Synthesis and Antiproliferative Activity of Steroidal Diaryl Ethers

Novel 13α-estrone derivatives have been synthesized via direct arylation of the phenolic hydroxy function. Chan–Lam couplings of arylboronic acids with 13α-estrone as a nucleophilic partner were carried out under copper catalysis. The antiproliferative activities of the newly synthesized diaryl ethers against a panel of human cancer cell lines (A2780, MCF-7, MDA-MB 231, HeLa, SiHa) were investigated by means of MTT assays. The quinoline derivative displayed substantial antiproliferative activity against MCF-7 and HeLa cell lines with low micromolar IC50 values. Disturbance of tubulin polymerization has been confirmed by microplate-based photometric assay. Computational calculations reveal significant interactions of the quinoline derivative with the taxoid binding site of tubulin.


Introduction
The development of highly efficient, environmentally friendly catalytic synthetic methods is one of the major goals of modern organic chemistry [1]. Carbon-heteroatom (C-X) bond formation is often a key challenge for organic chemists [2][3][4][5][6][7][8]. It is still desirable to develop mild but effective methods, which allow the construction of the C-X bond with high functional group tolerance. The Ullmann reaction is a traditional method using aryl halides and nucleophiles under transition metal catalysis [9,10]. Nevertheless, owing to its harsh reaction conditions, it is not applicable in certain cases. At the turn of the Millennium, Chan, Evans and Lam published the coupling reactions of arylboronic acids with nucleophiles under the copper salt catalysis, which were later called Chan-Lam coupling reactions [11][12][13]. These transformations are characterized by mild reaction conditions, low toxicity and appropriate stability. The extensions of Chan-Lam couplings allow the establishment of various C-X bonds. The proposed mechanism of the C-O coupling is depicted in Figure 1. After the coordination and transmetalation step (I.), disproportionation (II.) between a CuY 2 and a Cu II (Ar)Y occurs. A reductive elimination (III.) step provides the C-O coupled product. The terminal oxidant is responsible for the oxidation of Cu(I) salts. Application of such methodologies in selective transformations of biologically active compounds might allow feasible syntheses of drug candidates.
The literature describes certain copper-catalyzed methodologies for the synthesis of diphenyl ethers (DEs) [14]. The copper(II)-promoted coupling might even efficiently be carried out at room temperature using an organic or inorganic base in varied solvents. DE represents a compound group bearing two aromatic rings connected via a flexible oxygen bridge. The latter is an essential pharmacophore owing to its substantial hydrophobicity, good lipid solubility, cell membrane penetration and metabolic stability [15,16]. Both synthetic pharmaceuticals [17][18][19][20] and several biologically active natural products [21][22][23][24]  The literature describes certain copper-catalyzed methodologies for the synthesis of diphenyl ethers (DEs) [14]. The copper(II)-promoted coupling might even efficiently be carried out at room temperature using an organic or inorganic base in varied solvents. DE represents a compound group bearing two aromatic rings connected via a flexible oxygen bridge. The latter is an essential pharmacophore owing to its substantial hydrophobicity, good lipid solubility, cell membrane penetration and metabolic stability [15,16]. Both synthetic pharmaceuticals [17][18][19][20] and several biologically active natural products [21][22][23][24] contain the DE subunit. DEs, among others, possess anticancer [18][19][20][21], antiviral [25,26], anti-inflammatory [23], antibacterial [27], antiparasitic [28], fungicidal [29], herbicidal [30] or insecticidal [29] activities. Furthermore, the literature reports new efficient DE-based compounds for the therapeutic applications of devastating diseases affecting the central nervous or the cardiovascular system worldwide [14]. Figure 2 shows certain pharmacologically important DE-containing drugs, pesticides and natural products (1-4). Ibrutinib [17] and sorafenib [18] as small-molecule inhibitors of certain kinase enzymes belong to anti-cancer agents; however, nimesulide [20] is a nonsteroidal anti-inflammatory drug. Isoliensinine [21] displays a variety of biological activities, including anti-cancer, antioxidant and anti-HIV effects.  Cancer still remains the leading cause of death around the world. The development of new, highly effective anticancer agents with high selectivity is still one of the major goals of medicinal chemistry. Antitubulin compounds are considered to be the most effective tools for cancer chemotherapy nowadays [31]. Certain antitubulin agents have already been approved by the Food and Drug Administration (FDA) [32], but their efficacy might be limited by the development of multidrug-resistant (MDR) cancer cells. Due to  Cancer still remains the leading cause of death around the world. The development of new, highly effective anticancer agents with high selectivity is still one of the major goals of medicinal chemistry. Antitubulin compounds are considered to be the most effective tools for cancer chemotherapy nowadays [31]. Certain antitubulin agents have already been approved by the Food and Drug Administration (FDA) [32], but their efficacy might be limited by the development of multidrug-resistant (MDR) cancer cells. Due to their crucial role in cell division, αand β-tubulin are essential targets for the development of novel anticancer agents. Suppression of cell growth might be achieved by drugs that stabilize or destabilize microtubules (MTs) [33,34]. MT destabilizing agents (MDAs) prevent