Synthesis of 2,3-Diyne-1,4-naphthoquinone Derivatives and Evaluation of Cytotoxic Activity against Tumor Cell Lines

Uma série de derivados 2,3-diino-1,4-naftoquinona foi sintetizada a partir do 2,3-dibromo1,4-naftoquinona e diversos alquinos terminais funcionalizados usando a reação de acoplamento de Sonogashira catalisada por paládio. Os compostos foram submetidos à avaliação do potencial citotóxico em três linhagens de células tumorais, OVCAR-8 (ovário), PC-3M (próstata) e NCI-H358M (pulmão), apresentando, no geral, resultados satisfatórios para inibição do crescimento celular.


Introduction
Naphthoquinones are substances of great pharmacological interest as they exhibit a wide variety of biological activities, 1 such as antitumor, 2,3 antibacterial, 4 antifungal, 5 molluscicides, [6][7][8] antileishmanial 9 and anti-inflammatory. 10he mechanism of action of naphthoquinones involves oxidants and electrophile properties influenced by their chemical structure. 11There are two important mechanisms of quinone cytotoxicity: stimulation of oxidative stress and alkylation of cellular nucleophiles, which encompass a large range of biomolecules.Reactive oxygen species (ROS) may react directly with DNA, lipids and proteins, leading to cell damage 12,13 and shunting electrons toward oxygen, a futile pathway for reduction equivalents otherwise used for cytochrome P450 reductase-dependent reactions.8][19] 2,3-Diyne-1,4-naphthoquinones and related compounds exhibit an enediyne system that is capable of causing damage to cellular DNA through cycloaromatization, with the formation of diradical benzenoid intermediates that remove hydrogen atoms from the sugar skeleton-phosphate of the DNA double helix. 20nediyne compounds are among the most effective known chemotherapy agents, highly potent for cleavage of DNA.In recent years, considerable efforts have been invested in the development of drugs such as enediyne showing a maximum activity against tumor cells and minimal toxicity in normal cells. 21n this work, we report the synthesis of 2,3-diyne-1,4-naphthoquinones employing Sonogashira cross-coupling reaction 22 between 2,3-dibromo-1,4-naphthoquinone and various functionalized terminal alkynes.We also report the cytotoxic activity of these derivatives in three tumor cell lines: human ovarian adenocarcinoma (OVCAR-8), human metastatic prostate cancer (PC-3M) and human bronchoalveolar lung carcinoma (NCI-H358M), presenting, in general, satisfactory results for cell growth inhibition.

Synthesis of the compounds
The diyne derivatives (2a-i) were synthesized following a procedure described in the literature, 17 via cross-coupling between 2,3-dibromo-1,4-naphthoquinone (1) and several terminal alkynes catalyzed by palladium complexes and co-catalyzed by copper(I) iodide. 22Initially, the synthesis of compound 2a from the phenylacetylene alkyne and (1) was performed in a mixture of solvents, DMSO (dimethylsulfoxide) and DCM (dichloromethane) (1:1), using triethylamine, CuI and a palladium complex (Pd(PPh 3 ) 2 Cl 2 ) as catalyst.The product (2a) was obtained in 45% yield after isolation on silica gel column chromatography.In virtue of the importance of the catalyst to the formation of the coupling product, other palladium complexes were also tested under the same reaction conditions.Using Pd(PPh 3 ) 4 , compound 2a was obtained in only 27% yield (after consumption of 1 by TLC inspection).Other catalysts tested were unable to provide the product of interest (2a) (Table 1).
The preformed complex Pd(PPh 3 ) 2 Cl 2 (Table 1, entry 2) was chosen as catalyst in the coupling reaction of (1) with several terminal alkynes due to its higher stability and solubility and better performance for 2a formation.As a result, compounds 2a-i were obtained with 25-55% yields, as shown in Table 2. Compounds 2c-e were acetylated using acetic anhydride and montmorillonite clay K-10 under ultrasound treatment, 23 producing three new derivatives (2c'-e') in yields ranging from 56 to 71%.All compounds were characterized by 1 H and 13 C nuclear magnetic resonance (NMR), liquid chromatography-mass spectrometry (LC-MS) and Fourier transform infrared spectroscopy (FTIR).The characterization of 2a by 1 H and 13 C NMR (300 and 75 MHz, CDCl 3 ) analyses showed a simplified spectrum due to a planar symmetry.A signal centered at d 8.16 ppm (dd, 2H, J 3. DMSO was the solvent of choice for the preparation of compounds 2a-i due to the relatively good stability of 1 in this solvent, as reported by Romanov et al. 17 in contrast to what is observed in other solvents (pyridine, Dimethylformamide-DMF, Et 3 N, etc.).CH 2 Cl 2 was previously reported as a suitable co-solvent to reduce resinification produced in reaction. 17The use of THF (tetrahydrofuran) as solvent in the synthesis of 2a was unsuccessful, resulting in a rather complex mixture of products, not allowing isolation.Alternatively, when this reaction was performed in acetonitrile, 2a was produced in 20 min, but with 36% yield after column chromatography purification procedure.
The low yields in Sonogashira coupling products (2a-i) are related to several factors, e.g., low reactivity of 2,3-dibromo-1,4-naphthoquinone (1) in performing coupling reactions.In addition, vinyl bromides are less reactive than their iodide and triflate analogs in the Sonogashira type coupling reactions. 24Another problem is the undesirable formation of alkyne homocoupling due to reaction conditions and exposure to oxidizing agents. 24everal terminal alkynes with different substituents can be submitted to Sonogashira coupling, but some specific alkynes have a low reactivity or even do not react. 25he reaction with 4-methoxyphenylacetylene afforded compound 2b with 46% yield.It is possible to observe that the electron donor methoxy substituent increased the reactivity, and this reaction occurred two times faster than that from unsubstituted phenyl (entry 2, Table 2).Even though, the yields were not significantly different.The coupling with alkynes containing the alcohol function resulted in products 2c, 2d and 2e with yields of 42, 30 and 25%, respectively.It was observed that the hydroxyl group influences the yield of the coupling, so that the less hindered the hydroxyl group, the lower the reaction yields.The coupling of 1 with different alkyne alkyl (1-pentyne, 1-hexyne, 1-octyne and 1-decyne) resulted in products 2f, 2g, 2h and 2i yields with 38, 55, 38 and 30%, respectively.No obvious relation between the size of the alkyne chain and the reaction yield could be noticed.
Using the same above reaction conditions, the coupling between 2,3-dibromo-1,4-naphthoquinone (1) and some terminal alkynes were not successful.The reaction with alkyne prop-2-yn-1-ol (propargyl alcohol) resulted in a complex mixture of products which could not be isolated.The reaction with ethyl propiolate also did not result in the desired coupling product, probably due to electron-withdrawing alkyne conjugation, thus lowering the reactivity of the alkyne to Sonogashira coupling reaction. 26ttempts to react 1 with trimethylsilylacetylene were also unsuccessful, confirming previously reported results. 27

