Punctaporonins H–M: Caryophyllene-Type Sesquiterpenoids from the Sponge-Associated Fungus Hansfordia sinuosae

Abstract

Six new caryophyllene-based sesquiterpenoids named punctaporonins H–M (1–6), together with punctaporonin B (7) and humulane (8) were isolated from the fermentation broth of the sponge-derived fungus Hansfordia sinuosae. Their structures were determined by the extensive HRESIMS and NMR spectroscopic analysis, including the X-ray crystallographic data for the assignment of the absolute configurations of punctaporonins H–I (1–2). The isolated compounds were evaluated for antihyperlipidemic, cytotoxic and antimicrobial activities, and punctaporonin K (4) exhibited potent effects to reduce the triglycerides and total cholesterol in the intracellular levels.

1. Introduction

Caryophyllene-based sesquiterpenoids are a group of structurally unique natural products characterized by the presence of a bicyclo[2.7.0]undecane skeleton, in addition to the backbone-rearranged polycyclic derivatives [1]. The majority of caryophyllene-related analogues have been isolated from terrestrial plants [2,3,4] and the plant-associated fungal species [5,6,7,8,9,10,11,12]. Marine-derived caryophyllane-type compounds were found from the soft coral Sinularia nanolobata [13,14] and the gorgonian coral Subergorgia suberosa [15], while fuscoatrol A is the only caryophyllene sesquiterpene isolated from the marine fungus Humicola fuscoatra [16]. Some caryophyllene derivatives exhibited immunosuppressive [9], cytotoxic [15], and antibiotic activities [17,18]. As part of our ongoing search for the chemical diversity from marine-derived microorganisms, the sponge (Niphates sp.) associated fungus Hansfordia sinuosae (WGCA-25-3A) was isolated. Chemical examination of the ethyl acetate extract of the solid fermented H. sinuosae resulted in the isolation of six new sesquiterpenoids (1–6) together with two known analogues (Figure 1).

Figure 1. Structures of punctaporonins H–M (1–6), punctaporonin B (7) and humulane (8).

Figure 1. Structures of punctaporonins H–M (1–6), punctaporonin B (7) and humulane (8).

2. Results and Discussion

2.1. Structure Elucidation

The molecular formula of punctaporonin H (1) was determined as C₁₇H₂₆O₅ on the basis of its HRESIMS and NMR data, requiring five degrees of unsaturation. The IR absorptions at 3350 and 1708 cm⁻¹ suggested the presence of hydroxy and carbonyl groups. Inspection of the ¹³C NMR and DEPT spectra revealed seventeen carbon resonances characterized by four olefinic carbons for two double bonds and a carbonyl carbon, while the ¹H NMR spectrum displayed four methyl singlets, three olefinic protons, three methylene, and an oxymethine. Thus, the remaining degrees of the molecular unsaturation accounted for a bicyclic skeleton. A cyclobutane ring was evident from the COSY correlation between H-9 (δ_H 3.26, dd, J = 8.4, 11.0 Hz) and H₂-10 (δ_H 1.42, 2.02) and their HMBC interactions with C-1 (δ_C 80.9) and C-11 (δ_C 40.6). Additional COSY relationships established the segments from CH-5 to CH-7 and CH-2 to CH₂-3. The connection of the segments across a quaternary carbon C-4 (δ_C 72.1) was deduced by the HMBC correlations from H₃-15 (δ_H 1.08 s) to C-3 (δ_C 44.9), C-4 (δ_C 72.1) and C-5 (δ_C 143.2), while C-4 was co-positioned by a methyl and a hydroxy group. The linkage of C-2 (δ_C 74.6) to C-1 and C-8 (δ_C 139.0) to C-9 (δ_C 39.0) to form a cyclononene ring was ascribed to the HMBC relationships from H-9 to C-7 (δ_C 123.6), C-8 and C-2, in addition to the correlations from H-7 (δ_H 5.81, brs) to C-9 and H-2 (δ_H 4.90, brd, J = 3.6 Hz) to C-1 and C-9. The location of a hydroxymethylene at C-8 was confirmed through the HMBC correlations from H₂-14 (δ_H 3.87, 4.13, d, J = 13.2 Hz) to C-7, C-8 and C-9, whereas two methyl groups resonated at δ_H 1.03 (s) and 0.98 (s) were co-positioned at C-11 on the basis of their protons correlated to C-1, C-10 (δ_C 33.5) and C-11 in the HMBC spectrum. In addition, the HMBC correlation between H-2 and the acetyl carbonyl carbon (δ_C 170.8) clarified an acetoxy unit to be substituted at C-2. Thus, the gross structure was established as a caryophyllene-type sesquiterpene. The J_H-5/H-6 (13.2 Hz) value and the NOE interaction between H-7 and H₂-14 were in agreement with 5Z and 7E geometries. Additional NOE interactions from H₃-12 to H-9 and H-2 indicated the same orientation of H-9 and H-2. The orientation of OH-1 was supposed to be opposite to H-9 on the basis of the NOE interaction between H-9 and H-2. The NOE correlations observed from H-15 to H-3b (δ_H 2.75) as well as from H-2 to H-3a (δ_H 1.36) indicated the opposite face of H₃-15 toward H-2. Thus, the structure of 1 was determined as a C-2 acetoxylated punctaporonin B [6]. The absolute configuration of 1 was determined as 1S, 2S, 4S, and 9R by the results of the X-ray single-crystal diffraction using Flack parameters (Supplementary Information, Tables S1–S7 and Figure 2) [19].

