Half-Wave Potentials and In Vitro Cytotoxic Evaluation of 3-Acylated 2,5-Bis(phenylamino)-1,4-benzoquinones on Cancer Cells

A broad range of 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones were synthesized and their voltammetric values, as well as in vitro cancer cell cytotoxicities, were assessed. The members of this series were prepared from acylbenzoquinones and phenylamines, in moderate to good yields (47–74%), through a procedure involving a sequence of two in situ regioselective oxidative amination reactions. The cyclic voltammograms of the aminoquinones exhibit two one-electron reduction waves to the corresponding radical-anion and dianion, and two quasi-reversible oxidation peaks. The first and second half-wave potential values (E1/2) of the members of the series were sensitive to the push-pull electronic effects of the substituents around the benzoquinone nucleus. The in vitro cytotoxic activities of the 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones against human cancer cells (bladder and prostate) and non-tumor human embryonic kidney cells were measured using the MTT colorimetric method. The substitution of both aniline groups, by either methoxy (electron donating effect) or fluorine (electron withdrawal effect), decreased the cytotoxicity in the aminoquinones. Among the members of the unsubstituted phenylamino series, two of the 18 compounds showed interesting anti-cancer activities. A preliminary assay, looking for changes in the expression of selected genes, was performed. In this context, the two compounds increased TNF gene expression, suggesting an association with an inflammatory-like response.


Results and Discussion
The members of the series of 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones (Scheme 1) were prepared from 1,4-benzoquinone and a set of aldehydes and phenylamines. The acylhydroquinone precursors 1a-q were synthesized by the solar Friedel Crafts photoacylation of quinone 1 with aldehydes, according to our previously-reported procedure [24]. The preparation of 3-acyl-2,5-bis (phenylamino)-1,4-benzoquinones 2a-q was accomplished through a sequence involving: (a) Oxidation of the acylhydroquinones 1a-q, to the respective acylquinones, with silver (I) oxide (2.5 equiv.) in dichloromethane (DCM) and (b) oxidative amination of the 2-acylquinones resulting in (b) with phenylamines (2 equiv.) in ethanol under aerobic conditions. This procedure provides the substituted benzoquinones 2a-q in the yield range 47-74% (Table 1), and is a modification of the procedure, described by Shäfer and Aguado in [25], for the synthesis of compound 2a and some of their substituted phenylamino analogs. It is worth noting that synthetic 2,5-bis(arylamino)-1,4-benzoquinones containing acyl and alkoxycarbonyl substituents at C-3 have been used as precursor of benz-and naphthoisoxazolequinones [21][22][23]. Some of the members of these heterocycle series exhibit in vitro activity as radiosensitizers and cytotoxic properties. Here, we wish to report the synthesis of a broad variety of 3-acyl-2,5-bis(arylamino)-1,4-benzoquinones, in order to give information on their redox properties and in vitro cytotoxic activity on cancer cells.

