Potent Anti-Ovarian Cancer with Inhibitor Activities on Both Topoisomerase II and V600EBRAF of Synthesized Substituted Estrone Candidates

A series of 16-(α-alkoxyalkane)-17-hydrazino-estra-1(10),2,4-trien[17,16-c]-3-ol (3a–l) and estra-1(10),2,4-trien-[17,16-c]pyrazoline-3-ol derivatives (4a–d) were synthesized from corresponding arylidines 2a,b which was prepared from estrone 1 as starting material. Condensation of 1 with aldehydes gave the corresponding arylidine derivatives 2a,b which were treated with hydrazine derivatives in alcohols to give the corresponding derivatives 3a–l, respectively. Additionally, treatment of 2a,b with methyl- or phenylhydrazine in ethanolic potassium hydroxide afforded the corresponding N-substituted pyrazoline derivatives 4a–d, respectively. All these derivatives showed potent anti-ovarian cancer both in vitro and in vivo. The mechanism of anti-ovarian cancer was suggested to process via topoisomerase II and V600EBRAF inhibition.


Introduction
Synthetic alterations of estrone lead to discovering of compounds with diverse biological activities, for example with antitumor effect [1], as anti-breast cancer agent [2] and with antioxidant activity [3]. Estrone derivatives with antitumor activities must be devoid completely of the estrogenic activities [4][5][6]. The inversion of the configuration at C-13 lead to estrone derivatives with antitumor activities devoid from hormonal actions due to conformational change for the overall molecule resulting from the cis junction of rings C and D [4,7]. Some recent publications report on the syntheses and in vitro biological evaluation of several 13α-estrone derivatives [8][9][10][11]. These derivatives exhibited biological activities with substantial anti-proliferative or enzyme inhibitory potentials. Most literature data are mainly focusing on 13α-estrone substitution in ring D, while modified derivatives with ring A substitutions are rarely reported [12].
Cancer is considered one of the most significant causes of death worldwide. Ovarian cancer is one of the major causes for death among adult females [13]. Cancer is characterized by abnormal cell proliferation bypassing normal cell growth and death mechanisms. Recently, DNA topoisomerase II inhibitors have been researched as a potential target, which interferes with cancer growth and development [14]. Topoisomerase II inhibition leads finally to cell cycle inhibition and apoptosis in abnormal cancer cells [15]. BRAF kinase inhibition is another possible control mechanism, which was also developed to interfere with cancer cell growth [16]. BRAF kinase constitutes a major signaling process integrated in the RAS-MEK proliferation process. The activation of MEK by BRAF signals finally induces cell proliferation and survival [17]. Inhibiting BRAF activity results in inhibition of the proliferation signals, and thus inhibiting cancer cell development and growth [18].
In view of these observations and in continuation of our previous work in heterocyclic chemistry [19][20][21][22], the current work aimed at evaluating the effect or remote long cage distortion caused by introducing some moieties in both 16 and 17 locations of ring D. We screened some of synthesized estrone candidates for their anti-ovarian cancer potential both in vitro and in vivo. Furthermore, the inhibitory effects of the synthesized compounds were investigated against topoisomerase II and V600E BRAF kinase inhibitors.

Biological Screening
In Vitro Cytotoxic Activities The cytotoxicity of some synthesized derivatives against SKOV-3 cancer cell line in vitro was performed with the MTT assay. The obtained results showed that all synthesized compounds showed potent in vitro cytotoxic activities against SKOV-3 cells at the nanomolar level. Moreover, the effect of the synthesized compounds was dose-dependent. From the obtained IC 50 values (Figure 1), prepared compounds can be arranged discerningly in the following order: 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j, 3k, 3l, 4a, 4b, 4c, 4d ( Figure 1). Compounds 3a-l are characterized with the presence of hydrazine group rather than the pyrazoline group present in compounds 4a-d. Furthermore, the alkyl group is found to be more active than the aromatic group. Generally Cpd. 3a showed highest cytotoxic activity with IC 50 value of 4.23 ± 0.12 nM, while Cpd. 4d was the least active, where its IC 50 value was about 9.5% of that obtained for Cpd. 3a.
The effect of resveratrol (RES) and doxorubicin (DOX) on SKOV-3 showed IC 50 of 55 and 250 nM, respectively.