Figure 2. X-ray crystal structures of punctaporonins H (1) and I (2).

Figure 2. X-ray crystal structures of punctaporonins H (1) and I (2).

The NMR spectroscopic data of punctaporonin I (2) were closely related to those of 1, in fact both the compounds had the same molecular composition. However, C-6 (δ_C 66.1) was found to be a hydroxymethine group based on the HSQC correlation, and was located in the allylic position of the two double bonds according to the COSY and HMBC correlations observed. The latter displayed long range contacts from H₃-15 (δ_H 1.83, s) to C-3 (δ_C 40.3), C-4 (δ_C 127.4) and C-5 (δ_C 133.4), revealing one of the double bonds to be between C-4/C-5. The comparable NOE interactions of 1 and 2 in addition to the NOE correlations from H-6 (δ_H 4.70) to H-9 (δ_H 3.04) and H₃-15 and between H-5 (δ_H 5.17) and H-3b (δ_H 2.42, dd, J = 10.9, 10.8 Hz) confirmed a syn orientation for H-6 and H-9 and 4E geometry. Analysis of the X-ray single-crystal diffraction data (Supplementary Information, Tables S8–S14) revealed the absolute configurations of 2 to be 1S, 2S, 6R, and 9R. It is noteworthy that 2 is an unstable compound, which was partly converted to 4Z isomer 2a with an ratio of 5:4 (2/2a) during the measurement of 2D NMR spectra in DMSO-d₆ (Figure 3). This assignment was supported by the NOE interactions between H₃-15/H-5 and H-7/H₂-14 for 2a.

Figure 3. Auto-conversion of 2 to 2a in DMSO.

Figure 3. Auto-conversion of 2 to 2a in DMSO.

The molecular formula of punctaporonin J (3) was established as C₂₃H₃₅NO₇ by the HRESIMS data (m/z 438.2481 [M + H]⁺). The difference between 2 and 3 found in the NMR spectra was the presence of resonances of an exo-methylene at δ_H 4.79, 5.02/δ_C 119.6 and δ_C 143.5, an exchangeable proton at δ_H 7.79, and a side chain identified as (2E)-5-hydroxy-3-methylpent-2-enoyl group. The position of the exo-methylene group at C-4 was evident from its protons correlated to C-3 (δ_C 35.1), C-4 (δ_C 143.5) and C-5 (δ_C 62.5) in the HMBC spectrum. The side chain consisted of two olefinic carbons at δ_C 120.6 (C-2′) and 149.5 (C-3′), a carbonyl carbon at δ_C 166.4 (C-1′), two methylenes and a methyl carbon. The COSY relationship between H₂-5′ (δ_H 2.21, t) and H₂-6′ (δ_H 3.54, t), in association with the HMBC interactions from H₃-4′ (δ_H 2.05, s ) to C-2′, C-3′ and C-5′ (δ_C 44.1) and from H-2′ (δ_H 5.76, s) to C-1′, C-4′ (δ_C 18.2) and C-5′ and the NOE interaction between H-2′ and H₂-5′, established a (2E)-5-hydroxy-3-methylpent-2-enamide. The amide proton δ_H 7.79 (d) showing the COSY relationship with H-5 (δ_H 4.10, t) and the HMBC interactions with C-4, C-5 and C-6 confirmed the linkage of the amide moiety to C-5. The similar NOE interactions for the backbone of 3 and 2 in addition to the NOE correlations from H-9 (δ_H 3.26) to H-5 and H-6 (δ_H 4.36) assigned the same orientation of both H-5 and H-6 as H-9 (Figure 4).

