Anti-Protozoal Activities of Cembrane-Type Diterpenes from Vietnamese Soft Corals

Based on our previous finding that certain cembranoid diterpenes possess selective toxicity against protozoan pathogens of tropical diseases such as Trypanosoma and Plasmodium, we have subjected a series of 34 cembranes isolated from soft corals living in the Vietnamese sea to an in vitro screening for anti-protozoal activity against Trypanosoma brucei rhodesiense (Tbr), T. cruzi (Tc), Leishmania donovani (Ld), and Plasmodium falciparum (Pf). Twelve of the tested compounds displayed significant activity against at least one of the parasites. Specifically, 7S,8S-epoxy-1,3,11-cembratriene-16-oic methyl ester (1), (1R,4R,2E,7E,11E)-cembra-2,7,11-trien-4-ol (2), crassumol D (12), crassumol E (13), and (1S,2E,4S,6E,8S,11S)-2,6,12(20)-cembrantriene-4,8,11-triol (16) from Lobophytum crassum, L. laevigatum, and Sinularia maxima showed the highest level of inhibitory activity against T. b. rhodesiense, with IC50 values of about 1 µM or less. Lobocrasol A (6) and lobocrasol C (8) from L. crassum and L. laevigatum exhibited particularly significant inhibitory effects on L. donovani with IC50 values < 0.2 µM. The best antiplasmodial effect was exerted by laevigatol A (10), with an IC50 value of about 3.0 µM. The cytotoxicity of the active compounds on L6 rat skeletal myoblast cell was also assessed and found to be insignificant in all cases. This is the first report on anti-protozoal activity of these compounds, and points out the potential of the soft corals in discovery of new anti-protozoal lead compounds.


Introduction
Neglected tropical diseases (NTDs) are a group of seventeen mostly life threatening and/or disabling infections affecting more than a billion people worldwide.Most affected are poor populations in developing countries.People suffering from NTDs hence represent an unattractive market to private-sector research and development investment.Among the NTDs, infections caused by unicellular eukaryotic parasites ("protozoans"), Trypanosoma brucei (which causes human African trypanosomiasis (HAT) as well as Nagana affecting cattle), T. cruzi (causing Chagas disease), Leishmania spp.(causative agents for leishmaniases), most seriously demand the search for new effective, safe and affordable drugs.Malaria, caused by Plasmodium spp., is currently not classified as NTD but represents a major health threat to a large part of the world's population.The search for new drugs against these diseases is an urgent need and natural sources such as marine organisms with their extremely diverse secondary metabolites may play an important role [1].Nature is an attractive source of new therapeutic compounds, with a tremendous diversity found among millions of species of marine organisms, plants, and microorganisms.Intensive research over the last four decades has proven that marine organisms are magnificent sources of bioactive secondary metabolites [2].To date no marine natural products and/or derivatives have entered preclinical development specifically for trypanosomatid diseases [3].However, various marine natural products have been reported in the literature to possess anti-protozoan activity [4][5][6] but the full potential of marine organisms is still largely uninvestigated.Secondary metabolites produced by diverse marine organisms thus represent a huge repository of chemical structures for searching new drugs which can contribute to improved public health conditions in tropical developing countries [7,8].
In the course of our ongoing search for secondary metabolites with anti-protozoal activity from marine organisms [9], we recently reported on our study of methanolic extracts of the soft corals Lobophytum crassum, L. laevigatum, and S. maxima and some pure constituents isolated from them, which showed significant inhibitory in vitro activity against T. brucei (African sleeping sickness), with no toxicity on the mammalian HEK293T (human embryonic kidney) and HepG2 (human hepatoma) cell lines.Among the active compounds was a cembranoid diterpene, laevigatol B (11).Together with the previous observation that a plant-derived cembrane, serratol, isolated from Boswellia serrata (Burseraceae) also showed such activity [10], this finding prompted us to perform a more detailed study on the potential of this class of terpenoids against the mentioned diseases.We herein report on the results of in vitro activity tests of 34 cembranoid diterpenes (Figure 1), isolated as major pure constituents from Vietnamese soft corals, against the abovementioned protozoan parasites.

