It Takes Two to Tango, Part II: Synthesis of A-Ring Functionalised Quinones Containing Two Redox-Active Centres with Antitumour Activities

In 2021, our research group published the prominent anticancer activity achieved through the successful combination of two redox centres (ortho-quinone/para-quinone or quinone/selenium-containing triazole) through a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The combination of two naphthoquinoidal substrates towards a synergetic product was indicated, but not fully explored. Herein, we report the synthesis of 15 new quinone-based derivatives prepared from click chemistry reactions and their subsequent evaluation against nine cancer cell lines and the murine fibroblast line L929. Our strategy was based on the modification of the A-ring of para-naphthoquinones and subsequent conjugation with different ortho-quinoidal moieties. As anticipated, our study identified several compounds with IC50 values below 0.5 µM in tumour cell lines. Some of the compounds described here also exhibited an excellent selectivity index and low cytotoxicity on L929, the control cell line. The antitumour evaluation of the compounds separately and in their conjugated form proved that the activity is strongly enhanced in the derivatives containing two redox centres. Thus, our study confirms the efficiency of using A-ring functionalized para-quinones coupled with ortho-quinones to obtain a diverse range of two redox centre compounds with potential applications against cancer cell lines. Here as well, it literally takes two for an efficient tango!


Introduction
Cancer has become a global issue and represents nearly one in six worldwide annual deaths, according to the World Health Organization [1]. Different therapies are available nowadays for many types of cancer; however, the drugs currently applied commonly lead to painful side effects, in general due to the absence of a high degree of selectivity between a cancer cell on the one side and a healthy cell on the other [2,3]. In this context, the development and subsequential evaluation of new potential anticancer compounds have been explored extensively throughout the years [4][5][6][7]. From this perspective, important bioactive molecules with prominent antitumour activity have been described [8][9][10]. Amongst these molecules, quinones in general play an important role [11][12][13][14], since they actively participate in the molecular stress generated by reactive oxygen species (ROS) [15,16], culminating

Synthesis of the Azide Units
The construction of each family of products was based on the azide-containing quinone side of the molecule. For this matter, three different azides were achieved, all according to their respective sequential synthetic pathway (Scheme 2). Azide 4, the first one to be obtained in this investigation, was originated from nor-lapachol (3) through a cycloaddition with bromide and a nucleophilic substitution with sodium azide [43]. This process led to the desired quinone (4) in the quantitative yield. From this model, another azide (compound 7) was designed, also according to the previous knowledge of the group [44], starting from a C3-allyl lawsone derivative (5). The first step led to the formation of an iodinated 5-membered intermediate (6), in the presence of iodine and pyridine. From this isolated intermediate, a nucleophilic attack with sodium azide results in the desired azide 7, in a 91% yield. A six-membered azide (compound 10) may be also achieved when lapachol (8) itself is used as a substrate. A sequence of four distinguished steps, passing through an isolatable hydroxylated intermediate (9), leads to azide 10 [32], in a 93% yield. In all cases, lapachone products were obtained in good-to-excellent yields. Scheme 2. Synthetic pathway adopted to obtain the quinoidal azides.

Synthesis of the Aminoalkyne Units
To react properly with the above-depicted azides, five aminoalkynes (compounds 18a-e) were designed starting from their respective A-ring modified naphthoquinones (12)(13)(14)(15)17, Scheme 3A). These primordial modifications were also based on previous knowledge of the group, including an aromatic substitution from amine to iodine towards compound 12, Lewis acid-catalysed nucleophilic substitution aiming compounds 14 and 15 from juglone (13), and a reduction/oxidation from quinizarin (16) leading to compound 17.
Once the A-ring modified quinones were achieved, an amination was performed in the presence of propargylamine (Scheme 3B), based on a known procedure [45]. This process led to five A-ring modified alkyne quinones, from which interesting bioactive Scheme 2. Synthetic pathway adopted to obtain the quinoidal azides.

