Ochracenoids A and B, Guaiazulene-Based Analogues from Gorgonian Anthogorgia ochracea Collected from the South China Sea

Two new guaiazulene-based analogues, ochracenoids A (1) and B (2), along with four known analogues (3–6), were isolated from the gorgonian Anthogorgia ochracea collected from the South China Sea. The planar structures of the new compounds were elucidated by comprehensive spectroscopic data. The absolute configuration of 1 was determined as 3R by the comparison of TDDFT calculated electronic circular dichroism with its experimental spectrum. Compound 1 is a rare guaiazulene-based analogue possessing a unique C16 skeleton. The possible generation process of 1 through an intermolecular one-carbon-transfer reaction was also discussed. Compound 2 was previously described as a presumed intermediate involved in the biogenesis of anthogorgienes A and I. Compound 3 exhibited antiproliferative effects on the embryo development of zebrafish Danio rerio.


Introduction
Gorgonians have proven to be a rich source of guaiazulene-related pigments [1].Guaiazulene-based analogues are well recognized for their distinctive blue and purple colors, which are a part of the origin of the brilliant colors of gorgonians and other organisms [2].These compounds feature an azulene core, similar to the fused five-seven bicyclic aromatic ring system [3].They have received much attention due to their multiple potent biological activities including antifungal, antitumor, and immunoregulatory activities and antiproliferative effects on fertilized sea urchin eggs [4,5].In particular, a series of guaiazulene-based compounds were reported from gorgonians of Anthogorgia [2,6], and Acalycigorgia (synonymous with Anthogorgia) [4,7].Recently, in the course of our investigation on new bioactive substances from gorgonians and soft corals as well as their derived fungi from the South China Sea [8][9][10], the gorgonian Anthogorgia ochracea collected from the South China Sea attracted our attention because the EtOAc extract of the gorgonian showed the presence of guaiazulene-based sesquiterpenes with characteristic UV absorption spectra (UVA λ max 320-400 nm) [11].Chemical investigation on the extract led to the isolation of two new guaiazulene-based analogues, ochracenoids A (1) and B (2), and four known related analogues (3-6) (Figure 1).Herein, we report the isolation, structure elucidation, and biological activities of these compounds.As these compounds have been found to be quite labile and easily decomposable on exposure to air and light during the work-up [12], the possible generation process of 1 was also discussed.

