Potent anti-ovarian cancer with inhibitor activities on Both Topoisomerase II and V600EBRAF of synthesized substituted estrone candidates

Article abstract of DOI:10.3390/molecules24112054

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.

Full text of DOI:10.3390/molecules24112054

molecules
Article
Potent Anti-Ovarian Cancer with Inhibitor Activities
on Both Topoisomerase II and V600EBRAF
of Synthesized Substituted Estrone Candidates
Mohamed El-Naggar 1, Abd El-Galil E. Amr 2,3,
*
, Ahmed A. Fayed 3,4, Elsayed A. Elsayed 5,6
,
Mohamed A. Al-Omar 2 and Mohamed M. Abdalla 7
1
Chemistry Department, Faculty of Sciences, University of Sharjah, Sharjah 27272, UAE;
m5elnaggar@yahoo.com
Drug Exploration & Development Chair (DEDC), Pharmaceutical Chemistry Department,
2
College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia; malomar1@ksu.edu.sa
Applied Organic Chemistry Department, National Research Center, Cairo 12622, Egypt;
3
dr_ahmedfayed14@yahoo.com
Respiratory Therapy Department, College of Medical Rehabilitation Sciences, Taibah University,
4
Madinah Munawara 22624, Saudi Arabia
Zoology Department, Bioproducts Research Chair, College of Science, King Saud University,
5
Riyadh 11451, Saudi Arabia; eaelsayed@ksu.edu.sa
Chemistry of Natural and Microbial Products Department, National Research Centre, Cairo 12622, Egypt
Atos Pharma, Elkatyba Land, Belbis 44621, El Sharkya, Egypt; mmostafa201120@yahoo.com
6
7
*
Correspondence: aamr@ksu.edu.sa; Tel.: +966-565-148-750
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 25 April 2019; Accepted: 28 May 2019; Published: 29 May 2019
Abstract: A series of 16-(
estra-1(10),2,4-trien-[17,16-c]pyrazoline-3-ol derivatives (4a
arylidines 2a,b which was prepared from estrone 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 , respectively. Additionally,
treatment of 2a,b with methyl- or phenylhydrazine in ethanolic potassium hydroxide afforded the
corresponding N-substituted pyrazoline derivatives 4a , respectively. All these derivatives showed
α
-alkoxyalkane)-17-hydrazino-estra-1(10),2,4-trien[17,16-c]-3-ol (3a
–l) and
–
d) were synthesized from corresponding
1
–
l
–d
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.
Keywords: estrone derivatives; hydrazine; N-substituted pyrazoline; anti-ovarian cancer;
topoisomerase II inhibitor; kinase inhibitor
1. Introduction
Synthetic alterations of estrone lead to discovering of compounds with diverse biological activities,
for example with antitumor effect [
Estrone derivatives with antitumor activities must be devoid completely of the estrogenic activities [
1
], as anti-breast cancer agent [
2
] and with antioxidant activity [
3
6
].
].
4
–
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 [
evaluation of several 13 -estrone derivatives [
with substantial anti-proliferative or enzyme inhibitory potentials. Most literature data are mainly
focusing on 13 -estrone substitution in ring D, while modified derivatives with ring A substitutions
4,
7]. Some recent publications report on the syntheses and in vitro biological
α
8–11]. These derivatives exhibited biological activities
α
are rarely reported [12].
Molecules 2019, 24, 2054; doi:10.3390/molecules24112054
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2054
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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].
Inviewoftheseobservationsandincontinuationofourpreviousworkinheterocyclicchemistry[19–22],
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 V600EBRAF kinase inhibitors.
2. Results and Discussion
2.1. Results
2.1.1. Chenical Synthesis
A series of 17-hydrazino-estratrienol (3a–l) and pyrazolinestratrienol (4a–d) were synthesized
from corresponding arylidines 2a,b which was prepared from estrone 1 as starting material.
Treatment of aryledine derivatives 2a,b with hydrazine hydrate, in refluxing methanol or
ethanol afforded the corresponding 17-hydrazino-16-α-methoxymethane estrone derivatives 3a,b
and 17-hydrazino-16-α-ethoxyethane estrone derivatives 3c,d, respectively.
Also, reaction of compounds 2a,b with N-methyl hydrazine, in refluxing methanol or ethanol
afford 17-N-methyl hydrazino-16-α-methoxymethane estrone derivatives 3e,f and 17-N-methyl
hydrazino-16-α-ethoxyethane estrone derivatives 3g,h respectively.
Additionally, when compounds 2a,b reacted with N-phenyl hydrazine, under the same conditions
afford the corresponding 17-N-phenylhydrazino- 16-α-methoxymethane estrone derivatives 3i,j and
17-N-phenylhydrazino- 16- -ethoxyethane estrone derivatives 3k,l, respectively. Finally, condensation
α
of 2a,b with CH3NHNH2 or PhNHNH2 in refluxing dioxane afforded the corresponding N-substituted
pyrazoline derivatives 4a–d, respectively (Scheme 1).

Molecules 2019, 24, 2054
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Scheme 1. Synthetic route for compounds 2–4.
2.1.2. 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 IC50 values (Figure 1), prepared
compounds can be arranged discerningly in the following order: 3a
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 . Furthermore, the alkyl group is found
, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j, 3k,
,
,
,
,
–
d
to be more active than the aromatic group. Generally Cpd. 3a showed highest cytotoxic activity with
IC50 value of 4.23 ± 0.12 nM, while Cpd. 4d was the least active, where its IC50 value was about 9.5%
of that obtained for Cpd. 3a.
The effect of resveratrol (RES) and doxorubicin (DOX) on SKOV-3 showed IC50 of 55 and
250 nM, respectively.
The cytotoxicity of the newly synthesized compounds against fibroblast were as follow IC50
3a (1.2
3h(16.22
4c (25.55 µM), 4d (30.34 µM).
µ
M), 3b (2.45
M), 3i (15.13
µ
M), 3c (5.67
M), 3j (16.20
µ
M), 3d (7.44
M), 3k (17.78
µ
M), 3e (8.11
M), 3l (20.21
µ
M), 3f (9.20
M), 4a (23.34
µ
M), 3g (11.18
M), 4b (24.55 µ
µ
M),
M),
µ
µ
µ
µ
µ
µ

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Figure 1. In vitro Cytotoxicity activities of the tested compounds against SKOV-3 cells.
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
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%.
±
0.05% to 54.71
±
0.01%. The reduction in tumor growth increased significantly reaching
±
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.
Figure 2. Percentages of decrease in tumor volume in nude mice in response to different
synthesized derivatives.

