key: cord-0861002-4w0zw3br
authors: Macan, Andrijana Meščić; Harej, Anja; Cazin, Ines; Klobučar, Marko; Stepanić, Višnja; Pavelić, Krešimir; Pavelić, Sandra Kraljević; Schols, Dominique; Snoeck, Robert; Andrei, Graciela; Raić-Malić, Silvana
title: Antitumor and antiviral activities of 4-substituted 1,2,3-triazolyl-2,3-dibenzyl-L-ascorbic acid derivatives
date: 2019-12-15
journal: Eur J Med Chem
DOI: 10.1016/j.ejmech.2019.111739
sha: 23288b8c9fa1e91b2617ec589fdaf1902463ff3e
doc_id: 861002
cord_uid: 4w0zw3br

Two series of 6-(1,2,3-triazolyl)-2,3-dibenzyl-l-ascorbic acid derivatives with the hydroxyethylene (8a−8u) and ethylidene linkers (10c−10p) were synthesized and evaluated for their antiproliferative activity against seven malignant tumor cell lines and antiviral activity against a broad range of viruses. Conformationally unrestricted spacer between the lactone and 1,2,3-triazole units in 8a−8u series had a profound effect on antitumor activity. Besides, the introduction of a long side chain at C-4 of 1,2,3-triazole that led to the synthesis of decyl-substituted 2,3-dibenzyl-l-ascorbic acid 8m accounted for a selective and potent antiproliferative activity on breast cancer MCF-7 cells cells in the nM range. Further analysis showed that compound 8m strongly enhanced expression of hypoxia inducible transcription factor 1 α (HIF-1α) and to some extent decreased expression of nitric oxide synthase 2 (NOS2) suggesting its role in regulating HIF-1α signalling pathway. The p-methoxyphenyl-substituted derivative 10g displayed specific anti-cytomegalovirus (CMV) potential, whereas aliphatic-substituted derivatives 8l and 8m had the most potent, yet relatively non-specific, anti-varicella-zoster (VZV) activity.

Even though L-ascorbic acid (L-ASA) cancer therapy has a controversial history [1] , interest in L-ASA has been renewed due to its recently demonstrated ability to selectively kill cancer cells [2e5] . It was shown that L-ASA inhibits the growth of KRAS and BRAF mutant colorectal cancer cells by causing oxidative stress and accumulation of reactive oxygen species (ROS), the latter leading to inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an energy crisis and consequently cell death [6] . Lascorbic acid is an established activator of non-heme iron a-ketoglutarate (aKG) dioxygenases, such as hypoxia inducible factor prolyl hydroxylases (HIF PHD), and epigenetic ten-eleven translocation (TET) enzymes, which are directly relevant to cancer susceptibility and progression [7, 8] . HIF-1 is a critical mediator of the cellular response to hypoxia, making it an attractive molecular target for anticancer therapy. Hydroxylation of proline in HIF-1a under normoxia tags it for subsequent ubiquitinylation and proteasomal degradation [9] . Activation of the enzyme HIF prolyl hydroxylase inhibits the HIF-triggered antihypoxic program, the latter being the adaptive response of malignant tumors to hypoxic stress. Such activation was recently observed by L-ascorbic acid and its 2phosphate derivative that promoted tumor cells death [10, 11] . Furthermore, recent progress in the epigenetics identified a new role of ascorbate in the regulation of the demethylation of deoxyribonucleic acid (DNA) and histone proteins [12] . It was found that L-ASA acts as a direct regulator of TET activity and DNA methylation in mouse embryonic stem (ES) cells, thus indicating its potential application for treating cancers driven by aberrant DNA methylation in the clinic [13] . L-ascorbate also showed a selective killing effect against various human breast cancer cell lines which was dependent on the sodium-vitamin C transporter 2 (SVCT-2) expression responsible for L-ascorbate uptake [14] . Some 1,2,4-triazole and imidazole L-ASA derivatives exerted a pronounced cytostatic effect in human T-cell acute lymphoblastic leukemia cells [15] . Moreover, pyrimidine and purine derivatives of 6-deoxy-Lascorbic acid and 4,5-didehydro-5,6-dideoxy-L-ascorbic acid derivatives showed antiproliferative effect in a panel of tumor cells and antiviral potency against VZV and CMV ( Fig. 1) [16e19]. It was observed that 2,3-O,O-dibenzyl-L-ascorbic acid derivatives exhibited better growth inhibition of malignant tumor cells and antiviral activity than their 2,3-dihydroxy analogues.

1,2,3-Triazole has found increased application in the drug discovery because it has been recognized as good amide bioisostere that readily associates with biological targets through hydrogen bonding and dipoleÀdipole interactions [20] . Accordingly, a series of molecules containing a pentacyclic triterpene moiety connected to L-ASA via 1,2,3-triazole were shown to exhibit anti-influenza A/ WSN/33 virus activity, showing that 2,3-O,O-dibenzylated L-ASA skeleton was crucial for their antiviral potency, regardless of the triterpene fragment [21] . Additionally, several aspulvinone compounds containing g-butenolide moiety showed significant antiinfluenza A (H1N1) virus activity [22] .

Having in mind these effects and potential synergy of the antitumor and antiviral activity of L-ASA and 1,2,3-triazole moiety, we designed and synthesized L-ascorbic acid derivatives and their C4¼C5 unsaturated analogues with a 1,4-disubstituted 1,2,3triazole core on the C-6 atom hypothesizing that new chemical entities might exhibit a pronounced and selective antitumor and/or antiviral activity. To investigate the influence of substituents at the C-4 of 1,2,3-triazole conjugated with the L-ASA on biological activity, straight and branched alkyl chain of different lengths, cyclopropyl, variously substituted aromatic, benzenesulfonamide and dithiocarbamate moieties were introduced and their biological activity was compared to the unsubstituted triazole L-ascorbic acid conjugate (Fig. 1 ).

