Facile One-Pot Multicomponent Synthesis of Pyrazolo-Thiazole Substituted Pyridines with Potential Anti-Proliferative Activity: Synthesis, In Vitro and In Silico Studies

Pyrazolothiazole-substituted pyridine conjugates are an important class of heterocyclic compounds with an extensive variety of potential applications in the medicinal and pharmacological arenas. Therefore, herein, we describe an efficient and facile approach for the synthesis of novel pyrazolo-thiazolo-pyridine conjugate 4, via multicomponent condensation. The latter compound was utilized as a base for the synthesis of two series of 15 novel pyrazolothiazole-based pyridine conjugates (5–16). The newly synthesized compounds were fully characterized using several spectroscopic methods (IR, NMR and MS) and elemental analyses. The anti-proliferative impact of the new synthesized compounds 5–13 and 16 was in vitro appraised towards three human cancer cell lines: human cervix (HeLa), human lung (NCI-H460) and human prostate (PC-3). Our outcomes regarding the anti-proliferative activities disclosed that all the tested compounds exhibited cytotoxic potential towards all the tested cell lines with IC50 = 17.50–61.05 µM, especially the naphthyridine derivative 7, which exhibited the most cytotoxic potential towards the tested cell lines (IC50 = 14.62–17.50 µM) compared with the etoposide (IC50 = 13.34–17.15 µM). Moreover, an in silico docking simulation study was performed on the newly prepared compounds within topoisomerase II (3QX3), to suggest the binding mode of these compounds as anticancer candidates. The in silico docking results indicate that compound 7 was a promising lead anticancer compound which possesses high binding affinity toward topoisomerase II (3QX3) protein.


Introduction
The majority of the developed anticancer chemotherapies are not very effective, and side effects might concurrently occur, such as drug-induced impedance. Thus, there is still a critical need to develop novel effective and safe medicines with fewer side effects for the durable treatment of cancer [1,2]. Nitrogen-containing heterocyclic motifs are of high interest owing to their applications as pharmacologically active molecules. These molecules have gained cumulative attention, so they have contributed to the improvement of plentiful organic synthesis protocols and found ample applications in the chemical sciences [3][4][5][6][7]. Several N-heterocyclic conjugates are widely dispersed in nature and are constituents of many biologically important molecules, including several vitamins [8], antibiotics [9], nucleic acids [10], dyes and pharmaceuticals [11,12]. Moreover, the characteristics and utilization of N-heterocyclic skeletons (Figure 1) have gained a reputation in the rapidly growing fields of organic and therapeutic chemistry as well as the pharmaceutical industry [13][14][15]. On the other hand, the electron-donating heterocycle is not only able to readily receive or provide a proton, but it can also simply create various weak connections. Some of the intermolecular connections-for instance, hydrogen-bonding formation, van der Waals forces, dipole-dipole interactions, hydrophobic effects and π-stacking interactions-of N-heterocycles have amplified their significance in the field of therapeutic chemistry and allow them to efficiently adhere to a diversity of enzymes and receptors in drugs due to their improved solubility [16][17][18][19]. Among nitrogen heterocyclic analogues, pyrimidines have numerous applications in medicinal chemistry; the pyrimidine bases of uracil, cytosine and thymine are crucial building blocks of DNA and RNA [20]. In addition, pyrazole-containing scaffolds are a class of heterocycles that exhibit a wide range of biological effects, including anticancer [21], anti-HIV [22], antimalarial [23], anti-tubercular [24], anti-microbial [25] and diabetic activities [26,27].
Hybrid molecules containing thiazole scaffolds are a potential set of heterocyclic compounds; a thiazole core has been found in numerous biologically active natural drugs, such as thiamine (vitamin-B1), penicillin and luciferin [28]. There is a vast number of thiazoles that exhibit a wide range of pharmacological activities, including antibacterial [29], anticancer [30], antifungal [31], anti-inflammatory [32], antioxidant [33] and anti-tubercular activities [34]. Therefore, we believe that the merging of pyrazole and thiazole moieties will result in a class of pharmacophores that exhibit promising biological activities; in fact, several previous works have reported their potential activities, such as anticonvulsant, anti-HIV, anti-inflammatory and anticancer activities [35][36][37][38][39][40].