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2.1.3. 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 IC50 value of 3.45
of that obtained for the lest potent compound (IC50 for Cpd. 4d: 43.56 ± 0.98 nM).
±
0.13 nM, which was about 7,9%
Figure 3. IC50 of Topoisomerase II Inhibitor activities of the tested compounds.
2.1.4. In Vitro Kinase Assay
The in vitro kinase assay of synthesized derivatives was investigated against both WTBRAF
(BRAF kinase wild type) and V600EBRAF (mutant BRAF kinase). Results obtained in Figure 4 showed
that all synthesized compounds were highly active inhibitors for V600EBRAF compared with moderate
activities against WTBRAF. Again, the descending order of activities was as follow 3a
,
3b
4c, and 4d. Compound 3a recorded the most active inhibition
0.12 M for mutant and wild type BRAF kinase, respectively.
, 3c, 3d,
3e
,
3f
,
3g
,
3h
,
3i
,
3j
,
3k
,
3l
,
4a,
4b
,
(IC50: 0.041
±
0.0016 and 4.23
±
µ
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.
Figure 4. IC50 values of synthesized for both WTBRAF and V600EBRAF.

Molecules 2019, 24, 2054
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2.2. 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 V600EBRAF 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
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 -ethoxyl derivatives than the -methoxyl ones can be attributed
to their bulky nature. This effect can be observed among different series of compound . As the
α-methoxyl derivatives and the less active α-ethoxyl, and then the least active N-phenyl
α
α
3
bulky nature of the substituted groups increase, accordingly more steric hindrance can be observed,
leading to decreased cytotoxic activities.
3. Materials and Methods
3.1. 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 1H-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”.
3.1.1. Synthesis of 3-Hydroxy-16-[substituted]-estra-1(10),2,4-trien-17-ones (2a,b)
“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”.

Molecules 2019, 24, 2054
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3-Hydroxy-16-[ethylene]-estra-1(10),2,4-trien-17-one (2b)
Yield 97%, mp 198–200 ◦C, [α]D25 = + 71 (c 1, MeOH). IR (KBr, cm–1): 3340 (OH), 2944 (CH, aliphatic),
1743 (C=O), 1642 (C=C). 1H NMR: (600 MHz, CDCl3):
ppm 0.61 (1H, m, H-8 ), 0.90 (3H, s, CH3-18),
1.01 (1H, m, H-11 ), 1.12 (1H, m, H-7 ), 1.15 (1H, m, H-12 ), 1.25 (1H, m, H-14
1.63 (1H, m, H-15 ), 1.72 (1H, m, H-7 ), 2.13 (1H, m, H-11
2.56 (1H, m, H-6 ), 2.66 (1H, m, H-6
δ
β
β
α
α
α
), 1.44 (1H, m, H-15
β
),
),
α
β
), 2.01 (1H, m, H-9
α
α), 2.50 (1H, m, H-12
β
α
β
), 4.90 (1H, s, OH, disappeared with D2O), 5.75 (1H, dd, H-2),
6.67 (1H, d, H-4), 2.08 (3H, d, CH3, enone), 6.41 (1H, s, enone proton), 7.12 (1H, d, H-1). 13C NMR:
(150 MHz, CDCl3):
δ 13.52, 21.73, 25.51, 26.64, 27.48, 29.75, 36.35, 38.61, 43.81, 48.92, 50.43, 112.79,
115.53, 126.56, 128.48, 132.50, 137.02, 138.44, 153.82, 210.02 (20 C). MS (EI): m/z 296 (100%) [M+]. Anal.
Calcd for C20H24O2 (296.40): Calcd C, 81.04; H, 8.16. Found C, 81.00; H, 8.10.
3.1.2. Synthesis of 16-(α-alkoxy-alkane)-17-hydrazino-estra-1(10),2,4-trien[17,16-c]-3-ol and their
