Synthesis and Biological Evaluation of New Salicylate Macrolactones from Anacardic Acids

Lúcio P. L. Logrado, Dâmaris Silveira, Luiz A. S. Romeiro, Manoel O. de Moraes, Bruno C. Cavalcanti , Letícia V. Costa-Lotufo , Cláudia do Ó Pessoa d and Maria Lucilia dos Santos* Laboratório de Isolamento e Transformação de Moléculas Orgânicas, Instituto de Química and Faculdade de Ciências da Saúde, Universidade de Brasília, Campus Darcy Ribeiro, 70904-970 Brasília – DF, Brazil Núcleo de Química Bioorgânica e Medicinal, Universidade Católica de Brasília, 71966-700 Taguatinga – DF, Brazil


Introduction
Non-isoprenoid phenolic lipids exist in plants from a number of different families, notably the Anacardiaceae shrub, many small plants, and certain bacterial sources.The cashew tree (Anacardium occidentale), a species originally native to Brazil where it is still cultivated, also grown in a large number of other tropical and sub-tropical countries, represents one of the major and cheapest sources of this class of compounds. 1s the main component of natural cashew nut-shell liquid (CNSL), anacardic acids (1) are the most widely distributed phenolic lipids.Chemically, the anacardic acids feature a convenient salicylic acid system and a long side-chain at the 6-position, in which a double bond is found at C-8 in the monoene, diene and triene components (Figure 1).Anacardic acids are reported to exhibit a variety of biological activities, 2 and also have stimulated much research in order to prepare drug analogues for application in several fields. 3n the course of ongoing investigations aiming at new drug candidates, the particular structural behavior and abundance of anacardic acids have prompted us to search for a strategy to convert these materials into analogues of an emerging family of antitumor natural products, for example: oximidines I, II, and III, 4 apicularens A and B, 5 and salicylhalamides A and B 6 (Figure 2), which present a benzofused macrolactone bearing an unusual N-acylated enamide side-chain.][6][7] Other macrolactones structurally related to the above macrolides include lasiodiplodin, 8 cis-and transresorcylide, 9 and curvularin 10 (Figure 3).2][13] For instance, methyldehydrolasiodiplodin, a mixture of geometrical isomers used as a precursor for the synthesis of lasiodiplodin, exhibits a rather uniform in vitro activity against human tumors e.g.leukemia, lung, colon, melanoma, ovarian, renal, prostate and breast. 14few years ago, we reported the synthesis of lasiodiplodin from the non-isoprenoid phenolic lipids of cashew nut-shell liquid 15 as well as the salicylate macrolactone 2 (Figure 4). 16Supported by the structural relationship of the compound 2 with the naturally occurring macrolides shown in Figures 2 and 3, a cytotoxicity study on this simplified analogue was initiated.
Compound 2 showed pronounced cytotoxicity in the brine shrimp lethality assay (Artemia salina). 17Since this protocol has been used to access antitumor potential in preliminary test, this result has encouraged us to prepare structural variants of macrolactone 2 in order to establish the first structure-activity relationships based on cytotoxic assays.
Herein we provide a full account of the experimental details of earlier communications 16 and add new results.In particular, we report the synthetic protocols for the preparation of the salicylate macrolactone 2 and its  chemical transformations into related macrolides modified at either the C-13 or the C-14 positions (Figure 4, Scheme 1).Furthermore, we describe the results of the cytotoxicity screening performed on these analogues.

Chemistry
As shown in Scheme 1, our approach for the synthesis of the title compounds is an extension of a previous work involving the use of inexpensive and readily available anacardic acids (1).
The heterogeneous mixture of anacardic acids (1) was separated from fresh natural CNSL essentially by the same procedures described in the literature 18 and was quantitatively converted into 3, on a multigram scale, by treatment with dimethyl sulphate under phase transfer catalysis. 19The mixture of dialkylated anacardic acids was submitted to ozonolysis, followed by reductive cleavage of the derived ozonides with sodium borohydride, furnishing the alcohol 4 in 85% yield.Hydrolysis of the ester group in 4 required rather severe conditions and was accomplished by using aqueous potassium hydroxide (10 mol L -1 ) in ethylene glycol at 165 o C.
The resulting hydroxyacid 5 was converted into macrolide 2 under high dilution conditions and nitrogen atmosphere in 66% yield, employing Mukaiyama's procedure. 20The conversion of the macrolactone 2 into the analogues 9 and 10 commenced with benzylic bromination, according to classical conditions (NBS-benzoyl peroxide).After several attempts, we found that the bromine lactone 6 was quantitatively converted into trans-unsaturated lactone 7 by treatment with DBU in refluxing benzene under a nitrogen atmosphere.
Two distinct methods were explored toward the epoxidation of the double bond, either by treatment with performic acid or m-CPBA.Gratifyingly, in the latter case 7 was smoothly epoxidized to furnish 8 in 98% yield.The epoxide opening was investigated under various conditions by using heterogeneous catalytic hydrogenation in a Parr apparatus.The best result, i.e. 97%, was obtained with 5% Pd-C in ethanol, under 4 atm of hydrogen pressure.The oxidation of the hydroxyl group in 9 was achieved by the classical Jones protocol in ether at room temperature.By means of this methodology, compound 9 was successfully converted into the keto macrolactone 10, in 96% yield.
The spectral properties of the all acyclic precursors and the macrocyclic lactone 2 are in accordance to the data previously reported. 16The 1 H and 13 13 C NMR peaks for the acyclic precursors and macrocyclic lactones were described according to Figure 5.These spectra data will be further explored in connection with a study underway utilizing a NMR computer-aided approach and X-ray crystallography in order to establish the conformational structure of all new macrolactones.

