3,4-seco-Norclerodane Diterpenoids from the Roots of Polyalthia laui

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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3,4-seco-Norclerodane Diterpenoids from the Roots of Polyalthia laui Zhang-Xin Yu,†,§,# Cai-Juan Zheng,†,# Guang-Ying Chen,† Rong-Li Huang,† Xue-Ming Zhou,† Zhi-Gang Niu,† Xiao-Bao Li,† Chang-Ri Han,*,†,‡ and Xiao-Ping Song*,†,‡

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†

Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158, People’s Republic of China § Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Haikou 570311, People’s Republic of China ‡ Key Laboratory of Medicinal and Edible Plant Resources of Hainan Province, Hainan Institute of Science and Technology, Haikou 571126, People’s Republic of China S Supporting Information *

ABSTRACT: Ten new clerodane diterpenoids, polylauioids A−J (1−10), and five known analogues (11−15) were isolated from the roots of Polyalthia laui. Among the new compounds, 3 and 8 are artifacts. The structures were elucidated using spectroscopic methods and by comparison with published NMR spectroscopic data. The absolute configurations of 4, 5, and 7 were defined based on single-crystal X-ray diffraction and electronic circular dichroism data. Compounds 1 and 2 represent the first examples of rearranged 3,4-seco-norclerodane diterpenoids, and a putative biosynthesis pathway for these compounds is proposed. Compounds 1, 4, 6, 7, 9, and 10 showed anti-HIV activities with EC50 values ranging from 12.2 to 35.2 μM. (11),14 6α,16-dihydroxycleroda-4(18),13-dien-15-oic acid (12),15 6α,16-dihydroxycleroda-3,13-dien-15-oic acid (13),15 16-oxocleroda-3,13E-dien-15-oic acid (14),16 and kolavenic acid (15),17 were obtained from an EtOAc extract of the roots of P. laui. Among the new compounds, 3 and 8 are artifacts. Compounds 1 and 2 represent the first examples of rearranged 3,4-seco-norclerodane diterpenoids, and a putative biosynthesis pathway for these compounds is proposed. The relative configurations of 1 and 9 and the absolute configurations of 4 and 7 were confirmed by X-ray crystallography using Cu Kα radiation. Compounds 1, 4, 6, 7, 9, and 10 showed anti-HIV activities with EC50 values ranging from 12.2 to 35.2 μM. Herein, the structure elucidation, putative biosynthetic pathway, and the biological activities of these compounds are reported.

Polyalthia (Annonaceae) is a member of a genus with approximately 120 species, some of which are cultivated as both ornamental and economic plants in subtropical and tropical regions of the Eastern Hemisphere.1 The roots of several Polyalthia species are used in traditional medicine for the treatment of pharynx neurosis, high blood pressure, and febrifuge, among others.2,3 Previous phytochemical investigations on plants of this genus resulted in the isolation of a number of alkaloids, flavonoids, triterpenoids, diterpenoids, sesquiterpenoids, and acetogenins.4−10 Polyalthia laui is an endemic evergreen arbor plant comprising seven species growing in Hainan Island, People’s Republic of China.1 Previous studies focusing on the petroleum ether extract of this plant led to the isolation of clerodane diterpenoids and sesquiterpenes, and some of them showed cytotoxic or antibacterial activities.11−13 In a continuing search for novel, bioactive, and structurally diverse natural products from the medicinal plants of Hainan Island, 10 new clerodane diterpenoids, polylauioids A−J (1−10), as well as five known analogues, (4→2)-abeo-cleroda-2,13E-dien-2,14-dioic acid © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 22, 2018

A

DOI: 10.1021/acs.jnatprod.8b00243 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H NMR Data (δ) for 1−6 (400 MHz) (δ in ppm, J in Hz) position 1 3 6 7 8 10 11 12 14 16 17 18 19 20 2-OMe 15-OMe 16-OMe a

1a

2b

3b

4b

5b

6b

2.34, br d (4.8) 1.97, dd (16.8, 4.8)

2.32, br d (4.8) 1.98, dd (16.8, 4.8)

2.33, br d (4.8) 1.98, dd (16.8, 4.8)

1.51, m 1.87, m 1.49, m 1.52, m 1.70, m 2.56, t (4.8) 1.42, m 2.08, m 2.29, m 5.69, s 2.13, s 0.86, d (6.8) 2.20, s 1.14, s 0.79, s 3.61, s

1.52, m 1.87, m 1.48, m 1.51, m 1.72, m 2.56, t (4.8) 1.41, m 2.04, m 2.27, m 5.67, s 2.13, s 0.85, d (6.4) 2.20, s 1.14, s 0.79, s 3.60, s 3.67, s

