In Vitro and In Vivo Anti-Breast Cancer Activities of Some Synthesized Pyrazolinyl-estran-17-one Candidates

A series of estrone derivatives, 2–4, were synthesized from the corresponding arylidine estrone, 2a,b, as starting materials, which were prepared by condensation of estrone (3-hydroxy-estran-17-one, 1) with 4-bromobenzaldehyde and thiophene-2-aldehyde. Treating of 2a,b with hydrazine derivatives in acetic acid or propionic acid afforded pyrazoline derivatives, 3a–f and 4a–f, respectively. Furthermore, results proved the superiority of thienyl derivatives over 4-bromophenol derivatives in terms of cytotoxic effects on MCF-7 cancer cells. In vivo xenograft breast cancer animal model experiments revealed that the synthesized derivatives can be used for decreasing tumor volume, while the most potent derivative (4f) decreased the development of tumor volume by about 87.0% after 12 days.


Introduction
Estrone ( Figure 1) is a steroid, a weak estrogen, a minor female sex hormone [1], is synthesized from androstadienedione, and secreted mainly from the gonads, though it can also be formed from adrenal androgens in adipose tissue [2].

Introduction
Estrone ( Figure 1) is a steroid, a weak estrogen, a minor female sex hormone [1], is synthesized from androstadienedione, and secreted mainly from the gonads, though it can also be formed from adrenal androgens in adipose tissue [2].  Synthetic alterations of estrone have led to the discovery of compounds with diverse biological activities, for example antitumor effects [3][4][5]. Estrone derivatives with antitumor activities must be devoid completely of estrogenic activities [6][7][8]. Estrone derivatives have proved to be potent 17β-HSD1 inhibitors [9]. To reach estrone derivatives with antitumor activities devoid from hormonal actions, the following alterations were made. This was achieved via the inversion of the configuration at C-13 [6]. The influence of the inversion of the configuration at C-13 in 3,17-estradiols on their in vivo and in vitro estrogenic activity was shown by Ayan et al. [10]. Accordingly, the 13α-estrane core may serve as fundamental moiety for the design of hormonally inactive estrone derivatives bearing promising biological activities. Some have recently published the syntheses and in vitro biological evaluations of several 13α-estrone derivatives [11][12][13][14]. Certain compounds proved to be biologically active, bearing substantial anti-proliferative or enzyme inhibitory potential [11,14]. Most literature data are mainly about 13α-estrones substituted in ring D, with compounds modified in ring A being rarely described [15]. In view of these observations and in continuation of our previous work in heterocyclic chemistry [16][17][18], we screened some synthesized and fused pyrazoline derivatives with estrone ring as anti-breast cancer agents.

Chemistry
In the present study, condensation of estrone (3-hydroxyestran-17-one, 1) with 4-bromobenzaldehyde and thiophene-2-aldehyde afforded the corresponding arylidene derivatives (2a,b). Treating of arylidene derivatives 2a,b with hydrazine hydrate, methyl hydrazine, or phenyl hydrazine in glacial acetic acid afforded the corresponding pyrazoline derivatives, 3a-f, confined to ring D of the steroidal scaffold with acetylation of the 3-phenolic hydroxyl. Treating of arylidene derivatives, 2a,b, with hydrazine hydrate, methyl hydrazine, and phenyl hydrazine in propionic acid afforded the corresponding pyrazoline derivatives, 4a-f, confined to ring D of the steroidal scaffold ( Figure 2). Synthetic alterations of estrone have led to the discovery of compounds with diverse biological activities, for example antitumor effects [3][4][5]. Estrone derivatives with antitumor activities must be devoid completely of estrogenic activities [6][7][8]. Estrone derivatives have proved to be potent 17β-HSD1 inhibitors [9]. To reach estrone derivatives with antitumor activities devoid from hormonal actions, the following alterations were made. This was achieved via the inversion of the configuration at C-13 [6]. The influence of the inversion of the configuration at C-13 in 3,17-estradiols on their in vivo and in vitro estrogenic activity was shown by Ayan et al. [10]. Accordingly, the 13α-estrane core may serve as fundamental moiety for the design of hormonally inactive estrone derivatives bearing promising biological activities. Some have recently published the syntheses and in vitro biological evaluations of several 13α-estrone derivatives [11][12][13][14]. Certain compounds proved to be biologically active, bearing substantial anti-proliferative or enzyme inhibitory potential [11,14]. Most literature data are mainly about 13α-estrones substituted in ring D, with compounds modified in ring A being rarely described [15]. In view of these observations and in continuation of our previous work in heterocyclic chemistry [16][17][18], we screened some synthesized and fused pyrazoline derivatives with estrone ring as anti-breast cancer agents.

