Synthesis of Amino Acid–Naphthoquinones and In Vitro Studies on Cervical and Breast Cell Lines

We performed an extensive analysis about the reaction conditions of the 1,4-Michael addition of amino acids to 1,4-naphthoquinone and substitution to 2,3-dichloronaphthoquinone, and a complete evaluation of stoichiometry, use of different bases, and the pH influence was performed. We were able to show that microwave-assisted synthesis is the best method for the synthesis of naphthoquinone–amino acid and chloride–naphthoquinone–amino acid derivatives with 79–91% and 78–91% yields, respectively. The cyclic voltammetry profiles showed that both series of naphthoquinone–amino acid derivatives mainly display one quasi-reversible redox reaction process. Interestingly, it was shown that naphthoquinone derivatives possess a selective antitumorigenic activity against cervix cancer cell lines and chloride–naphthoquinone–amino acid derivatives against breast cancer cell lines. Furthermore, the newly synthetized compounds with asparagine–naphthoquinones (3e and 4e) inhibited ~85% of SiHa cell proliferation. These results show promising compounds for specific cervical and breast cancer treatment.


Introduction
Naphthoquinone (NQ) is a nucleus found in several natural and synthetic compounds that offers several applications, such as pigments, and many biological properties, including antibacterial, antifungal, anticancer, antimalarial, and antiviral, among others [1,2]. Regarding cytotoxic activity, several studies have reported that naphthoquinones with different substituents like paclitaxel, esters, metals, furans, carbazoles, or inclusive with carbohydrates in their structure, among others, present effects of diminishing cell proliferation [3][4][5].
These properties are principally attributed to the oxidant-reductive characteristics of the naphthoquinones, which allow the generation of dianions or semiquinone radicals. In the last decades, some publications have been dedicated to finding an explanation for the formation of these intermediates in the synthetic mechanism, and their properties that have produced different compounds with a plethora of applications of biological importance and effects that involve intra-or intermolecular interactions. One of the principal effects of these compounds is the generation of reactive oxygen species (ROS), producing cytotoxicity in different cell lines [6][7][8][9][10][11][12][13]. ROS generation with naphthoquinones represents a challenge in the design of new active compounds, principally with anticancer effect. In this context, the addition/substitution on naphthoquinone moiety by atoms or groups such as flour, oxygen, or amine can be modulated by redox properties, decreasing the toxicity levels and maintaining or potentiating the biological effect [14][15][16]. Several quinones from natural origin, like β-laphachone and menadione, among others, have been well characterized by their specific selectivity for cell lines, responding to temporal curse and dose response. Cell proliferation inhibition could be achieved by the induction of apoptosis, topoisomerase II-α inhibition, and ROS generation, among others [4]. Furthermore, it has been shown that β-lapachone inhibits epidermal growth factor (pEGFR), protein kinase B (pAKT/PKB), glycogen synthase kinase (pGsk-3β), cyclin D1, and cyclooxygenase-2 (COX-2) protein expression in a dose-dependent manner [17]. Interestingly, the naphthoquinone NSC 95,397 is used as a general potent inhibitor of cell cycle division 25C (Cdc25) [18]. Naphthoquinone regulation depends of the composition of its substituents, as well as targets and cellular components that could be inhibited and/or activated. Several naphthoquinones have been produced; however, they present non-desirable effects in cancer therapy use. Therefore, new compounds produced by alternative methods are imperative for cancer treatment.
In this regard, the synthesis and biological activities of some naphthoquinone-amino acids have been reported in several publications; nonetheless, all of these methodologies present different drawbacks, like low yields and/or long reaction times [19][20][21][22][23][24][25][26][27][28][29]. To our knowledge, the synthesis of naphthoquinone-amino acid derivatives has not been reported using microwave and ultrasound irradiation, which offer diverse advantages to conventional synthesis. In previous works, our group reported the synthesis of some Juglone and Lawsone derivatives using ultrasound and microwave irradiation under mild reaction conditions [30][31][32].
In this paper, we present the synthesis of a series of 1,4-naphthoquinone-amino acid (3a-e) and 2,3-dichloronaphhtoquinone (4a-4e) derivatives under several activation methods, such as room temperature synthesis (RTS), reflux synthesis (RS), microwave-assisted synthesis (MAS), and ultrasound-assisted synthesis (UAS), and determined the effectivity on the system. Newly synthesized compounds are promising in the cancer field. In the present work, we were able to incorporate alanine, phenylalanine, methionine, glycine, and asparagine to both naphthoquinones (1a,b) by MAS and evaluated their effect in the cervical cancer cell line, SiHa, and the breast cancer cell line, MCF-7. The incorporation of amino acids to naphthoquinones could enhance their cytotoxicity capacity, as well as their specificity.