Results and Discussion
Ochracenoid A (1) was obtained as a red-purple solid.Its molecular formula of C 16 H 16 O 2 (9 degrees of unsaturation) was determined by HRESIMS.The IR spectrum of 1 showed an absorption band corresponding to a formyl functionality (1655 cm -1 ).The 13 C NMR (Table 1) spectrum indicated the presence of 16 carbon resonances including a formyl carbon (δ C 187.0), seven quaternary olefinic carbons (δ C 166.7, 149.0, 138.5, 134.7, 130.3, 129.2 and 127.6), three olefinic methines (δ C 135.6, 131.0 and 116.0), one methine (δ C 39.4), one methylene (δ C 78.1), and three methyl groups (δ C 30.9, 20.5 and 13.1).The 1 H NMR (Table 1) spectrum showed signals corresponding to two aromatic methyls [δ H 3.11 (s, 3H) and 2.55 (s, 3H)] and three aromatic methines [δ H 8.12 (d, J = 1.2 Hz, 1H), 7.97 (s, 1H) and 7.09 (s, 1H)].These spectroscopic data indicated that the basic skeleton of 1 should be an azulene, and the 1 H NMR signals for the azulene portion of 1 were similar to those of linderazulene (Figure 1) [13].The remaining resonances, one methyl at δ H 1.49 (d, J = 6.6 Hz), one methine at δ H 3.81 (m), and one methylene at δ H 4.82 (t, J = 8.4 Hz) and 4.27 (dd, J = 8.4, 6.6 Hz), suggested that 1 was a 2,3-dihydro-derivative of linderazulene.The NMR spectra of 1 were nearly identical to those observed for 2,3-dihydrolinderazulene (Figure 1) [4], a guaiazulene-based analogue isolated from an Anthogorgia gorgonian.The main difference was the observation of resonances attributable to a formyl functionality [δ C 187.0 (CH) and δ H 10.61 (s, 1H)] connected to C-7 in 1, supported by the HMBC correlations from -CHO to C-7 and C-6 (Figure 2), rather than an olefinic proton in 2,3-dihydrolinderazulene.The planar structure of 1 was confirmed by 2D-NMR experiments including 1 H-1 H COSY, HMQC, and HMBC correlations (Figure 2).Compound 1 was therefore designated as 7-formyl-2,3-dihydrolinderazulene.The absolute configuration of C-3 in 1 was determined by theoretical calculation of electronic circular dichroism (ECD) spectrum.Arbitrarily assigned 3R-1 was employed to perform a random conformational search by using the Monte Carlo method with MMFF94 force field in the Spartan 08 software package [14].Only one lowest-energy conformer has been located within an energy cut-off of 2 kcal/mol (Figure 3).Full geometry optimization and harmonic vibrational frequencies calculation were performed at the B3LYP/6-31+G(d) level in the gas phase by using the Gaussian 09 software package [15].The energies, oscillator strengths, and rotational strengths of the first 30 electronic excitations were calculated using the TDDFT methodology at the B3LYP/6-311++G (2d,2p)/B3LYP/6-31+G(d) level in vacuum.The ECD spectra were then simulated by the overlapping Gaussian function (ζ = 0.5 eV) [16].In the 200-600 nm region, compared to the experimental ECD spectrum of 1, the calculated ECD spectrum of 3R-1 showed a similar curve with positive first, negative second, positive third, and negative fourth Cotton effects at 520 (+82), 375 (−1), 303 (+15), and 254 (−20) nm, respectively (Figure 3).Although there were some large wavelength differences between experimental Cotton effects and the corresponding theoretical ones, especially the first Cotton effect, due to idealized modeling and simulation, qualitative analyses of the results allowed the assignments of the experimental first positive Cotton effect around 438 nm to the predicted first positive electronic excitation at 491 nm, the experimental second negative Cotton effect around 376 nm to the predicted 2nd-5th negative electronic excitations at 391, 375, 339, and 339 nm, and so on (Figure 3).Base on the above assignments, the absolute configuration at C-3 in 1 was determined as R.
A literature survey revealed that guaiazulene and related compounds could gradually suffer autoxidation even on standing at room temperature to give various products [21].In the present study, guaiazulene-based analogues, including two C 16 -guaiazulene analogues (1 and 3), two indenone derivatives (2 and 6), and two common guaiazulenes (4 and 5), were obtained simultaneously.Compound 5 was reported to be a photo-oxidation product derived from linderazulene [22].Compounds 3, 4, and 6 were described to be generated from the same precursor guaiazulene (Figure 1) and followed by an intermolecular one-carbon-transfer reaction, a side-chain oxidation, and a rearrangement to indenone derivatives, respectively [23].Interestingly, guaiazulene-based sesquiterpenes commonly have fifteen carbon atoms, while 1, as well as 3, contained a unique C 16 skeleton.Based on the fact that 1 is constructed by a guaiazulene sesquiterpene moiety, 2,3-dihydrolinderazulene, with an additional formyl attached at C-7, a possible oxidative transformation process from 2,3-dihydrolinderazulene to 1 was proposed in Scheme 1.By the attack of oxygen, 2,3-dihydrolinderazulene was considered to initially form an electron-transfer complex, followed by the conformation of two tautomeric dimers in equilibrium [2,21,23].Then the unique tautomers constituted the important intermediates (Scheme 1, a and b) by exposure to air at room temperature.Finally, the intermediates were converted into 1 by an intermolecular one-carbon-transfer reaction to form the additional formyl group [21,23].In the literature, 2 was described as a presumed intermediate involved in the biogenesis of anthogorgienes A and I (Figure 1), and was depicted to be derived from guaiazulene via a C-1 and C-5 peroxidated intermediate to follow a 4,5,6-cyclopropane formation and then cleavage of the C-4/C-5 bond [2].The isolation of 2 gave evidence to the deduction of the generation process from guaiazulene to anthogorgienes A and I. Scheme 1. Possible transformation process from 2,3-dihydrolinderazulene to 1.
All the isolated compounds were evaluated for their antiproliferative effects on zebrafish embryo.It should be pointed out that the developing zebrafish Danio rerio is an in vivo pharmacologically relevant model which provides rapid and high throughput screening (HTS) for compounds with capability of reducing cell proliferation [24].Compound 3 showed strong antiproliferative effects leading to abnormal several aspects of the embryonic development including coagulated eggs (48 h), notochord malformation (72 h), and embryo death (72 h) with the EC 50 values of 3.98, 6.50, and 7.39 μM, respectively, while compounds 1, 2 and 4-6 exhibited no antiproliferative effects on zebrafish embryo.
The isolated compounds were also evaluated for their cytotoxicities and antibacterial activities.However, no compound showed any cytotoxicity against five human tumor cell lines (HeLa, A-549, HCT-116, HL-60, and K562) and antibacterial activity on six strains of pathogenic bacteria.
More details are available at the Supplementary Information.