Synthesis of the Aminoalkyne Units
To react properly with the above-depicted azides, five aminoalkynes (compounds 18a-e) were designed starting from their respective A-ring modified naphthoquinones (12)(13)(14)(15)17, Scheme 3A). These primordial modifications were also based on previous knowledge of the group, including an aromatic substitution from amine to iodine towards compound 12, Lewis acid-catalysed nucleophilic substitution aiming compounds 14 and 15 from juglone (13), and a reduction/oxidation from quinizarin (16) leading to compound 17. results may be observed once the desired products are accomplished. Since most of the Aring modified quinones possess a substituent at the C-5 position (with the exception of quinone 17), the amination procedure can happen on two different sites of the molecule, namely the C-2 or C-3 position, and this difference may generate a mixture of regioisomers. However, although this selectivity was expected to happen, the amination steps led to specific isomers in each case, from which the corresponding regioisomer was observed only as traces, and therefore was not isolated. To further understand this selectivity, it is important to understand the mechanism by which this reaction happens. The entrance of the aminoalkyne takes place through a nucleophilic attack of the nitrogen atom at one of the two carbons located each at the positions C-2 or C-3 of the B-ring. The selectivity of this attack depends on the relative Once the A-ring modified quinones were achieved, an amination was performed in the presence of propargylamine (Scheme 3B), based on a known procedure [45]. This process led to five A-ring modified alkyne quinones, from which interesting bioactive results may be observed once the desired products are accomplished. Since most of the A-ring modified quinones possess a substituent at the C-5 position (with the exception of quinone 17), the amination procedure can happen on two different sites of the molecule, namely the C-2 or C-3 position, and this difference may generate a mixture of regioisomers. However, although this selectivity was expected to happen, the amination steps led to specific isomers in each case, from which the corresponding regioisomer was observed only as traces, and therefore was not isolated.
To further understand this selectivity, it is important to understand the mechanism by which this reaction happens. The entrance of the aminoalkyne takes place through a nucleophilic attack of the nitrogen atom at one of the two carbons located each at the positions C-2 or C-3 of the B-ring. The selectivity of this attack depends on the relative intensity of the positive charge on each one of these carbon atoms and the corresponding negative charge on the opposite oxygen atom in their resonance contributors. In the case of quinone 12, it is possible to understand that the polarizability mediated by the large electronic cloud around the iodine made it more reasonable to stabilise a negative charge on vicinal-negative oxygen, consequently increasing the positive charge over the carbon C-2, which becomes more susceptible to a nucleophilic attack. Similar behaviour is expected to happen when the juglone (13) itself is used. A negative charge on an oxygen atom near the hydroxyl group can be stabilised easily through a hydrogen bond.
In the case of compounds 14 and 15, the resonance effect is no longer the main attributor to the observed phenomenon, but the indirect inductive effect instead, over the carbon atoms C-2 and C-3. Through this aspect, the C-3 carbons receive a slightly higher positive partial charge, leading to the C-3 aminated products 18c and 18d.