Chemistry
As an extension of our approach to the intended N-heterocycles [41][42][43][44][45][46][47], we studied the utilization of 2-(1,5-dimethyl-1H-pyrazol-3-yl)-5-acetyl-4-methyl-thiazole (2) to construct a potentially bioactive pyran and/or pyridine hybrids. Thus, coupling of the pyrazolecarbothiamide 1 with 3-chloropentane-2,4-dione in refluxing ethanol afforded the acetyl compound 2 in a 75% yield. Next, a multicomponent reaction of the acetyl compound 2 with thiophene-2-carbaldehyde and malononitrile under reflux in ethanolic piperidine solution yielded the pyran derivative 3. Meanwhile, performing the same reac-tion with NH 4 OAc yielded the pyridine analogue 4 in an 81% yield (Scheme 1). A plausible mechanism for the construction of 2-amino-3-cyano pyridine moiety 4 using NH 4 OAc is displayed in Scheme 2, where the intermediate 2-(thiophen-2-ylmethylene)malononitrile [A], which was obtained via the coupling of thiophene-2-carbaldehyde and malononitrile, was reacted with the thiazoylethenamine [B]; subsequently, an intramolecular cyclization, intermolecular rearrangement and auto oxidation yielded the pyridine analogue 4. The spectral as well as the analytical data of 3 and 4 were consistent with their own structures (see Materials and Methods section). Then, the reactivity of the o-aminonitrile tag in the pyridine analogue 4 was studied via cyclization with some active methylene compounds, to construct the envisioned pyridine and/or pyrrole nucleus-fused pyridine. Consequently, cyclization of the o-aminonitrile pyridine 4 under basic conditions in EtOH with diethylmalonate and malononitrile afforded 4-amino-1,8-naphthyridines (5 and 6) in fair yields (Scheme 3). Notably, the reaction product of compound 4 with ethyl cyanoacetate was extremely dependent on the reaction conditions. Thus, compound 4 refluxed with ethyl cyanoacetate in ethanolic piperidine solution yielded the 1,8-naphthyridine derivative 7 in a 73% yield, but when the same reaction was carried out under fusion, it yielded 2-cyanomethylpyrido[2,3-d]pyrimidin-4(3H)-one analogue (8) in an 82% yield. A reasonable mechanism for the formation of compound 8 is presented in Scheme 4. This reaction is assumed to proceed via an inter-molecular nucleophilic attack of the amino pyridine analogy 4, on the carbonyl carbon in the ethyl canoacetate, leading to the intermediate 4a, and subsequent intramolecular cyclization via further, nucleophilic attack by the -OH moiety on the nitrile group afforded the cyanomethylpyrido [2,3-  Moreover, fusing of a pyrrole nucleus with the pyridine moiety in compound 4 was achieved via the condensation of compound 4 with 2-chloroacetonitrile and/or ethyl bromoacetate in refluxing acetone and anhydrous K 2 CO 3 , which led to the new pyrrolo [2,3-b]pyridin-3-amine derivatives 9 and 10, respectively (Scheme 3). The IR spectrum of compound 10 disclosed the lack of nitrile absorption that was initially observed in compound 4 (IR), while new absorption bands at 1664 and 3341 cm −1 were assigned to C=O and NH 2 groups separately, and its mass spectrum showed an ion peak at m/z 478.12, which confirmed its molecular formula, C 23 H 22 N 6 O 2 S 2 . Moreover, the H-resonances of compound 10 showed triplet and quartet signals owing to the ethyl moiety at 1.26 and 4.32 ppm, respectively.

Cytotoxic Activity
The newly constructed derivatives 5-13 and 16 were evaluated in vitro for their cytotoxic potential towards a prostate cancer cell line (PC-3), lung cancer cell line (NCI-H460) and cervical cancer cell line (Hela) using etoposide as a standard and adapting the MTT assay protocol [48,49]. The obtained outcomes (Table 1, Figure 2) showed that all the   Anticancer activity was calculated using the MTT assay. Results are the average of three independent experiments run in triplicate.

