Synthesis, Antitumor and Antiviral In Vitro Activities of New Benzotriazole-Dicarboxamide Derivatives

Cancer and viral infections continue to threaten humankind causing death worldwide. Hence, the discovery of new anticancer and antiviral agents still represents a major scientific goal. Heterocycles designed to mimic the chemical structure of natural pyrimidines and purines have been designed over the years, exerting their activity acting as false substrates on several different targets. We reported a series of bis-benzotriazole-dicarboxamide derivatives which inhibit viral helicase of poliovirus, and hence we planned structure modifications to obtain different series of new dicarboxamides. Here, the synthesis and characterization of 56 new compounds: 31 bis-benzotriazole dicarboxamides and 25 mono-substituted acidic derivatives are reported. The synthesized compounds were tested for their antiviral and antitumor activity. Mostly, compounds 4a, 4c and 4d showed antiviral activity against tested Picornaviruses, Coxsackievirus B5 and Poliovirus-1. Likewise, four derivatives (3b, 3d, 4d, 9b) showed notable antiproliferative activity inhibiting cell growth in two distinct antitumor screenings. Compound 3b was selected as the antitumor lead compound for the wide range of activity and the potency proved. The lead compound was proved to induce apoptosis in SK-MES1 tumor cells, in a dose-dependent manner.


INTRODUCTION
Among the various diseases that affect humankind, cancer and viral infections remain great threats to human health worldwide. Therefore, the identification of novel anticancer and antiviral agents remains one of the most pressing health problems and the most stimulating cue for research. Polycyclic benzo-fused azoles can mimic the chemical structure of natural pyrimidines and purines, exerting their biological activity acting as false substrates, resulting in antitumor, antibacterial or antiviral agents. Considering the similar mechanism of action of the latter, cross-activity has been highlighted for several molecules (Kaufman, 1980;Matsuda et al., 1999;Ramachandran et al., 2011;Brüning et al., 2012). Therefore, anticancer drugs have been proved active against viruses (Kaufman, 1980) and antiviral agents have shown antimetabolite activity (Bergman et al., 2002;De Clercq, 2004;Brüning et al., 2012). 5-fluorouracil is a well-known example; it is a widely used anticancer agent which acts as antimetabolite. It was proved active against several viruses such as foot and mouth disease virus (Pariente et al., 2003;Agudo et al., 2009), herpes simplex virus (Dragún et al., 1990), poliovirus (Cooper, 1964). Purine-like antimetabolites enter the metabolic pathway acting as false substrates or mimicking the natural building blocks of RNA and DNA (De Clercq, 2013). For these features, besides humancell targets, viral enzymes involved in RNA and DNA synthesis, such as RNA-dependent-RNA-polymerases (RdRp) (Furuta et al., 2017), helicases (Carta et al., 2007), proteases (Kneller et al., 2019), may be targeted by this class of derivatives. Benzotriazole (BT) derivatives have been widely studied for their broad biological activity (Briguglio et al., 2015). BT derivatives may mimic purine ring acting as antimetabolites interfering with purine metabolism but may also bind the enzymes that naturally interact with purines, mimicking the natural substrate, resulting in potential antitumor (Al-Soud et al., 2003), antibacterial (Sanna et al., 1992;Carta et al., 2011;Ochal et al., 2013), antifungal (Patel et al., 2010) or antiviral (Borowski et al., 2003) agents. In the process of new drug discovery, we designed several promising antiviral active derivatives bearing the benzotriazole scaffold (Ibba et al., 2018;Piras et al., 2019a;Piras et al., 2019b;Sanna et al., 2020). Amongst them, benzotriazole derivatives depicted in Figure 1 have been designed, synthesized and proved as antiviral agents against two tested Picornaviruses (Coxsackievirus B2 and Poliovirus Sb-1) (Carta et al., 2007). Bis-benzotriazole-dicarboxamide derivatives ( Figure 1) were proved to exert their antiviral activity as false substrates binding the Poliovirus helicase in in silico assays, performed on the 3D model of the target protein (Carta et al., 2007). Bis-benzotriazole-dicarboxamide derivatives from series 2 turned out as the most active with EC 50 values ranging from 4 to 33 μM against Coxsackievirus B2.
Based on this, we designed and synthesized a new series of bisbenzotriazole-dicarboxamide derivatives as potential anti-Picornavirus agents, acting as false substrates. The structural modifications applied to the main scaffold are shown in red in Figure 2. We evaluated the substitution on the benzotriazole scaffold in positions 5 and 6 (R). We also considered the length and the role of the linker amidst the two amidic groups (X), including saturated or unsaturated aliphatic or small aromatic linkers. The insertion of a CH 2 bridge between the BT scaffold and the aromatic moiety directly connected to the BT nitrogen was also attempted.
All the synthesized compounds were tested against a panel of Picornaviruses and several cell lines to assess the cytotoxicity. Only a few of the designed molecules turned out active against tested viruses, while some were selected for antiproliferative assays, based on the known antiviral-anticancer correlations. Notably, four derivatives (3b, 3d, 4d, 9b) showed interesting antiproliferative activity inhibiting cell growth and inducing apoptosis. Compound 3b was selected as the lead compound to be subjected to further structure-modifications in order to improve the antiproliferative activity.

