Neo-clerodane diterpenoids from the whole plants of Scutellaria formosana

Phytochemistry 145 (2018) 1e9

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Neo-clerodane diterpenoids from the whole plants of Scutellaria formosana Xin Chen a, 1, Wenhao Chen a, 1, Guangying Chen a, Changri Han a, b, *, Johnny J. He a, Xueming Zhou a, Zhangxin Yu a, Chunyan Dai a, Xiaoping Song a, ** a

Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158, China Key Laboratory of Medicinal and Edible Plants Resources of Hainan Province, School of Chemical and Material Engineering, Hainan Institute of Science and Technology, Haikou 571126, China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 May 2017 Received in revised form 5 September 2017 Accepted 7 September 2017

Scuteformoids A-J, ten previously undescribed neo-clerodane diterpenoids along with one known analogue, were isolated from petroleum ether soluable fraction of the whole plants of Scutellaria formosana. The differences among these compounds are the substituents and stereochemistry at C-13. Their structures were elucidated by 1D and 2D NMR experiments, and the absolute configurations of Scuteformoids A, C, E, F, and I were further confirmed by single-crystal X-ray diffraction. Scuteformoids A, C, D, F, H, and I were evaluated for their inhibitory effects against HIV lytic replication and cytotoxic activities. All of them showed weak anti-HIV activities, with EC50 values ranging from 48.24 to 79.17 mg/mL. © 2017 Published by Elsevier Ltd.

Keywords: Scutellaria formosana Labiatae Neo-clerodane diterpenoids HIV lytic replication

1. Introduction Scutellaria is an unique cosmopolitan genus of the subfamily Scutellarioideae belonging to the Lamiaceae (Labiatae) family. Approximately 360 species are found spread throughout the world, including Europe, North America, and East Asia (Bruno et al., 2002; Paton, 1990). However the majority grow in Asia. The use of species of Scutellaria in Chinese popular medicine has a long tradition. They are still used for the treatment of several human diseases, which include respiratory and gastrointestinal bacterial infections (Tang and Eisenbrand, 1992). The genus Scutellaria is rich in flavonoids and diterpenoids, mainly neo-clerodane diterpenoids, with a variety of biological activities, such as anti-feedant (Raccuglia et al., 2010), anti-oxidant (Nguyen et al., 2009), cytotoxic (Kurimoto et al., 2015), anti-cancer (Wang et al., 2012), anti-influenzavirus FM1 (Gang et al., 2011), anti-EBV (Epstein-Barr virus) (Wu et al., 2015), and inhibition of nitric oxide production activities (Yeon et al., 2015). In the

* Corresponding author. Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158, China. ** Corresponding author. E-mail addresses: [email protected] (C. Han), [email protected] (X. Song). 1 Joint first author. https://doi.org/10.1016/j.phytochem.2017.09.002 0031-9422/© 2017 Published by Elsevier Ltd.

continuation of search for new neo-clerodanes from Scutellaria plants (Bozov et al., 1993; Bruno et al., 1993; de la Torre et al., 1992; Rodriguez et al., 1993; Rodriguez-Hahn et al., 1994; Wu et al., 2015; Yeon et al., 2015), we investigated Scutellaria formosana growing in southern China (Cui et al., 2010). In this paper, the isolation and structural elucidation of 10 previously undescribed neo-clerodane diterpenoids, scuteformoids A-J (1e10), and a known analogue, hastifolin A (11) were reported. In addition, compounds 1, 3, 4, 6, 8, and 9 were evaluated for their inhibitory effects against HIV lytic replication and cytotoxic activities. 2. Results and discussion An 85% EtOH extract of the whole plants of Scutellaria formosana was suspended in water and then partitioned successively with petroleum ether and EtOAc. As a result, 10 previously undescribed compounds (1e10) and a known compound, hastifolin A (11) (Fig. 1), were obtained from the petroleum ether soluable fraction. Their structures were elucidated on the basis of spectroscopic data interpretation, especially using 2D NMR (COSY, HSQC, HMBC, and NOESY) methods. The configurations of compounds 1, 3, 5, 6, and 9 were confirmed by single-crystal X-ray diffraction (Fig. 2). Compound 1 was obtained as a white crystal. Its molecular formula was established as C31H38O10 by its HRESIMS of the [M þ

2

X. Chen et al. / Phytochemistry 145 (2018) 1e9

Fig. 1. Structures of compounds 1e11.

Fig. 2. The Key 1H1H COSY (▬), HMBC (/) and NOESY (4) correlations of compound 1.

Na]þ peak at m/z 593.2366. The IR spectrum showed absorption signals for epoxide group (3001 cm1), g-lactone group (1785 cm1), ester carbonyl (1714 cm1), phenyl (1597 cm1), and acetoxyl (1248 cm1) functionalities. The 1H NMR spectrum of 1 also revealed the presence of a benzoyloxy moiety [dH 7.92 (2H, d, J ¼ 7.2 Hz, H-30 and H-70 ), 7.57 (1H, t, J ¼ 7.2 Hz, H-50 ), 7.44 (2H, t, J ¼ 7.2 Hz, H-40 and H-60 )], and acetoxyl groups [dH 1.98, s, (3H  2)]. The 13C NMR data of 1 revealed the presence of two acetoxyls (dC 170.1, 21.7 and 170.4, 21.4) and a benzoyloxy [dC 165.4 (PhCOO); 129.0 (C-20 ); 129.8 (C-30 and C-70 ); 128.8 (C-40 and C-60 ) and 133.8 (C-50 )] group. In addition to the acetoxyl and benzoyloxy groups, the 1H and 13C NMR spectra of 1 showed 20 carbon signals including three tertiary methyl groups [dH 0.96 (s, H3-20), 1.31 (s, H3-17), 1.37 (s, H3-19); dC 20.9 (C-20), 24.4 (C-17), 14.9 (C-19)], an epoxide methylene proton [dH 3.18 (br s, Ha -18), 2.46 (d, J ¼ 4.0 Hz, Hb-18); dC 51.7 (C-18), and 65.5 (C-4)], an oxygen bearing methylene group [dH 4.42 (d, J ¼ 9.6 Hz, Ha-16), 4.32 (d, J ¼ 9.6 Hz, Hb-16); dC

78.5 (C-16)], an isolated methylene group [dH 2.78 (d, J ¼ 16.8 Hz, Ha-14), 3.07 (d, J ¼ 16.8 Hz, Hb-14); 38.3 (C-14)], and two oxygenbearing quaternary carbons [dC 78.4 (C-13) and 80.7 (C-8)], which are characteristic signals for a neo-clerodane diterpenoid with a 4a,18; 8b,13-diepoxy-15,16-g-lactone moiety (Raccuglia et al., 2010). The locations of the substituents of the acetoxyls and benzoyloxy in 1 were established from the key HMBC correlations from H-1 (dH 5.36) to C-100 (dC 170.1), H-6 (dH 5.06) to C-1000 (dC 170.4), and H-12 (dH 5.60) to C-10 (dC 165.4). Furthermore, the HMBC correlations of H-12 with C-13 and C-16, H-14 with C-13, C-15, and C-16, and H-16 with C-15 supported the proposed 13-spiro-15,16-glactone skeleton of 1 (Fig. 2). The NOESY experiment (Fig. S8, Supporting Information) of 1 showed clear correlation peaks between Me-17 with H-14, indicating 13R* stereochemistry, and also between H-6 and H-18a, which confirmed, as in hastifolin B (Raccuglia et al., 2010), a 4R configuration. NOESY correlations were also observed between H-1 and Me-19, and between H2-7 and Me-

