Synthesis of quinone imine and sulphur-containing compounds with antitumor and trypanocidal activities: redox and biological implications

New quinoidal compounds were prepared and evaluated against T. cruzi and cancer cell lines for the identification of new bioactive agents.

Abstract

Ortho-Quinones represent a special class of redox active compounds associated with a spectrum of pronounced biological activities, including selective cytotoxicity and antimicrobial actions. The modification of the quinone ring by simple nitrogen and sulphur substitutions leads to several new classes of compounds with their own, distinct redox behaviour and equally distinct activities against cancer cell lines and Trypanosoma cruzi. Some of the compounds investigated show activity against T. cruzi at concentrations of 24.3 and 65.6 μM with a selectivity index of around 1. These results demonstrate that simple chemical modifications on the ortho-quinone ring system, in particular, by heteroatoms such as nitrogen and sulphur, transform these simple redox molecules into powerful cytotoxic agents with considerable “potential”, not only in synthesis and electrochemistry, but also, in a broader sense, in health sciences.

1. Introduction

The rapid expansion and simultaneous ageing of populations around the globe represent a demographical change and a challenge that, among many other issues, such as shortage of nutrition and increase of pollution, is also associated with higher incidence of new cases of cancer and related deaths. The projection for the year 2030 estimates 27 million new cases of cancer with 17 million cancer-related deaths worldwide.1 At the same time, an increased global population is also more susceptible to the outbreak and spread of infectious diseases, such as Chagas disease. This disease is caused by the protozoan Trypanosoma cruzi, which is considered by the World Health Organization (WHO) as one of the twenty neglected tropical diseases, affecting more than 5 million people worldwide. Its current chemotherapy is still restricted to the nitroderivatives benznidazole and nifurtimox, available for half a century, which present limited activity and severe adverse effects.2–8 These demographical developments and new ways of transmission, combined with the emergence of drug resistance, require new and effective drugs for the treatment of such diseases, i.e. agents which may be effective as (cyto)toxins and also selective for their targets.

Within this context, naphthoquinoidal compounds have been studied widely.9–13 The derivatization of naphthoquinones has been a subject of considerable interest among medicinal chemists and a wide variety of natural and synthetic naphthoquinones have already been reported as potent trypanocidal and anticancer agents.14–17 Mechanistically, the quinone core may undergo one-electron reduction under aerobic conditions to form a semiquinone radical which will redox cycle to release reactive oxygen species (ROS).10,18–20 Within the context of cancer, the cytotoxicity of quinones is therefore mainly associated with the catalytic generation of ROS and the alkylation of crucial proteins and nucleic acids, both processes, which provoke cell damage.14–16 Similar considerations also apply to pathogenic microorganisms, some of which are affected considerably by – similar – redox modulating agents thanks to their weak(er) cellular antioxidant defence systems. Indeed, the concept of catalytic “sensor/effector” agents, i.e. “smart” redox molecules responding to specific intracellular redox signatures and peculiarities, and thereby combining considerable activity with selectivity, has been evaluated for over a decade now, often with amazing results.21–23

For almost twenty years, our groups have been involved in the synthesis and biological evaluation of naphthoquinones.24–26 Whilst initial studies on such catalytic agents have employed comparably simple redox agents, subsequent ones have developed hybrid molecules with two or more redox sites integrated into one molecule. The hybridization of a triazole nucleus with β-lapachone, for instance, has already resulted in a derivative with high antitumor activity (IC₅₀ < 2 μM) for different cancer lineages and, more recently, we have also reported the antitumor activity of selenium-containing quinone-based triazoles, which were inspired by earlier quinone–chalcogen structures tested more than a decade ago (Scheme 1A).21,27–29 Quinone-based N-sulfonyl-1,2,3-triazoles were also prepared by our group using click chemistry reactions (Scheme 1B).30 In this work, we employed as a strategy the insertion of the 1,2,3-triazolic nucleus associated with a chalcogen atom, allowing the formation of a redox active system. The compounds were subsequently evaluated against eight types of cancer cell lines and some derivatives exhibited potent antitumor activity.30 Interestingly, the application of such “smart” catalytic agents is not limited to cancer research, and a pronounced – and selective – cytotoxicity also affects protozoan pathogens, such as T. cruzi, with some molecules exhibiting trypanocidal activity about two or six times higher than that of the standard drug benznidazole.31–35

Scheme 1. A schematic overview of the strategies employed in the preparation of bioactive compounds and transformation of ortho-quinones into para-quinone imines and sulphur containing compounds.

Interestingly, the ortho-quinone redox centre found in β-lapachone is prone to chemical modifications which impact considerably the redox behaviour of these agents.10 Here, we have investigated modifications with nitrogen and sulphur substituents, as both heteroatoms are redox active and, besides electronic effects, promise a major “transformation” of the original redox system into considerably more active species (Scheme 1C). We are now able to report the synthesis, electrochemistry, anticancer and trypanocidal evaluation of quinoidal derivatives containing alkynes and N-sulfonyl triazoles and propose a distinct relationship between the chemical structure and redox system, and their electrochemical potentials and biological activities.

2. Results and discussion

2.1. Chemistry

The first class of compounds with a nitrogen substituent at the ring was prepared from commercially available 1,2-naphthoquinone-4-sulfonic acid sodium salt (1). Initially, we prepared arylamino naphthoquinones (2a–h) by the reaction of 1 and the respective anilines following a procedure published previously in the literature with minor modifications.36–38 In general, the desired products were obtained in moderate to excellent yields (Scheme 2). It should be noted from the outset that these compounds now possess more complicated redox cores spanning the original ortho-quinone and the amine functionality in the para-position.

Scheme 2. Synthesis of arylamino ortho-naphthoquinones 2a–h.

With the quinoidal compounds 2a–h available, the changed “redox core” was modified further, this time by alkylation of one of the oxygen atoms. Notably, this kind of alkylation not only simply “blocks” one of the oxygen atoms of the ortho-quinone, it also transforms the electronic structure of the entire ring system by engaging the nitrogen in the para-position in the form of a distinctive para-quinone imine redox core (Scheme 3).39,40 The consequences of this apparently innocent O-alkylation for the redox and biological activity will be discussed later. Eight alkyne derivatives 3a–h were synthesized in moderate to excellent yields ranging from 48% to 99% according to Scheme 3 and in the presence of DMF as a solvent, excess K₂CO₃ as a base, and propargyl bromide.

Scheme 3. Synthesis of alkynes derived from arylamino ortho-naphthoquinones 3a–h.

Alkynes derived from ortho-naphthoquinones represent important intermediates for the synthesis of 1,2,3-triazole derivatives, and such molecules can be synthesized easily by means of a 1,3-dipolar cycloaddition reaction between an azide and alkyne using copper(i) as a catalyst, in a process known as a click chemistry reaction (Scheme 4). This type of reaction is considered an important tool to synthesize hybrid molecules with two or more redox centres and pronounced biological activities.41 In this context, the synthesis of the N-sulfonyl-1,2,3-triazoles was accomplished according to the methodology described by Fokin et al., employing copper(i)-thiophene-2-carboxylate (CuTC) as a catalyst, as this catalyst promises high yields and selective formation of the 1,4-regioisomer of the product.42 The reaction was carried employing the corresponding alkynes (3a–h) (Scheme 3) with an excess of tosyl azide in toluene, as a solvent, and at room temperature according to Scheme 4. In general, novel compounds were obtained in moderate to excellent (50–95%) yields.

Scheme 4. Synthesis of N-sulfonyl-1,2,3-triazoles 4a–h.

Nitrogen is not the only nucleophile able to replace the sulfonate group in compound 1. Sulphur has a similar nucleophilic character and is also redox active. Indeed, recent studies have shown that naphthoquinone compounds substituted with chalcogen atoms often exhibit considerable biological activity.17,43 Whilst these studies have focused primarily on para-quinones, compound 1 provides the basis for a series of similar ortho-quinones. For this synthesis, a methodology adapted from the literature36,37 yielded compounds 5a–5d in low to moderate yields (30–40%) (Scheme 5).

Scheme 5. Synthesis of chalcogenic aryl ortho-quinones 5a–d.