Scopes Achieved
A combination of the previously mentioned azides 4, 7 and 10 and the aminoalkynes 18a-e, through a copper-catalysed 1,3-dipolar cycloaddition, led to fifteen new triazoles, divided into three families according to the azide applied. This methodology was previously developed [39], and it requires pentahydrated copper sulfate (2 mol%) as a catalyst, and sodium L-ascorbate (5 mol%) as a reducing agent. A mixture of dichloromethane/water (1:1) was found to be a plausible solvent, used here to maximize the solubility of not only the quinoidal substrates, but also the ionic reactants. Therefore, it is important to maintain vigorous stirring during the reaction to provide the surface interaction required between the two phases. The reaction is performed at room temperature, for 24 h. In all cases, it was possible to successfully obtain bi-quinoidal structures presenting two redox centres in moderate-to-good yields.
It is not possible to directly link the structural substituents with their respective reactivity, since the reaction takes place on a site that is not chemically related to the influence of these substituents. It is reasonable to assume that, since it is an interface reaction, the solubility of the compounds involved plays a more important role here.
The first family (19a-e) was depicted using the azide 4, originated from nor-lapachol (3), and the aminoalkyne-quinones 18a-e (Scheme 4). In this first family, the best result was obtained when the aminoalkyne 18b was used, leading to product 19b, in a 71% yield. This result may be a consequence of the plausible solubility of the reactants (including the substrate 18b) in both solvents. The opposite behaviour was also observed, when the aminoalkyne 18e was used. In this particular case, this aminoalkyne does not present a perfect solubility in water or dichloromethane, therefore the product 20e was achieved in a lower yield (42%) when compared to the rest of the family, but still plausible for the obtention of the quinoidal product presenting two redox centres. Better results were observed for the construction of the second triazole family (Scheme 5). For this case, the azide 7 was combined with the aminoalkynes 18a-e, from which the products 20a-e were achieved in good yields (62-86%). The third scope involved using the azide 10 combined with the aminoalkynes 18a-e (Scheme 6). In this study, the best result was obtained when the aminoalkyne 18a was used, leading to compound 21a in a 64% yield. The anthraquinone-derived aminoalkyne 18e, which previously led to the final product in lower yields, was not different in this case, in which the desired product 21e was obtained in a 59% yield. 8
In this study, the IC50 was obtained in micromolar concentrations, using the colourimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)) assay, and doxorubicin was used as the positive control. The bioactivity was classified according to the IC50 value as follows: highly active (IC50 < 2 μM), moderately active (2 μM < IC50 < 10 μM) and inactive (IC50 > 10 μM). In most of the cases, a high-to-moderate activity was observed, especially against the HL60 cell line, for which IC50 values as low as 0.3 μM could be successfully achieved (compound 19d). The activity against the non-tumoural murine fibroblast cell line L929 was also evaluated in order to study the cytotoxicity behaviour of each compound and to understand their respective relative selectivity. The selectivity index was obtained using the ratio of measured cytotoxicity between the L929 cell line and each of the cancer cell lines, and the results are presented in Table 2. From a general point of view, every substrate submitted to this method led to the desired triazole with success, either in good or lower yields. This fact corroborates the large applicability of this method.
In this study, the IC 50 was obtained in micromolar concentrations, using the colourimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)) assay, and doxorubicin was used as the positive control. The bioactivity was classified according to the IC 50 value as follows: highly active (IC 50 < 2 µM), moderately active (2 µM < IC 50 < 10 µM) and inactive (IC 50 > 10 µM). In most of the cases, a high-tomoderate activity was observed, especially against the HL60 cell line, for which IC 50 values as low as 0.3 µM could be successfully achieved (compound 19d). The activity against the non-tumoural murine fibroblast cell line L929 was also evaluated in order to study the cytotoxicity behaviour of each compound and to understand their respective relative selectivity. The selectivity index was obtained using the ratio of measured cytotoxicity between the L929 cell line and each of the cancer cell lines, and the results are presented in Table 2. Table 1. Cytotoxic activity expressed as IC 50 µM (95% CI) against cancer and normal cell lines after 72 h exposure, obtained using nonlinear regression for all cell lines from three independent experiments. * Data previously described [32]. DOXO = doxorubicin. Table 2. Selectivity index calculated using the ratio of cytotoxicity between L929 cell line and each cancer cell lines. * Data previously described [32].   From a general point of view, azides 4 and 7 presented the best activity against all the cell lines studied when compared to azide 10. Most of the results observed for compounds 5 and 7 were around four times better than the results for azide 10. However, this result did not negatively affect the activity of the final triazole obtained from azide 10, since the third family (compounds 21a-e) still presented good results, as can be seen in Section 2.4.5. A very interesting result can be highlighted here, since azide 7, although moderately active against the PC3 cell line, presented a similar activity against the SNB-19 cell line when compared to the positive control, doxorubicin. Regarding the selectivity, compound 7 presented selectivity indexes near 2.0 related to its activity against the HCT-116, SNB-19, K-562 and B16 cell lines, which basically means that this compound hits these cancer cells twice as hard as non-tumoral cells. When compared to the positive control, doxorubicin (which presents a selectivity index of 1.4 against SNB-19 cells), compound 7 presents an even better selectivity (with an index of 2.4). The similarity of the structures of these azides makes it difficult to propose a direct correlation between structure and reactivity. Furthermore, the results presented by compounds 4 and 7 were similar for most of the cancer cell lines. However, since azide 10 presented a lower activity, it can be inferred that the presence of a six-membered ring might be an issue or an inhibiting factor.

