Design, Synthesis, Anticancer Evaluation and Molecular Modeling of Novel Estrogen Derivatives

A series of estrone derivatives 3–8 was designed and synthesized using estrone arylmethylenes 2a,b as starting materials and their structures were confirmed by different spectral data and elemental analyses. All the newly synthesized compounds exhibited potent in vitro and in vivo cytotoxic activities against breast cancer cell lines. In addition, all compounds were subjected to in vitro and in vivo inhibition assays for EGFR and VEGFR-2 kinases as well as p53 ubiquitination activity to obtain more details about their mechanism of action. Based on the promising results, a molecular docking study was investigated for the most representative compound 5a against the two targets, EGFR and VEGFR-2 kinases, to assess its binding affinity, hoping to rationalize and obtain potent anticancer agents in the future.


Introduction
Breast cancer is the most common malignant cancer for women, affecting about 1.7 million patients and causing 0.52 million deaths in 2012 [1]. Different factors may contribute to this encumbrance of breast cancer including genetics, life styles and environmental factors. The mechanism of breast cancer involved in cell proliferation, invasion and metastasis is not fully resolved [2][3][4]. In spite of enormous efforts in research to detect and treat cancer, discovery of optimal cancer therapies is still difficult due to the severe side effects associated with chemotherapeutics, as well as appearance of tumor resistance [5]. Recently, discovery of anticancer drugs has been stimulated from conventional cytotoxic drugs to those with multi-targeted mechanisms to produce potent anticancer agents with minimal side effects [6].
Receptor tyrosine kinases (RTKs), epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR-2) play vital roles in regulating tumor cell proliferation, differentiation, survival, angiogenesis and apoptosis [7][8][9]. Moreover, the tumor suppressor p53 is D In view of these reports and in continuation of our interest in the chemical and pharmacological properties of substituted heterocyclic derivatives [26][27][28][29][30][31][32], we report herein the synthesis of a new series of estrones fused with substituted pyrimidine and pyrazoline rings. The biological activities of the newly synthesized products were studied in vitro and in vivo against breast cancer, EGFR and VEGFR-2 kinases, and p53 ubiquitination. A molecular docking study has also been conducted to validate the findings. In view of these reports and in continuation of our interest in the chemical and pharmacological properties of substituted heterocyclic derivatives [26][27][28][29][30][31][32], we report herein the synthesis of a new series of estrones fused with substituted pyrimidine and pyrazoline rings. The biological activities of the newly synthesized products were studied in vitro and in vivo against breast cancer, EGFR and VEGFR-2 kinases, and p53 ubiquitination. A molecular docking study has also been conducted to validate the findings.

Chemistry
Arylmethylene of estrone derivatives 2a,b [33][34][35] was synthesized via Aldol condensation of estrone 1 and 2-chlorobenzaldehyde or 4-chlorobenzaldehyde in ethanol in the presence of 30% potassium hydroxide according to the previous reported procedure. Treatment of compounds 2a,b with urea or thiourea, in the presence of sodium ethoxide afforded the corresponding 2-oxopyrimidines 3a,b and 2-thioxopyrimidines 4a,b, respectively. Additionally, condensation of 2a,b with guanidine hydrochloride or 2-cyanoguanidine, in the presence of sodium ethoxide afforded the corresponding 2 -aminopyrimidines 5a,b and 2 -cyanoiminopyrimidines 6a,b, respectively. Finally, reaction of 2a,b with semicarbazide or thiosemicarbazide in refluxing dioxane afforded the corresponding 1-carbamoylpyrazolines 7a,b and 1-thiocarbamoylpyrazolines 8a,b, respectively (Scheme 1).   In vitro cytotoxic activities of the newly prepared derivatives toward MCF-7 cells were assessed. All the tested compounds showed potential cytotoxic activities towards MCF-7, at a micro molar level (Table 1, Figure 2). However, the descending order of activity was as follow 5a, 5b, 6a, 6b, 4a, 4b, 3a, 3b, 8a, 8b, 7a and 7b. Moreover, the most active compound (5a) was 2.5-fold more toxic than the least active one (7b) towards the MCF-7 cell line (IC 50 = 18.38 ± 0.13 and 46.17 ± 0.93 µM, respectively). The descending activities of the different prepared derivatives may be correlated with their structures. Generally, it can be observed that pyrimidine-containing derivatives (3)(4)(5)(6) are more active than those containing pyrazoline (7,8). Furthermore, substitution at p-2 on the pyrimidine moiety can affect the cytotoxic activity in descending order by different groups as follows: imino (5a, 5b) > cyanoimino (6a, 6b) > thioxo (4a, 4b) > oxopyrimidine (3a, 3b). On the other hand, the compounds bearing thiocarbamoyl pyrazoline fragments (8a, 8b) exhibited better activity than those bearing carbamoyl pyrazoline (7a, 7b). Finally, by screening the results of all tested compounds, it was noticed that derivatives having a 2-chlorophenyl moiety at p-6 of pyrimidine revealed higher potency than those having 4-chlorophenyl. Comparing IC 50 values obtained for the synthesized derivatives against MCF-7 with those obtained against non-tumorigenic MCF-10A cells, we can concluded that the synthesized derivatives have much less toxicity against normal cells. Generally, it can be seen that the IC 50 values obtained for normal non-tumorigenic MCF-10A cells were about 9.78 to 11.49-fold higher than those obtained by MCF-7 cells.

