3,4-Dimethoxybenzaldehyde,4-Chlorobenz-aldehyde, Ethyl acetoacetate, 4-Hydroxy- benzaldehyde were purchased from MERCK.4-Acetamidobenzaldehyde,4-Dimethylaminobenzaldehyde, 4-Methoxy- benzaldehyde, Dimethylamine taken from BDH.Benzaldehyde and Petroleum ether were obtained from APC pure. All chemicals were used as provided without more purification.

The Stuart SMP apparatus was employed to measure the melting point. Fourier transform infrared spectroscopy (FTIR) studies were completed by Schimadzu FTIR spectrometer (Japan) using potassium bromide (KBr) pellets for solid samples. ¹H-Nuclear magnetic resonance (NMR) and ¹³C-NMRspectra were recorded by a Bruker instrument (Brucker, Switzerland). CDCl₃ and DMSO-_d6 were employed as the solvent; however, tetramethyl silane served as the internal standard. Coupling constant (J) values were reported in hertz (Hz). A 5973 Agilent Mass spectrometer equipped with electron impact 70 eV(electron volt) was employed to obtain mass spectral measurements.

MCF-7 breast cell line was preserved in PRMI-1640 (Gibco-The National Cell Bank of Iran provided the human breast cancer cell line MCF7) complemented with 10% fetal bovine, 100 units/mL penicillin and 100 µg/mL streptomycin. Cells were reseeded at a 50% confluence twice a week using Trypsin/EDTA (Gibco) and incubation at 37 °C and 5% CO₂. The MTT cell viability assay was performed on 96-well plates to determine the cytotoxic effect. The MCF-7 cell line was seeded at around 1*10 ⁴ cells/well. After 24 hours or the formation of a confluent monolayer, the cells were treated with the produced substance at the final concentration (1000 g/mL). After 72 hours, cell viability was assessed by extracting the medium by applying 28 µL of 2 mg/ mL of MTT solution. Upon removal of the MTT solution, the crystal remaining in the well were solubilized by adding 100 µL of DMSO followed by an incubation at 37 °C for 15 minutes with shaking.

The absorbency was assessed on a microplate reader (Model WAVE XS2, Bio Tek, USA) at test wavelength 570 nm, the test was initiated at triplicate. The percentages of viable and killed cells were then calculated according to Eq. 1 and 2 respectively:

Proliferation Rate (PR) % = B / A * 100 Eq (1).

Inhibition Rate (IR) % = 100 – PR Eq (2).

Were:

A: is the mean optical density of untreated wells

B: is the optical density of treated wells.

The human ER-α ligand binding site with Raloxifene as nonsteroidal antagonist was used as a target protein. The crystal structure of human estrogenic receptor-α (PDB ID: 1ERR) was downloaded from protein data bank (www.rcsb.org). The water molecules were removed from protein PDB file (1ERR), then the side chain missing and residues were repaired and the energies of protein were minimized with the MMFF94s force field by applying MOE (Molecular Operating System) program 2015. The studied compounds were draw and their energies were minimized using the same force filed. A molecular docking study was undertaken between the resulted structure of the studied compounds and the crystal structure of the hER-α (1ERR) by using the MOE 2015.

Lipinski’s Rule of Five (Lipinski 2004) indicates a chemical compound’s potential as a medication with a specific biological activity that is intended for oral delivery (Chen et al. 2020). According to (RO5), a drug-like compound must have a molecular weight less than 500 g/mol, a log p value less than 5, representing its hydrophobicity, a number of hydrogen bond donors less than 5, and a number of hydrogen bond acceptors less than 10 (Chagas et al. 2018). Additional investigation has added two additional requirements: a polar surface area (PSA) less than 140 Ų which is associated with permeability, and the number of rotatable bonds less than 10, which is associated with flexibility.

The synthesis of coumarin-pyrazoline derivatives (9–16), was accomplished via the cyclocondensation reaction of appropriate coumarin-chalcone derivatives (1–8) with hydrazine hydrate in presence of formic acid, as shown in (Suppl. material 1: scheme 3).

