Synthesis and Anti-Cancer Activity of New Pyrazolinyl-Indole Derivatives: Pharmacophoric Interactions and Docking Studies for Identifying New EGFR Inhibitors

Newly designed series of indole-containing pyrazole analogs, pyrazolinylindoles, were synthesized, and their structures were confirmed based on the spectral data of the 1H NMR, 13C NMR, and HR-MS analyses. Preliminary anti-cancer activity testings were carried out by the National Cancer Institute, United States of America (NCI, USA). Compounds HD02, HD05, and HD12 demonstrated remarkable cytotoxic activities against nine categories of cancer types based cell line panels which included leukemia, colon, breast, melanoma, lungs, renal, prostate, CNS, and ovarian cancer cell lines. The highest cytotoxic effects were exhibited by the compounds HD02 [1-(5-(1-H-indol-3-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-phenylethanone], HD05 [1-(3-(4-chlorophenyl)-5-(1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)-2-phenoxyethanone], and HD12 [(3-(4-chlorophenyl)-5-(1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone] against some of the 56 types of NCI-based cell lines in different panels. Compound HD05 showed the maximum range of cancer cell growth inhibitions against all categories of the cell lines in all nine panels. On average, in comparison to the referral standard, imatinib, at a dose level of 10 µM, the HD05 showed significant activity against leukemia in the range of 78.76%, as compared to the imatinib at 9% of cancer cells’ growth inhibitions. Molecular docking simulation studies were performed in silico on the epidermal growth factor receptor (EGFR) tyrosine kinase, in order to validate the activity.


Introduction
Cancer is a global concern and draws major attention from governments worldwide. The World Health Organization (WHO) also published interval statistical reports for cancer incidences, preventions, and management protocols [1]. According to the latest WHO estimates, the global cancer burden is set to rise by over 18

Anti-Cancer Screening Methodology
The initial in vitro cytotoxicities of the compounds were evaluated according to the one-dose protocol assay method against a panel of fifty-six tumor cell lines at the NCI (National Cancer Institute), Bethesda, USA. The origins and processing of the cell lines used in the study have been previously discussed [21][22][23][24]. Briefly, the human tumor cell line of the cancer screening panel was grown in RPMI 1640 medium containing 5% FBS (fetal bovine serum), and 2 mM L-glutamine. For a typical screening experiment, cells were inoculated into a 96-well microtiter plate in 100 µL at plating densities ranging from 5000 to 40,000 cells/well, depending upon the doubling time of the individual cell line. After cell inoculation, the microtiter plates were incubated at 37 • C, with 5% CO 2 , 95% air, and 100% relative humidity for 24 hr prior to the addition of the test compound. After 24 h, two plates of each cell line were fixated in situ with TCA (trichloroacetic acid), in order to represent the measurements of the cell populations for each cell line at the time of each test compound's addition. The test compound was solubilized in DMSO (dimethyl sulfoxide) at 400x-folds of the desired final maximum test concentration, and was stored frozen prior to its use. At the time of test compound addition, an aliquot of frozen concentrate was thawed and was diluted to double the desired final maximum test concentration with complete medium, containing 50 µg/mL gentamicin. Following the test compound's addition, the plate was incubated for an additional 48 hr at 37 • C, 5% CO 2 , 95% air, and 100% relative humidity. For the adherent cells, the assay was terminated by addition of cold TCA. The cells were fixated in situ by gentle addition of 50 µL of cold 50% (w/v) TCA (final concentration, 10% TCA), and incubated for 60 min at 4 • C. The supernatant was discarded, and the plate was washed five times with water and air-dried. The sulforhodamine B (SRB) solution (100 µL) at 0.4% (w/v) in 1% acetic acid was added to each well, and the plate was incubated for 10 min at room temperature. After staining, unbound dye was removed by washing five times with 1% acetic acid, and the plate was air-dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology was the same except that the assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 µL of 80% TCA (final concentration, 16% TCA).

