Antiplasmodial Activities of Homogentisic Acid Derivative Protein Kinase Inhibitors Isolated from a Vanuatu Marine Sponge Pseudoceratina sp.

As part of our search for new antimalarial drugs in South Pacific marine sponges, we have looked for inhibitors of Pfnek-1, a specific protein kinase of Plasmodium falciparum. On the basis of promising activity in a preliminary screening, the ethanolic crude extract of a new species of Pseudoceratina collected in Vanuatu was selected for further investigation. A bioassay-guided fractionation led to the isolation of a derivative of homogentisic acid [methyl (2,4-dibromo-3,6-dihydroxyphenyl)acetate, 4a] which inhibited Pfnek-1 with an IC50 around 1.8 μM. This product was moderately active in vitro against a FcB1 P. falciparum strain (IC50 = 12 μM). From the same sponge, we isolated three known compounds [11,19-dideoxyfistularin-3 (1), 11-deoxyfistularin-3 (2) and dibromo-verongiaquinol (3)] which were inactive against Pfnek-1. Synthesis and biological evaluation of some derivatives of 4a are reported.


Introduction
Malaria is a major health problem in tropical and subtropical regions and up to 2.5 million people die from it each year, mostly children in sub-Saharan Africa. Plasmodium falciparum causes the majority of deaths, and the spread of its resistance to many antimalarials (quinine, chloroquine, and mefloquine) has increased the need for development of new drugs. Natural products have provided clinically used antimalarials such as quinine and artemisinin and continue to make an important contribution to the discovery of new lead compounds. In past few years, not only plants but marine organisms have also been intensively investigated for obtaining new therapeutic agents against malaria [1][2][3][4].
As part of our search of new drugs against malaria, we have looked for inhibitors of a Pfnek-1 [5], a NIMA-related protein kinase of Plasmodium falciparum, in South Pacific marine sponges. This strategy previously led to the isolation of xestoquinone from Xestospongia sp. and to the characterization of its antiplasmodial activity [6]. An ethanolic crude extract from a new species of Pseudoceratina collected in Vanuatu was selected for its promising activity against Pfnek-1. Marine sponges of the order Verongida are characterized by tyrosine metabolites. Among them, sponges of the genus Pseudoceratina were the source of many bromotyrosine metabolites with interesting biological activities [7][8][9][10][11][12][13][14][15][16][17][18].

Chemistry
The bromotyrosine metabolites, 11,19-dideoxyfistularin-3 (1), 11-deoxyfistularin-3 (2) and dibromoverongiaquinol (3) were isolated from an ethanolic extract of Pseudoceratina sp. which was subjected to solvent partitioning between CH 2 Cl 2 and H 2 O. The compounds were obtained from the CH 2 Cl 2 -soluble and -insoluble extracts by chromatography on silica gel and further purification by preparative thick layer chromatography or by reverse phase HPLC. The spectroscopic properties of the isolated bromotyrosine metabolites 1-3 were consistent with those previously published [19]. A bioassay-guided fractionation based on Pfnek-1 inhibition assay led us to isolate methyl (2,4-dibromo-3,6-dihydroxyphenyl) acetate (4a). An analogue of this compound, 4,6-dibromo-2,5dihydroxyphenylacetic acid amide, was already isolated from the sponge Verongia aurea [20]. Isolation of 4a from Verongia aurea was also briefly mentioned in a patent [21], but surprisingly, no spectroscopic data were reported. For the unambiguous identification of natural compound 4a as responsible for activity against Pfnek-1, it was necessary to perform its synthesis, which was achieved as reported in Scheme 1.
The principal impediment of this synthesis was the difficulty in achieving the regioselective bromination of the phenyl ring. First attempts at bromination of methyl (2,5-dihydroxyphenyl)acetate (8) yielded a mixture of mono-, di-and tri-bromo derivatives. In order to increase the regioselectivity, we used as starting material 5-hydroxy-3H-benzofuran-2-one (5) which bears only one hydroxyl group. A similar pathway has been used for the synthesis of 4,6-dibromo-2,5-dihydroxyphenylacetic acid amide and derivatives [21,22]. Compounds 6a and 6b were thus obtained concurrently by bromination of 5 in the presence of N-bromosuccinimide and a catalytic amount of p-toluenesulfonic acid in CH 2 Cl 2 , and subsequently separated on silica gel by column chromatography. Ring opening of the benzofuran-2-ones was achieved with p-toluenesulfonic acid in MeOH to give 4a and the monobrominated analogue 4b. The spectroscopic properties ( 1 H-, 13 C-NMR, MS) of the synthesized compound 4a were the same as those of the natural product, thus allowing its unambiguous identification (Table 1). In order to study the influence of the position of the two hydroxyl groups and the ester chain on the phenyl ring, we synthesized an analogue 12 of the natural product with hydroxyl groups in the meta position (Scheme 2).
Treatment of 2',4'-dihydroxyacetophenone (9) with potassium carbonate in the presence of benzyl chloride gave compound 10 in 73% yield. Willgerodt-Kindler rearrangement of 10 with thallium trinitrate and perchloric acid, followed by subsequent generation of hydroxyl groups in the presence of palladium 10% on carbon and ammonium formate gave compound 11. Electrophilic ring dibromination was then carried out with N-bromosuccinimide and p-toluenesulfonic acid as catalyst in MeOH under sonication to obtain 12 in good yield. Finally, to study the substitution of the two Furthermore, the synthesis of 12 and 4a allowed us to revise the structure of subreaphenol B, a natural compound isolated from the sponge Suberea mollis and described as 12 [23]. However, the spectroscopic data ( 1 H and 13 C NMR) published for the natural compound did not match with our data for 12, but rather with our data for 4a, showing that the structure of subreaphenol B was identical to 4a and not 12.