X. Chen et al. / Phytochemistry 145 (2018) 1e9

17 supporting the suggested a-orientation while the NOESY correlations between H-6 and H-10 and H-12 suggested b-orientation of the H at C-6, 10, 12 (Fig. 2). The above data and those reported in the literature for other pairs of C-13 stereochemistry (Hussein et al., 1998; Kizu et al., 1997; Raccuglia et al., 2010) allowed us to assign 1 a C-13 stereochemistry structure (13R*)-1b,6a-diacetoxy-12a-benzoyloxy-4a,18; 8b,13-diepoxy-neo-clerodan-15,16-olide. According to data reported in the literature, the absolute configurations of the neo-clerodane diterpenoid compounds were determined mainly by CD spectroscopy (Kurimoto et al., 2015; Nguyen et al., 2009; Wu et al., 2015). However, the absolute configurations of compound 1 could not be determined by this method due to the lack of a conjugated system (Fig. S81, Supporting Information). Luckly, a single crystal of 1 was cultured from a solution of MeOH by slow evaporation at room temperature. Hence, a single-crystal X-ray diffraction analysis was conducted using Cu Ka radiation, and the absolute configuration of 1 (Fig. 3) was determined as (1R,4R,5S,6S,8S,9R,10R,12S,13R)-1,6-diacetoxy-12-benzoyloxy-4,18; 8,13-diepoxy-neo-clerodan-15,16-olide by Flack's method with Flack's parameter determined as 0.01 (2) (Flank, 1983). The trivial name of compound 1 was assigned as scuteformoid A. The molecular formula of compound 2 was assigned as C31H38O8 by HRESIMS (m/z 561.3427, [M þ Na]þ). Its IR absorption features and 1H and 13C NMR spectra (Table 2) were similar to those of hastifolin B (Raccuglia et al., 2010), except for signals representative of an additional acetoxy group [dH 1.96, s (3H); dC 171.2; 20.9]. The acetoxy group was located at C-7 from a HMBC correlation (Fig. S13, Supporting Information) of H-7 [dH 5.28 (d, J ¼ 10.4 Hz)] with the carbonyl carbon C-1000. In the NOESY spectrum of 2 (Fig. S14, Supporting Information), the correlations of H-7 with Me-17 (dH 1.22, s), Me-19 (dH 1.45) and Me-20 (dH 0.98) suggested that these groups were cofacial, and they were arbitrarily assigned as a-oriented, whereas correlations of H-6 with H-10, and H2-18 suggested that these protons are b-orientation. NOESY correlations were also observed between H2-12 and H2-16 which supported the 13R configuration as 1. Thus, the structure of compound 2 was tentatively elucidated to be (4R,5S,6S,7R,8S,9R,10R,13R)-7-acetoxy-4,18; 8,13-diepoxy-6a-trans-cinnamoyloxy-neo-clerodan-15,16-olide, which was given the trivial name of scuteformoid B. As shown in Tables 3 and 4, the NMR data indicate that compounds 3e10 have similar structures to 1, except for the substituents at C-1, C-7, C-11, and C-12. Therefore, the structures of compounds 3e10 were identified by comparisons of their NMR data with those of 1. Compound 3 was isolated as a white powder and had the same molecular formula of C31H38O10 as 1, as established by HRESIMS (m/ z 593.2358, [M þ Na]þ). Its IR absorption features and 1H and 13C

3

Table 1 1 H and 13C NMR data (dH and dC in ppm) of compound 1.a Position

dH (J in Hz)

dC

Position

dH (J in Hz)

dC

1 2a 2b 3a 3b 4 5 6 7a 7b 8 9 10 11a 11b 12

5.36 1.69 2.24 1.08 2.33

71.0 31.3

2.78 d (16.8) 3.07 d (16.8)

38.3

71.5

14a 14b 15 16a 16b 17 18a 18b 19 20 10 20 30 ,70 40 ,60 50 1-OAc

78.4

6-OAc

ddd (10.8, 4.8, 4.4) m m m m

65.5 42.2 67.8 37.9

5.06 dd (11.2, 4.8) 1.81 m 1.73 m

2.41 2.58 1.69 5.60

29.7

80.7 41.9 45.8 34.4

d (11.2) dd (13.6, 4.0) m dd (12.4, 4.0)

4.42 4.32 1.31 3.18 2.46 1.37 0.96

d (9.6) d (9.6) s br s d (4.0) s s

7.92 d (7.2) 7.44 t (7.2) 7.57 t (7.2) 1.98 s

13

1.98 s

174.6 78.5 24.4 51.7 14.9 20.9 165.4 129.0 129.8 128.8 133.8 170.1 21.4 170.4 21.7

a 1

H NMR measured at 400 MHz, 13C NMR measured at 100 MHz, and spectra obtained in CDCl3 with TMS as internal standard. Assignments were supported with HSQC and HMBC NMR spectra.

Table 2 1 H and 13C NMR data (dH and dC in ppm) of compound 2.a Position

dH (J in Hz)

dC

Position

dH (J in Hz)

1 2a 2b 3a 3b 4 5 6 7 8 9 10 11a 11b 12a 12b 13 14a

1.55 1.97 1.40 0.95 2.14

21.0 25.2

14b 15 16a 16b 17 18a 18b 19 20 10 20 30 100 200 , 600 300 ,500 400 7-OAc

2.59 d (16.8)

m m m m m

5.23 d (10.4) 5.28 d (10.4)

2.08 1.77 1.62 1.81 1.58

m m m m m

2.74 d (16.8)

31.8 66.7 42.7 70.9 74.1 81.1 38.8 41.4 27.6 29.4 78.5 42.8

4.39 4.16 1.22 3.36 2.39 1.45 0.98

d (9.2) d (9.2) s (4.0, 2.4) d (4.0) s s

6.36 d (16.0) 7.60 d (16.0) 7.48 m 7.36 m 7.36 m 1.96 s

dC 174.8 79.9 20.2 52.2 15.4 20.7 165.8 118.3 145.0 134.7 128.3 128.9 130.3 171.2 20.9

a 1

H NMR measured at 400 MHz, 13C NMR measured at 100 MHz, and spectra obtained in CDCl3 with TMS as internal standard. Assignments were supported with HSQC and HMBC NMR spectra.