the polymerization of tubulin and they promote depolymerization, whereas MT stabilizing agents (MSAs) promote the polymerization of tubulin and they stabilize the polymer, preventing depolymerization. Six binding sites have been identified on tubulin [35,36]; however, MSAs generally bind reversibly to the taxoid binding site. Antimitotic drugs approved by FDA might be classified based on their binding site or their further modification (encapsulation or conjugation strategies) [32]. Vincristine sulfate (Oncovin), vinblastine sulfate (Velban) and vinorelbine (Navelbine) belong to the vinca alkaloid site binding drugs; however, colchicine binds to the site with the same name. Marquibo, as a vincristine sulfate liposome injection was developed to improve the pharmacodynamic properties of vincristine. T-DM1 is a maytansine derivative conjugated to trastuzumab applied in a second line breast cancer therapy. It can be stated that antitubulin agents bear a characteristic structure generally. It has two aryl rings and an ethylene, triazole or oxygen bridge, which determine the relative orientation of the rings [36,37]. The latter structural element, the diaryl ether scaffold, is present in certain potent antitubulin agents [15] including substituted or condensed variations (compounds 5-8, Figure 3). Nevertheless, the development of antitubulin compounds possessing improved potency and selectivity is still a leading challenge.  We recently published the syntheses and biochemical investigation of certain biologically active derivatives of the core-modified synthetic 13α-estrone 9. Inversion of the C-13 in natural estrone derivatives substantially reduces their estrogenic activity [38][39][40][41]. The group of 13α-estrone derivatives proved to be promising concerning their enzyme inhibitory and/or antiproliferative properties. However, their biological activity greatly depends on their structure [33,[42][43][44][45][46][47][48]. We have observed that the substitution pattern of ring A influences bioactivity markedly. The introduction of an apolar benzyl group onto the phenolic hydroxy function usually improves the cell growth-inhibitory action of 17keto or 17-hydroxy 13α-estrone derivatives [42]. Accordingly, the presence of the apolar ether moiety at C-3 seemed to be advantageous. The low micromolar antiproliferative action of benzyl ethers could further be enhanced by inserting a polar triazole ring between the 3-OH and the benzylic moiety [42,48]. Compound 11a displayed submicromolar IC50 values on HeLa, A2780, A431 and MCF-7 cancer cell lines. Introduction of a bromo sub- We recently published the syntheses and biochemical investigation of certain biologically active derivatives of the core-modified synthetic 13α-estrone 9. Inversion of the C-13 in natural estrone derivatives substantially reduces their estrogenic activity [38][39][40][41]. The group of 13α-estrone derivatives proved to be promising concerning their enzyme inhibitory and/or antiproliferative properties. However, their biological activity greatly depends on their structure [33,[42][43][44][45][46][47][48]. We have observed that the substitution pattern of ring A influences bioactivity markedly. The introduction of an apolar benzyl group onto the phenolic hydroxy function usually improves the cell growth-inhibitory action of 17-keto or 17-hydroxy 13α-estrone derivatives [42]. Accordingly, the presence of the apolar ether moiety at C-3 seemed to be advantageous. The low micromolar antiproliferative action of benzyl ethers could further be enhanced by inserting a polar triazole ring between the 3-OH and the benzylic moiety [42,48]. Compound 11a displayed submicromolar IC 50 values on HeLa, A2780, A431 and MCF-7 cancer cell lines. Introduction of a bromo substituent to the C-4 ortho position led to a compound (11b) with improved tumor selectivity, being most potent on A2780 cell line ( Figure 4) [48]. Mechanistic investigations suggest that compound 11b exerts a direct effect on microtubule formation. Molecular dynamics (MD, MMGBSA methods) were performed in order to calculate binding energy at an advanced level. Computational calculations revealed strong interactions of compound 11b with both colchicine (CBS) and taxoid binding sites (TBS) of tubulin with a stronger interaction at the TBS. Consequently, triazole derivative 11b might be considered as an MSA agent with remarkable tumor selectivity concerning the cell lines investigated. More recently we have shown that 3-deoxy-3-phenyl-13α-estra-1,3,5(10)-triene (12) displays weak antiproliferative action [47]. This observation suggests that direct phenylation at C-3 by the simultaneous removal of the hydroxy group is detrimental regarding the antiproliferative action. It follows that the oxygen-containing moiety at C-3 should be retained and its etherification with apolar benzyl or more polar triazolylbenzyl moieties might be highly beneficial. Our results indicate that the hormonally inactive 13α-estrane core with certain ring A modifications might be a suitable scaffold in the design of potent MT targeting agents.  With these considerations in mind, here we aimed to perform the direct arylation of 13α-estrone at C-3-O via Chan-Lam coupling using arylboronic acids as reagents. The set of boronic acid coupling partners included not only substituted phenyl but heteroaryl derivatives too. The evaluation of antiproliferative action of the newly synthesized compounds against five human cancer cell lines was also planned. Mechanistic investigations concerning the direct effect of the most potent compound on microtubule formation were also intended. To gain insight into the interaction of the selected potent compound with the taxoid binding site of tubulin, computational calculations were performed.