The structures of the novel compounds were determined initially by ¹H and ¹³C NMR, and were corroborated further using electrospray ionization mass spectra. In the case of compounds 3a, 3d, 3f, 3g, 3h and 5d, X-ray crystallographic analysis with suitable crystals obtained by the slow evaporation method was performed. Here, the bond lengths and angles are in good agreement with the expected values reported in the literature.44 The atoms of the naphthoquinonic ring (C1–C10) of all structures are coplanar and the largest deviation from the least-squares plane for each one is: 0.077(3) Å for atom C1 in 3a; 0.044(3) Å for atom C5 in 3d; 0.035(2) Å for atom C1 in 3f; 0.039(2) Å for atom C5 in 3g; 0.039(2) Å for atom C8 in 3h and 0.017(4) Å for atom C5 in 5d. The dihedral angles between the planes of the rings (C1–C10) and (C11–C12) are: 48.5(3)° for 3a; 57.4(2)° for 3d; 86.9(3)° for 3g; 57.1(4)° for 3h; 61.01° for 3f and 86.9° for 5d. ORTEP-3 diagrams of each molecule are shown in Fig. 1.

Fig. 1. ORTEP-3 projections of 3a, 3d, 3f, 3g, 3h and 5d, showing the atom-numbering and displacement ellipsoids at the 50% probability level.

2.2. Redox transformations

The process of substituting the sulfonate group in compound 1 for the nitrogen or sulphur in 2 and 5, respectively, and also by O-alkylation when moving from 2 to 3 and 4, changes the “redox core” of these molecules, as an additional redox active element is added to the initial ortho-quinone system. Some of the resulting redox systems therefore appear to differ significantly from the original structures, and these differences should also be reflected in the redox behaviour and biological activities associated with them. As such, cyclic voltammetry (CV) was employed to study redox changes, as it represents a readily applicable, fast, informative and also fairly reliable method to obtain initial information about reversible and irreversible reduction and oxidation processes associated with such molecules. CV was performed in mixed medium, i.e. phosphate buffer + 30% methanol, on a glassy carbon working electrode, with E vs. Ag/AgCl as a reference electrode (SSE), at 200 mV s^–1. In the presence of a protic organic solvent (methanol), the reduction occurs in two monoelectronic steps, different from the mechanism in totally aqueous medium, where the reduction occurs through the capture of 2e^– + 2H⁺.

The cyclic voltammograms of selected compounds belonging to class 1 (compounds 1 and 2), class 3 (compound 3a) and class 5 (compound 5b) were obtained and are shown in Fig. S39 (see the ESI† file), together with the values for the relevant reduction signals (E_pc) and corresponding oxidation (E_pa) ones for various quinones, in Table 1. We have focused here on the prime signals associated with the quinone redox centre, as these waves are important to rationalize the relevant biological activities (see below).

Table 1. Reduction potential values (E_pc1 and E_pc2) and corresponding oxidation potentials (E_pa1 and E_pa2), representing three different classes of compounds and their synthetic precursor (1).

Compounds	1st wave		2nd wave
	E pc1	E pa1	E pc2	E pa2
1	–250 mV	–340 mV	–602 mV	–28 mV
2a	–490 mV	–420 mV	–716 mV	–202 mV
3a	–429 mV	–493 mV	–669 mV	–259 mV
3b	–387 mV	–475 mV	–687 mV	–292 mV
3e	–339 mV	–415 mV	–690 mV	–235 mV
3h	–384 mV	–414 mV	–614 mV	–190 mV
5b	–210 mV	–304 mV	–552 mV	–132 mV
5c	–168 mV	–324 mV	–500 mV	–110 mV
5d	–254 mV	–360 mV	–574 mV	–178 mV

By analysis of the first reduction potential (E_pc1), the ease of reduction is the following: 5c > 5b > 1 ≈ 5d > 3e > 3h ≈ 3b > 3a > 2a.

As anticipated, the redox behaviour changes significantly, once the original ortho-quinone 1 is modified by a nitrogen substituent in the para-position to yield compounds of class 2, due to the electron donating character of the amino group. Interestingly, the subsequent O-alkylation converting 2 into 3 also – quite dramatically – changes the entire redox system, since there is a direct modification on the redox system. This significant change from an ortho-quinone to a para-quinone imine is also observed electrochemically, and is associated with a shift of the first reduction potentials to more negative potentials. Generally, the potentials for the para-quinone imine are 150 to 200 mV more negative, when compared to those for the ortho-quinones, pointing towards a less electrophilic compound, being reduced at more negative potentials. Modifying the compounds of class 2 (less electrophilic) to yield class 3 brings an anodic shift of the relevant potentials, however less intense, when compared to the original compound 1, and introducing another factor involved in the redox cycle with the quinone imines, once they are known to undergo a redox cycle through aminophenols. Another important fact should be considered: arylquinone imines can suffer from hydrolysis, to generate back the original quinone,45 possibly behaving as a pro-drug.40

In contrast, the substitution of the initial “redox core” with a sulfur atom seems to change the original redox properties to a quasi-reversible electron transfer with potentials in the range of –168 mV up to –254 mV (E_pc1) and from –500 mV up to –574 mV (E_pc2). A significant change from an ortho-quinone to a chalcogenic quinone is observed electrochemically, and is associated with a shift of the relevant potentials to more positive potentials. Generally, the potentials for the chalcogenic quinone are 20 to 100 mV more positive, when compared to those for the ortho-quinone, pointing towards a more oxidizing species. Hence, its influence on the overall redox behaviour of these compounds is primarily due to electronic effects.

2.3. Antiparasitic activity

Based on the strategy of modifying and hence modulating the initial “redox core” of the ortho-quinone, and the insights obtained by electrochemistry, it was then interesting to see if these modifications also translate into specific – changes in – biological activities. Here, two distinct models indicative of the activity of quinones have been selected, i.e. cancer cell lines and T. cruzi (see section 1 Introduction).

The selection of these models was based on studies previously described in the literature that demonstrate the antitumor and trypanocidal activity of quinoidal compounds containing the 1,2,3-triazole nucleus and the sulphur atom.27,29,30 As discussed in the Introduction, our research group demonstrated that quinone-based N-sulfonyl-1,2,3-triazoles exhibited cytotoxicity against various tumour cell lines. These compounds were not evaluated against T. cruzi, the parasite that causes Chagas disease, but it is well documented that active redox systems are potential candidates to present trypanocidal activity, since in general such activity may be intrinsically related to the generation of ROS.

Whilst none of the N-sulfonyl triazoles 4a–4h appears to be particularly active against T. cruzi, with IC₅₀ values higher than 500 μM, the sulphur-containing quinones 5a–5d exhibit rather promising IC₅₀ values between 24.3 and 112.0 μM (Table 2). It therefore appears that this particular microorganism is sensitive towards the substituted ortho-quinone series, and less so towards the quinone imines. From the perspective of redox chemistry, this may be explained by the fact that the ortho-quinones with their more positive electrochemical potentials are also generally more oxidizing and hence toxic, whilst the quinone imines, with their somewhat more negative potentials, are less oxidizing. Nonetheless, practical applications of such compounds may be compromised by a wider toxicity, as the selectivity index (SI) is rather low, with values ranging from 0.37 for compound 5d to 1.52 for compound 5a.

Table 2. Activity of the naphthoquinones on trypomastigote forms of T. cruzi ^a .

Compounds	IC50/24 h (μM)	LC50/24 h b (μM)	SI
4a	>500	80.8 ± 6.5	>6.19
4b	>500	44.3 ± 6.4	>11.29
4c	>500	88.5 ± 3.5	>5.65
4d	>500	26.9 ± 1.2	>18.59
4e	>500	>100	nd
4f	>500	>100	nd
4g	>500	>100	nd
4h	>500	40.6 ± 2.5	>12.31
5a	24.3 ± 7.9	36.9 ± 2.6	1.52
5b	88.7 ± 10.1	81.4 ± 3.7	0.92
5c	65.6 ± 10.2	67.3 ± 6.9	1.03
5d	112.0 ± 10.6	41.5 ± 0.2	0.37
Bz	103.6 ± 0.6	>4000	>38.6

^aMean ± SD of at least three independent experiments, 5% blood at 4 °C.

