Triad pyrazole–thiazole–coumarin heterocyclic core effectively inhibit HSP and drive cancer cells to apoptosis

Abstract Intensive studies on hepatocellular carcinoma (HCC), which is spreading rapidly around the world and has a high mortality rate, is due to the lack of adequate preventive or curative treatment methods. Treating patients with HCC has become very challenging because of the heterogeneity in the patient population lead activation of different signaling pathways, and pathway crosstalk for patients. Therefore, understanding these molecular mechanisms and combining drugs with molecular therapies to overcome these drawbacks has become an area of utmost importance. In this study, the biological activities of the designed and characterized triad Pyrazole–Thiazol–Coumarin (PTC) compounds were determined by performing cell viability, qPCR array, apoptosis and cell cycle assays. One of the compounds (PTC10) implicitly suppresses multiple pathways (RAS/MAP kinase and PI3K-AKT) simultaneously. This action is provided by (i) arresting cancer cells at G2 phase, (ii) driving cancer cells to apoptosis and (iii) inhibiting HSP network. Remarkably, HSP is an apoptotic factor and help cancer cell to survive. HSP90 also coordinates with Cdk4/Cdc37, therefore inhibiting HSP both drives cells to arrest and apoptosis. ATP hydrolysis and aggregation assay further displayed specific HSP inhibition. Therefore, PTC provides a unique drug template for HCC treatment. Communicated by Ramaswamy H. Sarma


Introduction
Cancer has the highest morbidity and mortality rate in the world.Several factors including genetic factors, gender, age, dietary habits, stress, radiation and chemical agents promote cancer.With the elucidation of the molecular mechanisms that play a role in the development and progression of cancer, targeted cancer drug design studies have gained momentum in recent years and several small molecule templates screened for drug development.With these developments and innovations, several drugs have been used routinely in the treatment of cancer.Although patient survival has increased significantly with the clinical success of these drugs, treatment success is not at the desired level.In order to reach the desired levels, researchers focused on explicit tumor targeted compounds in recent years (Moinul et al., 2022).Hybrid molecule design become a popular method in the development of these agents (Abulkhair et al., 2020).
Molecular hybridization, so called symbiotic approach, is based on the combination of pharmacophoric groups of different bioactive substances to produce a new hybrid compound with low toxicity, improved affinity and activity compared to currently used clinical drugs.Hybrid molecules find wide applications in various areas including chemistry, pharmacology and medicine.The synthesis of novel hybrid molecules by integration of different cores can provide effective anticancer activity as cancer may have pathway cross-talks at particular stage (Elkaeed et al., 2022;Lazar et al., 2004;Taghour et al., 2022).
Coumarins, also known as benzopyran-2-ones, are one of the most common molecules found in the structure of many natural products, as well as in synthetic bioactive molecules.They possess a variety of pharmacological properties such as anticancer (Zhang & Xu, 2019), antibacterial (Feng et al., 2020), antitubercular (Keri et al., 2015), anticoagulant (Mustafa et al., 2021), analgesic (Ghate et al., 2005), antiinflammatory (Bansal et al., 2013) and antiviral activities (Dorababu, 2021).Coumarins are used as medicinal drugs in the prevention and treatment of numerous diseases, as they easily interact with various enzymes and receptors in organisms.Considering the importance of coumarins, a number of synthetic strategies have been performed for the preparation of coumarin-containing hybrid compounds (Nasab et al., 2022).Azole heterocyclic compounds such as thiazole and pyrazole are among the nuclei that are frequently used in the synthesis of hybrid molecules with coumarin structure (Benazzouz-Touami et al., 2022).Many studies have been carried out on coumarin-based hybrid molecules that have a significant inhibitory effect on various cancer cell lines and carry hetero parts at the C-3 position, and they have attracted great interest among drug designers.The structures of some hybrid molecules having pyrazole and thiazole rings adjacent to coumarin core are given in Figure 1 (Emami & Dadashpour, 2015;Koca et al., 2015;Lee et al., 2006;Liu et al., 2010;Velpula et al., 2016).
Coumarin is known for its anticancer activity and the other moieties pyrazole and thiazole may complement the comarine part.Further, one core can display anticancer activity while the other cores may suppress the cross talking pathway simultaneously.Our group previously demonstrated anticancer properties of thiazolyl coumarin compounds for the treatment of human colon (DLD-1) and liver cancer (HepG2) (Koca et al., 2015).Heat Shock Proteins (HSPs) are key compounds in molecular mechanism.HSPs fold substrate proteins specially in cell cycle and in signaling.RAS-MAPK and PI3K/AKT pathways have several compounds which are folded by HSP90.Inhibition of HSP90 perturbs oncogenic pathways and drive cells to apoptosis.Since HSP forms distinct complexes (HSP90-HSP70-HSP40), different combinations of functional compounds may efficiently inhibit different cancer types.Additionally, cancer cells increases HSP90 expression and the increase varies depending on cancer type (Tutar & Tutar, 2010).Therefore, this study employs compounds with three core templates so called PTC to determine the anticancer activity.For this purpose, novel hybrid compounds containing pyrazole, thiazole and coumarin cores were synthesized to determine their anticancer activity.The synthesis of the target compounds (PTCs) was performed in four consecutive steps (Scheme 1).The structures of the synthesized novel compounds were characterized by FT-IR, 1 H NMR, 13 C NMR, HR-MS and elemental analysis studies.Different cancer cells used to screen anticancer efficiency of the PTCs and the compounds determined to be effective against hepatocellular carcinoma.Screening biochemical activity through cell cycle, apoptosis and array studies showed anticancer potential of the hybrid  (Emami & Dadashpour, 2015;Koca et al., 2015;Lee et al., 2006;Liu et al., 2010;Velpula et al., 2016).
compounds.Further, aggregation and ATP hydrolysis displayed inhibitory action of the compound against cancer cell survival HSP proteins.