Structure-Activity Relationship
From the data obtained, it is clear that the naphthyridine derivatives 5-7 were the most active compounds against PC-3, NCI-H460 and Hela cell lines (IC 50

Molecular Docking Study
To determine the mechanism of action behind the anticancer activity of the novel constructed compounds, these candidates were docked within topoisomerase II with the use of MOE software, 2010, version 8. An X-ray crystal of topoisomerase II with the cocrystallized ligand etoposide was attained from Protein Data Bank (code: 3QX3). Justification of the docking process was performed by redocking etoposide within topoisomerase II with RMSD = 0.9526. Etoposide formed two hydrogen bonds with AspB479 and DG C13 with binding score = −16.69 kcal/mol ( Table 2). The outcomes from this study illustrate that the novel compounds 5-13 and 16 fitted well within topoisomerase II. The most potent anticancer compound 7 recorded the highest binding energy score (−17.29 kcal/mol), showing two H-bond interactions with AspB479 with the carbonyl and amino groups of the pyridine ring. Moreover, the thiazole moiety of compound 7 recorded arene cation binding with ArgB503 ( Figure 3).
On the other hand, compound 6 performed two types of interactions with the topoisomerase II active site. One is hydrophobic binding of the thiophen moiety with DGC13, and the other binding involves hydrogen bond interactions as follows: (i) AspB479 with NH 2 , and (ii) LysB456 with CN ( Figure 4).
Moreover, compound 5 displayed hydrogen-bonding interactions with ArgB503 and DGC13 through binding with NH 2 and C=O groups. Moreover, this naphthyridine derivative recorded hydrophobic binding with a thiophen moiety with DAC12 with binding score = −16.16 kcal/mol ( Figure 5).
Moreover, the pyrimidine derivative 8 recorded binding energy score = −15.02 kcal/mol, forming three hydrogen bonds as follows ( Figure 6): On the other hand, the cyano group of compound 9 formed a hydrogen bond with ArgB820, with a binding score equal to −17.32 ( Figure 7).
The pyrrole moiety of pyrrolopyridine derivative 10 performed arene cation binding with ArgA820 with binding score = −14.06 kcal/mol. In addition, it formed two H-bonds with ArgB503 and DGC13 ( Figure 8).
Furthermore, the pyrrole derivative 12 formed two H-bonds with ArgB820 and SerB818 through the nitrogen atom of the pyrrole ring and the amino group with binding score = −14.22 kcal/mol ( Figure 9).
Compound 11 recorded a hydrophobic interaction with DAC12 and one hydrogen bond with AlaB816 ( Figure 10).
Moreover, the pyridine ring of candidate 13 showed a hydrophobic interaction with DAC12 in addition to forming only one H-bond with DGC13 ( Figure 11).
Finally, the least potent cytotoxic agent 16 formed only a hydrophobic interaction between the thiophen ring and DAC12, without displaying any hydrogen bond ( Figure 12). On the other hand, compound 6 performed two types of interactions with the topoisomerase II active site. One is hydrophobic binding of the thiophen moiety with DGC13, and the other binding involves hydrogen bond interactions as follows: (i) AspB479 with NH2, and (ii) LysB456 with CN ( Figure 4).

General Description of Materials and Methods
The chemicals used in this work were obtained from Sigma Aldrich (Palo Alto, CA, USA) and were used without any further purification.

Assessment of Anticancer Activity MTT Cytotoxicity Assay
The in vitro growth inhibitory activity of the ten achieved analogues was explored in comparison with a notable anti-malignancy standard medication, etoposide, adapting the colorimetric MTT assay in triplicate as described previously [48]. In brief, cells were seeded onto 96-well tissue culture plates in DMEM containing 10% FBS to a final volume of 0.2 mL. The cells were subjected to different treatments after 24 h of seeding. The cells were then incubated for 48 h with etoposide (positive controls), test drugs or vehicle (DMSO). The media were then removed, replaced with 200 lL DMEM containing 0.5 mg/mL of MTT and cells were incubated for 2 h. Next, the supernatants were removed and the precipitated formazan was dissolved by adding 200 lL of DMSO. Absorbance at 570 nm was determined using a microplate reader (Model 450 Microplate Reader; Bio-Rad). Results were calculated by subtracting blank readings.

Docking Study
Topoisomerase II crystal structure with the cocrystallized ligand (etoposide) was downloaded from Protein Data Bank (PDB code: 3QX3). The target derivatives 5-13 and 16 were docked within the topoisomerase II active site using the MOE 2010 program. To validate the docking step, etoposide was redocked with RMSD = 0.9526. Three-dimensional structures of the target derivatives were built, protonated and energy minimized and saved as mdb files to be docked within topoisomerase II. The results of the docking study are given in Table 2.

Statistical Analysis
The presented results are mean ± SD, and the statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparisons test. Differences were considered significant at p < 0.05. Statistical analysis was performed using SPSS for Windows (SPSS, Inc., Chicago, IL, USA).

Data Availability Statement:
The data presented in this study are available in this article.