Further New Xenicanes from a Chinese Collection of the Brown Alga Dictyota plectens

Abstract

Four new xenicanes, namely 4α-hydroxyisodictyohemiacetal (1), 4α-hydroxyisodictyoacetal (2), 13,18-diacetoxy-4-hydroxyisodictyo-19-al (3), and 4α-hydroxypachylactone (8), were isolated from a Chinese collection of the brown alga Dictyota plectens, along with four known analogues (4–7). The structures of the new diterpenes were determined by extensive spectroscopic data analysis. 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 inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages (PEMΦ).

Brown algae of the genus Dictyota (family Dictyotaceae) are significant producers of bioactive secondary metabolites, especially diterpenes. In the past four decades, about 400 diterpenes of more than 15 chemical classes have been isolated from Dictyota species.¹⁾ These diterpenes can be biosynthetically distributed into three groups: xenicanes, dolabellanes, and “extended sesquiterpenes.” Biological studies have confirmed that many of these dictyota diterpenes to possess significant ecological and pharmaceutical activities, such as antifouling, antifeeding, antibacterial, cytotoxic, and antiviral properties.^2–10)

In our previous work,¹¹⁾ we have reported the isolation of 27 diterpenes from the brown alga Dictyota plectens of the South China Sea and their anti-inflammatory and antiviral activities against human immunodeficiency virus type 1 (HIV-1) and the highly pathogenic avian influenza A (H5N1) virus. The significant biological activities, especially antiviral properties, of some of these metabolites encouraged our further chemical investigation on the same collection of this species and resulted in the isolation of another eight xenicanes including four new ones. Herein, we report the isolation, structural elucidation, and bioactivities of these xenicanes.

Results and Discussion

Further isolation of compounds from the EtOAc-soluble portion of the EtOH extract of the brown alga Dictyota plectens was accomplished by repeated column chromatography with the aid of ¹H-NMR guided fractionation, affording three new xenicane-type diterpenes 4α-hydroxyisodictyohemiacetal (1), 4α-hydroxyisodictyoacetal (2), and 13,18-diacetoxy-4-hydroxyisodictyo-19-al (3), a new xenicane derivative, crenulide-type diterpene 4α-hydroxypachylactone (8), along with four known analogues (4–7). The structures of known compounds were identified as isodictyohemiacetal (4),¹²⁾ isodictyoacetal (5),¹³⁾ (2S*,3S*,4R*,10R*,19R*)-19-deoxo-4-hydroxy-19-methoxydictyolactone (6),⁵⁾ and 4-hydroxydictyolactone (7)^14,15) by comparison of their ¹H- and ¹³C-NMR spectroscopic and optical rotations with those reported in the literature (Fig. 1).