Chemistry
In a general method to prepare 3a-e derivatives, the corresponding amino acid 2a-c was added in a 1,4-type bond form to the naphthoquinone 1a, using initially, the conditions reported in the literature (Figure 1) [19,23]. These conditions are shown as room temperature synthesis (RTS) and reflux synthesis (RS) in Table 1. Base effect was studied using potassium carbonate (K 2 CO 3 ) and trimethylamine (TEA) in equimolar proportion. However, RTS and RS methodologies showed trace or no production of the desired compounds when no base was used. Only for RS and base addition were the yields increased to a range of 20-50%. A modified methodology was performed with microwave (MAS) irradiation to determine the effect on the system. Interestingly, when the system was subjected to microwave radiation, the yield of the derivatives was in the range of 28-30% without base. Furthermore, the compound yields were increased when a base was used. The results indicate that TEA and potassium hydroxide (KOH) were better bases, among others, to produce the derivative compounds (Table 1). It was determined that the optimum pH for greater product formation was 9-10. At a lower pH, the reactions became very slow, and under higher pH, the presence of secondary products was observed, as when the K 2 CO 3 was used in an equimolar concentration (1 mmol) and not in solution.
Molecules 2019, 24, x FOR PEER REVIEW 3 of 14 products was observed, as when the K2CO3 was used in an equimolar concentration (1 mmol) and not in solution.  With these established conditions, we performed the synthesis of compounds 3a-e and 4a-e with different bases and stoichiometric proportions between NQ and amino acids under MAS and ultrasound (UAS) as an alternative method ( Table 2). It was determined that the best yields were obtained with KOH and TEA solutions. On the other hand, the yields were dependent of the proportion of each amino acid in the solution; besides, reaction times were reduced significantly to 25 min under MAS. In most cases, lower yields were obtained under UAS in comparison with MAS. Remarkably, the yield was dependent on pH, stoichiometry, and base used for each amino acid incorporation ( Table 2).   With these established conditions, we performed the synthesis of compounds 3a-e and 4a-e with different bases and stoichiometric proportions between NQ and amino acids under MAS and ultrasound (UAS) as an alternative method ( Table 2). It was determined that the best yields were obtained with KOH and TEA solutions. On the other hand, the yields were dependent of the proportion of each amino acid in the solution; besides, reaction times were reduced significantly to 25 min under MAS. In most cases, lower yields were obtained under UAS in comparison with MAS. Remarkably, the yield was dependent on pH, stoichiometry, and base used for each amino acid incorporation ( Table 2).
In Figure 2A,B, the CV profiles of the naphthoquinone derivative compounds mainly display one quasi-reversible redox reaction process as compared to the two reduction peaks of typical quinone derivatives in aprotic media [35,36]. Interestingly, compound 3a ( Figure 2A) does not show any significant electroactive processes in the studied potential range. The irreversible reduction peak observed near to −0.8 V for 3b-e and −0.9 V for 4a-e in Figure 2A,B, respectively, are related to the oxidation reactions occurring by electron dislocations of the quinone derivatives at more positive potentials, >−0.7 V or >−0.5 V for both 1,4-naphthoquinone and 2,3-dichloro-1,4-naphtoquinone-amino acid derivatives, respectively. Additionally, the irreversible reduction-oxidation peaks were not observed when further cyclic voltammetry studies were performed at different scan rates in the cathodic potential region for the evaluation of the redox reactions.  In several investigations, the generation of compounds 3a, 3b, 3d, 4a, and 4d has been reported [19][20][21][22][23][24][25][26][27][28][29], and their boiling point, infrared, and 1 H NMR spectra have been determined. Compounds 4b, 4c have been reported and used as intermediates for secondary reactions or biological evaluations, but we did not find their complete characterization [33,34], while 3c, 3e, and 4e have not been previously reported and, therefore, their complete characterization was carried out.
In Figure 2A,B, the CV profiles of the naphthoquinone derivative compounds mainly display one quasi-reversible redox reaction process as compared to the two reduction peaks of typical quinone derivatives in aprotic media [35,36]. Interestingly, compound 3a ( Figure 2A) does not show any significant electroactive processes in the studied potential range. The irreversible reduction peak observed near to −0.8 V for 3b-e and −0.9 V for 4a-e in Figure 2A,B, respectively, are related to the oxidation reactions occurring by electron dislocations of the quinone derivatives at more positive potentials, >−0.7 V or >−0.5 V for both 1,4-naphthoquinone and 2,3-dichloro-1,4-naphtoquinoneamino acid derivatives, respectively. Additionally, the irreversible reduction-oxidation peaks were not observed when further cyclic voltammetry studies were performed at different scan rates in the cathodic potential region for the evaluation of the redox reactions.
The main electrochemical parameters of the redox reaction of each naphthoquinone derivative are reported in Table 3. Overall, the half-wave potentials (E1/2) for the redox reaction peak were near −1.24 V for 3b-e and −1.12 V for 4a-e, respectively ( Table 3). The redox reaction is a diffusioncontrolled process, since the cathodic peak current density (ipc) or the anodic peak current density (ipa) are proportional to the square root of the scan rate (ipc vs. v 1/2 ) [37,38].  The main electrochemical parameters of the redox reaction of each naphthoquinone derivative are reported in Table 3. Overall, the half-wave potentials (E 1/2 ) for the redox reaction peak were near −1.24 V for 3b-e and −1.12 V for 4a-e, respectively (Table 3). The redox reaction is a diffusion-controlled process, since the cathodic peak current density (i pc ) or the anodic peak current density (i pa ) are proportional to the square root of the scan rate (i pc vs. v 1/2 ) [37,38].
In addition to this, the ratio of the cathodic peak current (i pc ) to the anodic peak current (i pa ) is close to one for the naphthoquinone-amino acid derivatives 3b-e and 4a-e, as described for reversible systems (Table 3). However, the potential values of cathodic peak (E pc ) to anodic peak (E pa ) separation (∆E p ) are quite large and disagree with the theoretical value reported for a one-electron reversible system, ∆E p > 60 mV, as shown in Table 3 [37]. Thus, the redox reaction process is considered quasi-reversible. Furthermore, it is unclear if the reduction reaction of the quinone moiety occurs via a single-step two-electron process as compared to the two successive one-electron transfer steps producing semiquinone (Q − ) and quinone dianion (Q 2− ), as typically observed for well-behaved quinone derivatives (Q) in nonaqueous media [35,[39][40][41][42][43]. Table 3. Electrochemical parameters of naphthoquinone derivatives with amino acid substituents a .

