Chlorinated Didemnins from the Tunicate Trididemnum solidum

Chemical investigation of the tunicate Trididemnum solidum resulted in the isolation of two new chlorinated compounds belonging to the didemnin class, along with two known compounds didemnin A and didemnin B. The structural determination of the compounds was based on extensive NMR and mass spectroscopic analysis. The isolated compounds 1–4 were tested for their anti-inflammatory activity using in vitro assays for inhibition of inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB) activity. The anti-cell proliferative activity of the above compounds was also evaluated against four solid tumor cell lines.


Introduction
Didemnins belonging to the class of depsipeptides were first isolated from the Caribbean tunicate Trididemnum solidum (Family Didemnidae) [1,2]. Didemnin B, a potent anticancer agent was the first marine natural product that entered into phase I clinical trials. Didemnin B exhibited a variety of biological activities which includes inhibition of the growth of both RNA and DNA viruses [3], in vitro and in vivo activity against B16 melanoma, in vivo activity against P388 leukemia and in vitro OPEN ACCESS activity against L1210 lymphocytic leukemia [4,5]. In the course of our search to identify new drug candidates and targets, the tunicate Trididemnum solidum (KY10508001) was collected from the Little Cayman Island and the freeze dried sample was extracted with CH 2 Cl 2 /MeOH (1:1 v/v). The extract was found to exhibit strong anti-inflammatory activity using in vitro assays for inhibition of inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB) activity. The IC 50 values were 0.2 µg/mL and 0.4 µg/mL for inhibition of iNOS and NF-κB, respectively. The crude extract was further subjected to a series of chromatographic separations to yield the didemnin class of pure compounds (1)(2)(3)(4), and the isolated compounds were tested for their in vitro anti-inflammatory and anticancer activities.

Bioassay-Guided Isolation
The crude DCM extract of the tunicate (5.5 g) was subjected to C 18 flash column chromatography using water and methanol mixtures. Based on the anti-inflammatory activity, fraction D (IC 50 of 0.14 µg/mL and 0.028 µg/mL for NF-κB and iNOS respectively) was further subjected to reversed phase HPLC (Phenomenex, Luna C 18 (2)), using a gradient mixture (60:40 MeOH: H 2 O to 100% MeOH with 0.05% TFA over 65 min) to afford four pure compounds (1-4) ( Figure 1).   Figure S1), which were identified to be 11× C (of which 8 were carbonyls); 17× CH; 8× CH 2 ; 13× CH 3 based on DEPT spectral data. Analysis of the 1 H NMR data (Supplementary Figure S2) revealed the structural similarities of compound 1 to didemnin A [6]. Mass spectral data (Supplementary Figure S3) in combination with the 1 H NMR spectrum (Table 1) Figure S4) of Marfey's derivatives [9]. The stereochemistry of chloro N,O-di Me Tyr was determined by synthesis of the same using N,O-di Me-L-Tyr and sulfuryl chloride [10]. Comparison of spectral data of 1 with the literature [11,12] revealed the stereochemistry of isoSta and Hip moieties to be similar to that in didemninA. Thus, the structure of compound 1 was established as N,O-diMe-o-chlorotyrosine derivative of didemnin A.  Several nordidemnins were reported previously [11] and analysis of 2D NMR data indicated the presence of norStatine unit in compound 2. Thus compound 2 was identified as nor-N,O-diMe-ochlorotyrosine derivative of didemnin A.