Fig. 1. Structures of Compounds 1–8

Compound 1 was isolated as a colorless oil. Its molecular formula was determined to be C₂₀H₃₂O₃ based on high resolution-electrospray ionization-mass spectrum (HR-ESI-MS) data (m/z 343.2244 [M+Na]⁺), implying five degrees of unsaturation. The ¹H-NMR spectrum of 1 showed four methyl signals including three olefinic methyl singlets at δ_H 1.92 (3H, s, H₃-20), 1.68 (3H, s, H₃-16), and 1.59 (3H, s, H₃-15) and one methyl doublet at δ_H 1.06 (3H, d, J=6.6 Hz, H₃-17) (Table 1), while the ¹³C-NMR spectrum exhibited 20 carbon signals including six olefinic carbons and three oxygenated carbons (Table 2). The six olefinic carbon signals at δ_C 145.5 (C, C-1), 134.3 (C, C-6), 131.3 (C, C-14), 128.2 (CH, C-7), 124.4 (CH, C-13), and 119.3 (CH, C-9), along with three olefinic proton signals at δ_H 5.60 (1H, dd, J=8.4, 3.6 Hz, H-9), 5.37 (1H, dd, J=10.8, 3.6 Hz, H-7), and 5.07 (1H, t, J=6.6 Hz, H-13), were attributed to three trisubstituted double bonds. The presence of a five-membered ring hemiacetal was revealed by the diagnostic NMR signals for an hemiacetal methine [δ_C 101.3 (CH, C-18); δ_H 5.61 (1H, br s, H-18)] and an oxymethylene [δ_C 71.7 (CH₂, C-19); δ_H 4.41 (1H, d, J=12.0 Hz, H-19a) and 4.38 (1H, d, J=12.0 Hz, H-19b)], in combination with the heteronuclear multiple bond connectivity (HMBC) correlations from the hemiacetal methine proton to the oxymethylene carbon and from the hemiacetal methine and oxymethylene protons to the same two carbons at δ_C 145.5 (C, C-1) and 46.9 (CH, C-2) (Fig. 2). Four degrees of unsaturation, accounted for by the functional groups from five in the molecular, indicated the remaining of a cyclic structure in 1. In addition, a 6-methylhept-5-en-2-yl side chain was established by the ¹H–¹H correlation spectroscopy (COSY) relationships of H-13/H₂-12 (δ_H 1.93, m), H₂-12/H₂-11 (δ_H 1.23, m; 1.13, m), H₂-11/H-10 (δ_H 1.90, m), H-10/H₃-17, and H-10/H-3 (δ_H 2.04, br s), and the HMBC correlations from H₃-15 and H₃-16 to C-13 and C-14 and from H₃-17 to C-10 (δ_C 31.4, CH) and C-11 (δ_C 38.5, CH₂). All of these NMR data are characteristic of a xenicane-type diterpene closely related to the co-occurring analogue isodictyohemiacetal (4).¹²⁾ The locations of two double bonds at C-6/C-7 and C-1/C-9 were confirmed by the HMBC correlations from H₃-20 and the oxymethylene protons H₂-19 to olefinic carbons C-6 and C-7, C-1 and C-9, as well as the ¹H–¹H COSY correlations between H-7/H₂-8 [δ_H 3.17 (1H, ddd, J=15.6, 10.8, 3.6 Hz, H-8a); 2.66 (1H, ddd, J=15.6, 8.4, 3.6 Hz, H-8b)] and H₂-8/H-9. Moreover, the ¹H–¹H COSY correlations between H₂-5 [δ_H 2.34 (1H, dd, J=13.2, 1.8 Hz, H-5a); 2.15 (1H, dd, J=13.2, 3.6 Hz, H-5b)] and an oxymethine proton at δ_H 4.29 (1H, dd, J=3.6, 1.8 Hz, H-4) disclosed that C-4 (δ_C 73.2, CH) was hydroxylated. The relative configurations at C-2, C-3, C-10, and C-18, and the geometries of C-6/C-7 and C-1/C-9 double bonds in 1 were consistent with those of 4 as revealed by the similar weak proton–proton couplings between H-2 (δ_H 2.93, br s)/H-18, H-2/H-3 (δ_H 2.04, br s), and H-3/H-10 and significant nuclear Overhauser effect spectroscopy (NOESY) correlations between H-2/H₃-20, H-3/H-7, and H-9/H₂-19 (Fig. 3), while the relative configuration at C-4 was assigned to be identical to that of (2S*,3S*,4R*,10R*,19R*)-19-deoxo-4-hydroxy-19-methoxydictyolactone (6)⁵⁾ by the compatible chemical shift of C-4 and weak coupling between H-3 and H-4. Thus, compound 1 was determined as 4α-hydroxyisodictyohemiacetal.

Table 1. ¹H-NMR Spectroscopic Data of Compounds 1–3 and 8 (600 MHz, CDCl₃)