N-substituted derivatives (3a–l)
“A mixture of 2a,b (14 mmol) and hydrazine derivatives, namely, hydrazine hydrate, methyl
hydrazine or phenyl hydrazine (8 mmol) in absolute methanol or ethanol (30 mL) was refluxed for
5–10 h. The solvent was concentrated under reduced pressure, the formed precipitate was filtered off,
washed with water, dried and crystallized from ethanol to give the corresponding derivatives 3a–l
,
respectively”.
16-(α-Methoxy-methane)-17-hydrazino-estra-1(10),2,4-trien-[17,16-c]-3-ol (3a)
Yield 80%, mp 199–201 C, [α]2D5 = + 81 (c 1, MeOH). IR (KBr, cm–1): 3444–3377 (NH, NH2),
3345 (OH), 2951 (CH, aliphatic), 1638 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.61 (1H, m, H-8β),
◦
0.90 (3H, s, CH3-18), 1.00 (1H, m, H-11
1.39 (1H, m, H-15 ), 1.58 (1H, m, H-15
16-H ), 2.11 (1H, m, H-11 ), 2.49 (1H, m, H-12
β
), 1.11 (1H, m, H-7
), 1.68 (1H, m, H-7
), 2.61 (1H, m, H-6
α
), 1.14 (1H, m, H-12
), 2.01(1H, m, H-9
), 2.66 (1H, m, H-6β
α
), 1.26 (1H, m, H-14
), 1.84 (1H, m, CH,
), 2.83 (1H, d,
α),
β
α
β
α
α
α
β
α
H-17), 3.48 (3H, s, OCH3), 4.65 (2H, m, NH2, disappeared with D2O), 4.80 (2H, d, CH2-O), 4.87 (1H,
s, OH, disappeared with D2O), 5.70 (1H, dd, H-2), 6.61 (1H, d, H-4), 7.10 (1H, d, H-1), 7.70 (1H, br.s,
NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3):
δ 13.58, 21.76, 25.57, 26.76, 28.00, 29.78,
36.38, 38.65, 43.89, 48.40, 50.45, 59.45, 69.11, 112.37, 115.56, 126.53, 132.81, 138.46, 153.84, 167.02 (20 C).
MS (EI): m/z 330 (90%) [M+]. Anal. Calcd for C20H30N2O2 (330.46): Calcd C, 72.69; H, 9.15; N, 8.48.
Found C, 72.60; H, 9.10; N, 8.40.
16-(α-Methoxy-ethane)-17-hydrazino-estra-1(10),2,4-trien[17,16-c]-3-ol (3b)
Yield 57%, mp. 308–310 ◦C, [α]D25 = + 112 (c 1, MeOH). IR (KBr, cm–1): 3448-3377 (NH, NH2),
3348 (OH), 2959 (CH, aliphatic), 1634 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.59 (1H, m, H-8β),
0.91 (3H, s, CH3-18), 1.01 (1H, m, H-11
1.39 (1H, m, H -15 ), 1.58 (1H, m, H-15
m, H-9 ), 1.83 (1H, m, CH, H-16 ), 2.11 (1H, m, H-11
2.66 (1H, m, H-6 ), 2.83 (1H, d, H-17), 3.48 (3H, s, OCH3), 4.65 (2H, s, NH2, disappeared with D2O),
4.84 (1H, d, CH-O), 4.99 (1H, s, OH, disappeared with D2O), 5.72 (1H, dd, H-2), 6.61 (1H, d, H-4),
7.11 (1H, d, H-1), 7.78 (1H, bs, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3):
13.58,
β
), 1.12 (1H, m, H-7
), 1.42 (3H, m, CH3,C-16 ethane), 1.69 (1H, m, H-7
), 2.49 (1H, m, H-12 ), 2.61 (1H, m, H-6
α
), 1.14 (1H, m, H-12
α
), 1.26 (1H, m, H-14
), 2.02(1H,
),
α),
β
α
β
α
α
α
β
α
β
δ
21.77, 22.22, 25.57, 26.47, 28.00, 29.78, 36.68, 38.47, 43.89, 48.69, 50.45, 59.45, 69.11, 112.37, 115.56, 126.55,
132.48, 138.46, 153.54, 167.76 (21 C). MS (EI): m/z 344 (11%) [M+]. Anal. Calcd for C21H32N2O2 (344.49):
Calcd C, 73.22; H, 9.36; N, 8.13. Found C, 73.18; H, 9.30; N, 8.10.
16-(α-Ethoxy-methane)-17-hydrazino-estra-1(10),2,4-trien-[17,16-c]-3-ol (3c)
Yield 56%, mp 219–221 C, [α]2D5 = + 77 (c 1, MeOH). IR (KBr, cm–1): 3445–3376 (NH, NH2),
◦
3346 (OH), 2951 (CH, aliphatic), 1638 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.61 (1H, m, H-8β),
0.90 (3H, s, CH3-18), 1.00 (1H, m, H-11β), 1.11 (1H, m, H-7α), 1.15 (1H, m, H-12α), 1.21 (3H, m, CH3,