NMR spectra (Table 3) showed high similarity with 1, indicating

Fig. 3. X-ray structures of compounds 1, 3, 5, 6 and 9.

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X. Chen et al. / Phytochemistry 145 (2018) 1e9

Table 3 1 H and 13C NMR data (dH and dC in ppm) of compounds 3e6.a Position

1a 1b 2a 2b 3a 3b 4 5 6 7a 7b 8 9 10 11a 11b 12 13 14a 14b 15 16a 16b 17 18a 18b 19 20 10 20 30 ,70 40 ,60 50 100 200 300 ,700 400 ,600 500 1-OAc

3

4

5

6

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

5.70 ddd (10.8, 4.8, 4.4)

71.4

25.0

5.40 ddd (10.8,4.8,4.4)

70.8

5.36 dd (10.8, 4.8)

70.8

1.80 2.22 1.01 2.38

31.6

1.92 1.54 2.13 1.01 1.58 2.39

32.0

2.27 1.72 1.08 2.38

31.3

2.27 1.67 1.09 2.33

31.3

m m m m

5.03 dd (10.8, 3.2) 1.82 m 1.70 m

2.46 1.51 2.22 5.64

m m m dd (12.4, 2.4)

2.84 d (16.8) 2.58 d (16.8) 4.21 d (8.8) 4.45 d (8.8) 1.22s 3.19 br s 2.49 br s 1.40 s 0.98 s

7.93 d (6.8) 7.38 t (6.8) 7.57 t (6.8)

29.7 65.5 42.3 67.7 37.9 80.5 41.9 45.8 34.2 69.8 78.5 37.9 174.6 78.0 24.2 51.7 14.9 21.4 165.6 130.1 129.9 128.6 133.6

m m m m m m

5.06 dd (11.2, 5.6) 1.80 m 1.76 m

2.26 m 5.62 d (11.6) 5.71 d (11.6) 2.78 d (16.8) 3.20 d (17.2) 4.47 4.32 1.43 3.19 2.42 1.28 0.99

d (9.6) d (9.6) s (3.6, 2.4) d (3.6) s s

7.90 dd (7.6, 1.2) 7.43 t (7.6) 7.57 t (7.6)

21.7 66.6 42.4 68.9 38.3 85.6 46.4 43.6 73.4 71.1 80.1 38.4 173.7 77.7 24.4 52.1 14.3 16.8 165.7 128.1 129.9 128.9 134.2

m m m m

5.33 d (10.4) 5.46 d (10.4)

2.56 2.70 1.75 5.67

m dd (14.4, 4.8) m dd (12.4, 4.8)

2.75 d (16.8) 3.07 d (16.8) 4.50 4.44 1.37 3.29 2.49 1.52 1.11

d (9.6) d (9.6) s (3.6, 2.4) d (3.6) s s

8.01 d (7.2) 7.43 t (7.2) 7.58 t (7.2)

7.93 d (7.6) 7.46 t (7.6) 7.58 t (7.6) 2.01 s

6-OAc 1.97 s

170.0 20.8

1.99 s

170.1 21.4

1.83 s

170.4 20.8

1.72 s

29.3 65.5 42.8 68.9 73.7 82.2 43.7 45.5 34.6 71.2 78.4 38.2 174.2 78.4 20.1 51.7 16.1 20.6 166.5 128.9 128.8 130.1 133.7 165.4 129.2 128.9 129.8 133.9 170.3 21.7 169.9 20.9

m m m m

5.13 d (10.4) 5.21 d (10.4)

2.49 2.64 1.70 5.63

m dd (14.0, 4.4) m dd (12.4, 4.8)

2.79 d (16.8) 3.07 d (16.8) 4.43 4.43 1.30 3.20 2.45 1.30 1.05

s s s (4.0, 2.4) d (4.0) s s

7.92 d (7.6) 7.44 t (7.6) 7.57 t (7.6)

2.00 s 1.97 s

7-OAc 2.06 s 11-OAc 12-OAc 1.97 s

29.2 65.4 42.8 68.9 73.1 82.0 43.6 45.4 34.6 71.2 78.3 38.2 174.2 78.4 19.9 51.7 16.1 20.8 165.3 129.0 129.8 128.8 133.9

170.2 21.6 169.7 21.0 170.8 20.5

170.3 20.8

a 1

H NMR measured at 400 MHz,13C NMR measured at 100 MHz, and spectra obtained in CDCl3with TMS as internal standard. Assignments were supported with HSQC and HMBC NMR spectra.

that compound 3 could be an isomer of 1. The only difference between these two compounds was the locations of the substituents. The HMBC correlations from H-1 [dH 5.70 (ddd, J ¼ 10.8, 4.8, 4.4 Hz)] to C-10 (dC 165.6), H-6 [dH 5.03 (dd, J ¼ 10.8, 3.2 Hz)] to C-1ʺ (dC 170.0), and H-12 [dH 5.64 (dd, J ¼ 12.4, 2.4 Hz)] to C-1000 (dC 170.3) indicated that the benzoyloxy group was located at C-1, and two acetoxy groups were located at C-6 and C-12, respectively. In the NOESY spectrum of 3 (Fig. S21, Supporting Information), the correlations of H-1 with H-17, H-19, and H-20 suggested that these groups were a-oriented, whereas the correlation of H-12 with H-10 indicated that were b-oriented. The configuration of 3 was deduced to be the same as that of 1 based on a comparison of their NMR chemical shifts, coupling constants, and NOESY data. In addition, an X-ray crystallographic study (Fig. 3) was performed to unambiguously confirm the structure and determine the absolute configuration of 3. Therefore, compound 3 (scuteformoid C) was determined to be (1R,4R,5S,6S,8S,9R,10R,12S,13R)-6,12-diacetoxy-1-

benzoyloxy-4,18; 8,13-diepoxy-neo-clerodan-15,16-olide. Compound 4 was isolated as a white powder. Its molecular formula was determined as C31H38O10, as established by HRESIMS, with a molecular ion at m/z 593.2354 [M þ Na]þ. Its IR absorption features and 1H and 13C NMR spectra (Table 3) showed high similarity with 1, indicating that compound 4 could be an isomer of 1. The remarkable difference occurred in the split pattern of an acylated CH proton which changed from a ddd-peak (dH 5.36, ddd, J ¼ 10.8, 4.8, 4.4 Hz, H-1) in 1 to a d-peak (dH 5.62, d, J ¼ 11.6 Hz, H11) in 4, implying that the location of the acetoxy group at C-1 in 1 was changed in 4. The 1He1H COSY cross peaks of H-11/H-12 and the HMBC correlations from H-11 to Me-20 (dC 16.8), C-10 (dC 43.6), C-12 (dC 71.1), C-13 (dC 80.1), C-8 (dC 82.6), and C-1000 (dC 170.4) further confirmed this speculation, and the acetoxy group was determined to be positioned at C-11 in compound 4. In the NOESY spectrum of 4 (Fig. S28, Supporting Information), the correlations of H-11 with Me-17 (dH 1.43), and Me-20 (dH 0.99) suggested that