Chemistry
Solvents and all other chemical reagents were purchased from commercial suppliers (Merck, Sigma-Aldrich) and used without further purification.All reactions were magnetically stirred and observed by thin-layer chromatography (TLC) on silica gel plates (Merck Kieselgel 60 F254) using UV light.Melting points (mp) were determined using a Electrothermal 9200 melting point apparatus and were not corrected.NMRspectra were recorded from CDCl 3 , or DMSO-d 6 solutions on Bruker Ultrashield 300 MHz and all 1 H-NMR and 13 C-NMR experiments were reported in d units, parts per million (ppm) relative to TMS as internal standard and coupling constants (J) were given in Hertz (Hz).Elemental analyses were performed with Leco-932 CHNS-O Elemental Analyzer.High resolution mass spectra were recorded with Agilent Technologies 6224 TOF LC/MS instrument.FT-IR spectra were registered on a Perkin Elmer Spectrum Two Model FT-IR Spectrophotometer using ATR method.

General procedure for the synthesis of compound PD
Compound P has been initially constructed through the binucleophilic cycloaddition reaction of thiosemicarbazide to the appropriate chalcone in refluxing ethanol (El-Shershaby et al., 2021).A mixture of compound P (5 mmol) and DMF-DMA (6 mmol) in dichloromethane (40 mL) was refluxed and stirred for 6h until the reaction was completed (followed by TLC).Then the solvent was evaporated under low pressure.Then, the obtained crude product was treated with diethyl ether.Without additional purification, the yellow crude product that precipitated was filtered and used in the subsequent reaction step (G€ um€ us ¸ et al., 2018).

General procedure for the synthesis of compound PT
A mixture of compound PD (1 mmol) and ethyl 4-chloro-3oxo butanoate (1 mmol) in ethanol (30 mL) was refluxed and stirred for 7h.The solvent was removed under reduced pressure.Then, diethyl ether was used to solidify the remaining oily residue.To obtain compounds PT, the yellow crude product that precipitated was filtered and refined by recrystallization from various solvents (G€ um€ us ¸ et al., 2018).

Cell culture and viability assay
HUH7 cells were maintained in 75 cm 2 sterile tissue culture flasks in 10% FBS, 100 U/mL penicillin and 100 lg/mL streptomycin.These cells were grown at 37 � C in a humidified atmosphere containing 5% CO 2 in air.MTT was used to measure the viability and proliferation of cells.Cells were seeded into 96-well plates at a density of 10 4 cells per well.The cells were then cultured for 24 h in 100 lL of DMEM complete medium.After pretreatment with different concentrations of Per Series for 48 h, 10 lL of MTT solution (5 mg/mL) was added to each well and incubated for 4 h at 37 � C, and 100 lL of DMSO (dimethyl sulfoxide) (Sigma-Aldrich, Darmstadt, Germany) was added to each well to dissolve the blue formazan crystals.The optical density was recorded at the wavelength of 570 nm with a microplate reader.

