Synthesis of Lapachol-Based Glycosides and Glycosyl Triazoles with Antiproliferative Activity Against Several Cancer Cell Lines

Lapachol ( 1 ), a natural naphthoquinone, presents several biological activities including antitumor activity, used as anticancer coadjuvant whose use was abandoned because of adverse effects. Herein, we reported the synthesis and cytotoxicity evaluation against cancer cell lines of a series of O glycosides and glycosyl triazoles derived from lapachol. In addition to the determination of IC 50 , the DNA fragmentation and clonogenicity were also evaluated. The glycoside derived from D -glucose ( 5 ) was far more active than lapachol ( 1 ) and more active in tumor cell lines HL60, Jurkat, THP-1 and MDA-MB-231 than to the non-tumoral PBMC cell line, indicating an improvement in activity and selectivity as compared with lapachol ( 1 ). Compound 5 and the glycosides derived from D -galactose ( 14 ) , D - N - acetylglucosamine ( 15 ) and L -fucose ( 16 ) showed good results in the DNA fragmentation and clonogenicity assays in the studies of subdiploid DNA content, indicating a pro-apoptotic potential and a good antiproliferative activity of these glycosides.


Introduction
Cancer is one of the principal causes of death worldwide and its incidence is expected to increase in the next years. According to WHO, about 30 million cases of cancer are expected to occur until 2040, with almost 50% taking place in developing countries [1]. Despite the great efforts in prevention and treatment of cancer there is a continuous need for new options. The lack of selective action towards the cancer cells of most current existing anticancer drugs results in toxicity to host tissues. Thus, the search for new potent and safer drugs of synthetic and natural origin is being pursued by several groups around the world.  Lapachol (1), 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthalenedione is a natural 1,4-naphthoquinone isolated from plants of the Bignoniaceae family, mainly Handroanthus impetiginosus. It presents several biological activities including antitumor activity [4,5]. This compound has been used as coadjuvant in the chemotherapy of certain tumors but its use was abandoned because of adverse effects, mainly related to blood clotting. [5,6] Some synthetic routes were established to get lapachol (1), firstly by Fieser [7], being obtained in low yields. Recently, lapachol was synthetized from lawsone in better yields [8]. Several derivatives of 1 were prepared with antifungal, antibacterial, antiviral and antitumor activity [5]. Eyong et al. described the synthesis of atovaquone, which was approved for treatment of pneumonia (Pneumocystis pneumonia), toxoplasmosis and malaria [4,9]. Atovaquone is a naphthoquinone as well as lapachol and studies carried out in the last years have shown that atovaquone has a potent antitumor activity [10]. The chemical structure of l and some of its derivatives with antitumor activity are shown below (Fig. 2).  [4,5].
The quinones are able to inhibit the mitochondrial oxidation and phosphorylation, as well to inhibit the enzyme succinate oxidase [11] which plays an important role in the citric acid cycle and the electron transport chain. Other mechanisms seem to be related to the intercalation of the naphthoquinones between the DNA base pairs [5] and inhibition of topoisomerases [12]. The main mechanism of action is related to the formation of reactive oxygen species (ROS), through semiquinone radicals. Both cause damage to cell macro molecules and consequently cell death [12].
The major problem for the clinical use of 1 is its low bioavailability, due to low water solubility, which implicated in the use of large doses for attaining plasmatic levels, causing severe side effects. The first attempt to enhance water solubility of lapachol was reported by Linardi and co-workers who described the synthesis of the β-D-glucoside of 1 (compound 6) and the corresponding peracetylated derivative 5 (Fig. 3) [13]. These two compounds were evaluated in vivo in mice bearing P-388 lymphocytic leukemia. The peracetylated glucoside 5 was active, enhancing the lifespan of mice by 80%, while the deacetylated derivative was inactive. According to the authors, the peracetylated glucoside was possibly acting as prodrug that could be absorbed by the cancer cells due to its lipophilic character. The unprotected derivative 6, being more hydrophilic, was possibly unable to cross the cell membranes being, therefore, inactive. [13]   Several anticancer drugs possess a carbohydrate moiety in their structures, as shown in Fig. 1 for etoposide and doxorubicin. The work of Linardi and co-workers [13] showed that the attachment of a glucosyl moiety to lapachol can be a good approach to obtain new anticancer compounds. Glycosidic derivatives of lawsone, another naphthoquinone, has been obtained and assayed for antitumor activity. The glycosides were cytotoxic against HL-60 (acute promyelocytic leukemia), SKBR-3, MCF-7 and MDA-MB-231 (breast cancer) cells indicating that the variation of the carbohydrate moiety and the anomer type (α or β-glycoside) influence the cytotoxicity [14,15]. Some these lawsone glycosides with antitumor activity are shown in  Besides classical glycosides, obtained by direct glycosylation, one strategy widely used to link a carbohydrate moiety to a natural or synthetic compound is the Cu(I)-catalyzed cycloaddition reaction between an alkyne derivative of the compound with a glycosyl azide, to get glycosyl triazoles [16]. Based on this, several glycosyl triazoles derived from naphthoquinones with antitumor activity are described in the literature (Fig. 5) [17][18][19]. Recently we described the synthesis and cytotoxic evaluation against HL-60 human leukemia cells of lapachol glycosides 5 and 15. These compounds showed low IC50 values, circa 5.0 µM. The mechanism of cytotoxic seems to involve the activation apoptosis signaling pathways, such as the DNA fragmentation, chromatin condensation and decrease of the mitochondrial transmembrane potential [20].
In the present work we describe the synthesis and cytotoxicity evaluation against cancer cell lines of a series of O-glycosides and glycosyl triazoles derived from lapachol. The structures of the synthesized compounds are shown in Fig. 6. The presence and orientation of groups (OH and NHAc) that can modulate the physico-chemical properties of the compounds was considered, taking into account that the parent carbohydrates have different solubility and that the O-acetyl groups confer lipophilic properties to the peracetylated derivatives. We also considered the presence of specific carbohydrate transporters in the cell surface [21], that should facilitate the transport of the deacetylated glycosides across the cancer cell membrane. 7 2 Results and discussion

