Leptogorgins A-C, Humulane Sesquiterpenoids from the Vietnamese Gorgonian Leptogorgia sp.

Leptogorgins A-C (1-3), new humulane sesquiterpenoids, and leptogorgoid A (4), a new dihydroxyketosteroid, were isolated from the gorgonian Leptogorgia sp. collected from the South China Sea. The structures were established using MS and NMR data. The absolute configuration of 1 was confirmed by a modification of Mosher's method. Configurations of double bonds followed from NMR data, including NOE correlations. This is the first report of humulane-type sesquiterpenoids from marine invertebrates. Sesquiterpenoids leptogorgins A (1) and B (2) exhibited a moderate cytotoxicity and some selectivity against human drug-resistant prostate cancer cells 22Rv1.

1. Introduction

Marine gorgonian corals have been reported to be a rich source of isoprenoids with unprecedented chemical structures and biological activities [1]. Species of the genus Leptogorgia (Gorgoniidae) have been shown to produce cembranoids [2,3,4,5,6,7], polyoxygenated steroids [8,9,10,11,12], alkaloids [13], fatty acids [14], homarine [15], thyroxine, and vitamin D [16]. To date, different humulane-type sesquiterpenoids have been found in plants [17,18,19], liverworts [20], and fungi [21,22,23]. However, until recently they were not found in marine invertebrates, including gorgonians. Interestingly, two new norhumulene were isolated from the soft coral Sinularia hirta [24]. In addition, one more norhumulene was found in a formazan soft coral Sinularia gibberosa [25]. Humulanes from the peeled stems of Syringa pinnatifida inhibit NO production in LPS-induced RAW264.7 macrophage cells and decrease the TNF-α and IL-6 levels in RAW264.7 cells [26]. Additionally, plant cytochrome P450 was reported to catalyse the conversion of α-humulene into 8-hydroxy-α-humulene [27].

For some humulenes, an antitumor activity was reported. Thus, zurumbone (2,6,9-humulatriene-8-one), as an active component of the Zingiber aromaticum extract, was shown to be active in human cancer HT-29, CaCO-2, and NCF-7 cell lines. Remarkably, it was more active than curcumin, which was used as a reference compound [28]. Herein, we report the structures and biological activities of three new humulane sesquiterpenoids, leptogorgins A–C (1–3), and a new steroid, leptogorgoid A (4), from the gorgonian Leptogorgia sp. (Figure 1).

Figure 1

The structures of 1−4.

2. Results and Discussion

The EtOH extract of the gorgonian Leptogorgia sp. (registration number O38-011) was concentrated and partitioned between aqueous EtOH and n-hexane. The EtOH-soluble materials were separated by silica gel flash chromatography, followed by Sephadex LH-20 column chromatography and normal and reversed-phase HPLC to give leptogorgins A–C (1–3, 2.5, 0.8, and 1.0 mg, respectively) and leptogorgoid A (4, 0.6 mg).

Compound 1 was isolated as a colourless oil. The HRESIMS of 1 showed an [M + Na]+ ion peak at m/z 273.1459 and an [M − H]− ion peak at m/z 249.1498, which indicated a molecular formula of C15H22O3. The 13C NMR spectrum displayed 15 signals, which could be assigned to a sesquiterpene substructure. Analysis of the 1H, 13C, and HSQC NMR spectra (Table 1) revealed signals indicative of one ketocarbonyl (δC 200.8, C-4), one oxymethine (δH 4.21/δC 71.7, C-7), one oxymethylene (δH 4.25; 4.38/δC 64.7, C-12), four methines (δH 6.32/δC 164.8, C-2; δH 5.97/δC 128.1, C-3; δH 5.75/δC 133.8, C-6, and δH 5.22/δC 125.9, C-10), two quaternary (δC 143.0, C-5; δC 132.4, C-9) olefinic carbons, and two methylene groups (δH 1.96 and 2.68/δC 45.3, C-8; δH 1.95 and 2.40/δC 40.7, C-11), as well as one quaternary carbon (δC 38.0, C-1), one corresponding vinylic methyl (δH 1.72/δC 20.1, CH3-13) and two methyl singlets (δH 1.18/δC 24.0, CH3-14; δH 1.13/δC 29.1, CH3-15). The 1H-1H COSY spectrum enabled three structural fragments to be established: CH=CH-, -CH-CH-CH2-, and -CH-CH2-, which could be connected by observing the correlations in the HMBC experiment (Figure 2). Thus, HMBC correlations from H-3 to C-1, C-4, and C-5, from H-6 to C-12 and C-8, from H-7 to C-5 and C-8, from H-8 to C-7, C-9, C-10, and C-13, from H-11 to C-10, C-9, and C-1, and from CH3-14 and CH3-15 to C-1, C-2, and C-11 established the planar structure of 1 (Figure 2).