Position	1	2	3	8
2	2.93 br s	2.91 br s	3.23 br t (7.2)
3	2.04 br s	2.02 br s	1.87 br s	2.83 d (11.4)
4	4.29 dd (3.6, 1.8)	4.28 dd (3.6, 1.8)	4.24 dd (3.6, 1.8)	4.30 br t (3.0)
5	2.34 dd (13.2, 1.8)	2.33 dd (13.2, 1.8)	2.43 dd (13.2, 1.8)	1.88 m
	2.15 dd (13.2, 3.6)	2.15 dd (13.2, 3.6)	2.08 dd (13.2, 3.6)	1.70 m
6				1.25 m
7	5.37 dd (10.8, 3.6)	5.37 dd (10.8, 3.6)	5.24 br d (11.4)	1.08 m
8	3.17 ddd (15.6, 10.8, 3.6)	3.17 ddd (15.6, 10.8, 3.6)	3.34 ddd (15.0, 11.4, 3.0)	0.91 m
	2.66 ddd (15.6, 8.4, 3.6)	2.65 ddd (15.6, 8.4, 3.6)	3.01 dd (15.0, 7.8)	0.17 dd (10.8, 5.4)
9	5.60 dd (8.4, 3.6)	5.52 dd (8.4, 3.6)	6.84 dd (7.8, 3.0)	1.70 m
10	1.90 m	1.94 m	2.32 m	2.49 m
11	1.23 m	1.23 m	1.11 m	1.29 m
	1.13 m	1.12 m	1.01 m	0.94 m
12	1.93 m	1.94 m	1.56 m	1.94 m
			1.47 m
13	5.07 t (6.6)	5.08 t (6.6)	5.06 t (6.6)	5.06 t (6.6)
15	1.59 s	1.59 s	4.92 br s	1.57 s
			4.87 br s
16	1.68 s	1.68 s	1.68 s	1.66 s
17	1.06 d (6.6)	1.07 d (6.6)	1.00 d (6.6)	1.01 d (6.6)
18	5.61 br s	5.09 br s	4.62 dd (10.2, 6.6)
			4.49 dd (10.2, 8.4)
19	4.41 d (12.0)	4.40 d (12.0)	9.32 s	4.77 d (18.0)
	4.38 d (12.0)	4.33 d (12.0)		4.55 d (18.0)
20	1.92 s	1.92 s	1.99 s	1.02 d (6.6)
OMe		3.31
13-OAc			2.04 s
18-OAc			1.99 s

Table 2. ¹³C-NMR Spectroscopic Data of Compounds 1–3 and 8 (150 MHz, CDCl₃)

Position	1	2	3	8
1	145.5 (C)	146.0 (C)	149.7 (C)	163.0 (C)
2	46.9 (CH)	45.7 (CH)	37.3 (CH)	132.2 (C)
3	49.2 (CH)	49.2 (CH)	49.9 (CH)	47.6 (CH)
4	73.2 (CH)	73.2 (CH)	74.9 (CH)	70.2 (CH)
5	49.1 (CH2)	49.2 (CH2)	48.5 (CH2)	46.7 (CH2)
6	134.3 (C)	134.3 (C)	138.1 (C)	28.7 (CH)
7	128.2 (CH)	128.1 (CH)	124.9 (CH)	26.6 (CH)
8	29.0 (CH2)	29.0 (CH2)	29.3 (CH2)	8.0 (CH2)
9	119.3 (CH)	118.4 (CH)	157.3 (CH)	13.2 (CH)
10	31.4 (CH)	31.3 (CH)	31.6 (CH)	30.3 (CH)
11	38.5 (CH2)	38.5 (CH2)	32.1 (CH2)	35.6 (CH2)
12	26.1 (CH2)	26.1 (CH2)	30.7 (CH2)	24.7 (CH2)
13	124.4 (CH)	124.5 (CH)	77.4 (CH)	124.4 (CH)
14	131.3 (C)	131.5 (C)	142.7 (C)	131.3 (C)
15	17.7 (CH3)	17.7 (CH3)	112.7 (CH2)	17.5 (CH3)
16	25.7 (CH3)	25.7 (CH3)	18.0 (CH3)	25.7 (CH3)
17	18.4 (CH3)	18.4 (CH3)	17.6 (CH3)	18.1 (CH3)
18	101.3 (CH)	107.8 (CH)	63.8 (CH2)	176.1 (C)
19	71.7 (CH2)	71.2 (CH2)	196.2 (CH)	72.2 (CH2)
20	20.0 (CH3)	20.0 (CH3)	20.1 (CH3)	23.8 (CH3)
OMe		54.5 (CH3)
13-OAc			170.1 (C)
			21.2 (CH3)
18-OAc			170.7 (C)
			21.0 (CH3)

Fig. 2. COSY and HMBC Correlations of Compounds 1, 3 and 8

Fig. 3. Key NOE Correlations and Computer-Generated Models Using MM2 Force Field Calculations for Compounds 1, 3 and 8

Compound 2 has a molecular formula of C₂₁H₃₄O₃ as determined by HR-ESI-MS data (m/z 357.2401 [M+Na]⁺), requiring five degrees of molecular unsaturation. Detailed analysis of one- and two-dimensional (1D)- and (2D)-NMR data revealed that 2 had a structure closely related to those of 1 and isodictyoacetal (5).¹³⁾ In comparison with 1, the only difference was found by the presence of a methoxy group [δ_C 54.5 (CH₃); δ_H 3.31 (3H, s)], indicating a methylated derivative of 1. The methoxy group was attached to the acetal carbon C-18 (δ_C 107.8) as evidenced by the HMBC correlations from the methoxy protons to the acetal methine carbon C-18 and from the acetal methine proton H-18 (δ_H 5.09, br s) to C-1 (δ_C 146.0, C), C-2 (δ_C 45.7, CH), C-3 (δ_C 49.2, CH), and the methoxy carbon. The relative configuration of 2 was suggested to be identical to that of 1 based on the similar NOE relationships. Thus, compound 2 was defined as 4α-hydroxyisodictyoacetal, which could be a methoxylated artifact of 1 formed during the isolation process.

