Synthesis, characterization, preliminary molecular docking, pharmacological activity, and ADME studies of some new pyrazoline derivatives as anti-breast cancer agents

This work studied the anti-breast cancer activities of pyrazoline-containing benzenesulfonamides (6–10) in vitro and silico. The GOLD suite performed molecular docking on the target human estrogen receptor and the PARPA1 antagonist crystal structure, yielding comparable results using tamoxifen as a reference drug. Researchers tested these compounds (6–10) on two types of breast cancer cells (MCF-7 and MDA-MB-468) in the lab and found an antiproliferative activity that depended on the dose. Compounds 9 demonstrated a high docking score on PARP1 antagonist crystal structure in triple-negative breast cancer with a PLP fitness score of 93.24 and potential antiproliferative activity with an IC50 of 2.79 µM on the MDA-MB-468 cell line. In contrast, compounds 8 and 10 showed promising activity on the MCF-7 cancer cell line with IC50s of 7.4 and 17.96, respectively.


Introduction
Cancer is universally acknowledged as one of the world's most serious health issues as well as a primary cause of mortality (Nepali et al. 2014).In the 21 st century, successful cancer treatment remains a challenge.There is an urgent need for newer, safer anticancer drugs with an excellent range of cytotoxicity to tumor cells (Jin et al. 2012).Breast cancer (BC) is one of the most lethal diseases in the world (Din et al. 2022).Estrogen receptor (ER) overexpression leads to the diagnosis of 80% of all new BC cases, classifying them as positive estrogen receptor (ER+) BC (Narzieyva and Jonibekov 2020;Tutzauer et al. 2020;Pham et al. 2021).The human estrogen receptor alpha (hER) is essential in physiology.BC ranks high among the world's most fatal diseases.The overexpression of ER categorizes most new BC cases, around 80%, as positive ER+ BC (Narzieyva and Jonibekov 2020;Tutzauer et al. 2020;Pham et al. 2021).Numerous physiological processes rely on the hER, including cell proliferation, survival, and cancer metastasis (Dika et al. 2019;Jacenik et al. 2019;Maity 2019).Tamoxifen-neoplastic breast cancer (TNBC) is characterized by an overexpression of poly ADP ribose polymerase 1 (PARP1) and does not express the ER, progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2).Of all BC, 10%-15% are TNBC (Zagami and Carey 2022).The inability to overcome multi-drug resistance and having poor selectivity are the primary obstacles to treating tumors.Consequently, there has been an all-out effort to find new, highly selective compounds that can halt tumor growth, thereby eradicating malignancy and reducing the severity of BC.Heterocyclic scaffolds are present in both naturally occurring and artificially produced organic compounds of biological importance, and aromatic heterocycles are predominant in numerous medicinal compounds and motifs with physiological activity.Because they are involved in many important biological reactions, heterocyclic molecules are essential to all living systems (Morais et al. 2021).With two nitrogen atoms in its five-membered heterocyclic ring, pyrazoline is a vital member of the heterocyclic family (Kiyani et al. 2015).It possesses a variety of chemical, biological, and medicinal characteristics (Lv et al. 2010).A previous comprehensive study suggests that the pyrazoline ring system can synthesize and design new biologically active molecules with promising anticancer treatment potential (Tok et al. 2010).Fig. 1 illustrates the anticancer effects of several drugs that have pyrazoline scaffolds in their chemical structure (Amin et al. 2013;Rathore et al. 2014;Hamblin 2016;Maity 2019;Fadhil et al. 2022).
Because of their cytotoxicity, compounds generated from sulfonamides, such as pyrazole and pyrazoline, have attracted considerable attention from researchers (Raauf et al. 2022).Researchers are designing, docking molecularly, synthesizing, characterizing, and testing new pyrazoline derivatives with a sulfonamide moiety in vitro for efficacy.

Chemicals and materials
All reagents and commonly used intermediates were obtained from commercial vendors (British company BDH and German company Sigma-Aldrich, hyper-chem, Hangxing RD., Hangzhou, China).

Characterization methods
The melting points were measured using the capillary method on an English-made Bamstead/Electrothermal 9100 instrument.The compounds as KBr disks were characterized using a Fourier transform infrared spectrophotometer (FTIR-6100 Type A).We conducted 1H-NMR at 500 MHz with a Variant Agilent instrument.The varying 13C NMR quality was assessed using a 125 MHz Agilent.