Table 1

1H (700 MHz) and 13C (175 MHz) NMR spectroscopic data for 1, 2 and 3 in CDCl3.

Position	1		2		3
	δC	δH mult (J in Hz)	δC	δH mult (J in Hz)	δC	δH mult (J in Hz)
1	38.0 C	-	38.1 C	-	40.4 * C	-
2	164.8 CH	6.32, d(16.3)	162.8 CH	6.24, d(16.3)	152.7 CH	6.29, d(16.1)
3	128.1 CH	5.97, d(16.3)	128.1 CH	6.07, d(16.3)	128.4 CH	5.76, d(16.1)
4	200.8 C	-	199.4 C	-	204.3 C	-
5	143.0 C	-	145.2 C		48.6 CH	3.38, m
6	133.8 CH	5.75, d(10.6)	129.5 C	5.70, dt(10.6; 1.3)	41.2 CH2	2.43, dd(16.9; 2.9)
						2.73, dd(16.9; 9.7)
7	71.7 CH	4.21 td(10.6; 5.4)	72.9 CH	5.28, td(10.6; 5.1)	204.3 C
8	45.3 CH2	1.96, m	42.7 CH2	2.03, m	54.1 CH2	3.00, d(12.4)
		2.68, dd(12.2; 5.4)		2.69, dd(12.5; 5.1)		3.15, d(12.4)
9	132.4 C	-	128.1 C	-	127.8 C	-
10	125.9 CH	5.22, brd(12.5)	127.1 C	5.32, m	129.0 CH	5.37, ddd(10.5; 5.7, 1.2)
11	40.7 CH2	1.95, m	40.7	1.97, m	40.2 * CH2	2.00, m
		2.40, t(12.5)		2.39, t(12.6)		2.07, m
12	64.7 CH2	4.25, d(13.3)	64.8 CH2	4.26, dd(13.2; 4.6)	63.0 CH2	3.78, m
		4.38, d(13.3)		4.40, dd(13.2; 6.3)		3.89, m
13	20.1 CH3	1.72, s	20.0 CH3	1.73, s	19.0 CH3	1.64, s
14	24.0 CH3	1.18, s	23.9 CH3	1.21, s	28.8 CH3	1.21, s
15	29.1 CH3	1.13, s	29.2 CH3	1.13, s	24.3 CH3	1.09, s
COCH3			169.7 C	-
COCH3			21.2 CH3	1.98, s

* Signals may be interchangeable.

Figure 2

Selected COSY and HMBC correlations for 1–4.

The geometry of the Δ2,3 double bond was further determined to be E by considering the coupling constant (J = 16.3 Hz) displayed in its 1H NMR spectrum. The NOE correlations of CH3-13 to H-2, H-6, and CH2-11, as well as H-10 with H-6 and H-6 with H-2 (Figure 3), suggested that the Δ5(6) and Δ9(10) double bonds in 1 were E configured.

Figure 3

Key NOE correlations for 1.

