Article pubs.acs.org/jnp
Diterpenes from a Chinese Collection of the Brown Alga Dictyota plectens Shimiao Cheng,† Min Zhao,† Zhongmin Sun,‡ Weiping Yuan,† Shuangshuang Zhang,† Zheng Xiang,† Yuepiao Cai,† Jianyong Dong,† Kexin Huang,*,† and Pengcheng Yan*,† †
School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325035, People’s Republic of China Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, People’s Republic of China
‡
S Supporting Information *
ABSTRACT: Twenty-seven diterpenes of six chemical classes, including seven new diterpenes (1, 2, 6, 10, 11, 16, and 19), have been isolated from a collection of the brown alga Dictyota plectens from the South China Sea. The structures of the new diterpenes were elucidated by extensive spectroscopic analysis and by comparison with reported data. In the in vitro assays, 9, 12, 14, 16, and 22 showed inhibitory activity against HIV-1 replication with IC50 values of 16.1−30.5 μM, compounds 5, 13, 24, and 26 exhibited anti-H5N1 activity with inhibition rates of 50%−62% at 30.0 μM, and 12 and 24 also showed potent inhibition against LPS-induced NO production with inhibition rates of 90% and 86%, respectively, at 10.0 μM.
■
B
RESULTS AND DISCUSSION Compound 1 has a molecular formula of C22H32O4 as determined by HRESIMS data, implying seven degrees of unsaturation. The IR absorptions at 3444 and 1735 cm−1 indicated the presence of hydroxy and carbonyl functionalities. The 13C NMR spectrum showed 22 carbon signals including a ketone (δC 205.8), an ester carbonyl (δC 170.4), and six olefinic carbons (four methines at δC 141.8, 141.3, 130.8, and 126.1 and two quaternary carbons at 136.2 and 133.5) (Table 2), which accounted for five of the seven degrees of unsaturation. Thus, a bicyclic structure could be assigned for 1. The six olefinic carbons, along with four olefinic proton signals at δH 7.35 (d, J = 7.2 Hz, H-7), 6.44 (dd, J = 10.2, 7.2 Hz, H-6), 6.29 (d, J = 10.2 Hz, H-5), and 5.47 (d, J = 6.6 Hz, H-3), were attributed to two trisubstituted and one 1,2-disubstituted double bond. The COSY correlations established the subunits from C-2 to C-3, C-5 to C-7, C-10 to C-11, and C-13 to C-14, while their connectivities were completed by HMBC relationships (Figure 1). The HMBC correlations from H3-15 (δH 1.18, s) to C-1 (δC 52.0), C-2 (δC 76.2), C-11 (δC 45.6), and C-14 (δC 35.9), from H3-16 (δH 1.84, s) to C-3 (δC 130.8), C-4 (δC 136.2), and C-5 (δC 141.3), from H3-17 (δH 1.76, s) to C-7 (δC 141.8), C-8 (δC 133.5), and C-9 (δC 205.8), from H2-10 (δH 3.03, dd, J = 13.8, 2.4 Hz; 2.60, dd, J = 13.8, 7.2 Hz) to C-9 and C-12 (δC 87.0), and from H2-13 (δH 1.63, m; 1.46, m) to C-11 and C-12 allowed connection of the subunits to form a 5,11-bicyclic ring system, in which C-1, C-4, and C-8 were substituted by
rown algae of the genus Dictyota (family Dictyotacea) are common inhabitants in the tropical and subtropical waters of the world and have proven to be a rich source of biologically active secondary metabolites, especially diterpenes. To date, about 400 diterpenes belonging to more than 15 chemical classes have been isolated from worldwide collections of Dictyota species.1 Some of these compounds are reported to possess a variety of significant ecological and pharmaceutical bioactivities, such as antifeeding, antifouling, cytotoxic, antibacterial, and antiviral properties.2−10 However, the Dictyota algae of the China Sea have been rarely chemically examined.11 In the course of our ongoing research on bioactive natural products from marine organisms of the South China Sea, the brown alga Dictyota plectens (Allender & Kraft) Kraft was collected. Chemical examination of this specimen led to the isolation of 27 diterpenes, including seven new diterpenes (1, 2, 6, 10, 11, 16, and 19), an ethoxylated artifact (20), and 19 known analogues. These diterpenes can be biosynthetically distributed into three groups: dolabellanes and derivatives (dolastanes) for 1−9, xenicanes and derivatives (crenulides) for 19−27, and prenylated guaianes for 10−18. All compounds were evaluated for their antiviral activities against human immunodeficiency virus type 1 (HIV-1) and the highly pathogenic avian influenza A (H5N1) virus and for inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages (PEMΦ). Herein we report the details of isolation, structure elucidation, and bioactivity of these diterpenes. © XXXX American Chemical Society and American Society of Pharmacognosy