X. Chen et al. / Phytochemistry 145 (2018) 1e9

5

Table 4 1 H and 13C NMR data (dH and dC in ppm) of compounds 7e10.a Position

1a 1b 2a 2b 3a 3b 4 5 6 7a 7b 8 9 10 11a 11b 12 13 14a 14b 15 16a 16b 17 18a 18b 19 20 10 20 30 ,70 40 ,60 50 100 200 300 ,700 400 ,600 500 1-OAc

7

8

9

10

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

2.46 1.56 1.99 1.25 1.02 2.15

21.5

5.35 dd (11.2, 5.2)

70.8

21.3

2.26 1.66 1.05 2.32

31.2

2.07 1.58 1.90 1.58 1.01 2.15

25.1

24.8

1.89 1.60 2.13 1.02 1.59 2.06

d (3.6) m m m m m

31.5

5.32 d (10.4) 5.45 d (10.4)

66.7 42.6 69.9 74.0

2.40 m 5.69 d (11.6)

83.6 47.4 43.2 73.0

5.79 d (11.6) 3.17 d (16.8) 2.78 d (16.8) 4.44 4.55 1.48 3.33 2.46 1.43 1.13

d (9.6) d (9.6) s br s d (3.6) s s

8.01 d (7.6) 7.45 m 7.59 d (7.6)

7.91 d (7.6) 7.45 m 7.59 d (7.6)

71.0 80.2 38.2 173.4 77.6 20.2 52.1 15.6 16.4 166.5 128.0 130.0 129.0 133.8 165.6 129.1 130.1 128.8 134.3

m m m m

5.27 d (10.4) 5.42 d (10.4)

2.47 2.42 1.62 5.47

m d (4.8) m dd (12.4, 4.8)

2.88 d (16.8) 2.58 d (16.8) 4.42 4.35 1.29 3.25 2.46 1.49 1.09

d (9.2) d (9.2) s br s m s s

7.98 d (7.2) 7.44 t (7.2) 7.58 t (7.2)

2.00 s 6-OAc 1.72 s

169.9 20.8

1.86 s

170.4 20.9

2.02 s

29.2 65.5 42.7 68.9 73.7 82.0 43.6 45.3 34.6 72.3 78.1 38.1 174.2 78.1 19.9 51.7 16.2 20.6 166.5 129.2 130.0 128.8 133.7

169.9 20.9 170.0 21.6

m m m m m m

5.09 d (10.4) 5.18 d (10.4)

2.14 2.33 1.59 5.20

m dd (14.0, 4.8) m d (14.0)

2.70 d (18.0) 3.22 d (18.0) 4.75 4.33 1.21 3.25 2.41 1.36 0.98

d (9.6) d (9.6) s (4.0, 2.4) d (4.0) s s

7.94 d (7.6) 7.43 t (7.6) 7.57 t (7.6)

1.96 s

7-OAc 2.07 s 11-OAc 12-OAc 1.70 s

31.7 24.9 66.7 42.7 70.6 73.5 81.4 42.9 42.6 34.4 72.2 78.7 42.3 172.7 73.3 20.2 52.3 15.3 20.5 165.8 129.1 129.8 128.9 133.9

169.9 21.0 170.9 20.8

m m m m m m

5.05 dd (10.4, 5.6) 1.75 ddd (13.2, 10.4, 5.8)

2.04 2.31 1.58 5.18

m dd (14.4, 4.8) m dd (12.4, 4.8)

2.69 d (17.6) 3.09 d (17.6) 4.79 4.34 1.21 3.19 2.41 1.27 0.91

d (9.6) d (9.6) s (4.0, 2.4) d (4.0) s s

21.3 32.1 66.8 41.6 69.6 37.8 80.2 41.9 43.2 32.5 72.5 78.8 42.4 173.0 73.7 24.6 52.4

7.95 d (7.6) 7.43 t (7.6) 7.57 t (7.6)

14.0 20.9 165.9 129.2 129.8 128.8 133.8

1.99 s

170.2 21.5

169.7 20.7

a 1 H NMR measured at 400 MHz, 13C NMR measured at 100 MHz, and spectra obtained in CDCl3 with TMS as internal standard. Assignments were supported with HSQC and HMBC NMR spectra.

these groups were a-oriented. Detailed analyses of the 2D NMR (1H-1H COSY, HMQC, HMBC, and NOESY) (Figs. S25-28, Supporting Information) data and a comparison with those of 1 revealed that compound 4 was (4R,5S,6S,8S,9R,10R,11R,12S,13R)-6,11-diacetoxy12-benzoyloxy-4,18; 8,13-diepoxy-neo-clerodan-15,16-olide, named scuteformoid D. Compound 5 was isolated as white needles. Its HRESIMS showed a molecular ion at m/z 713.3372 [M þ Na]þ, giving the molecular formula of 5 as C38H42O12. Its IR absorption features and 1H and 13C NMR spectra (Table 3) were similar to those of 1, except for signals representative of an additional benzoyloxy moiety [dH 8.01(2H, d, J ¼ 7.2 Hz, H-30 and H-70 ), 7.43 (2H, t, J ¼ 7.2 Hz, H-40 and H-60 ),7.58 (1H, t, J ¼ 7.2 Hz, H-5'); dC 166.5 (PhCOO), 128.9 (C-20 ), 128.8 (C-30 and C-70 ), 130.1 (C-40 and C-60 ), and 133.7 (C-50 )]. The HMBC correlations from H-1 (dH 5.40) to C-1000 (dC 170.3), H-6 (dH 5.33) to C10000 (dC 169.9), H-7 (dH 5.46) to C-1' (dC 166.5), and H-12 (dH 5.67) to C-100 (dC 165.4) suggested that the two benzoyloxy groups were

attached at C-7 and C-12, and two acetoxy groups were located at C-1 and C-6, respectively. In the NOESY spectrum of 5 (Fig. S35, Supporting Information), the correlations of H-7 with Me-19 (dH 1.52) and Me-20 (dH 1.11) suggested that these groups were a-oriented. Thus, the structure of 5 was conformed as (13R)-1b,6adiacetoxy-7b,12a-dibenzoyloxy-4,18; 8,13-diepoxy-neo-clerodan15,16-olide. In addition, single-crystal X-ray diffraction using Cu Ka radiation was performed to unambiguously identify the absolute configuration of 5 (Fig. 3) as (4R,5S,6S,7R,8S,9R,10R,11R,12S,13R)1,6-diacetoxy-7,12-dibenzoyloxy-4,18; 8,13-diepoxy-neo-clerodan15,16-olide. The trivial name of compound 5 was assigned as scuteformoid E. Compound 6 was obtained as a white amorphous powder. The molecular formula was established as C33H40O12 by HRESIMS, which displayed a quasi-molecular ion at m/z 651.2428 [M þ Na]þ. Its IR absorption features and 1H and 13C NMR spectra (Table 3) were similar to those of 1, except for signals representative of an

