It takes two to tango: synthesis of cytotoxic quinones containing two redox active centers with potential antitumor activity

Abstract

We report the synthesis of 47 new quinone-based derivatives via click chemistry and their subsequent evaluation against cancer cell lines and the control L929 murine fibroblast cell line. These compounds combine two redox centers, such as an ortho-quinone/para-quinone or quinones/selenium with the 1,2,3-triazole nucleus. Several of these compounds present IC₅₀ values below 0.5 μM in cancer cell lines with significantly lower cytotoxicity in the control cell line L929 and good selectivity index. Hence, our study confirms the use of a complete and very diverse range of quinone compounds with potential application against certain cancer cell lines.

Quinone-based frameworks with antitumor activities were described. We identified compounds with IC₅₀ values below 0.5 μM in cancer cells lines with significantly lower cytotoxicity in the control cell line L929.

1. Introduction

Quinones represent a particularly exciting class of natural products. These redox-active secondary metabolites are highly abundant in nature, and also exhibit pronounced redox modulatory activity in many target cells, which frequently translates into a pronounced and often also selective cytotoxic activity, for instance, against cancer cell lines under oxidative stress (OS).¹ Not surprisingly, lapachol and its derivatives, e.g., β-lapachone (β-lap), which occur naturally in the ipe tree, already present a pivotal role in cancer therapy, since studies have proven that β-lap showed significant activity against Sarcoma 180 ascite tumour cells (S-180 cells) in vitro, and in mice bearing S-180 tumors.² Antitumor activity of β-lap is based on its specialty to elevate endogenous levels of NAD(P)H: quinone oxidoreductase 1 (NQO1),³ and whereas the clinical uses of β-lap are limited due to its high hydrophobicity, the compound and its derivatives are one focus of ongoing research in this field.

For over a decade, our research groups have dedicated efforts to the synthesis and modification of quinone-based compounds, especially 1,2- and 1,4-naphthoquinone derivatives,⁴ using classical strategies such as molecular hybridization⁵ and bioisosterism.⁶ To achieve synthetic challenges and prepare selected molecules, robust methodologies such as C–H activation⁷ and Cu(i) catalysed azide-alkyne cycloaddition (CuAAC) reaction⁸ have been employed successfully, resulting in potent bioactive molecules. The triazole moiety is also considered a leading pharmacophore,⁹ and the use of “click” reactions to modify the furan and pyran ring, also named as “C-ring” of most common lapachol derivatives like β-lap and nor-β-lap have been employed by us for the synthesis of powerful multifunctional redox modulators.^10,11

Apart from the high potentiality against cancer cells, the toxicity of NQs to normal cells has also been reported.¹² Nonetheless, we recently discovered that the combination of a quinone-based compound and a selenium core could circumvent this problem, i.e., the hybrid compounds inhibited the growth of cancer cells more selectively, with keeping their potency.¹³ To expand our library of redox-sensitive compounds for the identification of more potent and selective antitumor agents, herein we report the synthesis and antitumour activity of novel hybrid molecules combining the quinoidal redox system of β-lap and nor-β-lap with either a second quinone or a redox active selenium atom to boost redox and catalytic activity of these natural products as depicted in Scheme 1.

Scheme 1. Overview of multifunctional redox agents derived from natural β-lap and nor-β-lap.

2. Results and discussion

2.1. Synthesis of multifunctional derivatives

In the first step, 3-azido-nor-β-lapachone (A) has been synthesized employing a previously described methodology.¹⁴ Six clickable alkynes,¹⁵ were subsequently obtained by the reaction of 1,4-naphthoquinone, propargyl bromine and six different para-substituted anilines, with moderate to high yields (Scheme S1†). Electron withdrawing groups (EWG) and electron-donating groups (EDG) were included as central features to obtain a diverse family of compounds for subsequent antitumour activity evaluation. Based on these clickable building blocks, the first class of novel triazoles 1–6 was prepared via the CuAAC “click” reaction,¹⁶ with moderate to high yields (40 to 90%) (Scheme 2).

Scheme 2. Synthetic route to the synthesis of new quinone-based 1,2,3-triazoles 1–18.