2.18, br d (12.4) 2.29, dd (15.2, 6.0)2.37 m 9.96, s 1.38, m 1.68, m 1.52, m 1.59, m 1.46, m 1.56, br d (6.0) 1.52, m 2.10, m 2.31, m 5.85, s 5.62, s 0.82, d (6.8) 2.03, s 0.94, s 0.90, s

2.24, dd (14.8, 6.8) 2.36, dd (14.8, 2.4)

1.52, m 1.86, m 1.48, m 1.51, m 1.69, m 2.57, t (4.8) 1.43, m 2.10, m 2.33, m 5.68, s 2.12, s 0.90, d (6.8) 2.22, s 1.13, s 0.84, s

2.16, br d (12.4) 2.32, dd (14.8, 6.8) 9.96, s 1.39, m 1.67, m 1.53, m 1.60, m 1.46, m 1.59, br d (6.0) 1.51, m 2.17, m 2.30, m 5.86, s 5.63, s 0.83, d (6.4) 2.03, s 0.93, s 0.89, s

3.55, s

3.56, s

1.36, m 1.68, m 1.53, m 1.62, m 1.46, m 1.61, br d (6.8) 1.48, m2.39 d (7.2) 2.22, m 2.35, m 5.84, d (1.6) 4.74, d (1.6) 0.82, d (6.4) 2.02, s 0.91, s 0.90, s

b

Recorded in methanol-d4. Recorded in CDCl3.

■

Table 2. 13C NMR Data (δ) for 1−6 (100 MHz) (δ in ppm)

RESULTS AND DISCUSSION Compound 1 was isolated as colorless crystals. Its molecular formula was determined as C19H30O5 (five indices of hydrogen deficiency) by HRESIMS m/z 339.2166 [M + H]+. The 1H NMR resonances (Table 1) were classified as the signals of five high-field methyl groups at δH 0.84 (3H, s), 0.90 (3H, d, J = 6.8 Hz), 1.13 (3H, s), 2.12 (3H, s), and 2.22 (3H, s), two methine protons at δH 2.57 (1H, t, J = 4.8 Hz) and 1.69 (1H, m), and an olefinic proton at δH 5.68 (1H, s, H-14). The 13C NMR and DEPT data (Table 2) displayed 19 carbon atoms, including three carbonyl groups, one trisubstituted double bond, and 14 sp3 carbon atoms (two quaternary carbons, two CH, five CH2, and five CH3). The 1D NMR data indicated that 1 was a secoclerodane-type diterpenoid,18−20 and the structure of 1 was similar to that of kolavenic acid17 except for the cleaved ring. The major differences in the 1H and 13C NMR spectroscopic data were the absence of the double-bond signals at δH 5.19 (1H, br s) and δC 120.6 and 144.6 and methylene signals at δH 2.01 (1H, m) and δC 27.0 in the spectra of kolavenic acid and the presence of two carbonyl signals at δC 176.9 and 217.0 in 1, indicating the double bond in kolavenic acid was oxidized to a dicarbonyl function and a methylene moiety had been lost in 1. The 1H−1H COSY correlation of H2-1/H-10 and the HMBC correlations (Figure 2) from H2-1 and H-10 to C-2 suggested that the carboxymethyl group was connected to C-10. Additionally, an acetyl group at C-5 was confirmed by the HMBC correlations from H-6, H-10, H3-18, and H3-19 to C-4 and from H3-18 to C5. Thus, the 2D structure of 1 was defined as a 3,4-seconorclerodane diterpenoid. The relative configuration of 1 was assigned by the NOESY experiment (Figure 3). The correlations of H-10 with H-8 and Me-18 indicated that these protons and the methyl group were cofacial, and they were designated to be β-oriented. The correlations of Me-19, Me-17, and Me-20 suggested that these three methyl groups were α-oriented. The E-configuration of the Δ13(14) double bond was defined by the NOESY correlation of H2-12/H-14. The relative configuration of 1 was defined as

a

position

1a

2b

3b

4b

5b

6b

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

33.1 176.9

32.3 174.2

32.3 174.2

217.0 54.5 36.7 27.3 38.0 41.1 41.3 38.3 35.2 162.4 116.8 170.5 19.1 16.1 25.9 17.6 18.6

214.3 53.2 36.0 26.2 36.9 40.1 40.3 37.0 34.4 163.8 115.3 171.4 19.4 15.9 25.5 17.4 18.3 51.9