Cytotoxicity screening
Lethality against brine shrimp has been successfully used in order to detect antitumor and pesticidal compounds.The Artemia salina assays of the novel synthesized macrolides 2 and 6-10 were performed essentially according to Meyer's method. 17This methodology has been described as a simple, inexpensive, and convenient bioassay system, which has mostly been employed for screening of general bioactivity or as a general measure of toxicity, as well as in structureactivity relationship studies.Furthermore, it is positively correlated to 9KB cell toxicity. 21Matthews suggests the use of this protocol for the discovery of compounds with ability to protect against damage by active oxygen species (AOS). 22ompounds 2 and 10 have been found to display significant toxicity against Artemia salina.As a matter of fact, 2 (DL 50 32.12ppm, 0.032 mg mL -1 ) is nearly 3-times more potent than 10 (DL 50 98.42 ppm, 0.098 mg mL -1 ).The latter compound is less active than the active controls e.g.lapachol (DL 50 76.82ppm, 0.077 mg mL -1 ) and potassium dichromate (DL 50 84.05ppm, 0.084 mg mL -1 ).On the other hand, compounds 6-9 showed no activity detected at concentration up to 200 ppm, indicating that modification at C-13 or C-14 decreased the shrimp lethality.In light of these results, we decided to examine further the cytotocity profile of the synthesized macrolactones.
Uncontrolled proliferation is an universal property of tumor cells, thus investigation of the cellular growth control mechanism has contributed to the understanding of carcinogenesis and the discovery of compounds with specific antineoplastic activity. 23For this purpose, all of the synthesized macrolides were submitted to human tumor cell growth inhibition screening.The cytotoxic evaluation against lymphoblastic leukemia (CEM), breast cell line (MCF-7), human colon (HCT-8), and murine skin (B16), was accomplished by the MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide] microassay described by Mosmann. 24In this method, the cell viability is determined by using a tetrazolium salt, which is reduced to a coloured product due to chemical interactions with enzymes present only in living metabolically active cells. 25etrazolium salts have also been used to improve detection of drug cytotoxicity in a soft agar colony formation assay 26 and exhibit the advantage over many other stains of being nonlethal, allowing cells to be recovered, if desired.As shown in Table 1, the assays shown that the compounds 6 and 7 inhibited the proliferation of all four tested cell lines, 2 was active against mammalian cells, while 8 was active against HCT8 and MCF-7 cell lines.Interestingly, the macrolactone 7 exhibited unique differential ex vivo activity against MCF-7 cells (Figure 6).These findings suggest that the double bond at the benzylic position may play an important role in assisting its cytotoxic profile, as in the truncated macrolides oximidines.Pure compounds could be considered satisfactory to warrant further studies as an antineoplastic drug when they present an IC 50 values lower than 1 μg mL -1 or 1 μmol L -1 . 27Efforts to expand the investigation around 7 are currently underway and will be reported in due course.
In order to verify whether the active substances acted through membrane disruption, all the new chemical entities (2 and 6-10) were tested for the ability to induce lysis in mouse erythrocytes. 28The results showed that none of them cause membrane damage.

Experimental Instrumentation, chemical and solvents
The oxidative cleavages were performed with a Welsbach T-408 ozonizator and the catalytic hydrogenations in a Parr apparatus.Flash Column Chromatography and Dry-Column Flash Chromatography were carried out using silica gel Merck 60 Å (70-230 mesh) and eluting with a gradient of hexane:ethyl acetate.Analytical thin layer chromatography was performed on precoated Merck silica gel (60F 254 /0.2 mm) plates using UV light, 5% ethanolic phosphomolybdic acid or iodine vapours to visualize the spots.Melting points were obtained on a Köffler apparatus and are uncorrected.IR-FT spectra were recorded on a Bomem Hartmann & Braun (MB -100) spectrometer. 1H and 13 C-NMR were recorded on Mercury plus Varian (7.05 T) and Bruker (17.60 T) spectrometers, using CDCl 3 /TMS. 1H and 13 C chemical shifts are reported in parts per million (ppm) relative to TMS. High pressure liquid chromatography electrospray mass spectroscopy (HPLC-ESIMS) experiments were performed using a Quattro II Micromass/Waters spectrometer.