The second class of quinone-based triazoles (compounds 7 to 12) were based on nor-β-lap. Initially, the azide derivative was synthesized from C-allyl-lawsone in a two-step reaction (Scheme S1†). At first, C-allyl-lawsone was submitted to a halo-cyclization,¹⁷ which provided the iodinated intermediate,¹⁸ which was then submitted to a nucleophilic substitution reaction with sodium azide affording the azide derivative B (Scheme S1†). The reaction of the respective alkynes by click chemistry reaction, in the presence of copper sulfate and sodium ascorbate afforded the desired triazoles 7–12 in 30 to 67% yield (Scheme 2).

The third class of triazole analogs is based on β-lap as the starting material. Initially, β-lap was subjected to a debromination reaction employing N-bromosuccinimide to afford the bromo derivative in 35% yield. In the next step, the bromo intermediate was treated with sodium azide to install an azide group in a regioselective manner to afford C in 35% yield (Scheme S1†).¹⁹ This intermediate was reacted with the alkynes in the presence of copper sulphate and sodium ascorbate to generate the triazoles 13–18, in moderate yields as outlined in Scheme 2.

The first series of compounds have combined the lapachone moiety with another quinone redox centre. As both quinones are likely to exert a similar redox activity and hence biological activity, the next step has involved adding another, distinctively different redox center in the form of a selenium atom. In this part of our study, we have varied the side chain on the quinone moiety to produce building blocks for click reactions with the azide group in different distance and position to the lapachone centre, as shown in Scheme 3. The first series in this part of the study is based on lapachol. This series of compounds commenced by a reaction of lapachol with paraformaldehyde and formic acid to obtain hydroxylated β-lap compound 19 in good yield (Scheme 3). The same formylation-cyclization methodology for compound nor-lapachol provided hydroxylated nor-β-lap 29 in 73% yield (Scheme 3).²⁰ Both hydroxylated lapachones were subjected to the reaction with MsCl at low temperature, followed by nucleophilic substitution with NaN₃ in DMF, resulting in novel quinone-azide compounds 20 and 30, which are similar in structure and subsequently employed as starting blocks to build two distinct series of multifunctional agents via click reaction,²¹ combining the biologically active triazole of the click reaction with a quinone and selenium redox centre (Scheme 3). The two selenated lapachone-triazole moieties were constructed by joining quinone-azides 20 and 30 with a series of organoselenium-based alkyne building block²²via a Cu(i)-catalyzed Hüisgen cycloaddition, affording triazoles 21–28 and 31–38 in good to excellent yields. The series of selenium-containing lapachones based on 20 and 30 feature a short methylene linker between the lapachone and triazole. To inch closer, we have prepared a series of compounds from the azide derivative A and D as outlined in Scheme 4. These more compact nor-β-lapachone-based 1,2,3-triazoles containing selenium and nor-α-lapachone derivatives were prepared following the previously discussed click method.⁸ Compounds 39–47 and 48–51 were obtained in moderate to good yields (Scheme 4).

Scheme 3. Synthetic route to the synthesis of novel selenated quinones 21–28 and 31–38.

Scheme 4. Synthetic route to the synthesis of novel selenated quinones 39–47 and 48–51.

As some of the compounds in the series of selenium-containing lapachone derivatives feature rather complex structures, single-crystal X-ray diffraction has been employed to confirm these structures experimentally in selected and representative compounds 20, 21 and 35. Single crystal X-ray diffraction analysis demonstrated that the three compounds 20, 21 and 35 crystallize in the triclinic crystal system with space group P1̄ and with two molecules in the asymmetric unit. The molecular structures of the compounds are shown in Fig. 1. Crystal data collection and structure refinement for the compounds 20, 21 and 35 are shown in the Table S1 (ESI†). The bond distances of Se–O are 1.910(4) and 1.957(4) Å in 35 and 1.920(5) and 1.937(5) Å in 21. The bond angle N(3)–N(2)–N(1) of 173.4(4)° was observed in the crystal structure of 20, with a characteristic double bond to N(2)–N(1) 1.190(4) Å and N(3)–N(2) of 1.119(4) Å. The X-ray diffraction crystallographic analyses also revealed unusual intermolecular weak hydrogen bonds in 20, whose bond lengths of C–H⋯O and C–H⋯N are approximately 2.61–2.65 Å.