Chemistry
In the present study, condensation of estrone (3-hydroxyestran-17-one, 1) with 4-bromobenzaldehyde and thiophene-2-aldehyde afforded the corresponding arylidene derivatives (2a,b). Treating of arylidene derivatives 2a,b with hydrazine hydrate, methyl hydrazine, or phenyl hydrazine in glacial acetic acid afforded the corresponding pyrazoline derivatives, 3a-f, confined to ring D of the steroidal scaffold with acetylation of the 3-phenolic hydroxyl. Treating of arylidene derivatives, 2a,b, with hydrazine hydrate, methyl hydrazine, and phenyl hydrazine in propionic acid afforded the corresponding pyrazoline derivatives, 4a-f, confined to ring D of the steroidal scaffold ( Figure 2

Cytotoxic Activity
The in vitro inhibitory effect of the newly prepared derivatives was evaluated against MCF-7 cells. Results revealed that all the tested compounds (except the starting arylidiene derivatives, 2a,b) exhibited potent cytotoxic activities in vitro against MCF-7 at the nano-molar level. In contrast, doxorubicin control resulted in a much higher IC 50 at the micro-molar level (2.2 ± 0.003 µM). Furthermore, MCF-7 cells responded to the synthesized compounds in a dose-dependent manner. Based on the obtained IC 50 values (Figure 3), it can be concluded that derivatives 4a and 4b (IC 50 , 69 ± 1.16 and 68 ± 1.0 nM, respectively) followed by 3a and 3b (IC 50 , 67 ± 1.26 and 55 ± 2.52 nM, respectively) were the least potent among all synthesized compounds. The most active compounds against MCF-7 cells were 4f, 4e, 4d, 4c, and 3f, where obtained IC 50 values were 43 ± 0.58, 45 ± 1.53, 56 ± 0.59, 47 ± 1.15, and 48 ± 1.52 nM, respectively. Generally, the synthesized compounds showed anticancer activities in the descending order of 4f, 4e, 4d, 4c, 3f, 3e, 3d, 3c, 3b, 3a, and 3a. Concerning the aryl substitutions, it can be observed that derivatives with thienyl moiety have more anticancer activity than the 4-bromophenyl derivatives. On the other hand, the pyrazoline substitution results revealed that the substitution increased the activity of the synthesized derivatives in the order of N-phenyl > N-methyl > N-acetyl > N-propionyl. The higher potency of the N-phenyl group can be explained due to the aromaticity of the ring and the resonance of the phenyl electrons. On the other hand, the superiority of N-methyl over N-acetyl and N-propionyl moieties confer that the presence of the C=O in the latter two substituted derivatives reduced the effect of the compound and, hence, its activity.

Cytotoxic Activity
The in vitro inhibitory effect of the newly prepared derivatives was evaluated against MCF-7 cells. Results revealed that all the tested compounds (except the starting arylidiene derivatives, 2a,b) exhibited potent cytotoxic activities in vitro against MCF-7 at the nano-molar level. In contrast, doxorubicin control resulted in a much higher IC50 at the micro-molar level (2.2 ± 0.003 uM). Furthermore, MCF-7 cells responded to the synthesized compounds in a dose-dependent manner. Based on the obtained IC50 values (Figure 3), it can be concluded that derivatives 4a and 4b (IC50, 69 ± 1.16 and 68 ± 1.0 nM, respectively) followed by 3a and 3b (IC50, 67 ± 1.26 and 55 ± 2.52 nM, respectively) were the least potent among all synthesized compounds. The most active compounds against MCF-7 cells were 4f, 4e, 4d, 4c, and 3f, where obtained IC50 values were 43 ± 0.58, 45 ± 1.53, 56 ± 0.59, 47 ± 1.15, and 48 ± 1.52 nM, respectively. Generally, the synthesized compounds showed anticancer activities in the descending order of 4f, 4e, 4d, 4c, 3f, 3e, 3d, 3c, 3b, 3a, and 3a. Concerning the aryl substitutions, it can be observed that derivatives with thienyl moiety have more anticancer activity than the 4-bromophenyl derivatives. On the other hand, the pyrazoline substitution results revealed that the substitution increased the activity of the synthesized derivatives in the order of Nphenyl > N-methyl > N-acetyl > N-propionyl. The higher potency of the N-phenyl group can be explained due to the aromaticity of the ring and the resonance of the phenyl electrons. On the other hand, the superiority of N-methyl over N-acetyl and N-propionyl moieties confer that the presence of the C=O in the latter two substituted derivatives reduced the effect of the compound and, hence, its activity.