Compound
E pa E pc

Effect of Naphthoquinone-Amino Acid Derivatives in Cell Proliferation of Cervical Cancer Cell Line SiHa and Breast Cancer Cell Line MCF-7
SiHa cells were treated with 0.75 mM of naphthoquinone-amino acid derivatives 3a-e and 4a-e; however, the concentration was extremely toxic for the cell line (data not shown). Therefore, 0.1 mM was used to register the proliferation effect on SiHa and MCF-7 cells. Compounds with glycine (3a) and asparagine (3e) substituents showed the most potent effect, inhibiting~80% of proliferation in SiHa cells, while in MCF-7, glycine (3a), chloride glycine (4a), and chloride asparagine (4e) showed a proliferation inhibition near 90% ( Figure 3A,B). Interestingly, the chloride substituent did not modify the effect in SiHa cells, suggesting an effect directly mediated by the amino acids glycine and asparagine (compare 3a and 3e versus 4a and 4e, Figure 3A), while in MCF-7, this substituent increased the effect of naphthoquinone-amino acid compounds (compare 3a, 3b, and 3c versus 4a, 4b, and 4c). In contrast, 4b increased 10% of inhibition compared with 3b (4b = 72.5% versus 3b = 61.3%; Figure 3A). However, chloride incorporation did not always diminish proliferation, as it could be observed in 3c versus 4c presenting 62.6% and 54.8% of proliferation inhibition in SiHa cells, respectively ( Figure 3A). Remarkably, in MCF-7 cells, chloride addition increased the proliferation inhibition effect of the compounds (Figure 3B), suggesting a specific and particular effect for breast cell line. The 3d compound with amino acid phenylalanine showed a bigger proliferation inhibition in MCF-7 cells (57%) over SiHa cells (40%). Interestingly, for the chlorine compound 4d, a similar effect was observed for both cell lines, presenting 69.8% of proliferation inhibition ( Figure 3A,B).

