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.


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).
The geometry of the ∆ 2,3 double bond was further determined to be E by considering the coupling constant (J = 16.3 Hz) displayed in its 1 H NMR spectrum. The NOE correlations of CH 3 -13 to H-2, H-6, and CH 2 -11, as well as H-10 with H-6 and H-6 with H-2 ( Figure 3), suggested that the ∆ 5(6) and ∆ 9(10) double bonds in 1 were E configured.
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 1 H and 13 C 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,9Edien-12-ol, as shown in Figure 1, and named leptogorgin C (3).   (Table 1) were similar to those of 1, suggesting that this compound possessed the same humulane skeleton. The key differences were in δ H for H-7 and δ C for carbon 7 in the spectrum of 2 (δ H 5.28/δ C 72.9). The corresponding signals were shifted downfield, compared to those of 1 (δ H 4.21/δ C 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).  (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 δ H for protons 6, 7, and 8 and δ C 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, Mar. Drugs 2020, 18, 310 5 of 10 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 C 27 H 42 O 3 . 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 (δ H 0.74/δ C 12.2, δ H 1.19/δ C 17.4) and three methyl groups of the side chain (δ H 1.04/δ C 20.3, δ H 1.15/δ C 23.8, and δ H 1.20/δ C 26.4), eight methylene groups, six methine groups, including one oxygenated methine (δ H 3.85/δ C 79.7), two quaternary sp 3 carbons (δ C 38.6, δ C 42.5), one quaternary sp 3 oxygenated carbon (δ C 72.8), one trisubstituted double bond (δ H 5.72/δ C 123.8 and 171.4), one disubstituted double bond (δ H 5.61/δ C 140.8 and δ H 5.43/δ C 126.0), and one conjugated with double bond ketone carbonyl (δ C 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 1 H 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). 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 (IC 50 = 31.0 µM and 35.8 µM, respectively), whereas 2 had IC 50 > 100 µM. Doxorubicine was used as a positive control and exhibited in 22Rv1 and PNT2 cells IC 50 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.

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).

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].

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 × 10 3 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 IC 50 of the compounds against the control cells treated with the solvent alone.

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 dH 2 O and air-dried. The number of cell colonies was counted with the naked eye.

Conclusions
In summary, 1 H 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.