Results and Discussion
The members of the series of 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones (Scheme 1) were prepared from 1,4-benzoquinone and a set of aldehydes and phenylamines. The acylhydroquinone precursors 1a-q were synthesized by the solar Friedel Crafts photoacylation of quinone 1 with aldehydes, according to our previously-reported procedure [24]. The preparation of 3-acyl-2,5-bis (phenylamino)-1,4-benzoquinones 2a-q was accomplished through a sequence involving: (a) Oxidation of the acylhydroquinones 1a-q, to the respective acylquinones, with silver (I) oxide (2.5 equiv.) in dichloromethane (DCM) and (b) oxidative amination of the 2-acylquinones resulting in (b) with phenylamines (2 equiv.) in ethanol under aerobic conditions. This procedure provides the substituted benzoquinones 2a-q in the yield range 47-74% (Table 1), and is a modification of the procedure, described by Shäfer and Aguado in [25], for the synthesis of compound 2a and some of their substituted phenylamino analogs. The structures of compounds 2a-q were established by 1 H-and 13 C-nuclear magnetic resonance (NMR), bi-dimensional nuclear magnetic resonance (2D-NMR), and high resolution mass spectroscopy (HRMS). Heteronuclear multiple bond correlation (HMBC) experiments of the members of the series, as those of the representative congeners 2b and 2p (Figure 2), allowed us to establish the location of the substituents around the benzoquinone nuclei. It is worth mentioning that inspection of minimal energy conformation of compounds 2b and 2p, performed by MM2 calculation (ChemBio3D 11.0, PerkinElmer, MA, USA), shows a co-planar orientation between the benzoquinone nucleus and the phenylamino substituent at C-5 (Supplementary materials). Furthermore, it was also observed that rotation of the substituents linked to the 2,3-quinone double bond are strongly hindered ( Figure 3). The structures of compounds 2a-q were established by 1 H-and 13 C-nuclear magnetic resonance (NMR), bi-dimensional nuclear magnetic resonance (2D-NMR), and high resolution mass spectroscopy (HRMS). Heteronuclear multiple bond correlation (HMBC) experiments of the members of the series, as those of the representative congeners 2b and 2p (Figure 2), allowed us to establish the location of the substituents around the benzoquinone nuclei. The structures of compounds 2a-q were established by 1 H-and 13 C-nuclear magnetic resonance (NMR), bi-dimensional nuclear magnetic resonance (2D-NMR), and high resolution mass spectroscopy (HRMS). Heteronuclear multiple bond correlation (HMBC) experiments of the members of the series, as those of the representative congeners 2b and 2p (Figure 2), allowed us to establish the location of the substituents around the benzoquinone nuclei. It is worth mentioning that inspection of minimal energy conformation of compounds 2b and 2p, performed by MM2 calculation (ChemBio3D 11.0, PerkinElmer, MA, USA), shows a co-planar orientation between the benzoquinone nucleus and the phenylamino substituent at C-5 (Supplementary materials). Furthermore, it was also observed that rotation of the substituents linked to the 2,3-quinone double bond are strongly hindered ( Figure 3). It is worth mentioning that inspection of minimal energy conformation of compounds 2b and 2p, performed by MM2 calculation (ChemBio3D 11.0, PerkinElmer, MA, USA), shows a co-planar orientation between the benzoquinone nucleus and the phenylamino substituent at C-5 (Supplementary Materials). Furthermore, it was also observed that rotation of the substituents linked to the 2,3-quinone double bond are strongly hindered ( Figure 3).  The members of the 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinone series 2a-q were evaluated for their half-wave potentials (E I 1/2 and E II 1/2). They were measured by cyclic voltammetry in acetonitrile at room temperature, using a platinum electrode and 0.1 M tetraethylamoniumtetrafluoroborate as the supporting electrolyte [26]. The voltammograms were The members of the 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinone series 2a-q were evaluated for their half-wave potentials (E I 1/2 and E II 1/2 ). They were measured by cyclic voltammetry in acetonitrile at room temperature, using a platinum electrode and 0.1 M tetraethylamoniumtetrafluoroborate as the supporting electrolyte [26]. The voltammograms were run in the potential range from 0.0 to −2.0 V versus non-aqueous Ag/Ag + . The cathodic peaks related to the reduction of quinone, and the anodic one due to its re-oxidation, were observed for the compounds as well-defined quasi-reversible waves.
The E I 1/2 values for the first one-electron, which is related with the formation of the semiquinone radical anion, fell within the range of −850 to −500 mV. The E I 1/2 values for the second one-electron transfer, corresponding to the dianion formation [27,28], were located within the range of −1170 to −860 mV (Table 1). Taking into account the notable differences of the E 1/2 values for each of the one-electron transfer processes, it is evident that they are due to the push-pull electronic effects of the substituents located in the 1,4-benzoquinone nucleus. Table 1, regarding the E I 1/2 and E II 1/2 values of the unsubstituted phenylamino members 2a-h, indicates that the nature of the acyl substituents mainly affects the second half-wave potential (∆E II 1/2 = 300 mV) over the first one (∆E II 1/2 = 90 mV). The effect of the insertion of methoxy substituents into the 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinone scaffold, as in compounds 2i-2m, was related to a significant cathodic shift of the E I 1/2 when compared to E II 1/2 . This fact could be attributed to the donor effect of the 3,4,5-trimethoxyphenyl group, which is transmitted to the quinonoid nucleus through the amino group. tetraethylamoniumtetrafluoroborate as the supporting electrolyte [26]. The voltammograms were run in the potential range from 0.0 to −2.0 V versus non-aqueous Ag/Ag + . The cathodic peaks related to the reduction of quinone, and the anodic one due to its re-oxidation, were observed for the compounds as well-defined quasi-reversible waves.
The E I 1/2 values for the first one-electron, which is related with the formation of the semiquinone radical anion, fell within the range of −850 to −500 mV. The E I 1/2 values for the second one-electron transfer, corresponding to the dianion formation [27,28], were located within the range of −1170 to −860 mV (Table 1). Taking into account the notable differences of the E1/2 values for each of the oneelectron transfer processes, it is evident that they are due to the push-pull electronic effects of the substituents located in the 1,4-benzoquinone nucleus. Table 1, regarding the E I 1/2 and E II 1/2 values of the unsubstituted phenylamino members 2a-h, indicates that the nature of the acyl substituents mainly affects the second half-wave potential (ΔE II 1/2 = 300 mV) over the first one (ΔE II 1/2 = 90 mV). The effect of the insertion of methoxy substituents into the 3-acyl-2,5-bis(phenylamino)-1,4benzoquinone scaffold, as in compounds 2i-2m, was related to a significant cathodic shift of the E I 1/2 when compared to E II 1/2. This fact could be attributed to the donor effect of the 3,4,5-trimethoxyphenyl group, which is transmitted to the quinonoid nucleus through the amino group.
The 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones 2a-q were evaluated for their in vitro cytotoxic activities against normal human embryonic kidney cells (HEK-293 cells) and two human cancer cell lines (T24 and DU-145 cells) in 72 h drug exposure assays. The cytotoxic activities of the new compounds were measured using conventional microculture tetrazolium reduction assays [29]. The cytotoxic activities are expressed in terms of IC 50 . Doxorubicin, a clinically used anti-cancer agent, was taken as a positive control. The cytotoxic activity data are summarized in Table 2. Regarding the cytotoxicity of aminoquinones, it should be noted that the DU-145 cells were more sensitive than T24 and HEK-293 cells. When comparing the IC 50 values calculated for the most active aminoquinones in both cancer cell lines, their activity ranged from 16.3 to 51.80 µM. Such values were at least one order of magnitude higher than that obtained with doxorubicin (0.43 and 0.93 µM in T24 and DU-145 cells, respectively). The unsubstituted phenylamino members 2a-2h were largely more active than the compounds 2i-2q, showing that the substitution of both aniline groups-by either methoxy (electron donating effect) or fluorine (electron withdrawal effect)-decreased the cytotoxicity of the aminoquinones. Among the series 2a-2h, the compounds 2c (IC 50 values of 16.3 and 45.2 µM in T24 and DU-145 cells, respectively) and 2d (IC 50 values of 34.0 and 23.5 µM in T24 and DU-145 cells, respectively) were the most active; displaying, in addition, a highly selective effect, as HEK-293 cells were affected only at doses higher than 100 µM. Moreover, within the same series, compounds 2f and 2g showed similar cytotoxic activities to previous compounds, but with a much lower selectivity. Outside this series, compound 2l was also active, but without any selectivity as it affected both HEK-293 and cancer cells. Thus, it may be concluded that, among all the tested aminoquinones, congeners 2c and 2d displayed the best cytotoxic activities, exhibiting high selectivity and lipophilicity values. Therefore, they represent lead-molecules for further investigations in exploring both intracellular targets as well as their molecular mechanism of action.
In order to gain information about potential molecular targets, a preliminary assay for gene expression was conducted. To this end, some genes were selected, regarding their key role in cell survival, for instance mTOR, TP53, TNF, and so on. Table 3 shows the relative expression levels of the genes implicated in anti-cancer effects in T24 cells after treatment with 2c and 2d. Compound 2c (and, to a lesser extent, 2d) enhanced the expression of the TNF gene. Prior to drawing a definitive conclusion, such an increase in gene expression should be further confirmed by measuring its protein levels and activity. Nevertheless, it should be emphasized that a local increase in TNF concentration is not only associated with an inflammatory response, but also with an immunogenic response able to activate tumor-specific cytotoxic T lymphocytes, which can seek out and destroy tumor cells and reduce tumor lesions [30]. In this context, the compounds 2c and 2d display a potential application in cancer immunotherapy that deserves to be further investigated.

