Novel thiadiazole-thiazole hybrids: synthesis, molecular docking, and cytotoxicity evaluation against liver cancer cell lines

One of the worst diseases, cancer claims millions of lives each year throughout the world, necessitating the creation of novel treatments. In this study, we designed a novel series of 1,3,4-thiadiazoles through the reaction of 2-(4-methyl-2-(2-(1-phenylethylidene)hydrazineyl)thiazole-5-carbonyl)-N-phenylhydrazine-1-carbothioamide (3) with the proper hydrazonoyl halides. Using the MTT assay, the newly synthesized thiadiazoles' growth-inhibitory potential against the liver cancer cell line HepG2-1 was assessed. In comparison to the standard drug doxorubicin (IC50 = 0.72 ± 0.52 µM), the results showed that two compounds, 16b and 21 (IC50 = 0.69 ± 0.41 and 1.82 ± 0.94 µM, respectively) had promising anticancer activity. The structural activity relationship (SAR) was investigated. In addition, molecular docking analysis onto quinone oxidoreductase2 (NQO2) receptor (PDB: 4ZVM) was investigated against the potent compounds to examine the reliability of the in vitro results. The newly prepared thiadiazole-thiazole hybrids are therefore regarded as potent anticancer drugs.


Introduction
Cancer is currently the second leading explanation for death worldwide [1]. Over 90% of primary liver tumours are hepatocellular carcinomas (HCC), which are primary tumours of the liver. Of patients with cirrhosis, HCC occurs in about 85% of cases [2]. HCC is currently the sixth most prevalent kind of cancer in the world, but it's also the third most frequent cause for cancer mortality [3]. HCC is that the second greatest reason for cancerrelated death in men, after carcinoma [3]. HCC incorporates a 5-year survival rate of 18%, which is second only to carcinoma [4]. Hepatitis (hepatitis B and C), alcoholic disease, and non-alcoholic steatohepatitis/nonalcoholic liver disease are all significant risk factors for hepatocellular cancer. HCC occurs in between 80% and 90% of cirrhotic patients. It is still a difficult task for medicinal chemists to research fresh leads for the development of novel structures that could be beneficial in developing more practical and fewer toxic new anticancer medicines. Medical researchers have focused on 1,3-thiazole and 1,3,4-thiadiazole systems in their quest to produce effective anticancer drugs.
As a follow-up to our studies [6][7][8][21][22][23][24][25] on the use of hydrazonoyl halides in the preparation of different bridgehead nitrogen polyheterocycles, we would like to present here a novel, simple method for producing a number of different functionalized 1,3,4-thiadiazoles derivatives having thiazole moiety as potent cytotoxic agents against liver cancer cell lines. Furthermore, GLIDE (Grid-based Ligand Docking with Energetics) of Schrödinger was used to explore the possible binding interactions of the most active derivatives onto the Protein quinone reductase-2 (4ZVM).
As shown in Scheme 1, the reaction of carbohydrazide derivative 2 with phenyl isothiocyanate in EtOH produced the N-phenylhydrazine-1-carbothioamide derivative 3. The spectral data (IR, NMR, and ms) for compound 3 and its structure were in agreement.
The thiosemicarbazone derivative 3, which is produced when 1,3,4-thiadiazoles combine with hydrazonoyl chlorides, offers synthetic methods for producing useful heterocycles. Thus, a catalytic quantity of Et 3 N allowed compound 3 to react with hydrazonoyl chloride 4 in EtOH to yield a single product, which was recognized as the respective 1,3,4-thiadiazole derivative 6 (Scheme 1). The structure 6 was elucidated based on study of spectral and elemental data (see Experimental part). The 1,3,4-thiadiazole derivative 6 was produced by cyclization through the elimination of the aniline molecule after the reaction was carried out by the nucleophilic displacement of the chlorine atom, which produced the thiohydrazonate intermediates.
Product 6's alternate technique provided more proof of the structure attributed to it. Thus, the hydrazine derivative 7 [28] and ester 1 were allowed to react producing product 6 (Scheme 1).
An alternative technique was used to demonstrate the chemical structure of 10 (Scheme 2). As a result, the reaction of compound 13 with 9a in refluxing EtOH containing Et 3 N produced a product that is completely identical to compound 11a.
Compounds 14a,b also interacted with derivative 3 to produce the corresponding thiadiazoles 16a,b (Scheme 3) in a similar manner. When 16a was used as an example of the constructed series for IR, 1 H-NMR and 13 C-NMR spectra, it could be seen that the hydrazone-NH function had disappeared while the ester CO 2 CH 2 CH 3 existed excluding the other structure 18a. A peak corresponding to each of the reaction products could be seen in each case's mass spectrum.
Additionally, a different technique was used to illustrate chemically the structure of 16 (Scheme 3). As a result, the reaction of compound 13 with compound 14a in refluxing EtOH/TEA produced a product that is completely identical to compound 16a in all respects.
Additionally, compound 19 and compound 3 were reacted to produce compound 21 (Scheme 4). Compound 21's 1 H-NMR spectrum showed a signal at 11.70 ppm corresponding to the carboxamide proton and an aromatic multiplet in the 7.12-7.74 ppm region. Compound 21's mass spectra showed a m/z = 568, which agrees with the suggested structure.
Also, compound 21 was proved chemically using a different technique from the reaction of compound 13 with 19 resulted in a product, which is identical to compound 21 in all aspects (Scheme 4).
The liver carcinoma (HepG2-1) cell line was used to test the antitumour effect of the newly prepared derivatives. As a reference standard, doxorubicin showed an  According to the results, as shown in Table 1, the most of the investigated compounds revealed a highly variable activity when compared to the reference drug. The descending order of activity of the newly synthesized compounds was as follow: 16b > 21 > 16a > 3 > 11b > 11a > 6.
The following conclusions are drawn from an examination of the SAR (Figure 1 group than the COCH 3 group, and finally the Ph group Figure 2.