Discussion
The naphthoquinone-amino acid derivatives have been previously reported; nonetheless, in this investigation, we performed a very extensive analysis about the reactivity of the equivalents of each amino acid used in the reactions. The results show a direct dependence of the pH on derivative production since, under pH = 9, reactions became very slow and above pH = 10.5, several secondary reactions appeared and reduced the yield, generating some different compounds that make difficult the purification. We optimized reactions under ultrasonic and microwave irradiation, particularly in the latter one, showing the highest efficiency obtaining the best yields 79-91% (3a-e) and 78-91% (4ae) compared with the literature reported data, and significantly reducing the reaction times from days or hours to only a few minutes under the optimized conditions.
A full description of the electron-transfer mechanism of the naphthoquinones with amino acid substituents is beyond the scope of the present paper. However, it is assumed that the quinone dianion is possibly stabilized by an intramolecular hydrogen bond, since the dissociation of proton donor groups, i.e., carboxyl group, in DMSO is not facile, thus, cannot protonate the dianion [44][45][46][47]. In such a case, the second redox process merges with the first (Figure 2), owing to the stabilization of the dianion by a hydrogen bond, as reported elsewhere [35,46,48,49]. For instance, the E1/2 values computed for compounds 3b-e (~1.24 V) are located at the mid-point of the half-wave potential values determined for the semiquinone (Q − ) and the dianion (Q 2− ) redox reaction peaks of the parental 1,4-naphtoquinone compound studied at same experimental conditions, −0.9 V or −1.6 V, respectively (data not shown). Likewise, the redox reaction process peak of the naphthoquinone-amino acid and chloride derivatives (4a-e) is positioned at E1/2 ~ −1.12 V, as shown in Table 3, while the E1/2 values for the Q − and Q 2-redox reaction steps of the 2,3-dichloro-1,4 naphthoquinone compound are -0.65 V and −1.37 V, respectively (data not show).
Particularly, naphthoquinone derivatives with glycine and chloride substituents (4a) present a noticeable electrochemical improvement regarding naphthoquinones with glycine (3a) (Figure 2). These results are associated to the electron-accepting capacity of the chloride substituent, and possibly to some conformational effects which modify the electronic configuration of the naphthoquinone-amino acid derivatives, facilitating the reduction processes [41,50]. It is then expected that naphthoquinone compounds with amino acids and chloride substituents (4a-e) present higher biological activity than derivatives (3a-e), as estimated by their E1/2 values, since reactive oxygen species (ROS) generation is facilitated at compounds with more positive E1/2 [39-42,49,51].