To evaluate the impact of substituents at the C-4 of 1,2,3-triazole and the hydroxyethylene and conformationally restricted ethylidene spacer between the lactone and 1,2,3-triazole moieties on biological activity, two series of 6-(1,2,3-triazolyl)-6-deoxy-(8aÀ8u) and 6-(1,2,3-triazolyl)-4,5-didehydro-5,6-dideoxy-Lascorbic acid (10cÀ10p) derivatives were synthesized as outlined in Scheme 1.

The isopropylidene protection of 5,6-hydroxyl groups, 2,3dibenzylation, and subsequent removal of 5,6-isopropylidene in Lascorbic acid to afford compound 3, were performed according to procedures described in the literature [18, 23, 24] . The tosylation of the 5,6-hydroxyl groups in compound 3 using tosyl chloride in pyridine afforded the 6-monotosylated 2,3-dibenzyl-L-ascorbic acid derivative 4 and the 5,6-ditosylated compound 5 in the same reaction, which were then used as parent molecules for the synthesis of the corresponding C-6 azido derivatives of 6-deoxy-(6) and 4,5didehydro-5,6-dideoxy-L-ascorbic acid (9) . The azido derivative 6 was synthesized by the reaction of the monotosylated derivative 4 with sodium azide in a yield of 77%. The 1,2,3-triazole was introduced at the C-6 position of the 2,3-dibenzyl-L-ascorbic acid via copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (CuAAC) of C-6-azido-6-deoxy-2,3-dibenzyl-L-ascorbic acid 6 and the corresponding terminal alkynes 7bÀ7u (method A, Scheme 1) in yields from 16% to 72%.

A Cu(II) salt in the presence of a reducing agent, metallic copper, was employed to generate the required Cu(I) catalyst in situ. Besides diverse aliphatic and aromatic substituents at C-4, benzenesulfonamide [25e28] and dithiocarbamate [29e31], recognized as important pharmacophores in the design of anticancer agents, were also introduced. Benzenesulfonamide-based terminal alkynes 7qÀ7t were prepared in a reaction of propargylamine and corresponding benzenesulfonyl chlorides [32] , while tert-butyl 4-((prop-2-ynylthio)carbonothioyl)piperazine-1-carboxylate (7u) was synthesized via reaction of piperazine with carbon disulfide (CS 2 ) and propargyl bromide in the presence of a base [33] . Following method A (Scheme 1), the best yields of 72% and 71% were obtained in the synthesis of compounds 8n and 8o with branched 4-tert-butyl and 4-(3-chloropropyl) side chains, respectively. Optimization of reactions was performed using another synthetic pathway (Scheme 1, method B). Method B represents a convenient one-pot two-step procedure to obtain the target compounds by the in situ generation of azide 6 via reaction of the monotosylated L-ascorbic acid derivative 4 with sodium azide, and followed by CuAAC reaction of azide 4 and corresponding alkyne to afford 1,4-disubstituted 1,2,3-triazole derivatives of L-ascorbic acid 8aÀ8u. For environmental reasons, the microwave-assisted reactions were performed in both methods over a period of 1 h for completion of the reaction. However, comparing the yields of the two synthetic pathways, it can be concluded that the method A, that included the isolation of the C-6 azido derivative 6, was generally more successful than the one-pot procedure (method B). 4-Substituted 1,2,3-triazolyl-4,5-didehydro-5,6-dideoxy-L-ascorbic acid (10cÀ10p) derivatives containing C-4¼C-5 double bond were prepared starting from the ditosylated compound 5 which, by elimination and subsequent nucleophilic substitution with NaN 3 , gave the 4,5-unsaturated azido derivative 9 [34] . The target compounds 10c¡10p were then obtained in a click reaction of the azido derivative 9 and the corresponding terminal alkynes 7cÀ7p using Cu(OAc) 2 ,-as a catalytic Cu(I) source, under both micro-flow and batch conditions following a previously described procedure [34] .

2.2.1. In vitro antiproliferative evaluations and in silico physicochemical profiling A series of 4-substituted 1,2,3-triazolyl 2,3-dibenzyl-L-ascorbic acid conjugates (8aÀ8u and 10cÀ10p) were evaluated against seven human tumor cell lines: lung adenocarcinoma (A549), ductal pancreatic adenocarcinoma (CFPAC-1), colorectal carcinoma (HCT-116), cervical carcinoma (HeLa), hepatocellular carcinoma (HepG2), breast adenocarcinoma (MCF-7) and colorectal adenocarcinoma, metastatic (SW620) cell lines as well as normal skin fibroblasts Scheme 1. Reagents and conditions: i) acetyl chloride, acetone; ii) benzyl chloride, potassium carbonate (K 2 CO) 3 ; iii) acetic acid, methanol; iv) tosyl chloride, pyridine, dichloromethane (CH 2 Cl 2 ); v) sodium azide (NaN 3 ), dimethylformamide (DMF), water; vi) Cu, 1 M copper (II) sulphate (CuSO 4 ), tert-butanol, DMF, water, microwave reactor; vii) NaN 3 , Cu, 1 M CuSO 4 , DMF, water, microwave reactor; viii) NaN 3 , acetonitrile; ix) copper (II) acetate (Cu(OAc) 2 ), methanol.