Chemistry
Reaction progression, retention factors (R f ) and purity of compounds were monitored by TLC using Merck F-254 commercial plates and a proper mixture of solvents as eluent. Melting points were taken in open capillaries in a Köfler hot stage or Digital Electrothermal melting point apparatus. 1 H-NMR spectra were recorded on a Nuclear magnetic resonance ( 1 H-NMR) spectra were determined in DMSO-d 6 and were recorded with a Bruker Avance III 400 NanoBay (400 MHz) and a Varian  instruments. Chemical shifts (δ scale) are reported in parts per million (ppm) downfield from tetramethylsilane (TMS) used as internal standard. The chemical shift values are reported in ppm (δ) and coupling constants (J) in

Cytotoxicity Assays
Vero-76 cells were seeded in 96-well plates at an initial density of 5 × 10 5 cells/ml, in Dulbecco's Modified Eagle Medium (D-MEM) with L-glutamine and 25 mg/L kanamycin, supplemented with 10% FBS. Cell cultures were then incubated at 37°C in a humidified and 5% CO 2 atmosphere, in the absence or presence of serial dilutions of test compounds. The test medium used for the cytotoxic assay as well as for antiviral assay contained 1% of the appropriate serum. Cell viability was determined after 48-96 h at 37°C by MTT method for Vero-76 (Pauwels et al., 1988).

Antiviral Assays
Compounds activity against CV-B5 and Sb-1 was determined by plaque reduction assays in infected cell monolayers. Briefly, monolayer of Vero-76 cells was grown overnight on 24-well plate. The cells were then infected for 2 h with 250 μl of proper virus dilutions to give 50-100 PFU/well. Following removal of unadsorbed virus, 500 μl of medium (D-MEM with L-glutamine and 4,500 mg/L D-glucose, supplemented with 1% inactivated FBS) containing 0.75% methyl-cellulose, with serial dilutions of test compounds, were added. The overlayed medium was also added to not treated wells as non-infection controls. Cultures were incubated at 37°C for 2 days for Sb-1 and 3 days for CV-B5 and then fixed with PBS containing 50% ethanol and 0.8% crystal violet, washed and air-dried. Plaques in the control (no inhibitor) and experimental wells were then counted (Sanna et al., 2015).

Antiproliferative Assays
Cell lines were purchased from American Type Culture Collection (ATCC). The absence of mycoplasma contamination was checked periodically by the Hoechst staining method. Exponentially growing leukemia and lymphoma cells were seeded at 1 × 10 5 cells/ml in 96 well plates in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin, and incubated at 37°C in a humidified, 5% CO 2 atmosphere in the absence or presence of serial dilutions of test compounds. Cell viability was determined after 96 h at 37°C by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method. Activity against solid-tumor derived cells was evaluated in exponentially growing cultures seeded at 5 × 10 4 cells/ml and allowed to adhere for 16 h to culture plates before the addition of the drugs. Cell viability was determined by the MTT method 4 days later. Vincristine and doxorubicin were used as reference drugs.

Linear Regression Analysis
The extent of cell growth/viability and viral multiplication, at each drug concentration tested, were expressed as percentage of untreated controls. Concentrations resulting in 50% inhibition (CC 50 or EC 50 ) were determined by linear regression analysis.