Received: September 5, 2014
A
dx.doi.org/10.1021/np5006955 | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Chart 1
Compound 2 has the same molecular formula as that of 1 according to its HRESIMS and NMR data (Tables 1 and 2). It was revealed to be a geometrical isomer of 1 with respect to the C-7/C-8 double bond on the basis of extensive 2D NMR spectroscopic analysis and by comparison of NMR data with those of 1. The difference was found by the significant downfield shift of H3-17 (δH 2.01, s) and C-17 (δC 21.8, CH3) in comparison with those of 1, suggesting the 7Z geometry13,14 in 2. This assumption was further confirmed by the NOE correlation of H3-17/H-7 (Figure 2). The relative configurations at C-1, C-2, C-11, and C-12 were assigned to be identical to those of 1 by the NOE correlations of H-2 (δH 5.08, d, J = 10.2 Hz)/H-11 (δH 2.04, d, J = 9.6 Hz), H3-15 (δH 1.20, s)/H-10a (δH 2.80, dd, J = 13.8, 9.6 Hz), and H-11/H3-19 (δH 1.03, d, J = 6.6 Hz). The coupling constant value JH‑2/H‑3 (10.2 Hz) and the NOE correlations of H-3 (δH 5.53, d, J = 10.2 Hz)/H3-15 and H-2/H3-16 (δH 1.77, s) indicated the trans-axial orientation of H-2 and H-3 and E geometry of C-3/C-4 double bond. In addition, the coupling constant value JH‑5/H‑6 (12.6 Hz) suggested the C-5/C-6 double bond to be Z. Thus, compound 2 was determined as (1S*,2S*,3E,5Z,7Z,11R*,12R*)-2-acetoxy-12-hydroxydolabella3,5,7-trien-9-one. Compound 6 was assigned a molecular formula of C22H32O4 according to the HRESIMS and NMR data, requiring seven degrees of unsaturation. The 13C NMR spectrum of 6 displayed six carbon signals for a ketone (δC 210.7), an ester carbonyl (δC 170.4), and four olefinic carbons (δC 131.9 (C), 121.0 (CH), 120.6 (CH), and 128.9 (CH)), indicating 6 to be tricyclic
methyls, C-2 and C-12 were oxygenated, while C-3/C-4, C-5/ C-6, C-7/C-8, and C-9 were present as an α,β,γ,δ,ε,ζunsaturated ketone. In addition, the linkage of an isopropyl group to C-12 was inferred by the HMBC correlations from H3-19 (0.73, d, J = 6.6 Hz) and H3-20 (0.92, d, J = 6.6 Hz) to C-12. Thus, 1 was revealed to be a dolabellane-type diterpene, similar to the co-occurring analogue (1S*,2S*,3E,7E,11R*,12R*)-2-acetoxy-12-hydroxydolabella3,7-dien-9-one (4).12 The location of an acetoxy group at C-2 was determined by the HMBC correlation from H-2 (δH 5.13, d, J = 6.6 Hz) to a carbonyl carbon (δC 170.4), and thus C-12 was ascertained to be hydroxylated by its chemical shift (δC 87.0) and the fact that an oxygen atom remained in the molecule according to the HRESIMS data. The relative configurations of the stereogenic centers and the geometries of the double bonds in 1 were assigned on the basis of NOESY analysis. The NOE correlations of H3-15/H-10a (δH 3.03, dd, J = 13.8, 2.4 Hz), H-11/H3-19, and H-11/H-2 suggested the trans fusion of the two rings and the same orientation of H-11, H-2, and the isopropyl group, while the NOE correlations of H3/H-11, H3-16/H-5, H-5/H-6, H-6/H3-17, and H-7/H-10a allowed the 3E, 5Z, and 7E assignment in 1 (Figure 2). Additionally, the geometries of 3E and 7E could be confirmed by the diagnostic chemical shifts of C-16 and C-17 (60.0 >60.0 >60.0 >60.0 UDc
4.7 1.8 1.7 1.0 1.3
a The other compounds were inactive at 30 μM. bCC50: cytotoxicity against human embryonic kidney 293T cells. cUD: undetected.