A modified Mosher ester analysis was obtained, and the negative ΔδSR (δS − δR) values of Ha-8, (ΔδH −0.01), Hb-8, (ΔδH −0.05) and CH3-13 (ΔδH −0.01), and positive ΔδSR values of H-6 (ΔδH +0.04) Ha-12 (ΔδH +0.01), and Hb-12 (ΔδH +0.04) (Figure 4) revealed the 7S configuration [25]. Thus, the structure of 1 was determined as 4-oxohumula-2E,5E,9E-trien-7S,12-diol, as shown in Figure 1, and named leptogorgin A (1).

Figure 4

Δδ (δS − δR) values (in ppm, CDCl3) for the MTPA esters of 1.

Compound 2 was obtained as a colourless oil. The HRESIMS of 2 showed an [M + Na]+ ion peak at m/z 315.1567 and an [M − H]− ion peak at m/z 291.1602, which indicated a molecular formula of C17H24O4. The 1H and 13C NMR spectra of 2 (Table 1) were similar to those of 1, suggesting that this compound possessed the same humulane skeleton. The key differences were in δH for H-7 and δC for carbon 7 in the spectrum of 2 (δH 5.28/δC 72.9). The corresponding signals were shifted downfield, compared to those of 1 (δH 4.21/δC 71.7). This characteristic difference and HRESIMS data were caused by the hydroxy group in 1 being displaced by an acetoxyl group in 2. The HMBC spectra of 2 demonstrated the expected key correlations. The ECD spectrum of compound 2 was compared with the ECD spectrum of leptogorgin A (1), in which the corresponding absolute configuration was established by modification of Mosher’s method. Both ECD spectra displayed similar Cotton effects (see Figure S27), allowing us to establish the same 7S configuration for compound 2. From these data, compound 2 was determined to be 4-oxohumula-2E,5E,9E-trien-7S-acetate,12-ol, as shown in Figure 1, and named leptogorgin B (2).

Compound 3 was isolated as a colourless oil. The HRESIMS of 1 showed an [M + Na]+ ion peak at m/z 273.1459 and an [M − H]− ion peak at m/z 249.1496, which indicated a molecular formula of C15H22O3. The 1H and 13C NMR spectra (Table 1) of 3 were similar to those of 1 and 2, suggesting that this compound also possessed the same humulane skeleton. Key differences concerned δH for protons 6, 7, and 8 and δC for carbons 4, 5, 6, 7, and 8 in the spectrum of 3, which were different compared to those of 1 and 2. This characteristic difference was caused by an absence of the hydroxy group, as in 1, or acetyl, as in 2 at position 7, being displaced by a ketogroup in 3, as well as by the absence of the 5,6 double bound in 3. The location of the ketogroup was further determined to be at C-7 by COSY, HSQC, and HMBC experiments. Thus, compound 3 was determined to be 4,7-dioxohumula-2E,9E-dien-12-ol, as shown in Figure 1, and named leptogorgin C (3).

Compound 4 was isolated as a colourless powder. The HRESIMS of 4 showed an [M + Na]+ ion peak at m/z 437.3026 and an [M − H]− ion peak at m/z 413.3061, which indicated a molecular formula of C27H42O3. The data of 1D- and 2D-NMR spectra of 1 (Table 2) indicated that this compound belonged to steroids. Its spectra contained five methyl groups, including two angular methyl groups in the steroid nucleus (δH 0.74/δC 12.2, δH 1.19/δC 17.4) and three methyl groups of the side chain (δH 1.04/δC 20.3, δH 1.15/δC 23.8, and δH 1.20/δC 26.4), eight methylene groups, six methine groups, including one oxygenated methine (δH 3.85/δC 79.7), two quaternary sp3 carbons (δC 38.6, δC 42.5), one quaternary sp3 oxygenated carbon (δC 72.8), one trisubstituted double bond (δH 5.72/δC 123.8 and 171.4), one disubstituted double bond (δH 5.61/δC 140.8 and δH 5.43/δC 126.0), and one conjugated with double bond ketone carbonyl (δC 199.5). The geometry of the 22,23 double bond was further determined to be E by considering the coupling constant (J = 15.3 Hz) displayed in its 1H NMR spectrum. The HMBC spectra of 4 demonstrated the expected key correlations. From these data, compound 4 was determined to be 3-oxocholesta-4E,22E-diene-24,25 dienol, as shown in Figure 1, and named leptogorgoid A (4).

