pharmaceuticals Article Synthesis of New 1, 3, 4-Oxadiazole-Incorporated 1, 2, 3-Triazole Moieties as Potential Anticancer Agents Targeting Thymidylate Synthase and Their Docking Studies 1 2 3, Mohammad Mahboob Alam , Abdulraheem SA Almalki , Thikryat Neamatallah y , 1 4 1, , Nada M. Ali , Azizah M. Malebari and Syed Nazreen * y 1 Department of Chemistry, Faculty of Science, Albaha University, Albaha-1988, Saudi Arabia; [email protected] (M.M.A.); [email protected] (N.M.A.) 2 Department of Chemistry, Faculty of Science, Taif University, Taif-21974, Saudi Arabia; [email protected] 3 Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia; [email protected] 4 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia; [email protected] * Correspondence: [email protected] These authors contributed equally to this work. y Received: 22 October 2020; Accepted: 12 November 2020; Published: 14 November 2020 Abstract: Thymidylate synthase (TS) has emerged as a hot spot in cancer treatment, as it is directly involved in DNA synthesis. In the present article, nine hybrids containing 1,2,3-triazole and 1,3,4-oxadiazole moieties (6–14) were synthesized and evaluated for anticancer and in vitro thymidylate synthase activities. According to in silico pharmacokinetic studies, the synthesized hybrids exhibited good drug likeness properties and bioavailability. The cytotoxicity results indicated that compounds 12 and 13 exhibited remarkable inhibition on the tested Michigan Cancer Foundation (MCF-7) and Human colorectal Carcinoma (HCT-116) cell lines. Compound 12 showed four-fold inhibition to a standard drug, 5-fluoruracil, and comparable inhibition to tamoxifen, whereas compound 13 exerted five-fold activity of tamoxifen and 24-fold activity of 5-fluorouracil for MCF-7 cells. Compounds 12 and 13 inhibited thymidylate synthase enzyme, with an half maximal inhibitory concentration, IC50 of 2.52 µM and 4.38 µM, while a standard drug, pemetrexed, showed IC50 = 6.75 µM. The molecular docking data of compounds 12 and 13 were found to be in support of biological activities data. In conclusion, hybrids (12 and 13) may inhibit thymidylate synthase enzyme, which could play a significant role as a chemotherapeutic agent. Keywords: thymidylate synthase; cytotoxicity; 1,2,3-triazole; 1,3,4-oxadiazole; 5-fluoruracil; pemetrexed; docking 1. Introduction Cancer is uncontrolled cell growth and cell proliferation, and remains a global burden despite the advancements in cancer diagnosis and treatment [1]. The available anticancer drugs on the market develop resistance against the chemotherapeutic agents and toxicity to normal cells [2,3]. Chemotherapy is the only effective treatment, causing the inhibition of cancer cell growth and induction of apoptosis, as the DNA levels in tumor cells are significantly higher than in normal cells [4,5]. Interestingly, thymidylate synthase (TS) has now emerged as an important target for chemotherapy for cancer treatment, as it is directly involved in DNA synthesis [6]. Pharmaceuticals 2020, 13, 390; doi:10.3390/ph13110390 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2020, 13, x FOR PEER REVIEW 2 of 15 Pharmaceuticals 2020, 13, 390 2 of 15 cells [4,5]. Interestingly, thymidylate synthase (TS) has now emerged as an important target for chemotherapyThymidylate for synthase cancer enzymetreatment catalyzes, as it is directly the reductive involved methylation in DNA synthesis of deoxyuridine [6]. monophosphate Thymidylate synthase enzyme catalyzes the reductive methylation of deoxyuridine (dUMP) to deoxythymidine monophosphate (dTMP) and 5,10-methylenetetrahydrofolate (CH2THF) [7, monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) and 8]. This dTMP upon phosphorylation changes into thymine triphospate (dTTP), which acts as a direct 5,10-methylenetetrahydrofolate (CH2THF) [7,8]. This dTMP upon phosphorylation changes into precursor for the synthesis of DNA [9,10]. The blocking of dTMP causes a decrease in deoxythymidine thymine triphospate (dTTP), which acts as a direct precursor for the synthesis of DNA [9,10]. The triphosphate (dTTP), thereby interrupting DNA biosynthesis causing DNA damage [11]. In addition, blocking of dTMP causes a decrease in deoxythymidine triphosphate (dTTP), thereby interrupting TSDNA inhibition biosynthesis causes causing an increase DNA in damage dUMP, [11]. leading In addition, to surge T inS inhibition the deoxyuridine causes an triphosphate increase in dUMP, (dUTP) levelleading [12]. to One surge of the in anticancerthe deoxyuridine drugs, triphosphate 5-fluoruracil (dUTP) (5-FU), actslevel as [12]. a strong One of thymidylate the anticancer inhibitor drugs, for various5-fluoruracil cancers (5 [13-FU)]. Also,, acts