In addition, all compounds were assayed for inhibitory activity against the highly pathogenic H5N1 (A/Viet Nam/ 1203/2004) infection. The strategy for this assay is to target the viral envelope glycoprotein, hemagglutinin (HA), which is involved in multiple steps during viral entry, including receptor binding, internalization, and fusion. An HIV-based pseudotyping system was used to screen the inhibitors against H5N1 entry,34 while VSVG/HIV was applied to evaluate the specificity of entry inhibitors.35 In this assay, 5, 13, 24, and 26 exhibited specific inhibition against HA-mediated viral entry with inhibition rates of 56%, 50%, 59%, and 62%, respectively, at 30.0 μM (Table 4), but the other compounds showed weak Table 4. Inhibition of HA-Mediated H5N1 Entrya compound
concentration (μM)
IRb (%)
CC50c (μmol/L)
5 13 24 26
30.0 30.0 30.0 30.0
56 50 59 62
>60.0 >60.0 >60.0 >60.0
The other compounds were inactive at 30 μM. bIR: inhibition rate against H5N1 entry. cCC50: cytotoxicity against 293T cells.
a
activity (inhibition rate
Diterpenes from a Chinese Collection of the Brown Alga Dictyota plectens Shimiao Cheng,† Min Zhao,† Zhongmin Sun,‡ Weiping Yuan,† Shuangshuang Zhang,† Zheng Xiang,† Yuepiao Cai,† Jianyong Dong,† Kexin Huang,*,† and Pengcheng Yan*,† †
School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325035, People’s Republic of China Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, People’s Republic of China
‡
S Supporting Information *
ABSTRACT: Twenty-seven diterpenes of six chemical classes, including seven new diterpenes (1, 2, 6, 10, 11, 16, and 19), have been isolated from a collection of the brown alga Dictyota plectens from the South China Sea. The structures of the new diterpenes were elucidated by extensive spectroscopic analysis and by comparison with reported data. In the in vitro assays, 9, 12, 14, 16, and 22 showed inhibitory activity against HIV-1 replication with IC50 values of 16.1−30.5 μM, compounds 5, 13, 24, and 26 exhibited anti-H5N1 activity with inhibition rates of 50%−62% at 30.0 μM, and 12 and 24 also showed potent inhibition against LPS-induced NO production with inhibition rates of 90% and 86%, respectively, at 10.0 μM.