Biological Properties
The ability of the pure natural products and of the synthetic compounds to inhibit Pfnek-1 activity was investigated (Table 2). Only compounds 4a and 4b exhibited a protein kinase inhibitor activity, particularly 4a (IC 50 = 1.8 µM) which was five times more active than 4b (IC 50 = 10 µM). 4,6-Dibromo-2,5-dihydroxyphenylacetic acid methyl ester (4a) was previously reported in a patent concerning homogentisic acid derivatives and their protein kinase C inhibitor activity [21]. The comparison of compounds 4a, 4b and 8 highlights the influence of the presence and number of bromine atoms on the phenyl ring. The non-brominated 8 was inactive, while monobrominated 4b was moderately active; the dibrominated compound 4a was the most active. Furthermore the presence of two free hydroxyl groups appears to be essential, as shown by: (i) the inactivity of the benzoyl analogues 13a and 13b and (ii) the inactivity of the lactone analogues 6a and 6b. In addition, the para position of the hydroxyl groups (hindered quinone system) is critical because the analogue 12 as well as the tyrosine metabolites 1-3 which have hydroxyl groups in the meta position are inactive. These results confirm the important role of quinone/phenolic part in the mode of action on Pfnek-1, a feature that is present in other Pfnek-1 inhibitors such as xestoquinone, halenaquinone, alisiaquinones A and B or alisiaquinol [6,24].

Compound number
Pfnek Compound number 1 Growth inhibition diameter (mm) S. aureus E. coli 1 14 In conclusion, three bromotyrosine metabolites 1-3 and the homogentisic acid derivative 4a have been isolated from the marine sponge Pseudoceratina sp. collected in Vanuatu. 11-Deoxyfistularin-3 (1) and dibromoverongiaquinol (3) showed strong antibacterial activities against S. aureus and E. coli like several bromotyrosine metabolites isolated from marine sponges belonging to the order Verongida. Homogentisic acid derivative 4a exhibited Pfnek-1 inhibitor activity (IC 50 = 1.8 µM) and its unambiguous identification has been performed by synthesis. The study of the structure-activity relationships for the natural product 4a and its synthetic analogues 4b, 5, 6a, 6b, 8, 12, 13a and 13b has highlighted the essential role of the bromine atoms and of the position of the hydroxyl groups for the inhibition of Pfnek-1. Consequently, the structural feature of homogentisic acid derivative 4a could serve as a model for the development of new Pfnek-1 inhibitors. Replacement of the bromine atoms by other halogens (Cl, F) and the elongation of the ester chain could be particularly interesting to enhance the Pfnek-1 inhibition of these homogentisic acid derivatives. Finally, the moderate activity against P. falciparum could result from the weak capacity of the inhibitor to reach the kinase (or other) target(s), and demonstrates the difficulties met when using an enzymatic test for the screening of natural extracts and for the discovering of new anti-infectious drugs.