aas 5-FU a strong metabolite, thymidylate fluorodeoxyuridine inhibitor for various monophosphate cancers [13]. (FdUMP), Also, a 5- bindsFU withmetabolite, a TS active fluor siteodeoxyuridine to form a stable monophosphate ternary complex, (FdUMP) thus blocking, binds with the binding a TS active of the site dUMP to form with a TS andstable leading ternary to the complex, inhibition thus of blocking dTMP synthesisthe binding [14 of]. the dUMP with TS and leading to the inhibition of HeterocycledTMP synthesis has [14]. been a main pharmacophore in drug discovery and development. In the last few years, thereHeterocycle is an emergence has been a in main the development pharmacophore of diinff druerentg discov 1,2,3-triazole-linkedery and development. heterocycles, In the duelast to theirfew excellent years, there pharmacological is an emergence properties, in the development including anticancerof different [ 151,2,3], antiviral-triazole-linked [16], antidiabetic heterocycles [17, ], antimicrobialdue to their [ 18excellent], anti-inflammatory pharmacological [19 ],properties and antitubercular, including anticancer [20]. Compounds [15], antiviral containing [16], 1,2,3-triazolesantidiabetic have[17], beenantimicro reportedbial [18], to exert anti- anticancerinflammatory effect [19] by, and inhibiting antitubercular TS enzymes [20]. [21Compounds–23]. On the othercontaining hand,1,3,4-oxadiazole 1,2,3-triazoles have plays been a reported crucial part to exert in medicinal anticancer chemistry. effect by inhibiting Zibotentan, TS an anticancer drugenzymes containing [21–23 1,3,4-oxadiazole]. On the other hand, as a pharmacophore, 1,3,4-oxadiazole isplays used a forcrucial various part cancersin medicinal [24]. Furthermore,chemistry. 1,3,4-oxadiazole-linkedZibotentan, an anticancer heterocycles drug containing have also 1,3,4 been-oxadiazole reported as toa pharmacophore act as potential, TSis used inhibitors for various [25,26 ]. Therefore,cancers [24]. combining Furthermore, these two 1,3,4 heterocycles-oxadiazole- inlinked one moleculeheterocycles may have lead also to development been reported of etoff ectiveact as TS inhibitorpotential (Figure TS inhibitors1). [25,26]. Therefore, combining these two heterocycles in one molecule may lead to development of effective TS inhibitor (Figure 1). O N O HN NH NO HN NH H3C N 2 O O O O O S O O S O F HN N S F O N N HN HN F O N N N O O Cl N NO2 IC = 0.057 M N N 50 N IC = 0.62 M N 50 Cl IC = 0.03 M F 50 IC50 = 0.11 M O HN N H3C O O O O O F N HN S HO N N N O O N O N O S N N O O N N N IC50 = 0.11 M OH N Cl R IC50 = 1.67 M Target compounds FigureFigure 1. 1.Reported Reported thymidylate thymidylate synthasesynthase inhibitorsinhibitors containing 1,3,4 1,3,4-oxadiazole-oxadiazole and and 1,2,3 1,2,3-triazoles.-triazoles. InIn our our previous previous work, work, we have we reportedhave reported thiazolidinedione-linked thiazolidinedione-linked 1,3,4-oxadiazole 1,3,4-oxadiazole as a promising as a TSpromising inhibitor [ 27TS]. inhibitor In continuation [27]. In ofcontinuation our work to of develop our work an etoff ectivedevelop TS an inhibitor, effective we TS tried inhibitor, to conjugate we 1,3,4-oxadiazoletried to conjugate and 1,3,4 1,2,3-triazole-oxadiazole pharmacophore and 1,2,3-triazole in a pharmacophore single hybrid, which in a single can inhibit hybrid the, which TS enzyme can effectively.inhibit the In TS this enzyme work, we effectively describe. theIn t synthesis,his work, we pharmacokinetic describe the synthesis, study, anticancer, pharmacokinetic and TS inhibitory study, activities.anticancer The, and active TS inhibitory compounds activities. were docked The active against compounds the thymidylate were docked synthase against enzymes the thymidylate to see the molecularsynthase interactions enzymes to see of active the molecular compounds interactions with binding of active proteins. compounds with binding proteins. 2. Results and Discussion Pharmaceuticals 2020, 13, 390 3 of 15 2. Results and Discussion Pharmaceuticals 2020, 13, x FOR PEER REVIEW 3 of 15 2.1. Chemistry 2.1. Chemistry Esterification of benzoic acid 1 in the presence of MeOH and concentrated H2SO4 yielded methyl Esterification of benzoic acid 1 in the presence of MeOH and concentrated H2SO4 yielded benzoate 2, which further reacted with hydrazine monohydrate in absolute ethanol to give acid methyl benzoate 2, which further reacted with hydrazine monohydrate in absolute ethanol to give hydrazide 3. To this acid hydrazide 3 were added ethanol, carbon disulphide, and KOH, and the acid hydrazide 3. To this acid hydrazide 3 were added ethanol, carbon disulphide, and KOH, and mixture was stirred for 24 h and then refluxed