214.4 53.2 36.0 26.3 36.9 40.0 40.3 37.0 34.2 161.2 115.2 167.4 19.4 15.9 25.6 17.4 18.4 51.9 53.2

26.5 137.1 188.7 171.8 51.0 34.1 28.3 37.9 38.1 54.4 36.8 22.0 168.1 118.0 170.7 104.5 15.3 9.80 17.2 17.7

26.2 137.1 188.7 172.0 51.0 34.2 28.3 37.4 38.0 54.0 36.4 21.8 168.1 117.9 170.8 104.6 15.2 9.81 17.2 18.1

29.7 125.6 170.9 168.7 50.8 34.5 28.4 37.7 38.0 54.3 37.2 22.9 171.1 115.3 174.2 73.2 15.3 11.9 17.0 17.9

57.1

57.4

Recorded in methanol-d4. bRecorded in CDCl3.

(5R*,8R*,9S*,10R*) by the X-ray diffraction analysis, with a Flack parameter of 0.3(3) (Figure 4). Therefore, the structure of polylauioid A (1) was defined as 4-oxo-3,4-seco-3-norclerodane13E-2,15-dioic acid. Compound 2 was obtained as a colorless oil, with the molecular formula C20H32O5, as established by HRESIMS data. Comparing the NMR data of 2 (Tables 1 and 2) with those of 1 suggested that 2 was also a 3,4-seco-norclerodane diterpenoid, but the molecular weight of 2 exceeds that of 1 by 14 units, B



Article

Compound 4 was obtained as a colorless oil. Its HRESIMS data exhibited a molecular ion at m/z 364.2479 [M + NH4]+ (calcd for 364.2482), indicating a molecular formula of C21H30O4. The IR spectrum showed absorption bands at 1731 and 1636 cm−1 indicating the presence of an unsaturated carbonyl group. The 1H NMR data (Table 1) displayed a formyl proton at δH 9.96 (1H, s), an olefinic proton at δH 5.86 (1H, s), three methine signals at δH 5.63 (1H, s), 1.59 (1H, br d, J = 6.0 Hz), and 1.46 (1H, m), a methoxy signal at δH 3.55 (3H, s), and four signals at δH 2.03 (3H, s), 0.93 (3H, s), 0.89 (3H, s), and 0.83 (3H, d, J = 6.4 Hz) indicating the presence of four methyl groups. The 21 carbon resonances (Table 2) were classified as a formyl, a carbonyl group, a tetrasubstituted and a trisubstituted olefinic bond, a methoxy, two quaternary carbons, three methines, four methyl groups, and five methylene groups by the 13C and DEPT NMR spectra. Analyses of the 1H and 13C NMR data suggested that 4 was a rearranged (4→2)-abeoclerodane-type diterpenoid.21 The 1D NMR data of 4 were similar to those of (4→2)-abeo-16(R and S)-2,13Z-kolavadien16,15-olide-3-al,22 except for the presence of a methoxy group at δH 3.55 (3H, s) and δC 57.1 (Me) in 4. The attachment of the methoxy group to C-16 was supported by the HMBC correlations of OMe-16/C-16 (δC 104.5) and H-16/OMe-16 (Figure 2). The NOESY correlations of Me-19 with Me-17 and Me-20 indicated that these methyl groups were cofacial, and they were assigned to be α-oriented, while H-8/H-10 were βoriented. To determine the C-16 absolute configuration, the experimental electronic circular dichroism (ECD) spectrum of 4 was measured, and it exhibited Cotton effects (CEs) for a positive n−-π* transition at 255.8 nm (Δε +1.57) and a negative π−π* transition at 224.5 nm (Δε −1.42) (Figure 5), suggesting the 16R absolute configuration.23,24 Furthermore, a single crystal of 4 was grown from a MeOH−H2O (3:1, v/v) solution (Figure 6). The absolute configuration of 4 was defined to be (5R,8R,9S,10R,16R) with a Flack parameter of 0.01(12) by Xray diffraction analysis. Therefore, the structure of polylauioid D (4) was identified as (4→2)-abeo-16β-methoxycleroda-2,13dien-15,16-olide. Compound 5, with a molecular formula of C21H30O4, the same as 4, was isolated as colorless crystals. The 13C NMR data (Table 1) closely resembled those of 4, except for small shifts in the signals for C-1 (ΔδC −0.3), C-8 (ΔδC −0.5), C-10 (ΔδC −0.4), C-11 (ΔδC −0.4), C-20 (ΔδC +0.4), and OMe-16 (ΔδC +0.3) in 5. Analysis of its 2D NMR spectra showed that 5 is a stereoisomer of 4. The ECD curve (Figure 5) of 5 showed positive 247.5 nm (Δε +3.9) and negative 216.9 nm (Δε −3.64) CEs, suggesting a (16S) absolute configuration.23,24 Thus, the structure of polylauioid E (5) was determined to be (4→2)abeo-16α-methoxycleroda-2,13-dien-15,16-olide. Compounds 4

