New 2-Acetyl-3-aminophenyl-1,4-naphthoquinones: Synthesis and In Vitro Antiproliferative Activities on Breast and Prostate Human Cancer Cells

The reaction of 2-acyl-1,4-naphthoquinones with N,N-dimethylaniline and 2,5-dimethoxyaniline, promoted by catalytic amounts of CeCl3·7H2O under “open-flask” conditions, produced a variety of 2-acyl-3-aminophenyl-1,4-naphthoquinones structurally related to the cytotoxic 2-acetyl-3-phenyl-1,4-naphthoquinone, an inhibitor of the heat shock chaperone protein Hsp90. The members of the 2-acyl-3-aminophenyl-1,4-naphthoquinone series were isolated in good yields (63-98%). The cyclic voltammograms of the 2-acyl-3-aminophenyl-1,4-naphthoquinone exhibit two one-electron reduction waves to the corresponding radical-anion and dianion and two quasireversible oxidation peaks. The first and second half-wave potential values (E1/2) of the members of the series are sensitive to the push-pull electronic effects of the substituents in the naphthoquinone scaffold. Furthermore, the in vitro antiproliferative properties of these new quinones were evaluated on two human cancer cells DU-145 (prostate) and MCF-7 (mammary) and a nontumorigenic HEK-293 (kidney) cell line, using the MTT colorimetric method. Two members, within the series, exhibited interesting cytotoxic activities on human prostate and mammary cancer cells.


Introduction
The electroactive naphthoquinone core is a common structural constituent of a variety of biochemical systems involved in the human defense system [1]. Several biological active quinones as ubiquinone and vitamin K are known to be electron transporters and are essential for many enzymatic processes. They can act as anti-or prooxidants depending on the nature of the media: this chemical versatility gives them an important role in different biochemical processes that are essential to living organisms [2]. The oxidation state of naphthoquinones allows them to act by different mechanisms, such as free radical scavengers, metal ion chelators, and also enzyme inhibitors for free radical production [3,4]. This imbalance between the formation and removal of ROS (reactive oxygen species) causes damage to the cells at nucleic acids, proteins, and membrane lipids associated with ageing, carcinogenesis, cardiovascular, and coronary diseases [5]. In this context, the naturally occurring 2-hydroxy-1,4naphthoquinone (lawsone) exhibits a number of interesting biological activities, such as antioxidant [6], antibacterial, antifungal [7], anti-inflammatory, antipyretic, analgesic [8], and anticancer cytotoxic [9]. Therefore, the quinones could play an important role in inhibiting or delaying oxidative stress that arises from an imbalance between free radical production and antioxidant and repair defenses [10]. by oxidation of acetylnaphthohydroquinone with sodium periodate, and the arylamines. Recent studies performed in our laboratory show that the reaction of diverse 2acylnaphthoquinones with 3,4,5-trimethoxyaniline in methanol yields amination products (C-N bond formation) together with two types of arylation-cyclisation products (C-C and two alternative N=C or N-C bond formations). We have not detected arylation products in these assays probably due to the fact that they undergo fast cyclisation reactions to produce their respective arylation-cyclisation products [26].
In this paper, we report flexible access to 2-acyl-3-aminophenyl-1,4-naphthoquinones prepared from 2-acylnaphthohydroquinones, N,N-dimethylaniline and 2,5dimethoxyaniline. The half-wave potentials of such new HTS1 analogues were calculated, and they were evaluated for their in vitro antiproliferative activities on two humanderived cancer cell lines DU-145 (prostate) and MCF-7 (breast) and nontumorigenic HEK-293 (kidney) cells using as endpoint the MTT colorimetric method.

Materials and Methods
2.1. 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 uncorrected. The record of IR, 1 H-and 13 C-NMR spectra, and chromatography procedures were done according to methods reported by Benites et al. [27]. Proton nuclear magnetic resonance ( 1 H NMR) spectra were measured at 300 MHz in a Bruker Ultrashield-300 spectrometer. Data for the 1 H NMR spectra are reported as follows: s = singlet, br s = broad singlet, d = doublet, and t = triplet, and the coupling constants (J) are in Hz. Carbon-13 nuclear magnetic resonance ( 13 C NMR) spectra were measured at 75 MHz in a Bruker Ultrashield-300 spectrometer. Bidimensional NMR techniques and distortion-less enhancement by polarization 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 high-resolution mass spectrometry (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 a positive mode. The acylnaphthohydroquinones (2)(3)(4)(5)(6)(7)(8) were prepared according to a previously reported procedure [28].