General Information
All the solvents and reagents were purchased from different companies, such as Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany), and were used as supplied. Melting points (mp) were determined on a Stuart Scientific SMP3 (Staffordshire, UK) apparatus and are un-corrected. The IR spectra were recorded on an FT IR Bruker spectrophotometer, model Vector 22 (Bruker, Rheinstetten, Germany), using KBr disks, and the wave numbers are given in cm −1 . 1 H-and 13 C-NMR spectra were recorded on a Bruker Avance-400 instrument (Bruker, Ettlingen, Germany) in CDCl 3 or DMSO-d 6 at 400 and 100 MHz, respectively. Chemical shifts are expressed in ppm downfield relative to tetramethylsilane, and the coupling constants (J) are reported in Hertz. Data for the 1 H-NMR spectra are reported as follows: s = singlet, br s = broad singlet, d = doublet, m = multiplet, and the coupling constants (J) are in Hz. Bi-dimensional NMR techniques and distortion-less enhancement by polarisation transfer (DEPT) were used for the signal assignment. Chemical shifts are expressed in ppm downfield relative to tetramethylsilane, and the coupling constants (J) are reported in Hertz. The HRMS data for all final compounds were obtained using a LTQ-Orbitrap mass spectrometer (Thermo-Fisher Scientific, Waltham, MA, USA) with the analysis performed using an atmospheric-pressure chemical ionization (APCI) source, operated in positive mode. Silica gel Merck 60 (70-230 mesh, from Merck) was used for preparative column chromatography and thin layer chromatography (TLC) aluminum foil 60F 254 was used for analytical thin layer chromatography. The acylbenzohydroquinones (1a-q) were prepared according to a previously-reported procedure [24].
Suspensions of the acylhydroquinones 1a-q (1 equiv.), Ag 2 O (2.0 equiv.) and MgSO 4 anhydrous (300 mg) in dichloromethane (30 mL) were left with stirring for 30 min at room temperature (rt). The mixtures were filtered, the solids were washed with dichloromethane (3 × 25 mL), and the filtrates were evaporated under reduced pressure. The residues were dissolved in ethanol, the phenylamines (2 equiv.) were added to the solutions, and the mixtures were left with stirring at rt for 24 h. The solvents were removed under reduced pressure and the residues were column-chromatographed over silica gel (petroleum ether/EtOAc) to yield the corresponding pure 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones 2a-q.