Fig. 1. ORTEP-3 of the compounds 20, 21 and 35, showing the atom-numbering and displacement ellipsoids at 50% probability level.

2.2. Biological activity

After successful synthesis of the various series of multifunctional lapachones, their respective activities have been assessed in HCT-116 (human colon carcinoma cells), PC-3 (human prostate cells), HL-60 (human promyelocytic leukemia cells) and SNB-19 (human glioblastoma cells) employing the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay, an absorbance based method - the measurement of the formazan salt, transformed from tetrazolium, directly correlates to the functioning metabolism of viable cells – and their activities have been compared to their activity in the reference murine fibroblast immortalized L929 cell line in order to establish an overview of activities between the different classes of compounds and also a possible selectivity for certain cell types. As described previously,²³ compounds were classified according to their activity as highly active (IC₅₀ < 2 μM), moderately active (2 μM < IC₅₀ < 10 μM), and inactive (IC₅₀ > 10 μM). The cytotoxicity of compounds is described in Table 1. The selectivity index for selected compounds is outlined in Table 2.

Cytotoxic activity was expressed as IC₅₀ μM (95% CI) against cancer and normal cell lines after 72 h exposure, obtained by nonlinear regression for all cell lines from three independent experiments.

Compounds	HCT-116	PC-3	HL-60	SNB-19	L-929
1	0.05 (0.02–0.13)	1.21 (1.07–1.36)	0.30 (0.25–0.37)	0.95 (0.79–1.13)	2.54 (2.29–2.83)
2	0.82 (0.75–0.90)	1.35 (1.28–1.42)	0.44 (0.38–0.51)	1.19 (1.01–1.38)	2.38 (2.16–2.63)
3	0.72 (0.66–0.78)	1.26 (1.15–1.38)	0.25 (0.20–0.31)	0.75 (0.67–0.84)	2.40 (2.11–2.73)
4	0.51 (0.44–0.59)	0.93 (0.85–1.02)	0.26 (0.22–0.30)	1.19 (1.07–1.33)	1.42 (1.27–1.58)
5	0.53 (0.45–0.63)	0.89 (0.84–0.94)	0.21 (0.17–0.26)	1.15 (1.03–1.29)	1.27 (1.16–1.39)
6	0.54 (0.40–0.74)	1.01 (0.91–1.11)	0.30 (0.24–0.38)	1.74 (1.60–1.89)	1.81 (1.68–1.95)
7	4.15 (3.63–4.74)	11.36 (10.42–12.38)	3.92 (3.07–5.01)	6.21 (5.19–7.43)	8.54 (6.44–11.34)
8	2.14 (1.93–2.37)	4.46 (3.96–5.01)	5.90 (4.74–7.34)	>17.98	14.39 (12.11–17.11)
9	2.59 (2.29–2.93)	5.36 (4.90–5.87)	0.75 (0.59–0.95)	2.79 (2.45–3.17)	4.16 (3.62–4.78)
10	2.08 (1.90–2.28)	2.68 (2.45–2.92)	0.74 (0.49–1.11)	2.98 (2.70–3.28)	2.93 (2.58–3.33)
11	1.12 (1.01–1.24)	2.13 (1.97–2.31)	0.51 (0.34–0.76)	1.82 (1.48–2.23)	2.25 (1.88–2.70)
12	1.34 (1.15–1.56)	2.60 (2.40–2.82)	1.15 (1.03–1.27)	4.33 (3.43–5.47)	4.56 (3.56–5.84)
13	0.81 (0.74–0.88)	1.80 (1.62–1.99)	0.50 (0.43–0.60)	1.93 (1.63–2.28)	1.66 (1.51–1.83)
14	0.68 (0.61–0.75)	1.71 (1.51–1.95)	0.40 (0.35–0.46)	1.52 (1.27–1.82)	2.01 (1.61–2.51)
15	0.78 (0.70–0.87)	2.40 (2.14–2.69)	0.55 (0.45–0.66)	1.96 (1.62–2.37)	2.42 (1.98–2.96)
16	0.49 (0.44–0.54)	1.38 (1.24–1.53)	0.15 (0.12–0.19)	1.26 (1.06–1.51)	1.19 (1.05–1.35)