Discussion
The naphthoquinone-amino acid derivatives have been previously reported; nonetheless, in this investigation, we performed a very extensive analysis about the reactivity of the equivalents of each amino acid used in the reactions. The results show a direct dependence of the pH on derivative production since, under pH = 9, reactions became very slow and above pH = 10.5, several secondary reactions appeared and reduced the yield, generating some different compounds that make difficult the purification. We optimized reactions under ultrasonic and microwave irradiation, particularly in the latter one, showing the highest efficiency obtaining the best yields 79-91% (3a-e) and 78-91% (4a-e) compared with the literature reported data, and significantly reducing the reaction times from days or hours to only a few minutes under the optimized conditions.
A full description of the electron-transfer mechanism of the naphthoquinones with amino acid substituents is beyond the scope of the present paper. However, it is assumed that the quinone dianion is possibly stabilized by an intramolecular hydrogen bond, since the dissociation of proton donor groups, i.e., carboxyl group, in DMSO is not facile, thus, cannot protonate the dianion [44][45][46][47]. In such a case, the second redox process merges with the first (Figure 2), owing to the stabilization of the dianion by a hydrogen bond, as reported elsewhere [35,46,48,49]. For instance, the E 1/2 values computed for compounds 3b-e (~1.24 V) are located at the mid-point of the half-wave potential values determined for the semiquinone (Q − ) and the dianion (Q 2− ) redox reaction peaks of the parental 1,4-naphtoquinone compound studied at same experimental conditions, −0.9 V or −1.6 V, respectively (data not shown). Likewise, the redox reaction process peak of the naphthoquinone-amino acid and chloride derivatives (4a-e) is positioned at E 1/2~− 1.12 V, as shown in Table 3, while the E 1/2 values for the Q − and Q 2redox reaction steps of the 2,3-dichloro-1,4 naphthoquinone compound are −0.65 V and −1.37 V, respectively (data not show).
Particularly, naphthoquinone derivatives with glycine and chloride substituents (4a) present a noticeable electrochemical improvement regarding naphthoquinones with glycine (3a) (Figure 2). These results are associated to the electron-accepting capacity of the chloride substituent, and possibly to some conformational effects which modify the electronic configuration of the naphthoquinone-amino acid derivatives, facilitating the reduction processes [41,50]. It is then expected that naphthoquinone compounds with amino acids and chloride substituents (4a-e) present higher biological activity than derivatives (3a-e), as estimated by their E 1/2 values, since reactive oxygen species (ROS) generation is facilitated at compounds with more positive E 1/2 [39][40][41][42]49,51]. However, additional factors such as solvents, nature of supporting electrolyte, protonation-deprotonation equilibrium, and intra-and intermolecular hydrogen bonding also play important roles in determining the half-wave potential of the redox reaction processes, probably affecting cellular biological processes.
Recently, a proliferation inhibition effect of naphthoquinone-amino acid derivatives in different cell lines was described. In the ileocecal adenocarcinoma cell line HCT-8, it was shown an 86, 84, and 59% of proliferation inhibition with phenylalanine, alanine, and proline naphthoquinone derivatives. In the breast cancer cell line MDAMB-435, it was shown a 100, 100, and 36%, while in human multiforme cell line SF-295 86, 83, and 59% of proliferation inhibition, respectively [23]. Glycine incorporation in naphthoquinone structure is used to add methyl groups that would extend the molecules to resemble other amino acids presenting different activities [17]. Naphthoquinones with amino acids could probably interact with proteins and/or complexes, inhibiting several molecular processes like replication, transcription, and translation. Moraes et al. [23] added three complete amino acids to naphthoquinone structure, showing similar cytotoxicity to doxorubicin. The effect of glycine, alanine, and phenylalanine derivatives synthesized by Moraes et al. was similar to our results. Marastoni et al. [18] incorporated leucine, asparagine, phenylalanine, and serine into naphthoquinone with diamine alkyl spacers, inhibiting three subunits of proteasome linked to proliferation inhibition of the breast cancer cell line MDA and ovarian cancer cell line A2780 in the range of 100 µM, similar as in the present study. However, in our study, we used a number of 1 × 10 5 cells, while they used 15 × 10 3 cells; therefore, the molecules per cell that we used in our study was less than Marastoni et al.'s study.
The newly synthetized compounds with asparagine-naphthoquinones inhibited~85% of SiHa cell proliferation. Inhibition grade of naphthoquinone-amino acid derivatives 3a, 3e, 3b, 3c, and 3d ranked from 85 to 40% in SiHa cells. In contrast, MCF-7 cells presented an effect ranking from 90 to 30% of proliferation inhibition. In order from higher to lower, the compounds 4a, 4e, 4c, and 4d and 4b showed their effect. It could be theorized that the differential effects observed may be dependent on hydropathy index and protein occurrence of the compounds. Glycine and asparagine present the negative hydropathy index −0.4 and −4.5, respectively. Nevertheless, it should be noted that asparagine's hydropathy index is more negative that glycine's hydropathy index, even though it is more abundant in proteins than asparagine-7.2 and 5.1, respectively. This hypothesis needs several experimental studies to be addressed. However, glycine-and asparagine-naphthoquinones, as well as dichloride compounds, show selectively antitumorigenic properties in cervix and breast cancer cell lines, respectively, positioning them as promising compounds for specific cancer treatment.