Table 2

1H (700 MHz) and 13C (175 MHz) NMR spectroscopic data for 4 in CDCl3.

Position	δC	δH mult (J in Hz)	Position	δC	δH mult (J in Hz)
1	35.7 CH2	1.70, m	16	28.5 CH2	1.29, m
		2.03, m			1.70, m
2	34.0 CH2	2.34, m	17	55.6 CH	1.19, m
		2.42, m
3	199.5	-	18	12.2 CH3	0.74, s
4	123.8 CH	5.72 s	19	17.4 CH3	1.19, s
5	171.4C	-	20	39.8 CH	2.14, m
6	32.9 CH2	2.27, ddd (14.7; 4.1; 2.4)	21	20.3 CH3	1.04, d (6.6)
		2.40, m
7	32.0 CH2	1.02, m	22	140.8 CH	5.61, dd (8.6; 15.3)
		1.84, m
8	35.7 CH	1.53, m	23	126.0 CH	5.43, dd (7.3; 15.3)
9	53.8 CH	0.94, m	24	79.7 CH	3.84, d (7.3)
10	38.6 C	-	25	72.8 C	-
11	21.0 CH2	1.44, ddd (13.6; 17.1; 4.2)	26	23.8 CH3	1.15, s
		1.54, m
12	39.5 CH2	1.20, m	27	26.4 CH3	1.20, s
		2.01, m
13	42.5 C	-
14	55.8 CH	1.04, m
15	24.2 CH2	1.11, m
		1.60, m

Next, we investigated the effects of the leptogorgins A (1) and B (2) on the viability of 22Rv1 cells (human drug-resistant prostate cancer cells) as well as on PNT2 cells (human prostate non-cancer cells). MTT assay revealed 1 to exhibit a moderate cytotoxicity to both cell lines (IC50 = 31.0 µM and 35.8 µM, respectively), whereas 2 had IC50 > 100 µM. Doxorubicine was used as a positive control and exhibited in 22Rv1 and PNT2 cells IC50 of 0.084 µM and 1.12 µM, respectively. Interestingly, both compounds were more active in human cancer 22Rv1 cells, in comparison with PNT2 cells (Figure 5). Additionally, we examined the ability of these compounds to inhibit the colony formation of 22Rv1 prostate cancer cells; however, no significant inhibitory activity was observed under the treatment with cytotoxic or non-cytotoxic concentrations of the compounds up to a concentration of 100 µM (data not shown). The isolated compounds may be considered as prototypes for future anticancer agents capable of selective inhibition of human drug-resistant prostate cancer cells. Note that we could not isolate enough leptogorgins C (3) and leptogorgoid A (4) to investigate the biological activity of these compounds.

Figure 5

The viability of 22Rv1 and PNT2 cells after 72 h of treatment with the indicated concentrations of the investigated compounds. The viability was evaluated using MTT assay.

3. Materials and Methods

3.1. General Procedures

Optical rotation was measured using a PerkinElmer 343 polarimeter. UV spectra were recorded on a Shimadzu UV-1601 PC spectrophotometer. ECD spectra were recorded with an Applied Photophysics Chirascan plus spectropolarimeter. IR spectroscopic data were measured using an IR spectrometer Equinox 55 (Bruker, Ettlingen, Germany) in CHCl3. The 1H and 13C NMR spectra were recorded on a Bruker Avance III-700 spectrometer (Bruker, Ettlingen, Germany) at 700 and 175 MHz, respectively, with Me4Si as an internal standard. ESI mass spectra (including HRESIMS) were obtained on a Bruker maXis Impact II Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) by direct infusion in MeOH. Low-pressure column liquid chromatography was performed using silica gel (Sigma-Aldrich Co., St. Louis, MO, USA) and Sephadex LH-20 (Sigma, Chemical Co., St. Louis, MO, USA) columns. HPLC was performed using a Shimadzu Instrument equipped with the differential refractometer RID-10A, a YMC-Pack ODS-A (250 × 10 mm) column (YM Co., Ltd., Kyoto, Japan), and a silica gel column (SUPELCOSILTM, 250 × 10 mm, 5 µm) (Sigma-Aldrich Co., USA). TLC was performed on silica gel plates (5–17 µm, Sorbfil, Russia).