Chemistry
First, the etherification of 13α-estrone 9 with phenylboronic acid 13a was carried out. Arylboronic acids are outstanding coupling partners in several metal-catalyzed reactions, owing to their broad availability, low toxicity, high stability and extensive functional group tolerance. Based on literature data [12], a copper-promoted C(sp 2 )-O coupling was performed, using 1 equiv. of Cu(OAc)2 as the catalyst in dichloromethane solvent. K2CO3, trimethylamine (NEt3) and diisopropylethylamine (DIPEA) were tested as bases but NEt3 proved to be the best option based on yields. The protocol was extended to couplings of different arylboronic acids (13a-l) with 13α-estrone (9) (Scheme 1). In order to get important structure-activity information, substituted phenyl (13a-h) or condensed carbo or heterocyclic derivatives (13i-l) were chosen as reagents. C(sp 2 )-O couplings proceeded with high isolated yields (Scheme 1). With these considerations in mind, here we aimed to perform the direct arylation of 13α-estrone at C-3-O via Chan-Lam coupling using arylboronic acids as reagents. The set of boronic acid coupling partners included not only substituted phenyl but heteroaryl derivatives too. The evaluation of antiproliferative action of the newly synthesized compounds against five human cancer cell lines was also planned. Mechanistic investigations concerning the direct effect of the most potent compound on microtubule formation were also intended. To gain insight into the interaction of the selected potent compound with the taxoid binding site of tubulin, computational calculations were performed.

Chemistry
First, the etherification of 13α-estrone 9 with phenylboronic acid 13a was carried out. Arylboronic acids are outstanding coupling partners in several metal-catalyzed reactions, owing to their broad availability, low toxicity, high stability and extensive functional group tolerance. Based on literature data [12], a copper-promoted C(sp 2 )-O coupling was performed, using 1 equiv. of Cu(OAc) 2 as the catalyst in dichloromethane solvent. K 2 CO 3 , trimethylamine (NEt 3 ) and diisopropylethylamine (DIPEA) were tested as bases but NEt 3 proved to be the best option based on yields. The protocol was extended to couplings of different arylboronic acids (13a-l) with 13α-estrone (9) (Scheme 1). In order to get important structure-activity information, substituted phenyl (13a-h) or condensed carbo or heterocyclic derivatives (13i-l) were chosen as reagents. C(sp 2 )-O couplings proceeded with high isolated yields (Scheme 1).
The structures of the newly synthesized diaryl ethers (14a-l) were deduced from 1 H and 13 C NMR spectra.
The structures of the newly synthesized diaryl ethers (14a-l) were deduced from 1 H and 13 C NMR spectra.