(HFF) (Tables 1-3) .

By varying the substituent at position C-4 of the 1,2,3-triazole ring, the library of 35 compounds has been created (Scheme 1, Fig. 2 ).

Structural similarity in aromatic and aliphatic fragments in 8aÀ8u and 10cÀ10p series has been recognized, whereas the introduction of a sulfonamide unit (8qÀ8t) and formation of C4¼C5 double bond (10cÀ10p) boosted the highest structural diversification (Fig. 2) . The synthetized compounds have spanned a wide range of lipophilicity (Tables 1 and 2 ). The n-octanol/water partition coefficients (clog P) of the dibenzylated derivatives 8a¡8u and 10c¡10p are within the range of 0.687¡6.359. The compounds 10c¡10p with a C4¼C5 double bond showed to be more lipophilic than their C-5-OH analogues 8c¡8p. In addition, although the dibenzylated derivatives are quite large molecules with molecular weight (MW) range from 407.4 to 665.8, only six of them (8qÀ8u and 10c) break the Lipinski rule of five filter for drug-like molecules ( Table S1 in Supplementary information) [35] .

In general, it can be observed that the 2,3-dibenzyl-L-ascorbic acid derivative 8a, with an unsubstituted 1,2,3-triazole ring, did not exhibit any cytostatic activity against evaluated tumor cell lines indicating that the nature of C-4 substituent had a crucial effect on antiproliferative activity (Table 1) . L-ASA itself was also lacking any cytostatic effect. Among L-ascorbic acid derivatives 8cÀ8j containing 4-aryl-substituted 1,2,3-triazole, the p-bromophenyl substituent in 8e caused a strong inhibitory effect on all tumor cell lines, particularly against lung adenocarcinoma cells (A549:

. While the electron-withdrawing trifluoromethylin 8c and fluoro-substituted aromatic moiety in 8d had a small influence on the antiproliferative effect, the electron-donating methyl group in 8f caused selectivity in the cytostatic activity. This compound strongly inhibited the growth of lung adenocarcinoma (A549: IC 50 ¼ 8.69 mM) and hepatocellular carcinoma (HepG2: IC 50 ¼ 8.77 mM) cell lines. A longer alkyl substituent, such as 4-pentyl, at the phenyl moiety in 8j also contributed to marked albeit non-specific cytostatic activity. On the contrary, p-methoxy and o-methoxy, as well as p-hydroxy substituents at the phenyl in 8g, 8h and 8i, respectively, did not have a significant impact on the antiproliferative effect on tumor cell lines. However, both pmethoxy and o-methoxy substituents had a pronounced cytotoxicity on normal fibroblasts (HFF). From compounds with a 4aliphatic-substituted 1,2,3-triazole, long straight 4-decyl and branched 4-tert-butyl in 8m and 8n, respectively, had a strong effect on cytostatic activity, particularly on breast adenocarcinoma cell line MCF-7. Thus, these compounds exhibited antitumor activity against MCF-7 cells in nM range (8m: IC 50 ¼ 0.08 mM; 8n: IC 50 ¼ 0.73 mM). However, compound 8n was also cytotoxic to normal fibroblasts (IC 50 ¼ 13.33 mM), while 8m showed to be nontoxic (IC 50 > 100 mM).

Among compounds containing a sulfonamide-substituted 1,2,3-triazole moiety, p-methylphenyl-(8q), p-chlorophenyl-(8r) and 4-fluoro-2-chlorophenylsulfonamide (8s) moieties in 1,2,3-triazolyl-2,3-dibenzyl-L-ascorbic acid derivatives affected marked inhibitory activities on MCF-7 cells, in the range from 3.39 mM to 6.4 mM. Compound 8r exerted also a strong antiproliferative activity on A549 cells. It is noteworthy that sulfonamide-substituted 1,2,3-triazolyl-2,3-dibenzyl-L-ascorbic acid derivatives (8qÀ8t) exhibited non-specific inhibitory effects and high cytotoxicity to normal fibroblasts (HFF). (Methylthiocarbothionyl)piperazine 2,3-dibenzyl-L-ascorbic acid derivative 8u did not have any impact on antiproliferative activity on evaluated tumor cell lines.

Some relationship between lipophilicity and biological activity of 4-substituted 6-(1,2,3-triazolyl)-6-deoxy-2,3-dibenzyl-L-ascorbic acid derivatives (8aÀ8u) has been observed, suggesting that compounds 8a and 8p with the lowest lipophilic properties (clogP 0.8) had a lack of antiproliferative activity on tumor cells. The most lipophilic compounds 8j (clogP ¼ 4.67) and 8m (clogP ¼ 5.25) showed cytostatic activity on tumor cells without being cytotoxic to normal skin fibroblasts.

By replacing the C-5-OH group with stereochemically constrained C4¼C5 double bond in Z-configuration [34] , cytostatic effect of compounds from the 10cÀ10p series were decreased against the majority of cancer cells lines (Tables 2 and 3) . However, 4,5-unsaturated analogues 10cÀ10p showed a higher cell specificity with the best activity against the growth of cells MCF-7 and HeLa, derived from female tumors ( Table 3 ).