Figure 4. Key HMBC, COSY and NOE correlations of 3 (ChemBioDraw Ultra 12.0, Cambridgesoft, Cambridge, MA, USA).

Figure 4. Key HMBC, COSY and NOE correlations of 3 (ChemBioDraw Ultra 12.0, Cambridgesoft, Cambridge, MA, USA).

The structure of punctaporonin K (4) was determined as 2-deacetoxy analogue of 3 on the basis of the similar NMR data of both compounds with the exception of the absence of acetoxy group and the presence of a methylene at C-2 (δ_C 32.9, δ_H 1.57, 1.94, m).

The molecular formula of punctaporonin L (5) was established as C₁₇H₂₆O₅ by its HRESIMS (m/z 333.1677 [M + Na]⁺) and NMR data. Comparison of the NMR data revealed the structure of 5 closely related to 6-hydroxypunctaporonin E [6]. The distinction was attributed to C-2 (δ_C 71.4) of 5 being substituted by an acetoxy group (δ_C 170.2, 21.4; δ_H 1.94, s) instead of a hydroxy group, as evident from the HMBC interaction between H-2 (δ_H 4.53, dd, J = 4.8, 11.2 Hz) and the acetyl carbonyl carbon (δ_C 170.2). The relative configurations of 5 were in accordance with those of 6-hydroxypunctaporonin E on the basis of the similar NOE interactions of both compounds. Since the absolute configurations of 6-hydroxypunctaporonin E were determined through X-ray diffraction, the same sign and similar magnitude of the specific optical rotations for both compounds led us to assign 5 as possessing the same absolute configurations as the known analogue.

The NMR data for punctaporonin M (6) were closely similar to those of 5, except that H₃-15 (δ_H 0.85, s) of 6 showed an HMBC correlation with an oxygenated methine carbon (δ_C 80.0, C-5) instead of an olefinic carbon, and H₂-14 (δ_H 3.46, 3.53) exhibited the correlation with an olefinic carbon C-7 (δ_C 132.2), indicating that C-5 of 6 was substituted by a hydroxy group and an olefinic bond resided at C-6/C-7. The similar ROE correlations of 5 and 6 (Figure 5), in association with the NOE interaction of H-2 (δ_H 4.94, dd, J = 5.0, 10.8 Hz) showing with H-5(δ_H 4.77, brs), H-9 (δ_H 1.98, m), and H₃-12 (δ_H 1.08, s), confirmed the same face of these protons. Additional NOE interaction between H₂-14 and H₃-15 was in agreement with the cis-fusion of the bicyclic ring (Figure 5).

Figure 5. Key NOE interactions of 5 and 6 (ChemBioDraw Ultra 12.0).

Figure 5. Key NOE interactions of 5 and 6 (ChemBioDraw Ultra 12.0).

Two known analogues were identical to punctaporonin B (7) [11] and humulane (8) [10] on the basis of the comparison of their NMR and specific optical rotations with those reported in literature.

2.2. Bioassay Results

All compounds showed weak cytotoxic activity against a panel of tumor cell lines including human colon carcinoma HCT-8, human hepatoma Bel7402, human gastric carcinoma BGC823, human lung adenocarcinoma A549, and human ovarian carcinoma A2780 with IC₅₀ values >10 μM. These compounds also showed weak inhibitory effects against the bacterial strains of Escherichia coli, Staphylococcus aureus, Bacillus thuringensis, and Bacillus subtilis with the MIC values more than 125 μM.

Compounds 1–4 and 7–8 were tested for the lowering effects against oleic acid (OA)-elicited lipid accumulation in HepG2 liver cells. Compound 4 significantly reduced the OA-elicited lipid accumulation as measured by the oil-red O staining (Figure 6) [20], while the lowering effects of the intracellular total cholesterol (TC) and triglyceride (TG) quantification of 4 (Figure 7) were comparable to those induced by the positive control lovastatin and were in a dose dependent manner.