Scheme 2.
Reaction conditions for the obtention of 2-O-propargyllapachol and the glycosyl azides.

11
The infrared spectra of the deprotected glycosyl triazoles 21-24 showed, as expected, absorption bands in the region of 3357-3281 cm -1 due to OH stretching of the carbohydrate moiety. The 1 H NMR and 13 C NMR spectra of deprotected glycosyl triazoles 21-24 agree with their chemical structures. The mass spectra of all lapachol derivatives showed molecular weight compatible with the proposed structures (supplementary data available).

Biological activities 2.2.1 Cytotoxic activity
Lapachol (1), its classical glycosides 5 and 14-16 and glycosyl triazoles derivatives 17-24 were evaluated for their cytotoxicity against six human cancer cell lines: HL60 (acute promyelocytic leukemia), Jurkat (acute T-cell leukemia), THP-1 (acute monocytic leukemia), MCF-7 (breast adenocarcinoma), MDA-MB-231 (triple-negative breast cancer) and HCT-116 (colorectal carcinoma). Cell viability was evaluated using the MTT method to evaluate cell viability as previously described [38][39][40]. As model of non-tumoral lineages, compounds were tested against human peripheral blood mononuclear cells (PBMC) and viability measured by resazurin assay [41]. Etoposide and lapachol were used as positive controls. Compared with lapachol (1), the majority of its glycosides were more cytotoxic towards one or more tumor cell lines, lapachol (1) being cytotoxic only against HL60, with poor activity when compared to its derivatives. To evaluate the toxicity to non-tumor cells, selected compounds were tested on peripheral blood mononuclear cells (PBMC) cells. The results are shown in Table 1. Table 1. Cytotoxicity of lapachol (1) and lapachol-based glycosyl triazoles 17-24 against four cancer cell lines and against human peripheral blood mononuclear cells ( a IC50, μM).

Evaluation of DNA fragmentation assay as indicative of cell death by apoptosis
The subdiploid DNA quantification accomplished in this work was used as strategy to measure the DNA fragmentation, being an indicative of cell death activation by apoptosis, according described by Nicoletti et al [51,52]. According to the protocol used, cells with DNA fragments by death apoptosis process can be evaluated by quantification of subdiploid DNA content. The classical glycosides (compounds 5 and 14-16) presented higher cytotoxicity against the majority of tumor cell lines and the best selectivity index (SI) for at least one tumor cell lines, so they were selected for the quantification of subdiploid DNA content as indicative of the pro-apoptotic potential [51]. There was an increase in subdiploid DNA content in the tumor cell lines treated with classical lapachol glycosides 5 and 14-16, but not with lapachol. The compounds were evaluated in the concentration of 50 µM and glycosides induced DNA fragmentation in all tumor cell line (Fig. 7).  A common characteristic of cell lines used in this work is related to presence or absence of checkpoint p53 protein activity. The leukemic cell lines HL60, Jurkat and THP-1 lack p53 protein [53][54][55]. cell line showed p53 wild type protein [59] and the activity of chemotherapeutics may be mediated by p53 and Bax pro-apoptotic proteins which activate the apoptosis mitochondrial pathway and can activate caspase-3 [58]. On this way, the MCF-7 cells are the only ones used in this work that lack caspase-3 [60].
For MCF-7 cells other mechanisms of apoptosis induced by different chemotherapeutic agents may occur independently of caspase-3 so that DNA fragmentation can be observed despite the absence of caspase-3 [60]. Therefore, different pathways should be involved in the pro-apoptotic potential observed for new lapachol glycosides.