Cytotoxicity assay
The cytotoxicity of the 2,3-diyne-1,4-naphthoquinone derivatives (2a-i; 2c'-e') were evaluated in vitro against three tumor cell lines of different histotypes, PC-3M, OVCAR-8 and NCI-H358M, using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay. 28Doxorubicin was used as a positive control.The cytotoxic activity of the samples is shown in Table 3, with their respective percentage of cell growth inhibition (GI in %).Substances which showed inhibition of tumor cell growth greater than 75% in at least two tumor cell lines tested in the preliminary test with a single dose of 25 µg mL -1 were selected to determine the IC 50 value.
Among the tested samples, ten (2a-f, 2h and 2c'-e') were selected as the most active as cytotoxic agents, as they presented cell growth inhibition greater than 75% in at least two tested tumor cell lines.In addition, compound 2g, 2,3-di(hex-1-yn-1-yl)-1,4-naphthoquinone, was also selected for IC 50 determination as it presented high specificity (95.83% inhibition) towards OVCAR-8 cell line.Only compound 2,3-di(dec-1-yn-1-yl)-naphthoquinone 2i showed no cytotoxic potential higher than 75% in any of the three tested tumor cell lines.In general, compounds having aliphatic side chains had lower percentage of cell  4. From the eleven selected samples for determination of IC 50 , eight (2b-f, 2c'-e') showed inhibitory concentration smaller than 10 µM for the three tested tumor cell lines, characterizing them as potent cytotoxic agents.The compounds (2c, 2d and 2e) which bear hydroxyl groups have approximately the same cell growth inhibition (IC 50 ) of acylated compounds (2c', 2d' and 2e').Thus, it is possible to see that the hydroxyl group in these compounds did not significantly influence the cytotoxic action for the three analyzed tumor cell lines.Samples 2a, 2g and 2h exhibited IC 50 higher than 10 µM for PC-3M, showing lower cytotoxic action for this cell line and higher selectivity for inhibitory action against OVCAR-8 and NCI-H358M lines.The increase of the aliphatic chain reduced the cytotoxic effect on PC-3M cell line.The 2,3-di(3-hydroxy-3-methylbut-1-in-yl)-1,4-naphthoquinone (2c) presented the lower IC 50 (2.28-4.28µM) for all three tested cell lines, characterizing this substance as a potent cytotoxic agent.Although the excellent cytotoxicity in cancer cell lines, all the compounds presented high inhibitory effect in peripheral blood mononuclear cells, which means non-selectivity effect and high cytoxicity.Further investigation aiming at mechanisms of action and hemolytic activity are required for a better understanding of the cytotoxic effects and for determining the potential of the compounds reported in this work as future antitumor agents.

Conclusions
In this work, new potential antitumor active diynenaphthoquinones were synthesized (twelve derivatives), nine of them new entities.In general, they exhibited inhibitory concentrations (IC 50 ) smaller than 10 µM for all three tested tumor cell lines, characterizing potent cytotoxic action.These results are significant for continuing studies of cytotoxicity in this class of bioactive compounds.

