Design, synthesis, molecular docking, and biological studies of novel phytoestrogen-tanaproget hybrids

ABSTRACT A diverse range of novel and highly functionalized flavonoid-based tanaproget hybrids were synthesized and evaluated in vitro for their antimicrobial and antiproliferative activities. Novel products were synthesized in good yields (81–95%) under Pd-catalyzed reaction from bromo flavones and tanaproget boronic acids within 18–20 min at 60 °C. Bioassay results exhibited excellent activities against both hormone-dependent and hormone-independent human breast cancer cells (MCF-7, MDA-MB-231, DU-145, PC-3, and HeLa). Among them, compounds 4e, 9a, 9c, 9e, 9 g, 9 h, 9 m, and 9n displayed excellent activity. Compounds 4d, 4o, and 9o were found equally potent against C. albicans compared to fluconazole. Compound 5c showed better antibacterial activity against S. aureus. Compounds 5a, 9i, 9o, and 10c have shown admirable antibacterial activity against E. coli. GRAPHICAL ABSTRACT


Introduction
Cancer is a generic term for a large group of diseases that can affect all parts of the body. There are 32.6 million people living with cancers in 2012 worldwide and 14.1 million new cases are reported every year, in which 8.2 million led to cancer death. Therefore, the search for anticancer agents that display a broader spectrum of cytotoxicity to tumor cells is of great interest. [1,2] Natural products obtained from plants, marine creatures, and microbes are the major resources for the bioactive agents and play a vital role in the discovery of lead molecules for new drugs. Flavonoids are polyphenolic phytochemicals that exist ubiquitously in the plant kingdom, which can be found in several food sources such as fruits, vegetables, whole grains, and legumes. [3,4] Earlier studies have explored flavone and flavanone moeities have an extensive variety of biological activities such as antioxidant, [5] anti-inflammatory, [6] antibacterial, [7] antifungal, [8] anticancer, [9] and antiestrogenic [10] activities as well as interaction with estrogen receptors [11] and inhibitory activities against aromatase enzyme. [12] Because of their broad spectrum of pharmacological activities, the flavonoids have been extensively studied as therapeutic agents, which is mainly of relevance to our study of anticancer and antimicrobial agents. In particular, because of their structural and functional resemblances to endogenous estrogens, flavonoids have attracted noteworthy interest as alternative estrogens, termed phytoestrogens, broadly studied for their potential role in many estrogen-dependent diseases including breast cancer. [13] Tanaproget is used as a nonsteroidal progesterone receptor agonist with high affinity and selectivity for the progesterone receptor [14] (Fig. 1). Generally, due to the four-ring structural motif found in natural steroid receptor ligands, steroidal progestins have higher cross-reactivity with other steroid hormone receptors. Currently, available progestins have side effects due to their lack of selectivity with other steroid hormone receptors. Recently, the novel classes of nonsteroidal progesterone receptor (PR) modulators with improved receptor selectivity have been reported. [15][16][17][18] These new compounds exhibit the potential for safer and wider pharmacological use as progestins.
Molecular hybridization is an effective tool to design and synthesize highly active novel molecules by covalently bonding two or more pharmacophores into a single entity [19,20] (Fig. 2). Moreover, the hybrid molecules may also minimize undesirable side effects and express synergic action. [21] These findings encouraged us to investigate the potential synergistic effect of phytoestrogen and tanaproget analog scaffolds on esterogen and progesterone receptors with respect to their parent core structures.
Herein, we report a series of hybrid compounds of these two pharmacophores with the intention to synergize the anticancer activity against human cancer cell lines (MCF-7, MDA-MB-231, DU-145, PC-3, and Hela). Our designed compounds have three parts, namely the flavonoid structure as a chief backbone, tanaproget-like moiety for enhancing desired pharmacophore behaviour with drug-like properties, and nonpolar butyl or pentyl chain for lipophilicity control. The excellent cytotoxicity of various compounds for hormone-dependent and hormone-independent breast cells pointed them as promising lead molecules for anticancer drug design. Furthermore, molecular modeling of these novel molecules was done to provide a comprehensive guide for further understanding of their synergistic effects on estrogen and progesterone receptors.
In the case of PC-3 cells, most of the compounds did not show considerable cytotoxicity except compounds 5b, 6a, and 6c. Compound 9i exhibited excellent cytotoxicity upto  13.49 � 2.72 and 11.09 � 1.32 µM) respectively. In the structure-activity relationship (SAR), it was found that the introduction of hydroxyl, methoxy, or amino group in the aza-flavone core with the vinyl or allyl substituent in tanaproget motif greatly enhanced their anticancer activities, which might be due to some carbon type and π-alkyl type interaction of electron donating groups with the amino acids, which is supported by docking study also. Compounds (4d, 4e, 9a, 9c, 9e, 9g, 9h, 9i, 9j, 9m, and 9n) that inhibited ≥50% cell proliferation in at least two different cell lines are shown in Fig. 3.
The antiproliferative activities expressed as IC 50 values are summarized in Table 2. This table includes IC 50 values of those compounds that at a 10 µM concentration caused a reduction �50% in the growth of breast tumor cell lines. However, IC 50 values corresponding to compounds that inhibited tumour cell proliferation less than 50% at 10 µM were not evaluated (Fig. 4).  We also compared the antitumor activity of two related prostate cell lines, DU-145 and PC-3 cells. A comparative analysis between these two cells showed that both cell lines behaved differently. On the other hand, HeLa cells were less responsive to the antiproliferative action of almost all the synthesized compounds except 4k, 4q, 4r, 5a, 6c, 9c, 9f, 9k, 9l, 9o, and 10a. Hence, the inspection of these results indicates the idea that cancerous tumours generally contain cell subpopulations with different biological properties.
Antibacterial activity. All the hybrid compounds underwent preliminary screening for the antibacterial assay and some of them were subjected to MIC (minimum inhibitory concentration) determination by microdilution method. [24] The results are given in Table 3. Reference drugs cefadroxil and ampicillin were chosen as positive controls in antibacterial activity assay.
Antifungal activity. Some previously screened compounds for the antifungal activity were subjected to MIC determination by microdilution method [24] and the results are given in Table 3.