Figure 6. Effects of compounds on oleic acid-elicited intracellular lipid accumulation. Cells were incubated with DMEM (Dulbecco’s Modified Eagle Medium) + oleic acid (OA, 100 μM) for 12 h, and then treated with 10 μM of each compound with lovastatin as a positive control. The blank group was tested in DMEM alone, while DMEM + 100 μM OA was used as a negative control. Neutral lipids were determined by spectrophotometry at 358 nm after oil-red O staining. Bars depict the means ± SEM (standard error of mean) of at least three experiments. *** p < 0.001, OA vs. Blank; ^††† p < 0.001, test group vs. OA group.

Figure 6. Effects of compounds on oleic acid-elicited intracellular lipid accumulation. Cells were incubated with DMEM (Dulbecco’s Modified Eagle Medium) + oleic acid (OA, 100 μM) for 12 h, and then treated with 10 μM of each compound with lovastatin as a positive control. The blank group was tested in DMEM alone, while DMEM + 100 μM OA was used as a negative control. Neutral lipids were determined by spectrophotometry at 358 nm after oil-red O staining. Bars depict the means ± SEM (standard error of mean) of at least three experiments. *** p < 0.001, OA vs. Blank; ^††† p < 0.001, test group vs. OA group.

High cholesterol and/or triglyceride problems are known to increase risk for hypertension, diabetes mellitus, obesity and others that impact on coronary artery disease [21]. Lipid overaccumulation in liver is also a key cause for the development of insulin resistance [22,23]. High triglyceride levels are also a risk factor for acute pancreatitis. Decreasing lipid accumulation in liver is therefore beneficial for the prevention and treatment of diabetes. Modulating the dysregulation of lipid metabolism and decreasing the elevated levels of serum TC and TG are helpful for the treatment and prevention of cardiovascular disease [24]. Natural molecules such as resveratrol, cordycepin and chlorogenic acid have been investigated to be the capability of suppressing the lipogenesis and fat accumulation in liver tissues that induced obesity and diabetes [25,26]. However, regulation of lipid accumulation in cell level by caryophyllene-based derivatives has not been reported. In the present work, we reported caryophyllene-type analogue 4 as a new natural scaffold which could potently induce the reduction of TC and TG in liver cells for the first time.

Figure 7. Inhibitory effects of 4 toward triglycerides and total cholesterol. Intracellular levels of triglycerides and total cholesterol were measured by kits according to the manufacturer’s instructions. Bars depict the means ± SEM of at least three experiments. *** p < 0.001, OA vs. Blank; ^†† p < 0.01, ^††† p < 0.001, test group vs. OA group. OA: oleic acid.

Figure 7. Inhibitory effects of 4 toward triglycerides and total cholesterol. Intracellular levels of triglycerides and total cholesterol were measured by kits according to the manufacturer’s instructions. Bars depict the means ± SEM of at least three experiments. *** p < 0.001, OA vs. Blank; ^†† p < 0.01, ^††† p < 0.001, test group vs. OA group. OA: oleic acid.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were measured using an Autopol III automatic polarimeter (Rudolph Research Co., Hackettstown, NJ, USA). Melting points were measured on JGW-360A melting point apparatus (Xmchongda, Tech. Co., Xiamen, China). IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer (Thermo Fisher Scientific Inc., New York, NY, USA). NMR spectra were measured on a Bruker Avance-500 FT NMR spectrometer (Bruker Co., Bremen, Germany) (500 MHz) using TMS as the internal standard. HRESIMS spectra were obtained on a FT-MS-Bruker APEX IV (7.0 T) (Bruker Co., Bremen, Germany). X-ray diffraction was obtained on Bruker D8 Advance single crystal X-ray Diffractometer (Bruker Co., Karlsruhe, Germany). TLC detection was carried out using precoated silica gel GF₂₅₄ plates (Yantai Chem. Ind., Yantai, China). Column chromatography was performed on Silica gel (200–300 mesh, Qingdao Marine Chemical Plant, Qingdao, China). Sephadex LH-20 (18–110 μm) was obtained from Pharmacia Co. (Peapack, NJ, USA), and ODS (50 μm) was provided by YMC Co. (Kyoto, Japan) High-performance liquid chromatography (HPLC) was performed on a Waters e2695 Separation Module (Waters, Milford, USA) coupled with a Waters 2998 photodiode array detector (Waters, Milford, CT, USA). A Kromasil C₁₈ semipreparative HPLC column (250 × 10 mm, 5 μm) (EKS Chemicals, Bohus, Sweden) was used for compound purification. All solvents were of analytical grade.