Molecular Modelling
Towards approaching the in silico studies, the X-ray crystal structure of EGFR tyrosine kinase (PDB: 2J5F) family protein, at a resolution of 3.00 Å, was downloaded ( Figure 1) from https://www.rcsb.org/structure/2j5f (accessed on 20 January 2022). The receptor orientations are displayed through the different colored coils (α and β coils) of chain A of the receptor, containing the EGFR kinase receptor domain in complex with an irreversible inhibitor, 34-JAB, which is shown as placed between the blue and the green coiled interaction area as part of the X-ray diffraction-based crystal structure of the tyrosine kinase receptor. It contains 2528 atoms, with 37.67 kDa structure weight where different structural domains of the receptor chain are shown with structural cross-links in different colors (blue, green, red, and yellow) of the structural domains. The protruding side chain (brown color) contains thioether (S-C) linkages with the cysteine residues. The receptor is classified as having global asymmetric-C1 symmetry, and global stoichiometry as monomer-A1 [(2J5F: FirstGlance in Jmol (proteopedia.org, accessed on 15 April 2022), and validation data 2j5f_full_validation.pdf (rcsb.org, accessed on 15 April 2022)]. The EGFR of the tyrosine kinase (TK) family, a transmembrane protein, the active site is defined by the presence of residues Phe 723 , Thr 790 , Lys 745 , Gln 791 , Gly 796 , Leu 792 , Met 793 , Asp 800 , and Asp 855 , which makes it a hydrophobic binding site along with the bound ligand 34-JAB [25].

Chemical Synthesis
The synthetic routes used to prepare the starting materials and the title compounds are outlined in Scheme 1. Aromatic acid hydrazide derivatives (1a-c) were prepared by the reaction between correspondingly substituted aromatic acid, ethanol, and hydrazine hydrate [26]. Indole carboxaldehyde was obtained on condensation with substituted acetophenone in absolute ethanol, and 10% NaOH afforded the corresponding 1-(substitutedphenyl)-3-(1H-indol-3-yl)-prop-2-en-1-one, chalcanoids 2a-d [27]. The chalcanoids are useful intermediates to generate various compounds with diverse biological activity. The titled indolopyrazolines, HD01-HD12 (Table 1), were synthesized by cyclo-condensation of the chalcanoids 2a-d with hydrazine hydrate derivatives (1a-c) in absolute ethanol, in the presence of glacial acetic acid, and molecular sieves, where reaction time varied from 18 to 22 h. The products were obtained in varying yields, purified, and characterized. All other aromatic and aliphatic protons were observed at expected chemical shift values. The 13 C-NMR spectra of the compounds exhibited carbonyl carbon signals between the ranges of δ 162.82-165.47 ppm. The chemical shift values of the carbon atoms varied between δ 43.34 and 43.42 ppm for C4, δ 60.04-60.24 ppm for C5, and δ 151.47-152.38 ppm for C3 carbon(s) presence, which corroborated the 2-pyrazoline structural part. The 1 H-NMR data also supported this. The mass spectral analyses data of the synthesized compounds were found to be in full agreement with the assigned structures. All the compounds showed satisfactory elemental analysis.  All other aromatic and aliphatic protons were observed at expected chemical shift values. The 13 C-NMR spectra of the compounds exhibited carbonyl carbon signals between the ranges of δ 162.82-165.47 ppm. The chemical shift values of the carbon atoms varied between δ 43.34 and 43.42 ppm for C4, δ 60.04-60.24 ppm for C5, and δ 151.47-152.38 ppm for C3 carbon(s) presence, which corroborated the 2-pyrazoline structural part. The 1 H-NMR data also supported this. The mass spectral analyses data of the synthesized compounds were found to be in full agreement with the assigned structures. All the compounds showed satisfactory elemental analysis.  All other aromatic and aliphatic protons were observed at expected chemical shift values. The 13 C-NMR spectra of the compounds exhibited carbonyl carbon signals between the ranges of δ 162.82-165.47 ppm. The chemical shift values of the carbon atoms varied between δ 43.34 and 43.42 ppm for C4, δ 60.04-60.24 ppm for C5, and δ 151.47-152.38 ppm for C3 carbon(s) presence, which corroborated the 2-pyrazoline structural part. The 1 H-NMR data also supported this. The mass spectral analyses data of the synthesized compounds were found to be in full agreement with the assigned structures. All the compounds showed satisfactory elemental analysis. All other aromatic and aliphatic protons were observed at expected chemical shift values. The 13 C-NMR spectra of the compounds exhibited carbonyl carbon signals between the ranges of δ 162.82-165.47 ppm. The chemical shift values of the carbon atoms varied between δ 43.34 and 43.42 ppm for C 4 , δ 60.04-60.24 ppm for C 5 , and δ 151.47-152.38 ppm for C 3 carbon(s) presence, which corroborated the 2-pyrazoline structural part. The 1 H-NMR data also supported this. The mass spectral analyses data of the synthesized compounds were found to be in full agreement with the assigned structures. All the compounds showed satisfactory elemental analysis.