Naphthoquinoidal Aminoalkyne Substrates (18a-e)
The quinoidal substrates 18a-e did not present potent anticancer activities. In most of the cases, the IC 50 values obtained were higher than 100 µM. These results, although not satisfactory, are a good example of the synergetic behaviour that quinoidal molecules can present. In most instances, the combination of the unactive naphthoquinoidal aminoalkyne with the previously mentioned azide quinones led to triazole products with a higher activity compared to their respective aminoalkyne precursor. Furthermore, regarding the bioactivity of the aminoalkynes, anticancer activity was observed when compound 18b was tested against the HCT-116 cancer cell line, presenting an IC 50 value of 12.73 µM.

First Family of Triazoles (19a-e)
As a general observation, the first family of triazoles presented the best anticancer activity. Within these results, the best anticancer activities were observed against the HL60 cancer cell line, with IC 50 values between 0.3 and 1.1 µM. These are impressive results when compared to the positive control, doxorubicin, which, under the same conditions, presented an IC 50 value of 0.02 µM. Regarding its selectivity, compound 19c presented good indexes against HCT-16, HL60 and RAJI cell lines (5.8, 8.6 and 9.9 respectively), whereas compound 19b presented a valuable index of 6.0 against the HL60 cell line. Furthermore, compound 19c also presented an IC 50 value of 0.9 µM against the RAJI cell line, more active than the positive control, resulting in the above-mentioned selectivity index of 9.9. Beyond that, combining both activity and selectivity, compound 19d also presented one of the best performances, with an impressive IC 50 of 0.3 µM and a selectivity index of 6.7 against the HL60 cell line. With these results in hand and further developments, compounds 19c and 19d might indeed become plausible alternatives for the treatment of human Burkitt's lymphoma and human pro-myelocytic leukaemia, respectively.

Second Family of Triazoles (20a-e)
The second family of triazoles presented a lower activity when compared to the other two families. Although the results were less impressive in this particular case, compound 20e can still be highlighted as a prominent molecule, regarding its IC 50 of 1.6 µM and its selectivity index of 10.1 against the HL60 cell line, being the most active compound in the second family of triazoles. Beyond that, compound 20a can also be cited here, since it presented moderate anti-cancer properties against all cancer cell lines studied here, and compound 20b, which presented an IC 50 value of 2.49 and an impressive selectivity index of 11.1 against the RAJI cell line.

Third Family of Triazoles (21a-e)
The third family of triazoles provided another good example of the applicability of quinones against the HL60 cell line, since some of its members presented IC 50 values as low as 0.5 µM. This result was achieved by compound 21e against the HL60 cell line, leading also to a high selectivity index of 6.6. The selectivity behaviour of this family was similar to the other ones, and impressive results were observed, for instance, for compound 21d, with an IC 50 of 0.89 µM against the HL60 cell line and a selectivity index of 5.9.