3.2. Fungal Material and Fermentation

The fungal strain Hansfordia sinuosae was isolated from the sponge of Niphates sp. collected from Southern China Sea. The fungus was identified by morphological observation and analysis of the ITS region of the rDNA, whose sequence data have been deposited at GenBank with the accession number KF877718. The strain (WGCA-23-3A) was deposited at the State Key Laboratory of Natural and Biomimetic Drugs, Peking University, China. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25 °C for 10 days. Spore inoculum was prepared by suspension in distilled H₂O to give a final spore/cell suspension of 1 × 10⁶/mL. A large scale fermentation was carried out in fifty 500 mL Fernbach flasks each containing 80 g of rice. Distilled H₂O with sterilization brine (100 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 lb/in² (pound per square inch) for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 25 °C for 35 days.

3.3. Extraction and Isolation

The fermented material was extracted with EtOAc (3 × 10 L), and the organic layer was evaporated to dryness under vacuum to afford a crude extract (99.0 g). The extract was fractionated by a silica gel packed vacuum liquid chromatography (VLC) using petroleum ether–EtOAc gradient (5:1 to 1:1) as the eluent to afford six fractions (F1–F6). F4 (2.1 g) was subjected to an ODS column (10 µm) eluting with a MeOH–H₂O gradient (from 30% to 100%) to yield five subfractions (F4A–F4E). F4A (150 mg) was subjected to a Sephadex LH-20 column eluting with MeOH to yield 8 (3.2 mg) and 7 (11.9 mg). F4E (300 mg) was separated on semipreparative HPLC (ODS) with 45% Acetonitrile-H₂O as a mobile phase to afford 1 (3.0 mg, Rt 38.8 min) and 2 (32.3 mg, Rt 32.0 min). F5 (557 mg) was subjected to Sephadex LH-20 column eluting with MeOH to give 3 (13.0 mg) and 4 (5.8 mg), while 5 (1.8 mg, Rt 45.4 min) and 6 (3.0 mg, Rt 29.4 min) were separated by semipreparative HPLC with 36% Acetonitrile-H₂O as a mobile phase.

Punctaporonin H (1). Colorless crystal. Mp. 152–154 °C, −83 (c 0.3, CH₃OH). UV (CH₃OH) λ_max 208, 198 nm; IR (KBr) ν_max 3350, 2961, 2934, 2870, 1708, 1530, 1383, 1263, 1122, 1024 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 333.1675 [M + Na]⁺ (calcd for C₁₇H₂₆NaO₅, 333.1672).

Punctaporonin I (2). Colorless crystal. Mp. 198–199 °C, −139 (c 0.22, CH₃OH). UV (CH₃OH) λ_max 216, 198 nm; IR (KBr) ν_max 3343, 2932, 2870, 1714, 1648, 1620, 1463, 1373, 1256, 1122 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 333.1669 [M + Na]⁺ (calcd for C₁₇H₂₆NaO₅, 333.1672).

Punctaporonin J (3). Colorless oil, −37 (c 0.65, CH₃OH). UV (CH₃OH) λ_max 221.1 196.5 nm; IR (KBr) ν_max 3326, 2939, 2873, 1715, 1664, 1633, 1537, 1438, 1373, 1260, 1047, 1026 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 438.2481 [M + H]⁺ (calcd for C₂₃H₃₆NO₇, 438.2486).

Punctaporonin K (4). Colorless oil. −64 (c 0.58, CH₃OH). UV (CH₃OH) λ_max 222, 195 nm; IR (KBr) ν_max 3295 (br), 2936, 2870, 1723, 1663, 1629, 1533, 1439, 1365, 1202, 1060 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 380.2429 [M + H]⁺ (calcd for C₂₁H₃₄NO₅, 380.2431).

Table 1. ¹³C and ¹H NMR data for 1–6 in DMSO-d₆.