Materials
The gorgonian Anthogorgia ochracea GXWZ-07 (1.9 kg, wet weight) was collected from the coral reef of Weizhou Island in the South China Sea, China, in April 2011, and was identified by Dr. Xiu-Bao Li, South China Sea Institute of Oceanology, Chinese Academy of Science.

Extraction and Isolation
The gorgonian A. ochracea was cut into small pieces and exhaustively extracted with CH 2 Cl 2 /MeOH (v:v, 1:1) three times (3 × 2000 mL) at room temperature, and the solvent was evaporated in vacuo.The organic layer was filtered and concentrated under reduced pressure to give a residue (36.2 g), which was partitioned between EtOAc and H 2 O (v:v, 2:1) for three times.The EtOAc extract was concentrated in vacuo to afford 20 g of EtOAc residue, which was subjected to column chromatography (CC) on silica gel, using EtOAc-petroleum ether (0%-100%) as eluent.By combining the fractions with TLC monitoring, six fractions (Fr.1-Fr.6)were obtained.Fr.3 (1.2 g) was fractionated over silica gel CC eluted with EtOAc-petroleum ether gradients (10%-90%) to afford four sub-fractions (Fr.

Biological Assays
The antiproliferative effects on zebrafish Danio rerio embryo were evaluated according to the described methods [25].3,4-Dichloroaniline was used as a positive control.
The cytotoxicities were evaluated for against human cervical carcinoma HeLa, human lung carcinoma A-549, and human colorectal cancer HCT-116 cell lines using SRB method [26] and human myeloid leukemia HL-60, and human leukemia K562 cell lines using MTT method [27].Adriamycin was used as a positive control.
The antibacterial activities against six bacterial strains, Staphylococcus epidermidis, S. aureus, Bacillus subtilis, B. cereus, Tetragenococcus halophilus, and Kocuria rhizophila, were determined by a serial dilution technique using 96-well microtiter plates [28].Ciprofloxacin was used as a positive control.

Conclusions
In summary, six guaiazulene-based analogues were obtained from a gorgonian Anthogorgia ochracea collected from the South China Sea.The planar structures of the new compounds (1 and 2) were elucidated by comprehensive spectroscopic data and the absolute configuration of 1 was determined by the comparison of TDDFT calculated electronic circular dichroism with its experimental spectrum.Compound 1 is a rare guaiazulene-based analogue possessing a unique C 16 skeleton.A possible generation process of 1 through an intermolecular one-carbon-transfer reaction was also discussed.Compound 2 was previously described as a presumed intermediate involved in the biogenesis of anthogorgienes A and I. Compound 3 showed strong antiproliferative effects on the embryo development of zebrafish Danio rerio.