Thiophene-based N -phenyl pyrazolines: Synthesis, anticancer activity, molecular docking and ADME study

This investigation aimed to synthesize and evaluate the anticancer potential of thiophene-based N -phenyl pyrazoline derivatives containing methoxy groups using both in vitro and in silico assays. The N-phenyl pyrazolines were synthesized by reacting phenylhydrazine with chalcone derivatives. Using the 3-(4,5-dimethyl thiazole-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay, we assessed the anticancer activity of the compounds that were synthesized in vitro against four human cancer cell lines (4T1 and T47D for breast cancer, HeLa for cervical cancer, and WiDr for colorectal cancer), as well as a normal cell line (Vero). The in silico evaluation was carried out through molecular docking and absorption, distribution, metabolism, and excretion (ADME) prediction. Among the synthesized pyrazolines, pyrazoline 2 was revealed as the most active anticancer against 4T1, HeLa, and WiDr cancer cell lines, with IC 50 values of 9.09, 9.27, and 0.25 µg/ml, respectively. Furthermore, pyrazoline 2 showed high selectivity with selectivity index values of more than 6 toward all tested cancer cell lines. In silico evaluation via molecular docking against epidermal growth factor receptor revealed that pyrazoline 2 had the lowest binding energy with a value of 8.8 kcal/mol, which was consistent with experimental data. The ADME study using the pkCSM webtool indicated that the N-phenyl pyrazoline derivatives had good pharmacokinetic properties. Therefore, the results of this study suggest that thiophene-based N -phenyl pyrazoline derivatives containing methoxy groups, especially pyrazoline 2 , could be potential anticancer agents.


INTRODUCTION
The World Health Organization has warned that cancer is a perilous disease that can result in a high number of fatalities.GLOBOCAN's 2020 report revealed that there were 19.3 million cancer cases globally, resulting in 10.0 million deaths.Among them, breast cancer in females (11.7%), lung cancer (11.4%), colorectal cancer (10.0%), prostate cancer (7.3%), and stomach cancer (5.6%) were the most-deadly types.Chemotherapeutic agents used to treat cancer can cause toxic and resistant effects [1], prompting researchers to develop potential compounds as anticancer agents.
Thiophene is a heterocyclic compound that shows promise as a drug component [2], and its bioactivity as an anticancer compound can be enhanced by combining it with other heterocyclic compounds [3,4].Pyrazoline is a heterocyclic compound consisting of a five-ring structure with two nitrogen atoms joined by an endocyclic double bond and has also demonstrated broad bioactivity in drug discovery, particularly when it contains phenyl groups [5,6].Pyrazoline derivatives have been shown to have significant potential as anticancer compounds [7].Among the various methods for synthesizing pyrazoline derivatives, the cyclo-condensation reaction between chalcones and hydrazine derivatives is one of the most commonly used methods [8].
Studies conducted previously reported the effective cytotoxicity of N-phenyl pyrazolines against various cancer cell lines [5,14].The present investigation centers on the synthesis of targeted thiophene-based N-phenyl pyrazoline (Scheme 1) and their evaluation for anticancer activity in vitro using colorectal (WiDr), breast (T47D and 4T1), cervix (HeLa), cancer cell lines, and normal (Vero) cell line.In addition, we validated our results through in silico assays, including molecular docking studies with EGFR receptors to determine their conformational pose and an ADME study to predict their pharmacokinetic properties.

Anticancer activity of pyrazolines 1-5
To culture cancer cells, Roswell Park Memorial Institute (RPMI) and Dubelccos's Modified Eagle's Medium (DMEM) media with 10% Fetal Bovine Serum (FBS) were used, and the cells were exposed to a water-saturated flow of 5% CO 2 .A cell-containing solution of 100 µl was added to each well of a 96-well plate, with each well containing 10 4 cells.Pyrazoline samples and commercial drugs (100 µl) were added to each 96-well plate, with seven concentrations tested and each sample tested four times.After 24 hours of incubation, a 1 ml aliquot of 3-(4,5-dimethyl thiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution in Phosphate Buffer Saline (PBS) (5 mg/ml) was prepared by dissolving it in 9.5 ml of culture medium.Subsequently, 100 µl of this mixture was added to each well and incubated for 4 hours.Then, 100 µl of sodium dodecyl sulfate (SDS) stopper solution was added, and the plate was incubated overnight.Finally, the absorbance of the solution was measured at 595 nm using an Enzymelinked immunosorbent assay (ELISA) reader.

Molecular docking study of pyrazolines 1-5
Pyrazoline derivatives were sketched in 2-D and then converted to 3-D structures using Chem 3-D Professional 16 software.The geometry optimization was then performed using the Density-functional theory (DFT) method basis set B3YLP 6-31G with Gaussian 09 software.All tested compounds were docked into EGFR with a PDB ID of 1m17 using a grid box size of 18 × 18 × 18 Å, suitable for validated protocol docking.The Root-mean-square deviation (RMSD) value was ensured O n l i n e F i r s t to be lower than 2 Å before conducting the docking protocol.The binding energy and chemical interaction were analyzed to assess the compounds' inhibitory activity compared to erlotinib, a native ligand.