In Vivo Anti-Ovarian Cancer
The in vivo anti-ovarian cancer activities of the tested compounds were evaluated using mouse xenograft model. Figure 2 represents the percentages of decrease in tumor growth upon treatment with different synthesized compounds. The obtained results showed that all compounds potentially reduced tumor growth over a period of 40 days of treatment, in comparison to control treatments, which showed normal tumor growth development. Moreover, it can be seen that the reduction in tumor size started directly from the 5th day of treatment, where the reduction percentage ranged from 45.7 ± 0.05% to 54.71 ± 0.01%. The reduction in tumor growth increased significantly reaching its maximal by the end of treatment period (40 days). The highest reduction in tumor growth was recorded for occurred Cpd. 3a, which significantly decreased tumor growth by about 93.61 ± 0.7%. It can also be seen that Cpd. 3a, was the most potent for in vivo treatment, where its effect was more obvious starting from day15 of treatment, where the reduction percentage in tumor size due to other compounds was more or less within the same range. We also can see that the in vivo results are coincided with those obtained for in vitro results, where the reduction in tumor size followed the same descending order: 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j, 3k, 3l, 4a, 4b, 4c, and 4d.

Inhibition of Topoisomerase II Activities
In order to further investigate the possible mechanism of action of the newly synthesized derivatives, we investigated their inhibitory effects on topoisomerase II. Results obtained ( Figure 3) showed that all synthesized compounds showed potential inhibitory effects against topoisomerase II. Furthermore, the inhibitory effect was also found to follow the same descending order obtained before during in vitro and in vivo investigation: 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j, 3k, 3l, 4a, 4b, 4c, and 4d. Cpd. 3a showed the most inhibitory action for topoisomerase II with IC 50 value of 3.45 ± 0.13 nM, which was about 7,9% of that obtained for the lest potent compound (IC 50 for Cpd. 4d: 43.56 ± 0.98 nM).

In Vitro Kinase Assay
The in vitro kinase assay of synthesized derivatives was investigated against both WT BRAF (BRAF kinase wild type) and V600E BRAF (mutant BRAF kinase). Results obtained in Figure 4 showed that all synthesized compounds were highly active inhibitors for V600E BRAF compared with moderate activities against WT BRAF. Again, the descending order of activities was as follow 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j, 3k, 3l, 4a, 4b, 4c, and 4d. Compound 3a recorded the most active inhibition (IC 50 : 0.041 ± 0.0016 and 4.23 ± 0.12 µM for mutant and wild type BRAF kinase, respectively. Furthermore, it can be seen that the inhibitory effect of Cpd. 3a was much more superior to that obtained for different positive control drugs (0.48, 3.87 and 0.97 µM for Sorafenib, Dabrafenib and Vemurafenib, respectively.