In addition, aliphatic-substituted compounds 10lÀ10p exhibited better antiproliferative effect compared to their aromaticsubstituted analogues 10cÀ10j. Among 4-aryl-substituted unsaturated 2,3-dibenzyl-L-ascorbic acid derivatives, compounds 10d, 10e and 10f with di(trifluoromethyl), p-bromo and p-methyl substituents, respectively, displayed a selective cytotoxic effect towards cervical carcinoma cell lines HeLa (Table 2 ). Besides, these compounds were not cytotoxic (IC 50 > 100 mM) to other tumor cell lines and normal fibroblasts HFF. 4,5-Unsaturated 2,3-dibenzyl-Lascorbic acid derivatives 10k, with a cyclopropyl and 10n, with tertbutyl side chain, exerted strong antiproliferative effect (10k: IC 50 ¼ 9.27 mM; 10n: IC 50 ¼ 4.93 mM) on breast adenocarcinoma cells MCF-7. Compound 10n exerted an approximate 7-fold reduced antiproliferative effect on MCF-7 cells compared to its saturated analogue 8n. This compound exhibited also a strong cytostatic activity on A549 cells and moderate cytotoxicity to normal fibroblasts. Within the 4,5-unsaturated series, 4-cyclopropyl-1,2,3triazole derivative 10k had a three-fold more pronounced inhibitory activity on MCF-7 cells than its saturated 2,3-dibenzyl-Lascorbic acid analogue 8k. On the opposite, 4-aliphatic-substituted 1,2,3-triazolyl 2,3-dibenzyl-L-ascorbic acid 10m had only marginal or no antiproliferative effect, whereas its analogue 8m, from 6deoxy-2,3-dibenzyl-L-ascorbic acid series, had a strong antiproliferative effect on MCF-7 in nM range.

The antiproliferative activity of synthetized compounds particularly for the saturated derivatives 8aÀ8u, against seven tested tumor cell lines were mutually quite linearly correlated, as it is illustrated by the PCA analysis (Fig. 3) .

The linear correlations coefficients between IC 50 values of various pairs of tumor cell lines for the compounds 8aÀ8u spanned range 0.632e0.968, with median coefficient value of 0.749. The greatest similarity in antiproliferative activity was observed between tumor cell lines A549, HeLa and HepG2 ( Table 3 ). The most growth inhibiting compounds for all tumor cell lines were 8e, 8j, and 8r, while 8m and 8n were particularly antiproliferative against tumor cell line MCF-7.

Lipophilicity comparison of the synthesized compounds from two series, 8 and 10, revealed that introduction of C-4¼C-5 double bond increased the lipophilicity of compounds 10cÀ10p in comparison to their analogues 8aÀ8u. Within the 4,5-unsaturated series, compound 10n with the best antiproliferative activity, has a clogP value of 3.55. The most hydrophilic compound within the 4,5unsaturated series 10p, containing 2-hydroxyethyl side chain at C-4, has a clogP value of 1.80 and showed a low to moderate inhibitory activity, while compounds exhibiting clogP >4.0 did not show appreciable antitumor activity.

Overall, the structureeactivity relationship of 4-substituted 1,2,3-triazolyl appended 2,3-dibenzyl-L-ascorbic acid derivatives (8aÀ8u and 10cÀ10p) indicates that both type of substituents at C-4 of 1,2,3-triazole scaffolds and the hydroxyethylene and ethylidene spacer connecting the lactone and 1,2,3-triazole moieties, had an impact on antiproliferative activity as presented in Fig. 4 . Table 1 The growth-inhibition effects in vitro of L-ascorbic acid and compounds 8aÀ8u on selected tumor cell lines and normal fibroblasts. 

>100 >100 a 50% inhibitory concentration or compound concentration required to inhibit tumor cell proliferation by 50%. NA ¼ not active. b Values of clog P were calculated by DataWarrior [36] .

The growth-inhibition effects in vitro of 1,2,3-triazolyl 4,5-unsaturated L-ascorbic acid derivatives (10cÀ10p) on selected tumor cell lines and normal fibroblasts. 

protein targets for compound 8m by Western blot analysis Due to the excellent selective cytostatic activities observed on breast adenocarcinoma MCF-7 cells, 4-decyl-substituted 1,2,3triazolyl 6-deoxy-2,3-dibenzyl-L-ascorbic acid 8m was chosen for further evaluation of its mechanisms of action in vitro. The mode of cell death induced by 8m was assessed by Annexin V assay, performed as described previously [37] (Table 4) .

Obtained results showed induction of apoptosis in MCF-7 cells treated with compound 8m. Compound 8m induced moderate increase in early and late apoptotic cell populations by 13.4% and 24.7% after 48 h, respectively (Table 4 ). A small increase of necrotic cell populations by 7.92% was also observed (Supplementary information, Fig. S22 ).

Since breast cancer and other solid tumors have long been known to be hypoxic, HIF-1a that is crucially involved in the response of mammalian cells to hypoxia has been recognized as a suitable target for development of antibreast cancer therapeutics [38e41]. Besides having pro-carcinogenic effects, some reports indicated that stabilization of HIF-1 in breast cancer cells decreases cancer cells growth by promoting the cell cycle arrest and autophagy [42e44]. It was also found that several cellular responses to HIF-1 are mediated by nitric oxide (NO) and vice versa. A complex relationship between HIF-1, NO and cancer has been thus explored (Tables 1 and 2 ). Coluoring denotes aromatic (red)/nonaromatic (blue) character of the R substituent (Scheme 1). More active compounds are grouped towards the left side of the plot. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) [44, 45] where it was shown that a biphasic response to NO occurs in HIF-1 regulation. Indeed, NO initially inhibits HIF PHD activity that leads to HIF-1 accumulation in the cell which in turn results in increased HIF PHD levels that then prevent HIF-1 accumulation at later stages [45] . In breast cancer tissues, nitric oxide synthase (NOS) activity is higher, especially in highly invasive tumors [46] . Accordingly, the relative expression analysis of HIF-1a (hydroxylated form at position Pro564) and NOS2 proteins' levels, as potential targets of the chosen candidate structure 8m, was performed (Fig. 5) . Proline hydroxylation of HIF-1a is known to lead to von Hippel-Lindau protein-dependent ubiquitination and rapid proteasomal degradation, which means that measurement of increased levels of this HIF-1a form is indicative for regulation of the HIF-1a levels in tumor cells.