Quantitative q-PCR analyses of oncogenic signaling pathways amplifications and gene enrichment analysis
Total RNA was extracted from cells using a commercial RNA isolation kit (Analytik Jena), and cDNA was synthesized using SensiFast cDNA Synthesis Kit according to the manufacturer's instructions.Reverse transcription was carried out as follows: 25 � C for 10 min, 42 � C for 15 min and 85 � C for 5 min (one cycle).cDNA was stored at À 20 � C for PCR.Real-time PCR was performed in 25 mL total reaction volume.The primer sequences synthesized by Sigma-Aldrich (Darmstadt, Germany).The optimum PCR concentrations of primers yielding the highest end-point fluorescence and the lowest Cp were experimentally determined for each set of primers.Real time PCR was carried out in optical grade 96-well plates at reaction volume of 25 mL, including SybrGreen Master Mix (EuroClone), 300 nM of each primer and 50 ng of template DNA.q-PCR was performed on qTOWER 3 thermocycler system.Threshold cycle (Ct) data were collected using qpcrSoft v4.0 (Analytik Jena, Jena, Germany).The conditions for cycling were 95 � C for 10 min (denaturation and Taq polymerase activation) followed by an amplification program of 40 cycles consisting of 95 � C for 15s, and 60 � C for 30s.All samples were run in duplicates.The relative gene expression was analyzed by the 2 À DDCt method.The fold change in target gene cDNA relative to the GAPDH internal control was determined by: Fold change ¼ 2 À DDCt where DDCt ¼ (Ct Target gene À Ct GAPDH) À (Ct Target gene À Ct GAPDH).Gene enrichment analysis was performed by employing reactome.

Apoptosis and cell cycle analysis
For apoptosis assay, FITC Annexin-V apoptosis Detection Kit with PI, ApopNexinTM FITC (APT750, Merk, Germany) was used.Total Huh7 cells were seeded into 6-well wells at a concentration of 1 � 10 6 cells per well.Then, the cells were cultured with the compounds at the cytotoxic concentration that was determined for each sample by cell viability assay.Apoptotic cells were cultured with the compounds, the medium was removed after incubation and the cells were washed twice with 5 mL of pre-chilled phosphate buffered saline.Cells washed with PBS were lifted with Trypsin-EDTA and centrifuged at 400 rpm for 5 min.The resulting cells were each treated with 1 mL of cold 1-X Binding Buffer (Sigma Aldrich, USA).Then, 200 lL of the mixture was taken and placed in a flow cytometry tube, and the other part containing cells was kept on ice.First, 3 mL of ApopNexinTM FITC and 2 mL of 100X propidium iodide (PI) (P4170, Sigma Aldrich, USA) were added to the solution taken into the sample tube and mixed.The resulting mixture was incubated for 15 min in the dark at room temperature.At the end of the incubation, the samples were measured in flow cytometry and data were obtained.Cell cycle analysis were performed using Sigma-Aldrich Mak344 Cell Cycle Analysis kit.The manufacturer protocol was followed.Huh7 cells were cultured at a concentration of 3 � 10 5 cells per well and treated at the IC 50 values determined for each sample by cell viability assay.At the end of the application, the cells were washed with 500 mL of phosphate buffered saline and trypsinized and the pellet was collected by centrifugation at 400 rpm for 5 min.After collecting the pellets, the cells were washed with 2 mL of 1X cell cycle assay buffer and centrifuged at 400 rpm for 5 min.The supernatant was discarded, and cells were fixed by slowly adding 2 mL of cold 70% ethanol to the pellets.Cells treated with alcohol were incubated on ice for a minimum of 30 min.Afterwards, the supernatant was discarded by centrifugation again.In the next step, the pellets were washed again with 2 mL of 1X cell cycle assay buffer and centrifuged again, and the supernatant was carefully discarded.100 mL of enzyme A solution and 500 mL of staining solution prepared in 400 mL of nuclear dye and 1X buffer solution were added onto the obtained cells and incubated for 30 minutes at room temperature in a dark environment.After incubation, cells were analyzed using flow cytometry.