General
Commercially supplied 1,4-naphthoquinone, 2,3-dichloronaphthoquinone, alanine, glycine, methionine, phenylalanine, and asparagine were used for the synthesis without further purification. 1 H and 1 3C nucleus magnetic resonance (NMR) spectra were recorded on a Bruker Fourier 300 MHz spectrometer ( 1 H at 300, 13 C at 75 MHz, Silberstreifen, Rheinstetten, Germany) and Bruker Avance III 400 MHz spectrometer ( 1 H at 400, 13 C at 101 MHz, Silberstreifen, Rheinstetten, Germany). The spectra were acquired from solution in dimethylsulfoxide-d 6 and methanol-d 4 at room temperature, TMS as internal reference, the chemical shifts (δ) are expressed in part per million (ppm) and the coupling constants (J) in Hz. High-resolution mass spectra (HRMS) were measured with a Jeol JMS-AccuTOF through DART (Direct Analysis in Real Time, Peabody, MA, USA) and by ESI (electrospray ionization) in an Agilent 6200 Series TOF (Santa Clara, CA, USA) and 6500 Series Q-TOF LC/MS System (Santa Clara, CA, USA). Infrared spectra were recorded on a Thermo Scientific NICOLET iS10 with ATR dispositive (SMART iTR, Madison, WI, USA). Melting points were determined using a Bicote-Stuart SMO 10 apparatus (Stone, Staffordshire, UK) and were uncorrected. Microwave-assisted synthesis (MAS) was performed on a CEM Mars 6 oven with a carrousel device (Matthews, NC, USA). Ultrasound-assisted synthesis (UAS) was carried out in an Autoscience Ultrasonic cleaner-AS2060B with 60 Watts of power (Lewisville, TX, USA). TLC was performed using silica gel 60 PF 254 containing gypsum (Merck, Darmstadt, Germany). The isolated reaction products were found to be >95% purity by NMR analysis (See Supplementary Materials).

General Procedure for the Optimization of the Reaction Conditions for the Synthesis of Compounds 3a-c
A solution of the appropriate amino acid 2a-c (1 mmol) in 40 mL of EtOH/H 2 O (4:1) was basified with several bases (pH = 9-10), and then a solution of 1 mmol of 1,4-naphthoquinone (1a) in ethanol (10 mL) was added. The mixture was activated by different methods, such as room temperature (RTS), reflux (RS), and microwave irradiation (MAS). Solvent and base effect were studied in the reaction yields, using K 2 CO 3 (3 N) and triethylamine (TEA) (Figure 1). The progress reaction was monitored by TLC, using MeOH/CHCl 3 (9:1) as eluent mixture. Finally, 20 mL of HCl (1 N) was added and the precipitated product was filtered and purified by flash column chromatography, starting with dichloromethane (DCM) and changing the polarity to finish with methanol.

Optimization of the Reaction Conditions for the Synthesis of Compounds 3a-c Under Microwave Irradiation
In 15 mL of dioxane/H 2 O (4:1) or 30 mL of EtOH/H 2 O (4:1), 1.5 to 2 mmol of respective amino acid was added, the mixture was basified with several bases (TEA, K 2 CO 3 aq., AcONa, or KOH aq.) to obtain a pH of 9-10, and activated under microwave irradiation with the following parameters: 110 • C, 250 W for a time of 10 min. Then, a solution of naphthoquinone (1a) in 5 mL or 20 mL of the respective mixture was added; the reaction solution was irradiated with the same parameters by 30 min. After the time of reaction, 20 mL of HCl (1 N) was added, and the compounds were purified with the methodology previously descripted.

Optimized Reaction Conditions Under Microwave Irradiation for the Synthesis of Compounds 3a-e and 4a-e
A solution of respective amino acid (1.5-2.0 mmol) in dioxane/H 2 O (4:1, 15 mL for 3a-e derivatives or 20 mL for 4a-e derivatives) was basified with TEA or KOH aq. (pH 9-10) and activated under microwave irradiation (110 • C, 250 W for a time of 10 min). Then, a solution of naphthoquinone 5 mL (1a) or 2,3-dichloro-1,4-naphthoquinone (1b) 10 mL of dioxane/water mixture was added; the reaction mixture was irradiated with the same parameters by 20 min. After the time of reaction, 20 mL of HCl (1N) was added, and the compounds were purified with the methodology previously descripted. Under this methodology, compounds 3a, 3d and 3e, 4b, 4c, and 4e were purified as was previously described, while 3b, 3c, 4a, and 4d were obtained by filtration without further purification.