Effect of lapachol glycosides in the clonogenic survival of solid tumor lineages
Is well known that the DNA damage inducing agents induce cell cycle arrest at checkpoints. This is a cell survival response that allows them to repair damaged DNA and is not directly related to cell death. Cells  performed in triplicate. Colonies of more than 50 cells were counted, and the surviving fraction was calculated relative to control (DMSO, 0.5%) to account for basal plating efficiencies 2. 3 Influences of the chemical structure of lapachol glycosides on bioactivity The cytotoxicity against the evaluated cancer cell lines was in general higher for lapachol glycosides (5,

14-16, 17-20, 22
and 24) than that of lapachol (1), indicating that carbohydrate moieties influenced in the cytotoxicity against tumoral and non-tumoral cells. The higher activity of peracetylated lapachol glycosides can be explained due the presence of the O-acetyl groups on saccharidic moiety, which impair appropriate liposolubility to the peracetylated glycosides enabling these compounds to cross the cell membrane [13].
The presence of hexose transporters on cell surface [21] may have facilitated the entry of the deacetylated lapachol glycosides that were active.
The nature of the carbohydrate moieties seems to influence on activity, wherein the variation of sugar improves or reduces the cytotoxic activity. For example, the peracetylated lapachol glycosyl triazole derived from L-fucose (20) was the most active against HCT-116 while its deacetylated analogue

Conclusions
The synthesis of 12 lapachol-based glycosides and glycosyl triazoes and evaluation against several cancer cell lines is described in the present work.

4.2.3
General procedure for the synthesis of lapachol glycosyl triazoles (17)(18)(19)(20)(21)(22)(23)(24) To a 50 mL round bottom flask was added 28 (0.30 mmol) dissolved in 1 mL of tetrahydrofuran, followed by the appropriate glycosyl azide (0.27 mmol), dissolved in 0.5 mL of tetrahydrofuran. Then, Cu(OAc)2.H2O50% mol, dissolved in 0.5 mL of water and sodium ascorbate 60% mol, dissolved in 1 mL of water were added in a stepwise manner. The reaction mixture was stirred at room temperature for 4 h and monitored by TLC analysis. The tetrahydrofuran was removed by distillation at reduced pressure. For 22 peracetylated glycosyl triazoles the reaction residues were solubilized in 50 mL CH2Cl2 and washed with 2 x 50 mL H2O and subsequently washed with 3 x 50 mL alkaline EDTA 20% w/v. The organic phase was dried over Na2SO4 and filtered. The organic phase was removed by distillation at reduced pressure. The deacetylated glycosyl triazoles (21)(22)(23)(24) were purified directly. The derivatives 17-20 were added to Florisil and purified with silica column with following mobile phase (CH2Cl2: ethyl acetate/4:6) and the deacetylated using ethyl acetate: MeOH/9:1 as mobile phase.

Evaluation of cell viability of human PBMC by resazurin assay
The cell viability assay was performed according to O'Brien et al. (2000), with modifications. [40] Resazurin is a blue dye and is weakly fluorescent until it is irreversibly reduced to pink and red fluorescent resorufin. It is used as an oxidation-reduction indicator in cell viability assays and its intensity is A blank of each sample was performed to avoid unspecified reactions of the compounds with resazurin (blank) and the results were analyzed using Prism 7.0 (GraphPad Software Inc).

Selectivity index (SI) determination
After determining the IC50 values for tumor (HL60, Jurkat, THP-1, MCF-7, MDA-MB-231 and HCT-116 cells) and non-tumor cells (PBMC), the selectivity index was calculated. Determination of the SI was performed by the ratio between IC50 of PBMC and IC50 tumor cell [65].

Clonogenic assay
The MCF-7, MDA-MB-231 and HCT-116 cells were seed in 6-well plates at a density of 400 cells/well. After 6 hours of incubation, cells were treated with the compounds (at their IC50 and IC80) or control (DMSO 0.5%). The cells were incubated with the compounds for 24 hours and then the medium was removed and replaced by supplemented DMEM medium without the compounds [66]. The cells were incubated for another 14 days and after incubation, the colonies were fixed in 70% alcohol for 15 minutes, stained with crystal violet (30% in ethanol) for 30 minutes and kept at room temperature overnight for drying. Colonies with 50 or more cells were counted. The survival fraction (ratio between the number of colonies treated with the compounds and the number of colonies counted in the control) were calculated and the results were analyzed by GraphPad Prism 7.0.

Statistical analysis
Data are expressed as the means± SD (standard deviation). Statistical analysis was conducted using the Prism 7.0 statistical package (GraphPad Software, USA). To ascertain significance, we used a one-way ANOVA with Bonferroni post-test. Statistical significance was considered at a limit of p<0.05 from three independent experiments conducted in triplicate.