Figure 1. Structures of compounds 1−10.

indicating that a hydroxy group in 1 was replaced by a methoxy group at δH 3.61 (3H, s) and δC 51.9 in 2. The HMBC correlations of H2-1, OMe-2/C-2 supported the attachment of the methoxy group to C-2. The NOESY correlations of Me-19/ Me-17 and Me-20 established the α-orientation of these three methyl groups, while H-8/H-10 and Me-18 were β-oriented. Hence, the structure of polylauioid B (2) was identified as shown in Figure 1. Compound 2 could be detected in the crude extract by UPLC-Q/TOF-MS (Supporting Information). Compound 3 was also isolated as colorless oil, with the molecular formula C21H34O5 according to the HRESIMS spectrum. The 1H and 13C NMR data of 3 (Tables 1 and 2) were similar to those of 1; the major differences included the presence of two methoxy signals at δH 3.60 (3H, s) and δC 51.9 and δH 3.67 (3H, s) and δC 53.2 in compound 3. These data indicated that the two hydroxy groups in 1 were replaced by methoxy groups in 3. This was corroborated by the HMBC correlations of H-1, OMe-2/C-2 and H-14, OMe-15/C-15. The relative configuration of 3 was assigned as (5R*,8R*,9S*,10R*), the same as that of 1, based on the NOESY correlations. Thus, the structure of polylauioid C (3) was established as shown in Figure 1. Compound 3 is likely an artifact because it was not detected in the crude extract by UPLC-Q/TOF-MS (Supporting Information), and methanol was used as eluent during chromatography.

Figure 2. 1H−1H COSY and key HMBC correlations of compounds 1, 4, 6, 7, and 10. C



Article

Figure 3. NOSEY correlations of compounds 1, 4, 6, 7, and 10.

Figure 4. ORTEP drawing of compound 1. Figure 6. ORTEP drawing of compound 4.

10 in the NOESY spectrum suggested that these two protons were β-oriented (Figure 3). Furthermore, the NOESY correlations between H2-12 and H-14 indicated the Zconfiguration of the Δ13(14) double bond. Accordingly, the structure of polylauiacid F (6) was identified as (4→2)-abeo-16hydroxycleroda-2,13Z-dien-2,14-dioic acid. Compound 7 was obtained as colorless crystals, giving the molecular formula C20H35O4, according to its HRESIMS. The 13 C NMR (Table 4) data were similar to those of 3,4dihydroxyclerodan-13E-en-15-oic acid,25 isolated from the fruits of Detarium microcarpum, except for the deshielding of the signals of C-6 (ΔδC +3.3), C-11 (ΔδC +2.4), and C-20 (ΔδC +11.1) and the shielding of the signals C-5 (ΔδC −1.1), C-10 (ΔδC −1.7), and C-19 (ΔδC −3.9) in 7. Analysis of its 1D and 2D NMR data indicated that 7 is a stereoisomer of 3,4dihydroxyclerodan-13E-en-15-oic acid. In the NOESY experiment (Figure 3), the correlations of H-10/H-3 and H-8 showed that these three protons were β-oriented, while Me-19/Me-17/ Me-18 and Me-20 were α-oriented. The absolute configuration of 7 was defined as (13E,3R,4R,5R,8R,9S,10R) with a Flack parameter of −0.03(7) by X-ray crystallographic analysis (Figure 7). Thus, the structure of polylauioid G (7) was assigned as (3R,4R,5R,8R,9S,10R)-3,4-dihydroxyclerodan-13Een-15-oic acid. Compound 8 showed an [M + H]+ ion at m/z 353.2688 (calcd for 353.2687) in its HRESIMS, suggesting the molecular formula C21H37O4. Comparison of the 1D NMR data of 8 with those of 7 indicated that 7 and 8 shared the same basic skeleton, but for the presence of one methoxy signal at δH3.68 (3H, s) and δC 51.0 in the data of 8 (Tables 3 and 4). These data indicated

Figure 5. Experimental ECD spectra of 4 and 5.