In MCF-7 cells treated with 8m, a significant increase of hydroxylated HIF-1a levels along with decreased levels of NOS2 were observed, that suggest involvement of 8m in regulation of HIF-1a signalling pathway in MCF-7, what is in accord with its strong growth-inhibition effect.

The 4-substituted-1,2,3-triazole-2,3-dibenzyl-L-ascorbic acid derivatives (8aÀ8u and 10cÀ10p) were evaluated against CMV, VZV, thymidine kinase deficient (TK À ) and thymidine kinase positive (TK þ ) strains of VZV, herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), vaccinia virus, adenovirus-2, vesicular stomatitis virus, human coronavirus, influenza A/H1N1, influenza A/H3N2, influenza B, Coxsackie virus B4, reovirus and Sindbis virus. Compounds that exhibited some antiviral activities are presented in Table 5 and their activity was compared with those of ganciclovir, cidofovir, acyclovir and brivudin. By comparison of the antiviral activities of two series of compounds (8aÀ8u and 10cÀ10p), we can infer that conformationally constrained 4,5-unsaturated derivatives from 10cÀ10p series showed a better antiviral potential against CMV and similar potential to VZV (Table 5) .

Among 6-(1,2,3-triazolyl)-6-deoxy-2,3-dibenzyl-L-ascorbic acid derivatives, only 8b, 8k, 8l, 8m and 8n exhibited antiviral activity. Compounds 8b and 8k, bearing 4-trimethylsilyl-and 4cyclopropyl-substituted 1,2,3-triazole, respectively, displayed moderate antiviral potency against CMV (AD-169 and Davis strains). 2,3-Dibenzyl-L-ascorbic acid derivatives 8lÀ8n, with 4aliphatic side chains, had antiviral activity against VZV (TK À ), with the best value of EC 50 ¼ 7.31 mM for compound 8l. However, 8l

and 8m produced an alteration of the cellular morphology at a concentration somewhat lower to that at which the compounds inhibited viral plaque formation (MCC ¼ 20 mM). Among 4,5-unsaturated structural analogues, it can be observed that the majority of 4-aromatic-substituted 2,3-dibenzyl-L-ascorbic Compounds did not show appreciable antiviral activity against HSV-1 and HSV-2, vaccinia virus, adenovirus-2, vesicular stomatitis virus, human coronavirus, influenza A/H1N1, influenza A/H3N2, influenza B, Coxsackievirus B4, reovirus and Sindbis virus. Only exceptions were 4-unsubstituted 1,2,3-triazolyl-2,3-dibenzyl-Lascorbic acid derivative 8a that exhibited slight activity against influenza A subtype H1N1 and H3N2 (Supplementary information, Tables S2) and 4,5-unsaturated 4-butyl-substituted 2,3-dibenzyl-Lascorbic acid derivative 10l that displayed modest activity against HSV-2, adenovirus-2, and coronavirus (Supplementary information, Table S3 ). . However, compound 8n was cytotoxic to normal fibroblasts, while 8m showed to be nontoxic and the most lipophilic compound from the 8aÀ8u series, with clogP 5.25. Within the 4,5-unsaturated 10cÀ10p series, 4-tert-butylsubstituted structural analogue 10n had the most potent inhibitory activity on A549 and MCF-7 cells. However, it exhibited considerably reduced antiproliferative activity on MCF-7 compared to its analogue 8n. A more detailed investigation of the mechanism of action for the selected candidate 8m showed that this compound induced cell death of breast cancer MCF-7 cells, partially due to apoptosis induction. Moreover, 8m was shown to affect the HIF-1a signalling pathway, as it enhanced expression of the hydroxylated HIF-1a form, tagged for degradation and concomitantly decreasing expression of NOS2.

Additionally, the specific antiviral effect against CMV (Davis) was observed for p-methoxyphenyl-substituted 4,5-unsaturated derivative 10g and its activity was 3-fold reduced (EC 50 ¼ 5.74 mM) in comparison with ganciclovir. Moreover, compounds 8l, 8m, 10i and 10n exhibited antiviral potential against VZV (07/1) with EC values similar to that of brivudin. However, they showed cytotoxicity at concentrations close to antiviral active concentrations.

In summary, our findings encourage further development of new structurally related conformationally unrestricted L-ascorbic acid analogues targeting the HIF-1a signalling pathway for breast cancer therapy. Also, additional efforts should be devoted to increase anti-CMV and anti-VZV potency and/or selectivity, along with a more detailed investigation of these compounds mechanisms of antiviral action that may be different from those of antiviral drugs ganciclovir and brivudin. (9) [34] and unsaturated 1,2,3-triazole-2,3-dibenzyl-L-ascorbic acid derivatives (10ce10p) [34, 47] were synthesized in accordance with procedures given in the literature. 

To the solution of 6-azido-2,3-O,O-dibenzyl-6-deoxy-L-ascorbic acid (6) (200e300 mg) in DMF (3 ml); tertbutanol (3 ml) and H 2 O (3 ml), Cu (0.8 eq), 1 M CuSO 4 solution (0.3 eq) and the corresponding alkyne (1.2 eq) were added. The reaction mixture was heated for 1 h using microwave radiation (300 W). The solvent was evaporated and the product was purified by column chromatography on silica gel and aluminium oxide. The obtained oily compound was further triturated using n-hexane to obtain white powder.