The molecular formula of compound 3 was determined to be C₂₄H₃₆O₆ by HR-ESI-MS data (m/z 443.2406 [M+Na]⁺), indicating seven degrees of unsaturation. The ¹H- and ¹³C-NMR spectra of 3 showed signals for one conjugated aldehyde [δ_C 196.2 (C, C-19); δ_H 9.32 (1H, s)], two acetyls [δ_C 170.7 (C), 170.1 (C), 21.2 (CH₃), 21.0 (CH₃); δ_H 2.04 (3H, s), 1.99 (3H, s)], and three olefinic double bonds including a terminal [δ_C 142.7 (C, C-14), 112.7 (CH₂, C-15); δ_H 4.92 (1H, br s, H-15a), 4.87 (1H, br s, H-15b)] and two trisubstituted [δ_C 157.3 (CH, C-9), 149.7 (C, C-1), 138.1 (C, C-6), 124.9 (CH, C-7); δ_H 6.84 (1H, dd, J=7.8, 3.0 Hz, H-9), 5.24 (1H, br d, J=11.4 Hz, H-7) ] ones. Thus, a monocyclic structure could be assigned for 3 according to the remaining one degree of unsaturation. In addition, two oxymethines [δ_C 77.4 (CH, C-13), 74.9 (CH, C-4); δ_H 5.06 (1H, t, J=6.6 Hz, H-13), 4.24 (1H, dd, J=3.6, 1.8 Hz, H-4)] and one oxymethylene [δ_C 63.8 (CH₂, C-18); δ_H 4.62 (1H, dd, J=10.2, 6.6 Hz, H-18a), 4.49 (1H, dd, J=10.2, 8.4 Hz, H-18b)] were present. These NMR data were very compatible with those of the co-occurring xenicane-type diterpene 18-acetoxy-4-hydroxydictyo-19-al.^11,12,16) The difference was attributed to the presence of an additional acetoxy and a terminal double bond as described above, but the absence of an olefinic methyl in comparison with the known analogue. Further detailed analysis of HMBC and ¹H–¹H COSY data revealed that 3 shared the same partial structure of 18-acetoxy-4-hydroxydictyo-19-al with the exception of the side chain (Fig. 2). The HMBC correlations from the olefinic methyl protons H₃-16 (δ_H 1.68, s) to an oxymethine carbon C-13 and the two carbons of a terminal double bond (C-14 and C-15) and from the oxymethine proton H-13 to a carbonyl carbon at δ_C 170.1 disclosed the presence of an isopropenyl at the end of the side chain and the attachment of an acetoxy to C-13 in 3. The relative configurations at C-2, C-3, C-4, and C-10, as well as the geometries of C-6/C-7 and C-1/C-9 double bonds were defined to be in agreement with those of 18-acetoxy-4-hydroxydictyo-19-al by the similar NOE relationships (Fig. 3) in association with the chemical shifts and coupling constants, while the configuration at C-13 remained to be determined. Thus, 3 was elucidated as 13,18-diacetoxy-4-hydroxyisodictyo-19-al.