^bCytotoxicity assays were performed using primary cultures of peritoneal macrophages obtained from Swiss Webster mice. nd: not determined. Bz: benznidazole. SI = selectivity index, represented by the ratio LC₅₀/IC₅₀.

It is already established in the literature that naphthoquinones are inactivated in the presence of certain components present in human blood.46,47 Compounds 5a–5d were therefore also assayed in the absence of blood at 37 °C (Table 3).46 These “blood-free” assays resulted in IC₅₀ values between 1.3 μM for compound 5a and 3.9 μM for compound 5d. The SI was also improved, with values above 10 observed for virtually all compounds of class 5 under investigation. When compared to the standard drug benznidazole, which under these experimental conditions shows an IC₅₀ of 9.7 μM, the quinones 5a–5d were even more active. Nonetheless, the selectivity for the parasite is notoriously low for the quinones when compared to benznidazole (SI > 413.2), regardless of the assay conditions.

Table 3. Activity of the synthetic derivatives against bloodstream trypomastigotes of T. cruzi (Y strain) at 37 °C in the absence of blood ^a .

Compounds	IC50/24 h (μM) at 37 °C, absence of blood	LC50/24 h b (μM) macrophages	SI at 37 °C, absence of blood
5a	1.3 ± 0.2	36.9 ± 2.6	28.38
5b	3.4 ± 1.0	81.4 ± 3.7	23.94
5c	4.6 ± 1.1	67.3 ± 6.9	14.63
5d	3.9 ± 1.4	41.5 ± 0.2	10.64
Bz	9.7 ± 2.4	>4000	>413.2

^aMean ± SD of at least three independent experiments, absence of blood at 37 °C.

^bCytotoxicity assays were performed using primary cultures of peritoneal macrophages obtained from Swiss Webster mice. nd: not determined. Bz: benznidazole. SI = selectivity index, represented by the ratio LC₅₀/IC₅₀.

The sulphur-containing quinones 5a–5d also exhibited some activity against macrophages with LC₅₀ values between 36.9 and 81.4 μM. The quinones 5a–5d were, however, more cytotoxic when compared to the standard drug benznidazole, which under these experimental conditions shows LC₅₀ > 4000 μM, and it is worth noting here the correlation between the observed activity and the recorded electrochemical potentials for the chosen compounds.

2.4. Cytotoxic activity

In the case of the cancer cell lines, cytotoxicity was evaluated in vitro by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay representative of six cancer cell lines, i.e. colon cancer cells (HCT-116), lung cancer cells (NCI-H460), prostate cancer cells (PC3), human promyelocytic leukemia cells (HL-60), human immortalized myelogenous leukemia cells (K-562) and multidrug resistant leukemia cells (Lucena 1) (Table 4). The murine, non-tumorigenic fibroblast immortalized cell line (L929) was employed as a control to evaluate the selectivity index of the compounds for tumour cells, once its IC₅₀ is compared to the corresponding values of neoplastic cell lines through selectivity index calculation. Doxorubicin was employed as a positive control and benchmark drug. The compounds were classified according to their activity as highly active (IC₅₀ < 2 μM), moderately active (2 μM < IC₅₀ < 10 μM), or inactive (IC₅₀ > 10 μM).48

Table 4. Cytotoxic activity of the compounds expressed as IC₅₀ (μM) in cancer and non-cancer cell lines after 72 h exposure, obtained by nonlinear regression for all cell lines from three independent experiments.

	HCT-116	NCI-H460	PC-3	HL-60	K-562	Lucena 1	L929
3a	>20.00	>20.00	14.10 (12.18–16.29)	17.44 (14.79–20.50)	>20.00	>20.00	>20.00
3b	10.71 (9.61–12.35)	9.45 (7.88–9.45)	8.82 (8.04–9.67)	7.18 (5.64–9.11)	>20.00	11.41 (9.36–13.90)	>20.00
3c	>20.00	16.92 (13.94–20.91)	18.45 (16.36–20.77)	11.98 (10.22–14.00)	>20.00	14.07 (12.51–15.80)	>20.00
3d	18.67 (16.51–21.13)	17.03 (15.07–19.32)	>20.00	9.20 (7.73–10.97)	13.48 (11.69–15.57)	17.00 (13.92–20.74)	>20.00
3e	18.87 (16.29–21.88)	16.16 (13.99–18.65)	14.64 (11.66–18.37)	11.03 (9.39–13.02)	12.80 (10.38–15.78)	16.43 (13.44–20.11)	>20.00
3f	>20.00	>20.00	15.87 (14.25–17.69)	14.20 (11.25–17.94)	>20.00	12.52 (10.31–15.20)	>20.00
3g	16.46 (13.07–20.81)	>20.00	>20.00	11.90 (10.38–13.65)	16.96 (14.33–20.09)	15.13 (12.27–18.66)	>20.00
3h	>20.00	>20.00	12.51 (10.93–14.33)	>20.00	>20.00	16.25 (11.87–22.23)	>20.00
4a	5.37 (4.75–6.40)	7.22 (6.40–8.05)	>20.00	6.71 (4.93–9.08)	12.24 (10.13–14.79)	3.67 (3.01–4.46)	>20.00
4b	7.43 (6.48–8.58)	8.39 (7.24–9.72)	>20.00	6.96 (5.75–8.46)	>20.00	4.58 (3.55–5.93)	>20.00
4c	3.61 (3.01–4.21)	8.02 (7.62–8.62)	2.65 (2.25–3.11)	5.42 (4.42–6.64)	6.22 (4.97–7.76)	1.44 (0.98–2.11)	17.73 (13.91–22.61)
4d	5.75 (4.96–6.69)	7.36 (6.77–7.96)	2.03 (1.65–2.49)	7.52 (6.07–9.33)	7.70 (6.39–9.27)	6.58 (5.57–7.78)	>20.00
4e	9.44 (8.48–10.79)	12.14 (10.98–13.30)	>20.00	6.92 (5.57–8.57)	9.80 (7.64–12.58)	3.16 (2.47–4.09)	>20.00
4f	9.25 (8.55–9.99)	9.41 (8.34–10.47)	3.41 (2.88–4.05)	4.22 (3.44–5.16)	5.40 (4.06–7.15)	3.32 (2.45–4.51)	>17.75
4g	>16.38	>16.38	>16.38	6.31 (5.29–7.49)	10.76 (8.08–14.32)	2.90 (2.44–3.44)	>16.38
4h	6.27 (5.32–7.41)	15.76 (14.43–17.47)	3.91 (3.42–4.50)	5.30 (4.51–6.28)	5.52 (4.77–6.39)	6.46 (5.05–8.26)	10.75 (8.77–13.17)
5a	12.77 (10.14–16.15)	>20.00	>20.00	6.08 (4.88–7.55)	15.71 (13.59–18.16)	15.58 (12.58–19.30)	>20.00
5b	>20.00	>20.00	>20.00	4.14 (3.21–5.31)	14.91 (11.38–19.54)	12.84 (10.52–15.66)	>20.00
5c	19.62 (16.77–22.83)	>20.00	>20.00	5.42 (4.21–6.99)	16.24 (12.86–19.87)	12.66 (10.20–15.73)	>20.00
5d	>20.00	>20.00	>20.00	9.38 (7.49–11.78)	13.33 (9.97–17.84)	>20.00	>20.00
Dox	0.21 (0.16–0.29)	0.15 (0.13–0.18)	0.76 (0.59–0.93)	0.02 (0.01–0.02)	0.49 (0.47–0.51)	0.18 (0.15–0.22)	1.72 (1.58–1.87)

As expected and reflected in Table 4, the redox active quinones exhibit significant cytotoxicity against a range of cancer cell lines, with IC₅₀ values for some compounds in the low micromolar range. For instance, some alkynes of class 3 are moderately active and generally present a non-selective cytotoxicity against the tumour cells evaluated. Compound 3d is an exception, as it is particularly active and selective against HL-60 cells, with an IC₅₀ of 9.20 μM (Table 4) and a selectivity index (SI) superior to 3.6 (Table 5). Compound 3b presents a broader cytotoxicity against NCI-H460 (IC₅₀ = 9.45 μM), PC-3 (IC₅₀ = 8.82 μM) and HL-60 (IC₅₀ = 7.18 μM) cells with a SI higher than 1.8. Generally, the members of class 4 exhibit a more selective cytotoxic activity against all the cell lines, with IC₅₀ values ranging from 1.44 to > 20 μM. For example, 4d presents IC₅₀ values between 2.03 and 7.70 μM (Table 4), with a SI higher than 2.6 (Table 5), while 4f presents an IC₅₀ from 3.32 to 9.41 μM (Table 4) and a SI higher than 1.9 (Table 5). Interestingly, the only difference between these molecules is the deactivating halogen substituent in the benzene ring, –F in 4d and –Br in 4f, suggesting that the more electronegative the halogen substituent, the more cytotoxic the compound. This pattern is followed by 4g, which possesses an –I substituent and is a less cytotoxic compound against the cell lines tested, except for a particularly high selectivity for Lucena 1, i.e. IC₅₀ of 2.90 μM and SI higher than 5.6 (Table 4).