In Vivo Xenograft Model Results
In this experiment, we assessed the in vivo anticancer potential of the synthesized derivatives using a breast cancer mouse xenograft model. Figure 4 represents different percentages of the

In Vivo Xenograft Model Results
In this experiment, we assessed the in vivo anticancer potential of the synthesized derivatives using a breast cancer mouse xenograft model. Figure 4 represents different percentages of the decrease in breast tumor volume obtained upon treating the animal model with different synthesized compounds for 20 days, with reference to control untreated animals. Results clearly show that the derivatives, 3a-f, exhibited their antitumor effect on the development of tumor volume after only six days of treatment, with their highest percentage of decrease in tumor volume obtained after 12 days of treatment (73.7%, 75.4%, 77.8%, 78.5%, 80.0%, and 81.9% for 3a, 3b, 3c, 3d, 3e, and 3f, respectively). Afterwards, the reduction percentage in tumor volume decreased and reached its minimum after 16 days of treatment for all synthesized derivatives. On the other hand, the derivatives, 4c-f, directly exhibited their antitumor effect on tumor volume after two days of treatment. Moreover, the percentage of reduction in tumor volume increased with treatment time and reached maximal reduction values (83.2%, 83.9%, 85.9%, and 87.0% for 4c, 4d, 4e, and 4f, respectively) after 12 days of treatment. Again, it can be observed that the development of the tumor volume was greatly affected by the derivatives, 4c-f, more than the derivatives, 3a-f, which confirms the cytotoxicity results obtained from the IC 50 data. Another striking evidence for the high potential present for derivatives, 4c-f, can be noticed from the fact that the antitumor effect of these derivatives is more persistent and can be sustained over the period of experiment (20 days). After 12 days of treatment, the percentages of reduction in tumor volume were constant until the end of the experiment and did not decrease obviously, which was the case for the derivatives, 3a-f. The potential effect of the newly synthesized derivatives may be attributed to the previously reported inhibitory effects of estrone derivatives for 17β-HSDs [9]. Day et al. [19,20] reported that in vivo estrone-dependent tumor growth was inhibited by the effect of their investigated β-m-pyridylmethylamindomethyl-estrone, which acts to inhibit the 17β-hydroxysteroid, dehydrogenase. Additionally, the synthesis of estrone derivatives has been investigated as a potential pathway for endocrine therapy of hormone-dependent breast cancer [21]. Again, it can be observed that the development of the tumor volume was greatly affected by the derivatives, 4c-f, more than the derivatives, 3a-f, which confirms the cytotoxicity results obtained from the IC50 data. Another striking evidence for the high potential present for derivatives, 4c-f, can be noticed from the fact that the antitumor effect of these derivatives is more persistent and can be sustained over the period of experiment (20 days). After 12 days of treatment, the percentages of reduction in tumor volume were constant until the end of the experiment and did not decrease obviously, which was the case for the derivatives, 3a-f. The potential effect of the newly synthesized derivatives may be attributed to the previously reported inhibitory effects of estrone derivatives for 17β-HSDs [9]. Day et al. [19,20] reported that in vivo estrone-dependent tumor growth was inhibited by the effect of their investigated β-m-pyridylmethylamindomethyl-estrone, which acts to inhibit the 17β-hydroxysteroid, dehydrogenase. Additionally, the synthesis of estrone derivatives has been investigated as a potential pathway for endocrine therapy of hormone-dependent breast cancer [21].

Chemistry
All melting points are uncorrected and were measured using an electro thermal capillary melting point apparatus. The infra-red spectra were detected on a Shimadzu FT-IR 8101 PC (Kyoto, Japan) infrared spectrophotometer. The 1 H and 13 C-NMR spectra were determined with bruker 600 MHz for ( 1 H-NMR) and 150 MHz for ( 13 C-NMR) NMR spectrometer by using CDCl3 as the solvent and using trimethylsilane as the standard reference. Mass spectra were recorded on Finnigan SSQ operating at 70 ev. Elemental analysis was determined on a Perkin Elmer 240 (microanalysis, Waltham, MA, USA), Microanalysis Center, Cairo University, Cairo, Egypt.