No	1		2		3		4		5		6
	δC	δH (J in Hz)		δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)
1	80.9		81.2		83.4		82.7		77.8		77.5
2	74.6	4.90, brd (3.6)	76.1	5.11, dd (10.9, 4.0)	77.4	5.08, dd (9.6, 5.2)	32.9	1.57, m; 1.94, m	71.4	4.53, dd (11.2, 4.8)	70.8	4.94, dd (10.8, 5.0)
3	44.9	1.36, d (15.8) 2.75, dd (15.8, 3.6)	40.3	2.13, dd (10.8, 4.0) 2.42, dd (10.9, 10.8)	35.1	2.19, dd (11.0, 9.6) 2.22, dd (11.0, 5.2)	28.7	1.95, brd (12.2) 2.05, m	39.0	1.66, dd (12.3, 4.8) 1.80, dd (12.3, 11.2)	36.5	1.62, dd (13.0, 10.8) 1.81, dd (13.0, 5.0)
4	72.1		127.4		143.5		148.3		52.6		54.4
5	143.2	5.75, d (13.2)	133.4	5.17, d (9.8)	62.5	4.10, t (8.0)	62.3	4.24, t (8.5)	145.6	5.71, d (5.6)	80.0	4.77, brs
6	124.3	5.63, brd (13.2)	66.1	4.70, dd, (9.8, 4.2)	69.5	4.36, dd (9.0, 8.0)	69.6	4.43, dd (8.5,9.2)	130.1	5.61, dd (5.6, 2.6)	136.1	5.52, brd (6.0)
7	123.6	5.81, brs	141.8	5.83, s	133.3	5.38, d (9.0)	131.5	5.36, d (9.2)	80.3	3.94, d (2.6)	132.2	5.48, d (6.0)
8	139.0		136.1		136.1		137.2		49.4		53.5
9	39.0	3.26, dd (11.0, 8.4)	40.6	3.04, dd (10.2, 7.9)	40.8	3.26, t (10.0)	43.9	3.16, dd (9.0, 8.7)	42.7	2.00, m	45.3	1.98, m
10	33.5	1.42, dd (9.1, 8.4) 2.02, dd (11.0, 9.1)	33.3	1.57, dd (12.0, 10.2) 1.92, dd (12.0, 7.9)	33.8	1.43, t (10.0) 2.04, t (10.0)	33.7	1.46, dd (9.0, 8.7) 2.02, t (9.0)	35.4	1.50, m 2.00, m	35.4	1.39, dd (5.0, 4.5) 2.00, m
11	40.6		41.6		41.0		40.9		41.6		42.5
12	24.5	1.03, s	24.5	0.94, s	24.7	1.08, s	24.3	1.07, s	23.6	1.02, s	23.7	1.08, s
13	24.1	0.98, s	25.5	1.02, s	24.0	0.98, s	22.5	0.86, s	24.3	0.98, s	24.3	0.97, s
14	63.4	3.87, d (13.2) 4.13, d (13.2)	65.4	3.78, d (11.2) 4.11, d (11.2)	64.3	3.76, d (11.0) 3.94, d (11.0)	64.3	3.74, d (12.4) 3.90, d (12.4)	60.3	3.50, d (11.0) 3.93, d (11.0)	61.9	3.46, d (11.0) 3.53, d (11.0)
15	31.9	1.08, s	17.5	1.83, s	119.6	4.79, s; 5.02, s	117.9	4.66, s; 4.86, s	27.3	1.15, s	17.8	0.85, s
1′					166.4		166.4
2′					120.6	5.76, s	120.6	5.76, s
3′					149.5		149.4
4′					18.2	2.05, s	18.2	2.07, s
5′					44.1	2.21, t (6.2)	44.1	2.20, t (6.2)
6′					59.6	3.54, t (6.5)	59.5	3.54, t (6.5)
Ac	170.8		170.1		169.8				170.2		170.1
	21.6	1.99, s	21.7	1.96, s	21.5	1.97, s			21.4	1.94, s	21.4	1.97, s
NH						7.79, d (8.0)		7.92, d (8.5)

Table 1. ¹³C and ¹H NMR data for 1–6 in DMSO-d₆.