To the solution of C-6 monotosylated Lascorbic acid derivative 4 (200e350 mg) in DMF (8 ml), Cu (0.8 eq), 1 M CuSO 4 solution (0.3 eq), NaN 3 (2.5 eq) dissolved in H 2 O (2 ml) and the corresponding alkyne (1.2 eq) were added. The reaction mixture was heated for 1 h using microwave radiation (300 W). The solvent was evaporated and the product was purified with column chromatography on silica gel and aluminium oxide. The obtained oily compound was further triturated using n-hexane to obtain white powder. 13 

The MACCS 166-bit fingerprints generation, clustering based on structural similarity with using a binary (Tanimoto) distance as a similarity metric, as well as principle component analysis (PCA) were done by R packages rcdk, factoextra and princomp, respectively [48, 49] . PCA analyses were performed in terms of measured IC 50 values and 1D/2D molecular descriptors. Simple structural parameters (molecular weight (MW), number of hydrogen-bond accepting/donating atoms (HBA/HBD), total/polar surface area (TSA/PSA), molecular flexibility (low < 0.5 < high), shape index (spherical < 0.5 < linear), numbers of rotable bonds, rings and specifically aromatic rings) and physicochemical properties (lipophilicity clogP, aqueous solubility clogS) were calculated by algorithms of DataWarrior (Table S1 in Supplementary information) [36] . No significant groupings and patterns, in terms of the molecular descriptors used, were revealed. Inputs for all algorithms were SMILES (simplified molecular-input line-entry system) representation of the compounds (Supplementary information, Table S1 ).

Human carcinoma cell lines A549, CFPAC-1, HCT-116, HeLa, HepG2, MCF-7, SW620 and normal fibroblasts HFF-1 were cultured in Dulbecco's modified Eagle medium (DMEM), supplemented with 10 FBS, 100 U/ml streptomycin and penicillin and 2 Mm L-glutamine (GIBCO, Invitrogen), at 37 C with 5 CO 2 in humidified atmosphere. Cell lines were obtained from American Type Culture Collection (ATCC).

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate anti-proliferative or proliferative effects on tumor and normal cell lines. Tumor cells and normal cells were seeded on 96-well microtiter plates at a seeding density of 5000 cells/well 24 h after seeding, cells were treated with freshly prepared compounds'dilutions in five concentrations (0.01e100 mM) and incubated for 72 h. Solvent DMSO, was tested for eventual cytotoxic effects and its concentration neverexceeded 0.1 mM. Following 72 h cells incubation with compounds, MTT measurment was performed. Measured absorbance of treated cells were transformed into a cell percentage growth (PG) using the formulas proposed by NIH and described previously [50] . Results of the test were obtained from two different experiments perfomed in quadruplicate and IC 50 values were calculated using linear regression, and were further statistically analyzed by ANOVA, Tukey posthoc test (at p < 0.5).

Programmed cell death and necrosis induction were tested by Annexin V assay. Briefly, 2 x 10 4 of A549, MCF-7 and HFF-1 cells were seeded onto 8-well Nunc Lab-Tek II Chamber Slide system (Thermo Fischer Scientific) and treated with compound 8m at 1 x IC 50 (0.08 mM) and 2 x IC 50 (0.16 mM) for 24 and 48 h. Cells were stained by use of reagents provided in the Annexin-V-FLUOS Staining kit (Roche Applied Science) according to the manufacturer recommendations. Cells were analyzed by fluorescence microscopy (Zeiss Axio Observer Z1 Inverted Phase Contrast Fluorescent Microscope) and 100 cells were counted for each condition. Results were statistically analyzed by ANOVA, Tukey posthoc test (at p < 0.5).

MCF-7 cells were seeded in Petri dishes, at 1 x 10 6 cells/dish and treated with compound 8m at IC 50 (0.08 mM) for 24 and 48 h.

Compound 8m was assessed as effective and selective on MCF-7 cells with low IC 50 . Protein lysates were prepared using a buffer containing 50 mM Tris HCl (pH 8), 150 mM NaCl, 1 NP-40, 0.5 sodium deoxycholate, 0.1 SDS and a protease and phosphatase inhibitor cocktail (Roche, Switzerland). A total of 50 mg of proteins were resolved on 12 SDS polyacrylamide gels using the Miniprotean cell (Bio-Rad, USA). The membranes were incubated with primary antibodies against CDC25 (1:1000, rabbit mAb, Abcam, UK), NOS2 (1:500, mouse mAb, SantaCruzBiotechnology, USA) and hydroxy-HIF-1a (Pro564) (HIF-1a, 1:1000, rabbit mAb, Cell Signaling Technology, NL) at 4 C overnight. Secondary antibody linked to anti-mouse (1:1000, Dako, USA) was used. The signal was visualized by Western Lightning Chemiluminescence Reagent Plus Kit (PerkinElmer, USA) on the ImageQuant LAS500 (GE Healthcare, USA) and a-tubulin (1:1000, mouse mAb, Sigma, USA) was used as a loading control. The signal intensities of particular protein lines on the blot were normalized with the intensity of the loading control and compared in Quantity One software (Bio-Rad, USA). The values are expressed as the average ± SD. Statistical analysis was performed in Microsoft Excel by using the ANOVA at p < 0.05.