Molecular structure synthesis
The following approaches were used to synthesize various novel pyrazoline derivatives, as shown in Scheme 1.

Synthesis of chalcone derivatives (1-5)
Intermediate compounds represented by different chalcone derivatives were synthesized via Claisen-Schmidt condensation of the respective 4-substituted benzaldehyde with 2-acetylpyridine, as described in the literature (Eid and George 2018).
Method A involves the production of molecules 2 and 4 that represent chalcone with nitro and chloro derivatives.
2-Acetylpyridine was slowly added over 2-3 hours to a solution containing 10% aqueous sodium hydroxide (10 mL), ethanol (5 mL), and the applicable aldehydes (4-nitrobenzaldehyde and 4-chlorobenzaldehyde) in the respective amounts of 17 mmol and 1.90 ml.The temperature was maintained at a steady 0 °C.After agitating the liquid for an additional 2 hours, we filtered it and rinsed it with abundant distilled water.Method A involves the production of molecules 2 and 4.
Method B involves the production of molecules 1, 3, and 5.
We introduced 17 mmol of 2-acetylpyridine and 17 mmol of the appropriate benzaldehydes (4-methoxybenzaldehyde, 4-dimethylaminobenzaldehyde, and benzaldehyde) into 100 mL of water when the temperature dropped below 5 °C.The mixture was vigorously agitated to achieve a highly dispersed emulsion, and then a 10 mL solution of 10% sodium hydroxide was added.The mixture was subjected to two rounds of mixing and then undisturbed overnight at a temperature of 4 °C.It is essential to avoid agitating the solution, as this might lead to phase separation and lower yields.The compound underwent phase separation upon agitation, transforming into an oily state that solidified.Perform filtration and wash with an abundant amount of distilled water.

The following is a general method for synthesizing pyrazoline derivatives (6-10)
Equal quantities of compounds (1-10) (2 mmol) and 4-hydrazine benzenesulfonamide hydrochloride (2 mmol, 0.8947 g) were combined in 5 ml of absolute ethanol.Potassium hydroxide (5 mmol, 0.28 g) was then added.The mixture was heated and refluxed for 8 hours.After cooling, the precipitate was separated, filtered, and washed with abundant distilled water (Daina et al. 2017).

The compound (9) is 4-(5-(4-chlorophenyl)-3-(pyridin-2-yl)-4,5-dihydro-1H-pyrazol-1-yl) benzenesulfonamide
We obtained a pale brown crystal with a melting point of 119-121 °C and a yield of 77%.The infrared spectrum has absorption peaks at 3365.9 and 3292.60 cm −1 for the NH 2 group, 3092.82cm −1 for aromatic C-H bonds, 1595.48 cm −1 for the C=N bond, and 1494.88 and 1404.21cm −1 for the C=C and C=N bonds in the aromatic ring.We observe additional peaks at 1327.07 and 1149.61cm −1 for the SO 2 N group and 1089.23 cm −1 for the C-Cl bond.The proton nuclear magnetic resonance ( 1 H NMR) spectrum, recorded at 500 MHz in DMSO, shows peaks at 3.23 and 3.98 ppm for the CH 2 groups in the pyrazoline moiety and 5.77 ppm for the CH group in the pyrazoline ring.The two protons of the SO 2 NH 2 group exhibit a singlet peak at 7.09 ppm.The aromatic protons appear as multiples in the 7.1-8.69ppm range, totaling 12 protons. 13C NMR shows peaks at 42.81 ppm for C-5 in the pyrazoline ring, 62.70 ppm for C-4 in the pyrazoline ring, and a range of peaks from 111.74 to 149.52 ppm for the 17 carbon atoms in the aromatic ring.The spectrum was recorded at 125 MHz in DMSO-d6.Additionally, the C=N bond in the pyrazoline ring exhibits a peak at 140.58 ppm.