Synthesis of N-phenyl pyrazolines
Pyrazolines 1-5 (Scheme 1) were synthesized from chalcones 1-5 and phenylhydrazine via a cyclocondensation reaction, utilizing the method described by Suma et al. [15].The physical data for pyrazolines 1-5 are presented in Table 1.The characterization of the synthesized products was carried out using FTIR, Gas chromatography-mass spectrometry (GC-MS), and 1 H-and 13 C-NMR spectrometers.The formation of N-phenyl pyrazoline products was confirmed by the appearance of three peaks in the δ 3, 4, and 5 ppm region as a doublet of a doublet, which comes from the methylene proton on the pyrazoline rings due to the diastereotopic effect [16].GC-MS was employed to verify the molecular weight and purity of the pyrazoline products.
The formation of N-phenyl pyrazoline was further confirmed by FTIR spectra showing the C=N stretching at 1,597 cm −1 and vibration of C-N aromatic and aliphatic stretching at 1,381 and 1,126 cm −1 , respectively, and 13 C-NMR spectra, which revealed the existence of carbon atoms within the pyrazoline ring.

In vitro anticancer activity
To evaluate the anticancer activity of pyrazolines 1-5, the MTT assay was used to perform in vitro cytotoxicity testing against cervical (HeLa), breast (T47D and 4T1), colorectal (WiDr), cancer cell lines, and a normal cell line (Vero).The percentage of cell growth inhibition was transformed into the probit numbers and plotted against log C to determine the IC 50 O n l i n e F i r s t respectively.Additionally, pyrazoline 2 exhibits high activity in inhibiting the cervical (HeLa), breast (4T1), and colorectal (WiDr) cancer cell lines.According to the research, pyrazoline 2 has been identified as the most effective candidate for fighting cancer compared to the other candidates.The variation in their effectiveness can be attributed to the location of the methoxy group within the pyrazoline structure.Pyrazoline 2, which contains a methoxy substituent at position 2 on the benzene ring, demonstrated greater potency.The current findings agree with Ciupa et al.'s [5] report, which suggested that adding methoxy groups to pyrazoline derivatives could affect their ability to inhibit cancer cells.

Molecular docking
Table 3 presents the results of a molecular docking study conducted on synthesized pyrazoline 1-5 against EGFR.To ensure accurate results, it is essential to validate the docking protocol.The superimpose of erlotinib, a native ligand, into redocking native ligand produced an RMSD of 1.304 Å (Fig. 1).In this study, erlotinib was used as a reference inhibitor for the synthetic compounds.The molecular docking of erlotinib showed a binding affinity of −7.9 kcal/mol.The chemical interaction between erlotinib and the amino acid residue Met769, which is involved in the catalytic reaction, was visualized (Fig. 2) [22].Furthermore, erlotinib is bound to Cys773 through the oxygen atom of the ether moiety, contributing to the inhibition of EGFR growth [23].
Upon inspection of the chemical interaction, it was observed that the nitrogen atom of quinazoline acted as an acceptor hydrogen bond to Met769.Erlotinib is bound to the amino acid residue Lys721, which connects the α-and value.Three commercial drugs, doxorubicin for breast cancer [17], cisplatin for cervical cancer [18], and 5-fluorouracil for colorectal cancer [19], were used as positive controls.
This study considers the compound's selectivity against the normal (Vero) cell line to identify the best candidate for an anticancer agent.According to Amin et al. [21], a compound with a SI value greater than 6 is considered highly selective.Compounds with SI values ranging from 3 to 6 are moderately selective, while those with SI values below 3 are considered to be non-selective.Based on O n l i n e F i r s t β-phosphate of the N-lobe to stabilize catalytic activity through hydrophobic interaction.Additionally, erlotinib also formed a pi-donor hydrogen bond with Thr830 and it has a role in the activation loop for the conformation of the phosphorylation process [24].Furthermore, the hydrophobic II areas of EGFR, where the catalytic protein kinase is located [25], were also bound by erlotinib through hydrophobic interactions with amino acid residues such as Leu694 and Leu820.The quinazoline ring facilitated this interaction with these hydrophobic residues.
The quinazoline ring facilitated this interaction with these hydrophobic residues.
The molecular docking study of pyrazoline derivatives containing a thiophene ring demonstrated good binding affinity.Compound 5 exhibited a slightly lower binding affinity (−7.6 kcal/mol) than erlotinib, while compound 4 was bound to EGFR with a similar affinity as the native ligand.Compounds 3, 1, and 2 showed strong binding affinity of −8.4,−8.8, and −8.8 kcal/ mol, respectively.All compounds were found to bind within the binding pocket of EGFR, with the N-phenyl substituent of the pyrazoline ring binding to the hydrophobic area and some residue amino acids, such as Leu694 and Leu820.Compounds 1, 2, and 3 contained an aromatic ring B that bound to Lys721 through hydrophobic interaction.This interaction destabilized the side chain of the N-lobe, resulting in a decrease in catalytic activity.
Although compounds 4 and 5 exhibited the lowest binding affinity among synthetic compounds, they both interacted with Met769.Compound 5 bound to Met769 through hydrophobic interaction on methoxy moiety may explain its lower binding affinity than compound 4. Similarly, compound 4 interacted with Met769 through a carbon-hydrogen bond, which is also a catalytic site of EGFR.Furthermore, this compound is bound to Cys773 through a hydrogen bond with a O n l i n e F i r s t is above 0.90 [26].The analysis showed that pyrazolines 1-5 have an HIA value above 90% and a CaCo-2 permeability value higher than 0.9, indicating that these compounds have an excellent absorption capacity.The steady-state volume of distribution (VDss) represents the capacity to distribute a complete blood plasma dose.According to Lombardo et al. [27], VDss values below −0.15 are considered low, while values above 0.45 are considered good.Blood-brain barrier (BBB) permeability refers to a drug's capability to traverse the barrier membrane of the brain, whereas central nervous system (CNS) permeability indicates a drug's ability to penetrate the CNS.Pires et al. [12] stated that a BBB permeability value is considered good to penetrate if log BB > 0.3 and poor to penetrate if log BB < −1, while CNS permeability is high if log PS > −2 and low if log PS < −3.Pyrazolines 1-3 have VDss value above 0.45 and log BB > 0.3, which suggests that these compounds are uniformly distributed in blood plasma and can cross the brain bond length of 3.19 Å.The methoxy moiety of aromatic ring B also contributed to the interaction with Leu694.The thiophene ring of this compound was linked to Lys721, resulting in the destabilization of catalytic activity.