Molecules 2019, 24, 2054
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ethoxyl), 1.25 (1H, m, H-14
α
), 1.36 (1H, m, H-15
), 2.10 (1H, m, H-11
), 6.61 (1H, d, H-4), 2.80 (1H, d, H-17), 3.50 (2H, m, OCH2), 4.70 (2H, m, NH2, disappeared with
D2O), 4.80 (2H, d, CH-O), 4.87 (1H, s, OH, disappeared with D2O), 5.70 (1H, dd, H-2), 7.10 (1H, d, H-1),
7.70 (1H, br.s, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3):
13.65, 18.74, 21.57, 25.17,
β
), 1.57 (1H, m, H-15
α
), 1.68 (1H, m, H-7
β), 2.60 (1H, m, H-6α), 2.70 (1H,
β
), 2.01 (1H, m,
H-9
α
), 1.84 (1H, m, CH, H-16
α
α
), 2.50 (1H, m, H-12
m, H-6
β
δ
26.38, 28.04, 29.64, 36.54, 38.15, 44.11, 48.44, 50.54, 68.45, 69.11, 112.44, 115.67, 126.34, 131.11, 138.46,
153.84, 161.47 (21 C). MS (EI): m/z 344 (35%) [M+]. Anal. Calcd for C21H32N2O2 (344.49): Calcd C,
73.22; H, 9.36; N, 8.13. Found C, 73.16; H, 9.30; N, 8.10.
16-(α-Ethoxy-ethane)-17-hydrazino-estra-1(10),2,4-trien-[17,16-c]-3-ol (3d)
Yield 90%, mp 223–225 C, [α]2D5 = + 95 (c 1, MeOH). IR (KBr, cm–1): 3440-3370 (NH, NH2),
◦
3350 (OH), 2951 (CH, aliphatic), 1631 (C=C). 1H NMR: (600 MHz, CDCl3): δ ppm 0.57 (1H, m, H-8β),
0.90 (3H, s, CH3-18), 1.00 (1H, m, H-11
ethoxyl), 1.24 (1H, m, H-14 ), 1.36 (1H, m, H-15
1.71 (1H, m, H-7 ), 1.96 (1H, m, H-9
H-12 ), 2.62 (1H, m, H-6
β
), 1.10 (1H, m, H-7
), 1.45 (3H, m, CH3,C-16 ethane), 1.59 (1H, m, H-15
), 1.82 (1H, m, CH, H-16 ), 2.10 (1H, m, H-11 ), 2.50 (1H, m,
), 2.86 (1H, d, H-17), 3.48 (2H, m, OCH2), 4.64 (2H, s,
α
), 1.15 (1H, m, H-12
α
), 1.20 (3H, m, CH3,
α
β
α
),
β
α
α
α
β
α
), 2.67 (1H, m, H-6
β
NH2, disappeared with D2O), 4.83 (1H, d, CH-O), 4.88 (1H, s, OH, disappeared with D2O), 5.74 (1H,
dd, H-2), 6.65 (1H, d, H-4), 7.12 (1H, d, H-1), 7.78 (1H, br.s, NH, disappeared with D2O). 13C NMR:
(150 MHz, CDCl3):
δ 13.55, 18.79, 22.07, 22.28, 25.47, 26.52, 27.77, 28.09, 36.68, 37.97, 44.09, 48.11, 51.41,
68.43, 69.13, 112.56, 115.57, 126.37, 133.18, 137.87, 155.56, 162.74 (22 C). MS (EI): m/z 358 (45%) [M+].
Anal. Calcd for C22H34N2O2 (358.51): Calcd C, 73.70; H, 9.56; N, 7.81. Found C, 73.64; H, 9.52; N, 7.75.
16-(α-Methoxy-methane)-17-[N-methyl-hydrazino]estra-1(10),2,4-trien[17,16-c]-3-ol (3e)
Yield 76%, mp 222–224 ◦C, [α]D25 = + 66 (c 1, MeOH). IR (KBr, cm–1): 3443–3371 (NH), 3344 (OH),
2953 (CH, aliphatic), 1634 (C=C). 1H NMR: (600 MHz, CDCl3):
δ
ppm 0.61 (1H, m, H-8
), 1.14 (1H, m, H-12 ), 1.25 (1H, m, H-14
), 2.00 (1H, m, H-9 ), 1.84 (1H, m, CH, H-16
), 2.32 (3H, s, NCH3), 2.48 (1H, m, H-12 ), 2.63 (1H, m, H-6 ), 2.68 (1H, m, H-6 ), 2.84 (1H, d,
β
), 0.92 (3H, s,
), 1.37 (1H, m,
), 2.13 (1H,
CH3-18), 1.03 (1H, m, H-11
H-15 ), 1.57 (1H, m, H-15
m, H-11
β
), 1.10 (1H, m, H-7
α
α
α
β
α
), 1.66 (1H, m, H-7
β
α
α
α
β
α
β
H-17), 3.49 (3H, s, OCH3), 4.86 (2H, d, CH-O), 4.87 (1H, s, OH, disappeared with D2O), 5.72 (1H, dd,
H-2), 6.63 (1H, d, H-4), 7.11 (1H, d, H-1), 7.76 (1H, brs, NH, disappeared with D2O), 7.99 (1H, bs, NH,
disappeared with D2O). 13C NMR: (150 MHz, CDCl3):
δ 13.53, 21.44, 25.67, 26.36, 28.03, 29.23, 36.33,
38.43, 43.83, 47.47, 48.43, 50.34, 59.35, 69.31, 112.36, 115.67, 126.52, 132.32, 138.21, 153.45, 162.32 (21 C).
MS (EI): m/z 344 (45%) [M+]. Anal. Calcd for C21H32N2O2 (344.50): Calcd C, 73.22; H, 9.36; N, 8.13.
Found C, 73.15; H, 9.31; N, 8.09.
16-(α-Methoxy-ethane)-17-[N-methyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol (3f)
Yield 57%, mp 310–312 ◦C, [α]D25 = + 112 (c 1, MeOH). IR (KBr, cm–1): 3443–3374 (NH), 3344 (OH),
2955 (CH, aliphatic), 1628 (C=C). 1H NMR: (600 MHz, CDCl3):
δ
ppm 0.58 (1H, m, H-8
), 1.14 (1H, m, H-12 ), 1.26 (1H, m, H-14
), 1.44 (3H, m, CH3, C-16 ethane), 1.70 (1H, m, H-7 ), 2.00 (1H, m,
), 2.11 (1H, m, H-11 ), 2.36 (3H, s, NCH3), 2.51 (1H, m, H-12 ), 2.62 (1H,
β), 2.85 (1H, d, H-17), 3.44 (3H, s, OCH3), 4.83 (1H, d, CH-O), 4.99 (1H, s,
β
), 0.90 (3H, s,
CH3-18), 1.00 (1H, m, H-11
m, H-15 ), 1.60 (1H, m, H-15
H-9 ), 1.82 (1H, m, CH, H-16
m, H-6 ), 2.65 (1H, m, H-6
β), 1.10 (1H, m, H-7
α
α
α
), 1.39 (1H,
β
α
β
α
α
α
β
α
OH, disappeared with D2O), 5.74 (1H, dd, H-2), 6.65 (1H, d, H-4), 7.13 (1H, d, H-1), 7.73 (1H, br.s,
NH, disappeared with D2O), 7.99 (1H, m, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3):
δ
13.78, 21.54, 22.27, 25.97, 26.69, 28.03, 29.43, 36.45, 38.54, 43.68, 47.88, 48.57, 50.86, 59.46, 69.15, 112.54,
115.69, 126.46, 132.90, 138.75, 153.45, 163.60 (22 C). MS (EI): m/z 358 (100%) [M+]. Anal. Calcd for
C22H34N2O2 (358.50): Calcd C, 73.70; H, 9.56; N, 7.81. Found C, 73.60; H, 9.49; N, 7.75.