Molecular Modelling Studies
The TK protein was prepared by deleting the covalent bond of the bound ligand with the receptor, using a preparation wizard (Maestro, version 8.5, Schrodinger, LLC, New York, NY, USA, 2008). The bond orders were assigned, hydrogen atoms were added, and the water molecules were deleted. The hetero state for the co-crystallized ligand was generated using Epik, protonation state. The optimization of H-bondings of the protein side chains were performed using Protassign ® . The energy minimizations were carried out using the OPLS-2005 force field. The receptor grid of 12 Å was generated around the active site of EGFR-TK defining the bound ligand by using GLIDE 5.0. The 3D structures of all the ligands were constructed using the Maestro 8.5, and were minimized using the Macromodel minimization panel with the OPLS-2005 force field, and GB/SA water model. LigPrep2.0 module of Schrodinger was used to generate the ionization states at the target pH of 7.0 ± 2.0. The twelve tautomers were generated at this pH, which were employed for ligand dockings. The Xtra precision (XP) mode protocol of GLIDE 5.0 was used for ligand dockings. The molecular docking simulations of the active 1-(5-(1H-indol-3-yl)-4,5-dihydro-1H-pyrazole-1-yl)-2-phenylethanoid derivatives were performed on the EGFR-TK protein base model (PDB: 2J5F).
A fragment-based development of the primary molecular template was appropriated. The choice of an indole-based chalcanoid infusion with the pyrazole ring as the starting molecular template was envisioned based on several studies on the anti-cancer activities of these two structural templates [28][29][30]. The role of the EGFR on certain chalcanoids has previously been discussed in an earlier report [31]. The modeled compounds, pyrazolinylindoles, at one end, were substituted by benzyl, phenoxy, and pyridyl groups, as the larger moieties selected for the binding at the EGFR-TK site possess the Met 793 residue. Simultaneously, the other set of substitutions, i.e., chlorophenyl, hydroxyphenyl, and toluyl, were attached nearly at the right angle set of the resultant geometry of the final template in order to interact with the Glu 762 amino acid residue of the receptor. The attached indole moiety was envisioned to be interacting with the Asp 855 residue. The binding deep pocket, and active sites containing other residues Phe 723 , Thr 790 , Lys 745 , Gln 791 , Gly 796 , Leu 792 , Met 793 , Asp 800 , and Asp 855 were also involved in the receptor-ligand interactions with the differently substituted templates. The molecular frameworks of pyrazolinylindoles and the substitutions thereupon helped to deduce the binding pocket disposition, and the geometric specifications of the site.
The binding of HD05 to the active site of the EGFR-TK protein, and the hydrogen binding sites are shown in Figure 2a,b, while the amino acid bindings, and the interactions as well the structural orientations, together with the 3D binding views of the compound, HD05, are visible in Figure 3. The active compound modeling confirmed the interaction sites, the participating sequence parts, and the geometric orientation of the active molecules. From the IFD (induced-fit docking) studies, the indole of HD05 depicted the π-cation, π-π, and H-bond (hydrogen bonding) interactions with key residues, such as, Arg 841 , Lys 745 , Phe 723 , and Gly 724 as depicted in Figures 2 and 3, whereas the chlorophenyl group of the compound HD05 structure also shown π-cation interacting with the residue at Lys 745 , halogen-based bond with Thr 790 , and carbamate backbone H-bond interaction with Met 793 residue. Moreover, the flexibility of Arg 841 , Lys 745 , and Phe 723 residues were observed in IFD studies, which also favored the bindings with other compounds of the currently synthesized series. It was also observed that the pyridine ring of the compounds exhibited backbone hydrogen bonding with crucial residue Met 793 as shown in Figures 4 and 5, and apart from that, the carbonyl oxygen and nitrogen atoms of the indole nucleus of compounds also showed side-chain hydrogen bonding with Thr 854 and Asn 842 . An indole nucleus also saw the π-cationic and π-π stacking interactions with residues Lys 745 and Phe 723 in the structures, while Figure 6 demonstrated the 3D in silico view at the active site of EGFR tyrosine kinase with the compounds, HD02, HD05, and HD12.