and 5 could be detected in the crude extract by UPLC-Q/TOFMS (Supporting Information). Compound 6 possessed a molecular formula of C20H30O5 based on its HRESIMS data, which corresponds to six indices of hydrogen deficiency. Comparison of the 1H and 13C NMR data of 6 with those of (4→2)-abeo-cleroda-2,13E-dien-2,14-dioic acid14 revealed that the vinylic methyl (C-16) signals at δH 2.15 (3H, s) and δC 17.7 for (4→2)-abeo-cleroda-2,13E-dien-2,14dioic acid were replaced by signals of a hydroxymethyl group at δH 4.74 (2H, d, J = 1.6 Hz) and δC 73.2 for C-16 in 6. The HMBC (Figure 2) correlations of H2-12, H-14/C-16 and H216/C-12, C-13, and C-14 supported the presence of a hydroxymethyl moiety. The correlation between H-8 and HD



Article

Table 3. 1H NMR Data (δ) for 7−10 (400 MHz) (δ in ppm, J in Hz) 7a

position 1

8b

1.29, m

1.38, m

1.61, m 1.64, m 2.01, m 3.49, t (2.4) 1.36, m 1.68, m 1.33, m; 1.57 m 1.48, m 1.48, m 1.83, dd (12.4, 2.0) 1.39, m; 2.39 d (7.2) 1.51, m

12 14 16 17 18 19 20 15OMe

2 3 6 7 8 10 11

9a

10a 1.45, m

1.60, m 1.67, m 2.01, m 3.58, t (2.8) 1.38, m 1.55, m 1.39, m; 1.57 m 1.45, m 1.47, m 1.73, dd (12.0, 2.4) 1.36, m; 2.39 d (7.2) 1.47, m

2.35, t (14.8); 2.37 m 2.68, br d (9.2) 2.37 m 5.71, s 143, m; 1.81 m 1.85, m; 1.49 m 1.52, m; 1.81 m 1.58, m; 1.49 m 1.45, m 2.01, br d (9.2)

6.67, br s 1.44, m 1.62, m 1.38, m 1.51, m 1.62, m 1.37, br d (12.4) 1.13, m

2.26, m

2.03, m

2.68, m

5.64, br s 2.14, d (1.2) 0.81, d (5.6) 1.19, s 1.12, s 0.77, s

5.67, br s 2.16, d (0.8) 0.79, d (6.0) 1.24, s 1.12, s 0.74, s 3.68, s

6.71, s

1.41, m 1.55, m

1.91, m 2.31, m

Figure 7. ORTEP drawing of compound 7.

2.37, br d (12.8) 2.62, br t (6.8) 6.69, s

0.90, d (6.0) 1.93, s 1.13, s 0.81, s

the crude extract by UPLC-Q/TOF-MS (Supporting Information), and methanol was used as eluent during chromatography. Compound 9 was obtained as colorless crystals. Its molecular formula was defined as C20H28O5 from its HRESIMS ion at m/z 349.2010 [M + H]+ (calcd for C20H29O5, 349.2011). The 1H NMR data of 9 (Table 3) contained two deshielded protons at δH 6.71 (1H, s) and 5.71 (1H, s), two methines at δH 2.01 (1H, br d, J = 9.2 Hz) and 1.45 (1H, m), and four high-field methyl groups at δH 1.93 (3H, s), 1.13 (3H, s), 0.90 (3H, d, J = 6.0 Hz), and 0.81 (3H, s) indicating the presence of two trisubstituted double bonds as in ent-clerodane diacid previously isolated by us.12 Comparison of the 13C NMR data of 9 with those of entclerodane diacid revealed that the methylene signal at δC 27.8 in ent-clerodane diacid was replaced by a carbonyl signal at δC 203.4 in 9, which was confirmed by the HMBC correlations from H2-1, H-3, and H-10 to C-2. The relative configuration of 9 was identical to that of ent-clerodane diacid based on the NOESY correlations. Slow evaporation of 9 from a MeOH− HOAc (20:1, v/v) solution afforded colorless crystals. The relative configuration of 9 was defined as (5R*,8R*,9S*,10R*) via the X-ray crystallographic analysis, with a Flack parameter of −0.25(15) (Figure 8). It also defined the E-configuration of the Δ13(14) double bond. Thus, the structure of polylauioid I (9) was identified as 2-oxo-3,13E-dien-clerodane-15,16-dioic acid. Compound 10 was isolated as a colorless oil and showed the molecular formula C20H28O6 based on the [M + H]+ ion at m/z 365.1960 (calcd for 365.1959) in its HRESIMS. Its 1D NMR (Tables 3 and 4) data also closely resembled those of entclerodane diacid,12 except for the absence of a methyl signal at δH 1.01 (3H, s) and δC 20.4 in ent-clerodane diacid and the presence of a hydroxycarbonyl carbon at δC 171.2 in 10. The long-range correlation of H-3/C-18 in the HMBC spectrum of 10 confirmed that the methyl at C-18 in ent-clerodane diacid was oxidized to a hydroxycarbonyl group (Figure 2). The relative configuration of 10 was established by its NOESY experiments (Figure 3). Thus, the structure of polylauioid J (10) was identified as 3,13E-dien-clerodane-15,16,18-trioic acid. Among the isolated compounds, polylauioids A−C (1−3) are novel rearranged 3,4-seco-norclerodane diterpenoids. Since most of the other compounds isolated from this plant were clerodane diterpenoids, we postulated that these clerodane diterpenoids served as precursors for this new type of 3,4-seco-norclerodane diterpenoid. Based on their structural similarities, a putative biosynthesis pathway for polylauioids A and B (1 and 2) is