6

X. Chen et al. / Phytochemistry 145 (2018) 1e9

additional acetoxyl moiety [dH 1.97 (3H); dC 170.8 and 21.0]. The HMBC correlations from H-7 [dH 5.21 (d, J ¼ 10.4 Hz)] to C-1ʺʺ (dC 170.8) and H-7 to C-17 (dC 19.9), C-11 (dC 34.6), and C-16 (dC 78.3) indicated that the acetoxy moiety is connected to C-7. In the NOESY spectrum of 6 (Fig. S42, Supporting Information), the correlations of H-7 with Me-19 (dH 1.30) and Me-20 (dH 1.05) suggested that these groups were cofacial and a-oriented. The structure and relative configurations of 6 were unambiguously confirmed by an X-ray crystallographic experiment (Fig. 3). In addition, the absolute configurations of 6 were deduced to be the same as those of 1 based on a comparison of their biogenetic grounds, NMR chemical shifts, coupling constants, NOESY data and optical rotation. Thus, the structure of 6 was tentatively elucidated to be (1R,4R,5S,6S,7R,8S,9R,10R,12S,13R)-1,6,7-triacetoxy-12-benzoyloxy4,18; 8,13-diepoxy-neo-clerodan-15,16-olide, and its trivial name was assigned as scuteformoid F. Compound 7 was isolated as a white powder. Its HRESIMS showed a molecular ion at m/z 713.3365 [M þ Na]þ, giving the molecular formula C38H42O12. Its IR absorption features and 1H and 13 C NMR spectra (Table 4) were similar to those of 4, except for signals representative of an additional benzoyloxy moiety [dH 8.01 (d, 2H, J ¼ 7.6 Hz, H-30 and H-70 ), 7.45 (m, 2H, H-40 and H-60 ), 7.59 (d, 1H, J ¼ 7.6 Hz, H-50 ); dC 166.5 (s, PhCOO), 128.0 (s, C-20 ), 130.0 (d, C30 and C-70 ), 129.0 (d, C-40 and C-60 ), and 133.8 (d, C-50 )]. The HMBC correlations from H-7 (dH 5.45) to C-6 (dC 69.9), and C-1ʹ (dC 166.5), and from Me-17 (dH 1.48) to C-7 (dC 74.0) further confirmed this speculation, and the benzoyloxy group was positioned to be at C-7 in compound 7. In the NOESY spectrum (Fig. S49, Supporting Information), the correlations of H-7 with Me-19 (dH 1.43) and Me-20 (dH 1.13) suggested that these groups are cofacial and aoriented. Thus, the structure of 7 was conjectured to be (4R,5S,6S,7R,8S,9R,10R,11R,12S,13R)-6,11-diacetoxy-7,12dibenzoyloxy-4,18; 8,13-diepoxy-neo-clerodan-15,16-olide. The trivial name of compound 7 was assigned scuteformoid G. Compound 8 was isolated as a white powder. Its HRESIMS showed a molecular ion at m/z 651.2428 [M þ Na]þ, giving a molecular formula of C33H40O12. The comparison of the 1D and 2D NMR spectra of 8 and 6 indicated that both compounds have the same 1b,6a,7b,12a-tetrasubstituted-4,18; 8,13-diepoxy-neo-clerodan-15,16-olide skeleton. The only difference between these two compounds was the locations of the substituents. The benzoyloxy group was determined to be positioned at C-7, and three acetoxy groups were determined to be positioned at C-1, C-6, and C-12 from the observed HMBC correlations from H-7 (dH 5.42) to C-1' (dC 166.5), H-1 (dH 5.35) to C-1ʺ (dC 169.9), H-6 (dH 5.27) to C-1000 (dC 170.0), and H-12 (dH 5.47) to C-10000 (dC 169.7). The configuration of 8 was deduced to be the same as that of 6 based on a comparison of their NMR chemical shifts, coupling constants, and NOESY data. Thus, the structure of 8 was identified to be (1R,4R,5S,6S,7R,8S,9R,10R,11R,12S, 13R)-1,6,12-triacetoxy-7,12dibenzoyloxy-4,18; 8,13-diepoxy-neo-clerodan-15,16-olide, and it was named scuteformoid H. Compound 9 was isolated as white needles and had the same molecular formula of C31H38O10 with 1 according to the HRESIMS, showing a molecular ion at m/z 593.2366 [H þ Na]þ. Its 1H and 13C NMR spectra (Table 4) showed high similarity to those of 1, indicating that compound 9 may be an isomer of 1. A remarkable difference occurred in the split pattern of an acylated CH proton which changed from a ddd-peak (dH 5.36, ddd, J ¼ 10.8, 4.8, 4.4 Hz, H-1) in 1 to a d-peak (dH 5.18, d, J ¼ 10.4 Hz, H-7) in 9, implying that the location of the acetoxy group at C-1 in 1 was changed in 9. The HMBC correlation from H-7 to C-1000 (dC 170.9) indicated that the acetoxy group [dH 2.07 (3H, s); dC 170.9; 20.8] was connected to C-7 in compound 9 (Fig. S62, Supporting Information). The NOESY correlations of H-6/H-10, H-7/Me-17, H-7/Me-19, H-7/Me-20 and

Table 5 In vitro anti-HIV-1 activities of selected isolated compounds (mg/mL). Compound

1

3

4

6

8

9

AZTc

EC50a CC50 SIb

56.89 >200 >3.52

48.24 >200 >4.14

77.53 >200 >2.58

79.17 >200 >2.53

54.64 >200 >3.66

67.27 >200 >2.97

0.0025 1291.00 516400

a The inhibitory effects of compounds against HIV lytic replication were tested and expressed as EC50 values (mg/mL). b SI ¼ CC50/EC50. c Positive control.