Aggregation assay
Aggregation assay performed as previously described (Cos ¸kun et al., 2021).Rhodanase protein (50 mM) denatured with 20 mM Hepes, 80 mM KCl, 10 mM dithiothreitol, 6 M guanidium-HCl buffer (pH: 7.4).Heat shock proteins added before rhodanese and aggregation rates were measured by monitoring light scattering with a spectrophotometer at 320 nm at 25 � C. Each data point is the average of two separate experiments.

ATP hydrolysis assay
ATP hydrolyses monitored by coupled enzymatic assay and ATP regeneration system as described earlier (Tutar & Tutar, 2008).ATP regeneration system was excluded from incubation.The assays performed in 10 mM Hepes, 80 mM KCl, 10 mM dithiothreitol, 1 mM MgCl 2 , 50 M ATP, pH: 7.4.Each data point is average of two separate experiments.

Synthesis and characterization
New hybrid compounds (PTC) containing triple heterocyclic moiety which are pyrazole, thiazole and coumarin were designed, synthesized and evaluated for their biological activity.PTCs were synthesized using 3,5-diaryl-4,5-dihydro-1H-pyrazole-1-carbothioamides (P) that were obtained from the reaction of chalcones with thiosemicarbazide in four steps as described in the Scheme 1.Then, as a result of compound P and N,N-dimethylformamide dimethyl acetal (DMF-DMA) was stirred in dichloromethane at the boiling temperature for 6h, PD derivatives were obtained.These compounds were directly used without further purification in the continuation reactions.In the next step, compounds PD were refluxed with ethyl 4-chloro-3-oxobutanoate in ethanol for 7h in the presence of 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) as a catalyst (G€ um€ us ¸ et al., 2018).
Synthesis of the hybrid compound (PTC) was performed benefiting the reactivity properties of the b-ketoester group situated in compounds PT.Compounds PT were converted to the compounds PTC through the knoevenagel reaction with salicylaldehyde derivatives.The reaction yields ranged from 64% to 84%.Some properties of new hybrid compounds (PTC) are illustrated in Table 1.The structures of compounds PTC were confirmed by their spectroscopic data as mentioned in the experimental section.For example, in the FT-IR spectrum of PTC-6, it was characterized by the presence of C ¼ O (lactone) and C ¼ O (ketone) at 1738 and 1618 cm À 1 , respectively.Stretching vibrations of aromatic and aliphatic groups of PTC-6 appeared as weak absorption bands at 3052, 3030, 2948 and 2923 cm À 1 .In addition, C ¼ N and C ¼ C stretching bands were appeared in the range of 1609-1494 cm À 1 .The 1 H-NMR spectrum of PTC-6 exhibited two singlets at 8.02 and 7.78 ppm due to the CH protons in coumarin and thiazole rings, respectively (Figure 2).In the 1 H-NMR spectrum of this compound gave multiplet belonging 12 of aromatic CH protons in the range of 7.69-7.15ppm, a doublet of doublet signals attributed to the resonance of the CH proton on five-numbered carbon atom of the pyrazole ring in the range of 5.71-5.65 ppm (dd, J cis ¼ 4.9 Hz, J trans ¼ 11.6 Hz, 1H, C 5 H-pyrazole).Besides, the diastereotopic protons (C 4 H-pyrazole) that gave ABX system showed two doublet of doublet signals in the range of 3.97-3.88ppm (dd, J gem ¼ 17.6 Hz, J trans ¼ 11.6 Hz, 1H, C 4 H-pyrazole) and 3.35-3.28(dd, J gem ¼ 17.7 Hz, J cis ¼ 4.9 Hz, 1H, C 4 H-pyrazole).The signals of two methyl groups appeared at 2.41 and 2.32 ppm as two singlet signals.
In the 13 C-NMR spectrum of this compound was observed at 180.7, 170.belonging to nineteen carbon atoms appeared at 154.3-111.0ppm.Besides, the signals of two Ar-CH 3 groups observed at 21.6 and 21.1 ppm.The spectroscopic data of all compounds PTC can be viewed in experimental part.