Cell-Line Based Anti-Cancers' Biological Testings
Preliminary anti-cancers' screenings were carried out according to the NCI institutional protocols, also described in the Materials and Methods section. The initial testings were performed at single dose levels on leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancer cell lines. The one-dose data are reported as the mean percent growth inhibition of the treated cancer cells. The reports are in comparison to the control of untreated cells, and are relative at the zero time's cells' numbers. The data of anti-cancer screening of the compounds HD02 (NSC: 778247/1), HD05 (NSC: 778249/1), and HD12 (778248/1) are presented in Table 2  Preliminary anti-cancers' screenings were carried out according to the NCI institutional protocols, also described in the Materials and Methods section. The initial testings were performed at single dose levels on leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancer cell lines. The one-dose data are reported as the mean percent growth inhibition of the treated cancer cells. The reports are in comparison to the control of untreated cells, and are relative at the zero time's cells' numbers. The data of anti-cancer screening of the compounds HD02 (NSC: 778247/1), HD05 (NSC: 778249/1), and HD12 (778248/1) are presented in Table 2  Preliminary anti-cancers' screenings were carried out according to the NCI institutional protocols, also described in the Materials and Methods section. The initial testings were performed at single dose levels on leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancer cell lines. The one-dose data are reported as the mean percent growth inhibition of the treated cancer cells. The reports are in comparison to the control of untreated cells, and are relative at the zero time's cells' numbers. The data of anti-cancer screening of the compounds HD02 (NSC: 778247/1), HD05 (NSC: 778249/1), and HD12 (778248/1) are presented in Table 2  Preliminary anti-cancers' screenings were carried out according to the NCI institutional protocols, also described in the Materials and Methods section. The initial testings were performed at single dose levels on leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancer cell lines. The one-dose data are reported as the mean percent growth inhibition of the treated cancer cells. The reports are in comparison to the control of untreated cells, and are relative at the zero time's cells' numbers. The data of anti-cancer screening of the compounds HD02 (NSC: 778247/1), HD05 (NSC: 778249/1), and HD12 (778248/1) are presented in Table 2, which is against nine categories of the cancer types' cell lines, i.e., leukemia (CCRF-CEM, MOLT4, and SR human lymphoma cancerous cell lines), lungs NSLC (NCI-H460 cell line), melanoma (LOX IMVI cell         The anti-cancer activity evaluated at NCI, USA, as per the NCI protocol at one dose level assay at 10 µM on nine panels of fifty-six cancer cell lines [24,25,28], are represented in terms of growth percent (GP) and percent growth inhibitions (%GI). The %GI was calculated from GP; that is, %GI = 100-GP. The averaged %GIs of the nine panels of cancer cell lines were calculated in order to compare the anti-cancer activity of compounds HD02, HD05, HD12, and imatinib, as shown in Figure 7. The anti-cancer data of imatinib were retrieved from the NCI website with the drug code NSC 759,854 [32].
Thus, the compounds HD02, HD05, and HD12 hold promise for the future and can serve as a template, and new molecular leads for further development. The compound HD05 showed higher anti-cancer activity than imatinib on all nine cell lines panels, leukemia, melanoma, ovarian cancer, CNS, renal cancer, lung cancer, colon cancer, and breast cancer.

Conclusions
Compounds HD02, HD05, and HD12, showed higher cell line growth inhibitions against nine panels of cell line categories constituting fifty-six cell lines. The compound HD05 was mostly active against all the cancer cell line types, and showed better activity against the leukemia cell line, MOLT4. This compound can serve as a template for new molecular leads toward further development.