H-12/H-10 in the NOESY spectrum of 9 suggested that H-7, Me-17, Me-19, and Me-20 are a-oriented and H-6, H-12 and H-10 are boriented (Fig. S62, Supporting Information). However, the configuration of C-13 was inverted in comparison with that of compound 1, which was determined to be S by the NOESY correlation between H2-16 and Me-17 (Fig. S63, Supporting Information) (Wang et al., 2010). This was further established by the comparison of the signals arising from H2-16 (dH 4.75, 4.33,each d, D ¼ 0.42 ppm) and C14 (dC 42.3, t) and C-16 (dC 73.3, t), which were markedly different from those observed in 1 and 3e8. Thus, compound 9 was determined to be (13S)-6a,7b-diacetoxy-12a-benzoyloxy-4,18; 8,13diepoxy-neo-clerodan-15,16-olide. A single-crystal X-ray diffraction analysis was conducted using Cu Ka radiation to define the absolute configuration of 9 (Fig. 3) as (4R,5S,6S,7R,8S,9R,10R,12S,13S)-6,7-diacetoxy-12-benzoyloxy-4,18; 8,13-diepoxy-neo-clerodan-15,16-olide, and it was named scuteformoid I. Compound 10 was obtained as a white powder. It was assigned the molecular formula C29H36O8, as established by HRESIMS (m/z 535.2420, [M þ Na]þ). The 1H and 13C NMR data (Table 4) of 10 were similar to those of 9, except for the absence of both an oxygenated functionality at the C-7 position and an acetoxy moiety. The HMBC correlations from H-6 (dH 5.05) to C-100 (dC 170.2), and H-12 (dH 5.18) to C-1ʹ (dC 165.9) suggested that the acetoxy and benzoyloxy groups are attached at C-6, and C-12 (Fig. S69, Supporting Information), respectively. The NOESY correlations of H-6/H-10, and H-12/H-10 in compound 10 indicated that these all have the same configuration, suggesting that H-6, H-12, and H-10 are b-oriented (Fig. S70, Supporting Information). In addition, the chemical shifts of H2-16 (dH 4.79, 4.34 D ¼ 0.45 ppm), C-14 (dC 42.4) and C-16 (dC 73.7) were similar to those of 9. Thus, compound 10 was assigned (4R,5S,6S,7R,8S,9R,10R,13S)-6-acetoxy-12-benzoyloxy-4,18; 8,13diepoxy-neo-clerodan-15,16-olide, and it was given the trivial name scuteformoid J. The known compound 11 was identified to be hastifolin A by spectroscopic analysis and comparing its NMR data with those reported in the literature (Raccuglia et al., 2010). 11 may conceivably be the biological precursor of those neo-clerodanes (1e10) formed by a nucleophilic addition of the 8b-hydroxyl group on the a,b unsaturated g-lactone (Raccuglia et al., 2010). Compounds 1, 3, 4, 6, 8, and 9 were tested for their anti-HIV-1 activities using a microtiter syncytium formation infectivity assay, with AZT used as the positive control (Table 5) (Bozov et al., 1993). Although the compounds showed no cytotoxicity against C8166 cell lines, with CC50 > 200 mg/mL, all the tested compounds showed weak anti-HIV activities, with EC50 values ranging from 48.24 to 79.17 mg/mL (AZT, EC50 ¼ 0.0025 mg/mL). Compounds 1, 3, 4, 6, 8, and 9 were also tested for their cytotoxicity against the Hela, A549, and MCF-7 human tumor cell lines by the MTT method. However, none of them showed inhibitory activity against any of the cell lines used (IC50 > 10 mg/mL).

X. Chen et al. / Phytochemistry 145 (2018) 1e9

3. Conclusions To the best of our knowledge, there has been no prior report on the phytocheimstry constitution of S. formosana. From this study, ten previously undescribed neo-clerodane diterpenoids have been isolated from the whole plants of S. formosana, one known analogue was also identified as part of this investigation. Their inhibitory effects against HIV lytic replication and cytotoxicity against the Hela, A549, and MCF-7 human tumor cell lines were evaluated. 4. Experimental 4.1. General experimental procedures Optical rotations were measured on a JASCO P-1020 digital polarimeter. UV data were obtained from online HPLC analysis. IR spectra were recorded on a Nicolet 6700 spectrophotometer. NMR spectra were recorded on a Bruker Avance spectrometer (400 MHz for 1H and 100 MHz for 13C). TMS was used as an internal standard. HRESIMS spectra were measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer. Silica gel (Qing Dao Hai Yang Chemical Group Co.; 100e200, 200e300, 300e400 mesh), octadecylsilyl silica gel (YMC; 12 nm-50 mm), and Sephadex LH-20 (GE) were used for column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin-layer chromatography (TLC). HPLC separations were carried out on an LC-20AT Shimadzu liquid chromatography system to an Agilent Eclipse XDB-C18 column (9.4  250 mm, 5 mm) connected with an SPD-M20A diode array detector. X-ray diffraction data were collected on an Agilent Technologies Gemini A Ultra system or collected on a Bruker APEX DUO diffractometer. 4.2. Plant material The whole plants of Scutellaria formosana were collected in Yingeling District (latitude 18 490 N; longitude 109110 E), Hainan Province, People's Republic of China, in September 2014 and were identified by Dr. Rongtao Li, Hainan Branch Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College. A voucher specimen (ZY-20151007) has been deposited at the Key Laboratory of Tropical Medicinal Plant Chemical of the Ministry of Education, Hainan Normal University. 4.3. Extraction and isolation The air-dried powder of the whole plant of S. formosana (2.0 kg) was extracted three times with 85% EtOH at 50  C to afford a crude extract. The extract (300.0 g) was suspended in 2 L of water and partitioned with petroleum ether (2 L  6). The combined petroleum ether layers were evaporated to dryness under reduced pressure to give a petroleum ether extract (21.0 g), which was subjected to silica gel CC (petroleum ether, EtOAc, MeOH v/v, gradient) to generate 12 fractions (Frs. 1e12). Fraction 4 (petroleum ether-EtOAc 85:15, v/v, 5 g) was then separated by silica gel CC, eluting with petroleum ether-EtOAc (10:1 to 1:3, v/v), to produce three fractions (4-14-8). Fraction 4-1 was isolated by CC on silica gel eluting with petroleum ether-EtOAc (6:1 to 1:5) to obtain 2 (20.0 mg). Fraction 4-2 was chromatographed by silica gel CC, eluting with ether-EtOAc (10:1 to 1:1, v/v), to obtain two major components, and both was further purified by semipreparative HPLC, eluted with methanol-water (55:45, v/v), to yield 1 (21.0 mg, 15.4 min), 6 (2.8 mg, 20.0 min), and 10 (2.0 mg, 23.2 min), respectively. By similar procedures, fraction 4-2 (100.0 mg) provided 3 (10.0 mg), and 4 (5.6 mg). Fraction 4-3 (2 g) was separated