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16-(α-Ethoxy-methane)-17-[N-methyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol (3g)
Yield 90%, mp 286–288 ◦C, [α]D25 = + 121 (c 1, MeOH). IR (KBr, cm–1): 3441–3379 (NH), 3348 (OH),
2957 (CH, aliphatic), 1636 (C=C). 1H NMR: (600 MHz, CDCl3):
ppm 0.61 (1H, m, H-8 ), 0.91 (3H, s,
CH3-18), 1.01 (1H, m, H-11 ), 1.13 (1H, m, H-7 ), 1.15 (1H, m, H-12 ), 1.22 (3H, m, CH3, ethoxyl),
1.25 (1H, m, H-14 ), 1.37 (1H, m, H-15 ), 1.58 (1H, m, H-15 ), 1.69 (1H, m, H-7 ), 2.00 (1H, m, H-9 ),
1.81 (1H, m, CH, H-16 ), 2.12 (1H, m, H-11 ), 2.33 (3H, s, NCH3), 2.54 (1H, m, H-12 ), 2.65 (1H, m,
H-6 ), 2.76 (1H, m, H-6 ), 2.87 (1H, d, H-17), 3.50 (2H, m, OCH2), 4.85 (2H, d, CH2-O), 4.98 (1H, s,
δ
β
β
α
α
α
β
α
β
α
α
α
β
α
β
OH, disappeared with D2O), 5.74 (1H, dd, H-2), 6.65 (1H, d, H-4), 7.10 (1H, d, H-1), 7.76 (1H, br.s,
NH, disappeared with D2O), 7.95 (1H, m, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3):
δ
13.35, 18.45, 21.43, 25.78, 26.80, 28.14, 29.88, 36.47, 38.90, 44.41, 47.37, 48.34, 50.54, 68.66, 69.67, 113.34,
115.55, 125.54, 131.53, 137.99, 154.44, 162.27 (22 C). MS (EI): m/z 358 (100%) [M+]. Anal. Calcd for
C22H34N2O2 (358.50): Calcd C, 73.70; H, 9.56; N, 7.81. Found C, 73.62; H, 9.50; N, 7.74.
16-(α-Ethoxy-ethane)-17-[N-methyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol (3h)
Yield 78%, mp 280–282 ◦C, [α]D25 = + 135 (c 1, MeOH). IR (KBr, cm–1): 3441–3371 (NH), 3351 (OH),
2951 (CH, aliphatic), 1631 (C=C). 1H NMR: (600 MHz, CDCl3):
δ
ppm 0.60 (1H, m, H-8
), 1.16 (1H, m, H-12 ), 1.19 (3H, m, CH3,ethoxyl),
), 1.46 (3H, m, CH3,C-16 ethane), 1.61 (1H, m, H-15 ), 1.72
), 1.93 (1H, m, H-9 ), 2.10 (1H, m, H-11 ), 2.50 (1H, m, H-12 ),
), 2.62 (1H, m, H-6 ), 2.85 (1H, d, H-17), 3.43 (2H, m, OCH2), 4.64
β), 0.93 (3H,
s, CH3-18), 1.04 (1H, m, H-11
1.23 (1H, m, H-14 ), 1.37 (1H, m, H-15
(1H, m, H-7 ), 1.82 (1H, m, CH, H-16
2.34 (3H, s, NCH3), 2.63 (1H, m, H-6
β), 1.11 (1H, m, H-7
α
α
α
β
α
β
α
α
α
β
α
β
(1H, s, OH, disappeared with D2O), 4.83 (1H, d, CH-O), 5.72 (1H, dd, H-2), 6.64 (1H, d, H-4), 7.10 (1H,
d, H-1), 7.75 (1H, br.s, NH, disappeared with D2O), 7.88 (1H, s, NH, disappeared with D2O). 13C NMR:
(150 MHz, CDCl3):
δ 13.53, 18.67, 22.43, 22.90, 25.67, 26.78, 27.45, 28.29, 36.89, 37.65, 44.65, 47.35, 48.68,
51.90, 68.78, 69.13, 112.56, 115.56, 126.34, 133.55, 137.43, 155.34, 163.34 (23 C). MS (EI): m/z 372 (100%)
[M+]. Anal. Calcd for C23H36N2O2 (372.54): Calcd C, 74.15; H, 9.74; N, 7.52. Found C, 74.05; H, 9.68;
N, 7.48.
16-(α-Methoxy-methane)-17-[N-phenyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol (3i)
Yield 67%, mp 312–314 ◦C, [α]D25 = + 95 (c 1, MeOH). IR (KBr, cm–1): 3430–3367 (NH), 3345 (OH),
3068 (CH, aromatic), 2951 (CH, aliphatic), 1638 (C=C). 1H NMR: (600 MHz, CDCl3):
δ ppm 0.61(1H, m,
H-8
H-14
CH, H-16
β
), 0.91 (3H, s, CH3-18), 1.00 (1H, m, H-11
), 1.40 (1H, m, H-15 ), 1.60 (1H, m, H-15
), 2.12 (1H, m, H-11 ), 2.49 (1H, m, H-12
β
), 1.12 (1H, m, H-7
), 1.68 (1H, m, H-7
), 2.59 (1H, m, H-6
α
), 1.15 (1H, m, H-12
α
), 1.28 (1H, m,
), 1.86 (1H, m,
α
β
α
β), 2.02 (1H, m, H-9
α
α
α
β
α
), 2.65 (1H, m, H-6
β), 2.84 (1H,
d, H-17), 3.47 (3H, s, OCH3), 4.80 (2H, d, CH-O), 4.87 (1H, s, OH, disappeared with D2O), 5.70 (1H, dd,
H-2), 6.61 (1H, d, H-4), 7.10 (1H, d, H-1), 7.28-7.48 (5H, m, Ar-H), 7.76 (1H, br.s, NH, disappeared with
D2O), 7.99 (1H, s, NH, disappeared with D2O). 13C NMR: (150 MHz, CDCl3):
δ 13.45, 21.76, 25.76, 26.67,
28.45, 29.66, 36.43, 38.65, 43.45, 48.40, 50.87, 59.45, 69.67, 112.43, 112.76, 115.66, 119.11, 126.68, 129.45,
132.67, 138.53, 152.55, 153.79, 161.02 (26 C). MS (EI): m/z 406 (77%) [M+]. Anal. Calcd for C26H34N2O2
(406.56): Calcd C, 76.81; H, 8.43; N, 6.89. Found C, 76.75; H, 8.40; N, 6.80.
16-(α-Methoxy-ethane)-17-[N-phenyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol (3j)
Yield 66%, mp 229–231 ◦C, [α]D25 = + 147 (c 1, MeOH). IR (KBr, cm–1): 3433–3363 (NH), 3346 (OH),
3066 (CH, aromatic), 2955 (CH, aliphatic), 1635 (C=C). 1H NMR: (600 MHz, CDCl3):
δ
ppm 0.62 (1H,
), 1.18 (1H, m, H-12 ), 1.26 (1H,
), 1.42 (3H, m, CH3, C-16 ethane), 1.58 (1H, m, H-15 ), 1.69 (1H, m,
), 2.03 (1H, m, H-9 ), 2.10 (1H, m, H-11 ), 2.52 (1H, m, H-12 ), 2.62 (1H,
β), 2.85 (1H, d, H-17), 3.49 (3H, s, OCH3), 4.86 (1H, d, CH-O), 4.89 (1H, s,
m, H-8
m, H-14
H-7 ), 1.83 (1H, m, CH, H-16
m, H-6 ), 2.65 (1H, m, H-6
β), 0.92 (3H, s, CH3-18), 1.02 (1H, m, H-11
β
), 1.13 (1H, m, H-7
α
α
α
), 1.39 (1H, m, H-15
β
α
β
α
α
α
β
α
OH, disappeared with D2O), 5.71 (1H, dd, H-2), 6.61 (1H, d, H-4), 7.12 (1H, d, H-1), 7.27-7.49 (5H, m,
Ar-H), 7.77 (1H, br.s, NH, disappeared with D2O), 7.97 (2H, s, NH, disappeared with D2O). 13C NMR:

Molecules 2019, 24, 2054
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(150 MHz, CDCl3):
δ
13.52, 21.78, 22.92, 25.67, 26.45, 28.10, 29.90, 36.17, 38.47, 43.80, 48.69, 50.55, 59.25,
69.11, 112.12, 112.78, 115.54, 119.17, 126.46, 129.53, 132.79, 138.78, 152.42, 153.76, 163.11 (27 C). MS (EI):
m/z 420 (81%) [M+]. Anal. Calcd for C27H36N2O2 (420.58): Calcd C, 77.10; H, 8.63; N, 6.66. Found C,
77.00; H, 8.58; N, 6.60.
16-(α-Ethoxy-methane)-17-[N-phenyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol (3k)
Yield 60%, mp 338–340 ◦C, [α]D25 = + 155 (c 1, MeOH). IR (KBr, cm–1): 3431–3362 (NH), 3343 (OH),
3064 (CH, aromatic), 2956 (CH, aliphatic), 1637 (C=C). 1H NMR: (600 MHz, CDCl3):
δ
ppm 0.63 (1H,
), 1.16 (1H, m, H-12 ), 1.21 (3H,
), 1.68 (1H, m, H-7 ),
), 2.61 (1H, m,
), 6. disappeared (1H, d, H-4), 2.80 (1H, d, H-17), 3.53 (2H, m, OCH2), 4.80 (2H,
m, H-8
m, CH3, ethoxyl), 1.24 (1H, m, H-14
2.00 (1H, m, H-9 ), 1.84 (1H, m, CH, H-16
H-6 ), 2.70 (1H, m, H-6
β), 0.90 (3H, s, CH3-18), 1.01 (1H, m, H-11
β
), 1.11 (1H, m, H-7
α
α
α
), 1.36 (1H, m, H-15
β), 1.59 (1H, m, H-15
α
β
α
α), 2.11 (1H, m, H-11α), 2.50 (1H, m, H-12β
α
β
d, CH-O), 4.84 (1H, s, OH, exchangeable with D2O), 5.70 (1H, dd, H-2), 7.15 (1H, d, H-1), 7.26-7.47
(5H, m, Ar-H), 7.76 (1H, br.s, NH, disappeared with D2O), 7.98 (1H, m, NH, disappeared with D2O).
13C NMR: (150 MHz, CDCl3):
δ 13.64, 18.74, 21.57, 25.17, 26.30, 28.05, 29.64, 36.54, 38.15, 44.18, 48.46,
50.55, 68.43, 69.13, 112.49, 112.72, 115.60, 119.11, 126.34, 129.50, 131.11, 138.49, 152.40, 153.84, 162.47
(27 C). MS (EI): m/z 420 (88%) [M+]. Anal. Calcd for C27H36N2O2 (420.58): Calcd C, 77.10; H, 8.63; N,
6.66. Found C, 77.02; H, 8.57; N, 6.61.
16-(α-Ethoxy-ethane)-17-[N-phenyl-hydrazino]-estra-1(10),2,4-trien-[17,16-c]-3-ol (3l)
Yield 96%, mp 288–300 ◦C, [α]D25 = + 95 (c 1, MeOH). IR (KBr, cm–1): 3431–3367 (NH), 3343 (OH),
3068 (CH, aromatic), 2951 (CH, aliphatic), 1631 (C=C). 1H NMR: (600 MHz, CDCl3):
δ
ppm 0.58 (1H, m,
), 1.14 (1H, m, H-12 ), 1.21 (3H, m,
), 1.44 (3H, m, CH3, C-16 ethane), 1.60 (1H, m,
), 2.11 (1H, m, H-11 ), 2.51 (1H,
), 2.88 (1H, d, H-17), 3.49 (2H, m, OCH2), 4.84 (1H, d,
H-8
CH3, ethoxyl), 1.24 (1H, m, H-14
H-15 ), 1.71 (1H, m, H-7
m, H-12 ), 2.62 (1H, m, H-6
β), 0.91 (3H, s, CH3-18), 1.00 (1H, m, H-11
β
), 1.10 (1H, m, H-7
α
α
α
), 1.36 (1H, m, H-15β
α
β), 1.97 (1H, m, H-9
α
), 1.83 (1H, m, CH, H-16
α
α
β
α
), 2.67 (1H, m, H-6
β
CH-O), 4.87 (1H, s, OH, disappeared with D2O), 5.73 (1H, dd, H-2), 6.62 (1H, d, H-4), 7.13 (1H, d, H-1),
7.23-7.47 (5H, m, Ar-H), 7.77 (1H, br.s, NH, disappeared with D2O), 7.87 (1H, m, NH, disappeared
with D2O). 13C NMR: (150 MHz, CDCl3):
δ 13.56, 18.77, 22.27, 22.32, 25.76, 26.78, 27.34, 28.29, 36.54,
37.90, 44.21, 48.23, 51.32, 68.34, 69.56, 112.67, 112.77, 115.56, 119.10, 126.45, 129.50, 133.47, 137.23, 152.55,
155.51, 163.73 (28 C). MS (EI): m/z 434 (93%) [M+]. Anal. Calcd for C28H38N2O2 (434.61): Calcd C,
77.38; H, 8.81; N, 6.45. Found C, 77.30; H, 8.75; N, 6.40.
3.1.3. Synthesis of pyrazoline-3-ol derivatives (4a–d)
A mixture of 2a,b (4 mmol) and hydrazine derivatives, namely, methy hydrazine or phenyl
hydrazine (16 mmol) in dioxane (25 mL) was refluxed for 5 h. The mixture was evaporated under
vacuum, the residue formed was triturated with water, filtered off, washed with water, dried and
crystallized from MeOH to give derivatives 4a–d, respectively.
1‘-Methyl-1‘H-estra-1(10),2,4-trien-[17,16-c]pyrazoline-3-ol (4a)
Yield 96%, mp 117–119 ◦C, [α]2D5 = + 109 (c 1, MeOH). IR (KBr, cm–1): 3351 (OH), 2946 (CH, aliphatic),
1627 (C=C), 1610 (C=N). 1H NMR: (600 MHz, CDCl3):
δ
ppm 0.61 (1H, m, H-8
), 1.18 (1H, m, H-12 ), 1.27 (1H, m, H-14
), 1.74 (1H, m, H-7 ), 1.84 (1H, m, CH, H-16 ), 2.01 (1H, m, H-9
NCH3), 2.22 (1H, m, H-11 ), 2.42 (1H, m, H-12 ), 2.52 (1H, m, H-6 ), 2.66 (1H, m, H-6
pyrazoline-50), 4.95 (1H, s, OH, disappeared with D2O), 5.75 (1H, dd, H-2), 6.65 (1H, d, H-4), 7.15 (1H,
d, H-1). 13C NMR: (150 MHz, CDCl3):
14.60, 22.56, 25.35, 26.46, 28.77, 29.56, 36.68, 38.68, 43.34, 45.36,
48.21, 50.24, 64.45, 112.46, 115.57, 126.44, 132.56, 138.45, 152.67, 156.46 (20 C). MS (EI): m/z 310 (100%)
β
), 0.94 (3H, s, CH3-18),
), 1.41 (1H, m, H-15 ),
), 2.10 (3H, s,
), 2.84 (2H, d,
1.08 (1H, m, H-11
β
), 1.13 (1H, m, H-7
α
α
α
β
1.66 (1H, m, H-15
α
β
α
α
α
β
α
β
δ