Cell viability assay
PTC compounds screened against various cancer cell lines at the one-does assay but only Huh-7 cell line displayed prominent inhibition.PTC compounds subsequently screened for six-dose assay (40, 20, 10, 5, 2.5 and 1.25 mM) and compared to untreated Huh-7 cell line for viability (Figure 2).PTC10 compound displayed promising activity and PTC15 activity was the lowest (Table 2).Therefore, two compounds employed in the next step for array studies to elucidate cancer pathways involved in PTC15 inhibition.
Why the compound PTC10 displayed prominent inhibition with lower IC 50 values against Huh7 cell line can be searched by employing PTC15 for negative control.To determine the effective cascade for the inhibitory action of PTC10, array studies employed and the results were examined with gene enrichment analysis.

Array studies and gene enrichment analysis
Effect of ligand on Huh7 cells was monitored by PCR array (Figures 3, 4).Expression pattern indicates that PTC10 use multiple pathways for cancer cell proliferation, mainly RAS/MAP kinase and PI3K-AKT (Chen et al., 2019;De Luca et al., 2012;Florkiewicz et al., 1991).RAS/MAPK is targeted in hepatocellular carcinoma and sorafenib is the only approved systemic therapy (Delire & St€ arkel, 2015).RAS/MAPK signal transduction pathway transduce signals from extracellular milieu to the nucleus and this transmission activates cell growth, division and differentiation genes.Further, PI3K-AKT pathway regulates intracellular signalling and controls cell survival, proliferation, cell cycle, growth and cancer in response to extracellular signals (King et al., 2015;Zhang et al., 2018).The pathways are deregulated due to genetic alterations and signalling pathway are regulated by crosstalk mechanism.The components of the signalling pathway are interesting targets for therapeutic approaches.However, generally more than one inhibitor is required for blocking the cross-talking pathways.It should be noted that MAPK and PI3K pathways are induced by oncogenes (Maik-Rachline et al., 2021).Thus, PTC in this regard offer personalized genomic information for anti-cancer efficiency.
PTC10 suppresses FOXC2, this gene is involved in metastases and in epithelial-mesenchymal transition (Fang et al., 2000).The inhibitory effect of the compound over proliferation, differentiation and transcription regulation is screened by decreased expression of MAP2K1 (Maik-Rachline et al.,   2021) and MAP2K3.MAP2K3 expression of RAS oncogene accumulation of the active form of this kinase (D� erijard et al., 1995).Decrease in MKI67 in the presence of PTC10 interfere with cellular proliferation as the gene codes a nuclear protein that is associated with cellular proliferation (Sobecki et al., 2016).HSP90 expression increases in different cancer types and HSP90 regulates signaling by folding key intermediates in the pathway.RAS-MAPK and PI3K/AKT pathways have several intermediates which are folded by HSP90.Perturbing the interactions drive cells to apoptosis.Since HSP family forms distinct complexes (HSP90-HSP70-HSP40), different combination of functional compounds may inhibit different cancer types.This is why it is difficult to estimate effect of compounds against distinct HSP complexes.For this purpose, novel hybrid compounds containing pyrazole, thiazole and coumarin cores were synthesized to determine their anticancer activity.The screening results indicated the triad core template PTC is effective in hepatocellular carcinoma.

Apoptosis
Flow cytometric studies indicate that PTC10 drives Huh7 cells to apoptosis.This is supported by PCR-array experiments (Figure 2).PTC10 efficiently inhibits the liver cancer cell line.This well differentiated hepatocyte-derived carcinoma cell line has the greatest challenge in clinical management due to high metastasis and invasion in clinical outcome (Liu et al., 2020).Therefore, targeting these signalling pathways is a promising approach for targeted therapy.However, single target interventions are ineffective as the data suggest cross-talking mechanism of Huh7.Since PTC10 targets multiple proteins and pathways in hepatocellular carcinoma cell line, PTC10 offers a promising anti-cancer activity as evidenced by flow cytometric assays (Figures 5, 6) and biochemical assays (Figures 7, 8).

Cell cycle
To explore the mechanism of PTC10 induced effect on Huh7 cells, cell cycle assay performed.PTC10 increased the population of G2/M phase cells and decreased the cell population in G1 phase (Figure 6).G2/M cell cycle arrest in the presence of PTC10 is further confirmed by array experiments.Reactome analysis indicate that WEE1, MCM2 play important role for the arrest.PTC10 leads G2/M cell cycle arrest and then induces apoptosis in Huh-7 cells in vitro (Hannak et al., 2001).