General Procedure for the Synthesis of Compounds 3a-e and 4a-6 Under Ultrasonic Irradiation
For 3a-e derivatives, 15 mL of dioxane/H 2 O (4:1) was used, and 20 mL for 4a-e compounds. Then, 1.5-2.0 mmol of respective amino acid in solution dioxane/H 2 O was basified with TEA or KOH aq. to pH 9-10, and activated by ultrasound at 25 • C and 15 min. Then, 5 mL a solution of naphthoquinone (1a) or 10 mL of 2,3-dichloro-1,4-naphthoquinone (same proportion dioxane/H 2 O) was added; the reaction mixture was irradiated again for 45 min (maximum temperature of 40 • C). After the time of reaction, 20 mL of HCl (1 N) was added, and the products were purified by column chromatography with the previously described methodology.

Cyclic Voltammetry
Cyclic voltammetry (CV) was performed in a conventional three-electrode electrochemical cell using a potentiostat/galvanostat (EG&G, PAR VersaStat 3). A polished glassy carbon disk electrode (GC) (0.07 cm 2 , BASi USA) was employed as working electrode. Prior to each experiment, the GC electrode was polished to mirror finishing with 0.3 mm and 0.05 mm alumina and washed with distilled water, and sonicated in ethanol for 5 min. The counter electrode was a Pt wire (Alfa-Aesar, USA), and a home-made Ag/Ag+ pseudo-reference electrode was used as a reference. The pseudo-reference electrode consisted of a silver wire in a body-glass (PINE research, USA) filled with a solution of 0.01 M silver nitrate (AgNO 3 , BASi, USA) in tetrabutylammonium tetrafluoroborate (TBABF 4 , Sigma Aldrich, USA) 0.1 M in dimethyl sulfoxide (DMSO, Sigma Aldrich, USA). The supporting electrolyte consisted of TBABF 4 0.1 M dissolved in DMSO. As received TBABF 4 and DMSO were used without further purification or drying processes. Furthermore, the testing solutions were prepared by adding compounds 3a-e or 4a-e to the supporting electrolyte for a final concentration of 5 × 10 −3 M. Then, cyclic voltammetry (CV) was carried out in a range from −2.0 to 0.75 V vs. Ag/Ag + at a scan rate of 100 mV s −1 in the cathodic direction to identify any electron transfer reaction. The redox reaction processes associated to compounds 3a-e or 4a-e were further studied by CV at a scan rate of 10, 25, 50, 100, 200, and 300 mV·s −1 .

Cell Proliferation Analysis
Cell proliferation was quantified by violet crystal dye in 1× phosphate-buffered saline (PBS) (2.7 mM KCl, 1.8 mM KH 2 PO 4 , 136 mM NaCl, 10 mM Na 2 HPO 4 pH 7.4). The treated cells were incubated in methanol for 15 min and washed two times with water. Cells were dyed with 0.1% crystal violet and washed three times with water, and finally, violet crystal was recovered with 10% acid acetic to be analyzed in microplate reader Multiskan GO Spectrophotometer (Thermo Scientific™, Ratastic, Finland).

Conclusions
As we previously mentioned, the synthesis of naphthoquinone-amino acid derivatives has been reported; nonetheless, neither microwave-or ultrasonic-assisted synthesis has been reported. The obtained results on the analysis of the synthetic methodology showed a direct dependence of the stoichiometry of the reactants and the pH, due to the need of the amino acids for their deprotonation, and the yield was increased with the microwave system. On the other hand, some products were obtained directly by precipitation, only with the acidification of reaction mixture after a few hours of rest without further purification. Since naphthoquinones possess redox properties, cyclic voltammetry studies were done, concluding that the redox reaction is a diffusion-controlled quasi-reversible process. The biological evaluation showed a potent proliferation inhibition in cervical and breast cancer cells lines. Remarkably, naphthoquinone-amino acid derivatives, as well as dichloride compounds, showed selectively antitumorigenic properties in cervix and breast cancer cell lines, respectively, positioning them as promising compounds for specific cancer treatment.