Discussion
Within the framework of the current work we synthesized new estrone derivatives from their corresponding arylidines. The newly synthesized derivatives showed potential cytotoxic activities against SKOV-3 cells. Moreover, in vivo investigations revealed that the synthesized compounds were able to potentially reduce tumor volume growth over a treatment period of 45 days.
The newly introduced 1-alkoxy benzyl moiety alone or combined with the 17-hydrazino-moities make remote destortion of the structure cage that completely elminate any hormonal activities of te estrone molceules and deviate the biological activities towards the aniticancer ones, so we investigated this property and tried to find the mechnisms of anticancer actions of these newly synthesized compounds.
In search for the possible mechanism of action of the anticancer activities of the tested compounds, we investigated the possibility of the compounds to inhibit both topoisomerase II and kinase enzymes. Results showed that the prepared compounds can potentially serve as inhibitors for these enzymes. The cytotoxic activities of the prepared estrone derivatives can be due to the inhibition of 17-hydroxysteroid dehydrogenases. We have previously reported on the anti-breast cancer activities of different estrone derivatives [23], and explained that their anticancer activities can be attributed to the inhibition of 17-hydroxysteroid dehydrogenase [24].
Concerning topoisomerase II and V600E BRAF kinase inhibition, our results suggest that the newly synthesized compounds exert their cytotoxic action against SKOV-3 cancer cells by interfering with the metabolic activity of these enzymes, thus preventing cancer cells from obtaining proliferation signaling molecules essential for their growth and survival [16,17]. Different activities of the prepared compounds may be attributed to the structure activity relationship of these compounds.
Hydrazine derivatives are generally potent than pyrazoline ones, due to their open chain structure as well as their higher electron density. Furthermore, it can be suggested that N-methyl derivatives are more active than N-phenyl ones. On the other hand, hydrazine derivatives can be divided into more active α-methoxyl derivatives and the less active α-ethoxyl, and then the least active N-phenyl derivatives. This can be explained due to the fact that phenyl group act as a withdrawing group. On the other hand, the weaker effect of α-ethoxyl derivatives than the α-methoxyl ones can be attributed to their bulky nature. This effect can be observed among different series of compound 3. As the bulky nature of the substituted groups increase, accordingly more steric hindrance can be observed, leading to decreased cytotoxic activities.

Chemical Synthesis
"Melting points reported are inaccurate. IR spectra were registered on Shimadzu FT-IR 8300 E (Shimadzu Corporation, Kyoto, Japan) spectrophotometer using the (KBr) disk technique. The 1 H-NMR spectra were determined with bruker 600 MHz NMR spectrometer. The chemical shifts are expressed on the δ (ppm) scale using TMS as the standard reference. Mass spectra were recorded on Finnigan SSQ operating at 70 ev. Elemental analysis determined on a Perkin Elmer 240 (Microanalysis), Microanalysis Center, Cairo University, Cairo, Egypt". "A mixture of (1) (0.54 g, 20 mmol) and formaldehyde or acetaldehyde (20 mmol) in EtOH (50 mL) and aqueous KOH (10 mL, 30%) was stirred for 24 h at room temperature. The formed solid was collected by filtration, crystallized from ethanol to give compounds 2a [25] and 2b, respectively".          1H, m, H-11α 1H, d, H-1

In Vitro Cytotoxic Activities
The cytotoxic activities of the synthesized derivatives against ovarian cancer cell lines (SKOV-3) were evaluated using standard MTT assay [26,27]. SKOV-3 cells cultivated on DMEM medium supplemented with 10% FBS, 100×, 1% antibiotic/antimycotic solution and 3.6 g/L NaHCO 3 were used. Cells were incubated under standard laboratory conditions [28,29]. Before testing compounds, cells were trypsinized, centrifuged and prepared as per our developed protocol [30,31]. MTT assay relies on the enzymatic conversion of the MTT substrate into purple formazan by living cells mitochondrial enzymes. Shortly, 96 well plates were seeded (10,000 cells/100 µL/well), then incubated for 24 h at standard conditions. Afterwards, cells were exposed to different concentrations from prepared compounds and incubated for another 24 h. Thereafter, MTT (10 µL, 5 mg/mL, PBS) were added to each well, and plates were incubated for 4 h. Supernatants were then replaced discarded with 200 µL of DMSO to dissolve the precipitated formazan crystals. Formazan absorbance, proportional to living cell number, was read at 550 nm using a microplate reader. Viability percentages were calculated and the corresponding IC 50 values were obtained from the linear regression of the calibration curve. For comparison, doxorubicin (DOX) and resveratrol (RES) were used as positive controls. Furthermore, the effect of the newly synthesized derivatives was tested against normal fibroblast cell line to assess their toxicities.