The structures of coumarin-pyrazoline derivatives(9–16), were established by numerous significant spectral changes, such as MS, IR, ¹H NMR and ¹³C NMR.

The molecular weight of synthesis compound detected by mass spectrum, and most derivatives determined by appearance as molecular ion (M^+.) peaks equal to molecular weight for each derivative, as noticed in (Suppl. material 3: table S1).

The synthesis compounds were established by the FT-IR spectra and showed a strong band at 1724–1732 cm^-1 attributed to carbonyl groups of coumarin part, while the absorption band at 1649–1674 cm^-1 related to aldehyde groups at pyrazoline ring (Singh et al. 2010; Ahmed et al. 2019). All synthetic compounds with Pyrazoline derivatives show the absorption bands in the regions 1608–1618 cm^–1 corresponding to the C═N stretching bands of pyrazoline ring (Ahmed et al. 2019). In addition, the absorption bands at regions around 1415–1481 cm^–1 were attributed to the (N–N) stretch vibrations, which also confirm the formation of the desired pyrazoline ring in all the derivatives (Celik et al. 2020), as shown in (Suppl. material 3: table S2).

The ¹H-NMR spectrum of coumarin- pyrazoline derivatives (9–16) showed singlet signal within the chemical shift about 8.9 ppm refers to the aldehyde (–CHO) proton that presence at pyrazoline ring (Ahmed et al. 2019). The methylene group (CH₂) of pyrazoline ring has two nonequivalent protons (Ha and Hb) appeared as pair of doublets of doublets signals. The Ha proton appeared at chemical shift 3.23–3.27 ppm with coupling constant about 18 Hz and 4.8 Hz, as geminal and vicinal coupling with Hb and Hc protons respectively, while the Hb proton appeared at chemical shift 3.89–3.98 ppm with coupling constant about 18 Hz and 11.8 Hz as geminal and vicinal coupling with Ha and Hc protons respectively. The doublet of doublets signal at chemical shift 5.39–5.54 ppm with coupling constants about 11.8 Hz and 4.8 Hz as a result of vicinal coupling with nonequivalent protons, Ha and Hb respectively. This signal refers to Hc proton of methine group (CH) of the pyrazoline ring (Patel 2019; Patel et al. 2021).Other chemical shift and their interpretation shown in (Suppl. material 3: table S3).

The ¹³C-NMR spectra of synthesis coumarin pyrazoline derivatives 9–16 showed two signals around 44 ppm and 59 ppm, referred to (-CH₂) and (-CH) of pyrazoline ring, that demonstrate the formation of the carbon skeleton of pyrazolines (Singh et al. 2010), a signal at about 153 ppm, refers to the C=N group as a part of the pyrazoline ring (Saroja et al. 2021).

The signal appears at 160 ppm, indicated the formation of an aldehyde group on the pyrazoline ring (Singh et al. 2010). While other aromatic carbons appeared between 116.5–158.6 ppm (Patel et al. 2021) as noticed in (Suppl. material 3: table S4).

All the synthesize coumarin pyrazoline compounds were studied using Lipinski’s Rule of Five. It indicates a chemical compound’s potential as a medication with a specific biological activity that is intended for oral delivery (Chen et al. 2020). The result shows that the calculated molecular weight of all compounds in the range 318.33–378.38 g/mol; the calculated number of hydrogen bond acceptors in the range 4–6; the calculated number of Hydrogen bonds donor in range 0–1; the calculated number of rotatable bonds in the range 3–5; the lipid–water partition coefficient was between 1.74 and 2.79; the calculated polar surface area in range 62.88–92.34. A substance’s pharmacokinetic characteristics and bioavailability throughout an organism’s metabolic process will be improved by complies with the five principles (Patel et al. 2021; Saroja et al. 2021), as shown in (Suppl. material 3: table S5).

The half maximal Inhibitory Concentration (IC₅₀) was considered depended on the dose-response curve created after finding the percent of the Cell death at several concentrations of the Composites as shown in Suppl. material 1: fig. S1. All the synthesized compounds displayed cytotoxicity and IC₅₀ estimated in a range of 22.6–318.32 µg/mL as shown in (Suppl. material 3: table S6).