In Vivo Anti-Breast Cancer
The in vivo anti-breast cancer activities of different synthesized derivatives were evaluated using a breast cancer mouse xenograft model. Figure 3 shows data obtained for inhibition percentages of tumor growth, i.e., decrease in tumor growth, after exposure to prepared derivatives for the whole period of experiment, in comparison to tumor development in control animals. Results revealed that all synthesized compounds showed potential inhibitory effects on tumor growth upon treatment from day 2. Furthermore, the degree of inhibition in tumor growth increased gradually over time until reaching maximal inhibition after 12 days; afterwards, inhibition percentages slightly decreased at 14 days, and then remained more or less constant for the rest of treatment period. Compound 5a showed the most promising effect in terms of growth inhibition, where tumor growth decreased by about 25.36% after only 2 days of treatment, and maximal inhibition of 91.1% was recorded after 10 days of treatment. After 14 days, the inhibition percentage decreased to 88.7% and then remained more or less constant up to 20 days of treatment. It can be observed that there is agreement between in vivo inhibitory patterns of the different derivatives and their in vitro anticancer patterns of activity. Also, the newly synthesized estrone derivatives described here coincide with those reported earlier [24]. Furthermore, estrone derivatives have been reported to inhibit in vivo tumor growth in a dose-dependent manner [36,37] through their inhibitory action on 17-hydroxysteroid dehydrogenase.

In Vivo Anti-Breast Cancer
The in vivo anti-breast cancer activities of different synthesized derivatives were evaluated using a breast cancer mouse xenograft model. Figure 3 shows data obtained for inhibition percentages of tumor growth, i.e., decrease in tumor growth, after exposure to prepared derivatives for the whole period of experiment, in comparison to tumor development in control animals. Results revealed that all synthesized compounds showed potential inhibitory effects on tumor growth upon treatment from day 2. Furthermore, the degree of inhibition in tumor growth increased gradually over time until reaching maximal inhibition after 12 days; afterwards, inhibition percentages slightly decreased at 14 days, and then remained more or less constant for the rest of treatment period. Compound 5a showed the most promising effect in terms of growth inhibition, where tumor growth decreased by about 25.36% after only 2 days of treatment, and maximal inhibition of 91.1% was recorded after 10 days of treatment. After 14 days, the inhibition percentage decreased to 88.7% and then remained more or less constant up to 20 days of treatment. It can be observed that there is agreement between in vivo inhibitory patterns of the different derivatives and their in vitro anticancer patterns of activity. Also, the newly synthesized estrone derivatives described here coincide with those reported earlier [24]. Furthermore, estrone derivatives have been reported to inhibit in vivo tumor growth in a dosedependent manner [36,37] through their inhibitory action on 17-hydroxysteroid dehydrogenase.