Molecular docking study
N-Ribosyldihydronicotinamide: quinone oxidoreduc-tase2 (NQO2/QR2) is a cytosolic enzyme, that is highly concentrated within the liver and can be found in various tissues throughout mammals [36]. It is a critical quinone's metabolism detoxification enzyme. Compared to other types of cancer, hepatocellular tumour tissue has a higher NQO2 activity [37]. Thus, in the present work, the NQO2 receptor PDB (4ZVM) was chosen from the protein database to explore possible binding interactions of the most active compounds using molecular modelling analysis [38,39]. Molecular docking simulations were done against the NQO2 receptor to achieve a multitarget approach and identify a promising candidate. The simulation was done using the GLIDE of Schrödinger [40]. The affinity of the novel synthesized compounds was investigated via SP Glide docking mode against the receptor (4ZVM). In terms of accuracy, we have re-docked the co-crystallized inhibitor doxorubicin into the receptor's active site using the generated grid for validation. The re-docked doxorubicin showed similar binding modes under the criterion of RMSD 1.6 A°as presented in Figure 3. The novel compounds were similarly docked in the quinone oxidoreductase2 4ZVM. The resulted SP docking score of the studied compounds was between −7.01 and −4.31 kcal/mol ( Table 2). Compound 16b, the most potent inhibitor against HepG2liver cell lines, showed the lowest docking score (−7.01 kcal/mol) with glide emodel −88.59. The 16b-4ZVM complex demonstrated four H-bonds with amino acids TRP 105, ASN161 and THR147 as presented in Figure 4. Previous research has indicated that the inhibitor resveratrol fitted deeply into the NQO2 catalytic site and potently inhibits NQO2 activity via forming a direct hydrogen bond with Asn116 [34] (1). This may justify the efficacy of compound 16b which displayed two hydrogen bonds with ASN161 (2.28 and 2.68 A°). The interaction network of 16a-4ZVM and 11b-4ZVM showed one hydrogen bond with ASN161 and GLU193, respectively. While the interaction mode of 3 presented two H-bonds against ASN161 (2.54 A°) and TRP105 (1.83A°). Compound 21 with a docking score of −6.42 kcal/mol, is located in the cleft of the active pocket but it did not establish any hydrogen interaction with the receptor's amino acids. It was bonded to the receptor via pi-pi stacking against TRP105 residue (Figure 4). The docked compounds 6 and 11a unveiled a similar pattern (two π-π stacking against TRP105) with a higher docking score of −4.31 and −5.10 kcal/mol, respectively.