Table 5. Selectivity index for the most active compounds ^a .

Compounds	HCT116	NCI-H460	PC3	HL-60	K-562	Lucena I
3a	1.2	1.6	2.5	2.0	1.0	1.5
3b	2.9	3.3	3.6	4.4	1.0	2.8
3c	1.6	2.0	1.8	2.8	1.5	2.4
3d	1.8	1.9	1.0	3.6	2.4	1.9
3e	1.6	1.9	2.1	2.8	2.4	1.9
3f	1.2	1.3	1.7	1.9	1.2	2.2
3g	1.5	1.0	1.0	2.0	1.4	1.6
3h	1.0	1.0	24	1.1	1.0	1.9
4a	3.8	2.9	1.0	3.1	1.7	5.6
4b	2.9	2.6	1.0	3.3	1.0	5.4
4c	4.9	2.2	6.7	3.3	2.9	12.3
4d	3.5	2.7	9.8	2.6	2.6	3.0
4e	2.0	1.6	1.0	2.8	2.0	6.1
4f	1.9	1.9	5.2	4.2	3.3	5.3
4g	1.0	1.0	1.0	2.6	1.5	5.6
4h	1.7	0.7	2.7	2.0	1.9	1.7
5a	2.2	0.7	0.7	4.6	1.8	1.8
5b	1.3	0.8	0.8	6.6	1.8	2.1
5c	1.8	1.0	1.0	6.6	2.2	2.8
5d	1.0	1.0	1.0	3.6	2.5	1.0

^aSelectivity index, represented by the ratio of cytotoxicities between normal cells and different lines of cancer cells.

Notable differences can be observed in the SI values shown in Table 5. The sulphur containing quinones seem to be fairly selective against certain cancer cell lines, such as HL-60, as observed in the case of compounds 5b and 5c. Indeed, compounds of class 5 are particularly attractive as far as observed activity and selectivity are considered, reflecting the potential of the redox core in concert with the sulfur atom added to this analogue. Compounds 5b and 5c present IC₅₀ values of 4.14 μM and 5.42 μM against HL-60, respectively, with an SI of 6.6.

In general, generation of intracellular ROS is a key mechanism associated with the cytotoxic effects of quinones in tumor cells. Thus, the redox status of treated K-562 cells was monitored using the oxidation-sensitive fluorescent dye CM-H₂DCFDA after 1 and 3 hours of incubation. Fig. 2 shows that all three tested compounds induced a significant increase in intracellular ROS levels, reaching a higher ROS + cell count at 3 hours. Compound 5a was the most potent in inducing significant changes (p < 0.05) in ROS levels, at both 1 and 3 hours. In contrast, compound 4f generated lower levels of ROS, but still significant (p < 0.05) at 3 hours. As expected, menadione and hydrogen peroxide increased ROS generation in treated cells.

Fig. 2. Effect of compounds 3e, 4f and 5a on ROS production in K-562 cells determined by flow cytometry using CM-H₂DCFDA, after 1 and 3 hours incubation. Menadione (MEN, 20 μM) and hydrogen peroxide (H₂O₂, 150 μM) were used as positive controls. A total of 10 000 events were analyzed per sample. Data are expressed as mean ± SEM from three independent experiments. *, p < 0.05 compared to the negative control by ANOVA followed by Tukey's test, performed using GraphPad Prism 8.

3. Conclusions

Considered together, the modification of the ortho-quinone lead 1 provides access to a wide range of different classes of compounds with distinctive “redox cores”, electrochemical potentials, biological activities and SI values. Here, the underlying mode(s) of action and also possible selectivity need to be investigated, together with potential applications against different human diseases. Whilst some of the compounds synthesised as part of this study already exhibit a combination of high activity and selectivity, these aspects may need to be further improved. It remains to be shown, for instance, against which cells or organisms these compounds are especially active, and if there is some selectivity associated with this activity, which may be exploited in practice.

Fortunately, the synthetic chemistry associated with these compounds is straightforward and provides wide opportunities for structural modifications, from the initial nucleophilic substitution and alkylation to the side chains and prospects provided by click chemistry reactions. Similarly, electrochemical investigations by cyclic voltammetry are fast and simple, and may provide initial information on the redox behaviour and activities one may expect.

4. Experimental section

4.1. Chemistry

Starting materials available from commercial suppliers were employed as received, unless stated otherwise. All other reagents requiring purification were purified by standard laboratory techniques, according to methods published by Perrin, Armarego and Perrin.49 Catalytic reactions were performed under nitrogen or argon atmosphere. Glassware, syringes and needles were either flame-dried immediately prior to use or placed in an oven (200 °C), for at least 2 h, and allowed to cool either in a desiccator or under an atmosphere of nitrogen or argon. Liquid reagents, solutions or solvents were added via a syringe through rubber septa. Melting points of solid compounds were measured on a Thomas Hoover melting point apparatus and are uncorrected. Column chromatography was performed on silica gel (SiliaFlash G60 Ultrapure 60–200 μm, 60 Å). ¹H and ¹³C NMR spectra were recorded at room temperature on a Bruker AVANCE DRX200 and DRX400, in the solvents indicated. Chemical shifts (δ) are given in parts per million (ppm) and coupling constants (J) are reported in Hertz (Hz). MS analyses were performed on a LC-MS/ESI TOF spectrometer (Model Xevo G2-XS QTof, Waters). The compounds are named in accordance with IUPAC rules as applied by ChemBioDraw Ultra (version 12.0).

General procedure for the synthesis of compounds 2a–h

The synthesis of compounds 2a–h was performed according to the methodology described in the literature by the Potter and Chen groups with minor modifications.36,37 To a solution of sodium 3,4-dioxo-3,4-dihydronaphthalene-1-sulfonate (1) (2.5 mmol) in EtOH : H₂O 1 : 1 (75 mL), the respective aniline (2.7 mmol) was added. The reaction mixture was stirred at room temperature monitored by TLC until the total consumption of the starting material. After completion of the reaction, the solvent was removed in vacuo and the resulting residue was purified by column chromatography over silica gel, using as the eluent a gradient mixture of hexane/ethyl acetate with increasing polarity. In some cases, ultrasound can be used to promote the reaction. The analytical data for compounds 2a–h are in accordance with those reported in the literature.36–38

General procedure for the synthesis of compounds 3a–h

To a stirred solution of a type 2 compound (2.0 equiv.) in DMF (4 mL), K₂CO₃ (250 mg, 0.18 mmol, 1.1 equiv.) was added and the reaction mixture was stirred at room temperature. After 10 min, propargyl bromide (80 mg, 0.7 mmol, 2.1 equiv.) was added dropwise and stirred under ultrasound for 40 min. The solvent was evaporated in vacuo and water (5 mL) was added to the crude reaction mixture. The aqueous phase was extracted with ethyl acetate (3 × 10 mL) and the organic phases were combined and dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography by eluting with an increasing polarity gradient mixture of hexane and ethyl acetate to afford the respective alkynes (3a–h).