Antiviral activity against CMV, VZV, HSV-1, HSV-2, vaccinia virus, vesicular stomatitis virus, adenovirus-2, vesicular stomatitis virus, human coronavirus, influenza A/H1N1, influenza A/H3N2, influenza B, Coxsackie virus B4, reovirus and Sindbis virus was determined essentially as described previously [50, 51] . Confluent HEL fibroblasts were grown in 96-well microtiter plates and infected with the human CMV strains Davis and AD-169 at 100 PFU per well. After a 2 h incubation period, residual virus was removed and the infected cells were further incubated with the medium containing different concentrations of the compounds tested (in duplicate). After incubation for 7 days at 37 C, virus-induced cytopathogenicity was monitored microscopically and after ethanol fixation and staining with Giemsa (for CMV and VZV). Antiviral activity was expressed as the EC 50 or concentration required to reduce virus-induced cytopathogenicy by 50%. EC 50 values were calculated from graphic plots of the percentage of cytopathogenicity as a function of the concentration of the compounds. Cytostatic measurements based on the inhibition of HEL cell growth were performed as follows: HEL cells were seeded at a rate of 5 x 10 3 cells/well into 96-well microtiter plates and allowed to proliferate for 24 h. Then, medium containing different concentrations of the test compounds was added. After 3 days of incubation at 37 C, the cell number was determined with a Coulter counter. The cytostatic concentration was calculated as the CC 50 , or the compound concentration required to reduce cell growth by 50% relative to the number of cells in the untreated controls. CC 50 values were estimated from graphic plots of the number of cells (percentage of control) as a function of the concentration of the test compounds. Cytotoxicity was expressed as minimum cytotoxic concentration (MCC) or the compound concentration that causes a microscopically detectable alteration of cell morphology of the confluent cell cultures that were exposed to the compounds.

The authors declare no conflict of interest.

Ph-q), 128.7 À 127.8 (Ph)

Preparation of 2,3-O,O-dibenzyl-6-deoxy-6-[(4-(4-methylbenzenesulfonamide)methyl-1,2,3-triazol-1-yl

34 mg; 0.530 mmol); 4-methyl-N-(2-propynyl)benzenesulfonamide (164.6 mg; 0.790 mmol)

DMF (3 ml) and H 2 O (3 ml). Compound 8q

mp ¼ 116e118 C) was isolated as white powder. 4.1.2.18.2. Method B. Compound 8q was synthesized following )

300 MHz, DMSO) d 8.01 (bs, 1H, NH), 7.86 (s, 1H, H-7), 7.67 (d, J ¼ 8.2 Hz, 2H, Ph'), 7.48e7.19 (m, 12H, CH 2 Ph, Ph'), 5.65 (d, J ¼ 7.0 Hz, 1 H, OH), 5.22 (q, J ¼ 11,6 Hz, 2H, CH 2 Ph), 5.04e4.94 (m, 2H, CH 2 Ph

3 -O , O -d i b e n z y l -6 -[ ( 4 -( 4 -chlorobenzenesulfonamide)methyl-1,2,3-triazol-1-yl

DMF (3 ml) and H 2 O (3 ml). Compound 8r (154 mg

mp ¼ 123e124 C) was isolated as white powder. 4.1.2.19.2. Method B. Compound 8r was synthesized following )

300 MHz, DMSO) d 8.21 (t, J ¼ 5.1 Hz, 1H, NH), 7.89 (s, 1H, H-7), 7.75 (d, J ¼ 8.6 Hz, 2H, Ph'), 7.61 (d, J ¼ 8.6 Hz, 2H, Ph'), 7.45e7.22 (m, 10H, CH 2 Ph), 5.63 (d, J ¼ 7.1 Hz, 1H, OH), 5.21 (dd, J ¼ 22.0 Hz, 11.7 Hz, 2H, CH 2 Ph), 5.03e4.91 (m, 2H, CH 2 Ph), 4.88 (d, J ¼ 1.1 Hz, 1H, H-4), 4.56 (dd, J ¼ 13.8 Hz, 3.9 Hz

530 mmol); 2-chloro-4-fluoro-N-(2-propynyl)benzenesulfonamide (194.8 mg; 0.787 mmol)

Compound 8s (239.5 mg; 59%; m.p. ¼ 120e121 C) was isolated as white powder

247 mmol)

2-chloro-4-fluoro-N-(2-propynyl)benzenesulfona

22 (dd, J ¼ 34.6 Hz, 11.6 Hz, 2H, CH 2 Ph), 4.99 (q, J ¼ 11.2 Hz, 2H, CH 2 Ph

CH 2 NH) ppm. Calcd for C 29 H

Preparation of 2,3-O,O-dibenzyl-6-deoxy-6-[(4-(4-fluorobenzenesulfonamide)methyl-1,2,3-triazol-1-yl

20 mg; 0.315 mmol); 4-fluoro-N-(2-propynyl) benzenesulfonamide (81.9 mg; 0.384 mmol)

DMF (3 ml) and H 2 O (3 ml). Compound 8t (62 mg; 38%

¼ 128e130 C) was isolated as white powder. 4.1.2.21.2. Method B. Compound 8t was synthesized following

Cu (30

481 mmol)

600 MHz, DMSO) d 8.17 (bs, 1H, NH), 7.91 (s, 1H, H-7), 7.84e7.78 (m, 2H, Ph'), 7.47e7.27 (m, 12H, CH 2 Ph, Ph'), 5.66 (d, J ¼ 7.1 Hz, 1H, OH), 5.34e5.13 (m, 2H, CH 2 Ph), 4.99 (q, J ¼ 11.1 Hz, 2H, CH 2 Ph

Ph-q)

À 127.8 (Ph)