Aggregation assay
Hsp90 plays key role not only in cell cycle but also in solubilization of protein aggregation.Aggregated rhodanase  solubilization by Hsp90 screened.Hsp90 efficiently solubilize aggregates in the presence of partner protein Hsp70 and its co-chaperone along with Hop (Hsp70-Hsp90 Organizing Protein).PTC10 inhibits the solubilization of the protein aggregation; however, PTC15 may not solubilize the aggregation.This assay indicates that PTC10 is highly effective in Hsp90 and possibly Hsp70 inhibition (Figure 7).

ATP hydrolysis assay
HSP90 consists of two distinct domains (Tutar, 2016).One domain is responsible for substrate protein folding while the other hydrolyzes ATP.The energy is coupled to substrate protein folding and to understand if the inhibitor perturbs this function, ATP rates of HSP90 with cochaperones measured.An ATP regenerative system employed, and the highest hydrolysis screened in the presence of HSP complex.The compound PTC10 decreases hydrolysis efficiently compared to that of PTC15 (Figure 8).These expected results indicated that PTC10 not only perturb HSP ATP hydrolysis but also substrate protein folding through interfering HSP ATP hydrolysis-substrate protein coupling function.
Biochemical assays further support array and flow cytometric analysis as inhibitor perturb HSP functions.HSPs employ ATP for their function.The hydrolysis energy is used to fold substrate proteins and solubilize aggregates.PTC10 efficiently inhibit ATPase activities of HSP90 and its cochaperone complexes (HSP70, HSP40 and HOP) as well as rhodanese solubilization.PTC inhibits HSP complex and drives the substrate protein to accumulate.The process eventually drives the cell to apoptosis as proteosomal system overloaded (Figures 7,8).

Molecular docking analysis
Small ligands binding to the receptor structures in a range of orientations, conformations and locations using molecular docking, provides information about molecular recognition and perturbation of receptor function (Sousa et al., 2006).In this perspective, the AutoDock Vina program (Trott & Olson, 2010) was used to undertake in silico-molecular docking investigations of newly synthesized compounds PTC1-15, which are regarded HSP90/PDB:1YC4 inhibitor.RCSB (Protein Data Bank) was used to obtain the associated receptors (Berman et al., 2000;Kreusch et al., 2005).
Here, 1YC4 receptor ligands (PTC1-15) were optimized by DFT/B3LYP theory/functional and 6-311þþG(d,p) basis set via Gaussian 09W package and Gauss View 5.0 programs (Dennington et al., 2009;Frisch et al., 2009).Discover Studio Visualizer 4.0 (DSV 4.0) software was used to perform   preparations such as removing hetero groups and adding polar hydrogen bonds for both receptors (Biovia, 2017).To begin, the active sites of PDB:1YC4 receptor were identified as VAL186, THR184, PHE138, MET98, GLY97, ILE96, ASP93, LYS58, ALA55 and ASN51, respectively.The grid parameters that were determined based on the active sites of the receptor are as follows: PDB:1YC4 has 50 � � 54 Å 3 x, y, z dimensions, 0.375 space, and 34.008, 31.908,À 4.657 x, y, z centers.The resulting docking scores or binding energies and inhibition constant (Ki) values calculated based on these values were given as in Table 3.The exp(DG/RT) equation was used to calculate K i values (G: binding energy, R: gas constant ¼ 1.987203610 À 3 kcal/mol, and T: room temperature ¼ 298.15K).
In addition, Figure 9 (PTC10þ PDB:1YC4) and Figs.S52-S65 (the other ligands þ PDB:1YC4) depict the molecular docking contact scenarios.When the obtained docking results were evaluated, it was determined that the PTC10 molecule has a better binding energy (the best binding) than the other molecules.Therefore, docking discussions in the manuscript will focus on the PTC10 molecule with 10.80 kcal/mol energy and 0.0121211 lm Ki value.van der Waals, conventional hydrogen bond, p-donor hydrogen bond, alkyl and p-alkyl interactions were detected when the result for the best binding in Figure 9 was analyzed.Furthermore, as shown in Figure 10, the optimum poses of the PTC10 molecule in the active site of HSP90/PDB: 1YC4 could be observed.
The interactions generated by active site residues are as follows: between VAL186 and Cl22 atom with 4.48 Å alkyl and between VAL186 and the center of benzene ring with 5.22 Å p-alkyl; between PHE138 and C 23 H 58,59,60 with 5.02 Å alkyl; between MET98 and the center of O-benzene ring with 3.70 Å p-alkyl; and between ASN51 and the center of O-benzene ring with 4.05 Å p-donor hydrogen bond interaction.Additionally, the conventional hydrogen bondings were observed between SER52 and Cl22 atom with 2.98 Å and between ASN106 and O46 atom with 3.05 Å.The ones formed outside of these interactions and their bond lengths could be easily seen in Figure 9.