General Remarks
The solvents were dried using molecular sieves in inert atmosphere storage. Lawsone, nor-lapachol (3), lapachol (8), juglone (13), and quinizarin (16) were used as purchased without further purification. 5-Amino-1,4-naphthoquinone (11) was synthesized according to a procedure already discussed in the literature [46]. The reaction concentration is expressed in molar (M); this concentration was calculated by the ratio of the amount of the main reactant (the limiting agent) in mmol and the volume of the solvent applied in mL. The presented yields refer to isolated compounds, estimated to be >95% pure as determined by 1 H-NMR. TLC: Merck, TLC Silica gel 60 F 254 , detection at 254 nm. Infrared spectra were recorded on a Bruker ATR FT-IR Alpha device and IR Prestige-21 Shimadzu using KBr plates. Mass-spectra: EI-MS: Jeol AccuTOF at 70 eV; ESI-MS: Bruker maXis and MicrOTOF. High-resolution mass spectrometry (HRMS): Bruker maXis, Bruker MicrOTOF and Jeol AccuTOF. Melting points: Büchi 540 capillary melting point apparatus; values are uncorrected. The NMR spectra were recorded on Avance III HD 400, Avance III 400, and Avance NEO 600 instruments. If not otherwise specified, chemical shifts (δ) are provided in ppm. 13 C-NMR shifts are classified as: C q (non-hydrogenated carbon), CH, CH 2 , and CH 3 , indicating the nature of the carbon assigned, according to what was observed by DEPT or ATP analysis. All of the structure names were given under IUPAC rules by the CS ChemDraw Ultra program. Single crystals were recrystallized from a mixture of acetonitrile and petroleum ether using a system of vapor diffusion. The crystals were analyzed on a XtaLAB Synergy Rigaku four-circle diffractometer. Using Olex2 [47], the structures were solved with the XT [48] structure solution program using Intrinsic Phasing and refined with the XL [49] refinement package using least squares minimization. (4, 7, and 10) 3-azido-2,2-dimethyl-2,3-dihydronaphtho[1,2-b]furan-4,5-dione (4): In a 100 mL roundedbottom flask, nor-lapachol (3, 456 mg, 2.0 mmol) and DCM (30 mL) were added. The mixture was cooled down to 0 • C, followed by the careful addition of bromine (1.0 mL, 3.12 g, 19.5 mmol). The reaction was kept under continuous stirring at 0 • C for 5 min. The excess bromine, along with the solvent, was removed under reduced pressure, resulting in an orange solid. This mixture was directly used without further purification in the next step through the addition of DCM (10 mL) and sodium azide (390 mg 6.0 mmol). The reaction was kept under continuous stirring at room temperature for 24 h. The final crude was suspended in 15 mL of distilled water, extracted with ethyl acetate (3 × 15 mL), and dried over Na 2

General Procedure for the Synthesis of Amino-Alkynes (18a-e)
The corresponding quinone (1.0 mmol) was dissolved in acetonitrile (3.0 mL, 0.3 m) at room temperature in a 10 mL rounded-bottom flask. N-propargylamine (128 µL, 110.2 mg, 2.0 mmol) was added to the mixture and it was kept under continuous stirring over 24 h at room temperature. The respective amino-alkyne was obtained by column chromatography (n-hexane/EtOAc 8:2). The correct position of the propargylamine substituent was determined over bidimensional NMR spectra analysis.

Conclusions
After the development of the research published by our research group in 2021 [32], it became clear that a sequel was necessary in order to fully evaluate and explore the anticancer activity that a synergetic combination of two naphthoquinoidal redox centres can offer. Through a copper catalysed 1,3-dipolar cycloaddition, fifteen new products were successfully achieved, each presenting astonishing anticancer activities against nine different cancer cell lines. Amongst those, the main activity was observed against the HL60 cell line, for which IC 50 values as low as 0.3 µM were observed. This is a good result when compared to the positive control, doxorubicin, which has an IC 50 value of 0.1 µM against the HL60 cell line. Along with these results, the cytotoxicity was also evaluated against the murine fibroblast cell line L929, in which it was possible to observe that compound 18b is one the most selective ones, presenting IC 50 values of 24 µM against the L929 and 1.8 µM against the HCT-116 cell line (selectivity index of 13.3), alongside compound 20e, which presents an IC 50 value of 15.9 µM against L929 and 1.6 µM against the HL60 cell line (selectivity index of 9.9). With these results in hand, the pathway towards a less aggressive additional therapy inches closer to reality, which may benefit thousands of people suffering from severe cases of cancer nowadays.