In Vivo and In Vitro Inhibition of p53 Ubiquitination Activities
p53 was found to play an important role in cancer prevention as a suppressor protein through variable pathways. Binding of p53 to E3 ubiquitin protein ligase HDM2 results in inhibiting its ability as a transcription activator, i.e., a negative regulatory mode of action. It was postulated that blocking p53 binding site on HDM2 is useful in obtaining potential antitumor agents. However, there are few reports on scaffolds having inhibitory HDM2 activity. Murine Double Minute 2 (MDM2) is a widely studied regulator that is used to inhibit p53 activity either by direct binding or by acting as an ubiquitin ligase (E3) catalyzing p53 ubiquitination and proteasome-mediated degradation [38].
All newly synthesized compounds exhibited in vitro suppression of p53 ubiquitination when incubated with GST-tagged HDM2, p53, ubiquitin or E1 and E2 (UbcH5B) ligases (IC 50 ranged from16.45 ± 0.23 to 77.56 ± 0.97 µM). Additionally, the evaluated compounds revealed excellent in vivo inhibition of p53 ubiquitination, with IC 50 ranging from 0.22 ± 0.0043 to 0.89 ± 0.0099 µM. By comparing the results with the standard diphenyl imidazole drug (Table 2, Figure 4), it was noticed that all tested compounds represented excellent and more potent activity than the reference for in vitro and in vivo inhibition of p53 ubiquitination with a descending order of activity as follow 5a, 5b, 6a, 6b, 4a, 4b, 3a, 3b, 8a, 8b, 7a and 7b. Also, compound 5a displayed the highest activities, which were 15.8-and 8.6-fold more active than the standard drug for in vitro and in vivo inhibition of p53 ubiquitination, respectively. Diphenyl Imidazole 260 ± 0.02 1.88 ± 0.0047 IC50: Compound concentration required to inhibit p53 ubiquitination by 50%, SEM = standard error of the mean; each value is the mean of three values; n = 6 in each group; Statistical analysis by one way analysis of variance (ANOVA) followed by Dunnet's test using Graphpad Instat software. (p < 0.05).

Inhibition of EGFR and VEGFR-2 Kinases
The same list of the tested compounds was screened for their in vitro inhibition activity against EGFR and VEGFR-2 kinases. IC50 values are reported in Table 3, Figure 5 and were compared with the positive control drug delphinidin. All examined compounds efficiently inhibited EGFR and VEGFR-2 kinases in a dose-dependent manner, with IC50 ranging from 0.086 ± 0.0032 to 0.227 ± 0.0004 µM for EGFR and from 0.027 ± 0.0012 to 0.057 ± 0.0005 µM for VEGFR-2 while compound 5a turned out to be most potent micromolar inhibitor. It was observed that the inhibitory activities for the new derivatives had a similar behavior in terms of descending order as 5a, 5b, 6a, 6b, 4a, 4b, 3a, 3b, 8a, 8b, 7a and 7b ( Figure 5) and had higher activities than the reference drug against both enzymes (IC50

Inhibition of EGFR and VEGFR-2 Kinases
The same list of the tested compounds was screened for their in vitro inhibition activity against EGFR and VEGFR-2 kinases. IC 50 values are reported in Table 3, Figure 5 and were compared with the positive control drug delphinidin. All examined compounds efficiently inhibited EGFR and VEGFR-2 kinases in a dose-dependent manner, with IC 50 ranging from 0.086 ± 0.0032 to 0.227 ± 0.0004 µM for EGFR and from 0.027 ± 0.0012 to 0.057 ± 0.0005 µM for VEGFR-2 while compound 5a turned out to be most potent micromolar inhibitor. It was observed that the inhibitory activities for the new derivatives had a similar behavior in terms of descending order as 5a, 5b, 6a, 6b, 4a, 4b, 3a, 3b, 8a, 8b, 7a and 7b ( Figure 5) and had higher activities than the reference drug against both enzymes (IC 50 of delphinidin = 6.27 ± 0.00076 µM against EGFR and 5.09 ± 0.0012 µM against VEGFR-2). Furthermore, the most potent derivative 5a was about 188.5-and 72.9-fold more active than delphinidin against EGFR and VEGFR-2 kinases, respectively. Furthermore, the most potent derivative 5a was about 188.5-and 72.9-fold more active than delphinidin against EGFR and VEGFR-2 kinases, respectively.