Experimental
General remarks 1 H and 13 C NMR spectra were recorded on a Varian unity plus-300 or 400 MHz spectrometer in CDCl 3 or DMSO-d 6 .All IR spectra were recorded on a Bruker IFS66 spectrophotometer using KBr pellets.High resolution mass spectra were obtained by electrospray on a Shimadzu LC-MS-IT-TOF spectrometer.Sonication was performed using a Model USC-1400A ultracleaner with a frequency of 40 kHz.Melting points are uncorrected and were determined with an electrically heated block apparatus.Monitoring of the course of the reactions was performed by GF 254 thin layer chromatography (TLC) and the column chromatography was performed on silica gel G 60 (70-230 mesh, Merck).All solvents and reagents were purchased from commercial suppliers (Sigma-Aldrich, Tedia, Merck, Cinética, Fluka), and used as received or purified by standard procedures.Data are presented as IC 50 values and 95% confidence intervals obtained by nonlinear regression for all cell lines.Doxorubicin was used as positive control.
Only compounds with an IC 50 value lower than 5 µg mL -1 for at least one cell line were considered active.ND: not determined.
Synthesis of the 2,3-diyne-1,4-naphthoquinone derivatives (2) A mixture of terminal alkynes (1.0 mmol), CuI (0.13 g, 0.68 mmol) and triethylamine (0.07 g, 0.8 mmol) in anhydrous DMSO (4 mL) and CH 2 Cl 2 (3 mL) was stirred for 2 min under argon atmosphere, then 2,3-dibromo-1,4-naphthoquinone (0.100 g, 0.32 mmol) and Pd(PPh 3 ) 2 Cl 2 (0.005 g, 0.007 mmol) were added to the reaction mixture.The stirring was continued until total consumption of the starting material (1), after that, CH 2 Cl 2 was removed by reduced pressure and water was added to the reaction mixture.The precipitate formed was separated by vacuum filtration and the compounds were isolated by silica gel column chromatography.Diol (2c, 2d or 2e) (0.125 mmol) was mixed with montmorillonite K-10 (200% by weight of substrate) and 1 mL of acetic anhydride.The reaction was performed under ultrasound irradiation at room temperature for 60 min.After this time, 15 mL of dichloromethane were added and montmorillonite K-10 was removed by simple filtration.The filtrate was neutralized with a concentrated solution of NaHCO 3 (3 × 20 mL) in a separatory funnel and the organic phase dried with anhydrous sodium sulfate.The product was purified by column chromatography on silica gel using a mixture of ethyl acetate/hexane.The cytotoxic effects of the synthesized compounds were evaluated against the following human cancer cell lines and non-tumor cells (PBMC), all transformed cell lines were obtained from the National Cancer Institute, Bethesda, MD, USA: PC-3M (prostate carcinoma), OVCAR-8 (ovarian carcinoma) and NCI-H358M (bronchoalveolar lung carcinoma).The cell lines were maintained in RPMI-1640 medium (cancer cells) supplemented with 10% fetal bovine serum, 2 mmol L -1 glutamine, 100 µg mL -1 penicillin and 100 µg mL -1 streptomycin at 37 °C with 5% CO 2 .
Cell growth was quantified by the ability of living cells to reduce a yellow dye (MTT) to a purple formazan product. 28or all of the experiments, the cells were seeded in 96-well plates (0.1 × 10 5 cells per well for adherent cells).After 24 h, the compounds (0.01 to 25 mg mL -1 ), dissolved in DMSO, were added to each well (using an HTS (high-throughput screening) Biomek 3000, Beckman Coulter, Inc. Fullerton, California, USA) and incubated for 72 h.Doxorubicin (Sigma-Aldrich Co., St. Louis, MO, USA) was used as a positive control.At the end of the incubation, the plates were centrifuged, and the medium was replaced by fresh medium (200 µL) containing 0.5 mg mL -1 MTT.After 3 h, the formazan product was dissolved in 150 µL DMSO, and the absorbance was measured using a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter, Inc., Fullerton, California, USA).The substance effect was quantified as the percentage of the control absorbance at 595 nm.
3 and 5.7 Hz) and at d 7.77 ppm (dd, 2H, 3.3 and 5.7 Hz) were attributed to orto-(5/8) and meta-benzenoid hydrogens.A multiplet centered at d 7.68 ppm (4H) and another at d 7.41 ppm (6H) were attributed to phenyl hydrogens.The 13 C NMR spectra presents a single carbonyl peak at d 181.1 ppm, and the sp carbons of triple bond at d 85.4 and 109.7 ppm, among the expected eight sp 2 signals.

Table 3 .
Percent inhibition of cell growth obtained with the samples in three tumor cell lines in a single dose of 25 mg mL-1 GI: growth inhibition percentage; SD: standard deviation.Vol. 24, No. 9, 2013 growth inhibition.The calculation of IC 50 was measured by MTT assay after 72 h of incubation at concentrations from 0.01 to 25 mg mL -1 .Peripheral mononuclear blood cells (PBMC) were tested to confront the data in normal cells.The results are shown in Table