ADME study
The ADME properties of pyrazolines 1-5 were further evaluated using the pkCSM webtool for ADME prediction.Table 4 presents the ADME properties results of N-phenyl pyrazoline derivatives.The absorption of drugs orally relies on two crucial factors, human intestinal absorption (HIA) and CaCo-2 permeability.HIA predicts the number of compounds that the human small intestine can absorb, whereas CaCo-2 serves as an in vitro model that stimulates the human intestinal mucosa and predicts oral drug absorption.A compound with an HIA value greater than 80% is considered highly absorbed, while the permeability of CaCo-2 is high if the predicted value The liver is a common site for the key enzyme Cytochrome P450, which plays a crucial role in the body by oxidizing xenobiotics to render drug compounds inactive.Cytochromes have isoform models, such as CYP3A4 and CYP2D6, and these processes are essential for drug metabolism and xenobiotic activities [28].Compounds that become substrates indicate that they can be metabolized by CYP450, while compounds that act as inhibitors can suppress the enzyme's metabolic activity.Table 4 shows that all compounds did not act as inhibitors of CYP2D6, whereas pyrazolines 2, 4, and 5 acted as inhibitors of the CYP3A4 enzyme.It can be concluded that pyrazolines 2, 4, and 5 exhibit poor metabolism with Cytochrome P450.
Proximal tubule cells contain a basolateral surface where it is located; the predominant human OCT transporter is OCT2.Shen et al. [29] established that OCT2 is crucial in removing and eliminating cationic drugs and endogenous substances.Additionally, administering OCT2 inhibitors with OCT2 substrates may result in unfavorable interactions.The total clearance value represents the characteristics of drug excretion, with the lowest total clearance indicating that the drug is susceptible to elimination.The data presented in Table 4 shows that all pyrazoline derivatives were not OCT2 substrates and had high total clearance values.

CONCLUSION
The novel thiophene-based N-phenyl pyrazolines 1-5 were successfully synthesized by reacting chalcones and phenylhydrazine.As determined by the MTT assay, Pyrazoline 2 displayed remarkable effectiveness as an anticancer agent targeting colorectal (WiDr), breast (4T1), and cervical (HeLa) cancer cell lines.It exhibited a high level of selectivity against the normal cell line (Vero).The molecular study revealed that N-phenyl pyrazoline 2 exhibited the lowest binding energy, with a value of 8.8 kcal/mol.Chemical interaction studies showed that this compound was located within the binding pocket and interacted with the essential amino acid of EGFR.The ADME study using the pkCSM program demonstrated that the N-phenyl pyrazoline derivatives had favorable pharmacokinetic properties.

ACKNOWLEDGMENT
The Indonesian Endowment Fund for Education (LPDP) Scholarship provided by the Ministry of Finance of the Republic of Indonesia is gratefully acknowledged by the authors for supporting Istna Chunaifah.Additionally, this study was supported by research funding through the Rekognisi Tugas Akhir (RTA-UGM) Project 2022.

AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the O n l i n e F i r s t

Table 1 .
Physical data of N-phenyl pyrazoline derivatives.

Table 2 .
IC 50 and SI values of N-phenyl pyrazoline derivatives.

Table 3 .
The binding affinity of chemical interaction by N-phenyl pyrazoline derivatives.

Table 4 .
ADME property values of N-phenyl pyrazoline.Conversely, pyrazolines 4 and 5 have VDss values below 0.45 and log BB < 0.3.Nonetheless, all pyrazoline derivatives have a log PS > −2, indicating that these compounds exhibit a robust capacity to infiltrate the CNS.