17	0.68 (0.64–0.73)	1.89 (1.69–2.12)	0.54 (0.47–0.61)	1.78 (1.60–1.98)	1.87 (1.57–2.23)
18	0.86 (0.76–0.97)	1.75 (1.55–1.98)	0.79 (0.68–0.93)	3.17 (2.67–3.76)	1.16 (1.01–1.33)
20	3.85 (3.70–4.00)	6.65 (5.68–7.78)	0.98 (0.55–1.71)	3.99 (3.88–4.10)	2.67 (2.51–2.82)
21	8.60 (7.92–9.34)	9.64 (8.02–11.59)	0.42 (0.31–0.55)	8.96 (8.23–9.75)	3.14 (2.91–3.37)
22	4.78 (4.50–5.08)	4.15 (3.31–5.20)	0.28 (0.13–0.60)	4.91 (4.69–5.13)	2.81 (2.55–3.09)
23	5.25 (4.96–5.54)	5.50 (4.59–6.57)	0.31 (0.15–0.61)	5.25 (4.41–6.22)	2.81 (2.54–3.11)
24	5.59 (5.12–6.10)	6.34 (5.06–7.93)	0.33 (0.14–0.75)	5.66 (5.16–6.20)	2.82 (2.51–3.17)
25	4.18 (3.53–4.95)	5.89 (5.38–6.43)	0.37 (0.19–0.69)	4.92 (4.33–5.58)	2.93 (2.59–3.29)
26	6.19 (5.58–6.86)	15.29 (14.84–15.75)	0.31 (0.21–0.43)	7.40 (7.00–7.80)	4.69 (4.45–4.94)
27	3.32 (2.81–3.91)	5.28 (4.91–5.66)	0.40 (0.37–0.43)	6.40 (5.57–7.35)	4.90 (4.44–5.41)
28	9.39 (8.25–10.67)	9.16 (8.10–10.34)	0.81 (0.65–1.00)	7.38 (6.99–7.77)	5.88 (4.66–7.42)
30	2.73 (2.33–3.19)	4.71 (4.22–5.25)	0.80 (0.74–0.85)	4.86 (4.32–5.46)	7.13 (6.65–7.63)
31	3.34 (2.10–5.30)	6.71 (5.769–7.79)	0.42 (0.37–0.46)	4.40 (3.67–5.26)	5.67 (4.45–7.21)
32	2.58 (2.09–3.17)	3.50 (3.28–3.71)	0.06 (0.02–0.16)	4.17 (3.44–5.04)	2.93 (2.08–4.11)
33	2.10 (1.74–2.52)	4.08 (2.94–5.65)	0.39 (0.03–4.99)	4.01 (0.54–29.63)	3.63 (3.38–3.88)
34	3.70 (3.38–4.05)	2.66 (2.14–3.29)	0.38 (0.27–0.51)	6.08 (5.91–6.25)	2.94 (2.32–3.70)
35	3.82 (3.43–4.26)	3.08 (2.61–3.64)	0.31 (0.30–0.31)	5.49 (4.45–6.77)	2.75 (2.49–3.04)
36	5.36 (4.87–5.88)	6.03 (4.73–7.69)	0.41 (0.39–0.43)	4.16 (3.74–4.63)	6.86 (6.40–7.34)
37	1.95 (1.54–2.47)	5.65 (5.17–6.16)	0.17 (0.16–0.18)	2.71 (2.60–2.81)	3.70 (3.25–4.21)
38	6.23 (5.48–7.08)	6.62 (5.71–7.66)	0.78 (0.65–0.93)	9.27 (8.56–10.03)	5.70 (4.99–6.50)
39	1.12 (0.93–1.34)	4.64 (3.68–5.86)	1.06 (0.95–1.19)	3.23 (1.81–9.23)	1.86 (1.65–2.10)
40	1.58 (1.29–2.08)	1.92 (1.25–3.10)	0.62 (0.57–0.68)	2.80 (2.35–3.34)	2.84 (2.43–3.32)
41	1.18 (1.05–1.34)	3.07 (2.46–3.87)	1.05 (1.05–1.23)	2.95 (2.45–3.57)	1.10 (0.74–1.65)
42	0.84 (0.74–0.96)	2.61 (2.09–3.27)	1.00 (0.90–1.11)	2.19 (1.96–2.45)	1.10 (0.81–1.50)
43	1.08 (0.87–1.34)	2.11 (1.82–2.43)	0.63 (0.45–0.93)	1.54 (1.10–2.19)	1.74 (1.38–2.22)
44	0.79 (0.69–0.91)	1.64 (1.40–1.93)	11.04 (8.37–15.00)	1.56 (1.31–1.85)	1.52 (1.13–2.04)
45	1.02 (0.88–1.17)	3.48 (2.82–4.29)	1.51 (1.38–1.66)	3.18 (2.82–3.59)	1.20 (0.97–1.51)
46	1.70 (1.47–1.98)	2.33 (1.67–3.41)	0.83 (0.74–0.94)	5.73 (3.89–9.65)	1.71 (1.19–2.54)
47	2.25 (1.96–2.60)	6.44 (5.40–7.68)	1.68 (1.53–1.86)	3.87 (3.01–4.98)	2.80 (2.35–3.33)
48	7.62 (6.49–8.95)	>10	4.09 (3.52–4.76)	>10	—
49	7.55 (6.76–8.44)	9.35 (8.50–10.30)	3.17 (2.78–3.60)	>10	>10
50	2.03 (1.88–2.20)	2.10 (1.71–2.59)	3.87 (3.41–4.40)	5.16 (4.61–5.79)	13.15 (11.20–15.43)
51	>10	>10	4.19 (3.42–5.14)	>10	>10
Doxorubicin	0.21 (0.16–0.29)	0.76 (0.59–0.93)	0.02 (0.01–0.02)	1.20 (1.03–1.39)	1.72 (1.58–1.87)