(E)-4-(Phenylimino)-2-(prop-2-yn-1-yloxy)naphthalen-1(4H)-one (3a)

The product was obtained as an orange solid (99% yield); mp 139–141 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.52 (1H, d, J = 7.8 Hz), 8.24 (1H, d, J = 7.8 Hz), 7.74 (1H, t, J = 7.7 Hz), 7.67 (1H, t, J = 7.7 Hz), 7.43 (2H, t, J = 7.8 Hz), 7.21 (1H, t, J = 7.4 Hz), 6.96 (2H, d, J = 7.4 Hz), 6.60 (1H, s), 4.61 (2H, d, J = 2.2 Hz), 2.55 (1H, t, J = 2.2 Hz); ¹³C NMR (100 MHz, CDCl₃): δ 180.3, 154.8, 154.3, 150.5, 134.8, 133.5, 131.3, 131.3, 129.2, 126.7, 125.5, 125.0, 120.6, 104.6, 77.5, 76.6, 56.2; IR v_max (cm^–1, KBr): 3264, 3067, 2974, 2117, 1670, 1612, 1483, 1383, 1332, 1255, 1189, 1044, 1019, 949, 857, 769, 694, 648; EI/HRMS (m/z) [M + H]⁺: 288.1023. Cald. for [C₁₉H₁₄NO₂]⁺: 288.1024.

(E)-4-((4-Methoxyphenyl)imino)-2-(prop-2-yn-1-yloxy)naphthalen-1(4H)-one (3b)

The product was obtained as a red solid (97% yield); mp 149–150 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.51 (1H, d, J = 7.8 Hz), 8.22 (1H, d, J = 7.8 Hz), 7.73 (1H, t, J = 7.9 Hz), 7.65 (1H, t, J = 7.9 Hz), 6.98 (4H, s), 6.72 (1H, s), 4.65 (2H, d, J = 2.3 Hz), 3.78 (3H, s), 2.59 (1H, t, J = 2.3 Hz); ¹³C NMR (100 MHz, CDCl₃): δ 180.4, 157.7, 154.3, 154.1, 143.6, 135.1, 133.4, 131.2, 131.0, 126.6, 125.3, 122.7, 114.6, 104.6, 77.5, 76.8, 56.2, 55.7; IR v_max (cm^–1, KBr): 3264, 2930, 2843, 2128, 1675, 1597, 1497, 1461, 1354, 1320, 1260, 1243, 1201, 1044, 1022, 847, 777, 682, 653; EI/HRMS (m/z) [M + H]⁺: 318.1122. Cald. for [C₂₀H₁₆NO₃]⁺: 318.1125.

(E)-2-(Prop-2-yn-1-yloxy)-4-(p-tolylimino)naphthalen-1(4H)-one (3c)

The product was obtained as an orange solid (95% yield); mp 148–150 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.50 (1H, d, J = 7.6 Hz), 8.21 (1H, d, J = 7.6 Hz), 7.71 (1H, t, J = 7.2 Hz), 7.64 (1H, t, J = 7.2 Hz), 7.22 (2H, d, J = 7.4 Hz), 6.88 (2H, d, J = 7.4 Hz), 6.65 (1H, s), 4.62 (2H, s), 2.59 (1H, s), 2.40 (3H, s); ¹³C NMR (100 MHz, CDCl₃): δ 180.1, 154.3, 153.9, 147.7, 134.8, 134.6, 133.2, 131.1, 130.9, 129.6, 126.4, 125.2, 120.7, 104.4, 76.8, 76.6, 56.0, 21.0; IR v_max (cm^–1, KBr): 3269, 2117, 1672, 1614, 1597, 1500, 1335, 1257, 1192, 1044, 1019, 772; EI/HRMS (m/z) [M + H]⁺: 302.1170. Cald. for [C₂₀H₁₆NO₂]⁺: 302.1176.

(E)-4-((4-Fluorophenyl)imino)-2-(prop-2-yn-1-yloxy)naphthalen-1(4H)-one (3d)

The product was obtained as an orange solid (79% yield); mp 147–149 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.45 (1H, d, J = 7.7 Hz), 8.18 (1H, d, J = 7.7 Hz), 7.70 (1H, t, J = 7.4 Hz), 7.63 (1H, t, J = 7.4 Hz), 7.10 (2H, t, J = 8.6 Hz), 6.93–6.90 (2H, m), 6.56 (1H, s), 4.61 (2H, d, J = 2.1 Hz), 2.57 (1H, t, J = 2.1 Hz); ¹³C NMR (100 MHz, CDCl₃): δ 180.1, 161.6, 159.2, 155.2, 154.3, 146.4, 134.6, 133.5, 131.3, 131.2, 126.6, 125.3, 122.2, 122.1, 116.1, 115.9, 104.1, 76.5, 56.2; IR v_max (cm^–1, KBr): 3296, 2990, 2123, 1667, 1624, 1597, 1497, 1339, 1264, 1221, 1204, 1184, 1044, 1019, 862, 772, 697, 648; EI/HRMS (m/z) [M + H]⁺: 306.0919. Cald. for [C₁₉H₁₃FNO₂]⁺: 306.0925.

(E)-4-((4-Chlorophenyl)imino)-2-(prop-2-yn-1-yloxy)naphthalen-1(4H)-one (3e)

The product was obtained as an orange solid (77% yield); mp 145–147 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.44 (1H, dd, J = 1.5 and 7.8 Hz), 8.19 (1H, dd, J = 1.5 and 7.8 Hz), 7.75–7.59 (2H, m), 7.36 (2H, d, J = 8.6 Hz), 6.89 (2H, d, J = 8.6 Hz), 6.51 (1H, s), 4.62 (2H, d, J = 2.4 Hz), 2.58 (1H, t, J = 2.4 Hz); ¹³C NMR (50 MHz, CDCl₃): δ 180.0, 155.2, 154.4, 148.8, 134.5, 133.5, 131.4, 131.1, 130.4, 129.3, 126.7, 125.4, 122.0, 104.0, 77.6, 76.5, 56.2; IR v_max (cm^–1, KBr): 3275, 1670, 1614, 1483, 1337, 1252, 1192, 1044, 1019, 854, 772, 639; EI/HRMS (m/z) [M + H]⁺: 322.0629. Cald. for [C₁₉H₁₃ClNO₂]⁺: 322.0629.

(E)-4-((4-Bromophenyl)imino)-2-(prop-2-yn-1-yloxy)naphthalen-1(4H)-one (3f)

The product was obtained as a red solid (72% yield); mp 156–158 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.48 (1H, d, J = 7.8 Hz), 8.23 (1H, d, J = 7.8 Hz), 7.75 (1H, t, J = 7.4 Hz), 7.68 (1H, t, J = 7.4 Hz), 7.54 (2H, d, J = 8.4 Hz), 6.85 (2H, d, J = 8.4 Hz), 6.54 (1H, s), 4.65 (2H, d, J = 2.1 Hz), 2.58 (1H, t, J = 2.1 Hz); ¹³C NMR (50 MHz, CDCl₃): δ 180.1, 155.3, 154.6, 149.4, 134.6, 133.6, 132.3, 131.5, 131.3, 126.8, 125.5, 122.4, 118.2, 104.2, 77.6, 76.5, 56.3; IR v_max (cm^–1, KBr): 3275, 2117, 1667, 1612, 1478, 1337, 1255, 1192, 1046, 1024, 852, 772, 685, 641; EI/HRMS (m/z) [M + H]⁺: 366.0120. Cald. for [C₁₉H₁₃BrNO₂]⁺: 366.0124.

(E)-4-((4-Iodophenyl)imino)-2-(prop-2-yn-1-yloxy)naphthalen-1(4H)-one (3g)

The product was obtained as a red solid (68% yield); mp 123–125 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.46 (1H, d, J = 7.8 Hz), 8.21 (1H, d, J = 7.8 Hz), 7.73–7.65 (4H, m), 6.72 (2H, d, J = 8.2 Hz), 6.52 (1H, s), 4.64 (2H, s), 2.59 (1H,s); ¹³C NMR (50 MHz, CDCl₃): δ 179.8, 154.9, 154.2, 149.7, 137.9, 134.3, 133.3, 131.2, 130.9, 126.5, 125.2, 122.5, 103.8, 88.6, 77.4, 76.2, 56.1; IR v_max (cm^–1, KBr): 3280, 1668, 1609, 1473, 1476, 1253, 1187, 1044, 1020, 770; EI/HRMS (m/z) [M + H]⁺: 413.9989. Cald. for [C₁₉H₁₃INO₂]⁺: 413.9985.