Materials
The sponge of the genus Pseudoceratina Carter, 1885 (order Verongida, family Pseudoceratinidae) was collected by scuba diving at 40 m depth at Rowa islands, Banks Territory (Vanuatu). A voucher specimen, voucher number G318491, is deposited with the Queensland Museum, Brisbane, Australia. The FcB1 strain of P. falciparum was kindly provided by Dr. A. Valentin, Laboratory of Parasitology, Faculty of Pharmacy, Toulouse, France. Solvents were purchased from Ajax (Australia), and distilled before use, except for the HPLC grade methanol; Biochemical reagents were purchased from Sigma-Aldrich and Cambrex. Radioactive γ-[ 33 P] ATP was purchased from Perkin Elmer (France). HPLC was performed on a Waters apparatus, (Waters 510 pumps; Waters 996 Photodiode Array Detector) using a μBondapack C 18 column (125 Å, 10 μm, 4.6 × 250 mm). NMR spectra were recorded on Bruker AC 250, Bruker Avance 300, 400 and 500 spectrometers. Mass spectra were recorded on an ion trap LCQ Finnigan spectrometer in APCI ionization mode (positive or negative), except for compounds 13a and 13b (ESI ionization mode, positive). High resolution mass spectra were recorded on a GCT Premier apparatus (Waters Micromass) in CI (CH 4 ) ionization mode. Radioactivity was measured using a liquid scintillation analyzer Packard Tri-carb 1600TR and Packard Ultima Gold MV scintillation cocktail.

Extraction and Isolation
The freeze-dried sponge was extracted twice overnight with fresh 95% EtOH at room temperature, filtered and the ethanol was evaporated. The residue was subjected to solvent partition between CH 2 Cl 2 and H 2 O to give the CH 2 Cl 2 soluble extract and the CH 2 Cl 2 /H 2 O insoluble extract.

Protein Kinase Assay
A GST-Pfnek-1 fusion protein was purified from the Escherichia coli BL21 strain carrying a Pfnek-1 expression plasmid harbouring an ampicillin resistance cassette (bacteria kindly provided by D. Dorin, INSERM 511) as described by Dorin et al. [5]. Pfnek-1 kinase activity was determined by measuring the 33 P incorporation in ß-caseine using γ[ 33 P]-ATP.
Briefly, test compounds were dissolved in DMSO and diluted in 20 mM Tris, pH 7.5 20 mM MgCl 2 , 10 mM NaF and 10 mM ATP. β-casein (3 mg/mL) and γ[ 33 P]-ATP were added prior to the addition of Pfnek-1 to start the kinase reaction. Approximately 5 µCi of γ[ 33 P]-ATP was used per reaction. After incubation at 30 °C for 30 min, each solution was blotted on a phosphocellulose filter paper (P81 Whatman-cation exchange chromatography paper). After four washes with 1% H 3 PO 4 at, the remaining radioactivity was measured using a liquid scintillation analyzer Packard 1600TR. The IC 50 is defined as the concentration of compound which inhibits 50% of enzyme activity compared to the control (reaction in the absence of inhibitor).

Activity against Erythrocytic Stages of Cultured P. falciparum
The antiplasmodial activity was studied in vitro against the chloroquine-resistant Plasmodium falciparum strain FcB1 by a micromethod using the lactate deshydrogenase (LDH) assay (Makler and Hinrichs [34]). Parasites were cultivated using the method of Trager and Jensen [35]. Erythrocytes infected with P. falciparum (ring stage, 1% of parasitaemia) were re-suspended in complete culture medium at a haematocrit of 1.5%. The suspension was distributed in 96-well microtitre plates (200 µL per well). Drug testing was performed in triplicate. For each assay, a parasite culture was incubated with the drug for 48 h in 5% CO 2 at 95% relative humidity, and frozen until the biochemical assay could be run. After defrosting, a 20 µL sub-sample of the contents of each well was mixed with 100 µL of a substrate solution containing 20 mg/mL of lithium L-lactate (Sigma), 5.5 mg/mL of TRIS (Sigma), and 3.7 mg/mL of 3-acetylpyridine adenine dinucleotide (APAD; Sigma), in the well of another microtitre plate. After incubation for 30 min, 25 µL of a mixture of NBT (1.6 mg/mL; Sigma) and PES (0.1 mg/mL; Sigma) were added to each well. After a further 35 min of incubation, the reaction was stopped by the addition of 25% acetic acid (25 µL per well). Accumulation of the reduced form of APAD was measured at λ = 650 nm, using a spectrophotometer (microplate reader, Metertech). IC 50 values were determined graphically in a concentration versus percent inhibition curve.