0.86, s 1.26, s 0.74, s

a


Table 4. 13C NMR Data (δ) for 7−10 (100 MHz) (δ in ppm) position

7a

8b

9a

10a

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

17.7 31.0 76.9 77.4 42.6 33.5 27.8 37.4 39.9 41.8 38.2 35.8 163.1 116.4 170.3 19.2 16.4 21.2 17.9 18.8

16.5 30.6 76.6 76.5 41.5 32.5 26.6 36.2 38.9 40.9 36.8 34.9 161.9 115.0 167.4 19.3 16.1 21.7 17.4 18.5 51.0

35.3 203.4 125.9 176.3 41.0 36.5 27.9 37.2 40.2 46.7 37.8 22.5 148.9 128.1 169.0 170.1 16.1 19.3 18.8 18.1

18.4 28.2 138.7 143.8 38.8 38.7 28.6 37.6 40.3 48.2 37.3 22.9 149.3 128.2 171.0 171.2 16.4 171.2 21.1 18.7

a


that the hydroxy group at C-15 in 7 was replaced by a methoxy group in 8, which was corroborated by the HMBC correlations of H-14, OMe-15/C-15. The relative configuration of 8 was identical to that of 7 by its NOESY correlations. Therefore, the structure of polylauioid H (8) was defined as shown in Figure 1. Compound 8 is likely an artifact because it was not detected in E



Article

Table 5. Anti-HIV Activities of Compounds 1, 4, 6, 7, 9, and 10 no.

CC50 (μM)a

EC50 (μM)b

TIc

1 4 6 7 9 10 AZTd

>200 >200 >200 >200 >200 >200 5231.6

12.2 16.8 23.1 27.4 35.2 19.8 0.02

>16.4 >11.9 >8.5 >7.3 >5.7 >16.4 337 605.3

a

CC50: 50% cytotoxic concentration. bEC50: 50% effective concentration. cTI (therapeutic index) = CC50/EC50. dAZT (3′-azido-3′deoxythymidine) was used as a positive control.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were recorded on a Bruker AV 400 NMR spectrometer by using CDCl3 or methanol-d4 as a solvent. Preparative HPLC was performed on an Agilent 1260 LC series equipped with a quaternary pump VL, a DAD detector, and a Waters XBridge C18 (250 × 10 mm, 5 μm) semipreparative column. The other experimental procedures, in this study, were performed as previously described.12,27 Plant Material. The roots of P. laui were collected in August 2012 from Bawangling Nature Reserve, Hainan Province, People’s Republic of China. A voucher specimen (HAN20120807) was deposited at the College of Chemistry and Chemical Engineering, Hainan Normal University. Extraction and Isolation. Dried roots of P. laui (16.5 kg) were extracted with 75% EtOH (3 × 20 L, each for 5 days) to yield a semisolid residue (618 g) that constituted the crude extract. After being suspended in water (3.0 L), the solution was successively extracted with petroleum ether and EtOAc. The EtOAc extract (260 g) was applied to silica gel column chromatography (CC), eluted with CHCl3−MeOH (100:1 to 1:100, v/v), to afford seven fractions (Frs. 1−7). Fr. 2 (28.5 g) was subjected to silica gel CC eluted with petroleum ether−EtOAc (100:1 to 1:100, v/v) to yield five subfractions, 2A−2D. Subfraction 2B (6.8 g) was eluted with petroleum ether−EtOAc (3:1, v/v) to give 15 (43.7 mg). Subfraction 2C (7.5 g) was eluted with petroleum ether− EtOAc (2:1, v/v) and purified by semipreparative liquid chromatography with MeCN−H2O (55:45, v/v) to obtain 6 (3.8 mg), 7 (9.3 mg),