Selectivity index for selected compounds [selectivity index, represented by the ratio of cytotoxicities between normal cells and different lines of cancer cells].

Compounds	HCT-116	PC-3	HL-60	SNB-19
1	51	2.1	8.4	2.7
2	2.9	1.8	5.4	2
3	3.3	1.9	9.6	3.2
4	2.8	1.5	5.5	1.2
5	2.4	1.4	6.1	1.1
6	3.4	1.8	6.1	1.1
7	2.1	0.8	2.2	1.4
8	6.7	3.2	2.4	0.8
9	1.9	0.8	5.5	1.5
10	1.4	1.1	3.9	0.9
11	2.0	1.1	4.4	1.2
12	3.4	1.7	3.9	1.1
13	2.1	0.9	3.3	0.8
14	2.9	1.2	5.0	1.3
15	3.1	1.0	4.4	1.2
16	2.4	0.8	7.9	0.9
17	2.8	0.9	3.5	1.1
18	1.3	0.7	1.5	0.4
20	0.7	0.4	2.7	0.7
21	0.4	0.3	7.5	0.4
22	0.6	0.7	10.0	0.6
23	0.5	0.5	9.1	0.5
24	0.5	0.4	8.5	0.5
25	0.7	0.5	7.9	0.6
26	0.8	0.3	15.1	0.6
27	1.5	0.9	12.2	0.8
28	0.7	0.6	7.2	0.8
30	2.6	1.5	8.9	1.5
31	1.7	0.8	13.5	1.3
32	1.1	0.8	48.8	0.7
33	1.7	0.9	9.3	0.9
34	0.8	1.1	7.7	0.5
35	0.7	0.9	8.8	0.5
36	1.3	1.1	16.7	1.6
37	1.9	0.7	21.7	1.4
38	0.9	0.8	7.3	0.6
39	1.6	0.4	1.7	0.6
40	1.8	1.5	4.5	1.0
41	0.9	0.4	1.0	0.4
42	1.3	0.4	1.1	0.5
43	1.6	0.8	2.8	1.1
44	1.9	0.9	0.1	0.9
45	1.2	0.3	0.8	0.4
47	1.0	0.7	2.1	0.3
Doxorubicin	8.2	2.3	86	1.4