■
B
RESULTS AND DISCUSSION Compound 1 has a molecular formula of C22H32O4 as determined by HRESIMS data, implying seven degrees of unsaturation. The IR absorptions at 3444 and 1735 cm−1 indicated the presence of hydroxy and carbonyl functionalities. The 13C NMR spectrum showed 22 carbon signals including a ketone (δC 205.8), an ester carbonyl (δC 170.4), and six olefinic carbons (four methines at δC 141.8, 141.3, 130.8, and 126.1 and two quaternary carbons at 136.2 and 133.5) (Table 2), which accounted for five of the seven degrees of unsaturation. Thus, a bicyclic structure could be assigned for 1. The six olefinic carbons, along with four olefinic proton signals at δH 7.35 (d, J = 7.2 Hz, H-7), 6.44 (dd, J = 10.2, 7.2 Hz, H-6), 6.29 (d, J = 10.2 Hz, H-5), and 5.47 (d, J = 6.6 Hz, H-3), were attributed to two trisubstituted and one 1,2-disubstituted double bond. The COSY correlations established the subunits from C-2 to C-3, C-5 to C-7, C-10 to C-11, and C-13 to C-14, while their connectivities were completed by HMBC relationships (Figure 1). The HMBC correlations from H3-15 (δH 1.18, s) to C-1 (δC 52.0), C-2 (δC 76.2), C-11 (δC 45.6), and C-14 (δC 35.9), from H3-16 (δH 1.84, s) to C-3 (δC 130.8), C-4 (δC 136.2), and C-5 (δC 141.3), from H3-17 (δH 1.76, s) to C-7 (δC 141.8), C-8 (δC 133.5), and C-9 (δC 205.8), from H2-10 (δH 3.03, dd, J = 13.8, 2.4 Hz; 2.60, dd, J = 13.8, 7.2 Hz) to C-9 and C-12 (δC 87.0), and from H2-13 (δH 1.63, m; 1.46, m) to C-11 and C-12 allowed connection of the subunits to form a 5,11-bicyclic ring system, in which C-1, C-4, and C-8 were substituted by
rown algae of the genus Dictyota (family Dictyotacea) are common inhabitants in the tropical and subtropical waters of the world and have proven to be a rich source of biologically active secondary metabolites, especially diterpenes. To date, about 400 diterpenes belonging to more than 15 chemical classes have been isolated from worldwide collections of Dictyota species.1 Some of these compounds are reported to possess a variety of significant ecological and pharmaceutical bioactivities, such as antifeeding, antifouling, cytotoxic, antibacterial, and antiviral properties.2−10 However, the Dictyota algae of the China Sea have been rarely chemically examined.11 In the course of our ongoing research on bioactive natural products from marine organisms of the South China Sea, the brown alga Dictyota plectens (Allender & Kraft) Kraft was collected. Chemical examination of this specimen led to the isolation of 27 diterpenes, including seven new diterpenes (1, 2, 6, 10, 11, 16, and 19), an ethoxylated artifact (20), and 19 known analogues. These diterpenes can be biosynthetically distributed into three groups: dolabellanes and derivatives (dolastanes) for 1−9, xenicanes and derivatives (crenulides) for 19−27, and prenylated guaianes for 10−18. All compounds were evaluated for their antiviral activities against human immunodeficiency virus type 1 (HIV-1) and the highly pathogenic avian influenza A (H5N1) virus and for inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages (PEMΦ). Herein we report the details of isolation, structure elucidation, and bioactivity of these diterpenes. © XXXX American Chemical Society and American Society of Pharmacognosy
Received: September 5, 2014
A
dx.doi.org/10.1021/np5006955 | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Chart 1