Molecular Modeling Studies
To explain and rationalize the experimental results obtained, molecular docking studies were conducted on the representative compound 5a using Molecular Operating Environment (MOE, 10.2008) software [39] against two targets, EGFR (PDB code: 1M17) [40] and VEGFR-2 (PDB code: 4ASD) [39] binding site structures. The root mean square differences (RMSD) between the top docking poses and original crystallographic geometry of co-crystallized ligands erlotinib for EGFR and sorafenib for VEGFR-2 were 0.90 and 0.88 A • , respectively. From Figure 6 and regarding the EGFR target, compound 5a showed two hydrogen bonds between the imino group at p-2 of the pyrimidine moiety and the sidechains of Arg817 and Asn818 (distance: 2.81 and 2.21 Å, respectively). Furthermore, the acidic Glu734 of the receptor formed a consistently stable hydrogen bond with the terminal hydroxyl group (distance: 3.37 Å). Moreover, compound 5a displayed two critical hydrophobic interactions, one between the centroid of 4-chlorophenyl and Phe699 as an arene-arene interaction and the other between the centroid of ring A of the steroidal moiety and Lys851 as an arene-cation interaction.

Molecular Modeling Studies
To explain and rationalize the experimental results obtained, molecular docking studies were conducted on the representative compound 5a using Molecular Operating Environment (MOE, 10.2008) software [39] against two targets, EGFR (PDB code: 1M17) [40] and VEGFR-2 (PDB code: 4ASD) [39] binding site structures. The root mean square differences (RMSD) between the top docking poses and original crystallographic geometry of co-crystallized ligands erlotinib for EGFR and sorafenib for VEGFR-2 were 0.90 and 0.88 A°, respectively.
From Figure 6 and regarding the EGFR target, compound 5a showed two hydrogen bonds between the imino group at p-2 of the pyrimidine moiety and the sidechains of Arg817 and Asn818 (distance: 2.81 and 2.21 Å, respectively). Furthermore, the acidic Glu734 of the receptor formed a consistently stable hydrogen bond with the terminal hydroxyl group (distance: 3.37 Å). Moreover, compound 5a displayed two critical hydrophobic interactions, one between the centroid of 4chlorophenyl and Phe699 as an arene-arene interaction and the other between the centroid of ring A of the steroidal moiety and Lys851 as an arene-cation interaction.   A docked model of 5a inside VEGFR-2 is shown in Figure 7. The pyrimidine ring was engaged in two hydrogen bond donors between N1 and the imino group at p-2 with the backbone of Asp1046 (distance: 2.25 and 1.42 Å, respectively). Additionally, the centroid of ring A of the steroidal scaffold was involved in an arene-cation interaction with the basic Arg1027.
Molecules 2019, 24 FOR PEER REVIEW 11 A docked model of 5a inside VEGFR-2 is shown in Figure 7. The pyrimidine ring was engaged in two hydrogen bond donors between N1 and the imino group at p-2 with the backbone of Asp1046 (distance: 2.25 and 1.42 Å, respectively). Additionally, the centroid of ring A of the steroidal scaffold was involved in an arene-cation interaction with the basic Arg1027.  The molecular docking results indicated that compound 5a was accommodated well in the binding sites of both enzymes through ring A of the steroidal scaffold and the pyrimidine moiety linked to ring D. Thus, the current results demonstrated that the estrone-pyrimidine hybride The molecular docking results indicated that compound 5a was accommodated well in the binding sites of both enzymes through ring A of the steroidal scaffold and the pyrimidine moiety linked to ring D. Thus, the current results demonstrated that the estrone-pyrimidine hybride structure is a considerable scaffold for subsequent optimization to develop novel effective inhibitors EGFR and VEGFR-2 kinases and anti-breast cancer drugs.