Ortho-quinone coupled para-quinones (1–6)

Compounds in this series presented IC₅₀ values ranging from 0.05 μM (compound 1, in HCT-116 cells) to 1.74 μM (compound 6, in SNB-19 cells). Although these compounds can be considered as highly active, their cytotoxicity against the reference L-929 cell lines has been considerably lower, pointing towards a good selectivity against cultured cancer cells. Compound 1, for instance, exhibited high activity against HCT-116 cells in the sub-micromolar range, and a 50-fold higher IC₅₀ of 2.54 μM against L-929 cells, hence combining high activity with considerable selectivity. In fact, of the six compounds tested, 1 showed excellent activity against two different cell lineages (HCT-116, IC₅₀ = 0.05 μM and HL-60, IC₅₀ = 0.30 μM) and a relatively high IC₅₀ value against normal cells, being considered an important prototype for further studies. All compounds in this group were especially active against human promyelocytic leukemia cells (HL-60) with IC₅₀ values between 0.21 μM and 0.30 μM.

Ortho-quinone coupled para-quinones (7–12)

This series of compounds differs from the one mentioned before by exchanging the nor-β-lap core for a C-allyl derivative core. This small structural modification led to a general increase in the IC₅₀ values in each one of the cell lines, with some minor exceptions. Compounds 7–10 were moderately active in general, with compound 7 being inactive against PC-3 cell lines and compound 8 inactive against SNB-19 and normal cell lines. Compounds 9–12 were highly active against HL-60 cells, presenting moderate values of IC₅₀ against L-929 cells and hence some selectivity. Compounds 11 and 12 also presented low IC₅₀ values against HCT-116 cells (11, IC₅₀ = 1.12 μM and 12, IC₅₀ = 1.34).

Ortho-quinone coupled para-quinones (13–18)

Members of this series of compounds, in which a β-lap model was employed as the lapachone core, led to a general increase in the antitumor activity when compared to C-allyl-lawsone-based compounds, although most compounds showed higher IC₅₀ values when compared to nor-β-lap derivatives. Nonetheless, most compounds in this series may also be considered highly active, with IC₅₀ values ranging from 0.15 μM (compound 16, HL-60) to 3.17 μM (compound 18, SNB-19). Interestingly, the new β-lap derivatives were especially active against HL-60 cell lines and can be considered promising prototypes in cancer treatment for this specific type of cell. Besides, compounds 14 and 15 also attracted attention because of their higher IC₅₀ values for normal cell lines (14, IC₅₀ = 2.01 μM and 15, IC₅₀ = 2.42) and certain selectivity against cancer cells.

Lapachone models containing selenium (21–28, 31–38)

Most of the compounds belonging to this series were less active, with higher IC₅₀ values for HCT-116, PC-3 and SNB-19 cell lines, from 2.10 μM (compound 33, HCT-116) to 15.29 μM (compound 26, PC-3). Nonetheless, these compounds were not wholly uninteresting either, as they exhibited higher IC₅₀ values for L929 cells, when compared to the previous results, de facto an improvement in selectivity. The most notable results are observed for the HL-60 lineage, where compounds presented low IC₅₀ values between 0.06 μM and 0.81 μM, and therefore are highly active for these cells. Compound 32, in particular, yielded an IC₅₀ (HL-60) of 0.31 μM, compared to an IC₅₀ of 4.49 against the reference L929 cell line. Therefore, several members of these series may be considered as promising prototypes in the treatment of human promyelocytic leukemia.

Furthermore, compounds 39–47 and 48–51 did not show any pronounced cytotoxic effects, and ortho-quinone derivatives generally were more active compared to para-quinones. The ortho-quinones may be more amenable to generate or employ reactive oxygen species (ROS) in their task to damage and subsequently destroy the cancer cells, an aspect, which needs to be explored in more detail. It should also be mentioned that many quinones are able to act catalytically inside cells and this kind of redox catalysis associated with many of these natural products and their derivatives may also explain the unique combination of high activity and high selectivity. In fact, the intracellular redox balance in cells tends to differ, and redox modulating catalysts in some ways sense these differences in ROS levels and exert their actions accordingly. Such catalytic sensor/effector agents are very promising and, in this concept, the presence of selenium is probably also beneficial for cytotoxic activity, and in any case, worth following up.