NCI60 Human Tumor Cell Lines Screen
The in vitro anti-cancer screening was performed through the NCI60 Human Tumor Cell Lines Screen, provided by the Developmental Therapeutics Program of the National Cancer Institute (NCI, Bethesda, United States). A single dose of 10 µM of each compound is tested in the whole NCI60 cell panel. More details about the NCI60 screen methods are reported on the NCI website (https://dtp.cancer.gov).

Apoptosis Assay
To assess which death mechanisms our compounds induced, cell apoptosis kit Annexin V/Propidium iodide (PI) double staining uptake (Invitrogen, Life Technologies, Italy) was used. 3b compound was selected as lead and employed in our assay. Human lung cancer cells (SK-MES 1) were seeded at the density of 8 × 10 5 cells/ml in 6-well plates (Corning, United States) with a complete medium (described in cell culture section). After overnight incubations, the cells were treated with or without different concentrations of 3b for 96 h. Cells were then labeled with Annexin V and PI as previously described (Ibba et al., 2021). Stained cells were then analyzed by flow cytometry, measuring the fluorescence emission at 530 and 620 nm using 488 nm excitation laser (MoFloAstrios EQ, Beckman Coulter). Cell apoptosis was analyzed using Software Summit Version 6.3.1.1, Beckman Coulter.

Antiviral activity
All the 56 synthesized compounds, depicted in Figure 3, were tested in a plaque-reduction assay for antiviral activity and in a cell-based assay for cytotoxicity. Viruses selected for the assay were Coxsackievirus B5 (CV-B5), a well-known human pathogen and the Poliovirus strain Sb-1, both belonging to the family of Picornaviridae. Compounds cytotoxicity was evaluated against three cell lines (Vero-76, MDBK, BHK-21).  ). Surprisingly, the mono-substituted derivatives obtained as secondary products were found more active than the corresponding dicarboxamides. Compound 4c was the most active against CV-B5, along with 4a and 4d with EC 50 values ranging from 9 to 13 µM. The latter two are also

Antitumor activity
All synthesized compounds was also tested for antitumor activity against 7 cancer cell lines. For this purpose, human leukemialymphoma-derived cell lines CCRF-CEM, WIL-2NS, CCRF-SB and solid tumor-derived cell lines SK-MEL28, SK-MES 1, DU145, HeLa were selected. Fibroblast CRL 7065 cell line was used as control. Table 2 shows the results of the most promising compounds, derivatives 3b, 3d among bis-benzotriazoledicarboxamides and 4d, 9b among mono-substituted acidic derivatives. In general terms, compound 3b showed the widest range of activity reducing cell proliferation of six out of seven cell lines with CC 50 ranging in the low micromolar values. Derivative 3d had the lowest CC 50 value against CCRF-CEM cell line (70 nM), while resulted inactive against most of the other tested cell lines. Among the mono-substituted acidic derivatives, 4d was proved active when tested on human leukemia-lymphoma-derived cell lines while completely inactive against solid tumors proving an interesting selectivity of action. Compound 9b is the one that turned out active against all the tested cancer cell lines, proving a wide range of action, showing, however, very low CC 50 values. Dicarboxamides turned out slightly more cytotoxic when tested on CRL 7065 control cell line than the tested mono-substituted derivatives. To determine the cytotoxic selectivity of tested compounds, the selectivity index (SI) was calculated as a ratio of CC 50 of non-tumor cells and CC 50 of tumor cells. Since the non-specific mechanism of action of designed compounds SI values turned out very low, except for compound 3d. The latter possessed CC 50 values of 70 nM against tumor CCRF-CEM cell line and 370 nM against non-tumor CRL 7065 cell line, with a resulting SI of 5.3. The above-mentioned four promising compounds (3b, 3d, 4d, and 9b) were then subjected to the NCI60 Human Tumor Cell Lines Screen (full tables of results in Supplementary Material). This screening was performed by the National Cancer Institute (NCI, Bethesda, United States), and evaluated our compounds in the anticancer assay at 10 μM concentration against a panel of 60 human tumor cell lines. The panel comprises a series of different cancer lines comprising hematological (leukemia) tumors and solid ones (non-small cell lung -NSCL, colon, central nervous system -CNS, renal, ovarian, breast and prostate cancers and melanoma). Among the tested compounds, 3b turned out as the most active with the widest range of action. Results from the assays are reported as Percentage of Growth Inhibition (PGI) and are graphed in bar charts depicted in Figure 4. Cell lines are grouped per type of cancer. Our lead compound 3b showed PGI values higher than 50% for 29 of the 60 cell lines, proving a wide antiproliferative activity. Best scores were recorded for breast cancer since most of the PGI values ranged from 48 up to 81%, and for prostate cancer (80 and 66% values of PGI against the two cell lines). Compound 3b can be considered as an interesting antiproliferative agent against ovarian (OVCAR-8, SK-OV-3), CNS (SF-295, SNB19, SNB75, U251) and NSCL (A549/ATCC, HOP-62, NCI-H226, NCI-H23, NCI-H460, NCI-H522) cancers, while the growth of leukemia, melanoma, renal and colon cancer cells was less affected by the administration of derivative 3b. The latter was also proved cytotoxic more than antiproliferative, against one ovarian cancer cell line (OVCAR-4) showing a PGI of 105.56%.