7

by silica gel CC, eluted with petroleum ether-EtOAc (10:1 to 1:2, v/ v), to obtain six fractions (4-3-1e4-3-6), and each of them was further purified by semipreparative HPLC, eluting with acetonitrilewater (70:30, v/v), to obtain 5 (18.9 mg, 23.1 min), 8 (2.5 mg, 34.3 min) and 11 (2.0 mg, 38.5 min). Fraction 5 (75%, 3.0 g) was subjected to silica gel CC, eluting with ether-EtOAc (6:1 to 1:3, v/v), to give five major fractions (5-15-5). Fraction 5-2 was subjected to repeated Sephadex LH-20 CC, eluting with CHCl3-MeOH (1:1) to obtain two major fractions (5-2-2 and 5-2-4), which were purified respectively by semipreparative HPLC, using acetonitrile-water (75:25, v/v) as the mobile phase, to afford 7 (15.3 mg, 21.0 min), and 9 (18.4 mg, 26.4 min), sequentially. Scuteformoid A (1): white crystal; [a]25 D -41.5 (c 0.35, MeOH); IR (KBr) nmax 3001e2959 (epoxide), 1785 (spiro g-lactone), 1714, 1597, 1459, 1368, 1248 (OAc), 1103, 1029, 968, 710 cm1; UV lmax (MeOH): 230 nm; 1H and 13C NMR data, see Table 1; positive ESIMS m/z 593.3 [M þ Na]þ; HRESIMS m/z 593.2366 [M þNa]þ (calcd 593.2357 for C31H38O10Na). Scuteformoid B (2): white amorphous powder; [a]25 D -56.1 (c 0.3, MeOH); IR (KBr) nmax 2938e2853 (epoxide), 1784 (spiro g-lactone), 1738, 1721, 1634, 1450, 1371, 1234 (OAc), 1031, 709 cm1; UV lmax (MeOH): 280 nm; 1H and 13C NMR data, see Table 2; positive ESIMS m/z 561.4 [M þ Na]þ; HRESIMS m/z 561.3427 [M þ Na]þ (calcd 561.3421 for C31H38O8Na). Scuteformoid C (3): shallow greenness oil; [a]25 D -5.3 (c 0.3, MeOH); IR (KBr) nmax 3011e2969 (epoxide), 1787 (spiro g-lactone), 1726, 1598 (phenyl), 1455, 1368, 1248 br (OAc), 1103, 1029, 708 cm1; UV lmax (MeOH): 230 nm; 1H and 13C NMR data, see Table 3; positive ESIMS m/z 593.3 [M þ Na]þ; HRESIMS m/z 593.2358 [M þ Na]þ (calcd 593.2357 for C31H38O10Na). Scuteformoid D (4): white, amorphous powder; [a]23 D -7.8 (c 0.4, MeOH); IR (KBr) nmax 3001e2959 (epoxide), 1785 (spiro g-lactone), 1727, 1600 (phenyl), 1459, 1369, 1248 (OAc), 1009, 1034, 710 cm1; UV lmax (MeOH): 230 nm; 1H and 13C NMR data, see Table 3; positive ESIMS m/z 593.3 [M þ Na]þ; HRESIMS m/z 593.2354 [M þ Na]þ(calcd 593.2357 for C31H38O10Na). Scuteformoid E (5): white needles; [a]25 D -8.4 (c 0.3, MeOH); IR (KBr) nmax 3002e2959 (epoxide), 1788 (spiro g-lactone), 1725, 1610 (phenyl), 1459, 1369, 1247 (OAc), 1009, 707 cm1; UV lmax (MeOH): 230 nm; 1H and 13C NMR data, see Table 3; positive ESIMS m/z 713.4 [M þ Na]þ; HRESIMS m/z 713.3372 [M þ Na]þ (calcd 713.3374 for C38H42O12Na). Scuteformoid F (6): white crystal; [a]23 D -3.2 (c 0.3, MeOH); IR (KBr) nmax 3001e2959 (epoxide), 1785 (spiro g-lactone), 1714, 1459, 1368, 1248 br (OAc), 1103, 1029, 968, 710 cm1; UV lmax (MeOH): 230 nm; 1H and 13C NMR data, see Table 3; positive ESIMS m/z 651.3 [M þ Na]þ; HRESIMS m/z 651.2428 [M þ Na]þ (calcd 651.2412 for C33H40O12Na). Scuteformoid G (7): white amorphous powder; [a]25 D -20.3 (c 0.2, MeOH); IR (KBr) nmax 2923e2857 (epoxide), 1787 (spiro g-lactone), 1734, 1621, 1429, 1381, 1255 (OAc), 1095, 1029, 711 cm1; UV lmax (MeOH): 230 nm; 1H and 13C NMR data, see Table 4; positive ESIMS m/z 713.4 [M þ Na]þ; HRESIMS m/z 713.3365 [M þ Na]þ (calcd 713.3374 for C38H42O12Na). Scuteformoid H (8): white amorphous powder; [a]23 D -15.8 (c 0.4, MeOH); IR (KBr) nmax 3001e2959 (epoxide), 1785 (spiro g-lactone), 1714, 1459, 1368, 1248 (OAc), 1003, 1029, 968, 710 cm1; UV lmax (MeOH): 230 nm; 1H and 13C NMR data, see Table 4; positive ESIMS m/z 651.4 [M þ Na]þ; HRESIMS m/z 651.2428 [M þ Na]þ (calcd 651.2412 for C33H40O12Na). Scuteformoid I (9): white needles; [a]25 D þ20.3 (c 0.35, MeOH); IR (KBr) nmax 3011e2959 (epoxide), 1787 (spiro g-lactone), 1726, 1598 (phenyl), 1455, 1368, 1247 br (OAc), 1103, 708 cm1; UV lmax (MeOH): 230 nm; 1H and 13C NMR data, see Table 4; positive ESIMS m/z 593.3 [M þ Na]þ; HRESIMS m/z 593.2366 [M þ Na]þ (calcd

8

X. Chen et al. / Phytochemistry 145 (2018) 1e9

593.2357 for C31H38O10Na). Scuteformoid J (10): white amorphous powder; [a]25 D þ9.3 (c 0.25, MeOH); IR (KBr) nmax 3001e2959 (epoxide), 1785 (spiro glactone), 1714, 1459, 1368, 1248 (OAc), 1103, 1029, 968, 710 cm1; UV lmax (MeOH): 230 nm; 1H and 13C NMR data, see Table 4; positive ESIMS m/z 535.3 [M þ Na]þ; HRESIMS m/z 535.2420 [M þ Na]þ (calcd 535.2408 for C29H36O8Na).

a ¼ b ¼ g ¼ 90 , V ¼ 2931.95(7) Å3, Z ¼ 4, T ¼ 293(2) K, Dc ¼ 1.293 g/ cm3, m (Cu Ka) ¼ 0.798 mm1, F (000) ¼ 1216, 18250 reflections measured, 4670 independent reflections (Rint ¼ 0.0294). The final R1 values were 0.0444 [I > 2s (I)]. The final wR2 (F2) values were 0.1078 [I > 2s (I)]. The final R1 values were 0.0395 (all data). The final wR2 (F2) values were 0.1043 (all data). Flack parameter ¼ 0.03(7).