In Vitro Cytotoxic Activity against MCF-7 Cancer Cells
MCF-7 (human breast cancer) cells, obtained from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany, were propagated in RPMI-1640 supplemented with 10% heat inactivated FBS, 2 mM L-glutamine, and 1% standard antibiotic solution. Cells were incubated in a 5% CO 2 humidified incubator at 37 • C and passaged bi-weekly. The in vitro anti-proliferative activity of the newly synthesized derivatives was assayed using standard MTT technique [39,41,42]. Results were expressed as IC 50 . Experiments were repeated at least in triplicate, to obtain good reproducibility between replicate wells with standard errors below 10%. Furthermore, the cytotoxic effects of the prepared derivatives were evaluated against normal nonmalignant cells, non-tumorigenic MCF-10A, in order to find out if the synthesized derivatives have toxicity against normal cells. Additionally, results were compared with reference compounds (cisplatin and milaplatin) as positive controls.

In Vivo Human Breast Cancer Xenograft Models and Animal Treatment
The breast cancer xenograft model protocol [24] was approved by the Institutional Animal Use and Care Committee of the University of Alabama at Birmingham (50-01-05-08B). Female athymic pathogen-free nude mice (nu/nu, 4-6 weeks) were used. Firstly, MCF-7 xenografts were initiated by implanting pellets slowly releasing estrogen for two months (1.7mg 17β-estradiol/pellet) subcutaneously in the female nude mice. After 24 h, confluent MCF-7 cells were harvested, washed two times with serum-free medium, re-suspended and injected subcutaneously (s.c.) (5 × 10 6 cells, total volume 0.2 mL) into the left inguinal area of the mice. Caliper measurement was used to measure tumor growth in two perpendicular diameters of the implant after 48 h, and its volume was determined [43]. Mice grafted with MCF-7 were divided into different groups (7-10 mice/group). Untreated mice received the solvent only. Treated groups received different prepared derivatives as previously described [24].

In Vitro p53 Ubiquitination Assay
Different prepared derivatives were evaluated for their inhibitory potential against p53 ubiquitination. The method is based on incubating different compounds with GST-tagged HDM2, immobilized on glutathione-Sepharose, p53, ubiquitin, E1 and E2 (UbcH5B) ligases. The buffer contained ATP as detailed by Gomha and Abdelaziz [44]. The reaction products were then resolved by SDSPAGE and p53 ubiquitination and were quantified by Western blotting using an anti-p53 antibody [45].

Statistical Analysis
Results are expressed as the mean ± standard error of mean (SEM), n = 6 in each group. Statistical analysis was performed by one way analysis of variance (ANOVA) followed by Dunnet's test using Graphpad Instat software, p < 0.05.

Molecular Modeling Studies
Molecular docking simulation was carried out using Molecular Operating Environment (MOE, 10.2008) software according to the reported method [39]. The three-dimensional X-ray structures of EGFR (PDB code: 1M17) [40] and VEGFR-2 (PDB code: 4ASD) [39] were obtained from the Protein Data Bank through the Internet.

Conclusions
In this study, a new series of estrone derivatives 3-8 was synthesized by heterocyclization at ring D of the steroidal moiety. All new compounds were evaluated for their in vitro and in vivo inhibitory activities against breast cancer, EGFR and VEGFR-2 kinases and p53 ubiquitination. Depending on their structural substitution, it was clear that all derivatives revealed promising and variable inhibitory activities following a similar descending order 5a, 5b, 6a, 6b, 4a, 4b, 3a, 3b, 8a, 8b, 7a and 7b in comparison to the known standard drugs (diphenyl imidazole and delphinidin). Such a finding was justified by molecular docking of the most potent compound 5a, which represented the importance of molecular hybridization of estrone with pyrimidine moiety at ring D, and this affords a new promising chemotherapeutic candidate that could be optimized for further development of potent anti-breast cancer agents.

Conflicts of Interest:
The authors declare no conflict of interest.