Apoptosis Assay
Aiming to investigate the mechanism wherewith the cells die after lead compound administration, an apoptosis assay was performed. Data plots were produced by employing normalized fluorescent expression of Annexin V and PI ( Figures 5A-C). Based on intensities distribution the percentage of living, early apoptotic, late apoptotic, and necrotic cells in untreated and treated conditions were analyzed. In the untreated cells (control) the majority (91.4%) of the cells were viable, the remaining cells died in the apoptotic and necrotic way (4.4 and 4.2%, respectively) ( Figure 5A). Compound 3b induced apoptosis after 96 h in a dose-dependent manner with 40% of apoptotic cells in 20 µm treated and 32% in 7 µm treated cells ( Figures 5B,C

CONCLUSION
We have designed and synthesized 56 new compounds, 31 bis-benzotriazole dicarboxamides (3a-g, 5e-g, 7a-g, 8ag, 9a-g) and 25 corresponding mono-substituted acidic compounds (4a-d,f, 6f, 10a-g, 11a-g, 12a-d,f) as potential antiviral and/or antitumor agents, acting as false substrates. They were purified, characterized and tested for antiviral and antitumor activity in properly selected assays. Only a few of the synthesized compounds turned out as active against Coxsackievirus B5. Compounds 4a, 4c, and 4d showed EC 50 values ranging from 9 to 13 µM against CV-B5, while derivative 9a is the sole compound among the bisbenzotriazole dicarboxamides that turned out quite active against both the tested Picornaviruses with EC 50 values of 23 and 43 µM, against CV-B5 and Sb-1, respectively. From a selection of representatives that were subjected to an antitumor in vitro assay, four of them (3b, 3d, 4d and 9b) resulted as the most interesting for their CC 50 values that Frontiers in Chemistry | www.frontiersin.org May 2021 | Volume 9 | Article 660424 mostly turned out to be in the micromolar range. The same derivatives were tested by the National Cancer Institute on a panel of 60 human tumor cell lines. All the newly synthesized compounds that showed antiviral or anticancer activity bear two chlorine atoms on the BT scaffold (series 3, 4 and 9). Concerning the linker, the medium-length (-(CH 2 ) 3 -) and the unsaturated (-CH CH-) ones showed the best antitumor results (3b, 3d, 4d, and 9b), while the aromatic linkers entail a complete loss of activity. 1H-BT moiety turned out to be the scaffold endowed with both biological activities (series 3, 4, and 9).
For the wide range of activity and the potency proved by the two screening assays, compound 3b was selected as lead compound. When derivative 3b was evaluated in apoptosis assay, results showed that the compound induced cell death by apoptosis in human lung tumor SK-MES 1. These findings will grant further studies involving this promising antiproliferative candidate. The latter will be used as a starting point for the next generation of compounds that will be the result of a proper structure modification process in order to increase the antiproliferative activity and potency. Alongside, compound 4c will be used as a base to design new mono-substituted acidic compounds endowed with selective anti-Coxsackievirus activity.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
SP and AC conceived and designed the experiments. RI, SP, PC, and FR synthesized the compounds. RL, ID, GC, GS, PC and TC performed antiviral, antiproliferative and apoptosis experiments. All authors analyzed the data. SP and RI wrote the paper. All authors reviewed and approved the manuscript.