3.2. Animal Material

The gorgonian Leptogorgia sp. (registration number PIBOC O38-011) was collected by dredging during the 38th scientific cruise of R/V “Academic Oparin”, May 2010, South China sea (09°08′2″ N; 109°01′7″ E, depth 134 m), in Vietnamese waters. A voucher specimen of 038-011 sample is stored in the Marine invertebrate collection of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry FEB RAS (Vladivostok, Russia).

3.3. Extraction and Isolation

The EtOH extract of the gorgonian (dry weight 170 g) was concentrated and partitioned between n-hexane and aqueous EtOH. The EtOH-soluble material was subjected to column chromatography on a silica gel column using CHCl3-EtOH (stepwise gradient, 1:0 1:1). Fractions eluted with CHCl3:EtOH (20:1) were concentrated and residue (171.3 mg) was subjected to column chromatography on a LH-20 column using CHCl3:EtOH, 2:1 to yield two fractions: F1 (46.6 mg) and F2 (61.4 mg). Preparative HPLC of the fraction F1 (SUPELCOSIL, n-hexane:EtOAc, 1:1) gave pure leptogorgin A (1, 2.5 mg, 0.002% based on dry weight of gorgonian). Preparative HPLC of the fraction F2 (YMC-Parck ODS-A, EtOH:H2O, 3:2) gave three sub-fractions: F2-1 (2.5 mg), F2-2 (6.4 mg), and F2-3 (8.0 mg). Preparative HPLC of the fraction F2-1 (SUPELCOSIL, n-hexane:EtOAc, 2:3) gave pure leptogorgin C (3, 1.0 mg, 0.001% based on dry weight of gorgonian). Preparative HPLC of the fraction F2-2 (SUPELCOSIL, n-hexane:EtOAc, 1:1) gave pure leptogorgin B (2, 0.8 mg, 0.001% based on dry weight of gorgonian). Preparative HPLC of the fraction F2-3 (SUPELCOSIL, n-hexane:EtOAc, 1:1) gave pure leptogorgoid A (4, 0.6 mg, 0.0006% based on dry weight of gorgonian).

3.4. Compound Characterization Data

Leptogorgin A (1): colourless oil; +38.7 (c 0.2, CHCl3); UV (EtOH) λmax (log ε) 195 (4.05), 229 (3.75) nm; ECD (c 1 × 10−3 M, EtOH) λmax (Δε) 194 (7.56), 228 (9.41), 274 (−3.52), 333 (1.30) nm; IR (CHCl3): νmax 3604, 2964, 2928, 2860, 1723, 1641, 1458, 1387, 1365, 1261, 1243, 1104, 1012 cm−1; 1H and 13C NMR data (CDCl3), Table 1; HRESIMS m/z 273.1459 [M + Na]+ (calcd for C15H22O3Na, 273.1461); HRESIMS m/z 249.1498 [M − H]− (calcd for C15H21O3 249.1496).

Leptogorgin B (2): colourless oil; +16 (c 0.1, CHCl3); UV (EtOH) λmax (log ε) 196 (3.23), 229 (3.07) nm; ECD (c 3 × 10−3 M, EtOH) λmax (Δε) 197 (2.90), 226 (1.41), 254 (−1.06), 336 (0.43) nm; 1H and 13C NMR data (CDCl3) Table 1; HRESIMS m/z 315.1571 [M + Na]+ (calcd for C17H24O4Na, 315.1567); HRESIMS m/z 291.1602 [M − H]− (calcd for C17H23O4 291.1602).