Molecules 2019, 24, 2054
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[M+]. Anal. Calcd for C20H26N2O (310.43): Calcd C, 77.38; H, 8.44; N, 9.02. Found C, 77.29; H, 8.36; N,
8.98.
1‘-Methyl -1‘H-5‘-methyl-estra-1(10),2,4-trien-[17,16-c]pyrazoline-3-ol (4b)
Yield 88%, mp 216–218 ◦C, [α]2D5 = + 111 (c 1, MeOH). IR (KBr, cm–1): 3351 (OH), 2946 (CH, aliphatic),
1627 (C=C), 1617 (C=N). 1H NMR: (600 MHz, CDCl3):
δ
ppm 0.62 (1H, m, H-8
), 1.05 (3H, s, CH3-50-methyl), 1.10 (1H, m, H-7
), 1.14 (1H, m, H-12
), 1.40 (1H, m, H-15 ), 1.66 (1H, m, H -15 ), 1.77 (1H, m, H-7
), 2.17 (3H, s, N-CH3), 2.20 (1H, m, H-11
β
), 0.92 (3H, s, CH3-18),
1.02 (1H, m, H-11
β
α
α
), 1.25 (1H,
m, H-14
α
β
α
β
), 1.88 (1H, m, CH, H-16
α
),
),
2.00 (1H, m, H-9
2.66 (1H, m, H-6
α
β
α
), 2.47 (1H, m, H-12
β
), 2.57 (1H, m, H-6
α
), 2.84 (1H, m, pyrazoline-50), 4.88 (1H, s, OH, disappeared with D2O), 5.79 (1H, dd,
H-2), 6.68 (1H, d, H-4), 7.19 (1H, d, H-1). 13C NMR: (150 MHz, CDCl3):
δ 13.35, 21.80, 24.27, 25.30,
26.07, 28.20, 29.77, 36.76, 38.70, 43.67, 45.67, 48.50, 50.70, 64.45, 112.37, 115.54, 126.46, 132.40, 138.77,
153.44, 155.42 (21 C). MS (EI): m/z 324 (100%) [M+]. Anal. Calcd for C21H28N2O (324.45): Calcd C,
77.74; H, 8.70; N, 8.63. Found C, 77.66; H, 8.64; N, 8.58.
1‘-Phenyl-1‘H-estra-1(10),2,4-trien-[17,16-c]pyrazoline-3-ol (4c)
Yield 70%, mp 255–257 ◦C, [α]D25 = + 128 (c 1, MeOH). IR (KBr, cm–1): 3347 (OH), 3071 and 3061
(CH, aromatic), 2931 (CH, aliphatic), 1633 (C=C), 1614 (C=N). 1H NMR: (600 MHz, CDCl3):
δ
ppm
), 1.20 (1H, m, H-12 ),
), 1.77 (1H, m, H-7 ), 1.86 (1H, m, CH,
), 2.48 (1H, m, H -12 ), 2.54 (1H, m, H-6 ), 2.64 (1H, m,
0.60 (1H, m, H-8
1.28 (1H, m, H-14
H-16 ), 2.04 (1H, m, H-9
H-6
d, H-4), 7.10 (1H, d, H-1), 7.28-7.40 (5H, m, Ar-H). 13C NMR: (150 MHz, CDCl3):
β), 0.90 (3H, s, CH3-18), 1.00 (1H, m, H-11
β), 1.10 (1H, m, H-7
α
α
α
), 1.49 (1H, m, H-15
β
), 1.68 (1H, m, H-15
α
β
α
α
), 2.26 (1H, m, H-11
α
β
α
β), 2.84 (2H, d, pyrazoline-5‘), 5.00 (1H, s, OH, disappeared with D2O), 5.71 (1H, dd, H-2), 6.70 (1H,
δ
13.49, 21.24, 25.55,
26.46, 28.51, 29.67, 36.64, 38.45, 43.35, 50.73, 58.26, 64.45, 112.10, 112.48, 115.46, 119.31, 126.10, 129.20,
132.52, 138.79, 152.20, 153.13, 163.48 (25 C). MS (EI): m/z 372 (81%) [M+]. Anal. Calcd for C25H28N2O
(372.50): Calcd C, 80.61; H, 7.58; N, 7.52. Found C, 80.52; H, 7.50; N, 7.48.
1‘-Phenyl-1‘H-5‘-methyl-estra-1(10),2,4-trien-[17,16-c]pyrazoline-3-ol (4d)
Yield 75%, mp 214–216 ◦C, [α]D25 = + 127 (c 1, MeOH). IR (KBr, cm–1): 3345 (OH), 3068 (CH, aromatic),
2949 (CH, aliphatic), 1625 (C=C), 1614 (C=N). 1H NMR: (600 MHz, CDCl3):
δ
ppm 0.62 (1H, m, H-8
), 1.05 (3H, s, CH3-5‘-methyl), 1.12 (1H, m, H-7 ), 1.17 (1H,
), 1.41 (1H, m, H-15 ), 1.71 (1H, m, H -15 ), 1.77 (1H, m, H-7 ), 1.87
), 2.02 (1H, m, H-9 ), 2.22 (1H, m, H-11 ), 2.46 (1H, m, H -12 ), 2.54 (1H, m, H-6 ),
), 3.83 (1H, s, pyrazoline-5‘), 4.91 (1H, s, OH, disappeared with D2O), 5.81 (1H, dd,
H-2), 6.73 (1H, d, H-4), 7.21 (1H, d, H-1), 7.28-7.48 (5H, m, Ar-H). 13C NMR: (150 MHz, CDCl3):
13.81,
β),
0.93 (3H, s, CH3-18), 1.02 (1H, m, H-11
m, H-12 ), 1.27 (1H, m, H-14
(1H, m, CH, H-16
2.65 (1H, m, H-6
β
α
α
α
β
α
β
α
α
α
β
α
β
δ
21.89, 24.25, 25.80, 26.35, 28.34, 29.23, 36.56, 38.67, 43.34, 48.55, 50.67, 64.44, 112.70, 115.34, 119.10, 126.76,
129.50, 132.45, 138.56, 152.40, 154.36, 163.56, 164.55 (26 C). MS (EI): m/z 386 (23%) [M+]. Anal. Calcd for
C26H30N2O (386.52): Calcd C, 80.79; H, 7.82; N, 7.25. Found C, 80.70; H, 7.76; N, 7.20.
3.2. Biological Screening
3.2.1. 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
supplemented with 10% FBS, 100 , 1% antibiotic/antimycotic solution and 3.6 g/L NaHCO3 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 L/well), then incubated for 24 h
at standard conditions. Afterwards, cells were exposed to different concentrations from prepared
,27]. SKOV-3 cells cultivated on DMEM medium
×
,
,
µ