(E)-4-((4-Acetylphenyl)imino)-2-(prop-2-yn-1-yloxy)naphthalen-1(4H)-one (3h)

The product was obtained as a yellow solid (48% yield); mp 172–174 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.49 (1H, dd, J = 1.0 and 7.7 Hz), 8.25 (1H, dd, J = 1.0 and 7.7 Hz), 8.05 (2H, d, J = 8.6 Hz), 7.77 (1H, t, J = 7.5 Hz), 7.70 (1H, t, J = 7.5 Hz), 7.01 (2H, d, J = 8.6 Hz), 6.44 (1H, s), 4.62 (2H, d, J = 2.3 Hz), 2.65 (3H, s), 2.56 (1H, t, J = 2.3 Hz); ¹³C NMR (50 MHz, CDCl₃): δ 197.0, 179.8, 155.1, 154.8, 154.5, 134.1, 133.6, 133.5, 131.4, 131.1, 129.7, 126.7, 125.6, 120.1, 104.0, 77.5, 76.1, 56.1, 26.5; IR v_max (cm^–1, KBr): 3264, 2925, 2134, 1679, 1614, 1592, 1560, 1359, 1267, 1209, 1019, 726, 685; EI/HRMS (m/z) [M + H]⁺: 330.1122. Cald. for [C₂₁H₁₆NO₃]⁺: 330.1125.

General procedure for preparing the 1,2,3-triazole derivatives

To a stirred solution of copper(i) thiophene-2-carboxylate (CuTC, 8 mg, 0.04 mmol, 0.08 equiv.) in toluene (5 mL), the respective propargylated aminonaphthoquinones (0.5 mmol, 1 equiv.) were added and the reaction mixture was cooled in an ice–water bath. Subsequently, the sulfonyl azide (0.5 mmol, 1 equiv.) was added dropwise. The reaction mixture was then allowed to warm to room temperature and stirred until the reaction was complete (as evidenced by TLC). The reaction mixture was extracted with EtOAc (3 × 30 mL). The combined organic layers were dried over sodium sulfate and filtered through Celite®. The eluent was concentrated under vacuum and the product was purified by silica gel column chromatography using eluents with an increasing polarity gradient mixture of hexane and ethyl acetate to afford compounds 4a–h.

(E)-4-(Phenylimino)-2-((1-tosyl-1H-1,2,3-triazol-4-yl)methoxy)naphthalen-1(4H)-one (4a)

The product was obtained as a red solid (60% yield); mp 153–155 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.48 (1H, d, J = 7.5 Hz), 8.22 (1H, s), 8.19 (1H, d, J = 7.5 Hz), 7.99 (2H, d, J = 8.0 Hz), 7.72 (1H, t, J = 7.5 Hz), 7.65 (1H, t, J = 7.5 Hz), 7.43–7.37 (4H, m), 7.19 (1H, t, J = 7.1 Hz), 6.84 (2H, d, J = 8.0 Hz), 6.52 (1H, s), 5.02 (2H, s), 2.44 (3H, s); ¹³C NMR (100 MHz, CDCl₃): δ 180.0, 154.6, 154.5, 150.2, 147.7, 141.8, 134.6, 133.4, 132.7, 131.1, 131.0, 130.5, 129.2, 129.2, 126.5, 125.3, 124.9, 123.2, 120.2, 120.1, 103.8, 61.7, 21.8; IR v_max (cm^–1, KBr): 3132, 2922, 1668, 1607, 1392, 1254, 1193, 1014, 667, 587; EI/HRMS (m/z) [M + H]⁺: 485.1208. Cald. for [C₂₆H₂₁N₄O₄S]⁺: 485.1278.

(E)-4-((4-Methoxyphenyl)imino)-2-((1-tosyl-1H-1,2,3-triazol-4-yl)methoxy)naphthalen-1(4H)-one (4b)

The product was obtained as a red solid (55% yield); mp 123–125 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.52 (1H, d, J = 7.8 Hz), 8.26 (1H, s), 8.22 (1H, d, J = 7.8 Hz), 8.02 (2H, d, J = 7.5 Hz), 7.74 (1H, t, J = 7.3 Hz), 7.66 (1H, t, J = 7.3 Hz), 7.41 (2H, d, J = 7.5 Hz), 6.98 (2H, d, J = 7.2 Hz), 6.86 (2H, d, J = 7.2 Hz), 6.67 (1H, s), 5.09 (2H, s), 3.89 (3H, s), 2.47 (3H, s); ¹³C NMR (100 MHz, CDCl₃): δ 180.0, 157.5, 154.4, 154.0, 147.7, 143.3, 142.0, 134.8, 133.3, 132.8, 131.0, 130.9, 130.5, 128.9, 126.5, 125.2, 123.1, 122.2, 114.6, 104.0, 61.7, 55.5; IR v_max (cm^–1, KBr): 3139, 2958, 2922, 1659, 1601, 1499, 1391, 1261, 1193, 1020, 670, 583; EI/HRMS (m/z) [M + H]⁺: 515.1372. Cald. for [C₂₇H₂₃N₄O₅S]⁺: 515.1384.

(E)-4-(p-Tolylimino)-2-((1-tosyl-1H-1,2,3-triazol-4-yl)methoxy)naphthalen-1(4H)-one (4c)

The product was obtained as a yellow solid (50% yield); mp 157–159 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.52 (1H, d, J = 7.8 Hz), 8.23–8.21 (2H, m), 8.03 (2H, d, J = 8.2 Hz), 7.75 (1H, t, J = 7.6 Hz), 7.67 (1H, t, J = 7.6 Hz), 7.41 (2H, d, J = 8.2 Hz), 7.23 (2H, d, J = 8.0 Hz), 6.78 (2H, d, J = 8.0 Hz), 6.59 (1H, s), 5.06 (2H, s), 2.47 (3H, s), 2.42 (3H, s); ¹³C NMR (100 MHz, CDCl₃): δ; 180.2, 154.8, 154.6, 147.9, 142.3, 135.0, 135.0, 133.6, 133.1, 131.3, 131.2, 130.8, 130.1, 129.1, 126.7, 125.5, 123.3, 120.6, 104.1, 62.0, 22.1, 21.2; IR v_max (cm^–1, KBr): 3132, 2917, 1665, 1597, 1396, 1260, 1197, 1015, 670, 587; EI/HRMS (m/z) [M + H]⁺: 499.1421. Cald. for [C₂₇H₂₃N₄O₄S]⁺: 499.1435.

(E)-4-((4-Fluorophenyl)imino)-2-((1-tosyl-1H-1,2,3-triazol-4-yl)methoxy)naphthalen-1(4H)-one (4d)

The product was obtained as a yellow solid (79% yield); mp 162–164 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.46 (1H, d, J = 7.6 Hz), 8.23 (1H, s), 8.19 (1H, d, J = 7.6 Hz), 8.00 (2H, d, J = 7.8 Hz), 7.72 (1H, t, J = 7.2 Hz), 7.65 (1H, t, J = 7.2 Hz), 7.39 (2H, d, J = 7.8 Hz), 7.11 (2H, t, J = 8.2 Hz), 6.83–6.81 (2H, m), 6.55 (1H, s), 5.06 (2H, s), 2.45 (3H, s); ¹³C NMR (100 MHz, CDCl₃): δ 179.9, 155.0, 154.6, 147.7, 141.7, 134.5, 133.5, 132.6, 131.2, 131.0, 130.6, 128.9, 126.5, 125.3, 123.3, 121.8, 121.8, 116.2, 116.0, 61.5, 21.8; IR v_max (cm^–1, KBr): 3127, 2925, 1670, 1607, 1495, 1388, 1260, 1197, 1015, 854, 670, 587; EI/HRMS (m/z) [M + H]⁺: 503.1154. Cald. for [C₂₆H₂₀FN₄O₄S]⁺: 503.1184.