4.4. X-ray crystal structure analysis of compounds 1, 3, 5, 6, and 9

4.5. Anti-HIV-1 assay

Crystal X-ray diffraction data were collected on a Bruker APEX DUO (Scuteformoid A and E) and Agilent Technologies Gemini A Ultra system (Scuteformoid C, F and I) diffractometer with Cu Ka radiation (l ¼ 1.5418 and 1.541 84 Å). The structure was solved by direct methods (SHELXS-97) and refined using full-matrix leastsquares difference Fourier techniques. Carbon and oxygen atoms were refined anisotropically. Hydrogen atoms were either refined freely with isotropic displacement parameters or positioned with an idealized geometry and refined riding on their parent C atoms. Crystals suitable for X-ray diffraction (1, 3, 5, 6, and 9) were obtained by slow evaporation of a solution in MeOHeCHCl3. Crystallographic data (excluding structure factors) for 1, 3, 5, 6, and 9 have been deposited with the Cambridge Crystallographic Data Centre: CCDC reference numbers 1473750, 1474088, 1473773, 1474073, and 1473759. These data can be obtained, free of charge, from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam. ac.uk/data_request/cif. Crystal data for 1: C32H39Cl3O10, M ¼ 689.98, space group P212121with a ¼ 10.78770 (10) Å, b ¼ 15.2391 (2) Å, c ¼ 20.7305 (3) Å, a ¼ b ¼ g ¼ 90 , V ¼ 3407.99 (7) Å3, Z ¼ 4, T ¼ 293 (2) K, Dc ¼ 1.345 g/cm3, m(Cu Ka) ¼ 2.895 mm1, F (000) ¼ 1448, 21919 reflections measured, 5499 independent reflections (Rint ¼ 0.0254). The final R1 values were 0.0601 [I > 2s (I)]. The final wR2 (F2) values were 0.1689 [I > 2s (I)]. The final R1 values were 0.0552 (all data). The final wR2 (F2) values were 0.1619 (all data). Flack parameter ¼ 0.01(2). Crystal data for 3: C31H38O10, M ¼ 570.61, space group P41212 with a ¼ 15.72243(11) Å, b ¼ 15.72243(11) Å, c ¼ 24.8041(2) Å, a ¼ b ¼ g ¼ 90 , V ¼ 6131.44(11) Å3, Z ¼ 8, T ¼ 293(2) K, Dc ¼ 1.236 g/cm3, m(Cu Ka) ¼ 0.764 mm1, F(000) ¼ 2432, 24549 reflections measured, 6064 independent reflections (Rint ¼ 0.0271). The final R1 values were 0.0323 [I > 2s(I)]. The final wR2 (F2) values were 0.0828 [I > 2s (I)]. The final R1 values were 0.0310 (all data). The final wR2 (F2) values were 0.0815 (all data). Flack parameter ¼ 0.09(10). Crystal data for 5: C38H42O12, M ¼ 690.72, space group P212121 with a ¼ 10.36800(10) Å, b ¼ 11.3380(2) Å, c ¼ 29.8682(4) Å, a ¼ b ¼ g ¼ 90 , V ¼ 3511.08(8) Å3, Z ¼ 4, T ¼ 293(2) K, Dc ¼ 1.307 g/ cm3, m(Cu Ka) ¼ 0.808 mm1, F(000) ¼ 1464, 25973 reflections measured, 6064 independent reflections (Rint ¼ 0.0271). The final R1 values were 0.0323 [I > 2s(I)]. The final wR2 (F2) values were 0.0828 [I > 2s(I)]. The final R1 values were 0.0310 (all data). The final wR2 (F2) values were 0.0815 (all data). Flack parameter ¼ 0.02(11). Crystal data for 6: C33H40O12, M ¼ 628.61, space group P212121 with a ¼ 11.1517(4) Å, b ¼ 7.5430(3) Å, c ¼ 19.5264(4) Å, a ¼ g ¼ 90 , b ¼ 104.047, V ¼ 1593.38(11) Å3, Z ¼ 2, T ¼ 293(2) K, Dc ¼ 1.312 g/cm3, m (Cu Ka) ¼ 0.806 mm1, F (000) ¼ 666, 8639 reflections measured, 4777 independent reflections (Rint ¼ 0.0560). The final R1 values were 0.0533 [I > 2s (I)]. The final wR2 (F2) values were 0.1196 [I > 2s(I)]. The final R1 values were 0.0471 (all data). The final wR2 (F2) values were 0.1267 (all data). Flack parameter ¼ 0.23(18). Crystal data for 9: C31H38O10, M ¼ 570.61, space group P212121 with a ¼ 7.85600(10) Å, b ¼ 18.4769(3) Å, c ¼ 20.1988(3) Å,

The cytotoxicity assay against C8166 cells (CC50) was assessed using an MTT method. Human T lymphocyte (C8166), MT-4, experimental strain of HIV-1IIIB were provided by British MRC, AIDS Reagant Project. C8166 cells were cultured with RPMI-1640 containing 10% fetal bovine serum for 1 day before the cells passaged, the cells were used at logarithmic phase of growth. The anti-HIV-1 activity was evaluated by the inhibition assay for the cytopathic effects of HIV-1 (EC50) (Wang et al., 2009). Zidovudine (AZT, Sigma) was used as a positive control. 4.6. Cytotoxicity assays The cytotoxicity assay was performed using an MTT method. The human tumor cell lines HeLa (ervical carcinoma), A549 (lung adenocar cinoma), and MCF-7 (breast) used were provided by College of Pharmacy, Hebei University and maintained in DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco) in a humidified atmosphere containing 5% CO2 at 37  C. Doxorubicin was used as a positive control. Author contributions X. Chen and W.-H. Chen contributed equally. Acknowledgments The work was financially supported by the State of International Science and Technology Cooperation Special (2014DFA40850), the National Natural Science Foundation of China (Nos. 81360478, 21202030, 21362009, 21662012), Program for Innovative Research Team in University (IRT-16R19), and the Hainan Province Natural Science Foundation of Innovatie Research Team Project (2016CXTD007). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.phytochem.2017.09.002. References Bozov, P.I., Malakov, P.Y., Papanov, G.Y., de la Torre, M.C., Rodriguez, B., Perales, A., 1993. Scutalpin A, a neo-clerodane diterpene from scutellaria alpina. Phytochemistry 34, 453e456. Bruno, M., de la Torre, M.C., Piozzi, F., Rodriguez, B., Savona, G., Arnold, N.A., 1993. A neo-clerodane diterpenoid from Scutellaria cypria var. Elatior. Phytochemistry 33, 931e932. Bruno, M., Piozzi, F., Maggio, A.M., Simmonds, M.S.J., 2002. Antifeedant activity of neoclerodane diterpenoids from two Sicilian species of Scutellaria. Biochem. Syst. Ecol. 30, 793e799. Cui, L., Gu, H.X., Lu, J.X., Lin, H.B., Lin, J.Q., Lin, J.Q., 2010. Analysis on the status quo of the germplasm resources and cultivation of Scutellaria baicalensis. Acta Chin. Med. Pharm. 38, 69e72. de la Torre, M.C., Bruno, M., Piozzi, F., Rodriguez, B., Savona, G., Servettaz, O., 1992. Neo-clerodane diterpenoids from Scutellaria columnae. Phytochemistry 31, 3639e3641. Flank, H.D., 1983. On enantiomorph-polarity estimation. Acta Cryst. A39, 876e881. Gang, X., Fang, Z., Yang, X.W., Zhou, J., Yang, L.X., Shen, X.L., Hu, Y.J., Zhao, Q.S., 2011. Neo-clerodane diterpenoids from Salvia dugesii and their bioactive studies. Nat.

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