Molecular docking study
To examine the synergistic effect of the hybrid molecules on binding with estrogen and progesterone receptors, an extensive docking study was done. Also, the majority of the flavonoid-tanaproget hybrid compounds with significant antiproliferative activities had shown distinct selectivity for the MCF-7 ERþve breast cancer cell line that expresses high levels of ER receptor. Therefore, to investigate whether the antiproliferative activity of these synthesized derivatives (against the MCF-7 cell line) is estrogen receptor mediated, the binding interactions of the products with the ER receptor were studied using molecular modelling (see supporting information). Genetic algorithm was implemented using AutoDock4.2 and all the final results were visually inspected using Discovery studio visualizer.
After the validation of the docking procedure (Fig. 5), the docking simulation suggested that the binding affinities of 9a-i and 10a-c were greater than those of 4a-r, 5a-c, and 6a-c.
Out of all the synthesized compounds 5b (PDB ID: 1ZUC) and 9h (PDB ID: 3EQM) were found to be the most potent hits with AutoDock binding energies of À 10.77 and À 12.09 kcal/mol respectively, which are significantly greater than the references used in the present study (Table 4). Visual analysis of docking pose indicated that compound 5b was fully buried inside the active site of progesterone receptor (Fig. 6). The aromatic ring of tanaproget, flavone, and furan seemed to interact completely with hydrophobic residues likes Met759, Leu763, Gly722, Leu718, Leu797, and Cys891 by π -alkyl interaction. The carbonyl and sec-amine group in tanaproget moiety of compound 9h were found to be hydrogen bonded with residue Arg145 and Arg435 of 3EQM. π-Alkyl interaction between aromatic ring of tanaproget and flavone moieties and residues such as Cys437, Phe430, Val369, and Met364 were also inspected and studied. π-Sulfur interaction between the aromatic ring of flavone and Met364 was also apparent by visualizers.
The present in silico work clearly indicated that all the tested molecules displayed greater affinity towards estrogen receptor (3EQM) than progesterone receptor (1ZUC). These docking results are also in sync with the outcome of in vitro antiproliferative activity against a panel of five human cancer cell lines.

Chemistry
All the required chemicals were purchased from Merck and Aldrich Chemical Company. Precoated aluminium sheets (silica gel 60 F254, Merck) were used for thin-layer chromatography (TLC) and spots were visualized under ultraviolet (UV) light. IR spectra were recorded with KBr on a Thermo Nicolet FT-IR spectrophotometer. 1 H NMR and 13 C NMR spectra were recorded respectively on Bruker Spectrospin DPX 500 and Jeol Resonance ECX 400II spectrometers using CDCl 3 or dimethylsulfoxide (DMSO) as solvent and trimethylsilane (TMS) as an internal standard. Spectra were processed using Bruker Topspin 3.0.b.8. Melting points were performed with Ambassador and a digital melting-point apparatus (Nutronics, Popular, India). Splitting patterns are designated as follows; s, singlet; d, doublet; m, multiplet. Chemical shift (δ) values are given in parts per million (ppm).