Compound 2 has the same molecular formula as that of 1 according to its HRESIMS and NMR data (Tables 1 and 2). It was revealed to be a geometrical isomer of 1 with respect to the C-7/C-8 double bond on the basis of extensive 2D NMR spectroscopic analysis and by comparison of NMR data with those of 1. The difference was found by the significant downfield shift of H3-17 (δH 2.01, s) and C-17 (δC 21.8, CH3) in comparison with those of 1, suggesting the 7Z geometry13,14 in 2. This assumption was further confirmed by the NOE correlation of H3-17/H-7 (Figure 2). The relative configurations at C-1, C-2, C-11, and C-12 were assigned to be identical to those of 1 by the NOE correlations of H-2 (δH 5.08, d, J = 10.2 Hz)/H-11 (δH 2.04, d, J = 9.6 Hz), H3-15 (δH 1.20, s)/H-10a (δH 2.80, dd, J = 13.8, 9.6 Hz), and H-11/H3-19 (δH 1.03, d, J = 6.6 Hz). The coupling constant value JH‑2/H‑3 (10.2 Hz) and the NOE correlations of H-3 (δH 5.53, d, J = 10.2 Hz)/H3-15 and H-2/H3-16 (δH 1.77, s) indicated the trans-axial orientation of H-2 and H-3 and E geometry of C-3/C-4 double bond. In addition, the coupling constant value JH‑5/H‑6 (12.6 Hz) suggested the C-5/C-6 double bond to be Z. Thus, compound 2 was determined as (1S*,2S*,3E,5Z,7Z,11R*,12R*)-2-acetoxy-12-hydroxydolabella3,5,7-trien-9-one. Compound 6 was assigned a molecular formula of C22H32O4 according to the HRESIMS and NMR data, requiring seven degrees of unsaturation. The 13C NMR spectrum of 6 displayed six carbon signals for a ketone (δC 210.7), an ester carbonyl (δC 170.4), and four olefinic carbons (δC 131.9 (C), 121.0 (CH), 120.6 (CH), and 128.9 (CH)), indicating 6 to be tricyclic
methyls, C-2 and C-12 were oxygenated, while C-3/C-4, C-5/ C-6, C-7/C-8, and C-9 were present as an α,β,γ,δ,ε,ζunsaturated ketone. In addition, the linkage of an isopropyl group to C-12 was inferred by the HMBC correlations from H3-19 (0.73, d, J = 6.6 Hz) and H3-20 (0.92, d, J = 6.6 Hz) to C-12. Thus, 1 was revealed to be a dolabellane-type diterpene, similar to the co-occurring analogue (1S*,2S*,3E,7E,11R*,12R*)-2-acetoxy-12-hydroxydolabella3,7-dien-9-one (4).12 The location of an acetoxy group at C-2 was determined by the HMBC correlation from H-2 (δH 5.13, d, J = 6.6 Hz) to a carbonyl carbon (δC 170.4), and thus C-12 was ascertained to be hydroxylated by its chemical shift (δC 87.0) and the fact that an oxygen atom remained in the molecule according to the HRESIMS data. The relative configurations of the stereogenic centers and the geometries of the double bonds in 1 were assigned on the basis of NOESY analysis. The NOE correlations of H3-15/H-10a (δH 3.03, dd, J = 13.8, 2.4 Hz), H-11/H3-19, and H-11/H-2 suggested the trans fusion of the two rings and the same orientation of H-11, H-2, and the isopropyl group, while the NOE correlations of H3/H-11, H3-16/H-5, H-5/H-6, H-6/H3-17, and H-7/H-10a allowed the 3E, 5Z, and 7E assignment in 1 (Figure 2). Additionally, the geometries of 3E and 7E could be confirmed by the diagnostic chemical shifts of C-16 and C-17 (60.0 >60.0 >60.0 >60.0 UDc
4.7 1.8 1.7 1.0 1.3
a The other compounds were inactive at 30 μM. bCC50: cytotoxicity against human embryonic kidney 293T cells. cUD: undetected.
In addition, all compounds were assayed for inhibitory activity against the highly pathogenic H5N1 (A/Viet Nam/ 1203/2004) infection. The strategy for this assay is to target the viral envelope glycoprotein, hemagglutinin (HA), which is involved in multiple steps during viral entry, including receptor binding, internalization, and fusion. An HIV-based pseudotyping system was used to screen the inhibitors against H5N1 entry,34 while VSVG/HIV was applied to evaluate the specificity of entry inhibitors.35 In this assay, 5, 13, 24, and 26 exhibited specific inhibition against HA-mediated viral entry with inhibition rates of 56%, 50%, 59%, and 62%, respectively, at 30.0 μM (Table 4), but the other compounds showed weak Table 4. Inhibition of HA-Mediated H5N1 Entrya compound
concentration (μM)
IRb (%)
CC50c (μmol/L)
5 13 24 26
30.0 30.0 30.0 30.0
56 50 59 62
>60.0 >60.0 >60.0 >60.0
The other compounds were inactive at 30 μM. bIR: inhibition rate against H5N1 entry. cCC50: cytotoxicity against 293T cells.
a
activity (inhibition rate