Pharmacology
Here we investigated the in vitro cell growth-inhibitory properties of the newly synthesized diaryl ethers (14a-l) by the MTT assay [49] on a panel of human adherent cancer cell lines. The panel included different cervical (HeLa and SiHa), breast (MCF-7 and MDA-MB-231) and ovarian (A2780) cancer cell lines [50][51][52][53][54][55][56]. The cervical and breast cancer cell line pairs were selected on the basis of their different HPV or receptorial status. NIH/3T3 mouse fibroblast cell line was used for the determination of cancer selectivity.

Pharmacology
Here we investigated the in vitro cell growth-inhibitory properties of the newly synthesized diaryl ethers (14a-l) by the MTT assay [49] on a panel of human adherent cancer cell lines. The panel included different cervical (HeLa and SiHa), breast (MCF-7 and MDA-MB-231) and ovarian (A2780) cancer cell lines [50][51][52][53][54][55][56]. The cervical and breast cancer cell line pairs were selected on the basis of their different HPV or receptorial status. NIH/3T3 mouse fibroblast cell line was used for the determination of cancer selectivity.
We have recently described the antiproliferative properties of 13α-estrone-3-benzyl ether (10, Figure 4) and its triazolyl derivatives (11a,b) against HeLa, MCF-7 and A2780 cell lines [42,48]. The etherified compounds (10; 11a,b) inhibited the growth of cells more effectively than their 3-OH counterpart (9). In both compound types (10; 11a,b), the carbo or the heteroaromatic ring was connected to C-3-O via a methylene linker. Here we were interested in the investigation of the antiproliferative properties of 13α-estrone derivatives arylated directly at the C-3-O function. Although compound structures 14a and 10 (IC 50 > 30 µM, HeLa [42]) differ only in a single methylene group, their antiproliferative action varies substantially (Table 1). Compound 14a is more active, especially on the HPV-18 positive HeLa cervical cell line, displaying a low micromolar IC 50 value (5.53 µM, Table 1). In addition, the estrogen receptor positive breast cancer cell line MCF-7 proved to be sensitive to 14a but the IC 50 value was twice as high as that mentioned previously. Interestingly, 14a behaved differently on the pairs of breast and cervical cell lines. All test compounds seemed to be active against the HeLa cell line with IC 50 values ranging from 5 to 10 µM with the exception of the 4-fluorophenyl derivative 14e (Table 1). Substitution of a hydrogen with its fluorine bioisostere usually leads to unique biological activity of the fluorinated derivative. The highest electronegativity and the small van der Waals radius make fluorine derivatives generally more active than their unsubstituted counterparts. Nevertheless, in the case of 14e the presence of fluorine seems to be disadvantageous. If we analyze the results obtained for para-tolyl (14b) and para-trifluoromethyl (14h) compounds, only slight differences appear. Accordingly, the effect of fluorine is more pronounced if it is connected directly to the phenyl group.
Introduction of a condensed bicyclic moiety onto the C-3-O site resulted in unique structure-activity results. The naphthyl derivative (14k) displayed growth-inhibitory actions similar to its phenyl counterpart (14a), except on cell lines MCF-7 and SiHa with SiHa being more sensitive to compound 14k. The presence of a nitrogen heteroatom in the introduced moiety (compound 14i) resulted in a significant improvement in the biological action. This compound should be highlighted as the most potent derivative on all cell lines investigated (except for SiHa). In contrast, the benzo-1,4-dioxane derivative (14l) seemed to be less potent than 14i on four cancer cell lines. The ovarian cell line A2780 could not distinguish between the two heterocyclic compounds 14i and 14l. The other nitrogen-containing heterocyclic derivative (14j) was less effective than 14i.
It should be emphasized that certain newly synthesized derivatives displayed more pronounced cell growth-inhibitory action than the reference compound cisplatin, especially on the HeLa cancer cell line.
In order to get information on the tumor selectivity of the antiproliferative action, the more promising derivatives were investigated on the mouse fibroblast cell line NIH/3T3. None of the compounds exerted inhibition above 50% even at a higher, 30 µM test concentration. In this regard, the reference agent cisplatin proved to be less selective.
It is particularly promising that slight structural modifications generated significant differences in inhibitory activities. This might be indicative of a cell type-dependent action. The broad toxic character of the test compounds might be excluded. On this basis, the compound group investigated might substantially contribute to lead-finding projects based on estrone derivatives. Nevertheless, the mechanistic investigation of antiproliferative action is necessary. The determination of the mechanism of action was inspired by our recent result [48]. We found earlier that certain 13α-estrone derivatives etherified on their phenolic hydroxy function might affect the microtubule formation. On this basis, here we performed an in vitro tubulin polymerization assay with the most potent compound 14i in two different concentrations (125 and 250 µM). Paclitaxel, a clinically applied anticancer agent was used as a reference compound.
The obtained absorbance values indicate that 14i disturbs the polymerization of tubulin by increasing the maximum rate of the procedure. This property was concentrationdependent and statistically significant even at the lower concentration range. The effect of 14i at 250 µM was lower than that of the reference agent. Based on these in vitro results it can be concluded that the antiproliferative action of 14i is elicited through the disturbance of tubulin polymerization ( Figure 5).