Compound 8 was assigned a molecular formula of C₂₀H₃₀O₃ according to the HR-ESI-MS data (m/z 341.2084 [M+Na]⁺), implying six degrees of unsaturation. The presence of an α,β-unsaturated γ-lactone was indicated by the NMR signals for an ester carbonyl (δ_C 176.1, C-18), two olefinic carbons at δ_C 163.0 (C, C-1) and 132.2 (C, C-2), and one oxymethylene [δ_C 72.2 (CH₂, C-19); δ_H 4.77 (1H, d, J=18.0 Hz, H-19a), 4.55 (1H, d, J=18.0 Hz, H-19b)], in association with the IR absorption bands at 1758 and 1662 cm⁻¹. A cyclopropane moiety was recognized by the diagnostic upfield proton signal at δ_H 0.17 (1H, dd, J=10.8, 5.4 Hz, H-8b). In addition, two olefinic carbons [δ_C 131.3 (C, C-14), 124.4 (CH, C-13)], two olefinic methyl singlets [δ_H 1.66 (3H, s, H₃-16), 1.57 (3H, s, H₃-15)], two methyl doublets [δ_H 1.02 (3H, d, J=6.6 Hz, H₃-20), 1.01 (3H, d, J=6.6 Hz, H₃-17)], and a secondary alcohol [δ_C 70.2 (CH, C-4); δ_H 4.30 (1H, br t, J=3.0 Hz, H-4)] were present in the ¹³C- and ¹H-NMR spectra. A 6-methylhept-5-en-2-yl side chain was further established on the basis of the ¹H–¹H COSY and HMBC correlations similar to those depicted in 1. All the above data are consistent with a crenulide-type diterpene, represented by pachylactone¹⁷⁾ and the co-occurring analogue isoacetoxycrenulatin.^11,17) Finally, the gross structure of 8 was established as 4-hydroxypachylactone by the HMBC correlations from the oxymethylene protons H₂-19 to an aliphatic methine carbon C-9 (δ_C 13.2), the carbonyl carbon C-18, and two olefinic carbons C-1 and C-2, from H-3 (δ_H 2.83, d, J=11.4 Hz) to the carbonyl carbon C-18, from the oxymethine proton H-4 to an olefinic carbon C-2 and an aliphatic methine carbon C-6 (δ_C 28.7), and from the methyl protons H₃-20 to a methylene carbon C-5 (δ_C 46.7) and two methine carbons C-7 (δ_C 26.6) and C-6, in combination with the ¹H–¹H COSY correlations between the oxymethine proton H-4 and H₂-5 [δ_H 1.88 (1H, m, H-5a), 1.70 (1H, m, H-5b)] (Fig. 2). The relative configurations of the stereogenic centers in 8 were suggested to be in agreement with those of isoacetoxycrenulatin based on the similar NMR data including the weak coupling between H-3 and H-4, the relatively large coupling constant (11.4 Hz) between H-3 and H-10, and NOE relationships of H-3/H-9 (δ_H 1.70, m), H-3/H-5a, H-5a/H-7 (δ_H 1.08, m), H-7/H-9, H-8b/H-19b, and H-4/H₃-17 (Fig. 3). Thus, 8 was elucidated as 4α-hydroxypachylactone.

All compounds were assayed for the in vitro anti-HIV-1 replication activity, the result showed that 1 and 6 were active against the replication of wild-type HIV-1 virus with inhibitory concentration 50% (IC₅₀) of 28.1 and 25.4 µM, respectively (the positive control nevirapine, IC₅₀ 0.05 µM), while the other compounds were inactive at concentration of 30.0 µM. In addition, all compounds were assessed for their inhibitory activities against the hemagglutinin (HA)-mediated highly pathogenic H5N1 (A/Viet Nam/1203/2004) infection using an HIV-based pseudotyping system.¹⁸⁾ In the primary assay, 7 showed specific inhibition against HA-mediated viral entry with an inhibition rate of 66.8% at 30.0 µM, however the other compounds were inactive (inhibition rate <50%) at this concentration. Moreover, all compounds were tested for the in vitro anti-inflammatory activity. The bioassay result revealed that 7 and 8 could effectively inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages (PEMФ) with inhibition rates of 76.0% and 53.2%, respectively, at 10.0 µM, whereas the other compounds showed weak activity (inhibition rate <50%) at this concentration.

Experimental

General Experimental Procedures

IR spectra were recorded on a Bruker Equinox 55 spectrometer. Optical rotations were obtained on a PoLAAR 3005 digital polarimeter. 1D- and 2D-NMR spectra were acquired on a Bruker Avance-600 FT NMR spectrometers using tetramethylsilane (TMS) as an internal standard. Chemical shifts (δ) were expressed in parts per million (ppm), and coupling constants (J) were reported in Hertz (Hz). HR-ESI-MS data were determined by a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer. Silica gel (200–300 mesh, Qingdao Marine Chemistry Co., Ltd.), Sephadex LH-20 (GE Healthcare Biosciences AB), and octadecyl silica (ODS) (50 µm, YMC) were used for column chromatography. TLC analysis was carried out using Precoated silica gel plates (Merck, kieselgel-60 F₂₅₄, 0.25 mm). Semipreparative HPLC was performed on an Agilent 1100 series instrument equipped with a YMC-Pack C₁₈ (10 µm, 250×10 mm) column. 293T cells were obtained from ATC C. Vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped HIV-1 vector NL4-3-luc was supplied by Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union College. A codon-optimized HA gene from A/Viet Nam/1203/2004 (H5N1) was cloned into pcDNA3. LPS (1 µg/mL), 4% sodium thioglycolate, fetal bovine serum (FBS), RPMI1640, phosphate buffered saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Griess reagents were supplied by Sigma-Aldrich, while penicillin/streptomycin, Dulbecco’s modified Eagle’s medium (DMEM) medium, and dexamethasone were purchased from Gibco.