The compound (10) is 4-(5-phenyl-3-(pyridin-2-yl)-4,5-dihydro-1H-pyrazol-1-yl) benzenesulfonamide
The brown crystal has a melting point of 96-98 °C and a yield of 62%.The infrared spectrum has absorption peaks at 3340.82 and 3288.74cm −1 for the NH2 group, 3061.13 cm −1 for aromatic C-H bonds, 1591.33 cm −1 for C=N bonds, and 1502.60 and 1464.02cm −1 for C=C and C=N bonds in the aromatic ring.Additionally, there are absorption peaks at 1332.68 cm −1 and 1153.47 cm −1 for the SO2 N group.The 1H NMR spectrum (500 MHz, DMSO) shows peaks at 3.22 and 3.99 ppm for CH2 groups in the pyrazoline moiety, 5.73 ppm for a CH group in the pyrazoline moiety, 7.07 ppm for two protons in the SO2NH2 group, and a range of peaks from 7.16 to 8.62 ppm for 12 aromatic protons.The 13C NMR spectrum (DMSO-d6, 125 MHz) shows peaks at 43.02 ppm for C-5 in the pyrazoline moiety, 63.35 ppm for C-4 in the pyrazoline moiety, and 17 carbon atoms in the aromatic ring with a range of peaks from 111.62 to 148.23 ppm.There is also a peak at 139.38 ppm for the C=N bond in the pyrazoline moiety.

Methods of computation
Fig. 2 illustrates the computational preparation of this study.We docked our compounds using the CCDC GOLD Suite (v.5.6.2).The CCDC Hermes visualizer (1.9.2) shows ligands, receptors, hydrogen bonding, short contacts, and bond length calculations.We used ChemBioOffice (v.17.1) to draw the structures of our ligands and reference medicine compounds.The synthetic drug ADME Was determined using the Swiss ADME server (Daina et al. 2017).

ADME procedures
The compounds (6-10) were formed using ChembioOffice software and subsequently transformed into SMILE names using the Swiss ADME tool.The Swiss ADME program also predicted the compounds' pharmacokinetic and physicochemical parameters.We evaluated the small molecule's lipophilicity and polarity using a boiled egg (Odoemelam et al. 2022).

Ligands and receptor preparation
The protein data bank (PDB) gave us the crystal structures of the human estrogen receptor and the PARP1 antagonist, and SwissPDB Viewer (SPDBV) version 3.7 was used to add any missing atoms.To create a protein's crystal structure, we remove water molecules that do not influence binding and retain those that do.Next, hydrogen was added to the amino acid residues to bring them into the correct ionized and tautomeric state.We minimized the energy of the generated molecules using CheBio3D (v.19.1) and the MM2 force field.

Molecular docking protocol
We performed the molecular docking using the licensed version of genetic optimization for ligand docking (GOLD) (v. 2020.3.0).The Hermes visualizer application within GOLD successfully executed the docking operation.Nucleotides and protein residues within 10 Angstroms of reference ligands found in protein structure complexes make up the docking binding site.The proteins were obtained from the PDB website, specifically from PDB entries 1ERR and 5HA9.The active site radius was determined to be 10 Angstroms (Å) using the protein reference ligand.Then, the chemscore kinase was used as a configuration template.We executed the scoring function using ChemPLP.The GOLD docking technique utilized default values for all parameters, and the solutions were evaluated based on the fitness function of piecewise linear potential (CHEMPLP).The docking findings, including the docked posture, binding mode, and binding free energy, were used to assess the ligands' interactions with the protein residues in the active site of the human estrogen receptor.

Antiproliferative ASSAY
We investigated the anticancer effect of (6-10) on the viability of breast cancer cell lines (MCF-7 and MDA) using the MTT test methodology.The College of Pharmacy at Mustansiriyah University conducted this investigation.

Determining the value of the half-maximum inhibitory concentration (IC50)
Based on the in vitro MTT experiment, the IC50 value indicates the concentration of the tested chemicals (ranging from 6 to 10) needed to decrease cell viability by 50%.Based on the findings of the in vitro MTT assay, the IC50 values were calculated for 6 to 10 compounds 72 hours after the cells were exposed to these compounds.

Quantitative analysis of data using statistical methods
Graph pad Prism and nonlinear curve fitting software were used to do statistical analyses of the IC50 and MTT test data for the medicines we looked at (6-10) on MCF-7 and MDA-MB-468 cells.A one-way analysis of variance (ANOVA) was conducted using the Tukey test to compare all groups within the same MTT plate (using both prism and software).P values exceeding 0.05 determined the statistical significance.