Isolation of anacardic acids (1)
The shells (500 g) of cashew nuts from Anacardium occidentale (Ceará, Brazil) were extracted in a Soxhlet extractor with commercial 95% ethanol (2.0 L) during 6 h, yielding a crude extract (CNSL, 157 g, 31% by weight).Anacardic acids (1) were removed in 61% from CNSL (15.25 g) either by precipitation with lead nitrate or calcium hydroxide. 18The spectral properties were identical to those reported in the literature. 29

Methyl-2-methoxy-6-(8-hydroxyoctyl) benzoate (4)
A solution of 3 (3.0g, 8.00 mmol average molecular wt 376) in dichloromethane (75 mL) and methanol (75 mL) at -70 o C was treated with ozone until the reaction was shown to be complete by thin layer chromatography (hexane-ethyl acetate, 9:1).The reaction mixture was purged with nitrogen, an excess of sodium borohydride (2.0 g) was added, and the resulting mixture was stirred at room temperature overnight.After addition of water (10 mL), the reaction mixture was hydrolysed with 10% hydrochloric acid (20 mL) and then extracted with ethyl acetate (3 x 30 mL).The combined extracts were washed with brine (3 x 30 mL), dried over sodium sulphate and evaporated.The residue was chromatographed on silica gel (hexane-ethyl acetate, 2:1) to afford the pure alcohol 4 16

Cytotoxicity assays
Brine shrimp lethality assay.Brine shrimp Artemia salina encysted eggs (Maramar) were incubated in simulated seawater at 28°C during 48 h with artificial light.The samples were dissolved in 200 μL of DMSO plus 20 μL of artificial seawater.Serial dilutions (triplicate) were prepared from the same solution.Metanauplii (10 units) was added to each set of tubes containing the samples and the cultures were incubated for additional 24 h.Controls containing DMSO were included in each set of experiments.Lapachol and potassium dichromate were used as reference standards.LC 50 (after 24 h) were calculated by Probit analysis.
Tumor cell growth inhibition assays.Adherent cells (0.2 x 10 5 cell 100 μL -1 ) and suspension cells (0.5 x 10 6 cells 100 μL -1 ) were seeded in 96 well microplates (Nucnck) and pre-incubated for 24 h in order to allow cell attachment.For the suspension cells this is not necessary.After plating the cells, fresh medium (100 μL) containing various concentrations (0.39 and 25 mg mL -1 ) of the samples was added to the cultures, then the cells were incubated for 72 h.Cell survival was evaluated by adding MTT tetrazolium salt solution with fresh medium.Doxorubicin (Doxolem ® , Zodiac Produtos Farmacêuticos S/A, Brazil) was used as positive control.After 3 h incubation at 37 o C, 150 μL of DMSO was added to dissolve the precipitate of reduced MTT.Microplates were then shaken for 15 min, and the absorbance was determined at 550 nm with a multiwell scanning spectrophotometer.CEM (human acute lymphoblastic leukemia), MCF-7 (breast cell line), HCT-8 (human colon) and B16 (murine skin) were maintained in RPMI 1640 (Gibco BRL), containing 10% fetal bovine serum (FBS; Gibco BRL), 1% penicillin and streptomycin solution (Life Technologies), and incubated at 37 °C in 5% CO 2 atmosphere.Hemolytic assay.The test was performed in 96-well plates.Each well received 100 μL of 0.85% saline containing 10 mmol L -1 calcium chloride solution.The first well was the negative control that contained only the vehicle (DMSO 10%) and, in the second well, 50 μL of the test sample and 50 μL of brine were added.The compounds were tested at concentrations ranging from 0.39 to 200 μg mL -1 .The serial dilution continued until the 11 th well.The last well received 20 μL of 0.1% triton X-100 (in 0.85% saline) to obtain 100% hemolysis (positive control).Then, each well received 100 μL of a 2% suspension of mouse erythrocytes in 0.85% saline containing 10 mmol L -1 calcium chloride solution.After incubation at room temperature for 30 min and centrifugation, the supernatant was removed and the liberated hemoglobin was measured spectroscopically as absorbance at 540 nm.

Figure 5 .
Figure 5. Numerical order of the atoms in acyclic and cyclic intermediates used for the NMR assignments.

Figure 6 .
Figure 6.Growth inhibition of the compound 7 against tumor cell lines.

Table 1 .
Cytotoxicity of compounds 2 and 6-10 based on tumor cell growth inhibition a Positive control.