiane sesquiterpenes from Teucrium leucocladum. Phytochemistry 34, 245–247. Burker, A.C., Huff, M.W., 2017. ATP-citrate lyase: genetics, molecular biology and therapeutic target for dyslipidemia. Curr. Opin. Lipidol. 28, 193–200. Carman, R., Marty, R., 1966. Diterpenoids. IX. Agathis microstachya oleoresin. Aust. J. Chem. 19, 2403–2406. Dellus, V., Mila, I., Scalbert, A., Menard, C., Michon, V., Herve du Penhoat, C.L.M., 1997. Douglas-fir polyphenols and heartwood formation. Phytochemistry 45, 1573–1578. Dewick, P.M., 2009. Medicinal Natural Products: A Biosynthetic Approach, third ed. John Wiley & Sons, Chippenham, pp. 228–230. Erdtman, H., Kimland, B., Norin, T., Daniels, P.J.L., 1968. The constituents of the "pocket resin" from Douglas fir Pseudotsuga menziesii (Mirb.) Franco. Acta Chem. Scand. 22, 938–942. Fang, J.-M., Sou, Y.-C., Chiu, Y.-H., Cheng, Y.-S., 1993. Diterpenes from the bark of Juniperus chinensis. Phytochemistry 34, 1581–1584. Flack, H.D., Bernardinelli, G., 2008. The use of X-ray crystallography to determine absolute configuration. Chirality 20, 681–690. Frija, L.M.T., Frade, R.F.M., Afonso, C.A.M., 2011. Isolation, chemical, and biotransformation routes of labdane-type diterpenes. Chem. Rev. 111, 4418–4452. Fu, L.-G., Jin, J.-M., 1992. China Plant Red Data Book: Rare and Endangered Plants. Science Press, Beijing and New York. Fu, L.-K., Li, N., Mill, R.R., 1999. In: In: Wu, Z.-Y., Raven, P.H. (Eds.), Flora of China, vol. 4. Science Press (Beijing) & Missouri Botanical Garden Press, St. Louis, pp. 11–52. Fujiwara, M., Yagi, N., Miyazawa, M., 2010. Acetylcholinesterase inhibitory activity of volatile oil from Peltophorum dasyrachis Kurz ex Bakar (Yellow Batai) and bisabolanetype sesquiterpenoids. J. Agric. Food Chem. 58, 2824–2829. Gan, X., Ma, L., Chen, Q., Chen, Q., Yu, Q., Hu, L., 2009. Terpenoids from roots of Chloranthus henryi. Planta Med. 75, 1344–1348. Grimblat, N., Zanardi, M.M., Sarotti, A.M., 2015. Beyond DP4: an improved probability for the stereochemical assignment of isomeric compounds using quantum chemical calculations of NMR shifts. J. Org. Chem. 80, 12526–12534. Golding, B.T., Pombo-Villar, E., 1992. Structures of α- and β-turmerone. J. Chem. Soc. Perkin Trans. I 12, 1519–1524. He, K., Zeng, L., Shi, G., Zhao, G.-X., Kozlowski, J.F., McLaughlin, J.L., 1997. Bioactive compounds from Taiwania cryptomerioides. J. Nat. Prod. 60, 38–40. Herz, W., Wahlborg, H.J., 1965. Resin acids. III. 9-hydroxyabietic acid and its transformation products. J. Org. Chem. 30, 1881–1886. Hu, C.-L., Xiong, J., Gao, L.-X., Li, J., Zeng, H., Zou, Y., Hu, J.-F., 2016a. Diterpenoids from the shed trunk barks of the endangered plant Pinus dabeshanensis and their PTP1B inhibitory effects. RSC Adv. 6, 60467–60478. Hu, C.-L., Xiong, J., Li, J.-Y., Gao, L.-X., Wang, W.-X., Cheng, K.-J., Yang, G.-X., Li, J., Hu,

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