Despite the complexity to establish structure–activity relationship (SAR) aspects due to the wide structural variety of the compounds and different tumor cell lines evaluated here, we draw a parallel of the potential of the new compounds in comparison with nor-β-lapachone-based 1,2,3-triazole derivatives, previously described in the literature.²⁴ For HL-60 and PC-3 tumor cell lines, the triazole derivative shows IC₅₀ values are equal to 2.10 and 2.58, respectively. The insertion of a second para-quinonoid redox center allowed more potent and selective compounds to be obtained. As an example, compound 1, with IC₅₀ values = 0.30 for HL-60 and 1.21 for PC-3, is seven-fold more active than the precursor triazole when considering the HL-60 tumor cell lines and two times more active against PC-3. We also evaluated the C-ring modification, insertion of a spacer and selenium as second redox center, to prepare new triazole derivatives containing two redox centers. This approach allowed us to maximize the activity of the compounds against HL-60 cell lines, but we observed a decrease in potency against PC-3, for example. The toxicity on L-929 cell lines remained similar in most cases. An important aspect of the strategy applied in this study is the observation that the compounds with two redox centers were more active against tumor cell lines but exhibited similar or lower toxicity against the non-tumor cell line L-929. For instance, the selectivity index for compound 1 was higher than the values observed for the triazole derivative previously reported²⁴ (Scheme 5). In general, we observed that the insertion of a second quinoidal or selenium redox center, enhanced the antitumor activity of the compounds against some tumor cell lines and decreased the cytotoxicity of the compounds. Naturally, there is a modification in the redox balance leading to these observations. Although, the nature of the mechanism of action involved in the antitumor activity of these compounds is not yet fully elucidated, we selected compound 42 and performed experiments of cell membrane integrity and viability, phosphatidylserine externalization, mitochondrial transmembrane potential and measurement of intracellular reactive oxygen species levels aiming to understand some aspects of the mechanism involving molecules containing two redox centers. Initially, the cytotoxic activity of compound 42 was evaluated in HCT-116 cells (0.85 μM, 1.7 μM and 3.5 μM). These concentrations were based on the IC₅₀ values obtained by the MTT assay after 24 h of treatment. Nor-β-Lapachone (NBL, 4 μM), a well-known naphthoquinone with antitumor activity, was used as a positive control.²⁵ As observed in Fig. 2, the quinone 42 led to a significant reduction in the number of viable cells when compared to both negative and positive (NBL) controls, corroborating the findings observed in the MTT assay. Our experiments also indicated that after 24 h of treatment, 42 induced significant apoptosis in the concentrations of 1.7 and 3.5 μM (Fig. 3).

Scheme 5. Nor-β-lapachone-based 1,2,3-triazole derivative and examples of compounds containing two redox centers: a comparative overview.

Fig. 2. Effect of compound 42 on cell membrane integrity in HCT-116 cells determined by flow cytometry, after 24 hours incubation. Nor-β-lapachone (NBL, 4 μM) was used as positive control. Data are expressed as mean ± SEM from three independent experiments. C-, negative control, was incubated with the vehicle used to dilute the tested compound. *, p < 0.05 compared to negative control by ANOVA followed by Dunnett's test. #, p < 0.05 compared to positive control by ANOVA followed by Dunnett's test.

Fig. 3. Effect of compound 42 on the PS externalization in HCT-116 cells determined by flow cytometry using Annexin V/7-AAD staining, after 24 hours incubation. Nor-β-lapachone (NBL, 4 μM) was used as positive control. 7-AAD-negative and Annexin V-negative cells were considered viable. Annexin V-positive cells and 7-AAD negative cells were considered early apoptotic cells. Annexin V-positive cells and 7-AAD positive cells were considered late apoptotic/necrotic cells. Data are expressed as mean ± SEM from three independent experiments. C-, negative control, was incubated with the vehicle used to dilute the tested compound. *, p < 0.05 compared to negative control by ANOVA followed by Dunnett's test. #, p < 0.05 compared to positive control by ANOVA followed by Dunnett's test.