Experimental
See supporting information.

Synthesis of compound 3
A mixture of carbohydrazide derivative 2 (2.89 g, 10 mmol) and phenyl isothiocyanate (1.35 g, 10 mmol) in ethanol (50 mL) was refluxed for four hours. The formed solid after cooling was recrystallized from EtOH to get the yellow product (

Alternate synthesis of 1,3,4-thiadiazole derivative 6
Equimolar concentrations of 1,3,4-thiadiazole 7 (0.268 g, 1 mmol) and ester 1 (0.303 g, 1 mmol) were mixed with 20 mL of EtOH and refluxed until no more H 2 S was emitted, and all of the starting materials had been consumed (12 h, monitored by TLC). The separated product was identified as compound 6.

Synthesis of compound 13
Addition of carbohydrazide 2 (2.89 g, 10 mmol) to a stirred KOH solution (0.56 g, 10 mmol) in DMF. After cooling, CS 2 (10 mmol) was progressively added, and the reaction mixture was heated under reflux for 2 h before adding CH 3 I (1.4 g, 10 mmol) dropwise while cooling with an ice bath. The precipitated product which formed after addition solution into 10 mL of H 2 O, was suction-isolated and crystallized from EtOH to yield compound 13 as a white solid in 68% yield; m.p.  synthesis of 1,3,4-thiadiazole  derivatives 11a, 16a and 21 Triethylamine (0.1 mL, 1 mmol) was added to an ethanolic solution of compound 13 (0.379 g, 1 mmol) and hydrazonoyl chloride 9a, 14a, or 19 (1 mmol). The resulting solid after removal of excess solvent and addition of ice/HCl solution, was recrystallized from DMF to provide the corresponding thiadiazoles 11a, 16a, or 21, which were similar in all aspects (IR spectra, mixed m.p. and m.p.) to those produced by the reaction of 9a, 14a, or 19 with compound 3.

Cytotoxic assay
The MTT assay was used to evaluate the cytotoxicity of the compounds under study. A complete cytotoxicity assay is given in the supplementary file [41,42].

Ligands preparation
First, the ligands were prepared using the LigPrep module of the Schrödinger package [43]. The novel synthesized compounds' final 3D structures were prepared for computational analysis under physiological pH circumstances. An OPLS3e force field algorithm was used to reduce the ligand geometry.

Protein preparation, grid generation and glide docking
The 3D structure of quinone oxidoreductase2 (PDB code: 4ZVM) in 1.97 Å resolution was downloaded from the RCSB Protein Data Bank [39]. The protein was prepared using a protein preparation wizard in the Schrödinger suite supported by the OPLS3e force field [44]. The protein was utilized to generate receptor grids for the docking procedure. The receptor grid was created by using the centroid of the co-crystallized inhibitor doxorubicin as the grid centre on the active site of the 4ZVM protein. The grid coordinates were 48.43 (X), 4.81 (Y), and 13.89 (Z), respectively. Rigid receptor docking protocol was run in Glide's standard precision (SP) mode based on the OPLS-3e force field [40]. During the process of docking, the protein was fixed, while ligands were flexible.

Conclusion
Novel 1,3,4-thiadiazole-based thiazoles have been synthesized, these newly prepared compounds were determined based on both spectroscopic and elemental data, as well as other methods when feasible. All of the synthesized compounds were tested for their anticancer effects using HepG2-1 cell line. It is found that the thiazoles 16b, and 21 having IC 50 values = 0.69 ± 0.41 and 1.82 ± 0.94 μM, respectively, were the compounds with the highest level of activity of the produced series. Moreover, SP Glide docking of these compounds revealed promising high affinities against NQO2 receptor (PDB: 4ZVM). On the basis of the experimental findings gathered from the anticancer activity, the structure-activity correlations were dissected and addressed.