Computational Investigations
Having a picture at the atomic level concerning the possible binding character of the 14i ligand, MD calculations have been performed. To sample the conformational space of the ligand binding to the taxoid binding site, five independent simulations were run providing a 2.5 µs-long trajectory in total. The resulting protein-ligand interaction diagrams of all five trajectories are presented in Supplementary Materials (Figures S1-S5). According to the simulations, a significant hydrogen-bonding interaction was formed between the ligand and two amino acids (Gln-282 and Thr-276). In one of the trajectories, the Thr-276 amino acid shows extra stabilization with the 17-keto function of the sterane skeleton when the modified steroid finds a deeper position in the taxoid binding site. Both amino acids are located in a flexible loop region near to the taxoid binding pocket and the motion of the loop can affect the binding of the modified steroid. Interestingly, despite that two Gln amino acids located next to each other in position 281 and 282, the ligand had frequent interaction mainly with the second one in the simulations.
Finally, demonstrating the relative position of those amino acids, which had significant interaction with the protein or the guanosine diphosphate (GDP) co-factor, we present a representative ligand-protein complex in Figure 6.
It is worth to mention that the above-mentioned special hydrogen bonds with Gln-282 and Thr-276 always formed between one of the oxygen atoms of the ligand and never with the nitrogen of the quinoline skeleton. Concerning other secondary interactions of the ligand, the analysis showed two further connection types, namely water bridges and hydrophobic interactions. Interestingly, the nitrogen atom in the quinoline ring does not show any specific binding to the tubulin protein.
Finally, we would like to point out that the ligand remained in the binder pocket all along the trajectories, while the GDP left its binding pocket in a number of cases.
In order to get information on the tumor selectivity of the antiproliferative action, the more promising derivatives were investigated on the mouse fibroblast cell line NIH/3T3. None of the compounds exerted inhibition above 50% even at a higher, 30 μM test concentration. In this regard, the reference agent cisplatin proved to be less selective.
It is particularly promising that slight structural modifications generated significant differences in inhibitory activities. This might be indicative of a cell type-dependent action. The broad toxic character of the test compounds might be excluded. On this basis, the compound group investigated might substantially contribute to lead-finding projects based on estrone derivatives. Nevertheless, the mechanistic investigation of antiproliferative action is necessary. The determination of the mechanism of action was inspired by our recent result [48]. We found earlier that certain 13α-estrone derivatives etherified on their phenolic hydroxy function might affect the microtubule formation. On this basis, here we performed an in vitro tubulin polymerization assay with the most potent compound 14i in two different concentrations (125 and 250 μM). Paclitaxel, a clinically applied anticancer agent was used as a reference compound.
The obtained absorbance values indicate that 14i disturbs the polymerization of tubulin by increasing the maximum rate of the procedure. This property was concentrationdependent and statistically significant even at the lower concentration range. The effect of 14i at 250 μM was lower than that of the reference agent. Based on these in vitro results it can be concluded that the antiproliferative action of 14i is elicited through the disturbance of tubulin polymerization ( Figure 5).