The coumarin pyrazoline derivatives 9–16 revealed little inhibition over the MCF-7 cell line as compared to Raloxifene (IC₅₀ = 7.29). Two of the all pyrazoline compounds, 4-hydroxy-3-methoxyphenyl substituted pyrazoline (cpd.13) and 4-chlorophenyl substituted pyrazoline (cpd.14), showed remarkable activity than other compounds with IC₅₀ = 22.6 µg/mL, and 25.99 µg/mL respectively against MCF-7 cell line.

Compound 9 without substituted showed moderate activity against MCF-7 cell line with IC₅₀ = 61.44 µg/mL. Compounds 12 (4-NHCOCH₃ substituted pyrazoline), showed moderate activity against MCF-7 at IC₅₀ = 70.11 µg/mL. The rest of the coumarin pyrazoline derivatives represented inactive compounds on the MCF-7 cell line.

The molecular docking study was made to understand in what manner coumarin- pyrazolines derivatives cooperate with the receptor.

The estrogen receptor (ER) was selected in this study since the majority of common kinds of breast cancer are determined by the expression of the ER-positive type, so to investigate the interaction of compounds 9–16 with the targeted proteins (ER-α), a molecular docking study was used.

To describe the better possible binding modes between studied compounds (9–16) and ER-α. The human ER-α ligand binding site with Raloxifene as nonsteroidal antagonist was used as a target protein. The crystal structure of human estrogenic receptor-α (PDB ID: 1ERR) was downloaded from protein data bank (www.rcsb.org). Before docking chain B and molecules of water were removed from PDB file of protein (ID: 1ERR). While the side chains missing and residues were corrected and the energy of protein were minimized using the force field MMFF94s. The studied compounds were draw and their energies were minimized with the same force field (MMFF94s) by applying MOE (Molecular Operating System) program 2015. The resulted structures of the studied compounds were used for docking analysis with the crystal structure of ER-α (1ERR) in MOE 2015 program.

Docking analysis provided several conformations that were scored to determine favorable binding modes with estrogenic receptor. The highest docking score for studied compounds and native ligand are summarized in (Suppl. material 3: table S7) in addition to the measured root mean square deviation (RMSD).

The docking studies of studied compounds were performed into the binding pocket of hER-α (PDB: 1ERR) protein (Met 343, Leu 346, Thr 347, Lue 349, Ala 350, Asp 351, Glu 353, Leu 354, Trp 363, Leu 384, Leu 387, Met 388, Leu 391, Arg 394, Phe 404, Met 421, Ile 424, Leu 428, Gly 521, His 524, Leu 525, Leu 536, Leu 539).

The results of docking studied revealed the following findings. The studied compounds were fitted in the active site of the native ligand (Raloxifene) in the hER-α (ID: 1ERR) as seen from the Suppl. material 2: figs S2–S10). But the binding energies of the studied compound were observed between -7.17308 to -6.71756 kcal.mol^-1, with a maximum score achieved by compounds 11 and 16 with-7.04260 and -7.17308 kcal.mol^-1 respectively, indicating the potency of these compounds as estrogenic receptor inhibitor (Savita et al. 2019). But, when these values were comparable to the binding energy obtained for Raloxifene co crystallized with the hER-α (1ERR), used here as a positive control (-9.61928 kcal/mol), and the comparison of the binding of studied compounds and Raloxifene with estrogenic receptor (1ERR), indicated that compound 11 form two π -H interactions, one between substituted aromatic ring and Ala350, but the other interaction was observed between the pyrone ring and Thr 347, while compound 16 form one π -H interaction between substituted aromatic ring and Leu525. Raloxifene form a strong ionic bonding with Asp 351 (Savita et al. 2019), which is absent in compounds 11 and 16, which means that a strong binding interaction with Asp 351 is important for activity. Furthermore, depending on the binding energy and binding mode of Raloxifene with the estrogenic receptor- α (1ERR), we can clearly see that all the studied compounds are less active than raloxifene as an estrogenic receptor inhibitor.