Cytotoxic Assays
The cytotoxicity of the quinones was assessed by following the reduction of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan blue [31]. Cells were seeded into 96-well plates at a density of 10,000 cells/well for 24 h and then incubated for 48 h, with or without the quinone derivatives. Doxorubicin was used as the standard chemotherapeutic agent (positive control). Cells were then washed twice with warm PBS and incubated with MTT (0.5 mg/mL) for 2 hours at 37 • C. Blue formazan crystals were solubilized by adding 100 µL DMSO/well, and the optical density of the coloured solutions was subsequently read at 550 nm. Results are expressed as percentage of MTT reduction, compared to untreated control conditions. The IC 50 values were calculated using the GraphPad Prism software (San Diego, CA, USA).

Quantitative real-time PCR (qPCR) Assay
The T24 cells were cultured as previously mentioned. They were seeded into 6-well plates (2 × 10 5 cells/well) and, after 24 h of incubation, they were treated for 48 h with 2c and 2d (at 32 and 68 µM, respectively). Afterwards, they were washed with phosphate-buffered saline. The cellular lysate was prepared with E.Z.N.A.®RNA-Lock Reagent (Omega Bio-tek, Norcross, GA, USA) to preserve and immediately stabilize the total RNA for the subsequent gene expression assays. The total RNA isolated from the cells using the E.Z.N.A.®HP Total RNA Isolation Kit (Omega Bio-tek) was reverse-transcribed to cDNA using the AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies, Santa Clara, CA, USA) and 1000 ng of the RNA sample.
The cDNA synthesized was employed for qPCR using Brilliant III Ultra-Fast SYBR®Green QPCR Master Mix (Agilent Technologies) in a Mx3000P qPCR System (Agilent Technologies), employing a 96-well plate with 20 µL of PCR reaction per well and 10 pmol each of forward and reverse gene-specific primers. Ten genes were analyzed (see Table 4). The relative gene expressions were determined using Beta-2-microglobulin (B2M) as housekeeping, and the delta-delta Ct method (2 −∆∆Ct method) with regard to the vehicle-treated group (i.e., the reference group). Five biological replicates were used from each group (treated and reference group). The qPCR reactions were run by duplicates and negative controls contained no cDNA, as previously reported [32,33]. The GraphPad Prism software was used for statistical analyses of the relative gene expressions. The comparisons between means were performed using one-way analysis of variance (ANOVA) and Dunnett's multiple comparisons test. All statistical analyses were performed with a significance level of p < 0.05.

Conclusions
In summary, we have synthesized of a number 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones and assessed their voltammetric values and cytotoxicities of cancer cells in vitro. The members of the series 2a-q were prepared from acylbenzoquinones and phenylamines, in moderate to good yields (47-74%). The first and second half-wave potential values (E 1/2 ) of the members of the series were sensitive to the push-pull electronic effects of the substituents around the benzoquinone nucleus, as shown by the cyclic voltammograms of the aminoquinones. The preliminary results of the biological evaluation of the compounds 2a-q showed interesting in vitro cytotoxic activity on cancer cells. In this context, there were two more active compounds which increased TNF gene expression, suggesting an association with an inflammatory-like response.