Results and Discussion
Thirty-four cembranoid diterpenes (for their structures see Figure 1), recently isolated from Vietnamese soft corals [11][12][13][14][15] were submitted to a multiple-target screening against T. brucei rhodesiense (Tbr), T. cruzi (Tc), L. donovani (Ld), and P. falciparum (Pf).Each compound was tested for growth inhibitory activity against each parasite at two concentrations, namely, 2.0 and 10.0 µM.The results are reported as supplementary information (Table S1).Quite noteworthy, Tbr appeared the most sensitive pathogen to the compounds under study most of which led to almost complete growth inhibition at 10.0 µM.In contrast, none of the compounds displayed any significant activity against the closely related Tc.Overall, twelve compounds (1, 2, 6, 8, 10, 12, 13, 15-18, and 21) showed a promising level of activity against at least one of the parasites, i.e., >50% growth inhibition against Tbr at 2.0 µM and/or >50% inhibition at 10.0 µM against one of the other parasites.In case of such active compounds, consequently, IC50 values were determined.In addition, the cytotoxic activity of these compounds was investigated by determining their IC50 values of cell viability in the rat skeletal myoblast cell line L6 allowing the calculation of selectivity indices (SI = IC50(L6)/IC50(parasite)).The results are reported in Table 1.In case of Ld, only two compounds showed a high level of activity.Compounds 6 (lobocrasol A) and 8 (lobocrasol C) presented very low IC50 values of 0.18 and 0.17 µM, respectively, against this pathogen.This appears to be a highly specific antileishmanial effect since both compounds displayed only a low level of activity against Tbr and were neither particularly active against Pf nor significantly cytotoxic.It is also highly interesting to note that lobocrasol B (7), differing only in the configuration at C-7 (orientation of a single OH group) from lobocrasol A (6), was devoid of antileishmanial activity, which also points towards a very specific mechanism.
The IC50 values of seven compounds 1, 2, 8, 10, 13, 15, and 17 with antiplasmodial activity against Pf were generally much higher.Compound 10 (laevigatol A) with an IC50 of 3.0 µM was found to be the most active one in this series.It is thus the second cembranoid, besides serratol [10], reported to possess some activity against the malarial parasite.In the cytotoxicity assay on L6 cells laevigatol A (10) showed an IC50 value of 28.75 µM and its SI was 9.52 (Table 1).
Although the level of activity was generally lower than that of the positive controls, it is worth noting that their cytotoxity against the mammalian control cells was very low (IC50 values all > 28 µM), so that all these compounds show very favourable SI.The most favourable SI with respect to activity against Tbr were found in case of compounds 1, 2, 12, 13, and 16, which were 54, 43, 96, 60, and 75 times more active against Tbr than against L6 cells, respectively.A plot of the logarithmic anti-Tbr activity data vs. the cytotoxicity data (Figure 2) for those compounds with an IC50(Tbr) < 20 µM clearly shows that there is no significant correlation between these activities and it may thus be expected that the antitrypanosomal effect is not due to general cytotoxicity but a specific and trypanosome-selective mechanism.Along the same lines, there is no clear correlation between the in vitro activities against the different parasites.This is much the same way as observed in other cases of other cembranoids reported to possess antiparasitic activity (such as serratol, laevigatol B, etc.) [9,10] and also points towards a specific activity towards Tbr.It appears difficult to recognize clear structure-activity relationships (SARs) among the present series.However, it is noteworthy that a clear correlation can be observed between the lipophilicity and anti-Tbr activity among the active compounds (Figure 3).Even though this correlation is not extremely strong, it appears obvious that a trend exists among these cembranoids to higher activity with increasing lipophilicity as would be expected in case that penetration of the compounds into the parasite plays a role.However, this correlation does not include the inactive compounds in the series, so that further factors must determine the general ability of such compounds to kill the parasites.First attempts to identify such structural properties by means of QSAR analyses using descriptors calculated with the software package MOE in a similar way as reported previously [16,17] were made but did not lead to satisfactory results so far.Further in-depth investigations in this direction are under way.