(E)-4-((4-Chlorophenyl)imino)-2-((1-tosyl-1H-1,2,3-triazol-4-yl)methoxy)naphthalen-1(4H)-one (4e)

The product was obtained as an orange solid (95% yield); mp 155–157 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.47 (1H, d, J = 7.6 Hz), 8.25 (1H, s), 8.21 (1H, d, J = 7.6 Hz), 8.03 (2H, d, J = 7.5 Hz), 7.75 (1H, t, J = 7.2 Hz), 7.68 (1H, t, J = 7.2 Hz), 7.43–7.39 (4H, m), 6.82 (2H, d, J = 7.2 Hz), 6.53 (1H, s), 5.07 (2H, s), 2.47 (3H, s); ¹³C NMR (100 MHz, CDCl₃): δ 180.1, 155.3, 155.1, 148.9, 147.9, 142.0, 134.7, 133.8, 133.0, 131.6, 131.3, 130.8, 130.6, 129.7, 129.2, 126.9, 125.6, 123.5, 121.9, 61.7, 21.9; IR v_max (cm^–1, KBr): 3138, 2919, 1670, 1609, 1391, 1260, 1194, 1088, 1010, 852, 670, 583; EI/HRMS (m/z) [M + H]⁺: 519.0883. Cald. for [C₂₆H₂₀ClN₄O₄S]⁺: 519.0888.

(E)-4-((4-Bromophenyl)imino)-2-((1-tosyl-1H-1,2,3-triazol-4-yl)methoxy)naphthalen-1(4H)-one (4f)

The product was obtained as a yellow solid (92% yield); mp 175–177 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.47 (1H, d, J = 7.7 Hz), 8.24 (1H, s), 8.21 (1H, d, J = 7.7 Hz), 8.03 (2H, d, J = 8.4 Hz), 7.75 (1H, t, J = 7.4 Hz), 7.68 (1H, t, J = 7.4 Hz), 7.54 (2H, d, J = 8.4 Hz), 7.42 (2H, d, J = 8.2 Hz), 6.76 (2H, d, J = 8.2 Hz), 6.52 (1H, s), 5.07 (2H, s), 2.48 (3H, s); ¹³C NMR (100 MHz, CDCl₃): δ 179.8, 154.9, 154.8, 149.1, 147.7, 141.7, 134.4, 133.5, 132.7, 132.3, 131.3, 131.0, 130.6, 128.9, 126.6, 125.4, 123.2, 122.0, 118.1, 103.4, 61.7, 21.9; IR v_max (cm^–1, KBr): 3127, 1670, 1609, 1393, 1260, 1192, 1015, 852, 774, 670, 587, 540; EI/HRMS (m/z) [M + H]⁺: 563.0364. Cald. for [C₂₆H₂₀BrN₄O₄S]⁺: 563.0383.

(E)-4-((4-Iodophenyl)imino)-2-((1-tosyl-1H-1,2,3-triazol-4-yl)methoxy)naphthalen-1(4H)-one (4g)

The product was obtained as a yellow solid (89% yield); mp 173–175 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.45 (1H, d, J = 7.8 Hz), 8.21 (1H, s), 8.19 (1H, d, J = 7.8 Hz), 8.01 (2H, d, J = 8.2 Hz), 7.75–7.69 (3H, m), 7.66 (1H, t, J = 7.4 Hz), 7.39 (2H, d, J = 8.2 Hz), 6.62 (2H, d, J = 8.3 Hz), 6.79 (1H, s), 5.05 (2H, s), 2.45 (3H, s); ¹³C NMR (100 MHz, CDCl₃): δ 179.8, 154.9, 149.7, 147.7, 141.7, 138.2, 134.4, 133.5, 132.7, 131.3, 131.0, 130.6, 128.9, 126.6, 125.4, 123.2, 122.3, 103.4, 88.8, 61.7, 21.9; IR v_max (cm^–1, KBr): 3138, 2919, 1670, 1609, 1396, 1260, 1194, 1017, 668, 585; EI/HRMS (m/z) [M + H]⁺: 611.0246. Cald. for [C₂₆H₂₀IN₄O₄S]⁺: 611.0244.

(E)-4-((4-Acetylphenyl)imino)-2-((1-tosyl-1H-1,2,3-triazol-4-yl)methoxy)naphthalen-1(4H)-one (4h)

The product was obtained as a yellow solid (87% yield); mp 174–176 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.46 (1H, d, J = 7.6 Hz), 8.22–8.20 (2H, m), 8.04–7.99 (4H, m), 7.76 (1H, t, J = 7.3 Hz), 7.68 (1H, t, J = 7.3 Hz), 7.40 (2H, d, J = 8.1 Hz), 6.91 (2H, d, J = 8.1 Hz), 6.39 (1H, s), 5.03 (2H, s), 2.65 (3H, s), 2.46 (3H, s); ¹³C NMR (100 MHz, CDCl₃): δ 197.0, 179.7, 155.0, 154.6, 147.7, 141.6, 134.1, 133.7, 133.6, 132.7, 131.5, 131.1, 130.6, 129.8, 128.9, 126.6, 125.5, 123.2, 119.9, 103.6, 61.8, 29.7, 26.5, 21.8; IR v_max (cm^–1, KBr): 3133, 2919, 2859, 1670, 1594, 1393, 1269, 1194, 1019, 670, 590; EI/HRMS (m/z) [M + H]⁺: 527.1378. Cald. for [C₂₈H₂₃N₄O₅S]⁺: 527.1384.

General procedure for the synthesis of compounds 5a–d

The synthesis of compounds 5a–d was performed according to the methodology described in the literature by the Potter and Chen groups with minor modifications.36,37 To a 25 mL round bottom flask, 5 mL of ethanol and 5 mL of water, and then sodium 3,4-dioxo-3,4-dihydronaphthalene-1-sulfonate (1) (1 mmol) and the corresponding thiophenol (2 mmol) were added. The reaction mixture was stirred at room temperature under ultrasound energy, and monitored by TLC until the total consumption of the starting material. After completion of the reaction, the organic phase was extracted with dichloromethane and dried over sodium sulphate. The solvent was removed under reduced pressure to afford the crude product, which was purified by column chromatography over silica gel, using as the eluent a gradient mixture of hexane/ethyl acetate with increasing polarity. The analytical data for compounds 5a are in accordance with those reported in the literature.17b

4-(p-Tolylthio)naphthalene-1,2-dione (5b)

The product was obtained as an orange solid (40% yield); mp 205–206 °C; ¹H NMR (200 MHz, CDCl₃): δ 8.09 (1H, d, J = 7.4 Hz), 7.87 (1H, d, J = 7.6 Hz), 7.65 (t, 1H, J = 7.2 Hz), 7.52 (1H, t, J = 7.4 Hz), 7.37 (2H, d, J = 8.0 Hz), 7.25 (2H, d, J = 8.0 Hz), 5.80 (s, 1H), 2.36 (s, 3H); ¹³C NMR (50 MHz, CDCl₃): 179.5, 176.6, 161.9, 141.7, 135.8, 135.0, 133.4, 131.1, 130,3, 129.4, 125.0, 122.6, 121.1, 21.3; EI/MS (m/z) [M + H]⁺: 281.0. Cald. for [C₁₇H₁₃O₂S]⁺: 281.0.

4-(m-Tolylthio)naphthalene-1,2-dione (5c)

The product was obtained as an orange solid (32% yield); mp 210–212 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.19 (1H, d, J = 7.4 Hz), 7.97 (1H, d, J = 7.6 Hz), 7.74 (1H, t, J = 7.2 Hz), 7.62 (1H, t, J = 7.4 Hz), 7.44–7.35 (m, 4H), 5.92 (s, 1H), 2.43 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): 179.5, 176.6, 161.9, 140.6, 136.5, 135.0, 133.4, 131.1, 130,3, 129.4, 125.0, 122.6, 121.1, 21.3; EI/MS (m/z) [M + H]⁺: 281.0. Cald. for [C₁₇H₁₃O₂S]⁺: 281.0.