No	1		2		3		4		5		6
	δC	δH (J in Hz)		δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)
1	80.9		81.2		83.4		82.7		77.8		77.5
2	74.6	4.90, brd (3.6)	76.1	5.11, dd (10.9, 4.0)	77.4	5.08, dd (9.6, 5.2)	32.9	1.57, m; 1.94, m	71.4	4.53, dd (11.2, 4.8)	70.8	4.94, dd (10.8, 5.0)
3	44.9	1.36, d (15.8) 2.75, dd (15.8, 3.6)	40.3	2.13, dd (10.8, 4.0) 2.42, dd (10.9, 10.8)	35.1	2.19, dd (11.0, 9.6) 2.22, dd (11.0, 5.2)	28.7	1.95, brd (12.2) 2.05, m	39.0	1.66, dd (12.3, 4.8) 1.80, dd (12.3, 11.2)	36.5	1.62, dd (13.0, 10.8) 1.81, dd (13.0, 5.0)
4	72.1		127.4		143.5		148.3		52.6		54.4
5	143.2	5.75, d (13.2)	133.4	5.17, d (9.8)	62.5	4.10, t (8.0)	62.3	4.24, t (8.5)	145.6	5.71, d (5.6)	80.0	4.77, brs
6	124.3	5.63, brd (13.2)	66.1	4.70, dd, (9.8, 4.2)	69.5	4.36, dd (9.0, 8.0)	69.6	4.43, dd (8.5,9.2)	130.1	5.61, dd (5.6, 2.6)	136.1	5.52, brd (6.0)
7	123.6	5.81, brs	141.8	5.83, s	133.3	5.38, d (9.0)	131.5	5.36, d (9.2)	80.3	3.94, d (2.6)	132.2	5.48, d (6.0)
8	139.0		136.1		136.1		137.2		49.4		53.5
9	39.0	3.26, dd (11.0, 8.4)	40.6	3.04, dd (10.2, 7.9)	40.8	3.26, t (10.0)	43.9	3.16, dd (9.0, 8.7)	42.7	2.00, m	45.3	1.98, m
10	33.5	1.42, dd (9.1, 8.4) 2.02, dd (11.0, 9.1)	33.3	1.57, dd (12.0, 10.2) 1.92, dd (12.0, 7.9)	33.8	1.43, t (10.0) 2.04, t (10.0)	33.7	1.46, dd (9.0, 8.7) 2.02, t (9.0)	35.4	1.50, m 2.00, m	35.4	1.39, dd (5.0, 4.5) 2.00, m
11	40.6		41.6		41.0		40.9		41.6		42.5
12	24.5	1.03, s	24.5	0.94, s	24.7	1.08, s	24.3	1.07, s	23.6	1.02, s	23.7	1.08, s
13	24.1	0.98, s	25.5	1.02, s	24.0	0.98, s	22.5	0.86, s	24.3	0.98, s	24.3	0.97, s
14	63.4	3.87, d (13.2) 4.13, d (13.2)	65.4	3.78, d (11.2) 4.11, d (11.2)	64.3	3.76, d (11.0) 3.94, d (11.0)	64.3	3.74, d (12.4) 3.90, d (12.4)	60.3	3.50, d (11.0) 3.93, d (11.0)	61.9	3.46, d (11.0) 3.53, d (11.0)
15	31.9	1.08, s	17.5	1.83, s	119.6	4.79, s; 5.02, s	117.9	4.66, s; 4.86, s	27.3	1.15, s	17.8	0.85, s
1′					166.4		166.4
2′					120.6	5.76, s	120.6	5.76, s
3′					149.5		149.4
4′					18.2	2.05, s	18.2	2.07, s
5′					44.1	2.21, t (6.2)	44.1	2.20, t (6.2)
6′					59.6	3.54, t (6.5)	59.5	3.54, t (6.5)
Ac	170.8		170.1		169.8				170.2		170.1
	21.6	1.99, s	21.7	1.96, s	21.5	1.97, s			21.4	1.94, s	21.4	1.97, s
NH						7.79, d (8.0)		7.92, d (8.5)

Punctaporonin L (5). White powder. −48 (c 0.10, CH₃OH); UV (CH₃OH) λ_max 199 nm; IR (KBr) ν_max 3336, 2952, 2933, 2870, 1729, 1682, 1529, 1454, 1350, 1251, 1028 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 333.1677 [M + Na]⁺ (calcd for C₁₇H₂₆O₅Na, 333.1672).