Figure 8. ORTEP drawing of compound 9.

proposed in Scheme 1. Taking polylauioid A (1) as an example, the precursor, kolavenic acid,17 could undergo oxidation, aldolization, and dehydration to yield solidagonal acid (i)14 that was also isolated from this plant by us. Solidagonal acid could undergo oxidation and decarboxylation to produce ii, which could undergo further oxidation to form the 3,4-seconorclerodane diterpenoid polylauioid A. Compounds 1, 4, 6, 7, 9, and 10 were tested for their anti-HIV bioactivities, and AZT was used as the positive control (Table 5).26 These compounds exhibited anti-HIV activities with E50 values ranging from 12.2 to 35.1 μM (AZT, EC50 = 0.02 μM) and displayed no cytotoxicity against C8166 cell lines (CC50 values >200 μM). In addition, the compounds were also tested for their cytotoxicity against three types of human tumor cell lines (MCF-7, A549, and HeLa) by the MTT method. However, none of them showed inhibitory activity (IC50 > 10 μM).

Scheme 1. Plausible Biosynthetic Pathway to the Formation of 1 and 2

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10 (5.5 mg), and 11 (4.6 mg). Fr. 3 (41.7 g) was subjected to silica gel CC eluted with CHCl3−MeOH (100:1 to 1:100, v/v) to afford six subfractions, 3A−3F. Fr. 3B (4.7 g) was successively separated by silica gel using petroleum ether−acetone (5:1, v/v), Sephadex LH-20 eluted with CHCl3−MeOH (1:3, v/v), and liquid chromatography (Waters XBridge C18 250 × 10 mm, 5 μm) with MeCN−H2O (70:30, v/v) to yield 2 (5.1 mg) and 3 (5.6 mg) and with MeCN−H2O (55:45, v/v) to afford 1 (6.4 mg), 8 (6.1 mg), and 9 (5.3 mg). Fr. 3C (5.2 g) was applied to silica gel CC using CHCl3−MeOH (8:1, v/v), then ODS eluting with MeOH−H2O (40:60, v/v), and purified by liquid chromatography (Waters XBridge C18 250 × 10 mm, 5 μm, MeCN−H2O, 58:42, v/v) to give 4 (6.3 mg) and 5 (7.2 mg). Compounds 12 (3.7 mg), 13 (3.9 mg) and 14 (5.8 mg) were obtained from Fr. 4 (11.2 g) by CC (petroleum ether−EtOAc, 2:1, v/v and CHCl3−MeOH, 10:1, v/v) and semipreparative HPLC (Waters XBridge C18 250 × 10 mm, 5 μm, MeCN− H2O, 60:40, v/v) in turn. Polylauioid A (1): colorless crystals; mp 158−160 °C; [α]25D −22 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 218 (3.53) nm; IR (KBr) νmax 2927, 1694, 1641, 1557, 1421, 1247, 1129, 751, 475 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 339.2166 [M + H]+ (calcd for C19H31O5, 339.2166). Polylauioid B(2): colorless oil; [α]25D −19 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 223 (3.46) nm; IR (KBr) νmax 2922, 1695, 1638, 1551, 1426, 1252, 1123, 755, 471 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 353.2324 [M + H]+ (calcd for C20H33O5, 353.2322). Polylauioid C (3): colorless oil; [α]25D −24 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 234 (3.35) nm; IR (KBr) νmax 2925, 1692, 1642, 1553, 1423, 1247, 1127, 747, 468 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 367.2479 [M + H]+ (calcd for C21H35O5, 367.2479). Polylauioid D (4): colorless crystals; mp 224−227 °C; [α]25D +21 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 216 (3.38), 235 (3.25) nm; ECD (c 1 × 10−4 mol/L, MeOH) λmax (Δε) 255.8 (+1.57), 224.5 (−1.42), 200.3 (−5.53); IR (KBr) νmax 2935, 1731, 1636, 1438, 1235, 1130, 752 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 364.2479 [M + NH4]+ (calcd for C21H34NO4, 364.2482). Polylauioid E (5): colorless oil; [α]25D −14 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 215 (3.50), 235 (3.42) nm; ECD (c 2 × 10−5 mol/L, MeOH) λmax (Δε) 247.5 (+3.9), 216.9 (−3.64), 195.8 (+0.73) nm; IR (KBr) νmax 2933, 1722, 1648, 1431, 1238, 1132, 756 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 347.2216 [M + H]+ (calcd for C21H31O4, 347.2217). Polylauioid F (6): colorless oil; [α]25D +7 (c 0.4, CHCl3); UV (MeOH) λmax (log ε) 238 (4.10) nm; IR (KBr) νmax 2932, 1657, 1387, 1123, 748 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 351.2167 [M + H]+ (calcd for C20H31O5, 351.2166). Polylauioid G (7): colorless crystals; mp 235−237 °C; [α]25D −21 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 225 (3.63) nm; IR (KBr) νmax 3446, 2968, 1735, 1638, 1224, 1119, 782, 481 cm−1; 1H and 13C NMR data, see Tables 3 and 4; positive-ion HRESIMS m/z 339.2531 [M + H]+ (calcd for C20H35O4, 339.2530). Polylauioid H (8): colorless oil; [α]25D −16 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 232 (3.38) nm; IR (KBr) νmax 3442, 2961, 1718, 1632, 1259, 1092, 786, 478 cm−1; 1H and 13C NMR data, see Tables 3 and 4; positive-ion HRESIMS m/z 353.2688 [M + H]+ (calcd for C21H37O4, 353.2687). Polylauioid I (9): colorless crystals; mp 255−257 °C; [α]25D −26 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 228 (3.75) nm; IR (KBr) νmax 3438, 2968, 1730, 1628, 1232, 1123, 763, 478 cm−1; 1H and 13C NMR data, see Tables 3 and 4; positive-ion HRESIMS m/z 349.2010 [M + H]+ (calcd for C20H29O5, 349.2011). Polylauioid J (10): colorless oil; [α]25D −25 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 234 (3.85) nm; IR (KBr) νmax 3442, 2961, 1718, 1632, 1259, 1092, 786, 478 cm−1; 1H and 13C NMR data, see Tables 3 and 4; positive-ion HRESIMS m/z 365.1960 [M + H]+ (calcd for C20H29O6, 365.1959). X-ray Crystal Structure Analysis of 1, 4, 7, and 9. X-ray crystallographic diffraction data were measured with Cu Kα radiation at