Computational Investigations
Having a picture at the atomic level concerning the possible binding character of the 14i ligand, MD calculations have been performed. To sample the conformational space of the ligand binding to the taxoid binding site, five independent simulations were run providing a 2.5 μs-long trajectory in total. The resulting protein-ligand interaction diagrams of all five trajectories are presented in Supplementary Materials (Figures S1-S5). According to the simulations, a significant hydrogen-bonding interaction was formed between the ligand and two amino acids (Gln-282 and Thr-276). In one of the trajectories, the Thr-276 amino acid shows extra stabilization with the 17-keto function of the sterane skeleton when the modified steroid finds a deeper position in the taxoid binding site. Both amino acids are located in a flexible loop region near to the taxoid binding pocket and the motion of the loop can affect the binding of the modified steroid. Interestingly, despite that two Gln amino acids located next to each other in position 281 and 282, the ligand had frequent interaction mainly with the second one in the simulations.
Finally, demonstrating the relative position of those amino acids, which had significant interaction with the protein or the guanosine diphosphate (GDP) co-factor, we present a representative ligand-protein complex in Figure 6. It is worth to mention that the above-mentioned special hydrogen bonds with Gln-282 and Thr-276 always formed between one of the oxygen atoms of the ligand and never with the nitrogen of the quinoline skeleton. Concerning other secondary interactions of the ligand, the analysis showed two further connection types, namely water bridges and hydrophobic interactions. Interestingly, the nitrogen atom in the quinoline ring does not

Chemistry
Melting points (Mp) were determined with a Kofler hot-stage apparatus. Perkin-Elmer CHN analyzer 2400 was used for the elemental analyses. Thin-layer chromatography was performed on silica gel 60 F 254 (layer thickness 0.2 mm, Merck); eluent (ss): 30% ethyl acetate/70% hexanes. The spots were detected with I 2 or UV (365 nm) after spraying with 5% phosphomolybdic acid in 50% aqueous phosphoric acid and heating at 100-120 • C for 10 min. Flash chromatography was performed on silica gel 60, 40-63 µm (Merck). 1 H NMR spectra were recorded in DMSO-d 6 or CDCl 3 solution with a Bruker DRX-500 instrument at 500 MHz. 13 C NMR spectra were recorded with the same instrument at 125 MHz under the same conditions. Full scan mass spectra of the newly synthesized compounds were acquired in the range of 50 to 1000 m/z with a Finnigan TSQ-7000 triple quadrupole mass spectrometer (Finnigan-MAT, San Jose, CA, USA) equipped with a Finnigan electrospray ionization source. Analyses were achieved in positive ion mode applying flow injection mass spectrometry with a mobile phase of 50% aqueous acetonitrile containing 0.1 v/v% formic acid (flow rate: 0.3 mL/min). Five µL aliquot of the samples were loaded into the flow. The ESI capillary was adjusted to 4.5 kV and N 2 was used as a nebulizer gas.

Determination of Antiproliferative Activities
The antiproliferative activities of the currently presented molecules 14a-l were determined against human adherent cancer cell lines of gynecological origin. MCF-7 and MDA-MB-231 cells were isolated from breast cancers, while A2780 is an ovarian cancer cell line. In addition, Hela and SiHa cells were isolated from cervical cancers containing HPV-18 and HPV-16, respectively. The tumor selectivity of molecules was determined using nonmalignant mouse embryo fibroblast cells (NIH/3T3). All cell lines were obtained from the European Collection of Cell Cultures (ECCAC, Salisbury, UK) except for SiHa (American Tissue Culture Collection, Manassas, VA, USA). Cells were maintained in minimal essential medium (MEM) completed with 10% fetal calf serum, 1% nonessential amino acids, and an antibiotic-antimycotic mixture. All media and supplements were purchased from Lonza Group Ltd., Basel, Switzerland. Near-confluent tumor and fibroblast cells were plated onto a 96-well microplate at 5000 cells/well density.
After overnight preincubation, a 200 µL new medium containing the tested compounds (at 10 or 30 µM) was added. After incubation (72 h, 37 • C, humidified air, 5% CO 2 ), the viable cells were determined by the addition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL, 20 µL). The reagent was metabolized by active mitochondrial reductase and precipitated as purple formazan during a 4 h contact period. Then the medium was discarded, and the crystals were solubilized in 100 µL of DMSO during a 60 min period of shaking at 37 • C. Finally, the produced formazan was assayed at 545 nm, using a microplate reader (SPECTROStar Nano, BMG Labtech, Offenburg, Germany), utilizing untreated cells as control [49]. In the case of the compounds eliciting higher than 50% growth inhibition at 30 µM, the assays were repeated with a set of dilutions and IC 50 values were calculated from the determined data (sigmoidal curve, GraphPad Prism 5.01, GraphPad Software, San Diego, CA, USA). All experiments were performed on two microplates with at least five parallel conditions. Stock solutions of 10 mM were prepared from the investigated items in DMSO and the highest concentration of the solvent in the medium (0.3%) did not elicit any considerable action on cell growth. Cisplatin (Ebewe Pharma GmbH, Unterach, Austria) was included as a reference agent.