Plant Material

The brown alga Dictyota plectens (Allender & Kraft) was collected in April 2013 from the Xuwen coast, Zhanjiang, China. The specimen was identified by Dr. Zhong-min Sun (Institute of Oceanology, CAS, Qingdao, China). A voucher specimen (GZ201301) is deposited at the Laboratory of Marine Natural Products Chemistry, Wenzhou Medical University, China.

Extraction and Isolation

The protocols for the extraction and prefractionation were described in the previous work.¹¹⁾ The air-dried algal material (160 g) was extracted with 95% EtOH at room temperature (r.t.) for three times. The concentrated extract (22.5 g) was partitioned between H₂O and EtOAc. The EtOAc fraction (5.0 g) was subjected to a silica gel vacuum column, eluting with a gradient of EtOAc–petroleum ether (1 : 20, 1 : 10, 1 : 5, 1 : 3, 1 : 2), to afford eight fractions (A1–A8). Fraction A2 (310.5 mg), found to contain terpenoids as detected by ¹H-NMR spectrum, was subjected to a Sephadex LH-20 column (70×2.5 cm), using CH₂Cl₂–MeOH (1 : 1) as the mobile phase, to afford three fractions (A2a–A2c). Fraction A2c (120.3 mg) was subsequently separated on an ODS column (C₁₈, 25×2 cm) to obtain five fractions (A2c1–A2c5). Fraction A2c3 (25.8 mg) was purified by semipreparative HPLC (MeOH–H₂O, 85 : 15) to yield 5 (4.7 mg). Fraction A3 (1.1 g) was fractionated on a Sephadex LH-20 column eluting with CH₂Cl₂–MeOH (1 : 1) to afford three fractions (A3a–A3c). Fraction A3b (450.2 mg) was subjected to an ODS column, using MeOH–H₂O (75 : 25, 80 : 20, 85 : 15, 90 : 10) as eluent, to yield five fractions (A3b1–A3b5). Fraction A3b4 (52.0 mg) was purified by HPLC (MeOH–H₂O, 70 : 30) to obtain 4 (6.5 mg). Fraction A4 (890.4 mg) was subjected to Sephadex LH-20 column, eluting with CH₂Cl₂–MeOH (1 : 1), to afford two fractions (A4a, A4b). Fraction A4b (582.0 mg) was subsequently separated on an ODS column, eluting with MeOH–H₂O (70 : 30, 75 : 25, 80 : 20), to obtain 11 fractions (A4b1–A4b11). Fraction A4b3 (63.5 mg) was further purified by HPLC (MeOH–H₂O, 65 : 35) to afford 3 (5.8 mg). In the same manner, fractions A4b6 (39.2 mg) and A4b9 (45.4 mg) were eluted with MeOH–H₂O (72 : 28, 80 : 20, respectively) to obtain 7 (5.0 mg) and 8 (6.7 mg), respectively. Fraction A7 (204.0 mg), also contained terpenoids as detected by ¹H-NMR spectrum, was separated on a Sephadex LH-20 column (CH₂Cl₂–MeOH, 1 : 1) to afford three fractions (A7a–A7c). Fraction A7b (92.4 mg) was purified by HPLC (MeOH–H₂O, 75 : 25) to obtain 1 (3.8 mg), 2 (5.7 mg), and 6 (7.9 mg).

4α-Hydroxyisodictyohemiacetal (1)

Colorless oil; [α]_D²⁵ −210.0 (c=0.10, CHCl₃); IR (KBr) ν_max 3431, 2956, 2923, 2858, 1456, 1383, 1095 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-ESI-MS (m/z) 343.2244 [M+Na]⁺ (Calcd for C₂₀H₃₂O₃Na, 343.2249).