Punctaporonin M (6). White powder. +49 (c 0.12, CH₃OH). UV (CH₃OH) λ_max 199 nm; IR (KBr) ν_max 3265, 2957, 2933, 2865, 1718 (br), 1463, 1377, 1249, 1017 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 333.1677 [M + Na]⁺ (calcd for C₁₇H₂₆O₅Na, 333.1672).

3.4. X-Ray Single Crystallographic Analyses

Punctaporonin H (1) and punctaporonin I (2) were crystallized from MeOH-CH₂Cl₂ (1:1) at room temperature. The X-ray crystallographic data of both compounds (see Supplementary information) were obtained on a Bruker SMART CCD detector employing graphite monochromated Cu-Kα radiation (operated in the φ-ω scan mode). The structures were solved by direct method using SHELXS-97 [27] and refined with full-matrix least-squares calculations on F2 using SHELXL-97 [27].

Crystal data of punctaporonin H: C₁₇H₂₆O₅, M = 310.38, monoclinic, a = 9.3925(11) Å, b = 9.3490(9) Å, c = 9.8735(9) Å, β = 107.503(12)°, U = 826.86(15) ų, T = 98.5, space group P2₁ (No. 4), Z = 2, μ(Cu Kα) = 0.741, 5653 reflections measured, 2970 unique (R_int = 0.0315) which were used in all calculations. The final wR(F₂) was 0.1288 (all data).

Crystal data of punctaporonin I: C₁₇H₂₆O₅, M = 310.38, orthorhombic, a = 11.2742(10) Å, b = 11.7414(11) Å, c = 12.020(2) Å, U = 1591.2(4) ų, T = 99.7, space group P2₁2₁2₁ (No. 19), Z = 4, μ(Cu Kα) = 0.771, 5650 reflections measured, 2964 unique (R_int = 0.0301) which were used in all calculations. The final wR(F₂₎ was 0.0970 (all data).

The crystallographic data for the structures of punctaporonins H (1) and I (2) have been deposited in the Cambridge Crystallographic Data Centre (deposition numbers: CCDC 974950 for 1 and CCDC 974951 for 2). Copies of the data can be obtained free of charge, on application to the director, CCDC [28], 12 Union Road, Cambridge CB21EZ, UK (Fax: +44-(0)1223-336033, or E-Mail: [email protected]).

3.5. Cell-Based Lipid Accumulation Assay

HepG2 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 μg/mL). The cells with 70%–80% confluence were incubated in DMEM + oleic acid (100 μM) for 12 h, then were treated with the compounds (each, 10 μM) and a positive control lovastatin in DMEM/100 μM oleic acid with DMEM/100 μM oleic acid as a blank for additional 6 h. Subsequently, the cells were subjected to oil-red O staining or TC and TG determination as described previously [20]. Each experiment (n = 8 for oil-red O staining or n = 3 for TC and TG determination) was repeated in triplication.

3.6. Cytotoxic and Antibacterial Assays

The cytotoxic assay was performed by a standard MTT method. Antimicrobial activities were measured against the bacterial strains of Escherichia coli, Staphylococcus aureus, Bacillus thuringensis, and Bacillus subtilis, by the broth microdilution method. The bacteria were grown for 16 h on a rotary shaker at 37 °C. Cultures were diluted with sterile medium to achieve an optical absorbance of 0.4–0.06 at 600 nm, then further diluted 10-fold before transferring into 96-well microtiter plates. Three replicates of each compound were tested in 125 μg/mL. The optical absorbance at 600 nm was measured after cultivation for 18 h. The lowest concentrations that completely inhibited visible growth of the tested strains were recorded from three independent experiments.

4. Conclusions

In biogenetic consideration, the tricyclic 5 was depicted to be derived from 1 through a transannular cyclization [29] by C-4/C-8 cycloaddition, whereas 6 was suggested to be derived from 5 via olefinic rearrangement. The 5-hydroxy-3-methyl-2-pentenamide unit in 3 and 4 was found from nature for the first time, while this unit was biogenetically considered to be generated through the mevalonate pathway. However, the mechanism for introducing nitrogen to form an amide instead of an ester is unknown. The significant effects of compound 4 to reduce the triglycerides and total cholesterol in the intracellular levels suggested it could be a potent lead compound for the development of a drug candidate for lowering lipids.