Analysis of the ADME findings
The drug-like characteristics of all the final compounds (610) were calculated using Lipinski's rule of five (Kadela-Tomanek et al. 2021).The current study routinely uses this procedure, known as Pfizer's rule of five (RO5), to screen compounds for drug design leads.The qualities of oral pharmaceuticals that Lipinski's rule of five requires include molecular weight 500, log p 5, hydrogen bond acceptor 10, and hydrogen bond donor 5. Also, the compounds' topological polar surface area (TPSA) and influencing bioavailability were calculated.As a result, passively absorbed drugs with TPSAs > 140 Ao have low bioavailability.According to ADME prediction data, all compounds except (7) had TPSA below 140 A, reaching 106.26, 142.85, 100.27, 97.03, and 97.03 A. All chemicals enter systemic circulation because of their 0.55 bioavailability.Table 1 shows Lipinski's rule of five (RO5).All derivatives met the key topological descriptors and fingerprints of the molecular drug-likeness structure, such as LogP and LogS.
All chemicals, except (7), had high GI absorption scores, which measure xenobiotic absorption from the gastrointestinal tract following oral administration.It has been expected that the present compounds have high absorption of intestinal solids, while compound 7 showed limited absorption.

Analysis of docking final results
The last compounds (6-10) were successfully docked using the GOLD Suite software.Ligands undergo energy minimization to correct distorted geometries by repositioning atoms to alleviate internal constraints.Energy reduction restores the geometry and leads to a minimum energy state.The complex's fitness function ability has been observed for all requested molecules.We rated the compounds (6-10) and tamoxifen's binding affinity to the human estrogen receptor with PDB (1ERR), as well as the compounds (6-10) and a new PARP1 an- tagonist's binding affinity with PDB (5HA9), based on their PLP fitness in forming complexes at the active sites.
The docked compounds on the ER had PLP fitness values ranging from 78.24 to 89.37.Table 2 shows that the PLP fitness of the docked compounds on the PARP1 antagonist ranged from 81.62 to 93.24.Table 2 displays the docking process, which involves the formation of hydrogen bonds and brief interactions with the current ligands.We use gold to determine the distance between the specified protein atoms and our manufactured molecules.The majority of these bonds are less than 3A in length.The short contacts refer to different interactions between molecules, such as van der Waals forces, electrostatic interactions, steric effects, pi-pi stacking, dipole-dipole interactions, and more.Through hydrogen bonds and short contacts, the docking analysis showed that several amino acid residues (ALA 350,ASP 351,GLU 353,TRP 383,LEU 384,LEU 387,MET 388,LEU 391,ARG 394,PHE 404,MET 421,ILE 424,PHE 425,LEU 428,GLY 521,HIS 524,and LEU 525) in the active site of the human estrogen alpha receptor interact with our final ligand's library.These interactions demonstrate promising anti-breast cancer activity.Compounds 13 and 15 have the most excellent PLP fitness values of 89.27 89.37, respectively.In comparison, tamoxifen and 4-hydroxytamoxifen (4-HRT) have PLP fitness values of 94.69 and 95.36, respectively.Fig. 2 illustrates the binding mechanism of 4-HRT, whereas Figs 3, 4 depict the bond interactions of compounds 8 and 10 with high scores.Fig. 5 illustrates the specific way in which chemical 9 binds to the PARP1 antagonist.

Analyzing results from anti-breast cancer tests
The compounds' cytotoxic findings (6-10) demonstrated their anti-BC activity.Compound (9), which had an IC50 value of 2.79 µM on MDA-MB-468, was found to have the most effective cytotoxic effect.This makes it about 5.4 times more active than tamoxifen, which had an IC50 value of 15.29 µM.Therefore, compound (9) requires a lower concentration than tamoxifen to inhibit the

Figure 1 .
Figure 1.Certain synthetic compounds containing pyrazoline motifs exhibit significant potential as anticancer agents.

13C
NMR spectrum (measured at 125 MHz) of the compound in DMSO-d6 shows the following chemical shifts in parts per million (ppm): 41.43 for the N[CH 3 ] 2 group, 46.22 for the C-5 atom in the pyrazoline moiety, 63.31 for the C-4 atom, and 108.21 to 144.11 for the 17 carbon atoms in the aromatic ring.Additionally, a peak at 148.21 ppm corresponds to the C=N bond in the diazole group.

Figure 2 .
Figure 2. Computational protocol for the desired compounds.

Figure 11 .
Figure 11.IC50 dose response curves for compound 10 on the MCF-7 cell line for compound 10, respectively.

Table 1 .
The ADME result for the final derivatives.

Table 3 .
Comparing tamoxifen to all compounds (C6-C10) at 72 hours for cell death in MCF-7 and MDA-MB-468 cell lines.