Psammaceratin A: A Cytotoxic Psammaplysin Dimer Featuring an Unprecedented (2Z,3Z)-2,3-Bis(aminomethylene)succinamide Backbone from the Red Sea Sponge Pseudoceratina arabica

Bioassay-guided partition of the extract of the Red Sea sponge Pseudoceratina arabica and HPLC purification of the active fraction gave a psammaplysin dimer, psammaceratin A (1), along with psammaplysin A (2). The dimer comprises two units of psammaplysin A (2) connected via the terminal amines with an unprecedented (2Z,3Z)-2,3-bis(aminomethylene)succinamide moiety, and it represents the first dimer to be identified among the psammaplysin family. Data from 1D- and 2D-NMR and HRMS supported the chemical structures of the compounds. Psammaceratin A (1) and psammaplysin A (2) exhibited significant growth inhibition of HCT 116, HeLa, and MBA-MB-231 cells down to 3.1 μM.

The sum of the elements of the assigned subunits A, A , B, and B was counted for C 42 H 44 Br 8 N 6 O 12 and for 20 degrees of unsaturation. The remaining elements of C 6 H 6 N 2 O 2 (Fragment C) were counted for the remaining four degrees of unsaturation in 1. These elements C 6 H 6 N 2 O 2 are assigned as 2,3-bis(aminomethylene)succinamide ( Figure 2 Table 2). An additional significant and expected downfield shift of H 2 -20 from 3.18 ppm (in 2) to 3.69 ppm in 1 (∆δ H = +0.51 ppm) was observed, confirming the effect of the substitution of the terminal amines in psammaplysin derivatives [11] ( Table 2).
In an MTT assay [13,24], psammaceratin A (1) displayed the highest activity against HCT 116 cells with an IC 50 value of 3.1 µM. On the contrary, psammaplysin A (2), with its free terminal amine moiety, was less active towards HCT 116, with an IC 50 value of 5.1 µM. On the other hand, compound 2 was more active against MDA-MB-231 (IC 50 = 3.90 µM), while 1 was less active against this cell line (IC 50 = 5.25 µM). These data suggest that MDA-MB-231 and HCT 116 have high sensitivities towards 1 and 2, respectively. Finally, psammaceratin A (1) and psammpalysin A (2) displayed close and similar activity towards HeLa cells (IC 50 = 8.50-9.40 µM) ( Table 3) suggesting lower sensitivity of this cell line towards 1 and 2. Thus, psammaceratin A and psammaplysin A are considered as potential leads for the establishment of novel anticancer entities.

Structure of Psammaplysin A (2)
Psammaplysin A (2) (Figure 1) was identified by interpretation of its NMR and MS data and by comparison of its NMR data with the literature [4].

General Experimental Procedures
Optical rotations were acquired on a digital DIP-370 polarimeter (JASCO). The UV spectra were measured on a Hitachi 300 spectrometer. NMR data were acquired on a Bruker Avance DRX 600 MHz spectrometer using CD 3 OD as the solvent. Positive ion HRESIMS spectra were collected on a Thermo LTQ Orbitrap XL mass spectrometer. A SiO 2 RP HPLC column (Merck, 70-230 mesh ASTM) and Sephadex LH 20 (0.25-0.1 mm, Pharmacia) were used for chromatography. An HPLC column (AR II Cosmosil, Waters, 250 × 10 mm, 5 µm) was used for purification of the compounds.

Biological Materials
The Red Sea Pseudoceratina arabica (Keller, 1883) was harvested by hand via scuba down to −17 m from Anas Reef, Obhur at the Saudi Red Sea coast (N 021 • 39 17.5 , E 038 • 52 26.3 ). The sponge consists of an encrusting mass of 1-2 cm with a conulose surface of yellowish green color underwater and greenish yellow interior. The sponge starts to turn blackish green in color after exposure to air. After storage in 70% ethanol solution, it turns completely into a black mass. The conules on the surface of the sponge are bluntly rounded, about 2-5 mm apart, and are of rubbery and compressible consistency. The specimen fragment measured about 12.0 × 5.0 × 1.5 cm. The sponge's skeleton contains spare unequal fibers containing only pith. The outline branching is irregular, and the thickness measures 80-300 µm. The specimen corresponded to an Eritrean Red Sea specimen of P. arabica. The voucher was stored in the Zoological Museum's collection at Amsterdam University under the code RMNHPOR 9161. Another specimen was stored at King Abdulaziz University under code #KSA-58.

Purification of 1 and 2
Freeze-dried sponge materials (230 g) were extracted thrice with 1000 mL MeOH. The combined extracts were dried under vacuum to afford 5.29 g of viscous residue. The extract was dissolved in MeOH-H 2 O (6:4) and successively extracted with hexane, CH 2 Cl 2 , and EtOAc. The cytotoxic CH 2 Cl 2 extract (2.1 g) was chromatographed over a SiO 2 VLC column using hexane/EtOAc/MeOH gradients to afford five fractions (Fr-1-Fr-5). Fractionation of the cytotoxic fraction (Fr-3) (270 mg) on a Sephadex LH 20 column with MeOH gave four major subfractions (Fr-3A-Fr-3C). The cytotoxic fraction Fr-3B (45 mg) was purified on an ODS HPLC column using 80% MeOH to yield compounds 1 (5.3 mg) and 2 (2.7 mg). Psammaplysin A (2) was identified by analyses of its 1D and 2D NMR data and by comparison of its spectroscopic data to those in the literature [4].

Cytotoxicity and Antiproliferative Activity
The evaluation of the cytotoxicity of the compounds was carried out via MTT assay as previously described [13,24]. Briefly, cells were incubated at 37 • C overnight in 5% CO 2 /air, followed by the addition of the compounds at the top row of a 96-well microtiter plate and descending serial dilution (1:4) of the concentration. The cells were incubated for 72 h with the compounds. Subsequently, the cell viability was estimated at 490 nm on a Molecular Devices Emax microplate reader using the Cell Titer 96 AQueous non-radioactive cell proliferation protocol. The IC 50 values of the compounds (expressed in micromoles) were evaluated using the program SOFTmax PRO. 5-Fluorouracil (5-FU) and DMSO were used as positive negative controls.

Conclusions
Bioassay-directed fractionation of the cytotoxic extract of the Red Sea sponge Pseudoceratina arabica afforded an unprecedented psammaplysin dimer, psammaceratin A (1), along with psammaplysin A (2). Psammaceratin A, with its unique (2Z,3Z)-2,3bis(aminomethylene)succinamide backbone connecting two units of psammaplysin A, represents the first dimer of this type within the psammaplysin family. This previously unknown functional group, 2,3-bis(aminomethylene)succinamide, offers a novel synthetic moiety that could be utilized as an isostere in synthetic chemistry, as a novel connecting moiety, or for other design and derivatization purposes. Accordingly, psammaceratin A and psammaplysin A are potential scaffolds for the development of novel antitumor leads.