Biological Assays
Human breast cancer cell line (MCF-7), purchased from ATCC, Rockville, MD, USA, was used for the in vitro evaluation of the toxicity of the synthesized compounds and the determination of IC 50 values of the tested compounds using the MTT Assay (colorimetric assay). RPMI-1640 containing 10% FBS, L-glutamine, and 1% penicillin-streptomycin antibiotic solution was used to cultivate the cells in a 5% CO 2 humidified incubator at 37 • C. The FBS used in the work was, firstly, stripped according to standard protocol of Green and Leake [22]. The method depends on incubating the serum with dextran-coated charcoal at 4 • C to remove hormones naturally present in the serum before being used.

In Vitro Cytotoxicity Assay
The cytotoxic effect of the prepared derivatives on MCF-7 cells was evaluated using the Mosmann's-MTT assay [23,24]. The methodology depends on the reductive activity of living cells, resulting in the formation of purple formazan from the MTT substrate. Cells were prepared for the assay by culturing them in RPMI 1640 medium. Cells were plated in 96-well culture plates at a concentration of 2 × 104/mL and incubated for 24 h to allow cell adherence. Different concentrations of the synthesized derivatives (0-1 µM) were prepared using dimethysulfoxide (DMSO). Afterwards, the test samples (2 µL) were added to the 96-well plates and the plates were further incubated for three days. 20 µL of MTT solution (5 mg/mL in PBS) were added to the cultured cells and the incubation was further continued for another 4 h. The plates were carefully aspirated and then DMSO was added (100 µL/well), with further shaking for 5 min. The absorbance was then read using a microplate reader at 570 nm [25]. IC 50 values corresponding to the concentration producing a 50% reduction in cell growth was used to express the resulted cell toxicity compared to control cells. The IC 50 values were calculated from the linear regression of the dose-response curve using Origin ® 6.1 software (OriginLab Corporation, Northampton, MA, USA). Triplicate runs were carried out to ensure result reproducibility. Doxorubicin was used as positive control in the experiments.

Human Breast Cancer Xenograft Models and Animal Treatment
The in vivo anticancer potential of the synthesized derivatives was investigated using an MCF-7 mouse xenograft model of breast cancer. The animal protocol was approved by the Institutional Animal Use Ethics 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 purchased from Frederick Cancer Research and Development Center (Frederick, MD, USA). To establish MCF-7 human breast cancer xenografts, each of the female nude mice was first implanted with a 60-day (subcutaneously, s.c.) slow release estrogen pellet (SE-121, 1.7 mg 17β-estradiol/pellet; Innovative Research of America, Sarasota, FL, USA). After 24 h, grown cells were harvested, washed twice with serum-free medium, resuspended, and injected subcutaneously (5 million cells/0.2 mL) into the left inguinal area of the mice. During the experiment, animals were checked periodically and the percentages of tumor growth, as well as animal weights, were recorded. Every 48 h, the size of the tumor was recorded by measuring two perpendicular diameters of the tumor and tumor volume was calculated according to Wang et al. [26]. Treated animals and control groups (7-10 mice/group) received differently prepared derivatives, as well as vehicles, respectively. The tested compounds were dissolved in PEG400:ethanol:saline (57.1:14.3:28.6, v/v/v), and injected intraperitoneal (i.p.) at doses of 5 and 10 mg/kg/d, 3 d/wk for 3 weeks. The higher dose (10 µM/kg/d, 3 d/wk) inhibited MCF-7 xenograft tumor growth.

Conclusions
A series of estrone derivatives, 2-4, were synthesized from the corresponding arylidine estrone, 2a,b, as starting materials, which were prepared by condensation of estrone (3-hydroxy-estran-17-one, 1) with 4-bromobenzaldehyde and thiophene-2-aldehyde. Treating of 2a,b with hydrazine derivatives in acetic acid or propionic acid afforded the pyrazoline derivatives, 3a-f and 4a-f, respectively. All the compounds were tested and showed potent cytotoxic activities in vitro against MCF-7. Further experiment in in vivo xenograft animal models revealed that the derivatives can be used for decreasing tumor volume, with the most potent derivative (4f) decreasing the development of the tumor volume by about 87.0% after 12 days.

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