Anticancer studies usually count on selective induced apoptosis as a tool for the fight against cancer cell lines. As well discussed by Galluzzi and co-workers, from a biochemical point of view, apoptosis is defined as a caspase-dependent variant of regulated cell-death (RCD).²⁷ Apoptosis can be triggered by intracellular (intrinsic) or extracellular (extrinsic) stimuli.^26,27 The intrinsic apoptosis is initiated by perturbations of the extracellular or intracellular microenvironment and this process is commonly known as mitochondrial outer membrane permeabilization (MOMP).^28,29 In the other hand, the extrinsic apoptosis is a specific variant of RCD initiated by perturbations of the extracellular microenvironment, and is resulted by the activation of CASP8/CASP3, which, in some cell types, also involves MOMP, due to the CASP8-dependent activation of BH3-interacting domain death agonist (BID).³⁰

Aware of the importance of apoptotic events related to antitumor studies, we performed mitochondrial transmembrane potential experiments with the quinoidal derivative 42. According to the results observed in Fig. 4, after 24 h of treatment, 42 induced a concentration-dependent alteration in the mitochondrial transmembrane potential, indicating that the molecule might be inducing an apoptotic cell death through the intrinsic pathway.

Fig. 4. Effect of compound 42 on the mitochondrial transmembrane potential in HCT-116 cells determined by flow cytometry using rhodamine 123, after 24 hours incubation. Nor-β-lapachone (NBL, 4 μM) was used as positive control. Data are expressed as mean ± SEM from three independent experiments. C-, negative control, was incubated with the vehicle used to dilute the tested compound. *, p < 0.05 compared to negative control by ANOVA followed by Bonferroni's test. #, p < 0.05 compared to positive control by ANOVA followed by Bonferroni's test.

In general, the antitumor activity of quinonoid compounds is intrinsically related to the generation of reactive oxygen species (ROS).³¹ In this sense, we also evaluated the ability of compound 42 to generate ROS. The naphthoquinone 42 induced a significant increasing in intracellular ROS levels (Fig. 5), in a time- and concentration-dependent manner, reaching a higher ROS + cell count at 5 hours. As expected, the positive control nor-β-lapachone also induced significant (p < 0.05) ROS generation.

Fig. 5. Effect of naphthoquinone 42 on ROS production in HCT-116 cells determined by flow cytometry using CM-H₂DCFDA, after 1-, 3- and 5 hours incubation. Nor-β-lapachone (NBL, 4 μM) was used as positive control. A total of 10 000 events were analyzed per sample. Data are expressed as mean ± SEM from three independent experiments. C-, negative control, was incubated with the vehicle used to dilute the tested compound. *, p < 0.05 compared to negative control by ANOVA followed by Dunnett's test.

Further studies to understand more details on the mechanism of action of the compounds outlined here are in progress in our laboratories and will be published in due course.

3. Conclusions

In this study, we have described the synthesis and cytotoxic evaluation of 47 new quinones, each containing two redox centers and a triazole centre as a result of our click approach to assemble the individual redox building blocks. Whilst some of these compounds have combined ortho- and para-quinone redox sites, others contain selenium. With these compounds in hand, we have been able to confirm that our strategy of joining two redox centers is effective in delivering highly active compounds against several tumor cell lines, frequently with activities in the mid nanomolar range and considerable selectivity when compared to the L929 reference cell line. Indeed, we have obtained more than 22 compounds with IC₅₀ values below 2 μM, demonstrating their cytotoxic potential as new prototypes for subsequent studies to identify new antitumor drugs. Together with the accessible synthetic approaches described herein, our studies provide a wide range of compounds with powerful cytotoxic activity and represent an avenue of possibilities when searching for new drugs to be inserted in the therapeutic arsenal against cancer and similar diseases.

4. Experimental section

For all experimental details, including general procedures, synthesis of substrates, the general procedure for Cu(i) catalyzed “click” cycloadditions, crystallographic data, biological assays, NMR spectra, HRMS and elemental analysis, see ESI.†

Conflicts of interest

The authors declare no conflict of interest.