Design and antiproliferative and antioxidant activities of furan-based thiosemicarbazides and 1,2,4-triazoles: their structure-activity relationship and SwissADME predictions

Due to the limited number of drugs in current clinical use, the diverse biological applications of furan have encouraged the preparation of a wide variety of thiosemicarbazide and triazole derivatives for the purpose of developing new drug agents. This study aimed to investigate the antiproliferative and antioxidant activities of thiosemicarbazides (1–12) and 1,2,4-triazoles (13–24). Out of the synthesized target compounds, 3, 4, 6, 7, 8, 9, 10, 11, 12, 15, 16, 18, 19, 20, 21, 22, 23, and 24 are novel while the synthesis of the remaining compounds is present in the literature. Compound 15 (IC50: 8.81 ± 0.28 µM) showed the highest antiproliferative activity against the cervical (HeLa) cancer cell line among the compounds. In the lipid peroxidation inhibitory activity, thiosemicarbazide derivatives 3, 10, and 9 showed highest activity with IC50 of 21.80 ± 0.69, 26.49 ± 0.61, and 29.07 ± 0.52 µM, respectively, while triazole derivatives 15, 18, 19, 20, 21, and 22 exhibited the highest activity. Moreover, physicochemical properties, pharmacokinetic properties, and drug-likeness of all synthesized products were calculated using SwissADME. In addition, the effect of the structure–activity relationships of the 1,2,4-triazole derivatives (13–24) on the results of antiproliferative and antioxidant activity assays was evaluated.


Introduction
Cancer, which has been one of the world's biggest health problems for many years, is a malignant disease of the cell cycle that involves uncontrollable mitosis of abnormal cells, which invade surrounding tissues and often spread to other parts of the body [1,2]. About 11 million cases of cancer are diagnosed each year. Cancer, if not treated correctly, is likely to become widespread in a large proportion of the world's population [3,4]. The cervical cancer cell line known as HeLa, which was taken from Henrietta Lack, who passed away in 1951, is the oldest and most common cell line affecting women worldwide and cervical cancer ranks fourth in terms of both incidence and mortality [5,6]. The main treatments of the disease are surgery and radiotherapy. Surgery is performed if the disease is caught in the early stage of the disease, while radiotherapy is used for advanced stages.
In the many clinical studies on cervical cancer, satisfactory results have been obtained with chemotherapy such as increasing the five-year survival rate of patients [7,8]. Since the incidence of cervical cancer is at an undeniable level in young women, chemotherapeutic agents are needed [9]. However, despite superior studies on the design of effective chemotherapeutic drugs, there are still drawbacks involving toxicity and selectivity [10,11]. The problem of toxicity and resistance of cancer cells to anticancer agents has led to a continuous search for new chemotherapy agents. High amounts of reactive oxygen species (ROS) have been reported to promote many aspects of tumor development and progression in almost all types of cancer [12].
ROS produced by the cellular metabolism in living cells, while essential for life, can adversely cause the destruction of tissues or affect their normal functioning [13]. By attacking healthy cells, ROS can change cell structure or cause the cell to lose its ability to function [14]. All ROS form strand breaks or damaged bases because they have the potential to interact with DNA cellular components found in the genetic material to match its unstable free electron [13]. ROS-induced oxidative damage plays an important role in the development of diseases such as neurodegenerative disorders, arthritis, arteriosclerosis, inflammation, weakening of the immune system, liver disease, brain dysfunction, cardiovascular events, diabetes, and kidney failure [15]. Furthermore, many researchers suggest that ROS damage plays a vital role particularly in the development of malignant cancer and the initiation of proliferation of cancerous cells [13]. Antioxidant defense system agents developed for the elimination or cleaning of ROS in order to prevent permanent diseases caused by ROS can prevent oxidative chain reactions, direct quenching of reactive oxygen species, enzyme inhibition, reactions responsible for chelating metal ions such as Fe +2 and Cu + , and free radicalmediated oxidative damage of biomolecules such as proteins, nucleic acids, polyunsaturated lipids, and sugars even at low concentrations [16]. Therefore, there is a need to develop radical scavenging antioxidant agents to control the harmful effects of free radicals in the human body.
Heterocycles are compounds where the cyclic ring contains heteroatoms i.e., nitrogen, oxygen, and sulfur. They are capable of various supramolecular interactions including hydrogen bonding, self-assembly, pi-stacking, ability to bind to enzymes, forming coordination bonds with metals and Van der Waals and hydrophobic forces [17,18]. Many enzyme binding pockets undergo interactions with heterocycles as they possess versatile functionalities and can plays important roles in the biological pathways. There are various studies where heterocycles have been found to treat cancer or disrupt the continuity of the cancer progression [18]. In particular, furan-derived rings are an important class of heterocyclic compounds with very important biological properties. Many researchers have shown intense interest in the synthesis of a new furan-derived scaffold with different pharmacological activities for the discovery of new drugs in recent decades [19]. On the other hand, 1,2,4-triazole (C 2 N 3 H 3 ) derivatives containing three nitrogen atoms from five-membered ring systems, which were firstly described by Blodin, have become the focus of attention of many research teams for obtaining synthetic agents with high bioavailability due to the known antidepressant, anticancer, anti-inflammatory, analgesic, antiviral, and antioxidant effectiveness of these compounds [20][21][22].
To date, all triazoles recorded in the literature have been of synthetic origin, and there is no study showing that a triazole and its derivative isolated from natural products have been detected [23]. The synthesis strategy of the present study, inspired by the known anticancer therapeutic effect of 1,2,4-triazole, due to the need to discover alternative agent(s) for cancer, which is the biggest health problem of the age, started with the synthesis of some thiosemicarbazide derivatives (1-12) from heterocyclicbased 2-furanoylhydrazide, followed by the synthesis of the activity potential agent 1,2,4-triazole derivatives (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). After the structures of all the derivatives synthesized were elucidated, their in vitro antiproliferative activity against the HeLa cancer cell line and in vitro antioxidant activity were tested. In addition, the physicochemical properties (including Lipinski), pharmacokinetics, drug-likeness, and medicinal chemistry properties of products synthesized  were calculated using the program SwissADME.
In the 13 C NMR spectra, the C-5 carbon of azomethine (-C = N-) and the C 8   protons and carbons were shifted up or down by the influence of electron-withdrawing or electron-donating groups connected to the aromatic ring. Three spin systems were observed in the COSY spectrum (H-14-H-15, H-11-H-12) of compound 24, which was chosen as a model compound for the 2D NMR spectrum (Fig. S1).
The values obtained from the elemental analysis of thiosemicarbazides 1-12 and 1,2,4-triazole derivatives 13-24 were compatible with the calculated values.