Tubulin Polymerization Assay
The effect of 14i on tubulin polymerization was determined by means of a commercially available assay kit as described previously [48]. Briefly, 10 µL of a 125 or 250 µM solution of the tested analog was placed on a prewarmed (37 • C), UV-transparent microplate. Paclitaxel and general tubulin buffer were used as positive and negative control, respectively. A total volume of 100 µL of 3.0 mg/mL tubulin in 80 mM PIPES with 2 mM MgCl 2 , 0.5 mM EGTA with 1 mM GTP at pH 6.9, was added to the samples to initiate the polymerization. A 60 min kinetic measurement protocol was used to describe the absorbance of the reaction mixture per minute at 340 nm (SpectoStarNano, BMG Labtech, Ortenberg, Germany). The maximum reaction rate (Vmax: ∆absorbance/min) was determined by calculating the moving averages of absorbances at three consecutive time points. The highest difference between two succeeding moving averages was considered as the Vmax of the tested molecule. Each sample was prepared in two parallels and the measurements were repeated twice. For statistical evaluation, Vmax data were analysed by the one-way ANOVA test with the Newmann-Keuls post-test by using Prism 5.01 software (GraphPad Software, San Diego, CA, USA).

Computational Simulations
For molecular dynamics (MD) simulations, a taxol-stabilized microtubule complex (PDB entry: 5SYF) was selected [57]. The original PDB structure was prepared for further calculations using the Protein Preparation Wizard module of the Schrodinger Maestro program [58,59]. It consists of the addition of missing hydrogen atoms under physiological pH condition as well as the missing loops or side chains. The prepared pdb structure was relaxed by a short (5 ns) MD simulation and the last frame of the relaxation running was selected as the target protein for the docking calculations. Docking was performed with the Glide package of the Schrödinger suite [60], using the extra-precision docking protocol. To sample the conformational space, 5 independent MD simulations of 500 ns length were run in a cubic box with a 10 Å buffer size and 0.15 M salt concentration by the Desmond package [61]. The initial structure of the production MD run was selected as the outcome of the docking calculation with the best docking score. In all MD runs, the OPLS4 force field and a simple point charge (SPC) water model were applied [62].

Conclusions
Antitubulin compounds represent the most effective class of anticancer agents. The more we understand about the structure-activity relationship of these compounds, the better options we have in utilizing them in fight against cancer. The literature reveals characteristic structural elements responsible for antitubulin effect, including the diaryl ether scaffold. In order to develop novel potential antitubulin compounds, we synthesized 13α-estrone 3-O-aryl derivatives via Chan-Lam coupling reactions. The copper-catalyzed etherification of the steroidal phenolic hydroxy function was achieved using arylboronic acids as coupling partners. Carbo or heterocyclic rings, bearing different substituents were introduced. The antitumoral properties of the newly synthesized 13α-estrone derivatives were investigated in vitro on a panel of human adherent cancer cell lines (A2780, MCF-7, MDA-MB 231, HeLa and SiHa). Certain compounds exerted more pronounced antiproliferative action than the reference agent cisplatin. The HeLa cancer cell line seemed to be the most sensitive to test compounds. The quinoline derivative 14i should be highlighted as the most potent steroid against MCF-7 and HeLa cell lines. The tumor selectivity of the test compounds proved to be higher than that of the reference agent cisplatin. Compound 14i might be regarded as a MT stabilizing agent, since it exerted its antiproliferative effect through the disturbance of tubulin polymerization. Significant interactions of the 14i derivative with the taxoid binding site of tubulin were identified by computational simulations. Our results might contribute to the development of more potent antitubulin agents with high selectivity.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28031196/s1, Table S1: Antiproliferative activities of compounds 14a-l. Figure S1: Presence of secondary interactions between the protein amino acids and 14i ligand along the 1st trajectory. Figure S2: Presence of secondary interactions between the protein amino acids and 14i ligand along the 2nd trajectory. Figure S3: Presence of secondary interactions between the protein amino acids and 14i ligand along the 3rd trajectory. Figure S4: Presence of secondary interactions between the protein amino acids and 14i ligand along the 4th trajectory. Figure S5: Presence of secondary interactions between the protein amino acids and 14i ligand along the 5th trajectory. 1 H and 13 C NMR spectra of the newly synthesized compounds.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.