Drug-likeness and ADME/T investigations
Pfizer's rule of five is a rule of thumb for determining druglikeness and determining whether or not an inhibitor with specific biological and pharmacological features would be an   orally active drug in the human body (Lipinski et al., 1997).If two or more of these thresholds are met, a ligand or inhibitor can be orally absorbed/active.These rules could be classified as MW� 500 g/mol, MlogP � 5, HBA� 10 and HBD� 5, n Rot � 10, TPSA < 140 Å 2 .In this state, these parameters were with the help of the SwissADME site (Daina et al., 2017), taking into account the Lipinski's five rules and the obtained results were presented in Table S1 (Supporting Information).As seen in Table S1, molecular weight (MW) results were calculated as 477.53 (PTC1)-580.87 (PTC15); the n-octanol/water partition coefficient (MlogP) as 3.02 (PTC4)-4.47(PTC11); number of H-bond acceptor and number of H-bond donors are 5-6 and 0-1, respectively; number of rotatable bonds are 5-6; finally topological polar surface area (TPSA) values in the 104.01 (PTC1, PTC5, PTC6, PTC10, PTC11 and PTC15)-124.24 (PTC4, PTC9 and PTC14) ranges.In addition, the violation numbers and violation states of the molecules according to the Lipinski rules (physicochemical properties) were given in Table S1.Additionally, log S (Ali) aqueous solubility values, which are property of water solubility were calculated in the range of À 8.23 (PTC1) and À 10.18 (PTC15).The MR: molar refractivity values were computed as in the range of 143.85 (PTC1)-160.36 (PTC12 and PTC13).The results in Table S1 can be better appreciated if these physicochemical parameters are represented graphically in the form of a bioavailability radar, which takes into account six physicochemical properties: lipophilicity, size, polarity, solubility, flexibility and saturation.For each description, a physicochemical range is displayed as a pink area inside which the molecule's radar plot must fall wholly to be termed drug-like.Figure 11 illustrates these findings.
Using the SwissADME web page (Daina et al., 2017), inhibitor (þ) and non-inhibitor (À ) properties of CYP450 enzymes such as CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4 were evaluated.Inhibitory states were represented by þ/positive and non-inhibitor states were shown by À /negative in the Table S1.
In drug discovery and development, chemical absorption, distribution, metabolism, excretion and toxicity (ADMET) play critical roles.A high-quality drug candidate should not only be effective against the therapeutic target, but also have the right ADMET characteristics at a therapeutic dose.Some essential ADMET features of the PTC1-15 molecules were acquired using AdmetSAR 2.0 (Daina et al., 2017) over the output forms in this part of the research, and the results were given in Table S2 (Supporting Information).As seen from the Table S2, BBB values were found positive for our series except PTC14 molecule (its values is À and 0.5335) and the positive values were determined in the range of 0.5644 (PTC12)-0.8949(PTC1).The second parameter is human intestinal absorption and this parameter was determined as positive in all compounds and in the range of 0.9250 (PTC4)-0.9915(PTC10).As the third, P-glycoprotein inhibitor properties were investigated and it was determined that all molecules show non-inhibitor properties range from 0.59787 (PTC2) to 0.9049 (PTC4) except PTC7 and PTC14 molecules show inhibitor effect their values were found as 0.7375 and 0.6954, respectively.
To forecast human intestinal absorption (HIA) and blood brain barrier (BBB) access, the compounds' WLogP and TPSA values were shown (Figure 12).The boiled-egg plot is separated into three sections: grey (no HIA or BBB access), white (HIA) and yolk (BBB access).With the boiled-egg model was also predicted whether PTC 1-15 were P-glycoprotein substrates (PGP), and the PGP value of all molecules was negative (red dot).A gene called hERG (KCNH2) produces the potassium ion channel Kv11.1.By mediating the repolarizing IKr current in the cardiac action potential, the protein aids in the coordination of the heart's beating.hERG inhibition, which can lead to irregularity of the heartbeat and sudden death, should be avoided during drug discovery (Munawar et al., 2018;Priest et al., 2008;Roy et al., 1996;Sanguinetti & Tristani-Firouzi, 2006;Warmke & Ganetzky, 1994).Based on the literature expressed here, it can be said that the drug candidate to be designed should have weak inhibition of hERG.Our results support argument.When human ether-a-go-go-related gene (hERG) values were examined, it was seen that all molecules showed weak inhibitory effects and their values ranged from 0.8604 (PTC13) to 0.9671 (PTC9).Later, it was seen that all molecules showed non-carcinogen effects and their scales ranged from 0.7081 (PTC10) to 0.8395 (PTC2).Finally, it was determined that acute oral toxicity values belong to class III for all molecules and their values ranged from 0.5821 (PTC8) to 0.6685 (PTC10).