SwissADME Prediction of synthesized compounds
The data predicted for the physicochemical characteristics, lipophilicity, solubility, pharmacokinetics, drug likeness, and medicinal chemistry of synthesized compounds evaluated by SwissADME are given in Table S1 (see: supplementary file). According to Lipinski's rule of five, the molecular weights of thiosemicarbazide and 1,2,4-triazole derivatives were 261.30-363.74 and 243.28-345.73 Da, respectively, and within the limits of 200-600 Da. The logP values of the thiosemicarbazide (1-12) and 1,2,4-triazole (13-24) derivatives were less than 5, in the range of 2.24-3.11. The HBA number of all synthesis products was between 2 and 5 and less than 10; the number of HBD atoms for the thiosemicarbazide (1-12) and 1,2,4-triazole (13-24) derivatives was 3 and 1, respectively, less than 5 [28]. Brain Or IntestinaL EstimateD permeation (BOILED-Egg) method is a graphical model that works by calculating the polarity and lipophilicity of small molecules. This prediction provides a visual clue to the synthesis design of new compounds in terms of the oral absorption potential of drug candidates [29]. Graphical estimations of gastrointestinal absorption and blood-brain barrier (BBB) penetration of the synthesized thiosemicarbazide and 1,2,4-triazole derivatives are shown in Fig. 2. According to the BOILED-Egg plot, none of the compounds synthesized were located in the BBB (except compound 13 of the 1,2,4-triazole derivatives) with the yellow circle expressing good intestinal absorption and the gray region representing poor intestinal absorption. All compounds (except compound 13) are contained within the while ellipse representing human intestinal absorption.
Compound 10 was found to be the blue spot, evidence of its good bioavailability. Thus, compound 10 demonstrated that it could be a substrate for P-glycoprotein and it could reduce its absorption and penetration in the brain. All compounds except compound 13 can be promising agents that can very easily be absorbed by the gastrointestinal tract without potential BBB permeability. Since these compounds cannot cross the BBB, they do not cause central nervous system depression or drowsiness as side effects.

General
Analytical grade chemicals and solvents were purchased from Acros, Alfa Aesar, Sigma-Aldrich, and Merck. Thin layer chromatography (TLC, Merck 60 F 254 ) was used to monitor the chemical reactions. The melting points were examined using an SMP20 melting point apparatus and were uncorrected. The FTIR spectra were acquired using a

Synthesis
General synthetic procedure for compounds 1-12 2-Furoic acid hydrazide (0.01 mol) and substituted phenylisothiocyanate (0.01 mol) were dissolved in methanol and the mixture was refluxed overnight. After completion of the reaction, the mixture was left to cool down in order to precipitate the solids. Finally, ethanol was used to purify the precipitate [31].

General synthetic procedure for compounds 13-24
A mixture of thiosemicarbazides 1-12 (0.45 mmol) and 2 N NaOH solution (10 mL) was refluxed for 4 h. The reaction mixture was filtered, allowed to cool, and then brought to pH 5.0-6.0 with a dilute solution of HCl. The crude product was dried, washed with water, and recrystallized from ethanol [31].

Biological studies
Antiproliferative activity assay The antiproliferative assessment of the compounds was conducted on HeLa (cervical) cells using the BrdU ELISA [33][34][35][36]. The results of this in vitro investigation were given as means ± SEM of six parallel measurements (p < 0.01).
IC 50 values were calculated using the online software ED 50 plus v1.0.

In silico ADME prediction
Computational studies of the synthesized compounds 1-24 were performed to predict molecular properties using the SwissADME online server [41]. The molecular volume (Mv), molecular weight (Mw), logarithm of partition coefficient (milog P), number of hydrogen-bond donors (HBDs), number of hydrogen-bond acceptors (HBAs), topological polar surface area (TPSA), number of rotatable bonds (Nrotbs), and Lipinski's rule of five of the synthesized compounds were determined.

Statistical analysis
The antiproliferative and antioxidant activity data were given as the means of six and three parallel measurements, respectively. All biological activity assays were carried out at four different concentrations, and the results were presented as IC 50 values. The data were recorded as mean ± SEM (standard error of the mean); p < 0.01.