Ascorbate as a co-factor for fe-and 2-oxoglutarate dependent dioxygenases: physiological activity in tumor growth and progression

Sodium ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake

Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues

High-dose parenteral ascorbate enhanced chemosensitivity of ovarian cancer and reduced toxicity of chemotherapy

Enhanced antitumor activity of vitamin C via p53 in Cancer cells, Free Radic

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH

L-ascorbic acid: a true substrate for HIF prolyl hydroxylase

Natural product-derived small molecule activators of hypoxia-inducible factor-1 (HIF-1)

A conserved family of prolyl-4-hydroxylases that modify HIF

Intracellular ascorbate enhances hypoxia-inducible factor (HIF)-hydroxylase activity and preferentially suppresses the HIF-1 transcriptional response, Free Radic

Ascorbic acid and ascorbate-2-phosphate decrease HIF activity and malignant properties of human melanoma cells

The epigenetic role of vitamin C in health and disease

Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells

SVCT-2 in breast cancer acts as an indicator for L-ascorbate treatment

Novel 1,2,4-triazole and imidazole derivatives of l-ascorbic and imino-ascorbic acid: synthesis, anti-HCV and antitumor activity evaluations

The novel pyrimidine and purine derivatives of l-ascorbic acid: synthesis, one-and two-dimensional 1H and 13C NMR study, cytostatic and antiviral evaluation

Rai c-Mali c, Synthesis and antiviral and cytostatic evaluations of the new c-5 substituted pyrimidine and furo[2,3-d] pyrimidine 4 0 ,5 0 -didehydro-L-ascorbic acid derivatives

Rai c-Mali c, Synthesis, structural studies, and cytostatic evaluation of 5,6-di-O-modified L-ascorbic acid derivatives

The novel C-5 aryl, alkenyl, and alkynyl substituted uracil derivatives of l-ascorbic acid: synthesis, cytostatic, and antiviral activity evaluations

The growing impact of click chemistry on drug discovery

Design, synthesis and biological evaluation of novel l-ascorbic acid-conjugated pentacyclic triterpene derivatives as potential influenza virus entry inhibitors

Aspulvinones from a mangrove rhizosphere soil-derived fungus Aspergillus terreus Gwq-48 with anti-influenza A viral (H1N1) activity

Derivate der L-Ascorbins€ aure 1, Darstellung und Eigenschaften der O2,O3-Ethandiyl-und der O2,O3-Dibenzyl-L-Ascorbins€ auren

Derivate der L-Ascorbins€ aure 2, Darstellung von Deoxy-L-Ascorbins€ auren

4-Hydroxy-3-nitro-5-ureido-benzenesulfonamides selectively target the tumor-associated carbonic anhydrase isoforms IX and XII showing hypoxia-enhanced antiproliferative profiles

An update on anticancer drug development and delivery targeting carbonic anhydrase IX

Fluorinated benzenesulfonamide anticancer inhibitors of carbonic anhydrase IX exhibit lower toxic effects on zebrafish embryonic development than ethoxzolamide

Synthesis and anticancer activity of some novel trifluoromethylquinolines carrying a biologically active benzenesulfonamide moiety

A novel dithiocarbamate analogue with potentially decreased ALDH inhibition has copper-dependent proteasome-inhibitory and apoptosis-inducing activity in human breast cancer cells

Application of organic carbamates in drug design. Part 1: anticancer agents -recent reports

Organic carbamates in drug development. Part II: antimicrobial agents -recent reports

Rai c-Mali c, Synthesis, in vitro anticancer and antibacterial activities and in silico studies of new 4-substituted 1,2,3-triazoleecoumarin hybrids

Design and synthesis of novel 1,2,3-triazole-dithiocarbamate hybrids as potential anticancer agents

Continuous flowultrasonic synergy in click reactions for the synthesis of novel 1,2,3-triazolyl appended 4,5-unsaturated l -ascorbic acid derivatives

Drug-like properties and the causes of poor solubility and poor permeability

DataWarrior: an open-source program for chemistry aware data visualization and analysis

Novel pyrimidine-2,4-dionee1,2,3-triazole and furo[2,3-d]pyrimidine-2-onee1,2,3-triazole hybrids as potential anti-cancer agents: synthesis, computational and X-ray analysis and biological evaluation

Hypoxia-inducible factor-1 in human breast and prostate cancer

Hypoxia-inducible factor-1a is associated with angiogenesis, and expression of bFGF, PDGF-BB, and EGFR in invasive breast cancer

Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases

Expression of hypoxia-inducible factor-1a and cell cycle proteins in invasive breast cancer are estrogen receptor related

CTGF drives autophagy, glycolysis and senescence in cancer-associated fibroblasts via HIF1 activation, metabolically promoting tumor growth

Shaping genetic alterations in human cancer: the p53 mutation paradigm

Crosstalk between nitric oxide and hypoxiainducible factor signaling pathways: an update

Interactions between nitric oxide and hypoxiainducible factor signaling pathways in inflammatory disease

HIF-1a induces cell cycle arrest by functionally counteracting Myc

Synthesis and anticancer activity of g-(triazolyl ethylidene) butenolides and polyfunctional pyrrolinones

Reoptimization of MDL keys for use in drug discovery

R: the R project for statistical computing

Synthesis, cytostatic and anti-HIV evaluations of the new unsaturated acyclic C-5 pyrimidine nucleoside analogues

A novel selective broad-spectrum anti-DNA virus agent

Financial support from the Croatian Science Foundation under the project HRZZ-IP-2018-01-4682 is gratefully acknowledged.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.111739.