Cytotoxic
Assays. The cytotoxicity of the quinones was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) reduction assays [29], according to Valderrama et al. [30]. Briefly, adherent cells were detached by using trypsin/EDTA solution. The culture medium was removed, and cells were washed with a free Ca-Mg salt solution to remove all traces of serum. After removing salt solution, trypsin/EDTA solution was added to completely cover the monolayer of cells for 2-3 min at 37°C. When the trypsinization process was completed, trypsin/EDTA was removed by aspiration and cells were resuspended, diluted in fresh medium, and seeded for 24 h into 96-well plates at a density of 10,000 cells/well. Then, they were further incubated for 48 h, with or without the quinone derivatives. Doxorubicin was used as the standard chemotherapeutic agent (positive control) in a dose range of 0.01 to 10 μM. Cells were then washed twice with warm PBS, and they were further 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 colored solutions was subsequently read at 550 nm. Results are expressed as a percentage of MTT reduction, compared to untreated control conditions. The IC 50 values were calculated using the GraphPad Prism software (San Diego, CA, USA).
Among the members of the acylnaphthoquinone series, compound 14 was selected to get preliminary insights into its reactivity to undergo arylation reaction with N,Ndimethylaniline. In a preliminary assay, acylnaphthoquinone 14, prepared from acylnaphthohydroquinone 7, was reacted with N,N-dimethylaniline in methanol at rt. The reaction carried out under open-flask conditions to favor aerobic oxidation reactions takes place slowly to give, after 72 hours, the arylation product 20 in 73% yield, referred to precursor 7. Furthermore, it was observed that the reaction of 14 with N,N-dimethylaniline in refluxing ethanol takes place relatively faster (33 hours) than in methanol, but compound 20 is produced in moderate yield (51%). The validity of the arylation mechanism of acylquinone 9 with arylamines proposed by Pardo et al. [24] was assumed to proceed through a sequence of a Michael addition reaction followed by aerobic oxidation of the adduct intermediate. Therefore, we envisaged to improve the arylation reaction of 14 (Table 1) with N,N-dimethylaniline by using a green Lewis acid catalyst such as CeCl 3 ·7H 2 O [31] or IBr 3 [32]. Successful results on the use of the Ce(III) catalyst in the oxidative amination reaction of quinones with arylamines have been reported in literature [32][33][34][35][36][37][38]. These acid catalysts probably increase the electrophilic character of the enone system of the quinones, via coordination with the oxygen atom of the carbonyl group, thus promoting the Michaeltype addition [35,39,40].
We examined the reactions of 14 with N,N-dimethylaniline in the presence of catalytic amounts of CeCl 3 ·7H 2 O and IBr 3 (5%) in methanol, under open-flask conditions at room temperature. Both reactions occurred faster (28 h) than in the absence of these Lewis acids (72 h), and the arylation product 20 (Table 1) was isolated in 98 and 78% yield, respectively. The successful results obtained in the assay employing Ce(III) led us to evaluate the scope of the arylation reaction of the remaining members of the series with the selected arylamines, under the above optimized conditions. The results of the assays are summarized in Table 1.
The data in Table 1 show that the arylation reaction of 2acylnaphthoquinones 9-15 with the arylamines, catalyzed with Ce(III), yields the corresponding 2-acetyl-3-aminoaryl-1,4-naphthoquinones with good to excellent yields ranging from 63 to 98%, except in the case of product 17 (33%). Comparison of the reaction time formation of compounds 16-28 reveals two facts: the first one was linked to the lower reactivity of N,N-dimethylaniline compared to 2,5-dimethoxyaniline and secondly the strong influence of the stereoelectronic nature of the acyl substituents on the  nucleophilic attack of the arylamines. It has to be mentioned that no attempts were made in order to decrease the reaction time formation of the arylation products. Based on our recent results [41] and that reported by Liu and Ji [42], on the arylamination of quinones, we will attempt, in future researches, the use of ultrasound to accelerate the Ce(III)-promoted arylation reaction of acylnaphthoquinones with arenes in order to expand the 2-acetyl-3-aminoaryl-1,4-naphthoquinone series for further biological studies. The acid-induced formation of the arylation compounds 16-28 from acylquinones 9-15 and their reaction with N,Ndimethylaniline are proposed to occur according to the mechanism of reaction depicted in Scheme 3. This approach is based on the reaction mechanism for the Ce(III)-promoted phenylamination reaction of 1,4-naphthoquinone with 2fluoroand 2-methoxyanilines reported by Leyva et al. [35]. Initially, a selective conjugate Michael-type addition of the arylamine across the enone system C 3 =C 2 -C 1 =O seems plausible. This reaction, which involves the high electrophilic C-3 of the acylquinones, provides the respective C-C Michael adduct intermediates. Further enolization of these species, followed by aerobic oxidation, gives compounds 16-21. It should be noted that the electrophilicity of the C-3 in these acylquinones is mainly due to the electron-withdrawing effects of the acyl substituents attached to the 2-position. The coordination of Ce(III) to the oxygen atom of the enone system should contribute to increasing the electrophilicity of the C-3.
An interesting feature of the synthetized arylation products is their strong purple color of the chromophores that, according to Pardo et al. [24], is due to the strong donoracceptor interactions between the quinoid and the electronrich nitrogen substituents. Figure 3 shows the hybrid structures  of compounds 16 and 23 where such interactions are clearly observable. Inspection of minimal energy conformation of compounds 16 and 23 performed by MM2 calculation (Chem-Bio3D 11.0, PerkinElmer, MA, USA) shows a noncoplanar orientation between the acyl-carbonyl groups and the naphthoquinone framework. This data agrees with the infrared absorption of the acyl-carbonyl groups of these compounds 16-28 that appeared within the range: ν máx 1706-1735 cm -1 .