Structural Elucidation of the New Compounds
Compounds 3 & 4 were isolated as white solids and identified as didemnin A and didemnin B by comparison of spectral data to the literature [11][12][13].
The isolated compounds 1-4 were tested in a series of in vitro cell-based assays for their effects on selected targets involved in the process of inflammation and cancer. The results are presented in Table 2. Inflammation and oxidative stress are known to be linked to the development of many disorders such as cancer, organ damage, and neurodegenerative conditions. The NF-κB family of transcription factors plays a key role in inflammation, cell cycle regulation, apoptosis, and oncogenesis by controlling gene network expression [14,15]. The activation of NF-κB involves many cellular processes leading to inflammation and development of cancer [16,17]. In the assay for NF-κB activity, a luciferase construct with binding sites for specificity protein (SP-1) was used as a control because this transcription factor is relatively unresponsive to inflammatory mediators such as phorbol myristate acetate (PMA). Inducible nitric oxide synthase (iNOS) plays a key role in regulation of blood pressure, the immune system, infection, and inflammation [18]. Overproduction of nitric oxide (NO) by iNOS has been implicated in various pathological processes such as septic shock, inflammation, rheumatoid arthritis, cancer, and tissue damage [19,20]. The increase in NO response caused mainly by endotoxins and proinflammatory mediators such as lipopolysaccharide (LPS) can be reduced by anti-inflammatory agents acting as iNOS inhibitors. The isolated metabolites were also evaluated for their cytotoxicity towards four human solid tumor cell lines (SK-MEL: melanoma; KB: epidermal carcinoma; BT549: breast carcinoma and SK-OV-3: ovarian carcinoma) and non-cancer kidney cells (Vero: monkey kidney fibroblasts). The IC 50 values for cell growth inhibition of compounds (1-4) are presented in Table 3.  All the compounds (1-4) showed strong activity against iNOS and NF-κB in LPS induced macrophages and PMA induced chondrocytes, respectively. The inhibition of NF-κB was not associated with the inhibition of SP-1 indicating that inhibition of NF-κB mediated transcription by these compounds was specific. Compound 4 was the most potent in inhibiting iNOS activity compared to 1-3, but its effect could be related to strong cytotoxicity towards all cell lines. All four compounds strongly inhibited cell proliferation of all four cancer cell lines in comparison to doxorubicin as the control drug (Table 3). Similar to the effect of doxorubicin, the compounds (1-3) were less toxic towards noncancerous cells (Vero cells) which were included for comparison. The strong anticancer activity can be partly explained due to a strong inhibition of NF-κB mediated transcription that may cause enhanced apoptosis and cell death [21].

General
Optical rotations were measured using a JASCO DIP-370 digital polarimeter. UV spectra were recorded on a Hewlett-Packard 8452A diode array spectrometer. NMR spectra were measured on Bruker Avance DRX-500 and Varian 600MHz spectrometer. 1 H and 13 C NMR spectra were measured and reported in ppm using d 5 -pyridine solvent peak (δ H 8.74 and δ C 150.4) as an internal standard. ESI-FTMS analyses were measured on a Bruker Magnex BioAPEX 30es ion cyclotron HR HPLC-FT spectrometer by direct injection into an electrospray interface. HPLC purifications were carried out on a Waters 2695 model system equipped with dual absorbance UV detector.

Tunicate Material
The tunicate (ID: KY10508001) was collected from Little Cayman Island at a depth of 10 m. A voucher specimen of the sample was deposited at the NOAA Ocean Biotechnology Center and Repository, Oxford, MS, USA.

Biological Assays
Inhibition of NF-κB mediated transcription was determined in SW1353 cells by a reporter gene assay as described by Ma et al. [21]. Inhibition of iNOS activity was determined in RAW264.7 cells as described earlier [22]. In vitro cytotoxicity was determined against a panel of four solid tumor cell lines and compared with noncancerous kidney cells (Vero) [23]. Parthenolide and doxorubicin were used as positive controls for anti-inflammatory and cytotoxic activity, respectively.

Conclusions
The investigation of the tunicate Trididemnum solidum for bioactive natural products resulted in the isolation of two new chlorinated didemnin classes of compounds in addition to two known compounds. The recent reports of didemnin B from the α-proteobacterium Tistrella mobilis by Tsukimoto et al. [13], didemnin B and nordidemnin B from several species of Tistrella by Xu et al. [24] suggests that the chlorinated derivatives reported in this paper could also be of bacterial origin. Compound (1-4) exhibited significant inhibition of NF-κB and iNOS activity, as well as strong anticancer activity towards four cancer cell lines. Compound 1, the chlorinated derivative was less active compared to 3 in anti-inflammatory and cytotoxicity assays.