Long-Chain Acetylenic Ketones from the Micronesian Sponge Haliclona sp. Importance of the 1-yn-3-ol Group for Antitumor Activity.

Two new long-chain C33 polyacetylenic compounds, halicynones A and B were isolated from the marine sponge Haliclona sp. along with known analogs. The known compound pellynol A possessing a 1-yn-3-ol terminus, exhibited strong antitumor activity against the human colon tumor cell line HCT-116 (IC50 0.026 μg/mL), however, the corresponding 1-yn-3-one, halicynone A, was inactive, which suggests an important role for the terminal 1-yn-3-ol functional group in mediating cytotoxic activity.


Introduction
Long-chain polyacetylenes are known from marine sponges of the order Haplosclerida [1]. The molecular structures vary in chain length from C 33 -C 46 and are substituted with varying numbers of hydroxyl groups, typically at C 1 and ω-3 (third last carbon from the terminus). The long-chain alkynes found in Petrosia, Pellina, Haliclona and other genera often possess remarkable cytotoxic activity against tumor cells that is not easily explained by the simple functionality that adorns the structures of these molecules, nor dismissed by non-specific lipid aggregations under assay conditions (e.g. micelles). A recent report that demonstrates that analogs of the polyacetylene petrosynol, from Petrosia sp., directly inhibit DNA replication at the level of initiation supports a suggestion that the compounds interfere with proteins required to establish initiation forks [2]. Indeed, earlier studies show that petrosynol inhibits both RNAand DNA-dependent DNA polymerase [3]. Nevertheless, a unifying structural model to explain these unusually high activities has not been advanced. In our screening for compounds that induce apoptosis (programmed cell-death) in tumor cell lines, an extract of the marine sponge Haliclona sp., collected in Micronesia, showed weak antifungal activity against Candida glabrata and high cytotoxicity against human colon tumor cells, HCT-116 (1.4% survival at 26 µg/mL). We report here two new acetylenic ketones, 1 and 2, that are related to the highly cytotoxic long-chain polyacetylenes, pellynols [4] and triangulynes [5] from Pellina triangulata. Measurement of structure-activity relationships of 1, 2 and related compounds 3-9 shows that a terminal 1-yn-3-ol group [6] -but not a 1-yn-3-one -imparts cytotoxic activity to this class of molecules.

Scheme 1.
( The configuration of the Edouble bond at C29-30 and Zdouble bond at C18-19 in 1 followed from observation of a large vicinal coupling constant between the vinyl protons H29 and H30 (J= 14.8 Hz) and the upfield shifts of the allylic CH 2 groups C17 and C20 (δ C 27.2, t; 27.2, t), respectively. Although location of the Z-double bond was not explicitly indicated by the spectroscopic data, it was supported by selective oxidation (MnO 2 , CH 2 Cl 2 ) of pellynol A (5) to 1. The product of oxidation was identical to natural 1 by 1 H NMR, ESIMS, HPLC retention time and co-injection with an authentic sample (C 18 HPLC, Dynamax, 4.6x250 mm, 88:12 MeOH/H 2 O, rt 32.1 min).
Compound 2, C 32 H 50 O 3 was obtained in only small amounts and the structure assigned on the basis of comparisons with pellynol I (9). The Zdouble bond present in 1 was replaced in 2 by signals due to a methyl branch (δ H 0.83, d, J= 6.8 Hz, 3H); a substitution pattern that is also seen in 9. All other signals were identical with those of 9. Although insufficient material was available for 13 C-NMR or chemical correlation, the assignments of the functional groups were strongly supported by the presence of identical 1 H NMR signals in 1 and 2 for the cross-conjugated acetylenic enone and the terminal propargylic CH 2 OH group at C1. Thus, the structure of 2 is highly suggestive of dehydro-pellynol I, however, as with the known parent compound 9 [4c], we were unable to assign the position of the methyl branch from electron impact MS fragmentation data.
Pellynols A-D, F and I showed strong cytotoxicity against several melanoma and ovarian cancer cell lines (IC 50 0.08-2.0 µM) [4c], but no simple correlation with chain-length or positions of chain oxidation was established. In the present work we reveal that the terminal 1-yn-3-ol functionality is critical in eliciting in vitro antitumor activity as oxidation of the terminal propargylic alcohol in 5 to the conjugated acetylenic ketone 1 abolishes activity.

Conclusions
Two new long-chain unsaturated acetylenic ketones, 1 and 2, were identified from the marine sponge Haliclona sp. and correlated with known compounds. Both compounds exhibit unusually high cytotoxicity similar to that reported for other long-chain ene-ynes from Porifera, however, the critical role of the 1-yn-3-ol for cytotoxic activity is now revealed. It is unlikely that long-chain ene-ynes will find utility as antitumor drugs, but the intriguing recurrence of reports of cytotoxicity within this class of compounds merits investigation of their cytological effects.

Experimental
General 1 H-and 13 C-NMR spectra were obtained using a Varian Inova 400 NMR spectrometer at 400 and 100 MHz, respectively. Solvents used in extraction or chromatography were HPLC-grade or distilled from glass. ESIMS was carried out on a ThermoFinnigan Surveryor LC and LC Deca ion-trap with infusion in MeOH (0.1% acetic acid). HRMS results were obtained from University of California, Riverside Mass Spectrometry Facility. General experimental procedures are described elsewhere [12]. Antifungal assays were carried out using a modification of a standard microtiter broth dilution assay [13]. Cytotoxicity assays against human colon tumor cells were performed at Scripps Institution of Oceanography (La Jolla, California) using cultured HCT-116 cells incubated with MTS [14]. The endpoint and cell viabilities were determined by measurement of the soluble formazan product (λ 490 nm) using a Molecular Devices Spectramax microplate reader [15].