4α-Hydroxyisodictyoacetal (2)

Colorless oil; [α]_D²⁵ −164.0 (c=0.10, CHCl₃); IR (KBr) ν_max 3448, 2960, 2918, 2857, 1453, 1381, 1099 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-ESI-MS (m/z) 357.2401 [M+Na]⁺ (Calcd for C₂₁H₃₄O₃Na, 357.2406).

13,18-Diacetoxy-4-hydroxyisodictyo-19-al (3)

Colorless oil; [α]_D²⁵ −43.3 (c=0.12, CHCl₃); IR (KBr) ν_max 3447, 2923, 1732, 1692, 1377, 1239, 1027 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-ESI-MS (m/z) 443.2406 [M+Na]⁺ (Calcd for C₂₄H₃₆O₆Na, 357.2410).

4α-Hydroxypachylactone (8)

Colorless oil; [α]_D²⁵ +42.7 (c=0.16, CHCl₃); IR (KBr) ν_max 3450, 2924, 2859, 1758, 1662, 1457, 1380, 1241, 1036 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-ESI-MS (m/z) 341.2084 [M+Na]⁺ (Calcd for C₂₀H₃₀O₃Na, 341.2093).

Assay for Anti-HIV-1 Activity

A cell-based VSVG/HIV-1 pseudotyping system was used for evaluating the anti-HIV replication activity as described previously.¹⁹⁾ Briefly, vesicular stomatitis virus glycoprotein (VSV-G) plasmid was cotransfected with env-deficient HIV-1 vector (PNL4-3.luc.R⁻E⁻) into human embryonic kidney 293T cells based on a modified Ca₃(PO₄)₂ method.²⁰⁾ Sixteen hours post-transfection, plates were washed by PBS, and fresh medium (DMEM with 10% FBS) was added into the plates. Forty-eight hours post-transfection, supernatant containing VSVG/HIV-1 virions was harvested and filtered through a 0.45 µm filter. Viral solution was quantified in terms of p24 concentrations, which were detected by ELISA, then diluted to 0.2 ng p24/mL for direct use or storage at −80°C.

For the infection assay, 293T cells were plated on 24-well plates at the density of 6×10⁴ cells per well, one day prior to infection. Compounds, dissolved in DMSO, were added into target cells and incubated for 15 min prior to adding VSVG/HIV-1. The same amount of solvent was used as blank control, while nevirapine was employed as positive control. Forty-eight hours post-infection, infected cells were lysed in 50 µL of Cell Lysis Reagent. Luciferase activity of the cell lysate was determined by a Sirius luminometer according to the manufacturer’s instructions.

Assay for Inhibition of H5N1 Entry

A previously established protocol¹¹⁾ was followed. Hemagglutinin envelope expression plasmid was co-transfected with NA and Env-deficient HIV vector, PNL4-3.luc.R⁻E⁻, into 293T cells by a standard Ca₃(PO₄)₂ protocol. Sixteen hours post-transfection, cells were washed by PBS without Ca²⁺ and Mg²⁺, and then 10 mL of fresh medium was added into each plate. Forty-eight hours post-transfection, the supernatants were harvested and filtered through a 0.45 µm filter, and the pseudovirions were ready for infection. In addition, the 293T cells and test compounds were prepared for infection assay following the protocol described in the anti-HIV-1 activity assay. Then, the prepared HIV pseudovirions (0.5 mL/well) were incubated with the targeted cells. For HIV-Luc virions, the targeted cells were lysed in 50 µL of cell culture lysis reagent, 48 h post-transfection. The luciferase activity was measured by the same method as described in the anti-HIV-1 activity assay.

Assay for Inhibition of LPS-Induced NO Production

A previously established protocol²¹⁾ was employed except that 30 mM of dexamethasone in DMSO was used as the positive control and each test compound (30 mM in DMSO) was diluted to 1–30 µM range at r.t. before the experiment.

Acknowledgments

This research was supported by Grants from ZJNSFC (No. LQ12B02002), CSC (No. 201408330121), NSFC (No. 21202123 and 32100163), and Start-Up Funding from Wenzhou Medical University (No. QTJ10018). We are grateful to the Key Laboratory of Laboratory Medicine, Ministry of Education, China, for the measurements of HR-ESI-MS spectra.

Conflict of Interest

The authors declare no conflict of interest.

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