Microwave irradiation experiment
All microwave experiments were carried out in a dedicated Anton Paar Monowave-300 reactor, operating at a frequency of 2.455 GHz with continuous irradiation power of 0 to 850 W. The reactions were performed in a G-10 borosilicate glass vial sealed with Teflon septum and placed in a microwave cavity. Before microwave heating, oxygen was purged into the reaction vial. Initially, microwave of required power was used and temperature was ramped from room temperature to the desired temperature. Once this temperature was attained, the process vial was held at this temperature for the required time. The reactions were continuously stirred. Temperature was measured by an IR sensor. After the experiments a cooling jet cooled the reaction vessel to ambient temperature. (4a-r, 5a-c, 6a-c, 9a-o, 10a-c) 3-Bromo flavone (0.5 mmol), boronic acid (0.6 mmol), Pd-complex C (0.2 mol%, 1.0 mg), TBAB (0.12 mmol), K 2 CO 3 (1.2 mmol), and 6 ml of ethanol were added to a G-4 process vial capped with a Teflon septum. After a prestirring for 1 min, the vial was subjected to microwave irradiation for 18-20 min at 60 °C. It was then cooled to room temperature, diluted with water, and extracted with EtOAc three times. The organic phase was dried with Na 2 SO 4 and concentrated to yield the product. The crude material was chromatographed on a silica-gel column eluted with a mixture of hexane and ethyl acetate (8:2) to afford the product in 81-95% yield. The purified products were identified by FTIR, NMR, and HRMS spectra.  bovine serum (heat inactivated) and 1% antibiotic (100 U/ml of penicillin and 100 µg/ml streptomycin) (Himedia, Mumbai, India) mix at 37 °C in humidified atmosphere in a CO 2 incubator.

In vitro antimicrobial assay/studies
Test strain. The clinical isolates of C. albicans were obtained from the Department of Microbiology, IITR, India. The isolate was subcultured on Sabouraud dextrose agar at 37 °C for 48-72 h. Both Gram (þ) bacteria (S. aureus) and Gram (-) bacteria (E. coli) were also obtained from the Department of Microbiology, IITR, India, and used to evaluate the antibacterial effectiveness of the test compounds.
Microdilution assay. Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial compound that inhibits the visible growth of a microorganism. MIC values of the compounds against bacterial and fungal isolates were determined on the basis of micro-well-dilution method following NCCLS recommendations. [26] In this method we made stock of chemically synthesized compounds at a concentration of 10 mg/ml in DMSO, which was converted to working solution of concentration 1 mg/ ml solution in methanol. Using a multichannel micropipette, 100 µl of media into all wells of presterilized microtitre plate was dispensed (experiment was done in triplicate). Twofold serial dilutions were carried out from the well 1 to well 10 and excess media (100 µl) was discarded from the last well (no. 10). Liquid culture of test organisms were grown to a suitable phase in corresponding medium (YPD; yeast extract peptone dextrose, for fungal growth, and LB; Luria Bertani broth, for bacterial growth) for 12-18 h at 37 °C. Then optical density (OD) of liquid culture was determined at 600 nm and diluted in such a way that each well received 10 4 cfu/100 µl of fungal suspension and 10 7 cfu/100 µl of bacterial culture. Appropriate positive and negative controls were also included in the study. Positive control contained only microbial cells whereas negative control contained only standard drug solution (fluconazole for fungus and ampicillin and cifadroxil for bacteria). All experimental procedures were performed under sterile condition using bio-safety hood (ESCO, USA). The plates were incubated at 30 °C (fungal culture) and 37 °C (bacterial culture) for 12-16 h of growth phase.

Docking studies
AutoDock4.2 was used for docking simulation. [27] In AutoDock, we employed Lamarckian genetic algorithm (LGA), which is the hybrid of genetic algorithm (GA) and local search algorithm (LSA), for conformation searching. The RMSD between the docked conformation, as generated by the docking algorithm and the native cocrystallized ligand conformation, which is well within the grid spacing used in the docking procedure, shows that the docking method used was trustworthy and valid. For protein preparation, the crystal structure of molecular and pharmacological properties of a potent and selective novel nonsteroidal progesterone receptor agonist tanaproget, at resolution 1.80, was retrieved from protein data bank (PDB code: 1ZUC) and structural basis for androgen specificity and oestrogen synthesis in human aromatase (PDB code: 3EQM). The inhibitor, progesterone, ligand, and all water molecules were removed, leaving only the residues of the receptor. Preparation of the target protein with ADT involved the addition of polar hydrogens to the macromolecule, an essential step to correct the calculation of partial charge. Finally, Gasteiger charges were calculated for each atom of the macromolecule. The docking area was assigned visually around the apparent active site. A grid of 80 Å � 80 Å � 80 Å with grid spacing of 0.375 Å was positioned around the active site with all the ligand atom types using AutoGrid. All parameters were set to defaults except ga_run (100). The docking results were clustered on the basis of root mean square deviation (rmsd) and were ranked on the basis of free energy of binding. All compounds were drawn and then converted to three dimensions (3D) by ChemDraw (Cambridgesoft Inc.). Each structure was then energy minimized by AM1 force field. Discovery Studio Visualizer (DSV) was used for 3D visualization. [28] Funding