4-((4-Methoxyphenyl)thio)naphthalene-1,2-dione (5d)

The product was obtained as a red solid (30% yield); mp 215–216 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.17 (1H, d, J = 7.6 Hz), 7.95 (1H, d, J = 7.7 Hz), 7.73 (1H, t, J = 7.6 Hz), 7.60 (1H, t, J = 7.5 Hz), 7.48 (2H, d, J = 8.4 Hz), 7.03 (2H, d, J = 8.4 Hz), 5.88 (1H, s), 3.89 (3H, s); ¹³C NMR (100 MHz, CDCl₃): 179.5, 176.6, 162.0, 137.5, 135.8, 135.0, 133.4, 131.1, 130,3, 129.4, 125.0, 122.6, 121.1, 55.6; EI/MS (m/z) [M + H]⁺: 297.0. Cald. for [C₁₇H₁₃O₃S]⁺: 297.0.

4.2. Biological assays

4.2.1. Animals

All experiments dealing with animals were performed in accordance with the Brazilian Law 11.794/2008 and regulations of the National Council of Animal Experimentation Control under the license L038/2018 from the Ethics Committee for Animal Use of the Oswaldo Cruz Institute (CEUA/IOC).

4.2.2. Cytotoxicity assays

The compounds were tested for cytotoxic activity in cell culture in vitro employing several human cancer cell lines obtained from the National Cancer Institute, NCI (Bethesda, MD). Cytotoxicity was investigated against six cancer cell lines, i.e. HCT-116 (human colon carcinoma cells), NCI-H460 (human lung cancer cells), PC3 (human prostate cells), HL-60 (human promyelocytic leukemia cells), K-562 (myelogenous leukemia cell line) and Lucena 1 (MDR-cell line). The murine fibroblast immortalized cell line (L929) was used as control lineage. All culture media were supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 IU mL^–1 penicillin, and 100 μg mL^–1 streptomycin at 37 °C with 5% CO₂. In cytotoxicity experiments, cells were plated in 96-well plates (0.1 × 10⁶ cells per well for leukaemia cells, 0.7 × 10⁵ cells per well for HCT-116, and 0.1 × 10⁶ cells per well for PC3, L929 and NCI-H460). All the compounds tested were dissolved in DMSO. The final concentration of DMSO in the culture medium was kept constant (0.1%, v/v). Doxorubicin (0.001–1.10 μM) served as positive control, and negative control groups received the same amount of vehicle (DMSO). The cell viability was determined by reduction of the yellow dye 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue formazan product as described by Mosmann.50a At completion of the incubation after 72 h, the plates were centrifuged and the medium was replaced by fresh medium (200 μL) containing 0.5 mg mL^–1 MTT. Three hours later, the MTT formazan product was dissolved in DMSO (150 μL) and the absorbance was measured on a multiplate reader (SpectraCount, Packard, Ontario, Canada). The influence of the compound on cell proliferation and survival was quantified as the percentage of control absorbance of the reduced dye at 550 nm. The results were obtained by nonlinear regression for all the cell lines from three independent experiments. All cell treatments were performed with three replicates. All cells were mycoplasma-free.

4.2.3. Measurement of intracellular reactive oxygen species levels

Intracellular reactive oxygen species (ROS) accumulation was monitored using 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), which is converted to the highly fluorescent dichlorofluorescein (DCF) in the presence of intracellular ROS.50b K-562 cells (myelogenous leukemia cell line) were pre-loaded with 10 μM CM-H₂DCFDA and incubated for 1 hour, in the dark, at 37 °C/5% CO₂. After that time, the cells were centrifuged, the medium containing CM-H₂DCFDA was removed and the cells were washed with PBS buffer. From this stage, the cells were always protected from light. Fresh medium containing compounds 3e, 4f and 5a was added and the cells were incubated at the times of interest (1 and 3 hours). After the incubation time, the cells were centrifuged, washed and re-suspended in PBS containing propidium iodide (PI) to a final concentration of 1 μg mL^–1. Tubes were placed on ice and immediately analyzed by flow cytometry. Living cells, which are PI negative, were selected by gating. In those living cells, the DCF fluorescence was recorded using excitation and emission wavelengths of 490 and 525 nm, respectively. Menadione (MEN, 20 μM) and hydrogen peroxide (H₂O₂, 150 μM) were used as positive controls. A total of 10 000 events were analyzed per sample. Data are expressed as mean ± SEM from three independent experiments.

4.2.4. Trypanocidal assay and selectivity index

Stock solutions of the compounds were prepared in dimethyl sulfoxide (DMSO), with the final concentration of the latter in the experiments never exceeding 0.4%. Preliminary experiments showed that DMSO has no deleterious effect on the parasites when its concentration is up to 4%. T. cruzi bloodstream trypomastigotes (Y strain) were obtained at the peak of parasitaemia from infected albino mice, purified by differential centrifugation and resuspended in RPMI to a parasite concentration of 10⁷ cells per mL in the presence of 10% mouse blood. This suspension (100 μL) was added in the same volume of each compound previously prepared at twice the desired final concentrations for 24 h at 4 °C. Cell quantification was performed in a Neubauer chamber and the trypanocidal activity was expressed as IC₅₀/24 h, corresponding to the concentration that leads to lysis of 50% of the parasites. In order to determine the SI, cytotoxicity assays were performed with primary cultures of peritoneal macrophages obtained from Albino Swiss mice. For the experiments, 2.5 × 10⁴ cells in 200 μL of RPMI-1640 medium (pH 7.2 plus 10% foetal bovine serum and 2 mM glutamine) were added to each well of a 96-well microtitre plate and incubated for 24 h at 37 °C. The treatment of the cultures was performed in fresh supplemented medium (200 μL per well) for 24 h at 37 °C. After this period, 110 μL of the medium was discarded and 10 μL of PrestoBlue (Invitrogen) was added to complete the final volume of 100 μL. Thus, the plate was incubated for 2 h and the measurement was performed at 560 and 590 nm, as recommended by the manufacturer. The results were expressed as the difference in the percentage of reduction between treated and untreated cells being the LC₅₀ value, corresponding to the concentration that leads to damage of 50% of the mammalian cells.

5. X-ray crystallography

X-ray diffraction data collection for the compounds was performed on an Enraf-Nonius Kappa-CCD diffractometer (95 mm CCD camera on a κ-goniostat) and XtaLAB Mini (ROW) two-circle diffractometer employing graphite monochromated MoK_radiation (0.71073 Å), at room temperature. Data collection was carried out using the COLLECT software and CrysAlisPro.51,52 Integration and scaling of the reflections, and correction for Lorentz and polarization effects were performed with the HKL DENZO-SCALEPACK and the CrysAlisPro system of programs.53 The structure of the compounds was solved by direct methods with SHELXS-97.54 The models were refined by full-matrix least squares on F² using SHELXL-97.54 The program ORTEP-3 was used for graphic representation and the program WINGX was used to prepare materials for publication.55,56 All H atoms were located by geometric considerations placed (C–H = 0.93–0.96 Å; N–H = 0.86 Å) and refined as riding with U_iso(H) = 1.5U_eq(C-methyl) or 1.2U_eq(other). Crystallographic data for the structures were deposited in the Cambridge Crystallographic Data Centre, with CCDC numbers ; 1928261 (3a), ; 1928296 (3d), ; 1928326 (3f), ; 1928361 (3g), ; 1928382 (3h) and ; 1928407 (5d).

6. Electrochemical studies

Cyclic voltammetry was performed on a 100B/W electrochemical workstation (BASI®, West Lafayette, USA) at ambient temperature. Cyclic voltammograms of the compounds (1 mM) were recorded in phosphate buffer (pH 7.4), employing a glassy carbon working electrode cleaned and polished with Al₂O₃ after each scan, an Ag/AgCl reference electrode (SSE) and a platinum wire counter electrode, at a potential range between –1 and +1 V. Quinone derivatives required 30% methanol due to limited solubility in aqueous media. Buffers were purged with nitrogen for 30 min prior to use. The sensitivity of the cell was adjusted to 10 μA V^–1 and the compounds were scanned at the rate of 200 mV s^–1.

Conflicts of interest

Authors declare no conflict of interest.