Voltammetric Measurements and Antioxidant Activities.
In order to assess the redox properties of the members of the 2-acyl-3-aminoaryl-1,4-naphthoquinone series 16-28, their half-wave potentials E I 1/2 and E II 1/2 were measured by cyclic voltammetry. The measurement was conducted in acetonitrile at room temperature, using a platinum electrode and 0.1 M tetraethylamoniumtetrafluoroborate as the supporting electrolyte [19]. The voltammograms were recorded in the potential range from 0.0 to -2.0 V vs. nonaqueous Ag/Ag + . Figure 4 shows the typical electrochemical behavior of the arylation compound 23 that proceeded in two one-electron diffusion stages.
The cathodic peaks related to the reduction of quinone nucleus, and the anodic one due to its reoxidation, were observed for compound 23 as well-defined quasireversible waves. The E 1/2 values for the first one-electron correspond to the semiquinone radical anion formation and the second one-electron transfer to the dianion formation [17]. The magnitude of these values falls within the ranges −875 to −620 mV/−1295 to −1125 mV for the members of the 2acyl-3-(4-N,N-dimethylaminophenyl)naphthoquinones 16-21 and -890 to -685 mV/-1410 to -1180 mV for the members of the 2-acyl-3-(4-amino-2,5-dimethoxyphenyl)naphthoquinones 22-28 ( Table 2).
3.3. Antitumor Activity. The 2-acyl-3-aminophenylnaphthoquinones 16-28 were evaluated for in vitro cytotoxic activities against nontumorigenic human embryonic kidney cells (HEK-293 cells) and two human cancer cell lines DU-145 (prostate) and MCF-7 (mammary) in 72 h drug exposure assays. The cytotoxic activities of the new compounds were measured using conventional microculture tetrazolium reduction assays [29]. Such activities are expressed in terms of IC 50 . Doxorubicin, a well-known anticancer agent currently used in clinical practice, was taken as a positive control. The cytotoxic activity data are summarized in Table 3. The data indicated that, in general, the DU-145 cells are more sensitive than MCF-7 cells to the compounds. In addition, the 2-acyl-3-(4-amino-2,5-dimethoxyphenyl)naphthoquinone members 22-28 exhibited higher activity than their corresponding analogues 16-21 but lower than those displayed by doxorubicin. Among the 2-acyl-3-(4-dimethylaminophenyl)naphthoquinone members, those containing the furan-2carbonyl and thiophene-2-carbonyl groups, as compounds 20 and 21, are the most active on the DU-145 cancer cells. Inspection of the biological activity of 2-acyl-3-(4-amino-2,5dimethoxyphenyl)naphthoquinone members reveals that those containing acetyl and furan-2-carbonyl groups, as 22 and 27, exhibited the higher activity on DU-145 and MCF-7 cancer cell lines. However, by taking the mean IC 50 value and calculating a selectivity index (IC 50 HEK-293 value/IC 50 cancer cell value), compound 22 displays a better index (1.71) than 27 (0.65), suggesting that this latter quinone did not discriminate between cancer and healthy cells. By increasing the number of the aliphatic chain within the acyl substituents, a decrease in the antiproliferative activity is observed: compounds 22 and 23 are more active than 24. The screening also shows that the members having 4-methoxy-and 2,5dimethoxybenzoyl substituents, as compounds 18, 19, and 25 show weak or almost nonexistent cytotoxic activity. Finally, an additional element may be proposed to explain why compound 22 displays the best activity among the series: namely, its strong electronegative redox potential. In this regard, it is tempting to speculate that compound 22 has higher redox cycling ability, and therefore, it may generate the largest amount of ROS. Indeed, quinone redox cycling has been evoked as the main molecular mechanism explaining numerous deleterious effects by such molecules [43][44][45][46].

Conclusions
We have developed a simple and flexible route for the synthesis of novel 2-acyl-3-(aminophenylamino)-1,4-naphthoquinones whose structure is related to the HTS1, an inhibitor of the heat shock protein Hsp90. The members of the series were prepared in moderate to good yields (63-98%) by reaction of acylnaphthoquinones with phenylamines, catalyzed by CeCl 3 ·7H 2 O. The new quinones were characterized by their spectral data and half-wave potentials. The first and second half-wave potential values (E 1/2 ) of the members of the series are sensitive to the acceptor and donor electronic effects of the substituents located in the quinone double bond. The in vitro cytotoxicities of the compounds on cancer cells were determined by using the MTT colorimetric method. The results of the biological evaluation of the 2acyl-3-(aminophenyl)-1,4-naphthoquinone series show interesting in vitro cytotoxic activity on DU-145 and MCF-7 cancer cell lines for the member 22 making it a suitable new chemical entity to be further developed.

Data Availability
All data to be shared are included in the main text as well as in Supplementary Materials.

Conflicts of Interest
The authors declare no conflict of interest.