Conclusion
In this study, hybrid compounds containing thiazole, coumarin and pyrazole triad core synthesized to screened anticancer activity specifically against hepatocellular carcinoma which has complex pathway crosstalk mechanism.In silico experiments showed that PTC derivatives met the requirements of Lipinski's rules and among them PTC10 inhibits HSP90 efficiently as determined by binding energy and Ki values.
Further, Hepatoma carcinoma cell line treated with PTC10 directly effect two main signaling pathways as evidenced by gene enrichment analysis (Reactome and KEGG) of hub genes.The analysis results indicated RAS-MAPK and PI3K/AKT as the key pathways involved in PTC activity.It should be noted that inhibiting both pathways simultaneously is difficult due to possible pathway crosstalk and the two center must be suppressed to inhibit the hepatoma carcinoma.In addition, a synergetic effect is required between these two distinct centers for effective cancer treatment.PTC10 may not only target these two pathways but inhibit HSP as evidenced by aggregation and ATPase assays.HSP90 plays crucial roles especially in cell cycle.HSP90 forms complex with its cochaperone Cdc37 and control Cdk4 Cdk4 regulates G1 and G2 phase of cell cycle.Cell cycle control for cancer cells are important and this is why expression of HSP90 increases in cancer cells.HSP90 not only controls cell cycle but also prevents apoptosis.Therefore, HSP inhibition is interest to drug designers during the last decade.PTC not only inhibits HSP90 but also suppresses XIAP which stops apoptotic cell death.Similar array expression levels support effectiveness of PTC10 compound.
PTC further decrease AURKA concentration and this gene activity peaks during the G2 phase to M phase transition in the cell cycle.Similar trend is observed in CCND3.CCND3 or cyclin D3 forms a complex with CDK4 and functions as a regulatory subunit.Additionally, CCND3 is responsible for cell cycle progression through G2 phase.The compound further inhibits WEE1 which inhibits Cdk1.Cdk1 is essential for cyclin-dependent passage at different cell cycle checkpoints.
The inhibitor decreases expression of FGF2 which provides mitogenic and cell survival activities, and is involved in cell growth, morphogenesis, tumor growth and invasion.Flow cytometry experiments and biochemical assays supports effectiveness of compound PTC10.HSP cooperates and coordinates with distinct partner proteins.Further, at different stages cancer may introduce complex molecular interactions as pathway crosstalk.Triad compounds may specifically inhibit key interactions.Thus, PTC10 provides a unique platform for specific clinical drug template over hepatoma carcinoma.

Figure 2 .
Figure 2. PTC 10 displayed promising activity against Huh7 cell lines with IC 50 value of 3.77 mM at 48 and 1.64 mM at 72 h.

Figure 5 .
Figure 5. Huh7 cells are driven to apoptosis in the presence of PTC10.

Figure 6 .
Figure 6.Cell cycle analysis of PTC10 indicates that the compound arrests the Huh7 cells in G2/M stage.

Figure 7 .
Figure 7. Effect of PTC inhibiton over substrate protein aggregation in the presence of HSP90 and its co-chaperone complex (HSP70, HSP40 and HOP).

Figure 8 .
Figure 8.Effect of PTC inhibition over ATP hydrolysis in the presence of HSP90 and its co-chaperone complex (HSP70, HSP40 and HOP).

Figure 10 .
Figure 10.The placement of PTC-10 molecule in active site of PDB: 1YC4 protein (the yellow parts represent the active region).

Table 1 .
The structures, yields and melting points of newly synthesized compounds PTC.