293(2) K. The structures were solved with direct methods (SHELXS97) and refined by full-matrix least squares difference Fourier techniques. Crystallographic data for 1, 4, 7, and 9 have been deposited with the Cambridge Crystallographic Data Centre: Deposition Nos. CCDC 1816078, 1816077, 1816076, and 1860199, respectively. Crystallographic data for 1, 4, 7, and 9 are given in the Supporting Information. The data can be obtained, free of charge, from the CCDC via https://www.ccdc.cam.ac.uk/structures-beta/. Anti-HIV Assay. Human T-lymphocyte (C8166), MT-4, an experimental strain of HIV-1IIIB, was provided by British MRC, AIDS Reagent Project. The cytotoxicity against C8166 cell lines (CC50) was assessed using an MTT method.28 The anti-HIV activity was evaluated by an assay of the inhibition of the cytopathic effects of HIV-1 (EC50).29 AZT (3′-azido-3′-deoxythymidine) was used as a positive control. The therapeutic index (TI) was calculated from the ratio of CC50/EC50. Cytotoxicity Assays. The cytotoxicity assay was performed using the MTT method.28 Three human tumor cell lines, MCF7 (breast), HeLa (cervical carcinoma), and A549 (lung adenocarcinoma), were used. Doxorubicin was used as a positive control.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00243. MS, 1D and 2D NMR spectra for 1−10, and the TIC chromatograms of compounds 1, 2, 4, 5, and 7 of the EtOAc extract of P. laui by UPLC-Q/TOF-MS (DOC) X-ray crystallographic data for 1 (CIF) X-ray crystallographic data for 4 (CIF) X-ray crystallographic data for 7 (CIF) X-ray crystallographic data for 9 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: 86-0898 65730237. E-mail: [email protected]. *E-mail: [email protected]. ORCID

Zhang-Xin Yu: 0000-0001-6973-5142 Cai-Juan Zheng: 0000-0003-1779-8736 Author Contributions #

Z. X. Yu and C. J. Zheng contributed equally to this study.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Key R&D Projects in Hainan Province-Social Development (ZDYF2018164), Hainan Institutions of Higher Learning Scientific Research Project (Hnky2017-87), the National Natural Science Foundation of China (Nos. 21362009 and 81360478), the Program for Innovative Research Team Project of Ministry (IRT-16R19), and the International S&T Cooperation Program of China (ISTCP) (2014DFA40850).

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REFERENCES

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3,4-seco-Norclerodane Diterpenoids from the Roots of Polyalthia laui

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