Samples for Biological Tests
Stock solutions (concentration 10.0 mM) of the tested compounds were prepared in DMSO, kept at −20 °C, and diluted to the final concentration in fresh media before each experiment.In order to ensure unaffected cell growth, the final DMSO concentration did not exceed 0.5% in all experiments.

Anti-Trypanosomal Activity on T. brucei rhodesiense (Tbr)
Minimum Essential Medium with Earle's salts (50.0 µL) supplemented with 0.2 mM 2-mercaptoethanol, 1.0 mM Na pyruvate and 15% heat-inactivated horse serum was added to each well of a 96-well microtiter plate.Serial drug dilutions of eleven 3-fold steps covering a range from 100 to 0.002 µM were prepared.Then 10 4 bloodstream forms of T. b. rhodesiense STIB 900 in 50.0 µL of medium were added to each well and the plate incubated at 37 °C under a 5% CO2 atmosphere for 72 h.Alamar blue solution (10.0 µL of a solution of 12.5 mg resazurin dissolved in 100 mL distilled water) were then added to each well and incubation continued for a further 2-4 h.The plate was then read in a Spectramax Gemini XS microplate fluorometer (Molecular Devices Corporation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and emission wavelength of 588 nm [18].Fluorescence development was measured and expressed as percentage of the control.Data were transferred into the graphic programme Softmax Pro (Molecular Devices), which calculated IC50 values.

Antileishmanial Activity on L. donovani (Ld; Axenic Amastigote Assay)
Fifty µL of SM medium [19] at pH 5.4 supplemented with 10% heat-inactivated FBS, was added to each well of a 96-well microtiter plate.Serial drug dilutions were prepared covering a range from 100.0 to 0.002 µM.Then 10 5 axenically grown L. donovani amastigotes (strain MHOM/ET/67/L82) in 50.0 µL medium were added to each well and the plate incubated at 37 °C under a 5% CO2 atmosphere for 72 h.Resazurin solution (10.0 µL, 12.5 mg resazurin dissolved in 100 mL distilled water) were then added to each well and incubation continued for a further 2-4 h.The plate was then read in a Spectramax Gemini XS microplate fluorometer (Molecular Devices, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and emission wavelength of 588 nm [18].Fluorescence development was measured and expressed as percentage of the control.Data were transferred into the graphic programme Softmax Pro (Molecular Devices) which calculated IC50 values from the sigmoidal inhibition curves.

Antiplasmodial Activity on P. falciparum (Pf) NF54 Strain
Antiplasmodial activity was determined using the NF54 strain of P. falciparum (drug sensitive).A modification of the [ 3 H]-hypoxanthine incorporation assay was used [20].Briefly, infected human red blood cells (final parasitaemia and haematocrit were 0.3% and 1.25%, respectively) in RPMI 1640 medium with 5% Albumax were exposed to serial drug dilutions in microtiter plates.After 48 h of incubation at 37 °C in a reduced oxygen atmosphere, 0.5 μCi 3 H-hypoxanthine was added to each well.Cultures were incubated for a further 24 h before they were harvested onto glass-fiber filters and washed with distilled water.The radioactivity was counted using a Betaplate™ liquid scintillation counter (Wallac, Zurich, Switzerland).The results were recorded as counts per minute (CPM) per well at each

Table 1 .
In vitro antiprotozoal activities and cytotoxic activity of marine metabolites.All data represent the mean ± margin of deviation of two independent determinations; b Positive controls; ND: IC 50 not determined due to low activity in initial screening; NT: not tested.
aT. b. rhodesiense L. donovani P. falciparum a