In Vitro Anti-Ovarian Xenograft Model
Anti-ovarian cancer xenograft animal model was developed in nude mouse using SKOV-3 cells according to McCauley et al. [32]. Treatment protocol was approved by the University of South Dakota, Institutional Animal Care and Use Committee (study protocol 50-01-05-08B). Immunodeficient (athymic nude-Foxn1nu) female mice were inoculated (1 × 10 6 cells/site). Upon tumor development, (av. volume~50 mm 3 ), 10-mice groups were implanted s.c. with constant release pellets containing tested compound (1 µM/gm) or placebo. Animal and tumor development were monitored measured every 5 days after pellet implantation, using standardized Caliper in two perpendicular diameters of the implant according to our previous work [2]. Due to large tumor volumes, (5-fold difference between largest and smallest tumor), relative tumor volumes were used for comparison. Relative tumor volume = Vt volume at time t /V0 initial volume .

Topoisomerase II Inhibition
The inhibitory effects of the synthesized compounds against topoisomerase II were investigated using relaxation assay according to Goyeneche, et al. [33]. Topoisomerase II was purified from P388 cells, where enzyme unit corresponds to the activity required complete relaxation of 0.125 g of supercoiled pBR-322 DNA at 30 • C for 1 h. The assay was performed as per protocol and samples were subjected to electrophoresis (0.7% agarose gels, TBE buffer. Ethidium bromide-stained DNA was examined under UV light.

In Vitro Kinase Inhibition
Protein Expression and Purification BRAF wild-type and V600E mutant kinase domains were expressed and purified according to Nakamura et al. [34]. Shortly, Sf 9 cells infected with BRAF kinase domain containing baculovirus were resuspended, sonicated, and then cleared using ultracentrifugation. Afterwards, the cleared lysate with mixed with equilibrated Talon resin, and the resin then washed with 10 column volumes of wash buffer and then eluted with buffer (25 mM Tris, pH 7.0, 250 mM NaCl, 160 mM imidazole, 10% glycerol). The eluant was then diluted, concentrated using Superdex 200 gel filtration column, purified as previously described and finally stored at -80 • C until use.
In Vitro ELISA-Based Kinase Assay "This method was adopted from Nakamura et al. [34] and Qin et al. [35]. The previously prepared GST MEK-His protein was diluted in TTBS buffer (100 µL, 50 µg/mL), and then bound to 96-plate wells coated with glutathione (Pierce Biotechnology). Two DMSO-dilutions of each compound (1 µL) were added to 50 µL (50mM HEPES, pH7.0, 0.7 pmol of BRAF V600E kinase). Tubes were incubated at ambient temperature for 1 h, then added to GST-MEK-His-plate wells. Afterwards, 50 µL of phosphorylation buffer were added to start kinase reaction (37 • C/30 min/shaking). Reaction was stopped, then substrate was added, and the signal was recorded. The high throughput inhibitor screening was carried out according to standard assay conditions at different concentrations, to generate a sigmoidal dose response curve, a four-parameter logistic model using GraphPad Prism, for BRAF proteins, which was used to obtain corresponding IC 50 values".

Conclusions
During the current investigation, we synthesized a series of 17-hydrazino-and 17,16-c-pyrazoline estrone derivatives. Prepared compounds exhibited potential in vitro and in vivo anti-ovarian cancer activity against SKOV-3 cells. Compound 3a was found to be the most potent with an IC 50 value of 4.23 ± 0.12 nM. Additionally, in vivo xenograft ovarian cancer model showed that Cpd. 3a was able to reduce tumor growth by 93.61 ± 0.7% after 40 days of treatment. Furthermore, the newly synthesized compounds were able to interfere with tumor proliferation through inhibiting the activity of topoisomerase II and V600E BRAF, where the obtained IC 50 values for both enzymes were 3.45 ± 0.13 nM and 0.041 ± 0.0016 µM, respectively.