Molecules 2019, 24, 2054
12 of 15
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 IC50 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.
3.2.2. 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
×
106 cells/site). Upon tumor development,
(av. volume ~50 mm3), 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 = Vtvolume at time t/V0initial volume
.
3.2.3. 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.
3.2.4. 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
coated with glutathione (Pierce Biotechnology). Two DMSO-dilutions of each compound (1
added to 50
L (50mM HEPES, pH7.0, 0.7 pmol of BRAFV600E kinase). Tubes were incubated at ambient
temperature for 1 h, then added to GST-MEK-His-plate wells. Afterwards, 50 L of phosphorylation
µL, 50
µg/mL), and then bound to 96-plate wells
µL) were
µ
µ
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

Molecules 2019, 24, 2054
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response curve, a four-parameter logistic model using GraphPad Prism, for BRAF proteins, which was
used to obtain corresponding IC50 values”.
3.2.5. Statistical Analysis
“Measurements were carried out in thrice and data are represented as means
±
SEM. Significance
was calculated by Student’s t-test using SPSS software (SPSS Inc., Paris, France). * p <0.05, ** p < 0.01,
***
p < 0.001. IC50 values were calculated using GraphPad Prism software, GraphPad Software,
San Diego, CA, USA [36]”.
4. 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 IC50 value
of 4.23
±
0.12 nM. Additionally, in vivo xenograft ovarian cancer model showed that Cpd. 3a was
0.7% after 40 days of treatment. Furthermore, the newly
able to reduce tumor growth by 93.61
synthesized compounds were able to interfere with tumor proliferation through inhibiting the activity of
topoisomerase II and V600EBRAF, where the obtained IC50 values for both enzymes were 3.45
and 0.041 ± 0.0016 µM, respectively.
±
±
0.13 nM
Author Contributions: M.E.-N., A.E.-G.E.A., A.A.F. performed most of the experiments; A.E.-G.E.A., M.A.A.-O.,
A.A.F. analyzed the data; E.A.E. and M.M.A. the contributed to the anticancer activity assays; All authors read
and approved the final manuscript.
Funding: The authors are grateful to the Deanship of Scientific Research, king Saud University for funding
through Vice Deanship of Scientific Research Chairs.
Acknowledgments: The authors are grateful to the Deanship of Scientific Research, king Saud University for
funding through Vice Deanship of Scientific Research Chairs.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).