Leptogorgin C (3): colourless oil; 1H and 13C NMR data (CDCl3) Table 1; HRESIMS m/z 273.1463 [M + Na]+ (calcd for C15H22O3Na, 273.1461); HRESIMS m/z 249.1496 [M − H]− (calcd for C15H21O3 249.1496).

Leptogorgoid A (4): colourless powder; +33 (c 0.05, CHCl3); 1H and 13C NMR data (CDCl3) Table 1. HRESIMS m/z 437.3021 [M + Na]+ (calcd for C27HO3Na, 437.3026); HRESIMS m/z 413.3060 [M − H]− (calcd for C27H4103 413.3061).

MTPA esterification of1. To a part of 1 (0.6 mg) in dry C5H5N (1 µL), R-(−)-α-metoxy-α-trifluoromethylphenylacetyl chloride (10 µL) was added. The mixture was stirred on one hour at room temperature.and evaporated in vacuo to give (S)-MTPA diester 1a. By the same procedure, (R)-MTPA diester 1b was prepared.

(S)-MTPA diester (1a): Select 1H NMR data (CDCl3) see Table S1. HRESIMS m/z 707.25 [M + Na]+ (calcd for C35H38F6O7Na, 707.25).

(R)-MTPA diester (1b): Select 1H NMR data (CDCl3) see Table S1. HRESIMS m/z 707.25 [M + Na]+ (calcd for C35H38F6O7Na, 707.25).

3.5. Bioactivity Assay

3.5.1. Reagents

The MTT reagent (Thiazolyl blue tetrazolium bromide) was purchased from Sigma (Taufkirchen, Germany).

3.5.2. Cell Lines and Culture Conditions

The human prostate cancer cells 22Rv1 and human prostate non-cancer cells PNT2 were purchased from ATCC. Cell lines were cultured according to the manufacturer’s instructions in 10% FBS/RPMI media (Invitrogen, Carlsbad, CA, USA) and handled as described in [29].

3.5.3. In Vitro MTT-Based Drug Sensitivity Assay

The in vitro cytotoxic activities of the isolated substances were evaluated by MTT assays. The assays were performed as described previously [30]. In brief, cells were seeded in 96-well plates (6 × 103 cells/well), incubated overnight, and treated with the tested compounds for 72 h. Next, 10 μL/well of MTT reagent was added and the plates were incubated for 2 h. The media were aspirated and the plates were dried. The formed formazan crystals were dissolved in DMSO and the cell viability was measured using an Infinite F200PRO reader (TECAN, Männedorf, Switzerland). Results were calculated by the GraphPad Prism software v. 7.05 (GraphPad Prism software Inc., La Jolla, CA, USA) and are represented as the IC50 of the compounds against the control cells treated with the solvent alone.

3.5.4. Colony Formation Assay

Colony formation assay was performed as described before, with slight modifications [30]. Cells were treated with the drug for 48 h; then, cells were trypsinized and the number of alive cells was counted with the trypan blue exclusion assay as described before [31]. One hundred viable cells were plated into each well of 6-well plates in complete drug-free media (3 mL/well) and were incubated for 14 days. Then, the media were aspirated, surviving colonies were fixed with 100% MeOH, followed by washing with PBS and air-drying at RT. Next, cells were incubated with Giemsa staining solution for 25 min at RT, the staining solution was aspirated, and the wells were rinsed with dH2O and air-dried. The number of cell colonies was counted with the naked eye.

4. Conclusions

In summary, 1H NMR-guided chemical investigation led to the isolation of three new humulane-type sesquiterpenoids and one new steroid. The structures of the new compounds were elucidated via analyses of their MS, NMR, and ECD spectroscopic data, as well as using the Mosher’s esters analysis. These molecules represent the new humulenes possessing an oxygenation pattern which was significantly different from those found in plants, liverworts, and fungi. Leptogorgin A (1) exhibits a moderate cytotoxicity to human prostate cancer 22Rv1 cells.